Heat As Medicine: Omicron / Flu Cure in 2 Hours - Training Manual by India Book of Records

H E AT AS M E D I C I N E Training Manual

OMICRON / FLU

CURE IN 2 HOURS Dr. Biswaroop Roy Chowdhury

T R A I N I N G M A N UA L i

Pain Management Cancer Therapy Self Dialysis Heart Attack prevention Curing Insomnia & Depression

To design your own personal GRAD-Dialysis Tub

go to www.biswaroop.com/Dialysistub The bath tub that cleanses your body from inside

Note:

This Training Manual will be meaningful only after watching the video “Heat As Medicine”

Section-I

Training Manual

Heat As Medicine SARCoV-2 survives for 28 days at 20°C Less than 24 hours at 40°C Reference: 1 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Heat As Medicine Corona virus become inactive 60°C for 30 min 65°C for 15 min 80°C for 1 min Reference: 2 

Training Manual

Mechanism of Heat Heat inactivates virus

 Denaturing structure of protein  Alter configuration of protein in attachment & replication Reference: 3 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Killing Human

Killing Virus

100 years

Normal Lifespan

3-4 weeks

No food

Paracetamol Full Lifespan Antibiotics Steroids 60°C 30 min

3-4 days

No water

65°C

15 min

3-4 min

No Oxygen

80°C

1 min

4-30 sec

Beheaded

100°C

Few seconds

Training Manual

In vaccine manufacturing, virus is deactivated by heating the virus Reference: 4 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Heat & Humid Cold & Dry

Reference: 5 

Training Manual

Barrier for Virus Nasal Cavity Mucosal Barrier

Heat exchange

Reference: 6, 7 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Heaven for Virus Winter → Restricted Sunlight → Dry Air ↓ Nasal cavity becomes coldest part of the body Reference: 8 

Training Manual

Heat applied to the body  Direct inhibition of pathogen  Stimulation of innate & adaptive arm of immune system.  Reduce inflammatory response  Prevents tissue damage Reference: 9 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Heat stress applied on body to mimic fever thus supporting and activating immune system, the second line of defense. Reference: 10, 11 

Training Manual

Defense – Stage 1 Direct application of heat to upper airways, at the first sign of infection inhibits virus replication. Reference: 12 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Defense – Stage 2 Heat stress (on body) alters blood pH, creating alkaline condition – favourable condition for immune system. Reference: 13 

Training Manual

Skin – The Third Kidney Blood supply – 22% of cardiac output Ability of eliminate – 12 L liquid Skin excretes sodium, potassium, urea, creatinine, calcium, phosphorous, glucose, drug substance and heavy metals. Reference: 14 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Key to activate skin Rise is temperature & rise in sweat (10°C rise in temperature triples sweating)

Reference: 15 

Training Manual

Lower Leg Hot Water Immersion    

Improves immune response Improves blood circulation Counteracting infection Effective in reducing body temperature Reference: 16 , 17 

Heat As Medicine – Omicron / Flu Cure in 2 hours

Warm Water v/s Paracetamol

Rate of reduction of body temperature is faster in warm water therapy in first 30 min.

Reference: 18 

Training Manual

Nasal hyperthermia can give immediate relief from symptom. Reference: 19 

Heat As Medicine – Omicron / Flu Cure in 2 hours

With 1st sign of Flu / 1st 30 min Step-I

Lower leg hot water immersion (42°C to 43°C)

With lavender oil 3 Step Heat Protocol

Reference: 20 

Training Manual

With 1st sign of Flu / 1st 30 min Step-II

Sipping very-very hot water (>80°C) about 500 ml with lemon drops 3 Step Heat Protocol

Heat As Medicine – Omicron / Flu Cure in 2 hours

With 1st sign of Flu / 1st 30 min Step-III

Nasal irrigation (Jal neti) (40°C to 42°C) about 200 ml with salt (one spoon) 3 Step Heat Protocol

Training Manual

3 Step Heat Protocol 200 ml warm water (40°C to 42°C) for Jal neti (Nasal irrigation) with salt

Hot water (>80° C) with lemon drops (to drink) Lower leg hot water (42°C to 43°C with lavender oil) immersion for 30 min

Electric kettle Salt Pomegranate (to roast) Lavender oil Water thermometer

Extra hot water (>50°C)

Lemon

For details, go to: www.biswaroop.com/heatprotocol

Heat As Medicine – Omicron / Flu Cure in 2 hours

3 Step Heat Protocol – 30 min

Step I → LL HWI (Lavender oil) Step II → Sipping very hot water (500 ml + Lemon) Step III → Jal neti (200 ml water + salt)

Training Manual

Whooping Cough

Roast pomegranate for 4 to 5 minutes + Eat while its hot Heat As Medicine

Heat As Medicine – Omicron / Flu Cure in 2 hours

Prevention

Repeat the 3 Step Heat Protocol 2-3 times the same day + Follow 3 Step Flu Diet Read N.I.C.E way to Cure COVID-19

Training Manual

Treatment of respiratory illnesses (pneumonia, tuberculosis, asthma) 3 Step Heat Protocol + DIP Diet (minus plate 2 in lunch)

For one to 3 months

Heat As Medicine – Omicron / Flu Cure in 2 hours

If you are ready with 3 Step Heat Protocol, then register with us at www.biswaroop.com/heatprotocol & give N.I.C.E service to the nation

Section-II

Reference 1 (2020) 17:145 Riddell et al. Virol J https://doi.org/10.1186/s12985-020-01418-7

Open Access

RESEARCH

The effect of temperature on persistence of SARS-CoV-2 on common surfaces Shane Riddell* , Sarah Goldie, Andrew Hill, Debbie Eagles and Trevor W. Drew

Abstract Background: The rate at which COVID-19 has spread throughout the globe has been alarming. While the role of fomite transmission is not yet fully understood, precise data on the environmental stability of SARS-CoV-2 is required to determine the risks of fomite transmission from contaminated surfaces. Methods: This study measured the survival rates of infectious SARS-CoV-2, suspended in a standard ASTM E2197 matrix, on several common surface types. All experiments were carried out in the dark, to negate any effects of UV light. Inoculated surfaces were incubated at 20 °C, 30 °C and 40 °C and sampled at various time points. Results: Survival rates of SARS-CoV-2 were determined at different temperatures and D-values, Z-values and half-life were calculated. We obtained half lives of between 1.7 and 2.7 days at 20 °C, reducing to a few hours when temperature was elevated to 40 °C. With initial viral loads broadly equivalent to the highest titres excreted by infectious patients, viable virus was isolated for up to 28 days at 20 °C from common surfaces such as glass, stainless steel and both paper and polymer banknotes. Conversely, infectious virus survived less than 24 h at 40 °C on some surfaces. Conclusion: These findings demonstrate SARS-CoV-2 can remain infectious for significantly longer time periods than generally considered possible. These results could be used to inform improved risk mitigation procedures to prevent the fomite spread of COVID-19. Keywords: Environmental stability, SARS-CoV-2, COVID-19, Survivability Background The World Health Organization (WHO) declared SARSCoV-2 a pandemic on 11th March 2020 and as at the 7th August 2020, there have been over 18.8 million confirmed cases with more than 708,000 reported deaths from SARS-CoV-2 [1]. The transmission of SARS-CoV-2 appears to be primarily via aerosols [2–4] and recent studies have shown that SARS-CoV-2 is able to remain infectious in airborne particles for greater than 3 h [5, 6]. The role of fomites in the current pandemic is yet to be fully determined, although they have been suggested as a potential mode of transmission [7] also reflected by the strong focus on *Correspondence: Shane.Riddell@csiro.au Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Centre for Disease Preparedness, Geelong, VIC, Australia

hand-washing by WHO and national control schemes. Broadly, viruses have been shown to be readily transferred between contaminated skin and a fomite surface [8], with high contact surfaces such as touchscreens on mobile phones, bank ATMs, airport check-in kiosks and supermarket self-serve kiosks all acting as fomites for the transmission of viruses [9]. Fomite transmission has previously been shown to be a highly efficient procedure, with transmission efficiencies of 33% for both fomite to hand and fingertip to mouth transfer for bacteria and phages [10]. With the high efficiency of fomite transfer, the persistence of SARS-CoV-2 on environmental surfaces is therefore a critical factor when considering the potential for fomite transmission for this virus. Currently, there are conflicting reports on the survivability of SARS-CoV-2, with data ranging from 3 to 14 days at room temperature for a single surface type, stainless steel

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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(2020) 17:145

cabinet (BSCII) at room temperature and humidity prior to inoculation.

[5, 11]. This study aims to provide environmental stability data for SARS-CoV-2 under controlled temperature and humidity conditions for a range of common surfaces.

Surface inoculation and sampling

Methods

Stock virus was diluted in a defined organic matrix, consisting of bovine serum albumin (BSA), mucin and tryptone, following international standard ASTM E2197 [15], designed to mimic the composition of body secretions. Briefly, 360 µL of virus stock was added to 160 µL of a solution consisting of 2.5 mg/mL BSA, 3.5 mg/mL tryptone and 0.8 mg/mL mucin. Ten microlitres of the resulting suspension (final concentration of 3.38 × 105/10 µL) was inoculated onto the centre of the coupon and allowed to dry in a BSCII for 1 h. Once dry, the coupons were placed into a humidified climate chamber (Memmert HPP110) for specified time points. Samples were incubated in the dark to limit any effect light might have on viral decay. A single humidity set point (50% relative humidity) was maintained for each of three separate temperature experiments (20 °C, 30 °C, 40 °C). For the 20 °C and 30 °C temperature experiments, three replicates of each surface type were inoculated and sampled at the following time points; 1 h, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days post inoculation. For the 40 °C experiment, triplicate samples were inoculated for the following time points; 1 h, 1 day, 2 days, 3 days, 4 days, and 7 days. For non-porous surfaces, for each replicate, virus was eluted in 2 × 115 µL volumes of DMEM with repeated pipetting then titrated individually, in quadruplicate wells on a 96-well plate. For recovery from cotton cloth, inoculated swatches of the cloth were individually submersed in 500 µL DMEM and pipetted repeatedly for at least 1 min before 230 µL of the recovered eluent from each swatch was titrated separately, in quadruplicate. Suspensions of Vero E6 cells (3 × 105/mL) were added to the wells and the plates were incubated for 3 days at 37 °C with 5% CO2. Wells were scored for the presence of cytopathic effect and titres calculated using the Spearman–Karber method.

Virus isolate

The SARS-CoV-2 isolate (Betacoronavirus/Australia/ SA01/2020) used in this study was kindly supplied by the Peter Doherty Institute (Victoria, Australia) on behalf of South Australian Health (South Australia). The virus was passaged four times through Vero E6 cells (ATCC CRL1586) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Penicillin, Streptomycin, Fungizone and 10% fetal calf serum and pelleted via ultracentrifugation at 100,000×g for 90 min. The virus was resuspended in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) and stored at − 80 °C. The virus stock was titrated on Vero E6 cells and the TCID50 was determined to be 4.97 × 107/mL by the Spearman–Karber method [12, 13]. All work with infectious SARS-CoV-2 was conducted in the high containment laboratory (Biosafety level 4) at the Australian Centre for Disease Preparedness. Surfaces

Australian polymer bank notes, de-monetised paper bank notes and common surfaces including brushed stainless steel, glass, vinyl and cotton cloth were used as substrates in this study. Both polymer and paper banknotes were included in the study to gather information on the possible roles of note based currency in general for the potential for fomite transmission. Stainless steel is used in kitchen areas and public facilities and is the substrate used in some disinfectant testing standards [14, 15]. Glass was chosen due to its prevalence in public areas, including hospital waiting rooms, public transport windows and shopping centres, and high contact surfaces such as mobile phone screens, ATMs and self-serve check-out machines. Vinyl is a common substrate used in social settings, tables, flooring, grab handles on public transport, as well as mobile phone screen protector material. Cotton was chosen as a porous substrate, often found in clothing, bedding and household fabrics. All surfaces were prepared by cutting into approx. 1–1.5 cm2 coupons, non-porous surfaces were disinfected prior to use by washing in a mild detergent (Beckman 555), rinsing in distilled water and then immersing in 80% v/v ethanol. Paper bank notes (in very good condition) were heated in a dry oven to 75 °C for 1 h to reduce bacterial/viral contamination. The 100% cotton cloth was steam sterilised prior to use. Following preparation, all surfaces were placed into a petri dish and allowed to dry in a class II biological safety

Statistical analysis

Data analysis (regression analysis) and graphical representations were performed using GraphPad Prism (version 5). Decimal reduction time (D value—time at which there was a one log/90% reduction in titre) was calculated using

t logN0 − logNf

Z-values (temperature change required to achieve a tenfold (i.e. 1 log10) change in the D value) was calculated

et al. Virol– JOmicron (2020)/17:145 Heat Riddell As Medicine Flu Cure in 2 hours

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by plotting log D values against temperature. Calculated using: Z = (t2 − t1 )/ logD1 − logD2 The half-life of each surface was calculated using;

t1/2 =

log10 2 k

Results At 20 °C, infectious SARS-CoV-2 virus was still detectable after 28 days post inoculation, for all non-porous surfaces tested (glass, polymer note, stainless steel, vinyl and paper notes). The recovery of SARS-CoV-2 on porous material (cotton cloth) was reduced compared with most non-porous surfaces, with no infectious virus recovered past day 14 post inoculation. The majority of virus reduction on cotton occurred very soon after application of virus, suggesting an immediate adsorption effect. The calculated D values for surfaces at 20 °C ranged from 5.5 days for cotton to 9.1 days for paper notes and are shown in Table 1. At 30 °C, infectious virus was recoverable for 7 days from stainless steel, polymer notes and glass, and 3 days for vinyl and cotton cloth. For paper notes, infectious virus was detected for 21 days, although there was less than 1 log of virus recovered for both 14 day and 21 day time points. The D values for surfaces at 30 °C ranged from 1.4 days for vinyl to 4.9 days for paper notes (Table 1). At 40 °C, virus recovery was significantly reduced compared to both 20 °C and 30 °C experiments. Infectious SARS-CoV-2 was not recovered past 24 h for cotton cloth and 48 h for all remaining surfaces tested. Greater than

Table 1 Calculated D values (time taken to achieve a 90% reduction in titre) and half-life (time taken to achieve a 50% reduction in titre—in parentheses) for all surfaces at 20 °C, 30 °C and 40 °C D values (half-life)

Z value

20 °C—days 30 °C—days 40 °C – hours (°C) Stainless steel

5.96 (1.80)

1.74 (12.6 h)

4.86 (1.5 h)

13.62

Polymer note

6.85 (2.06)

2.04 (14.7 h)

4.78 (1.4 h)

13.02

Paper note

9.13 (2.74)

4.32 (32.7 h)

5.39 (1.6 h)

12.43

Glass

6.32 (1.90)

1.45 (10.5 h)

6.55 (2.0 h)

14.65

Cotton

5.57 (1.68)

1.65 (11.0 h)

–

18.91

Vinyl

6.34 (1.91)

1.40 (10.1 h)

9.90 (3.0 h)

16.86

Calculated Z values (temperature shift required to alter D value by 1 log). No infectious virus was recovered for cotton cloth at 40 °C at 24 h, D values were not able to be calculated

4-log reduction (99.99% reduction from starting titre) was observed in less than 24 h at 40 °C on all surfaces. The D values for surfaces at 40 °C have been converted to hours as they were all less than 1 day, values ranged from 5 h for polymer notes to 10.5 h for vinyl (Table 1). For each temperature and substrate material, the mean titre from three replicates of recovered virus was plotted against time, with standard deviations included. Linear regression was used to calculate a line of best fit. Plots showing virus survival on each substrate at the three temperatures investigated are shown in Fig. 1. Plots presenting this data grouping all substrates at each of the three temperatures are given in Fig. 2. Calculated D-value, Half Life and Z-value are presented in Table 1. An additional table containing average titre and standard deviation for all substrates, time points and temperatures is available (See Additional file 1).

Discussion While the primary spread of SARS-CoV-2 appears to be via aerosols and respiratory droplets, fomites may also be an important contributor in transmission of the virus. Fomite transmission has been demonstrated as an important factor in the spread other coronaviruses such as porcine epidemic diarrhea virus [16], as well as being suspected for Middle East Respiratory Syndrome coronavirus [17], human coronavirus 229E and OC43 [18] and SARS-CoV-2 [7]. This study utilised a virus concentration of 4.97 × 107/ mL diluted into a standard solution which mimics body fluid composition (final concentration of 3.38 × 105/10 µL inoculum), which equates to a cycle threshold (CT) value of 14.2, 14.0 and 14.8 for N gene, E gene and RdRp gene real time RT-PCR, respectively (unpublished data). Previous studies have shown some patients with high viral loads have recorded CT values of between 13 and 15 [19– 21]. van Doremalen et al. [5] described their test material (105 TCID50/mL) as having a CT of 20–22, which compared similarly to CTs reported from clinical patients [5, 22]. While the titre of virus utilised in this study is high it represents a plausible amount of virus that may be deposited on a surface. The present study has demonstrated that in controlled conditions, SARS-CoV-2 at a starting viral load and in a fluid matrix equivalent to that typically excreted by infected patients, remains viable for at least 28 days when dried onto non-porous surfaces at 20 °C and 50% relative humidity. Research on the original SARS virus also showed recovery of infectious virus when dried on plastic for up to 28 days at room temperature and 40–50% RH [23]. Recent data published on SARS-CoV-2 survivability on hospital PPE observed viable virus up to 21 days post inoculation on both plastic and N95 mask material when

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Fig. 1 Recovery of infectious SARS-CoV-2 for all surfaces and temperatures over time, TCID50 data is plotted in log10 intervals. No infectious virus was recovered at 24 h at 40 °C for cotton cloth. LoD (limit of detection) is recorded as 0.8 Log10 TCID50

Fig. 2 Grouping of each surface for individual temperatures. Trend lines for 20 °C show similar slopes, including for cotton cloth (although a reduced recovery was observed). A single well of virus was observed for paper banknotes in one out of three replicates for both 14 days and 21 days.

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held at room temperature [11], correlating with the data presented in this study. The persistence of SARS-CoV-2 on surfaces presented here and from Kasloff et al. [11] demonstrate significantly longer time points than previously published data for SARS-CoV-2 [5, 24]. These earlier studies reported recovery of infectious SARS-CoV-2 up to 3 days post inoculation and 4 days on non-porous surfaces, respectively. The titre of virus used in this study is at least 2 logs higher than used in the paper by van Doremalen et al. [5], which may account for the longer survivability. Work by Lai et al. has shown that stability of SARS virus was enhanced with higher concentrations [25]. Temperature and humidity are both critical factors in viral survivability with an increase in either being detrimental to virus survival [23, 26, 27]. Survivability on stainless steel coupons for transmissible gastroenteritis virus and murine hepatitis virus (both coronaviruses) was reduced with higher humidity’s and temperature [28] and survivability of Middle East Respiratory Syndrome coronavirus also followed a similar pattern [29]. The higher humidity of ~ 65% RH used by Chin et al. [24] may explain the shorter persistence of virus when compared to the data presented here. SARS-CoV-2 has been shown to be rapidly inactivated under simulated sunlight [30, 31]. To remove any potential decay by light sources, inoculated coupons were held in the dark for the duration of the experiment. Decimal reduction (D value; the timetaken to reduce the titre by 1 log) for SARS-CoV-2 at 20 °C and 50%RH ranged from 5.57 to 9.13 days (average 6.82) for all surfaces tested. This data is significantly longer than modelling predications performed by Guillier et al. [32]. The data presented here was performed under controlled conditions with fixed temperatures, relative humidity, suspension matrix and in the absence of light, which may explain the enhanced survivability observed in this study. The generation of Z values at different temperatures also allows for extrapolation of D values for each surface at other temperatures. The Z value represents the temperature change required to alter the D value by 1 log. For stainless steel, the D value was determined to be 6.48 days at 20 °C, and the Z value of 13.62 °C, therefore if the temperature was to drop by 13.62 °C from 20 °C (i.e. to 6.38 °C), then the D value would increase from 6.48 days to over 64 days. This data could therefore provide a reasonable explanation for the outbreaks of COVID-19 surrounding meat processing and cold storage facilities. The data also supports the findings of a recent publication on survival of SARS-CoV-2 on fresh and frozen food [33]. Stainless steel is a common surface for study of viral stability, and has been used to study the persistence on a number of viruses such as Ebola virus, hepatitis virus,

Influenza A and Coronaviruses [28, 34–37]. This study demonstrates that SARS-CoV-2 is extremely stable on stainless steel surfaces at room temperature (> 28 days at 20 °C/50%RH) however, is less stable at elevated temperatures (7 days at 30 °C and < 48 h at 40 °C). Recovery of infectious virus on stainless steel has been observed for murine hepatitis virus and transmissible gastroenteritis virus for up to 28 days albeit at a lower humidity 20%RH [28]. Interestingly, the same study showed survivability at 20 °C and 50%RH was significantly less (4–5 days), further suggesting the humidity may play a significant role in virus survival. The persistence of virus on both paper and polymer currency is of particular significance, considering the frequency of circulation and the potential for transfer of viable virus both between individuals and geographic locations. While other studies have shown that paper notes harbour more pathogens than polymer notes [38], this data demonstrates that SARS-CoV-2 persists on both paper notes and polymer notes to at least 28 days at 20 °C, albeit with a faster rate of inactivation on polymer notes. Data presented in this study for banknotes is significantly longer than reported for other respiratory viruses such as Influenza A (H3N2) which demonstrated survival up to 17 days at room temperature [39]. It is also noted that prior to SARS-Cov-2 being declared a pandemic, China had commenced decontamination of its paper based currency, suggesting concerns over transmission via paper banknotes existed at the time [40, 41]. The United States and South Korea have also quarantined bank notes as a result of the pandemic [42, 43]. It is important to note that after 28 days, infectious SARS-CoV-2 was also recovered from stainless steel, vinyl and glass, suggesting survivability on paper or polymer banknotes was not very different from the other non-porous surfaces studied. The persistence on glass is an important finding, given that touchscreen devices such as mobile phones, bank ATMs, supermarket self-serve checkouts and airport check-in kiosks are high touch surfaces which may not be regularly cleaned and therefore pose a transmission risk of SARS-CoV-2. It has been demonstrated that mobile phones can harbour pathogens responsible for nosocomial transmission [44], and unlike hands, are not regularly cleaned [45]. The data presented in this study correlates well with previously published data for Influenza A (H1N1) which recovered infectious virus up to 22 days at 22 °C and 7 days at 35 °C [37]. The persistence of SARS-COV-2 on glass and vinyl (both common screen and screen protector materials, suggest that touchscreen devices may provide a potential source of transmission, and should regularly be disinfected especially in multi-user environments.

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The persistence of both SARS and SARS-CoV-2 on cotton has been demonstrated to be significantly shorter than on non-porous surfaces [11, 25]. The data presented here also shows a significant decrease in titre of recovered virus after just 1 h drying at room temperature (20 °C) the amount of virus recovered from cotton swatches was approximately 99% less than for comparable virus recovery time points for non-porous material. To verify the reduced recovery on cotton, virus was eluted 5 min after depositing on the cotton, as well as 1 h, the titre of recovered virus after 5 min was similar to that of non-porous surfaces (data not shown) suggesting the process of drying down was a significant factor for cotton material but not from the non-porous surfaces. Recovery of virus from porous substrates is also likely to be reduced compared to nonporous substrates due to adherence of the virus to the fabric fibres. When the rate of viral inactivation is considered over time rather than the gross reduction from the initial inoculum there is a more subtle difference from the non-porous surfaces. The D values for cotton at 20 °C, when compared other materials, are not significantly different from other substrates (eg. 5.6 days for cotton vs. 6.3 days for vinyl), and the slopes of the line which suggests the decay rate of virus is similar across substrates. This study also demonstrates significantly longer survival times on cotton (7 days) than previous reported [11, 25]. This difference could be due to differences in the types of cotton material used, the current study used 100% cotton cloth, while previous studies used either a cotton gown or cotton t-shirt.

Abbreviations ASTM: American Society for Testing and Materials; ATM: Automatic teller machine; BSCII: Biological Safety Cabinet, Class 2; BSA: Bovine serum albumin; CO2: Carbon dioxide; CT: Cycle threshold; DMEM: Dulbecco’s Modified Eagle Medium; E gene: Envelope gene of SARS-CoV-2; N gene: Nucleocapsid gene of SARS-CoV-2; N95: Non-oil 95 mask; PBS: Phosphate buffered saline; RH: Relative humidity; RT-PCR: Reverse transcription polymerase chain reaction; RdRp: Ribonucleic acid dependant ribonucleic acid polymerase; SARS: Severe Acute Respiratory Syndrome; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2; TCID50: Tissue Culture Infectious Dose—Fifty; U/V: Ultraviolet; WHO: World Health Organisation; V/V: Volume per volume.

Conclusions The data presented in this study demonstrates that infectious SARS-CoV-2 can be recovered from nonporous surfaces for at least 28 days at ambient temperature and humidity (20 °C and 50% RH). Increasing the temperature while maintaining humidity drastically reduced the survivability of the virus to as little as 24 h at 40 °C. The persistence of SARS-CoV-2 demonstrated in this study is pertinent to the public health and transport sectors. This data should be considered in strategies designed to mitigate the risk of fomite transmission during the current pandemic response.

Received: 7 August 2020 Accepted: 22 September 2020

Supplementary information Supplementary information accompanies this paper at https://doi. org/10.1186/s12985-020-01418-7. Additional file 1. Table of average titre and standard deviation for recovery of infectious SARS-CoV-2 for all substrates, time points and temperatures.

Acknowledgements Virus strain used in this study was generously provided by SA Health through the Victorian Infectious Disease Research Laboratory, Peter Doherty Institute, Melbourne, Victoria. Authors’ contributions SR—Conceptualisation, Data curation, Formal analysis, investigation, methodology, writing—original draft. SG—Conceptualisation, Data curation, Formal analysis, investigation, methodology, writing—review and editing. AH—Conceptualisation, Data curation, Formal analysis, writing—review and editing, supervision. DE—Conceptualisation, writing—review and editing, funding acquisition. TD—Conceptualisation, analysis methodology, writing—review and editing. All authors read and approved the final manuscript. Funding Funding for the climate chambers utilised in this study was provided by a SARS-CoV-2 collaboration grant from the Defence, Science and Technology Group (Australia). Availability of data and materials All data generated or analysed during the study is included in the Additional file 1. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests.

References 1. Coronavirus disease (COVID-19) pandemic. https://www.who.int/emerg encies/diseases/novel-coronavirus-2019. 2. Stadnytskyi V, Bax CE, Bax A, Anfinrud P. The airborne lifetime of small speech droplets and their potential importance in SARS-CoV-2 transmission. Proc Natl Acad Sci U S A. 2020;117(22):11875–7. 3. Morawska L, Milton DK. It is time to address airborne transmission of COVID-19. Clin Infect Dis. 2020. https://doi.org/10.1093/cid/ciaa9 39/5867798. 4. Zhang R, Li Y, Zhang AL, Wang Y, Molina MJ. Identifying airborne transmission as the dominant route for the spread of COVID-19. Proc Natl Acad Sci. 2020;117(26):202009637. 5. van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, Williamson BN, et al. Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1. N Engl J Med. 2020;382(16):1564–7. https://doi. org/10.1056/NEJMc2004973. 6. Smither SJ, Eastaugh LS, Findlay JS, Lever MS. Experimental aerosol survival of SARS-CoV-2 in artificial saliva and tissue culture media at medium and high humidity. Emerg Microbes Infect. 2020;9(1):1415–7. https://doi. org/10.1080/22221751.2020.1777906.

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16. 17.

18. 19.

20. 21. 22. 23. 24. 25. 26.

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Reference 2

Heat As Medicine – Omicron / Flu Cure in 2 hours

Reference 3 & 4

Heat As Medicine – Omicron / Flu Cure in 2 hours

References

Heat As Medicine – Omicron / Flu Cure in 2 hours

References

Heat As Medicine – Omicron / Flu Cure in 2 hours

Reference 5 Low ambient humidity impairs barrier function and innate resistance against influenza infection Eriko Kudoa,1, Eric Songa,1, Laura J. Yockeya,1, Tasfia Rakiba, Patrick W. Wonga, Robert J. Homerb,c, and Akiko Iwasakia,d,e,2 a Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; bDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06520; cDepartment of Pathology and Laboratory Medicine, Veterans Affairs Connecticut Healthcare System, West Haven, CT 06516; d Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511; and eHoward Hughes Medical Institute, Chevy Chase, MD 20815

In the temperate regions, seasonal influenza virus outbreaks correlate closely with decreases in humidity. While low ambient humidity is known to enhance viral transmission, its impact on host response to influenza virus infection and disease outcome remains unclear. Here, we showed that housing Mx1 congenic mice in low relative humidity makes mice more susceptible to severe disease following respiratory challenge with influenza A virus. We find that inhalation of dry air impairs mucociliary clearance, innate antiviral defense, and tissue repair. Moreover, disease exacerbated by low relative humidity was ameliorated in caspase-1/11–deficient Mx1 mice, independent of viral burden. Single-cell RNA sequencing revealed that induction of IFNstimulated genes in response to viral infection was diminished in multiple cell types in the lung of mice housed in low humidity condition. These results indicate that exposure to dry air impairs host defense against influenza infection, reduces tissue repair, and inflicts caspase-dependent disease pathology.

flu season interferon disease tolerance

| mucosal immunity | respiratory tract |

Mx1 congenic mice to study host response to IAV infection. Most laboratory mouse strains are highly susceptible to IAV infection due to a defective Mx1 gene, an important ISG against IAV (13, 14). Mx1 congenic mice reveal the necessity of RIG-I signaling through MAVS and TLR7 for inducing type I IFN response and controlling viral replication (15). In the absence of these sensors, compensatory activation of caspase-1/11 induces lung pathology and mortality, due to the formation of neutrophil extracellular traps (15). Thus, the Mx1 congenic mice provide a physiologically relevant model to study IAV infection and disease. Here, we examined the impacts of relative humidity (RH) on host response to IAV infection and disease outcomes in Mx1 congenic mice. We show that mice kept at low relative humidity (10–20% RH) experience more severe symptoms than those kept at higher relative humidity (50% RH). Lower RH impaired mucociliary clearance and tissue repair and blocked the induction of ISGs known to restrict IAV, resulting in higher viral burden. Further, disease exacerbation at low RH was facilitated Significance

nfluenza A viruses (IAVs) cause seasonal infections worldwide, leading to half a million deaths annually (1, 2). IAV outbreaks occur during the winter months in temperate regions, peaking between November and March in the Northern Hemisphere and between May and September in the Southern Hemisphere (3–5). Several factors are thought to contribute to this seasonality, including fluctuations in temperature, humidity, indoor crowding, and sunlight or vitamin D exposure (5–8). A key epidemiological study analyzing data collected over 30 y across the continental United States showed that a drop in absolute humidity, which is dependent on relative humidity and temperature, correlates most closely with the rise in influenza-related deaths (9). Experimental studies in guinea pigs demonstrate that low temperature and low humidity enable aerosol transmission of influenza virus, providing one explanation for the seasonality of viral transmission (10). While these studies clearly show that environmental conditions affect transmission of influenza virus, the impact of ambient humidity on host response to influenza virus infection and disease outcome remains unclear. During influenza infection, the respiratory mucosal barrier provides the first line of defense. The mucus layer, the surface liquid layer, and the cilia of the surface of the bronchus epithelia promote mucociliary clearance (MCC) of invading pathogens and particles. If the virus breaches these layers, the innate immune defense mechanisms triggered through recognition of viral pathogen-associated molecular patterns (PAMPs) by RIG-I and TLR7 will induce secretion of type I interferons (IFNs) to turn on hundreds of IFN-stimulated genes (ISGs) to block virus spread (11). If the virus manages to breach the innate defense layer, the adaptive immune system is engaged to induce virusspecific T and B cell immune responses critical for the clearance of IAV (12). A recent study highlights the importance of using

Influenza virus causes seasonal outbreaks in temperate regions, with an increase in disease and mortality in the winter months. Dry air combined with cold temperature is known to enable viral transmission. In this study, we asked whether humidity impacts the host response to influenza virus infections. Exposure of mice to low humidity conditions rendered them more susceptible to influenza disease. Mice housed in dry air had impaired mucociliary clearance, innate antiviral defense, and tissue repair function. Moreover, mice exposed to dry air were more susceptible to disease mediated by inflammasome caspases. Our study provides mechanistic insights for the seasonality of the influenza virus epidemics, whereby inhalation of dry air compromises the host’s ability to restrict influenza virus infection. Author contributions: E.S., L.J.Y., T.R., and A.I. designed research; E.K., E.S., L.J.Y., T.R., and P.W.W. performed research; E.S. and R.J.H. contributed new reagents/analytic tools; E.K., E.S., L.J.Y., T.R., P.W.W., R.J.H., and A.I. analyzed data; and E.K., E.S., L.J.Y., and A.I. wrote the paper. Reviewers: G.N., University of Michigan; and P.P., Icahn School of Medicine at Mount Sinai. Conflict of interest statement: This work was in part supported by a gift from the Condair Group. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. PRJNA528197). 1

E.K., E.S., and L.J.Y. contributed equally to this work.

To whom correspondence should be addressed. Email: akiko.iwasaki@yale.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1902840116/-/DCSupplemental. Published online May 13, 2019.

PNAS | May 28, 2019 | vol. 116 | no. 22 | 10905–10910

www.pnas.org/cgi/doi/10.1073/pnas.1902840116

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Contributed by Akiko Iwasaki, April 4, 2019 (sent for review February 19, 2019; reviewed by Gabriel Núñez and Peter Palese)

Heat As Medicine – Omicron / Flu Cure in 2 hours by inflammasome caspase activity. Thus, our data suggest that controlling the relative humidity is important to prevent influenza infection and disease outcomes in the dry winter season. Results Low Ambient Humidity Leads to More Severe Disease in Mx1 Congenic Mice. Mx1 mice at 50% RH were infected with vary-

ing doses of highly virulent IAV PR8 strain (hvPR8) to determine the LD50 (SI Appendix, Fig. S1). Based on this dose– response, we decided to use 2 × 105 pfu for aerosol challenge to approximate LD50. To investigate the impact of ambient humidity on the host response to influenza infection, we employed environment chambers to precondition mice at different RH for 4–5 d at 20% or 50% RH while holding temperature constant at 20 °C before respiratory challenge with hvPR8. Immediately following infection, mice were returned to the environmental chambers and maintained in the respective RH for up to 7 d after infection. When challenged with 2 × 105 pfu/mL of aerosolized hvPR8, Mx1 mice housed at 20% RH suffered a worse disease course compared with those kept at 50% RH, with more rapid weight loss, drop in body temperature, and shortened survival (Fig. 1). These data indicated that low humidity renders Mx1 mice more susceptible to IAV disease.

Low Ambient Humidity Predisposes Mice to Caspase-1/11-Dependent Influenza Disease. Our previous study showed that caspase-1/11–

dependent inflammation underlies disease pathogenesis of influenza virus in Mx1 congenic mice (15). Thus, we examined the role of these inflammasome caspases on IAV disease in mice exposed to different humidity levels. Notably, unlike WT Mx1 mice, caspase-1/11 KO Mx1 mice were spared from influenza disease exacerbation even when preconditioned in low ambient humidity of 10% RH (Fig. 2). These results indicated that dry air exposure predisposes mice to severe IAV disease in a caspase-1/11–dependent manner.

Low Humidity Exposure Impairs Viral Clearance Independent of T Cell Immune Responses. To determine whether ambient humidity al-

ters respiratory viral clearance, we measured viral titers in the

Fig. 1. Low relative humidity predisposes Mx1 congenic mice to influenza disease. Mx1 congenic mice were preconditioned at 20% and 50% RH for 5 d and then challenged with aerosolized hvPR8 at 2 × 105 pfu/mL. (A) Weight loss, (B) core body temperature, and (C) survival were monitored for 11 d (n = 10 mice per group, pooled from two independent experiments). Data are representative of five experiments and means ± SEM *P < 0.05; one-way ANOVA; log-rank (Mantel–Cox).

Fig. 2. Low humidity increases influenza disease through caspase-1/11 activation. WT Mx1 mice or caspase-1/11 KO Mx1 mice were preconditioned at 10% and 50% RH for 5 d and challenged with aerosolized hvPR8 at 2 × 105 pfu/mL for 15 min (n = 6 mice per group). (A and B) Weight loss was monitored for 14 d. Data are representative of four experiments and means ± SEM *P < 0.05; one-way ANOVA; Student’s t test.

lungs of mice exposed to 10% vs. 50% RH and infected with influenza virus. While similar infectious virus doses are found in the lung at 2 d postinfection (d.p.i.), mice housed in 10% RH sustained slightly higher viral titers at 6 d.p.i. (indicative of continued viral growth), while those kept at 50% RH had reduced titers at 6 d.p.i. (indicative of viral control) (Fig. 3A), suggesting that higher humidity increased resistance to influenza virus. Interestingly, similar viral titers were observed in WT and caspase-1/11 KO Mx1 mice in 10% RH (Fig. 3A). However, caspase-1/11 KO Mx1 mice showed no significant weight loss (Fig. 2) despite the impaired resistance at 10% RH. When weight loss was plotted against viral titers, WT, but not caspase-1//11 KO Mx1 mice, showed positive correlation (SI Appendix, Fig. S2). These data indicated that low humidity renders mice less able to control IAV infection and that disease mediated at low humidity requires active inflammasome caspases. To examine the cell types infected by IAV, we harvested the lungs at 6 d.p.i. and stained for viral antigen. Influenza protein was detected in both the alveolar epithelial cells and in alveolar macrophages throughout the lungs of mice exposed to 10% RH, in both WT and caspase-1/11 KO Mx1 mice (Fig. 3 B and C). In contrast, virus staining was mostly confined to the alveolar macrophages of mice housed in 50% RH, with little staining observed in the epithelial cells in both genotypes (Fig. 3 B and C). Given the enhanced viral infection and delayed clearance at later time points in low humidity, we next examined whether 50% RH promotes a more robust adaptive immune response to confer protection against influenza infection. Since we observed differences in disease as early as 5 d.p.i. (Fig. 1), before the onset of a protective antibody response, we focused on T cell immunity to IAV. Analysis of IAV PA- or NP-specific tetramer+ CD8 T cells in the mediastinal lung draining lymph nodes (SI Appendix, Fig. S3 A and B) showed less IAV-specific CD8 T cells in mice exposed to 50% RH than 10% RH. This is presumably due to more IAV antigens being generated from higher viral burden at the 10% RH (Fig. 3). In contrast, we detected similar numbers of IAV-specific CD8 T cells in the lungs of mice infected at different humidity levels (SI Appendix, Fig. S3 C and D). These results collectively indicate that the protection provided by 50% RH is not through enhanced induction of T cell immune responses but more likely due to increased clearance of infectious virus by innate immune mechanisms. Low Humidity Exposure Impairs Tissue Repair of the Airway Epithelia.

Our data thus far indicated that exposure to low RH impairs antiviral resistance. Viral spread in the lung airway epithelia is

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Fig. 3. Low humidity impairs viral clearance independent of the adaptive immune response. Mx1 congenic WT mice or caspase-1/11 KO Mx1 mice were preconditioned at 10% and 50% RH for 5 d and challenged with aerosolized hvPR8 at 2 × 10 5 pfu/mL for 15 min (n = 4–6 mice per group). (A) Bronchoalveolar lavage collected at 2 and 6 d.p.i. Data are representative of four experiments and means ± SEM. There are not significant differences except between 10% WT and 50% WT. (B and C) Mice were killed on day 6 and lung sections from each group were subjected to immunohistochemistry with an antiinfluenza A antibody (B). Percentage of influenza positive area was assessed by image analysis (C). Data are means ± SEM *P < 0.05; one-way ANOVA. n.s., not significant.

S4B). The scRNA-seq of whole lung single-cell suspension revealed 22 distinct cell types belonging to epithelial, endothelial, phagocytes, and lymphocyte groups based on the t-distributed stochastic neighbor embedding (tSNE) clustering (SI Appendix, Fig. S5). An abundance of cell types shifted in a predictable manner, such as the influx of neutrophils in infected mice (SI Appendix, Fig. S6A, blue box). In addition, we detected changes in alveolar macrophage phenotype in infected mice (SI Appendix, Fig. S6A, red box). The changes in the alveolar macrophages in infected mice were due to genes up-regulated in pathways related to defense responses [Gene Ontology (GO):0006952], response to other organisms (GO:0051707), and other expected responses against flu (Fig. 6A and SI Appendix, Fig. S6F). Otherwise, no large changes in the composition of immune cells, endothelial, or epithelial cell populations were detected in response to infection or exposure to different levels of humidity (SI Appendix, Figs. S5 and S6). Strikingly, ISGs known to be critical in antiviral defense against IAV, namely, Mx1 (19), IFITM3 (20), IFITM2 (21), BST2 (22), Viperin (23), ISG15 (24), and ZAP (25) were all elevated in response to IAV infection across different cell types in mice exposed to 50% RH condition but not those kept in 10% RH environment (Fig. 6B). Of the cells that contained IAV viral RNA, a higher proportion of cells expressed Mx1 in mice housed at 50% RH compared with 10% RH (Fig. 6C, Left). Similarly, of the cells that were devoid of viral RNA, higher proportions also expressed Mx1 at 50% RH, suggesting that IFN-induced Mx1 expression is also more robust at the higher humidity (Fig. 6C, Right). Collectively, our data show that exposure of the host to low ambient humidity results in impaired MCC, reduced ISG

expected to result in tissue damage (16, 17). To investigate whether tissue repair mechanisms are impacted by the humidity of the inhaled air, we examined the proliferative response of lung epithelial cells before and after influenza infection. Exposure to low or normal humidity air in uninfected mice showed very little difference in epithelial proliferation (Fig. 4). In contrast, on day 6 after IAV infection, a much higher proportion of airway epithelial cells of mice housed in 50% RH were proliferative (Ki67+) compared with those kept in 10% RH (Fig. 4). These results suggest that the tissue repair function of epithelial cells might be impaired at 10% RH. Low Humidity Exposure Decreases Mucociliary Clearance in Mouse Trachea. Given that 10% RH exposure results in impaired viral

clearance, we next examined the impact of humidity on MCC. MCC is an important innate defense mechanism which removes pathogens, allergens, and debris by ciliary action (18). To determine the impact of low humidity exposure on the function and efficiency of the mucociliary transport, we measured MCC of the trachea of mice exposed to 10% vs. 50% RH. Tracheal MCC was significantly reduced in 10% RH compared with 50% RH (Fig. 5 and Movies S1–S3). Both the directionality of flow (Fig. 5C) and the flow speed generated (Fig. 5D) were severely impaired in the trachea of mice exposed to 10% RH (Movie S1) compared with 50% RH air (Movie S2).

Low Ambient Humidity Blocks IFN-Stimulated Gene Expression in the Lung. Finally, to examine the impact of humidity on host re-

sponse to IAV across multiple different cell types, we performed single cell RNA-sequencing (scRNA-seq) using samples obtained from the whole lung tissue of uninfected and IAVinfected mice housed in 10% and 50% RH. The weight of mice was monitored after infection (SI Appendix, Fig. S4A) and cell populations were analyzed by flow cytometry (SI Appendix, Fig.

Fig. 4. Low humidity impairs tissue repair of airway epithelial cells. (A and B) WT Mx1 and caspase-1/11 KO Mx1 mice were preconditioned at 10% and 50% RH for 5 d and challenged with aerosolized hvPR8 at 2 × 105 pfu/mL for 15 min (n = 4–6 mice per group). Mice were killed on day 6 and lung sections from each group were subjected to immunohistochemistry with an anti-Ki67 antibody (A). Ki67+ cells were assessed by image analysis (B). Data are means ± SEM **P < 0.01; one-way ANOVA. PNAS | May 28, 2019 | vol. 116 | no. 22 | 10907

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References

Heat As Medicine – Omicron / Flu Cure in 2 hours

Fig. 5. Low humidity decreases MCC. (A–D) WT Mx1 mice were preconditioned at 10% and 50% RH for 7 d, and tracheas were collected for MCC assay (n = 3 mice per group). Frequency of directionality and cilia-generated flow rate were measured by microscopy. (A) Maximum projected images of particle diffusion over a span of 1 s. (B) Representative particle trajectory over a span of 1 s. (C) Frequency chart of the directionality of particles in tracheas of Mx1 mice preconditioned at different humidity. (D) Cilia-generated flow was measured by multiple particle tracking. Water control was measured by diluting particles in water and loading them onto slides to simulate Brownian motion. Trachea control represents tracheas from WT mice that were collected and imaged 1 h later to ensure no flow was generated by dead tissue. The 10% and 50% RH tracheas were imaged within 5 min of being excised from mice. Data are means ± SEM ****P < 0.0001; one-way ANOVA. n.s., not significant.

expression and antiviral resistance, increased viral spread, tissue damage, impaired epithelial repair, and loss of disease tolerance to pathology mediated by the inflammasome caspases, leading to lethality from influenza infection. Discussion Seasonality of the influenza epidemic is linked to environmental factors such as the drop in ambient humidity and temperature. Low humidity has been shown to impact the transmission of IAV from an infected host to an uninfected host (7). In this study, we examine whether relative humidity impacts host responses to influenza infection using Mx1 congenic mice. Exposure of mice to low relative humidity increased their susceptibility to more severe IAV disease and faster lethality. Low relative humidity exposure rendered mice less competent to cope with the pathological consequences of inflammasome caspase activation. Moreover, low humidity exposure impaired the MCC function of the trachea, and tissue repair function of the airway epithelial cells, resulting in viral spread and tissue damage.

One of the likely reasons by which high relative humidity prevents influenza disease is through increasing the expression levels of anti-IAV ISGs, including Mx1 (19, 26–28), IFITM3 (20, 29, 30), IFITM2 (21), BST2 (22), Viperin (23), ISG15 (24), and ZAP (25). How does inhalation of dry air result in the inability of mice to induce antiviral ISGs in response to IAV infection? One possibility is the host stress response to cope with the stress of dehydration and loss of MCC and accumulation of particles in the respiratory tract may be incompatible with IFN and ISG induction. Our recent work showed that activation of the NRF2-dependent antioxidant pathway is incompatible with the induction of antiviral IFN and restriction of respiratory virus replication (31). Future work is needed to determine the precise mechanism by which dry air exposure leads to impaired antiviral signaling. Another reason for the inability of the host animal exposed to low humidity to clear the virus (Fig. 3A) may be due to the reduced MCC (Fig. 5) and removal of airway virus particles up the trachea. Our data showed that exposure to low relative humidity decreased MCC, both with respect to the direction of flow and the speed (Fig. 5). These results are consistent with the observations made in humans, that under low humidity and temperature, the length of the periciliary layer is reduced, movement of ciliary cells becomes decreased, and MCC slows down (32–34), possibly resulting in increased pathogen spread. Mice housed in 10% RH suffered from IAV disease that was driven by caspase-1/11. Of note, caspase-1/11–deficient mice housed at 10% RH harbored similar viral burden (Fig. 3A), viral spread (Fig. 3 B and C), and loss of epithelial proliferation (Fig. 4) to the WT mice housed at 10% RH. However, weight loss (i) did not correlate with viral titers (SI Appendix, Fig. S2) and (ii) was significantly less in caspase-1/11 KO mice compared with WT mice kept at low humidity (Fig. 2), indicating that caspase-1/11 KO mice were better able to cope with the same infection and were disease tolerant. These data show that caspase-1/11–mediated pathology is downstream of viral replication and epithelial repair. Our previous study showed that in Mx1 congenic mice, caspase-1/11–driven disease pathology was only evident when the host mice were deficient in innate resistance (TLR7 and MAVS deficient) (15). Our current study showed that low humidity impaired ISG induction across different cell types of the lung, resulting in higher viral burden and caspase-1/11– dependent pathology. Taken together, disease seen in the low humidity condition may be the result of a combination of factors, including reduced ISG expression, impaired antiviral resistance enabling higher viral burden, and caspase-1/11–dependent host damage. In summary, our study provides mechanistic insights through which ambient humidity impacts physical and innate immune defense against a respiratory viral infection. These mechanisms, such as impaired MCC and ISG induction may in part underlie the epidemiological correlation of a drop in absolute humidity preceding death from seasonal influenza infection in temperate regions (9). It is worth noting that humidity does not seem to affect host defenses against influenza viruses in all situations, as tropical and subtropical climate regions, which are wet and warm, allow for influenza virus to thrive (35, 36). Future studies will be required to understand why certain regions of the world may be affected differently by humidity and temperature. Our study suggests that increasing ambient humidity may be a viable strategy to reduce disease symptoms and to promote more rapid recovery in influenza-infected individuals. Materials and Methods Mice. C57BL/6 mice carrying a functional Mx1 allele (37) were used. Caspase-1/11 KO mice crossed to Mx1 congenic mice have been previous reported (15). Mice were maintained in our facility until the ages described. All procedures used in this study complied with federal and institutional policies of the Yale Animal Care and Use Committee. The study was approved by an institutional review board.

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References

Fig. 6. Analysis of low humidity impact by scRNA-seq. WT Mx1 mice were preconditioned at 10% and 50% RH for 7 d, and challenged intranasally with 750 pfu/mL of hvPR8. Uninfected and infected mice were killed on day 2 and lung tissue was subjected to scRNA-seq. (A) Differentially expressed genes in alveolar macrophages before and after infection. (B) Heatmap tSNE of flu-associated ISGs in different biological conditions. (C) Percentage of cells with Mx1 expression among the cells that are either positive or negative for influenza viral RNA.

Environmental Conditioning and Infection of Mice. Sex-matched 8- to 12-wk-old mice were kept in an environmental chamber (model 7000-10, Caron) for 4–5 d in which the RH was maintained at 10–20% RH for low humidity or 50% RH for normal humidity conditions. The temperature was maintained at 20 °C throughout the study. Mice were kept in the environmental chambers for up to 7 d.p.i. then moved to the ambient animal room conditions, which had a RH of 50–60% and temperature of 20–22 °C. For infection, highly virulent A/PR/8/ 34 (H1N1; hvPR8) IAV was delivered via intranasal or aerosol administration as indicated. The hvPR8 strain was adapted in Mx1 mice (38). Aerosol challenge was performed using an MPC aerosol nebulizer with aerosol pie cage (Braintree Scientific). Infection was performed in a biosafety cabinet at ambient animal room conditions. Before intranasal inoculation of IAV, mice were anesthetized by i.p. injection of ketamine and xylazine. Body weight, survival, and rectal temperature (MicroTherma 2T hand-held thermometer, Braintree Scientific) were monitored throughout the course of infections. Mucociliary Clearance Measurement. MCC was measured according to previous publications (39, 40). Custom slides with 2-mm indentations were made

to allow for whole trachea mounting. Mice were killed and tracheas were immediately removed, cut laterally to allow for the cilia to face the coverslip, and placed on the slide. Fifty microliters of FluoSpheres carboxylate, 0.2 μm crimson, 625/645 (Life Technologies) diluted 1:1,000 in PBS was placed on top of the trachea and mounted with coverslip. Within 5 min of trachea excision, the sample was imaged using an Opterra confocal microscope (Bruker) at 100 frames per second. All focus was matched to be ∼1 μm on top of the surface of the trachea, as flow rate decreases away from the surface of the tissue. Images were processed using ImageJ. Single-Cell RNA-Seq. Mice were housed at either 10% or 50% RH in an environmental chamber for 5 d and infected with intranasal hvPR8 or mock infected with PBS. At 2 d.p.i., mice were killed, perfused with PBS, and lungs were isolated. The lung tissues were dissociated using 20 μg/mL DNase I, 1 mg/mL Collagenase A in RPMI at 37 °C for 30 min, followed by passage through a 70-μm filter and ACK buffer to remove any residual red blood cells. The cells were loaded onto the chromium controller (10× Genomics). Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3′ v2 Reagent Kit (10× Genomics) PNAS | May 28, 2019 | vol. 116 | no. 22 | 10909

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according to the manufacturer’s protocol. Samples were sequenced on the HiSeq4000 with 28-bp read 1, 8 bp i7 index, and 98-bp read 2. Sequencing results were demultiplexed into Fastq files using Cell Ranger (10× Genomics, 2.2.0) mkfastq function. Samples were aligned to mm10-2.2.0 10× genome with custom flu virome annotations that included all eight segments of the influenza A PR8. The count matrix was generated using the count function with default settings. An estimate of 10,976 cells were sequenced (four conditions, duplicates) with a mean read number of 266,989 and median gene number per cell of 2,069. Matrices were loaded into Seurat v2 (41) for downstream analysis. Cells with fewer than 500 unique molecular identifers (UMIs) or high mitochondrial content were discarded. Cell types were determined using previously published datasets as refs. 42 and 43. For differential expression of genes in alveolar macrophages, cells from 10% and 50% uninfected were pooled and 10% and 50% infected were pooled. This differentially expressed gene list was used to create an expression heatmap including each of the four conditions to highlight the 1. Wong GH, Goeddel DV (1986) Tumour necrosis factors alpha and beta inhibit virus replication and synergize with interferons. Nature 323:819–822. 2. Johnson NB, et al.; Centers for Disease Control and Prevention (CDC) (2014) CDC National Health Report: Leading causes of morbidity and mortality and associated behavioral risk and protective factors–United States, 2005-2013. MMWR Suppl 63:3–27. 3. Tamerius JD, et al. (2013) Environmental predictors of seasonal influenza epidemics across temperate and tropical climates. PLoS Pathog 9:e1003194, and erratum 2013 Nov;9(11). 4. Alonso WJ, et al. (2015) A global map of hemispheric influenza vaccine recommendations based on local patterns of viral circulation. Sci Rep 5:1–6. 5. Tamerius J, et al. (2011) Global influenza seasonality: Reconciling patterns across temperate and tropical regions. Environ Health Perspect 119:439–445. 6. Cannell JJ, et al. (2006) Epidemic influenza and vitamin D. Epidemiol Infect 134:1129– 1140. 7. Lowen AC, Steel J (2014) Roles of humidity and temperature in shaping influenza seasonality. J Virol 88:7692–7695. 8. Eccles R (2002) An explanation for the seasonality of acute upper respiratory tract viral infections. Acta Otolaryngol 122:183–191. 9. Shaman J, Pitzer VE, Viboud C, Grenfell BT, Lipsitch M (2010) Absolute humidity and the seasonal onset of influenza in the continental United States. PLoS Biol 8: e1000316. 10. Lowen AC, Mubareka S, Steel J, Palese P (2007) Influenza virus transmission is dependent on relative humidity and temperature. PLoS Pathog 3:1470–1476. 11. Iwasaki A, Pillai PS (2014) Innate immunity to influenza virus infection. Nat Rev Immunol 14:315–328. 12. Chen X, et al. (2018) Host immune response to influenza a virus infection. Front Immunol 9:320. 13. Garber EA, Hreniuk DL, Scheidel LM, van der Ploeg LHT (1993) Mutations in murine Mx1: Effects on localization and antiviral activity. Virology 194:715–723. 14. Staeheli P, Grob R, Meier E, Sutcliffe JG, Haller O (1988) Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation. Mol Cell Biol 8: 4518–4523. 15. Pillai PS, et al. (2016) Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease. Science 352:463–466. 16. Taubenberger JK, Morens DM (2008) The pathology of influenza virus infections. Annu Rev Pathol 3:499–522. 17. Short KR, et al. (2016) Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions. Eur Respir J 47:954–966. 18. Bustamante-Marin XM, Ostrowski LE (2017) Cilia and mucociliary clearance. Cold Spring Harb Perspect Biol 9:a028241. 19. Verhelst J, Parthoens E, Schepens B, Fiers W, Saelens X (2012) Interferon-inducible protein Mx1 inhibits influenza virus by interfering with functional viral ribonucleoprotein complex assembly. J Virol 86:13445–13455. 20. Everitt AR, et al.; GenISIS Investigators; MOSAIC Investigators (2012) IFITM3 restricts the morbidity and mortality associated with influenza. Nature 484:519–523. 21. Brass AL, et al. (2009) The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:1243–1254. 22. Swiecki M, Wang Y, Gilfillan S, Lenschow DJ, Colonna M (2012) Cutting edge: Paradoxical roles of BST2/tetherin in promoting type I IFN response and viral infection. J Immunol 188:2488–2492. 23. Wang X, Hinson ER, Cresswell P (2007) The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts. Cell Host Microbe 2:96–105.

intensity difference of each gene in the relative humidity. The dataset is available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA528197 (44). Statistical Analysis. The data were analyzed by Student’s t test, one-way ANOVA with either Tukey’s or Kramer’s multiple comparison test, or log-rank (Mantel–Cox) tests. All statistical tests were calculated using GraphPad Prism (GraphPad software). A P value of <0.05 was considered statistically significant. ACKNOWLEDGMENTS. We thank Melissa Linehan for technical and logistical assistance, Guilin Wang and Christopher Castaldi (Yale Center for Genome Analysis) for 10× Chromium library preparations and sequencing help, and Dr. Adriano Aguzzi for scientific advice. This work was supported in part by the Howard Hughes Medical Institute (A.I.), a gift from the Condair Group, the Naito Foundation (E.K.), and National Institutes of Health Grants T32GM007205 (Medical Scientist Training Program training grant to L.J.Y. and E.S.) and F30 HD094717-01 (to L.J.Y.). 24. Lenschow DJ, et al. (2007) IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses. Proc Natl Acad Sci USA 104: 1371–1376. 25. Tang Q, Wang X, Gao G (2017) The short form of the zinc finger antiviral protein inhibits influenza A virus protein expression and is antagonized by the virus-encoded NS1. J Virol 91:e01909-16. 26. Arnheiter H, Skuntz S, Noteborn M, Chang S, Meier E (1990) Transgenic mice with intracellular immunity to influenza virus. Cell 62:51–61. 27. Kolb E, Laine E, Strehler D, Staeheli P (1992) Resistance to influenza virus infection of Mx transgenic mice expressing Mx protein under the control of two constitutive promoters. J Virol 66:1709–1716. 28. Haller O, Staeheli P, Kochs G (2007) Interferon-induced Mx proteins in antiviral host defense. Biochimie 89:812–818. 29. Allen EK, et al. (2017) SNP-mediated disruption of CTCF binding at the IFITM3 promoter is associated with risk of severe influenza in humans. Nat Med 23:975–983. 30. Randolph AG, et al.; Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network Pediatric Influenza (PICFLU) Investigators; Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network Pediatric Influenza (PICFLU) Investigators (2017) Evaluation of IFITM3 rs12252 association with severe pediatric influenza infection. J Infect Dis 216:14–21. 31. Mihaylova VT, et al. (2018) Regional differences in airway epithelial cells reveal tradeoff between defense against oxidative stress and defense against Rhinovirus. Cell Rep 24:3000–3007.e3. 32. Clary-Meinesz CF, Cosson J, Huitorel P, Blaive B (1992) Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells. Biol Cell 76:335–338. 33. Daviskas E, et al. (1995) Changes in mucociliary clearance during and after isocapnic hyperventilation in asthmatic and healthy subjects. Eur Respir J 8:742–751. 34. Oozawa H, et al. (2012) Effect of prehydration on nasal mucociliary clearance in low relative humidity. Auris Nasus Larynx 39:48–52. 35. Moura FE, Perdigão AC, Siqueira MM (2009) Seasonality of influenza in the tropics: A distinct pattern in northeastern Brazil. Am J Trop Med Hyg 81:180–183. 36. Shek LP, Lee BW (2003) Epidemiology and seasonality of respiratory tract virus infections in the tropics. Paediatr Respir Rev 4:105–111. 37. Horisberger MA, Staeheli P, Haller O (1983) Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza-virus. Proc Natl Acad Sci USA 80:1910–1914. 38. Grimm D, et al. (2007) Replication fitness determines high virulence of influenza A virus in mice carrying functional Mx1 resistance gene. Proc Natl Acad Sci USA 104: 6806–6811. 39. Mastorakos P, et al. (2015) Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy. Proc Natl Acad Sci USA 112:8720–8725. 40. Francis R, Lo C (2013) Ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia. J Vis Exp, e50343. 41. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36:411–420. 42. Cohen M, et al. (2018) Lung single-cell signaling interaction map reveals basophil role in macrophage imprinting. Cell 175:1031–1044.e18. 43. Steuerman Y, et al. (2018) Dissection of influenza infection In Vivo by single-cell RNA sequencing. Cell Syst 6:679–691.e4. 44. Kudo E, et al. (2019) Low ambient humidity impairs barrier function, innate resistance against influenza infection. NCBI BioProject. Available at https://www.ncbi.nlm.nih.gov/ bioproject/PRJNA528197. Deposited March 20, 2019.

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Effect of inspiration cycle and ventilation rate on heat exchange in human...

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Journal of Thermal Biology Volume 84, August 2019, Pages 357-367

Eﬀect of inspiration cycle and ventilation rate on heat exchange in human respiratory airways Bharat Soni , Ameeya Kumar Nayak

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https://doi.org/10.1016/j.jtherbio.2019.07.026

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Highlights •

Variations in ventilation rate and breathing time aﬀect the heat exchange rate.

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Airway and its adjacent mucus-tissue layer play their role to condition the air.

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Inhaling air with high temperature and frequency may cross G16-17.

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Ill conditioned air crosses G16-17 of bronchial tree and interrupts gas exchange.

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Assumption of regular geometry is valid only for slow breathing.

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Abstract A transient three dimensional (3D) theoretical axisymmetric model is developed for heat exchange across the human respiratory tract during inspiration phase and applied to study the changes in the airway temperature and velocity profile for varying ventilation rates and inhalation temperatures. A multi-compartment approach is used to study the same to avoid the airway scaling problem from micro to nano scale. This analysis also includes the role of water evaporation in mucus and non perfused tissue layers and the role of capillary bed in thermal variations during respiration. The results of heat transfer in airway and mucus layer depend on the local morphological parameters. The results are compared with the case of hypothetical regular geometry to show the significance of local morphology. The location where the inhaled air gets saturated with the body core temperature is computed to estimate the saturation distance of air. The complete analysis is made for two breathing cycles with different inhalation to exhalation ratios. The results indicate that decreasing the ventilation rate and increasing the respiration cycle can avoid the deep penetration of heat into the tract and consequently tissue thermal injury can be avoided. We have also explained numerically the role of mucus layer in avoiding tissue injury in intra-thoracic airways. We have also observed a significant difference in results for high ventilation rates between the cases of actual (cast replica) and regular airway geometry. The numerical results are in good adjustment with existing experimental data and thus validate our approach. Previous

Keywords Air conditioning; Multi-compartment mathematical model; Mucus layer; Respiration cycle

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Reference 7 NIH Public Access Author Manuscript

N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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Published in final edited form as: N Engl J Med. 2010 December 2; 363(23): 2233–2247. doi:10.1056/NEJMra0910061.

Airway Mucus Function and Dysfunction John V. Fahy, M.D. and Burton F. Dickey, M.D. Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, San Francisco (J.V.F.); and the Department of Pulmonary Medicine, University of Texas M.D. Anderson Cancer Center, Houston (B.F.D.).

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The lungs are remarkably resistant to environmental injury, despite continuous exposure to pathogens, particles, and toxic chemicals in inhaled air. Their resistance depends on a highly effective defense provided by airway mucus,1–7 an extracellular gel in which water and mucins (heavily glycosylated proteins) are the most important components. Airway mucus traps inhaled toxins and transports them out of the lungs by means of ciliary beating and cough (Fig. 1). Paradoxically, although a deficient mucous barrier leaves the lungs vulnerable to injury, excessive mucus or impaired clearance contributes to the pathogenesis of all the common airway diseases.1–4 This review examines the normal formation and clearance of airway mucus, the formation of pathologic mucus, the failure of mucus clearance that results in symptoms and abnormal lung function, and the therapy of mucus dysfunction.

STRUCTURE AND FUNCTION OF THE NORMAL AIRWAY Epithelial surfaces in contact with the outside environment are protected by mechanical barriers (e.g., keratinized skin) and chemical barriers (e.g., gastric acid). Mucosal surfaces are wet epithelia that have a mucous barrier as part of their protective mechanism.1–7 Mucus layers vary widely in composition and structure; for example, they are thick and adherent to the epithelium in the gut, but thin and mobile in the airway.

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SURFACE EPITHELIAL CELLS The surface epithelium of intrapulmonary airways is composed of two principal cell types — ciliated and secretory (Fig. 2). These cells are present in similar numbers and form a mosaic. Secretory cells have been further divided into subtypes based on their microscopical appearance (e.g., Clara, goblet, and serous cells). However, studies indicate great structural, molecular, and functional plasticity in secretory cells.10–14 Therefore, it is simplest to refer to them generically as “secretory cells.” Besides mucins, secretory cells release a variety of antimicrobial molecules (e.g., defensins, lysozyme, and IgA), immunomodulatory molecules

Copyright © 2010 Massachusetts Medical Society. Address reprint requests to Dr. Dickey at the M.D. Anderson Cancer Center, P.O. Box 301402, 1515 Holcombe Blvd., Houston, TX, 77030-4009, or at bdickey@mdanderson.org. No other potential conflict of interest relevant to this article was reported. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

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(e.g., secretoglobins and cytokines), and protective molecules (e.g., trefoil proteins and heregulin) constitutively and inducibly; these can become incorporated into mucus.15,16

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SUBMUCOSAL GLANDS In large airways (luminal diameter, >2 mm), submucosal glands contribute to the secretion of mucins and liquid (Fig. 1). Each gland is connected to the airway lumen by a superficial ciliated duct that propels secretions outward and a deeper nonciliated collecting duct.17,18 The body of the gland is located between the spiral bands of smooth muscle and the cartilage plates. Mucous cells constitute approximately 60% of the gland volume, and based on studies in primates, it has been estimated that half as much intracellular mucin is stored in submucosal glands as is stored in surface epithelial cells.19 Serous cells, located distally, make up the remaining approximately 40% of the gland and secrete proteoglycans and numerous antimicrobial proteins. In pathologic states, the volume of submucosal glands can increase to several times the normal volume.20,21 MUCUS GEL LAYER

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A gel is a dilute network that holds shape; thus, although it is composed mostly of liquid, it has many physical characteristics of a solid. Mucus is a gel with properties of both a soft (deformable), elastic solid and a viscous fluid.1,4,5,22,23 Normal mucus is 97% water and 3% solids (mucins, non-mucin proteins, salts, lipids, and cellular debris). Mucins, exceedingly large glycoproteins (up to 3×106 D per monomer) with regions rich in serine and threonine residues linked by their hydroxyl side groups to sugar chains (O-glycosylation), account for less than 30% of the solids.3,4,6,15,24 Mucins are 50 to 90% carbohydrate, and they are highly anionic because most of their terminal sugars contain carboxyl or sulfate groups. There are 17 genes encoding mucins in the human genome, of which the gene products of seven are secreted and the remainder is membrane-bound.3,4,6 Five of the secreted mucins have terminal cysteinerich domains that can form disulfide bonds resulting in polymers that impart the properties of a gel (Fig. 2). Two of these polymers, MUC5AC and MUC5B, are strongly expressed in the airways and are detected in similar quantities in human mucus.3,4

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MUC5AC and MUC5B form homotypic polymers (i.e., MUC5AC monomers bond only with MUC5AC, and MUC5B monomers bond only with MUC5B), structured as long single chains rather than branches (Fig. 2). They form the mucus gel both by entanglement in a mesh and by noncovalent calcium-dependent cross-linking of adjacent polymers.1,3 The glycan side chains bind large amounts of liquid (hundreds of times their weight), which allows mucus to act as a lubricant and the gel layer to serve as a liquid reservoir for the periciliary layer.2 The hydration of mucus dramatically affects its viscous and elastic properties, which in turn determine how effectively it is cleared by ciliary action and cough.1–5,22 Healthy mucus contains 3% solids, with the consistency of egg white. However, mucin hypersecretion or dysregulation of surface liquid volume may increase the concentration of solids up to 15%, resulting in viscous and elastic mucus that is not easily cleared.1,5,22 In addition, dehydrated mucus adheres more readily to the airway wall.23,25 Since infection is often initiated by the recognition of host epithelial surfaces by microbial sugar-binding proteins, mucin glycans help sequester pathogens by providing a diverse N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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“glycoprotein landscape” for interaction with these microbial proteins, and patterns of glycosylation can change during inflammation.3,5,26 In addition, the mucus gel layer acts as a solid physical barrier to most pathogens.1,3,5,7 However, the pore size of the gel mesh is sufficiently large (approximately 500 nm) that it is readily penetrated by small viruses with hydrophilic capsids; this has implications for microbial infection and gene therapy.5 MUCIN PRODUCTION In healthy persons, MUC5AC is produced predominantly in proximal airways by surface goblet cells, whereas MUC5B is produced in surface secretory cells throughout the airways and by submucosal glands.3,4,14,27–29 In the airways of normal mice, which resemble human distal airways, almost no Muc5ac is produced,10–12,30–32 and mice with Muc5ac deletion are healthy, whereas Muc5b is produced constitutively in airway surface secretory cells,11–13,29 and mice with Muc5b deletion die from lung inflammation (Evans CM: personal communication). This finding suggests that Muc5b mediates baseline barrier and clearance functions in mice, and MUC5B probably does the same in human distal airways.28,33 Since MUC5AC is produced constitutively in human proximal airways, it may augment proximal barrier and clearance functions.

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The proportion of MUC5AC and MUC5B varies with the state of health. For example, during allergic mucous metaplasia of the surface epithelium in humans, the production of MUC5AC increases greatly (40 to 200 times as high as normal levels) within the same cells that produce MUC5B at baseline, with similar findings in mice.11,12,14,24,29,30,33–36 Muc5b production increases moderately (3 to 10 times as high as normal levels) during allergic inflammation in mice,14,30 and MUC5B transcripts and protein increase in the distal airways of patients with asthma and smokers,28,33 though MUC5B transcripts actually decrease in proximal airways.35,37,38 Hyperplasia plays only a minor role in augmented surface epithelial mucin production, since epithelial-cell numbers increase by 30% or less during inflammation.11,12,27,39 However, hyperplasia may play a major role in augmented submucosal gland MUC5B production in chronic obstructive pulmonary disease (COPD) and cystic fibrosis, since gland volume increases by up to four times the normal volume,20,21 though the relative contributions of hyperplasia and hypertrophy have not been defined. The resolution of surface epithelial mucous metaplasia occurs when secretory cells downregulate mucin production after the withdrawal of inflammatory stimuli, and the resolution of hyperplasia occurs through apoptosis of excess secretory cells.4,11,27 Since MUC5AC and Muc5ac production is highly regulated at the transcriptional level, its control is of great clinical interest. ErbB-receptor signaling appears to play a ubiquitous role, since inhibitors block MUC5AC and Muc5ac up-regulation by diverse stimuli.10,36,40–42 Interleukin-13 greatly increases MUC5AC and Muc5ac expression,34,36,43,44 and key downstream transcription factors have been identified,30,39,44,45 although the pathways that connect them are not yet fully established (Fig. 3). Many other stimuli that increase MUC5AC and Muc5ac expression, such as viruses,31 the smoke component acrolein,40 and the cytokines interleukin-4, 9, 17, 23, and 25,52–54 do so, at least in part, through interleukin-13. Overexpression of proinflammatory cytokine interleukin-1β or interleukin-17 increases Muc5ac expression,32,54 whereas interleukin-6 and tumor necrosis factor α do not

N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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themselves increase Muc5ac expression but do so indirectly by augmenting the intensity of allergic inflammation.55,56 The control of MUC5B and Muc5b expression is less well understood.57 MUCIN SECRETION

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The secretion of polymeric mucins is regulated separately from mucin production.50,58 The most important secretagogue for surface epithelium appears to be ATP, which acts on apical membrane P2Y2 receptors.59–61 It is not yet clear whether other agonists such as acetylcholine or histamine directly activate receptors on airway epithelial cells or induce airway smooth-muscle contraction leading to ATP release.59,62–64 The continuous presence of low levels of ATP in airway-surface liquid (see below) causes continuous low activity of the secretory machinery, resulting in the steady release of mucins that provide a normal barrier. When mucin production is increased so that mucins accumulate intracellularly (Fig. 3B), and secretion of a large number of granules is then triggered (mucus hypersecretion) (Fig. 3C), airway luminal occlusion can occur.13,65–67 It might seem that the secretion of a mucin granule would result in no net change in the volume of luminal air space if epithelialcell volume decreased by the same amount as the volume of secreted mucin. However, mucins are stored in dehydrated form within secretory granules, and they swell to several hundred times their dehydrated volume after secretion as a result of hydration and the exchange of each calcium counterion within the granule for two sodium ions in the extracellular space.9,68 Rapid secretion can deplete airway-surface liquid, resulting in the formation of concentrated, rubbery mucus that is resistant to dilution once the mucin network is formed.1,5,17 Submucosal glands continuously secrete polymeric mucins at a low level and can be further stimulated by adrenergic, cholinergic, and nonadrenergic, noncholinergic nerves.17 PERICILIARY LAYER

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The airway mucus gel lies atop a periciliary layer approximately 7 µm deep (Fig. 2A). The depth of this layer is critically important for mucociliary clearance (see below). Since the airway epithelium is highly permeable to water, liquid volume is determined by the amount of sodium chloride in the airway lumen.63 In turn, the amount of sodium chloride is regulated primarily by sodium absorption through the epithelial sodium channel and chloride extrusion through the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels.63,69 As mucus is propelled proximally, there is net salt and water absorption (>90%) commensurate with the decreasing total cross-sectional area of the airways.2 Locally, the depth of the periciliary layer is fine-tuned by the concentrations of adenine and uridine nucleotides and the metabolite adenosine. Adenine nucleotides are released through channels from ciliated cells that sense mechanical stress during ventilation63,70 and by exocytosis along with uridine nucleotides from secretory cells.60,61 These nucleotides activate P2Y2 receptors and adenosine activates A2b receptors on the apical membrane of ciliated cells, causing changes in intracellular second messengers that promote chloride release and inhibit sodium absorption; as a result, water moves into the airway lumen.60,61

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Membrane-bound mucins contribute to the physical properties of liquid near the cell surface, conferring features of a “grafted gel” rather than a fluid on the periciliary layer (Boucher RC, University of North Carolina: personal communication). MUC4 is densely expressed on cilia, configured like parallel bottle brushes, where it prevents penetration by the mucus gel layer and provides lubrication through bound water.4,6 MUC1 is much smaller than MUC4 and is present on the cell surface and microvilli of both ciliated and secretory cells. It has a cytoplasmic tail capable of intracellular signaling, and it modulates pathogen defense and inflammation.6,71 MUC16, the largest mucin, is expressed by both ciliated and secretory cells, and it can be cleaved and incorporated into the mobile gel layer.6,72 CLEARANCE MECHANISMS

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The mucus gel is propelled in a proximal direction by ciliary beating, clearing inhaled particles, pathogens, and dissolved chemicals that might damage the lungs.2 Polymeric mucins are continuously synthesized and secreted to replenish the gel layer. Normal cilia beat 12 to 15 times per second, resulting in a velocity of the gel layer of approximately 1 mm per minute.73 The rate of mucociliary clearance increases with greater hydration,2,73 and the rate of ciliary beating can be increased by purinergic, adrenergic, cholinergic, and adenosine-receptor agonists,60,73 as well as irritant chemicals.74 A second mechanism for the expulsion of mucus from the airways is cough clearance. This may help explain why lung diseases caused by impaired ciliary function are less severe than those caused by dehydration, which impedes both clearance mechanisms.2 Although cough contributes beneficially to the clearance of mucus in diseases of excessive production or impaired ciliary function, it can also be a troublesome symptom.75,76

MUCUS DYSFUNCTION IN DISEASE

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Effective mucus clearance is essential for lung health, and airway disease is a consistent consequence of poor clearance. Healthy mucus is a gel with low viscosity and elasticity that is easily transported by ciliary action, whereas pathologic mucus has higher viscosity and elasticity and is less easily cleared.5,38 The conversion from healthy to pathologic mucus occurs by multiple mechanisms that change its hydration and biochemical constituents; these include abnormal secretion of salt and water, increased production of mucins, infiltration of mucus with inflammatory cells, and heightened bronchovascular permeability (Fig. 4). The accumulation of mucus results from some combination of overproduction and decreased clearance, and persistent accumulation can lead to infection and inflammation by providing an environment for microbial growth. The principal symptoms of impaired mucus clearance are cough and dyspnea. Cough is caused by the stimulation of vagal afferents in the intrapulmonary airways or the larynx and pharynx.75,76 Patients often infer that laryngopharyngeal stimulation, described as “a tickle in the throat,” results from “postnasal drip,” since they recognize that gravity causes mucus to descend from the nasopharynx but are generally unaware that it also ascends from the lungs by ciliary action. Dyspnea is caused when mucus obstructs airflow by occupying the lumen of numerous airways.21,65–67 Physical signs of impaired mucus clearance include cough, bronchial breath sounds, rhonchi, and wheezes. Retained mucus and inflammatory N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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exudates may appear as localized atelectasis or linear or branched opacities on plain radiographs of the chest, and as luminal filling in proximal airways or tree-in-bud opacities in peripheral airways on computed tomographic examination.77 It is important to recognize the role of mucus in clinical presentation. It is necessary to clear mucus from the airway lumen in order to resolve symptoms and allow effective delivery of aerosol therapies. In addition, the presence of mucus may be a sign of underlying inflammation or infection that may warrant additional treatment. CYSTIC FIBROSIS

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Cystic fibrosis is caused by mutations in the gene encoding CFTR, which result in reduced chloride secretion and increased sodium absorption. Together, these result in insufficient airway luminal liquid.78,79 In addition, reduced bicarbonate secretion may result in excessive mucin cross-linking by calcium.80 In transgenic mice with overexpression of a subunit of the epithelial sodium channel in airway epithelial cells, airway luminal liquid is insufficient and a cystic fibrosis–like phenotype develops.81 This finding, coupled with a large amount of supportive data from in vitro studies of human airway epithelial-cell function, has led to a broad consensus that the major consequences of CFTR dysfunction in the airway are dehydration of mucus and reduction in the height of the periciliary layer, particularly in response to infectious or toxic insults.63,69,78,79 These changes result in poor mucus clearance, which sets up a vicious cycle of infection, inflammation, and injury. In patients with cystic fibrosis, mucus has the following characteristics: infiltration with neutrophils and high concentrations of neutrophil-derived DNA and filamentous actin22,82,83; infection with organisms such as Pseudomonas aeruginosa, Staphylococcus aureus, and aspergillus species, often in biofilms at the epithelial-cell surface; and dehydrated, highly entangled polymeric macromolecules that form a gel matrix with a pore size reduced from the normal size of approximately 500 nm to approximately 150 nm.5 Decreased pore size is postulated to immobilize microbes within the mucus gel, thereby promoting biofilm formation, and to inhibit the movement of neutrophils that might otherwise clear the infection.5,69 The net effects of these processes are manifested radiographically as bronchiectasis; pathologically as neutrophilic inflammation, airway fibrosis, and increased numbers of mucin-secreting cells, especially in the submucosal glands; and clinically as cough, purulent sputum, hemoptysis, dyspnea, recurrent lung infections, and rapid loss of lung function.78,79,84

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ASTHMA The central role of diffuse mucus plugging of the airways in the pathophysiology of asthma has been recognized by pathologists for more than 100 years.65,66,85 However, mucus dysfunction in asthma is often underappreciated by clinicians, possibly because cough in asthma infrequently results in expectoration or because the unavailability of therapies to clear mucus plugs has diverted attention exclusively toward reversing bronchoconstriction and inflammation.86 Mucous metaplasia (i.e., increased surface epithelial mucin production) and an increased number of bronchial microvessels are important components of the airway remodeling in asthma that confers a predisposition to mucus dysfunction.86 These changes occur in patients with airway inflammation characterized by infiltration of the airway wall and luminal mucus with CD4+ T cells, eosinophils, and innate immune cells that secrete N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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Th2 cytokines,85,87 although neutrophil infiltration may also be prominent in acute exacerbations.84 Airway occlusion by mucus plugs can cause localized atelectasis that is evident radiographically in patients with acute asthma exacerbations, and widespread mucus plugging is consistently detected at autopsy in patients with fatal asthma.65,66 Diffuse airway narrowing from a combination of concentric smooth-muscle contraction and luminal obstruction with mucus makes asthma uniquely dangerous among airway diseases in its propensity for sudden exacerbations.

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Airway mucus in severe asthma has a rubbery quality that contributes to impaired clearance and plug formation. Biochemical analysis of these plugs shows high concentrations of mucins and plasma proteins,84,88 and biophysical analysis shows high entanglement density and elastic modulus.89 Another important pathologic role of plasma proteins in forming these highly elastic mucus plugs is shielding mucins from protease digestion.89 Since neutrophil elastase activity is increased in the airways of patients with asthma in the recovery phase of near-fatal exacerbations, it may help to digest mucus plugs in these patients.90 A history of persistent symptoms related to sputum is associated with more severe disease phenotypes in chronic asthma,91 and mucus hypersecretion is especially problematic in allergic bronchopulmonary aspergillosis.85 CHRONIC OBSTRUCTIVE PULMONARY DISEASE Small-airway mucus obstruction is characteristic of COPD, even in patients who do not expectorate sputum or who have an emphysematous phenotype.92,93 Conversely, patients with COPD who have copious expectoration may have little airflow obstruction, probably because the mucus comes from large airways and causes minimal occlusion. Despite this weak correlation with sputum production, airflow obstruction does correlate with changes in mucin gene expression,38 increases in goblet-cell number and size,38 the occlusion of small airways with mucus,88 and expansion of the submucosal glands.21,92 Mucus dysfunction induced by cigarette smoke is complex and incompletely understood, but it involves adverse effects on the structure and function of cilia,94–96 activation of ErbB receptors,41 decreased function of CFTR,97 and proinflammatory effects that increase mucin production while decreasing mucus hydration and clearance. Cigarette smoke contains multiple toxins, including particulate matter, oxidative chemicals, and organic compounds, among which acrolein is important because it potently induces mucin production.40

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Increased mucin production and decreased luminal liquid in COPD have deleterious consequences for airway health, as they do in asthma and cystic fibrosis, including mucus stasis and airway infection. Haemophilus influenzae, P. aeruginosa, Streptococcus pneumoniae, and Moraxella catarrhalis are detected in sputum in 25 to 50% of adults with COPD. The infection rate increases with increasing disease severity, and the acquisition of new bacterial strains is associated with COPD exacerbations.98 On the basis of studies in a mouse model in which H. influenzae lysate elicited airway inflammation and fibrosis but not mucous metaplasia,56 one may speculate that in COPD, a reduction in mucus clearance that is related to cigarette smoke leads to airway infection, which, in turn, leads to inflammation and fibrosis.

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OTHER AIRWAY DISEASES ASSOCIATED WITH MUCUS DYSFUNCTION

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Mucus dysfunction occurs in virtually all inflammatory airway diseases. Acute viral and bacterial infections and chronic diseases such as primary ciliary dyskinesia, non–cystic fibrosis bronchiectasis (which is often caused by atypical mycobacterial infection), panbronchiolitis, and immunodeficiency states (e.g., hypogammaglobulinemia, human immunodeficiency virus infection, organ transplantation, and hematologic malignant conditions) all have a component of mucus dysfunction. In addition, retained mucus is a problem in intubated patients and those in whom lung mechanics are disrupted as a result of paralysis, immobilization, or surgery; atelectasis and pneumonia are common complications in such patients. Genomic markers in chromosomal region 11p15.5 (which encompasses MUC5AC and MUC5B) have been reported to be associated with asthma severity,99 and panbronchiolitis,100 although mechanisms leading to disease susceptibility have not yet been defined.

TREATMENT

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The development of rationally designed treatments for pathologic mucus has been hindered by a lack of understanding of the mechanisms of mucus dysfunction. Over-the-counter medications for airway mucus dysfunction, including guaifenesin, have not been rigorously evaluated in clinical trials, and they are not recommended in treatment guidelines for cystic fibrosis, asthma, or COPD.65,85,93,101–103 Multiple additional agents with uncertain mechanisms are used worldwide without clear evidence of a benefit.104,105 Asthma, COPD, and cystic fibrosis have important differences in pathologic mucus, and mucus treatment should be tailored accordingly. Current therapies do this to an extent, but they may be facilitated by therapies that are currently under development. Therapies can be subdivided into those that decrease mucin production, those that decrease mucin secretion, those that promote mucus clearance, and those that treat airway infection (Table 1).

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Recent insights into the formation of pathologic mucus in disease have led to the introduction of tailored therapies such as hydration by means of aerosolized hypertonic saline solutions or the reduction of mucus viscosity and elasticity by aerosolized dornase alfa. Targeted treatment of pathologic airway mucus not only improves symptoms of cough and dyspnea but also decreases the frequency of disease-related exacerbations and slows disease progression. Elucidation of how mucin production is controlled is still needed, since that might allow the development of additional therapies to prevent overproduction.

Acknowledgments Dr. Dickey reports receiving consulting fees from BioMarcks Pharmaceuticals; Dr. Fahy, serving on a scientific advisory board for Cytokinetics and receiving consulting fees from Five Prime Therapeutics, Amira, Oxagen, Gilead, GlaxoSmithKline, and Amgen, grant support to the University of California, San Francisco, from Genentech, Boehringer Ingelheim, and Aerovance, and travel fees from GlaxoSmithKline, Merck, Amira, and Amgen. Dr. Fahy reports being named on a provisional patent application submitted for a gene signature for type 2 helper T cell–high asthma (with Genentech). We thank Kenneth B. Adler, Richard C. Boucher, Stephen D. Carrington, C. William Davis, Kyubo C. Kim, Christopher M. Evans, Susan J. Muller, Jay A. Nadel, Mary C. Rose, David J. Thornton, and Jeffrey J. Wine for their careful reading of an earlier version of the manuscript; and members of the Fahy and Dickey laboratories for helpful insights.

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88. Sheehan JK, Richardson PS, Fung DC, Howard M, Thornton DJ. Analysis of respiratory mucus glycoproteins in asthma: a detailed study from a patient who died in status asthmaticus. Am J Respir Cell Mol Biol. 1995; 13:748–756. [PubMed: 7576713] 89. Innes AL, Carrington SD, Thornton DJ, et al. Ex vivo sputum analysis reveals impairment of protease-dependent mucus degradation by plasma proteins in acute asthma. Am J Respir Crit Care Med. 2009; 180:203–210. [PubMed: 19423716] 90. Ordoñez CL, Shaughnessy TE, Matthay MA, Fahy JV. Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma: clinical and biologic significance. Am J Respir Crit Care Med. 2000; 161:1185–1190. [PubMed: 10764310] 91. Siroux V, Boudier A, Bousquet J, et al. Phenotypic determinants of uncontrolled asthma. J Allergy Clin Immunol. 2009; 124:681–687. [PubMed: 19665764] 92. Hogg JC, Chu F, Utokaparch S, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004; 350:2645–2653. [PubMed: 15215480] 93. The Global Initiative for Chronic Obstructive Lung Disease. 2009 updates. (http:// www.goldcopd.com.). 94. Leopold PL, O’Mahony MJ, Lian XJ, Tilley AE, Harvey BG, Crystal RG. Smoking is associated with shortened airway cilia. PLoS ONE. 2009; 4(12):e8157. [PubMed: 20016779] 95. Tamashiro E, Xiong G, Anselmo-Lima WT, Kreindler JL, Palmer JN, Cohen NA. Cigarette smoke exposure impairs respiratory epithelial ciliogenesis. Am J Rhinol Allergy. 2009; 23:117–122. [PubMed: 19401033] 96. Verra F, Escudier E, Lebargy F, Bernaudin JF, De Crémoux H, Bignon J. Ciliary abnormalities in bronchial epithelium of smokers, ex-smokers, and nonsmokers. Am J Respir Crit Care Med. 1995; 151:630–634. [PubMed: 7881648] 97. Cantin AM, Hanrahan JW, Bilodeau G, et al. Cystic fibrosis transmembrane conductance regulator function is suppressed in cigarette smokers. Am J Respir Crit Care Med. 2006; 173:1139–1144. [PubMed: 16497995] 98. Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med. 2008; 359:2355–2365. [PubMed: 19038881] 99. The Collaborative Study on the Genetics of Asthma (CSGA). A genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nat Genet. 1997; 15:389–392. [PubMed: 9090385] 100. Kamio K, Matsushita I, Hijikata M, et al. Promoter analysis and aberrant expression of the MUC5B gene in diffuse panbronchiolitis. Am J Respir Crit Care Med. 2005; 171:949–957. [PubMed: 15709052] 101. Flume PA, O’Sullivan BP, Robinson KA, et al. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health. Am J Respir Crit Care Med. 2007; 176:957–969. [PubMed: 17761616] 102. Flume PA, Mogayzel PJ Jr, Robinson KA, et al. Cystic fibrosis pulmonary guidelines: treatment of pulmonary exacerbations. Am J Respir Crit Care Med. 2009; 180:802–808. [PubMed: 19729669] 103. Flume PA, Robinson KA, O’Sullivan BP, et al. Cystic fibrosis pulmonary guidelines: airway clearance therapies. Respir Care. 2009; 54:522–537. [PubMed: 19327189] 104. Boogaard R, de Jongste JC, Merkus PJ. Pharmacotherapy of impaired mucociliary clearance in non-CF pediatric lung disease: a review of the literature. Pediatr Pulmonol. 2007; 42:989–1001. [PubMed: 17902149] 105. Rogers DF. Mucoactive agents for airway mucus hypersecretory diseases. Respir Care. 2007; 52:1176–1193. [PubMed: 17716385] 106. Southam DS, Ellis R, Wattie J, Glass W, Inman MD. Goblet cell rebound and airway dysfunction with corticosteroid withdrawal in a mouse model of asthma. Am J Respir Crit Care Med. 2008; 178:1115–1122. [PubMed: 18849499] 107. Kapur N, Bell S, Kolbe J, Chang AB. Inhaled steroids for bronchiectasis. Cochrane Database Syst Rev. 2009; 1:CD000996. [PubMed: 19160186]

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108. Woodruff PG, Wolff M, Hohlfeld JM, et al. Safety and efficacy of an inhaled epidermal growth factor receptor inhibitor (BIBW 2948 BS) in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010; 181:438–445. [PubMed: 20007923] 109. Singer M, Martin LD, Vargaftig BB, et al. A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med. 2004; 10:193–196. [PubMed: 14716307] 110. Chen S, Barbieri JT. Engineering botulinum neurotoxin to extend therapeutic intervention. Proc Natl Acad Sci U S A. 2009; 106:9180–9184. [PubMed: 19487672] 111. Foster KA, Adams EJ, Durose L, et al. Re-engineering the target specificity of Clostridial neurotoxins — a route to novel therapeutics. Neurotox Res. 2006; 9:101–107. [PubMed: 16785105] 112. Fahy JV, Steiger DJ, Liu J, Basbaum CB, Finkbeiner WE, Boushey HA. Markers of mucus secretion and DNA levels in induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis. 1993; 147:1132–1137. [PubMed: 8484621] 113. O’Donnell AE, Barker AF, Ilowite JS, Fick RB. Treatment of idiopathic bronchiectasis with aerosolized recombinant human DNase I. Chest. 1998; 113:1329–1334. [PubMed: 9596315] 114. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med. 2006; 354:241–250. [PubMed: 16421365] 115. Elkins MR, Robinson M, Rose BR, et al. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med. 2006; 354:229–240. [PubMed: 16421364] 116. Levin MH, Sullivan S, Nielson D, Yang B, Finkbeiner WE, Verkman AS. Hypertonic saline therapy in cystic fibrosis: evidence against the proposed mechanism involving aquaporins. J Biol Chem. 2006; 281:25803–25812. [PubMed: 16829520] 117. Shridharani M, Maxson TR. Pulmonary lavage in a patient in status asthmaticus receiving mechanical ventilation: a case report. Ann Allergy. 1982; 49:156–158. [PubMed: 7114588] 118. Decramer M, Janssens W. Mucoactive therapy in COPD. Eur Respir Rev. 2010; 19:134–140. [PubMed: 20956182] 119. Rao S, Wilson DB, Brooks RC, Sproule BJ. Acute effects of nebulization of N-acetylcysteine on pulmonary mechanics and gas exchange. Am Rev Respir Dis. 1970; 102:17–22. [PubMed: 5427399] 120. Minasian C, Wallis C, Metcalfe C, Bush A. Comparison of inhaled mannitol, daily rhDNase and a combination of both in children with cystic fibrosis: a randomised trial. Thorax. 2010; 65:51–56. [PubMed: 19996349] 121. Seemungal TA, Wilkinson TM, Hurst JR, Perera WR, Sapsford RJ, Wedzicha JA. Long-term erythromycin therapy is associated with decreased chronic obstructive pulmonary disease exacerbations. Am J Respir Crit Care Med. 2008; 178:1139–1147. [PubMed: 18723437] 122. Daniels JM, Snijders D, de Graaff CS, Vlaspolder F, Jansen HM, Boersma WG. Antibiotics in addition to systemic corticosteroids for acute exacerbations of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2010; 181:150–157. [PubMed: 19875685] 123. Anwar GA, Bourke SC, Afolabi G, Middleton P, Ward C, Rutherford RM. Effects of long-term low-dose azithromycin in patients with non-CF bronchiectasis. Respir Med. 2008; 102:1494– 1496. [PubMed: 18653323] 124. Smyth AR, Bhatt J. Once-daily versus multiple-daily dosing with intravenous aminoglycosides for cystic fibrosis. Cochrane Database Syst Rev. 2010; 1:CD002009. [PubMed: 20091528] 125. McCoy KS, Quittner AL, Oermann CM, Gibson RL, Retsch-Bogart GZ, Montgomery AB. Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis. Am J Respir Crit Care Med. 2008; 178:921–928. [PubMed: 18658109] 126. Retsch-Bogart GZ, Quittner AL, Gibson RL, et al. Efficacy and safety of inhaled aztreonam lysine for airway pseudomonas in cystic fibrosis. Chest. 2009; 135:1223–1232. [PubMed: 19420195]

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Figure 1. Mucus Clearance in Normal Airways

Mucus is continuously swept from distal to proximal airways. In the most distal bronchioles, epithelial cells are cuboidal and do not produce mucin (bottom box), and bronchiolar patency is stabilized by surfactant from adjacent alveoli.8 In the adjacent small airways, a thin mucus gel layer is produced by columnar secretory (Clara) cells that do not stain for intracellular mucins because they are produced in low amounts and steadily secreted. In the large airways lined by a pseudostratified epithelium, a thick mucus gel layer (up to 50 µm) accumulates from mucus transported from distal airways and additional mucins are N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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produced by surface secretory cells and glands. After mucus ascends the trachea, it is propelled through the vocal cords by ciliary epithelium in the posterior commissure of the larynx. It then enters the pharynx and is swallowed, with approximately 30 ml of airway mucus eliminated by the gastrointestinal tract daily. The vocal cords are covered by squamous epithelium, so they do not participate in ciliary clearance, although they promote cough clearance by closing while expiratory pressure builds and then opening suddenly so airflow is forceful.

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Figure 2. Structure of Airway Mucus

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Panel A shows a distal bronchus with a single layer of columnar epithelial cells. The ciliated cells express approximately 200 cilia that are about 7 µm in length. The secretory cells show mixed features of Clara cells (small, black, apical granules containing proteins) and goblet cells (large granules up to 1 µm in diameter containing yellow mucins and black proteins). The mucus gel layer increases in thickness from distal to proximal airways, whereas the periciliary layer is approximately 7 µm deep throughout the conducting airways. Panel B shows an electron micrograph of a partially expanded MUC5B polymer after secretion, intermediate between its condensed form within a secretory granule and its expanded linear structure.9 Nodes where monomers are bonded appear as white globules (arrow). Panel C shows an electron micrograph of an extended MUC5B polymer. A MUC5B monomer is approximately 450 nm in length, and polymers contain 2 to 20 subunits. Panel D shows the structure of MUC5B. It is organized into an N-terminal region containing von Willebrand factor D1–3 domains involved in N–N polymerization (blue), a central region containing glycosylated mucin domains (pale yellow), and a C-terminal region containing von Willebrand factor D4, B, C, and CK domains involved in C-C polymerization.1,3 The structure of MUC5A is similar (not shown). Electron micrographs provided courtesy of Mehmet Kesimer and John K. Sheehan, University of North Carolina.

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Panels A through C show axial sections through bronchi of mice, which are similar in size and structure to human bronchioles. Panel A shows an airway under healthy conditions, in which polymeric mucin production is low so that the secretory cells (arrowhead) do not show mucin granules when stained with Alcian blue and periodic acid–Schiff reagent. Nonetheless, antibodies indicate that the cells do produce small amounts of Muc5b (not shown), although Muc5ac is undetectable.14,29 Ciliated cells are interspersed among the secretory cells (arrow). Panel B shows an airway 2 days after the induction of mucous metaplasia by asthmalike allergic inflammation due to sensitization and challenge with ovalbumin.11 Mucin-containing granules are visible in the secretory cells as a result of greatly increased Muc5ac and moderately increased Muc5b production.14,29,30 Panel C shows a metaplastic airway 10 minutes after stimulation of mucin secretion by an ATP aerosol (scale bar, 10 µm). Panel D shows ligands and transcription factors that are important in Muc5ac expression. Interleukin-13 binds to a receptor that includes the interleukin-4Rα subunit, activating Janus kinase 1 (Jak1), leading to the phosphorylation of Stat6. There is no consensus Stat6 binding site in the MUC5AC and Muc5ac promoter, but Stat6 activation leads to increased expression of SPDEF (SAM pointed domain-containing N Engl J Med. Author manuscript; available in PMC 2014 June 08.

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Ets transcription factor), which up-regulates multiple genes involved in mucous metaplasia,39 and inhibits expression of Foxa2, which negatively regulates Muc5ac.45 Several ligands bind ErbB receptors, including epidermal growth factor, transforming growth factor α, amphiregulin, and neuregulin, activating mitogen-activated protein kinases (MAPK).42,46 Hypoxia-inducible factor 1 (HIF-1) also can be activated downstream of ErbB receptors, and there is a conserved HIF-1 binding site in the proximal MUC5AC and Muc5ac promoter,30 but whether this is the dominant mechanism of up-regulation by ErbB ligands is not known. Not shown are complement C3 and β2-adrenergic–receptor signaling, which amplify Muc5ac production,29,47–49 or transcription factors such as Sox2, Notch, E2f4, and Math, which primarily regulate development. Panel E shows the molecular mechanism of mucin exocytosis. A mucin-containing secretory granule is docked to the plasma membrane by the interaction of a granule-bound Rab protein with an effector protein that acts as a tether to Munc18, which binds the closed conformation of Syntaxin anchored to the plasma membrane. Secretion is triggered when ATP binds to P2Y2 purinergic receptors (P2Y2R) coupled to Gq, activating phospholipase C (PLC), which generates the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). DAG activates Munc1314 to open Syntaxin so it can form a four-helix SNARE (soluble N-ethylmaleimide– sensitive factor attachment protein receptor) complex with SNAP-23 (synaptosomalassociated protein 23) and VAMP (vesicle-associated membrane protein), drawing together the granule and plasma membranes.50 IP3 induces the release of calcium from IP3 receptors (IP3R) in the endoplasmic reticulum (ER), activating Synaptotagmin51 to induce final coiling of the SNARE complex, which results in fusion of the membranes and release of the mucins. The photomicrographs are courtesy of Dr. Michael J. Tuvim.

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Figure 4. Airway Mucosal Disease and Mucus Characteristics

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The top panels show the contribution of mucosal disease to abnormal mucus. In asthma, airway remodeling is characterized by increases in epithelial mucin stores because of surface epithelial mucous metaplasia with modest hyperplasia and increased numbers of subepithelial bronchial microvessels that become leaky during inflammation. Changes in submucosal glands are not prominent except in severe disease. In chronic obstructive pulmonary disease (COPD), increased mucin stores occur because of surface epithelial mucous metaplasia and some hyperplasia, together with increases in the volume and number of the submucosal glands. Bronchial microvessel remodeling is not as prominent as in asthma. In cystic fibrosis, epithelial mucin stores are similar to normal levels (possibly because of increased secretion), but submucosal glands are very prominent. Bronchial microvessel remodeling is not as prominent as in asthma. Not shown in the top panels are the increased numbers of inflammatory cells in the airway wall and lumen, which occur in all airway diseases. Products of inflammatory cell death include DNA and actin polymers, which are important constituents of pathologic mucus. The bottom panels list some of the constituents of mucus in health and in airway disease. The degree of cellular inflammation and biochemical constituents of mucus differ among airway diseases. The data for asthma

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reflect changes that occur in acute severe asthma. The number of Xs indicates the relative abundance of the constituents in each disease state.

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Table 1

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Treatment of Mucus Hypersecretion. Purpose and treatment

Comments

Decrease mucin production Available treatments Glucocorticoids

Allergen-induced increases in numbers of airway goblet cells are inhibited by glucocorticoids. In acute severe asthma, glucocorticoids may also reduce bronchovascular permeability and promote neutrophildriven mucus turnover.89 Glucocorticoids are much less effective in treating mucus in other airway diseases.93,107

Agents in development ErbB-receptor inhibitors

A recent trial of inhaled BIBW 2948 BS, an epidermal growth factor receptor inhibitor (ClinicalTrials.gov number, NCT00423137), did not show a benefit in reducing airway mucin gene expression or epithelial mucin stores in patients with COPD, and the treatment was associated with adverse effects on lung and liver function.108

Decrease mucin secretion Available treatments None Agents in development

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MARCKS inhibitors

MARCKS regulates the reconfiguration of actin filaments at the apical pole of goblet cells during mucin secretion.50,109 A peptide derived from the MARCKS N-terminal that is myristoylated so that it enters cells inhibits stimulated mucin secretion in mice.109 In a phase 2 trial (NCT00648245), this peptide is being administered by aerosol in patients with COPD who have mucus hypersecretion.

Botulinum neurotoxins

Botulinum neurotoxins are zinc proteases that cleave SNARE proteins to inhibit release of synaptic vesicles. The C and E neurotoxins have been engineered so that they are active against non-neuronal SNARE isoforms, inhibiting epithelial-cell mucin secretion, and the modified C toxin has been conjugated to epidermal growth factor to promote delivery to goblet cells, but no related clinical trial has been registered.110,111

Promote mucus clearance Available treatments

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Physical measures

Chest percussion and postural drainage improve clearance of purulent airway mucus in cystic fibrosis.103 The value of alternative methods, including positive expiratory pressure, flutter valves, or high-frequency chest-compression vests, is difficult to assess objectively, although trials are in progress (e.g., NCT01057524). Mucus clearance is probably aided by any maneuver that promotes coughing and increased minute ventilation, including exercise. Since airflow generates shear stress on airway cell surfaces that stimulate release of nucleotides that interact with P2Y2 receptors to regulate mucus hydration, there are both mechanical and biochemical mechanisms of benefit from nonpharmacologic approaches to mucus clearance.63,103

Bronchodilators

Bronchodilation with beta-adrenergic agonists or anticholinergic drugs may improve mucus clearance in the short term because of an enlarged luminal diameter. In addition, beta-adrenergic agonists increase the frequency of ciliary beats, and anticholinergic drugs may decrease surface mucin secretion and mucin secretion from the submucosal gland.17,62,64,73 However, betaadrenergic agonists up-regulate mucin production in animal models,29,49 so they are not recommended for long-term treatment of mucus hypersecretion. The long-term effects of both classes of bronchodilators on mucus clearance warrant further study.

Inhaled dornase alfa

Inhaled dornase alfa hydrolyzes DNA, improves lung function, and decreases the frequency of exacerbation in patients with cystic fibrosis, in whom airway mucus concentrations of DNA are very high (5–10 mg/ml).84,101 The concentration of DNA in other airway diseases, including non −cystic fibrosis bronchiectasis, COPD, and asthma, is 1/5 to 1/10 as great84,88,112; dornase alfa does not have beneficial effects in these diseases and may even be harmful.113

Inhaled hypertonic saline

Treatment twice daily with aerosolized 7% hypertonic saline solution is associated with significant improvements in mucus clearance, modest improvements in airflow, and clinically meaningful reductions in rates of exacerbation among patients with cystic fibrosis.114,115 The mechanism of benefit is thought to be rehydration of the periciliary layer through the drawing of water from epithelial cells, but other mechanisms such as promotion of cough and direct effects on mucus elasticity and entanglement may also contribute.69,116 Trials are under way for the treatment of other airway diseases with 3 to 7% hypertonic saline; these diseases include infantile bronchiolitis (NCT01016249, NCT00677729, NCT00729274, NCT00619918, NCT00151905,

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Purpose and treatment

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Comments and NCT00696540), COPD (NCT00639236), atelectasis (NCT00671723), non−cystic fibrosis bronchiectasis (NCT00484263), and asthma (NCT01073527). N-acetylcysteine breaks the disulfide bonds that link mucin monomers to polymers, and it is very effective in vitro in solubilizing sputum. Case reports attest to its usefulness when applied through the bronchoscope to break up mucus plugs.117 Clinical studies of N-acetylcysteine and carbocysteine in COPD have shown some promise.118 Aerosolized N-acetylcysteine also can be irritating to the airway,119 so its routine use is not recommended. Oral N-acetylcysteine is under study as an antiinflammatory (glutathione-replenishing and antioxidant) treatment in cystic fibrosis and COPD (NCT00969904 and NCT00809094).

N-acetylcysteine

Agents in development

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Mannitol

Nonabsorbable osmotic agents have a theoretical advantage of drawing liquid into the periciliary layer for longer periods than sodium chloride. Inhaled mannitol is undergoing safety and efficacy testing in cystic fibrosis, COPD, and bronchiectasis (NCT00446680, NCT00117208, and NCT00669331). Its use in children with cystic fibrosis is associated with bronchoconstriction and cough, but a 3-month treatment protocol showed similar efficacy to that of dornase alfa.120

P2Y2 agonists

P2Y2 agonists promote the activity of calcium-activated chloride channels and inhibit the activity of epithelial sodium channels, so they may normalize the height of the periciliary layer and improve mucus clearance, especially in cystic fibrosis. Phase 2 and 3 trials of denufusol in cystic fibrosis are ongoing (NCT00625612 and NCT00357279).

CFTR modulation

Several therapeutic agents are in development to augment the function of mutant CFTR, including ataluren to promote read-through of premature termination codons (NCT00803205), VX-809 to promote transport of misfolded CFTR protein to the cell surface (NCT00865904 and NCT00966602), and VX-770 to promote the opening of CFTR proteins expressed on the cell surface (NCT00909532 and NCT00966602).

Epithelial sodium-channel modulation

An aerosolized inhibitor of epithelial sodium-channel function, GS-9411, is being evaluated as a potential therapy to improve airway hydration and mucociliary clearance in cystic fibrosis (NCT00800579, NCT009999531, NCT01025713, and NCT00951522).

Actin filament depolymerizing agents, proteases, and antiproteases

Gelsolin and thymosin β4 depolymerize actin filaments,23,83 which could be helpful in promoting mucus clearance in cystic fibrosis; however, the results of clinical studies of recombinant human plasma gelsolin in cystic fibrosis in the 1990s were not promising, and no trials are currently registered. Proteases that digest gel-forming mucins could be helpful in treating mucus plugs in severe asthma,89 although no trials are yet under way. Inhaled alpha1-antitrypsin is being studied to prevent lung parenchymal damage from leukocyte proteases in cystic fibrosis (NCT00499837).

Treat airway infection Available treatments

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Inhaled antibiotics

Treatment with inhaled tobramycin is associated with improved lung function and reduced exacerbation frequency in patients with cystic fibrosis.101

Oral antibiotics

Prolonged use of azithromycin improves lung function in cystic fibrosis, although it is associated with increased nausea and diarrhea.101 Long-term treatment with oral erythromycin reduces the frequency of exacerbation in COPD,121 and several groups, including the COPD Clinical Research Network (NCT00119860), are studying azithromycin in patients with this condition. The value of oral antibiotics in the short-term treatment of COPD exacerbations is questionable.122 In patients with bronchiectasis who do not have cystic fibrosis, long-term treatment with low-dose azithromycin reduces the frequency of exacerbation and improves lung function.123

Intravenous antibiotics

Intravenous antibiotics directed against pseudomonas species infections are effective in the treatment of pulmonary exacerbations of cystic fibrosis.102,124

Agents in development Inhaled antibiotics

Inhaled aztreonam reduced the time to exacerbation in cystic fibrosis in two phase 3 studies (NCT00112359, NCT00104520),125,126 and a study comparing inhaled aztreonam with inhaled tobramycin in cystic fibrosis is ongoing (NCT00757237). Also under study are inhaled ciprofloxacin (NCT00645788), liposomal amikacin (NCT00558844), and tobramycin combined with fosfomycin (NCT00794586).

CFTR denotes cystic fibrosis transmembrane conductance regulator, COPD chronic obstructive pulmonary disease, MARCKS myristoylated alanine-rich C-kinase substrate, and SNARE soluble N-ethylmaleimide–sensitive factor attachment protein receptor.

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Reference 8 & 9 HHS Public Access Author manuscript

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Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10. Published in final edited form as: Nat Rev Immunol. 2015 June ; 15(6): 335–349. doi:10.1038/nri3843.

Fever and the thermal regulation of immunity: the immune system feels the heat Sharon S. Evans, Elizabeth A. Repasky, and Daniel T. Fisher Department of Immunology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY, USA

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Abstract Fever is a cardinal response to infection that has been conserved in warm and cold-blooded vertebrates for over 600 million years of evolution. The fever response is executed by integrated physiological and neuronal circuitry and confers a survival benefit during infection. Here, we review our current understanding of how the inflammatory cues delivered by the thermal element of fever stimulate innate and adaptive immune responses. We further highlight the unexpected multiplicity of roles of the pyrogenic cytokine interleukin-6 (IL-6), both during fever induction as well as during the mobilization of lymphocytes to the lymphoid organs that are the staging ground for immune defence. Finally, we discuss the emerging evidence that suggests the adrenergic signalling pathways associated with thermogenesis shape immune cell function.

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The fever response is a hallmark of infection and inflammatory disease and has been shaped through hundreds of millions of years of natural selection. Febrile temperatures are so closely linked to the inflammatory response that heat (calor) is one of the four cardinal signs of inflammation, along with pain (dolor), redness (rubor), and swelling (tumour), as described by Celsus in ~30 BC.1 The induction of fever in endothermic (warm-blooded) animals occurs at a high metabolic cost such that a 1°C rise in body temperature requires a 10–12.5% increase in metabolic rate.2 There is mounting evidence that the increase of 1 to 4°C in core body temperature that occurs during fever is associated with improved survival and resolution of many infections. For example, the use of antipyretic drugs to diminish fever correlates with a 5% increase in mortality in human populations infected with influenza virus and negatively affects patient outcomes in the intensive care unit.3–5 Preclinical studies in rabbits infected with rinderpest virus also found an increase in mortality when fever was inhibited with the antipyretic drug acetylsalicylic acid — 70% of acetylsalicylic acid-treated animals died as a result of infection as compared with only 16% of animals with a normal febrile response.6 However, fever is not universally beneficial, particularly in cases of extreme inflammation where lowering, rather than raising body temperature has evolved as a protective mechanism.7–10 Thus, uncontrolled fever is associated with worse outcomes in patients with sepsis or neurological injuries, whereas treatments that induce hypothermia can have a clinical benefit.11,12 A challenge in

Please address all correspondence to Sharon S. Evans, Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263. Phone: (716) 845-3421 Fax: (716) 845-1322 sharon.evans@roswellpark.org. Conflict of interest statement: The authors have no conflicting financial interests.

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ascertaining the precise value of fever in endotherms is that the antipyretics used to inhibit fever target multiple aspects of the inflammatory response besides temperature regulation.11

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Ectothermic (cold-blooded) vertebrates, which last shared a common ancestor with mammals over 600 million years ago, provide an ‘experiment in nature’ in which to examine the direct impact of febrile temperatures on survival. Ectotherms as diverse as reptiles, fish, and insects raise their core temperature during infection through behavioural regulation, which leads to their seeking warmer environments (despite the risk of predation) or, in the case of bees, raising the local temperature of the hive through increased physical activity.2,13–19 Landmark studies published 40 years ago by Kluger’s laboratory showed that survival of the desert iguana Dipsosaurus dorsalis is reduced by 75% if prevented from behaviourally raising its core temperature by approximately 2°C after infection with the Gram-negative bacterium Aeromonas hydrophila.2,13,14 The heat-seeking behaviour of the desert iguana, blue-finned tuna and leech is negated by antipyretic drugs, indicating that common biochemical pathways drive fevers in ectothermic and endothermic animals.14,16,20 Surprisingly, the correlation between infection and increased temperature extends even to plants, which arose 1.5 billion years ago. For example, the temperature of the leaves from the bean plant Phaseolus vulgaris increases by around 2°C following infection with the fungus Collectotrichum lindemuthianum.21 Thermoregulation in plants occurs through mitochondrial respiration22, although it is not known whether these fever-like responses have a direct impact on clearance of infection.

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The fact that fever has been retained throughout vertebrate evolution strongly argues that febrile temperatures confer a survival advantage. A long-standing mystery relates to the protective mechanisms by which fever wards off attacks by invading pathogens. One mechanism involves direct effects of febrile temperatures on the infectious potential of pathogens.23 For example, temperatures in the febrile range (40–41°C) cause a greater than 200-fold reduction in the replication rate of poliovirus in mammalian cells and increase the susceptibility of Gram-negative bacteria to serum-induced lysis.24,25 In this Review, we discuss the evidence suggesting that febrile temperatures boost the effectiveness of the immune response during infections by stimulating both the innate and adaptive arms of the immune system. We will highlight the role of the pyrogenic cytokine interleukin-6 (IL-6) in two key phases of the febrile response: firstly in driving the rise in core temperature, and secondly as a downstream effector cytokine orchestrating lymphocyte trafficking to lymphoid organs. We also describe febrile temperature as a ‘rheostat’, dialling down systemic inflammation during the return to homeostasis. Finally, we highlight new data demonstrating the overlapping signalling pathways that are involved in thermogenesis and in the regulation of the immune response. We only briefly discuss the neuronal circuitry that drives fever and the evolutionarily conserved heat shock protein (HSP) response (BOX 1), but refer the reader to recent comprehensive reviews for additional information on these topics as well as on the contributions of hypothermia to limiting inflammation.26–30

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Induction of fever The IL-6–COX2–PGE2 axis drives fever

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The induction and maintenance of fever during infection involves the tightly coordinated interplay between the innate immune system and neuronal circuitry within the central and peripheral nervous systems. Immune sensing of infection begins with the binding of pathogen-associated molecular patterns (for example, lipopolysaccharide ((LPS)), viral RNA, or fungal sugars) to pathogen recognition receptors (PRRs), such as Toll-like receptors (TLs), which are expressed by innate immune cell populations, including macrophages, neutrophils and dendritic cells (DCs) (FIG. 1). Much of our current understanding of the molecular mechanisms underlying fever stems from studies in which rodents were injected with LPS, a component of Gram-negative bacterial cell walls, to model immune-induced thermoregulation. In this model, prostaglandin E2 (PGE2) produced by brain vascular endothelial cells is considered a major pyrogenic mediator of fever.31–33 This lipid effector molecule integrates input signals from pyrogenic cytokines produced in response to pathogenic stimuli, with output signals involving neurotransmitters that raise core body temperature (FIG. 1). PGE2 is also synthesized in the periphery early in this response – that is, prior to the detection of circulating cytokines. It is produced by hematopoietic cells following LPS-mediated activation of TLR4 and travels through the blood/brain barrier to initiate fever.26,30,34–38 LPS-induced fever occurs via autonomic mechanisms driven by PGE2 binding to EP3 prostaglandin receptors expressed by thermoregulatory neurons in the median preoptic nucleus within the hypothalamus.8,39–41 Endotherms elevate body temperature through the release of norepinephrine, which increases thermogenesis in brown adipose tissue and induces vasoconstriction in the extremities to reduce passive heat loss.26,27 In addition, signalling through the neurotransmitter acetylcholine stimulates the musculature to convert stored chemical energy into thermal energy and increases overall metabolic rates.2,26,42,43 Endotherms, like ectotherms, also engage in heat-seeking behavioral thermoregulation which does not require median preoptic neurons although the pathways involved are largely unknown.8–10

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LPS-induced TLR4 signalling stimulates the synthesis of pyrogenic cytokines (namely IL-1, tumour necrosis factor ((TNF)) and IL-6) at the site of infection as well as within the brain, and it is becoming clear that IL-6 is as an important mediator of fever induction.26,44–47 Notably, multiple cell types within the brain (for example, astrocytes, microglial cells and neurons) have the capacity to synthesize IL-6 in response to local inflammatory stimuli48–53. Although the direct administration of TNF, IL-1, or IL-6 into the brain leads to a febrile response, several lines of evidence point to a requisite role for IL-6 in sustaining fever. In this regard, LPS-induced fever does not occur in the presence of IL-6-specific neutralizing antibody or in IL-6-deficient mice, even though TNF and IL-1 upregulation is normal in these settings.54–58 Moreover, direct intracerebroventricular injection of IL-6, but not IL-1, restores febrile responses in IL-6-deficient mice.55 Febrile temperatures have further been implicated in a positive feedback loop during the early stages of infection. Specifically, passive elevation of the core body temperature of mice to the febrile range using whole body hyperthermia substantially augments circulating levels of TNF, IL-1, and IL-6 during LPSinduced inflammation.26,59–61 The pyrogenic role of IL-6 has recently been corroborated in Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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patients with paediatric leukaemia, where treatment with the IL-6 receptor antagonist tocilizumab was found to reverse the high fevers that develop during T cell basedimmunotherapy (specifically, following the administration of chimeric antigen receptorexpressing T cells or a CD19/CD3-bispecific antibody).62,63

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Systemic or locally produced cytokines act in the brain to augment the synthesis of cyclooxygenase 2 (COX2), the enzyme responsible for oxidizing arachidonic acid to produce PGE2 (FIG. 1). For instance, IL-1 receptors that mediate COX2 induction have been identified on brain endothelial cells within the preoptic region of the hypothalamus.64,65 Although the specific cell types that upregulate COX2 in response to IL-6 remain to be identified, blood vessels in the brain reportedly express the IL-6 receptorα subunit,53 which together with the ubiquitously expressed gp130 subunit forms the functional IL-6 receptor. Several studies have shown that cerebral COX2, PGE2, and fever are not induced during LPS-driven inflammation in IL-6-deficient mice or in the presence of IL-6-specific neutralizing antibody.66–68 Alternatively, IL-6 cannot initiate a febrile response in the absence of COX2 or PGE2, and intracerebroventricular delivery of PGE2 bypasses the requirement of IL-6 for fever induction in IL-6-deficient mice.69,70 Collectively, these observations establish that COX2 and PGE2 are crucial mediators that can operate downstream of IL-6 in the LPS-induced febrile response. RANKL and fever induction

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An open question is whether IL-6 is the direct regulator of COX2 and PGE2 induction during the febrile response or whether other intervening cytokines are involved. The latter possibility is suggested by an elegant study by Hanada et al. showing that, similarly to IL-6, the cytokine known as receptor-activator of NF-kB ligand (RANKL) converges on the COX2–EP3–PGE2 pathway leading to fever induction in the LPS-induced model of inflammation47 (FIG. 1). RANKL is best known as a regulator of bone remodelling and lymph node organogenesis.71 However, mRNA encoding RANKL is also produced in the lateral septal nucleus region of the brain that interconnects with the hypothalamus and the RANKL receptor, RANK, is found on astrocytes in the preoptic region of the hypothalamus.47 Further support for a role of this cytokine in thermoregulation is provided by findings that children with RANK mutations exhibit impaired fever responses during pneumonia.47 Although the potential interplay between IL-6 and RANKL–RANK during fever has not been explored, it is tempting to speculate that RANKL is a downstream mediator of IL-6-induced pyrogenesis based on evidence that IL-6 directly stimulates RANKL synthesis by synovial fibroblasts in mouse models of rheumatoid arthritis.72

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Immune stimulation by thermal stress One benefit widely attributed to fever is the enhancement of immune-protective mechanisms during infection. Defence against pathogens involves tight spatial and temporal regulation of the immune system, and the same pyrogenic cytokines that are produced during the induction of fever also operate locally to orchestrate immunity within infected tissues.73 Innate immune cells are the ‘first responders’, arriving within hours to directly destroy pathogens via phagocytic or cytotoxic activities. These activities limit infection until a peak adaptive immune response is generated, normally around one week later. Macrophages and Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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DCs bridge the gap between innate and adaptive immunity by taking up pathogens in peripheral tissues and then relocating to draining lymph nodes where they drive expansion of pathogen-specific effector T cells.74,75 Crucial for this process is the co-localization of DCs and T cells near high endothelial venules (HEVs) that are the major portals for entry of blood-borne lymphocytes.74–76

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Given the complexity of these immune mechanisms, it is remarkable that fever-range temperatures stimulate almost every step involved in this process, promoting both innate and adaptive immunity. The potential impact of the thermal element of fever has primarily been explored using hyperthermic temperatures within the febrile range for mammals (that is, ranging from 38–41°C; ΔT~1–4°C above baseline) in the various in vitro and in vivo studies described below. Experimental hyperthermia is a powerful approach to study the impact of febrile-range temperatures on immunity, which is otherwise difficult to discriminate during natural fever because of the attendant inflammatory programme (comprised of lipid and cytokine mediators) that regulate both fever and immunity. However, an important caveat from a physiological perspective, is that the heat conservation associated with natural fever differs fundamentally from cooling mechanisms enacted by thermoregulation following exogenous heat application. Impact of febrile temperatures on innate immunity

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Previous research using animal models of hyperthermia treatment alone, or with LPS challenge or bacterial infection, strongly supports the idea that fever-range temperatures elevate the respiratory burst typically associated with activation and bacteriolytic activity of neutrophils77,78 (FIG. 2a). Thermal stress further increases neutrophil recruitment to local sites of infection and other distant tissues61,79 (FIG. 2a) including tumours77 Neutrophil localization in peripheral tissues is due, at least in part, to heat-induced increases in circulating neutrophils which are dependent upon granulocyte colony-stimulating factor (GCSF).80,81 G-CSF is also central in a model of radiation-induced neutropenia where feverrange whole body hyperthermia substantially increases the rate of neutrophil recovery in the blood, and augments the number hematopoietic stem cells and neutrophil progenitors within the bone marrow82 (FIG. 2a). This effect is dependent upon enhanced production of IL-17, IL-1β and IL-1α preferentially in intestinal tissue. Importantly, the precise outcome of the thermal effect depends on the heating protocol used and the geography of cell recruitment (FIG. 2a). Indeed, temperatures above the normal febrile range impair neutrophil accumulation and function.83 Moreover, Hasday and colleagues found that fever, or exposure to fever-range hyperthermia, in an LPS model increases neutrophil localization to the lung, which can have negative consequences due to inflammation-induced local tissue damage.61,84 Heat-induced neutrophil recruitment in the lung depends on the non-canonical chemotactic HSP, CXCL8 (also known as IL-8), which is expressed under the control of the heat-inducible transcription factor heat shock factor 1 (HSF1) (BOX 1).61,84,85 Neutrophil recruitment in the lung also involves a decrease in endothelial barrier integrity through a mechanism depending on p38 MAPK and ERK1–ERK2 signalling.84 The impact of heat on natural killer (NK) cells has been most extensively studied in the context of tumour immunity. It has been shown that NK cell cytotoxic activity and

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recruitment to tumour sites is increased by fever-range hyperthermia in vivo86–89 (FIG. 2b). This enhanced cytotoxicity depends upon heat-induced upregulation of the NKG2D ligand MICA (MHC class I polypeptide-related sequence A) on tumour cells as well as on the clustering of NKG2D receptors on the surface of NK cells.90 Elevated temperatures also decrease MHC class I expression by tumour cells while simultaneously increasing HSP70 production, and both of these responses are linked to enhanced cytotoxic potential in NK cells.91 The upregulation of HSPs in tumour cells in response to thermal stress is also likely to be involved in the enhanced cross-priming of antigen-specific cytotoxic T lymphocytes that was observed when DCs were loaded with lysate from heated melanoma cells.92

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Macrophages have served as a major model for the study of febrile-range hyperthermia. Early studies demonstrated that whole-body heating (to ~39.5°C) improves bacterial clearance and also increases serum concentrations of TNF, IL-1, and IL-6 in mice challenged with LPS.59,60,93,94 The source of these cytokines was found to be the macrophages of the liver (that is, Kupffer cells) as well as macrophages in other organs. Later work by Lee et al. showed that hyperthermia induces the upregulation of HSP70 and this ‘reprogrammes’ macrophages to show sustained activation in response to LPS.95 The mechanism involves the phosphorylation of the IKK and IκB kinases, the nuclear translocation of NFκB and its binding to the Tnfpromoter.95,96 HSP70 is also required for enhancing the expression of nitric oxide and inducible nitric oxide synthase by peritoneal macrophages following exposure to fever-range temperatures together with LPS and IFNγ.97 Although HSPs are usually assumed to be intracellular, heat stress can induce HSP70 release from cells into the extracellular environment where it can act as a damage-associated molecular pattern (DAMP) to stimulate macrophages and DCs.98–100 Extracellular HSP70 and other HSPs engage multiple surface receptors, including CD91, scavenger receptor A, CD40, TLR2 or TLR4, leading to the release of nitric oxide, TNF, IL-6, IL-1β and IL-12.100–110 Of note, some investigators have paradoxically observed an anti-inflammatory role for HSPs.111–113 It is suggested that these differences result from the precise location of HSPs within macrophages: extracellular HSPs provide danger signals to enhance inflammation whereas intracellular HSP could help to suppress inflammatory signalling.114 Taken together, the data regarding innate immune cells, body temperature and HSPs reveal fascinating, yet still poorly understood, layers of interdependency between the febrile response and the more ancient HSP response. Fever enhances DC functions

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Several studies demonstrate that elevated temperatures substantially enhance the phagocytic potential of DCs, in addition to augmenting interferon-α (IFNα) production in response to viral infection (FIG. 2c).115–118 Heating of immature DCs also upregulates their expression of TLR2 and TLR4, suggesting a role for thermal signals in enhancing pathogen sensing by innate immune cells.119,120 Febrile temperatures further increase DC expression of MHC class I and class II molecules and co-stimulatory molecules, including CD80 and CD86, and can augment the secretion of the Th1 cell-polarizing cytokines IL-12 and TNF.102,117,119–123 Additional reports point to a role for febrile-range temperatures in augmenting the migration of antigen-presenting cells (APCs), such as skin Langerhans cells, to draining lymph nodes124 (FIG. 2c). These data may help to explain the fact that febrile Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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temperatures can accelerate the swelling phase of a contact hypersensitivity reaction when heat is delivered to mice shortly after the application of the elicitation dose of a skin sensitizer, fluorescein isothiocyanate (FITC).124 The underlying mechanism directing DC migration to draining lymph nodes likely involves increased responsiveness of CCchemokine receptor 7 (CCR7) to its ligands, which has been described for heat-treated mature DCs in chemotaxis assays in vitro.121 CCR7 senses CCL21 chemokine gradients in vivo, thereby guiding DC entry into afferent lymphatics and their subsequent migration near HEVs within draining nodes.125–128 Thus, febrile temperatures appear to regulate the CCR7–CCL21 axis in order to optimally position DCs in lymphoid organs at sites where they can present antigen to lymphocytes upon their arrival via HEVs.

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Given these observations, it is not surprising that fever-range thermal stress enhances the ability of DCs to stimulate T cells as well as DC cross-presenting functions (FIG. 2c). In mixed lymphocyte reactions, applying thermal stress ex vivo to LPS-pulsed mature human monocyte-derived DCs led to enhanced proliferation of naïve CD4+ T cells and promoted their differentiation towards a Th1 cell phenotype.121 Similarly, DCs isolated from heated mice exhibit a superior ability to activate T cells.102 In studies where DCs from patients with medullary thyroid cancer were preheated prior to co-culture with T cells, the T cells showed enhanced cytotoxicity against tumour targets.119 This increased cytotoxicity of effector T cells correlated with heat-induced upregulation of both MHC class I and HSP70 expression in mature, but not immature, DCs. Together, these findings demonstrate that systemic feverrange temperatures can target different components of the innate immune system, including the HSP response, in order to enhance effector T cell responses. Thermal mechanisms boost adaptive immunity

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A crucial determinant for the generation of adaptive immunity is the high rate of lymphocyte trafficking through lymphoid organs. The entire pool of naive T cells in a mouse lymph node turn overs ~2–3 times per day as a result of T cell recirculation.75,129 This dynamic flux increases the probability that rare antigen-specific T cells (present at a frequency of only ~1 in 105–106)130,131 will receive activating signals from DCs. The entry of bloodborne T and B cells into lymph nodes and Peyer’s patches occurs preferentially at HEVs through a well-defined adhesion cascade that involves; one, L-selectin and/or α4β7 integrin initiated tethering and rolling; two, CCL21-dependent activation of CCR7 on adherent lymphocytes; three, LFA1–mediated firm arrest via binding to its endothelial counterreceptors intercellular adhesion molecule-1 (ICAM1) and ICAM2; and four, LFA1– ICAM1–2-directed transendothelial migration.74–76,132,133 As described below, we have shown that fever-range thermal stress targets multiple steps in this cascade by invoking a wide array of lymphocyte and endothelial trafficking molecules134–142 (FIG. 3a). An early indication that fever could control lymphocyte trafficking emerged from studies showing transient decreases in circulating T cells in mice or patients with cancer following elevation of core body temperatures to ~39.5°C by febrile-range whole body hyperthermia.83,137,143 Reductionist studies found that direct heat treatment of T or B cells ex vivo for 6 hours resulted in an approximately 2-fold increase in their ability to bind to HEVs in vitro or to home to lymph nodes or Peyer’s patches in vivo.134–139 Lymphocytes

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isolated from heated mice exhibit similar enhancement of homing properties.138 It is worthwhile noting that this represents a substantial increase above the already efficient rate of homeostatic trafficking whereby ~1 in 4 lymphocytes initiate the adhesive events that precede extravasation.75,129

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Fever-range temperatures augment trafficking through a lymphocyte-autonomous mechanism by targeting the binding activity of both L-selectin (FIG. 3a) and α4β7 integrin without altering their density.134,136–139 In lymph node HEVs, fever-range hyperthermia promotes L-selectin-dependent lymphocyte rolling along vessel walls through the formation of short-lived catch-bonds with its endothelial counter-receptor, peripheral node addressin (PNAD).74,75 Febrile temperatures also enhance α4β7 integrin binding to mucosal addressin cell adhesion molecule 1 (MADCAM1) in Peyer’s patch and mesenteric lymph node HEVs.144 Direct exposure of lymphocytes to heat does not alter LFA1 affinity for its endothelial ligands.134,136 It remains an open question whether the chemokine receptor, CCR7, is responsive to febrile temperatures.

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The intrinsic binding function of HEVs is also enhanced approximately 2-fold in LPS- or turpentine-induced mouse models of fever as well as during exposure of mice to fever-range whole body hyperthermia.136,137,140–142 (FIG. 3a). As in lymphocytes, maximal enhancement of HEV adhesion requires sustained temperature elevation (more than 6 hours),136,137,140–142 recapitulating the extended time-frame of physiological fever responses. Chen et al. visualized lymphocyte interactions in mouse HEVs using intravital microscopy (FIG. 3b), together with quantitative image analysis of trafficking molecules, to pinpoint the thermally responsive trafficking mechanisms in HEVs.140,141 Thermal stress does not alter the ability of HEVs to support rolling, nor does it change the intraluminal density of the prototypical rolling molecules, PNAD or MADCAM1.140,141 Instead, exposure to febrile temperatures profoundly increases the ability of HEVs to support the stable arrest of lymphocytes and this can be attributed to heat-induced increases in the intravascular density of CCL21 and ICAM1140,141 (FIG. 3a). Of note, the level of HEV adhesiveness and ICAM1 expression induced by thermal stress is equivalent to that observed in response to the potent pro-inflammatory cytokine, TNF.140 Thermal upregulation of CCL21 and ICAM1 in HEVs is consistent with the known concentrationdependent roles of these molecules in augmenting LFA1 affinity (~10,000-fold), thereby supporting stable adhesion of lymphocytes within vessel walls.145,146 Additionally, ICAM1 elevation in response to hyperthermia likely promotes LFA1-dependent transendothelial migration in HEVs and the formation of ICAM1-dense adhesive patches that guide lymphocyte diapedesis into underlying tissues.133,147,148

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Once lymphocytes gain entry into lymphoid organs, there is evidence that their ability to respond to stimulatory signals is also enhanced by febrile temperatures. Direct exposure of T cells to fever-range hyperthermia increases their proliferation in response to mitogens.149,150 Furthermore, in both in vitro and in vivo models of antigen-driven T cell activation by APCs, thermally treated CD8+ T cells show greater differentiation towards an effector phenotype, with pronounced L-selectin downregulation, enhanced cytotoxic function and increased production of IFNγ151,152 (FIG. 3a). Enhanced stimulation of naive CD8+ T cells is aligned with temperature-dependent PKCβ activation, prolonged stable contacts with Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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APCs, and transient clustering of components of the immunological synapse (TCRβ and CD8) in cholesterol-enriched microdomains,.151,152 Similar heat-induced changes in membrane fluidity and macromolecular clustering in the plasma membrane occur in CD4+ T cells which reduce the requirement for CD28 stimulation for IL-2 production.153 These findings suggest that febrile temperatures lower the threshold for T cell signalling and effector T cell differentiation by pre-associating the signalling components of the TCR complex. IL-6 is a thermally sensitive effector of trafficking

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Investigation into the mechanisms underlying thermal regulation of trafficking led to the unexpected discovery that the same pyrogenic cytokine responsible for inducing fever, namely IL-6,135,137,138 also controls both lymphocyte and endothelial adhesion.132,138–140,142 The thermal response further depends on a second soluble factor, the soluble form of the IL-6 receptor α subunit (sIL-6Rα), which acts cooperatively with IL-6 and the membrane-anchored gp130 signal transducing molecule through a well-defined mechanism termed trans-signalling138–140,154,155 (FIG. 4a). This thermally sensitive mechanism was identified in vitro and in vivo using recombinant soluble gp130138,140, which is a competitive antagonist of IL-6 trans-signalling but does not affect classical signalling involving membrane-anchored IL-6Rα.154,156

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In lymphocytes, the MEK1–ERK1/ERK2 signalling pathway, but not p38 MAPK or JNK, operates downstream of IL-6–sIL-6Rα trans-signalling in response to heat.138 This promotes L-selectin interactions with actin-based cytoskeletal scaffolding elements, thereby enhancing its apparent tensile strength (FIG. 4b). IL-6-induced activation of STAT3 also occurs in lymphocytes in response to thermal stress138, although it is not known whether this contributes to lymphocyte adhesion, or delivers survival signals157,158 that aid the expansion of populations of effector lymphocytes within lymphoid organs. Consistent with the evolutionary conservation of the febrile response, L-selectin adhesion is induced by feverrange temperatures through a common IL-6 trans-signalling mechanism in animals representing four taxa of jawed vertebrates that includes endothermic mammals (for example, human, rodents, dog, cow, tiger, elephant, and rhinoceros) and birds (chicken), as well as ectothermic amphibians and fish.134,135,137–139 These observations strongly suggest that conservation of IL-6-regulated lymphocyte trafficking mechanisms over hundreds of millions of years of evolution confers a survival benefit during fever.

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Gp130 ligation by IL-6–sIL-6Rα also upregulates the intravascular density of ICAM1 in HEVs during heat treatment of mice132,140 (FIG. 4b). The dual requirement for IL-6 and sIL-6Rα for ICAM1-dependent trafficking in HEVs during thermal stress is in line with the prevailing view that endothelial cells generally lack membrane-anchored IL-6Rα, and thus are refractory to IL-6 unless sIL-6Rα is available.132 STAT3 and MEK1–ERK1/ERK2 signalling have been implicated in transcriptional regulation of ICAM1132 and, thus, are potential mediators of the thermal response in HEVs. By contrast, CCL21 induction is not dependent on IL-6 trans-signalling,140 suggesting an additional molecular pathway is induced by febrile temperatures.

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One of the most intriguing findings to emerge from intravital imaging relates to the tight spatial regulation of IL-6–sIL-6Rα responses in vascular beds during thermal responses. In this regard, Chen et al. showed that HEVs respond to IL-6 trans-signalling during thermal stress, but contiguous vascular segments that are not comprised of high endothelial cells (HECs) are completely refractory to thermally induced IL-6 trans-signalling (FIG. 4b).140 Similarly, non-HEVs in other organs are not responsive to febrile temperatures although heat-shock (which occurs at temperatures greater than 43°C) reportedly stimulates ICAM1 expression in normal vascular endothelium137,140–142,159,160 This restricted vascular response to physiological temperature elevation is proposed to maintain focal trafficking of lymphocytes at HEVs in lymph nodes and Peyer’s patches located throughout the body, thus maximizing their opportunity to scan pathogen-derived antigens from peripheral sites of infection.137,140,141

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The mechanism maintaining spatial resolution within venular segments over distances spanning the width of a single HEC (~30 µm)161 remains to be resolved, but clues have emerged from recent transcriptional profiling of various cell subsets in lymphoid organs. HECs are distinguished from their normal endothelial cell counterparts by elevated expression of Il6st (which encodes gp130)162 that could theoretically predispose them to be highly sensitive to IL-6–sIL-6Rα in the local milieu (FIG. 4b). Although the overall nodal concentrations of IL-6–sIL-6Rα are unchanged by thermal stress,140,163 heat could theoretically induce their synthesis by discrete cell populations or lower the threshold for signalling in HECs. Fibroblastic reticular cells (FRCs) are a possible source of IL-6 during fever based on their high expression of Il6 mRNA relative to hematopoietic cells or blood endothelial cells within skin-derived lymph nodes.164 Unlike other vascular beds that are circumscribed by pericytes, HEVs are in direct contact with FRCs, and thus are optimally positioned to receive instructions from FRC-derived cytokines.74,75,165 Of particular relevance is a report that IL-6 synthesis by fibroblasts can be induced by the heat-inducible transcription factor, HSF1.166 The sIL-6Rα necessary for trans-signalling is likely provided by neighbouring leukocytes including DCs, monocytes, and/or T cells.138,164 Recent studies have shown that febrile temperatures can also act through IL-6 trans-signalling to augment the recruitment of cytotoxic CD8+ T cells across tumour-associated vessels.142 These studies are highly relevant to the use of thermal medicine as an adjuvant for cancer immunotherapy (BOX 2) and raise the possibility that fever could invoke similar mechanisms to amplify effector T cell trafficking at sites of infection.

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The immune response must be tightly regulated to avoid excessive tissue damage after infection. By extension, it makes sense that the effects of febrile temperatures on the immune system are also temporally regulated during the resolution phase of inflammation although a full picture of the underlying mechanisms is yet to emerge. One example is the rapid restoration of lymphocyte trafficking in HEVs to basal levels within 6 hours following cessation of fever-range hyperthermia.134,137,141 Normalization of HEVs is mediated by zinc-dependent metalloproteinases that cleave endothelial ICAM1 while sparing other trafficking molecules (such as PNAD),141 although it is not known if heat stimulates the catalytic activity of these enzymes. In line with a potential anti-inflammatory role of Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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hyperthermic temperatures, heat shock (42°C for 15 min) has been found to blunt leukocyte adhesion within vessels if administered 2 day prior to the intravascular delivery of the neutrophil attractant FMLP in vivo.167

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Although febrile temperatures initially increase the production of pro-inflammatory cytokines by macrophages at sites of inflammation,59–61,95,96 there is also evidence that thermal stress dampens cytokine synthesis once macrophages become activated. This sequence of events is analgous to natural fever, which often occurs after macrophages and other innate immune cells initially encounter PAMPs. In this regard, human monocytederived macrophages with an activated phenotype produce less TNF, IL-6, and IL-1β when exposed to febrile temperatures than heat-inexperienced cells.95,96,168–170 Heat reduces transcription of pro-inflammatory cytokines through repressive activities of HSF1, together with diminished recruitment of NF-κB to the promoter regions of cytokine-encoding genes, and also lowers cytokine mRNA stability.171–173 Thermal treatment of LPS-activated macrophages also appears to dial down inflammation by inhibiting the release of the inflammatory DAMP known as high mobility group box 1 (HMGB1), which is a ligand for TLR2 and TLR4.170,174 Inhibition of HMGB1 release prevents the subsequent activation of NF-κB, which controls the synthesis of pro-inflammatory cytokines in innate immune cells.169,170,174 The idea that heat can dampen an on-going pro-inflammatory condition in vivo has recently been tested in a murine model of collagen-induced arthritis.175 Mice exposed to fever-range hyperthermia had significantly less joint damage, correlated with a reduction in serum TNF levels and increased IL-10 production in inflamed joints. Collectively, these findings suggest that strategic temperature shifts contribute to a biochemical negative feed-back loop that protects tissues against damage from excessive cytokine release following infection.

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Thermogenesis and adrenergic signalling in immunity– an emerging concept

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Neural components of the thermoregulatory system continuously monitor temperature changes throughout the body and initiate integrated responses that either increase internal heat content (for example, through thermogenesis in brown adipose tissue) or increase the dissipation of heat (for example, following intense exercise).8 Given the homeostatic importance of thermoregulation, it is all the more remarkable that fever has been so long maintained in evolution, as natural thermoregulatory signals must be suppressed in order to increase body temperature. Although the examples discussed earlier in this review demonstrate that the immune system is responsive to elevated temperatures, new studies reveal that this system is also highly sensitive to the metabolic stress associated with thermogenesis. Emerging evidence strongly supports a direct role for cold stress-induced norepinephrine production and its interaction with β-adrenergic receptors on immune cells as a major mechanism for immune modulation by environmental cold stress. It is well established that norepinephrine-driven stimulation of β-adrenergic receptors is crucial for the release of additional heat from mitochondria in brown adipose tissue during cold stress to maintain a normal core body temperature.176,177 Moreover, the ubiquitous presence of βadrenergic receptors has been observed on the surface of immune cells, and there is a

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growing appreciation of the functional consequences of signalling through these receptors.178–180 Even more recent studies demonstrate a crucial role for β-adrenergic receptor signalling by norepinephrine for control of lymphocyte egress from lymph nodes and modulation of cytokine production and proliferation in memory CD8+ memory T cells.181,182

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These parallel lines of research have now been joined in studies that demonstrate marked alterations in immune cell activity during cold stress. Nguyen et al. discovered that cold stress stimulates IL-4 and IL-13–driven differentiation of macrophages in brown fat toward an ‘alternative activation’ programme leading to their production of norepinephrine.183 (FIG. 5a). Surprisingly, data obtained using various knock-out mice (deficient in IL-4, IL-13, STAT6 or IL-4 receptor) revealed that the norepinephrine produced by these macrophages is critical for maintaining sufficient thermogenesis in the face of cold stress.183,184 Kokolus et al. further demonstrated that DCs exhibit a reduced ability to stimulate T cells if they are from cold-stressed mice that have a normal body temperature due to increased thermogenesis.183,184 Cold stress is also associated with accelerated tumour growth in murine models, which reflects enhanced tumour cell survival pathways as well as a shifted balance toward an immunosuppressive microenvironment with elevated intratumoral myeloid-derived suppressor cells and regulatory T cells together with reduced CD8+ effector T cells184–186 (FIG. 5b).

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An intriguing aspect is that the presence of cancer creates a notable heat seeking behavioural response in animals.184 These data support the conclusions drawn by Romanovsky and colleagues who have contended that endothermic animals, including humans, exhibit heatseeking behaviour even before other fever-generating symptoms occur.9,10 Findings in this exciting area contribute additional molecular detail to the fundamental role of temperature stress in influencing the functional balance between various arms of the immune system.187,188

Concluding remarks and future directions

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The evolutionary conservation of the fever response over millions of years is in line with its protective role — the survival benefit conferred on the host outweighs the metabolic cost of elevating core body temperatures during infection. Cellular components of the immune system have emerged as central players that actively drive fever induction in addition to serving as thermally sensitive effectors. Moreover, the complexity of the molecular pathways that coordinate a febrile response is mirrored by the diverse cell types that are affacted by hyperthermic temperatures; these include DCs, macrophages, NK cells, neutrophils, T and B lymphocytes, and vascular endothelial cells. The picture that emerges is one in which febrile temperatures serve as a systemic alert system that broadly promotes immune surveillance during challenge by invading pathogens. Furthermore, mechanistic insight into the immune-protective nature of fever has opened new avenues to exploit the immunostimulatory activities of thermal stress in the context of cancer therapy. Fundamental questions remain regarding the nature of the temperature-sensing machinery that triggers changes in immune cell behaviour. HSF1-regulated HSPs are strong candidates

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in view of their rapid induction even at the relatively modest temperature elevation (ΔT~1– 4°C) that accompanies fever.26,122,189–191 Also intriguing are reports that HSF1 regulates additional genes relevant to the induction and/or effector phases of fever, including IL6 and COX2.166,192 Notably, HSP90 and the JAK1–JAK2–STAT3 signalling axis triggered by IL-6 are participants in a feed-forward loop — IL-6-–STAT3 signalling stimulates HSP90 production while JAK2 and STAT3 are established client proteins that are chaperoned by HSP90.193–197 Thus, it is tempting to speculate that induction of HSP90 or other HSPs by febrile temperatures lowers the threshold for IL-6 signalling. Additionally, a class of temperature-sensing transient receptor potential (TRP) cation channel proteins expressed on immune cells and endothelial cells are likely to coordinate responses to febrile temperatures and inflammatory cytokines such as IL-6 and lipid mediators.26,198,199 There are also outstanding questions regarding the mechanisms underlying the spatial regulation by IL-6 during fever induction and lymphocyte trafficking in HEVs. Although brain endothelial cells and HECs are predicted to be main targets for IL-6, contributions of intermediary cells have not been excluded.

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Another unresolved question is whether febrile temperatures mobilize innate and adaptive immune cells to sites of infection. Observations that administration of fever-range hyperthermia is effective in boosting E-selectin, P-selectin and ICAM1-dependent trafficking of cytotoxic CD8+ T cells in tumour tissues142 raise the strong possibility that similar mechanisms are enacted by fever in infected tissues. Similarly to CXCL8, several inflammatory chemokines that recruit NK cells, CD4+ and CD8+ T cells, and monocytes (CXCL9, CXCL10, CXCL11, CXCL12) contain putative HSF1-binding sites within their gene promoters and, consequently, may be induced by thermal stress.26 An important caveat is that information regarding the cytokine circuitry (for example, IL-6, RANKL and IL-1) leading to fever as well as the impact of temperature on immune function is largely based on experimental models employing LPS or fever-range hyperthermia as surrogates for pathogen-induced fever. Although these studies provide insight into the mechanistic underpinnings for immune regulation by temperatures within the febrile range, lessons learned from studies of thermogenesis183–185 indicate that overall temperature sensing (cold or hot) in the absence of disease can have unexpected outcomes on innate and adaptive immunity. The next frontier will be to establish whether the same mechanisms identified during challenge of healthy animals with LPS or fever-range hyperthermia are operative during febrile responses to pathogens.

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We thank M. Appenheimer, J. Black, and M. Messmer for helpful comments on the manuscript, E. Smith and UC Berkeley Natural Resources Library for assistance with archived citations, and J. Muhitch for providing the photomicrograph depicting lymph node HEV. This work was supported by the US National Institutes of Health (CA79765, CA085183, and AI082039) and the Jennifer Linscott Tietgen Family Foundation. We also acknowledge the significant contributions of colleagues in the field that could not always be cited due to space limitations.

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140. Chen Q, et al. Fever-range thermal stress promotes lymphocyte trafficking across high endothelial venules via an interleukin 6 trans-signaling mechanism. Nat Immunol. 2006; 7:1299–1308.

[PubMed: 17086187] This study establishes that fever-range thermal stress acts through an IL-6 trans-signaling dependent mechanism to upregulate ICAM1 expression selectively in HEVs.

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BOX 1 Thermal regulation of heat shock proteins

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Heat shock proteins (HSP) are cytoprotective proteins that are constitutively expressed and also rapidly induced under proteotoxic stress conditions such as heat, hypoxia, oxidative stress, toxin exposure, nutrient deprivation, and infection.26–29,200–202 Although HSPs were originally discovered in the context of heat shock (42–45°C), they are also inducible by febrile temperatures in mammalian cells (38–41°C).26,122,189–191 Stress-induced transcription of HSPs is driven by post-translational modifications (sumoylation and phosphorylation) of heat shock factor protein 1 (HSF1) which release it from a complex with HSP70 and HSP90.29,200,201 This results in the formation of HSF1 homotrimers that translocate to the nucleus and activate transcription of genes including HSPs that contain ‘heat shock element’ sequences.29,200,201 The major function of HSPs is to maintain appropriate folding of their client proteins, thereby protecting them from proteolysis. HSPs have key roles in regulating multiple signalling pathways under constitutive and stress conditions. For example, there are more than 200 established client proteins of HSP90, including members of the MAPK, JAK–STAT and CDK1 signalling pathways.29,200,202–204 Cancer cells under chronic proteotoxic stress conditions often become ‘addicted’ to HSPs and high intratumoral expression of HSP70 or HSP90 is a poor prognostic indicator in cancer patients, suggesting HSP inhibitors as a treatment option in cancer.200,203,204 There are also non-canonical HSPs that do not have traditional chaperoning/protein folding activity but whose expression is nonetheless tightly regulated by HSF1. One example is the chemokine CXCL8 (also known as IL-8) that mediates recruitment of neutrophils upon exposure to fever-range hyperthermia in LPS instillation models of acute lung inflammation.84,85 Active areas of investigation in the HSP field are considering the physiological impact of the multiple post-translational modifications of HSFs and HSPs (for example, phosphorylation, acetylation, Snitrosylation, ubiquitination, and sumoylation) as well as the interplay between these molecules and positive and negative immune regulation.29,200,205

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BOX 2 Thermal therapy and cancer

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Thermal therapy is administered at a wide range of temperatures for cancer treatment. High temperature focal hyperthermia (> 45°C) and ablation therapy (> 70°C) directly destroy cancer cells and can indirectly boost antitumour immunity, while moderate hyperthermic therapy (38–42°C) is used mainly in an adjuvant setting to target the tumour microenvironment.206–208 Temperature effects on blood flow, vascular permeability, interstitial pressure and hypoxia are implicated in enhanced chemo- and radiosensitization in cancer patients treated with hyperthermia.209–215 Thermal therapy also holds promise for improving delivery of chemotherapeutic drug cargo by heatsensitive liposomes.216 Recent preclinical studies suggest that the immunostimulatory activities of febrile temperatures can be exploited therapeutically in combination with promising cancer treatments. Emerging immunotherapies such as dendritic cell (DC) vaccination, adoptive transfer of ex vivo activated T cells, or checkpoint blockade inhibitors (for example, drugs targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PD1)) have shown benefit in generating antitumour immunity.217–220 Notably, the efficacy of DC vaccines in patients with advanced melanomas or mouse tumour models is substantially improved with the use of hyperthermia as an adjuvant therapy.221,222 Moreover, fever-range thermal therapy overcomes impediments to trafficking in mouse tumour vessels through an interleukin-6 (IL-6) trans-signalling mechanism that stimulates E-selectin and P-selectin-dependent rolling and intracellular adhesion molecule-1 (ICAM1)-dependent firm adhesion of adoptively transferred CD8+ cytotoxic T cells (see figure).142 Increased T cell entry into tumours is further linked to improved antitumour immunity and delayed tumour growth.142 The antitumour immune effects of IL-6 unleashed by thermal therapy are counterintuitive in light of substantial evidence that IL-6 signalling exerts protumourigenic activities by stimulating the survival and proliferation of tumour cells as well as angiogenesis.155,223 Together, these studies highlight a unique role for thermal therapy in modulating the tumour microenvironment that can be co-opted to increase the efficacy of diverse anti-cancer therapies.

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Figure 1. The induction of fever during infection

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The recognition of damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), by Toll-like receptors (TLRs) and other pattern recognition receptors drives the activation of dendritic cells (DCs) and macrophages. These innate immune cells release prostaglandin E2 (PGE2) as well as pyrogenic cytokines (namely, interleukin-1 (IL-1) IL-6, and tumour necrosis factor (TNF)) that act systemically to induce fever. IL-6 operates downstream of IL-1 in the median preoptic nucleus region within the hypothalamus to induce the synthesis of cyclooxygenase 2 (COX2), the enzyme responsible for production of additional PGE2.64,65 PGE2 is considered the major pyrogenic mediator of fever.31–33 Receptor activator of NF-κB (RANK) expressed by astrocytes also acts via the COX2–PGE2 pathway to induce fever.47 However, it is not known whether this pathway parallels the IL-6 response or if the IL-6 and RANKL pathways converge, potentially via IL-6 regulation of RANKL expression in vascular endothelial cells in the hypothalamus. Neurons expressing PGE2 receptor 3 (EP3) trigger the sympathetic nervous system to trigger norepinephrine release, which elevates body temperature by increasing thermogenesis in brown adipose tissue as well as by inducing vasoconstriction to prevent passive heat loss.2,26,27,42,43 Additionally, acetylcholine contributes to fever by stimulating muscle myocytes to induce shivering.

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Figure 2. Response of innate immune cells to thermal stress

(a) Fever-range temperatures drive several crucial aspects of innate immunity. Fever-range hyperthermia stimulates the release of neutrophils from the bone marrow in a granulocyte– colony-stimulating factor (G-CSF)-driven manner.80–82 Febrile-range temperatures also promote neutrophil recruitment to the lungs and other local sites of infection in a CXCchemokine ligand 8 (CXCL8)-dependent fashion that additionally involves decreased barrier function of vessels.61,84,85 Upon arriving in the site of infection, thermal stress further elevates the respiratory burst which increases the bacteriolytic activity of neutrophils.77,78 Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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(b) Thermal treatment improves natural killer (NK) cell cytolytic activity through induction of MHC class I polypeptide-related sequence A (MICA) expression on target cells (for example, tumour cells) as well as by inducing the clustering of the MICA counter-receptor NKG2D on the surface of NK cells.90 (c) Temperatures in the febrile range increase the ability of antigen-presenting cells to support the formation of the adaptive immune response. Heat improves the phagocytic potential of macrophages and dendritic cells (DCs) and increases their responsiveness to invading pathogens by upregulating their expression of both Toll-like receptor 2 (TLR2) and TLR4.119,120 Thermal treatment also induces the release of immunomodulatory molecules such as cytokines (for example, TNF), nitric oxide (NO) and heat shock protein 70 (HSP70). Additionally, heat increases expression of MHC class I and II molecules as well as co-stimulatory molecules (CD80 and CD86) by mature DCs and augments their CC-chemokine receptor 7 (CCR7)-dependent migration via the afferent lymphatics that serve as a conduit to draining lymph nodes.117,121–124 DCs exposed to febrile temperatures are also more efficient at cross-presenting antigens and inducing T helper 1 (Th1) cell polarization.121

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Author Manuscript Author Manuscript Author Manuscript Figure 3. Fever-range thermal stress and the adaptive immune response

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(a) Fever-range thermal stress supports increased adaptive immunity by targeting two distinct aspects of T cell activation in lymph nodes. Heat enhances the rate of lymphocyte trafficking across high endothelial venules (HEVs) in peripheral lymph nodes through effects on each step of the adhesion cascade. Heat treatment of lymphocytes increases the frequency of L-selectin-dependent tethering and rolling interactions.134,135,137–139 Febrilerange temperatures independently act on HEVs to enhance the transition of lymphocytes from transient rolling to stable arrest by increasing the intravascular density of CCchemokine ligand 21 (CCL21) and intracellular adhesion molecule 1 (ICAM1).140–142 ICAM1 also supports lymphocyte crawling to inter-endothelial cell junctions as well as transendothelial migration.131,145,146 Heat also acts directly on the T cells within lymphoid organs by pre-clustering components of the immunological synapse (TCRβ and CD8) into lipid rafts. This prolongs stable contacts with APCs and increases CD8+ T cell Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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differentiation towards an effector phenotype characterized by enhanced L-selectin downregulation, cytotoxic function, and production of interferon-γ (IFNγ).151,152 (b) Epifluorescence whole-mount confocal microscopy imaging of HEVs that are actively supporting lymphocyte trafficking in a mouse lymph node. HEVs are stained in red with PEconjugated MECA-79 antibody that recognizes peripheral lymph node addressin (PNAD) whereas lymphocytes are labelled in green using carboxyfluorescein succinimidyl ester (CFSE).

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Figure 4. Thermal stress acts through IL-6 trans-signalling to improve lymphocyte trafficking into lymph nodes

(a) Heat-dependent interelukin-6 (IL-6) trans-signalling is initiated by binding of the soluble form of the IL-6 receptor α subunit (sIL-6Rα) to both IL-6 and membrane-anchored gp130.154,155 Soluble gp130 functions as a selective antagonist of IL-6 trans-signalling and downstream activation of canonical JAK–STAT and MEK1–ERK1/ERK2 signalling pathways but does not interfere with classical signalling by membrane-anchored IL-6Rα and transmembrane gp130.156 (b) Febrile temperatures act on lymphocytes and high endothelial cells (HECs) to improve lymphocyte trafficking exclusively across high endothelial venules Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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(HEVs) in lymph nodes. Vessel segments immediately proximal to HEVs are refractory to thermal treatment, which may reflect the lower expression of gp130 by non-specialized squamous endothelial cells that line non-HEVs.162 Left inset, fever-range temperatures act directly on lymphocytes through IL-6 trans-signalling to stimulate the MEK1–ERK1/ERK2 signalling pathway, promoting L-selectin adhesion as well as intermolecular interactions between the actin-based cytoskeleton, α-actinin, and the cytoplasmic tail of L-selectin.138 Right inset, IL-6 trans-signalling upregulates the intravascular density of ICAM1 in HEVs during heat treatment of mice, although the downstream signalling mediators remain unknown. Fibroblastic reticular cells that are in direct contact with HECs165 are a possible source of the IL-6 while proximal dendritic cells (DCs) and T cells could provide the sIL-6R138,164 required to enhance the adhesive properties of HEVs during thermal stress.

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Figure 5. Cold stress stimulates nerve-driven modulation of thermogenesis and anti-tumour immunity

(a) Exposure to cold stress drives the release of neurotransmitters, such as norepinephrine, by neurons. This initiates the interleukin-4 (IL-4) and IL-13-driven ‘alternative activation’ programme of differentiation in macrophages, resulting in additional production of norepinephrine, which stimulates β-adrenergic receptors (βAR) expressed on brown adipose cells driving thermogenesis.183 (b) Cold stress in tumour-bearing mice maintained at standard housing temperatures (20–26°C) tilts the balance towards an immunosuppressive local tumour microenvironment. This is characterized by a substantial increase in Nat Rev Immunol. Author manuscript; available in PMC 2016 March 10.

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populations of intratumoral myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells and a concomitant decrease in the number of CD8+ T cells when compared to tumours that develop in mice housed under thermoneutral ambient temperature (30– 31°C).184 Tumour cell survival and tumour growth are also accelerated by cold stress. 184–186

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Reference 10 Pathogens and Disease, 74, 2016, ftw106 doi: 10.1093/femspd/ftw106 Advance Access Publication Date: 3 November 2016 Minireview

MINIREVIEW

Alexandria M. Palaferri Schieber and Janelle S. Ayres∗ The Salk Institute for Biological Studies, Immunobiology and Microbial Pathogenesis, 10010 North Torrey Pines Road, San DIego CA, USA Corresponding author: The Salk Institute for Biological Studies, Immunobiology and Microbial Pathogenesis, 10010 North Torrey Pines Road, USA. Tel: (858) 453-4100; E-mail: jayres@salk.edu One sentence summary: In this review, the concept of disease tolerance is applied to thermoregulation during infection, inflammation and trauma, and the authors discuss the physiological consequences of thermoregulation during disease including tissue susceptibility to damage, inflammation, behavior and toxin neutralization. Editor: Brooke Napier

∗

ABSTRACT Physiological responses that occur during infection are most often thought of in terms of effectors of microbial destruction through the execution of resistance mechanisms, due to a direct action of the microbe, or are maladaptive consequences of host–pathogen interplay. However, an examination of the cellular and organ-level consequences of one such response, thermoregulation that leads to fever or hypothermia, reveals that these actions cannot be readily explained within the traditional paradigms of microbial killing or maladaptive consequences of host–pathogen interactions. In this review, the concept of disease tolerance is applied to thermoregulation during infection, inflammation and trauma, and we discuss the physiological consequences of thermoregulation during disease including tissue susceptibility to damage, inflammation, behavior and toxin neutralization. Keywords: tolerance; fever; hypothermia

INTRODUCTION Perhaps because our view of host defenses is shaped by the importance of anti-microbial strategies, we assume that any physiological change that occurs during an infection does so to support the immunological response is a direct action of microbial products, or is simply a maladaptive consequence of the immune or other host response. Thermoregulation during infection is a prime example of this. Infection elicits changes in the thermoregulatory strategies of a host that occurs through the integration of signals from the immune, metabolic and neural systems to change the thermal regulatory set point and change body temperature (Kluger 1980). In many cases, a higher body temperature is achieved—a response known as fever or hyperthermia. However, with certain infection types, particularly during sepsis or severe systemic inflammation, a lower

body temperature is selected for a response known as hypothermia. In certain organisms, such as songbirds, a single infection type can cause circadian-associated patterns of hypothermia and fever (Skold-Chiriac et al. 2015). Infection-associated changes in body temperature had long been viewed as an undesirable consequence of host–pathogen interplay. However, because of the widespread occurrence of fever and hypothermia among many different animals in the context of many different types of infectious diseases, and because of the costs to the host associated with thermoregulatory changes, we support the idea that thermoregulation is likely an evolved mechanism that has adaptive value for the host in fighting infections (Kluger 1980, 1986). This initial proposal made by Kluger in the 1970s only considered that the fever response is beneficial for host defenses (Kluger, Ringler and Anver 1975).

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against infections cannot be readily explained only with the traditional paradigms of microbial killing mechanisms (Table 1). Infectious diseases cause significant physiological damage to the host. In addition to executing resistance mechanisms to kill microbes, a host must deal with the collateral damage that occurs during host–pathogen interplay in order to survive. Ecologists have long recognized that genetic variation in disease susceptibility exists in plants that can be dissociated from the plants ability to kill a pathogen or pest. This variation is due to a distinct defense strategy encoded by plants, called ‘tolerance’ that promotes plant fitness in the presence of a given level of pathogen or herbivore. In recent years, the concept of tolerance defenses has been introduced into the field of animal immunology (Raberg, Sim and Read 2007; Ayres and Schneider 2008, 2009, 2012; Ayres, Freitag and Schneider 2008; Ayres, Trinidad and Vance 2012; Medzhitov, Schneider and Soares 2012; Schieber et al. 2015). In this context, tolerance is a defense strategy that minimizes the physiological damage that occurs during infection without having a negative impact on microbial fitness. We propose that thermoregulatory mechanisms that both increase and decrease body temperature are adaptive strategies of the host to promote tolerance defenses and survival following infections. There is evidence that fever and heat shock induce cyto- and tissue-protective responses that will work to limit tissue susceptibility to damage. In addition to fever, the beneficial effects of hypothermia in defense against tissue damage have long been recognized. For example, the ancient Greeks used hypothermia for treating various conditions including hemorrhaging (Diller and Zhu 2009). Hibernating animals are less susceptible to damage induced by ischemia due to their low body temperatures causing reduced oxygen consumption, cardiac function and metabolism rendering tissues less susceptible to damage (Dave et al. 2012). In current medical practices, hypothermia is used for its cyto- and tissue-protective effects in the context of trauma in which the metabolic and inflammatory states of the body and microenvironments change frequently making tissues and cells vulnerable to damage, compromising the normal function of organs (Andresen et al. 2015; Saigal et al. 2015; Usach, Sakopoulos and Razavi 2015; Alkabie and Boileau 2016). Behavioral experiments demonstrated that animals that chose to move to colder environments under conditions in which tissue homeostasis is compromised are healthier than those that do not develop behavioral hypothermia (Romanovsky et al. 2005; Almeida et al. 2006b). In infections, hypothermia is most often associated with advanced stage septic patients and those with severe systemic inflammation, whom are vulnerable to similar forms of cellular and physiological damage as trauma patients (Perman et al. 2014; Chisholm et al. 2016; Kohlhauer et al. 2015). Hypothermia may be viewed as a host’s final effort to limit physiological damage during these disease states but over longer periods of time become maladaptive to the host as is the case with prolonged hyperthermic responses. The function of thermoregulation during infections is almost inevitably defined in terms of microbial killing mechanisms. Evidence shows, however, that several effects of temperature cannot be readily explained within the paradigm of resistance defenses. In this review, we apply the conceptual framework of tolerance to thermoregulation in animals to illustrate how the physiological responses that occur during hyperthermia and hypothermia can promote health and survival during infectious diseases in animals (Fig. 2).

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However, we suggest that both fever and hypothermic response are evolved strategies of the host to optimize host defenses during infection and non-infectious diseases in which thermoregulatory strategies of the host are employed. Indeed, there is evidence to suggest that both fever and hypothermic responses are beneficial for host health in the context of an array of diseases. In pioneering studies using the ectotherm Dipsosaurus dorsalis, which uses behavioral thermoregulation to generate environmental fevers, Kluger demonstrated that iguanas infected with the pathogen Aeromonas hydrophila that were prevented from seeking warmer environments showed significantly increased mortality compared to infected iguanas that were allowed to generate fevers (Kluger, Ringler and Anver 1975; Bernheim and Kluger 1976). The snail, Lymnaca stagnalis, exhibited behavioral hypothermia when infected with the trematode species Diplostomum pseudospathaceum or Plagiorchis elegans. Achieving behavioral hypothermia increased longevity in infected snails compared to those that were maintained at ambient temperatures during infection (Zbikowska 2005). Similar benefits for infection-associated thermoregulation in endotherms have also been established. Treatment of Pasteurella multocida-infected rabbits with an antipyretic was associated with greater mortality than infected rabbits that were allowed to develop fevers (Vaughn, Veale and Cooper 1980). In humans, the therapeutic use of fevers demonstrates the importance of fever in host defenses. Plasmodium, the causative agent of malaria, causes cyclical fevers due to the rupture of erythocytic stage schizonts. Wagner-Jauregg discovered that upon infection of neurosyphilus, patients with Plasmodium infection developed very high fevers and were cured of the neurosyphilis (Karamanou et al. 2013). In the zebra finch, injection with the immune elicitor lipopolysaccharide (LPS) induces a fever response at night but a hypothermic response during the day (Skold-Chiriac et al. 2015). These day-to-night differences in body temperature may indicate that there is a tradeoff between the benefit of fever and the possibility of overheating. During the night, when body temperature is lower, the birds are able to achieve a fever to promote host defense but during the day, when body temperatures are normally higher, the birds induce a hypothermic response to offset the costs of fever and prevent overheating. Thus, an additional thermoregulatory strategy may be to use both fever and hypothermic responses in complimentary ways over the course of a single infection to offset the potential maladaptive effects they may have on host health. Why infection-induced alterations in thermoregulatory strategies are beneficial for the host in combatting infections and the mechanisms by which these protective responses occur remain unknown. The widespread assumption is that thermoregulation protects the host by having a negative impact on pathogen fitness. For example, pathogens may be less able to replicate if the host body temperatures are above or below the optimal temperature for the pathogen. During fever, iron, which is used by many microbes, is sequestered by host tissues (Hacker, Rothenburg and Kluger 1981; Zinchuk and Borisiuk 1997). Thus, changes in body temperature may make the host a less hospitable niche for microbes, yet pathogens vary in their temperature preferences and dependency on nutrient utilization. It has also been proposed that thermoregulation is important for shaping the immune response by optimizing resistance mechanisms and inducing the remobilization of energy stores to fuel microbial killing mechanisms (Hart 1988; Evans, Repasky and Fisher 2015). However, cellular and physiological evidence suggest that temperature-associated defense

References

Table 1. Contribution of thermoregulation to resistance and tolerance defenses. This table summarizes in vitro and in vivo observations of how temperature influences host defense. This table also summarizes data describing how temperature sensitive factors influence various processes and the observed or predicted effects this would have on resistance and tolerance.

Thermal sensitivity

Apparent effects on host defense strategies/pathogen fitness

Aeromonas hydrophila-infected iguanas that seek warmer environments were better able to survive infection

Heat sensitive

Undetermined

Trematode-infected snails that seek colder environments were better able to survive infection

Cold sensitive

Tolerance

Blocking fever in Pasteurella multocida infected rabbits increased mortality

Heat sensitive

Undetermined

LPS challenged zebra finches develop cyclical hypothermia and fever responses

Heat and cold sensitive

Tolerance

Elevated or reduced temperatures impair pathogen growth

Heat and cold sensitive

Impair pathogen fitness

Observation

Reference Kluger, Ringler and Anver (1975); Bernheim and Kluger (1976) Zbikowska (2005) Vaughn, Veale and Cooper (1980) Karamanou et al. (2013) Carmichael, Barnes and Percy (1969); MacKowiak et al. (1981)

Sequestration of iron by host

Heat sensitive

Impair pathogen fitness

Ancient Greeks used hypothermia to treat hemorrhage

Cold sensitive

Tolerance

Diller and Zhu (2009)

Hacker, Rothenburg and Kluger (1981); Zinchuk and Borisiuk (1997)

Hibernating animals are less susceptible to ischemic tissue damage

Cold sensitive

Tolerance

Dave et al. (2012)

Honeybees develop a brood-comb fever by exerting movement in response to a colonial infection with the fungus Ascosphaera apis

Heat sensitive

Impair pathogen fitness

In LPS-treated human monocytes, HSF-1 inhibits transcription of genes encoding proinflammatory cytokines

Heat sensitive

Tolerance

Cahill et al. (1996)

Listeria monocytogenes infected hsf-1-/- mice exhibited significantly higher levels of serum TNF-α and IFN-γ and rapidly succumbed to infection without a significant difference in liver and spleen Listeria burdens

Heat sensitive

Tolerance

Murapa et al. (2011)

Heat exposure prevented transcriptional upregulation of proinflammatory cytokines by preventing the release of the damage associated molecular pattern, HMGB1

Heat sensitive

Tolerance

Fiuza et al. (2003); Lee and Repasky (2012)

In a mouse model of arthritis, mice exposed to higher temperatures had reduced joint damage

Heat sensitive

Tolerance

Lee et al. (2015)

IL-1b causes insulin resistance and the resulting impairment in glucose uptake can limit further IL-1b transcription and ROS production.

Heat sensitive

Tolerance

Jager et al. (2007); Wen et al. (2011); Benetti et al. (2013)

Larsen et al. (2010); Ferreira et al. (2011)

Starks, Blackie and Seeley (2000)

Heat sensitive

Tolerance

Heat sensitive

Tolerance

Koyama et al. (2002); Beumer et al. (2003); Tuin et al. (2006)

HSP72 inhibits proteotoxicity Hypothermia induces expression of cold shock proteins that activate the UPR

Heat sensitive Cold sensitive

Tolerance Tolerance

Mayer and Bukau (2005) Rzechorzek et al. (2015)

HSP72 maintains cardiac function during sepsis

Heat sensitive

Tolerance

Robert et al. (2014)

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HSP32 detoxifies free heme during malaria and sepsis Alkaline phosphatase detoxifies LPS

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Heat As Medicine – Omicron / Flu Cure in 2 hours Table 1. (Continued).

Shift from carbohydrate to lipid metabolism during hibernation and hypothermia limiting ischemic injury

Apparent effects on host defense strategies/pathogen fitness

Cold sensitive

Tolerance

Reference Wong (1983); Drew et al. (2007); Suozzi, Malatesta and Zancanaro (2009); Hindle et al. (2011); Darwazeh and Yan (2013); Xu et al. (2013); Quinones et al. (2014)

Hypothermia improved coagulopathy in septic patients

Cold sensitive

Tolerance

Johansen et al. (2015)

Cold temperatures delay initiation of thrombus formation and speed of clot formation

Cold sensitive

Tolerance

Valeri et al. (1987); Patt, McCroskey and Moore (1988); Michelson et al. (1994); Ruzicka et al. (2012)

Endotherms and ectotherms lower body temperatures under hypoxic conditions

Cold sensitive

Tolerance

Hicks and Wood (1985); Dupre and Owen (1992)

Survival of hypoxic animals increased when put at lower temperatures

Cold sensitive

Tolerance

Gollan and Aono (1973); Hicks and Wood (1985); Gordon (2001)

Hypothermia protects from reperfusion tissue injury

Cold sensitive

Tolerance

Ning et al. (2007); Lampe and Becker (2011)

Reduced sensitivity of fibrosarcoma cells to TNFα-mediated cell lysis

Heat sensitive

Tolerance

Gromkowski, Yagi and Janeway (1989)

Incubation of HeLa cells at mild hyperthermic temperatures rendered them resistant to apoptosis when incubated at lethal hyperthermic temperatures

Heat sensitive

Tolerance

Bettaieb and Averill-Bates (2008)

HSP32-deficient mice infected with Plasmodium had increased liver damage caused by increased sensitivity of hepatocytes to TNF-mediated apoptosis

Heat sensitive

Tolerance

Seixas et al. (2009)

HSP70 and HSP90 inhibit apoptosome formation

Heat sensitive

Tolerance

Beere et al. (2000); Pandey et al. (2000); Saleh et al. (2000)

HSP60 affects caspase-3 activation to inhibit apoptosis

Heat sensitive

Tolerance

Xanthoudakis et al. (1999)

Hypothermia has been shown to alleviate pump dysregulation and help maintain intracellular Ca2+ homeostasis and may prevent cell death

Cold sensitive

Tolerance

Siesjo et al. (1989); Hall (1997)

Cold temperatures delay apoptosis in response to stress by delaying cytochrome c release

Cold sensitive

Tolerance

Goldstein et al. (2000)

Heat can directly activate Bax and Bak to induce cytochrome c release

Heat sensitive

Tolerance

Pagliari et al. (2005)

HSP90 is required for the induction of necroptosis

Heat sensitive

Tolerance

Jacobsen et al. (2016); Zhao et al. (2016)

Increased phagocytosis by macrophages

Heat sensitive

Resistance

Evans, Repasky and Fisher (2015)

Increased expression and release of cytokines

Heat sensitive

Resistance or tolerance

Hypothermia protected rats from endotoxemia

Cold sensitive

Trade-offs in resistance and tolerance

Evans, Repasky and Fisher (2015) Liu et al. (2012)

Increased neutrophil release, infiltration and microbial killing

Heat sensitive

Resistance

Evans, Repasky and Fisher (2015)

Enhanced lymphocyte trafficking and proliferation in response to antigens

Heat sensitive

Resistance

Hart (1988)

Increased antibody synthesis

Heat sensitive

Resistance

Hart (1988)

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Observation

Thermal sensitivity

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THERMOREGULATORY MECHANISMS IN ANIMALS

when examining how such drugs promote host defenses during infection. Recent evidence suggests that the microbiota, the trillions of beneficial microbes that colonize the body surfaces exposed to the environment, can also regulate hyperthermic responses. Cold temperatures can induce ecological perturbations in the intestinal microbiota composition that are apparently adaptive for the host. These ecological changes are associated with intestinal tissue remodeling and changes in adipose tissue physiology that cause white adipose tissue to become beige, which has thermogenic properties similar to brown adipose tissue, and cause a rise in body temperature (Chevalier et al. 2015). This is a complex relationship in that environmental conditions cause a host response that perturbs the microbiota, which in turn influences host physiology to return to homeostasis. While ectotherms such as amphibians, reptiles and insects do not generate endogenous pyrogenic chemical responses, they can generate fevers through behavioral regulation of body temperature during infections. For example, ectotherms will move to warmer environments in order to exogenously induce fever in response to an LPS injection and this movement is required to increase survival during challenge (Vaughn, Bernheim and Kluger 1974). Honeybees develop a brood-comb fever by exerting movement in response to a colonial infection with the fungus Ascosphaera apis. This pathogen is heat sensitive and in this case it seems that this social fever response promotes host defense by creating a less hospitable niche for the pathogen (Starks, Blackie and Seeley 2000). Thus, the recognition of infections and microbial-derived products in ectotherms appears to induce behavioral changes that result in thermoregulatory responses to promote host survival by promoting mechanisms that can have either a negative or neutral/positive impact on pathogen fitness. There is also evidence to suggest that in addition to endotherms, ectotherms use PGE2 in order to induce behavioral fevers. Treatment of the toad, Bufo paracnemis, with the COX inhibitor indomethacin rendered the toads unable to produce fevers in response to an LPS challenge (Bicego et al. 2002). Histamine, hemoglobin, corticosterine and temperature-sensitive transient receptor potential channels have also presented as possible factors in mediating ectothermal fever (Hutchison and Spriestersbach 1986; Wiggins and Frappell 2000; Preest and Cree 2008; Saper, Romanovsky and Scammell 2012). Thermoregulatory behavior requires a complex neuronal circuitry of initiating, organizing, performing and controlling motor actions. Our current understanding of the neural pathways regulating behavioral thermoregulation is limited, and neural pathways controlling thermoregulatory behaviors differ from those controlling autonomic thermoeffectors, making work in these areas more complex (Nagashima et al. 2000; Flouris 2011). Recent studies involving neural thermal stimulation have provided evidence as to which central thermosensors are involved with behavioral thermoregulation; these include the medulla oblongata, pons, midbrain, and amygdala, and the orbitofrontal, insular and somatosensory cortex. Further studies in the toad B. paracnemis have demonstrated that lesions in the POA of the hypothalamus prevented toads from developing a febrile response triggered by LPS challenge, although regular thermoregulatory abilities were not affected. These data suggest that the POA may be important in fever generation in ectotherms (Bicego and Branco 2002). Hypothermia or anapyrexia has been most studied as a clinical therapeutic and less focus has been placed on mechanistically understanding the induction of the cryogenic response triggered by infection and immune stimulation. Studies of hypoxia-induced anapyrexia, which may serve as a strategy

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Initiation of fever in endotherms occurs upon central and peripheral recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors on innate immune cells including macrophages and dendritic cells (Jounai et al. 2012). In addition, multiple cell types in the brain including endothelial cells, microglial cells and neurons can respond to PAMPs (Rivest 2009; Hernangomez et al. 2014). The fever response has been best characterized in the context of LPS challenge and we will focus our discussion around these studies. Activation of Toll-like receptor 4 (TLR4) by LPS centrally and peripherally induces the production of host-derived pyrogens including IL-1β, TNF-α and IL-6. These cytokines circulate through the lymphatic system and signal to the brain (Kluger et al. 1995; Romanovsky, Steiner and Matsumura 2006; Zhang et al. 2008; Yamawaki et al. 2010; Nakano et al. 2015; Poon et al. 2015). Il-1β has been long thought to be the primary pyrogen responsible for fever induction; however, accumulating evidence using genetic knockout and neutralization studies as well as clinical data now suggests that IL6 is the essential mediator for the febrile response (Hart 1988; Evans, Repasky and Fisher 2015). The febrile response is due to these systemic and locally produced cytokines acting on the hypothalamus in the brain to change the thermoregulatory set point. This set point is raised so that what was once a thermal neutral temperature is now subjectively cold to the host. The host reaches a new thermal equilibrium by a number of mechanisms. Among these are brown adipose tissue thermogenesis driven by noradrenaline release and increasing metabolism to induce shivering to produce heat. Vasoconstriction is also induced to reduce blood flow to peripheral fevers preventing heat loss (Almeida et al. 2006a; Nakamura and Morrison 2011). Endotherms will also exhibit behavioral changes including seeking warmer environments and curling up to reduce the amount of body surface exposed to the environment to minimize heat loss. The mechanism by which endogenous pyrogenic cytokines induce the hypothalamus to raise the thermogenic set point involves their induction of cyclooxygenase 2 (COX2) to induce the conversion of arachidonic acid into prostaglandin E2 (PGE2). Peripheral synthesis of COX2 and PGE2 is important for initiation of fever. In a LPS fever model, recognition of LPS by TLR4 on lung and hepaptic hematopoietic cells induces transcriptional upregulation of COX2 leading to production of PGE2 to signal to the brain (Steiner et al. 2006). Injection of a cyclooxygenase inhibitor in the preoptic area (POA) has shown inconsistent results, but seems to mainly attenuate activation of median preoptic and arcuate hypothalamus suggesting that there may also be a central mechanism that regulates COX2 activity in fever induction (Nadjar et al. 2010). PGE2 binds to the PGE2 receptors (EP), on thermoregulatory neurons in the hypothalamus (Oka et al. 2000; Nakamura et al. 2002; Oka 2004; Lazarus et al. 2007). Signals are then sent through neurons to the dorsomedial hypothalamic nucleus to elicit sympathetic thermogenesis in peripheral effector organs (Nakamura et al. 2005). The cytokine receptor activator of NF-kB, TNFSF11, found on astrocytes in the hypothalamus, also activates COX2 and PGE2 production to activate EP3 (Hanada et al. 2009). Other EP receptors including EP1 and EP4 may contribute to different phases of fever suggesting functional redundancy of EP receptors in fever induction (Oka et al. 2000). Drugs that suppress fever including salicylic acid do so through the inhibition of prostaglandin synthesis (Vane and Botting 2003). Antipyretics are non-specific and can also influence resistance mechanisms. This must be considered

References

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Fraifeld 2004). A few cryogens have been identified in the context of hypoxic-induced hypothermia. Nitric oxide in the POA is a potential promoter of hypothermia induced by hypoxia (Steiner and Branco 2003). Simultaneous increases in the levels of cyclic adenosine monophosphate (cAMP) and cGMP in the POA may play a role in hypoxia-induced hypothermia due to increases in production and/or release of serotonin and nitric oxide (Steiner and Branco 2003). An intracellular cascade of adenylate cyclase, protein kinase A and cAMP caused by high levels of hydrogen sulfide in the POA may play an essential role in the occurrence of the hypothermia in response to hypoxia (Kwiatkoski et al. 2012). It appears that carbon monoxide may downregulate hypoxia-induced hypothermia, as studies using heme-oxygenase (CO-synthesizing enzyme) inhibition showed that hypothermia was attenuated (Paro et al. 2002). There have been indications of human cryogens, but the specific identity of these molecules remains to be determined (Shido et al. 2004; Shido and Sugimoto 2011). Methods by which tolerance can be measured have been described in several recent reviews (Raberg, Sim and Read 2007; Ayres and Schneider 2008, 2011). The relative contributions of resistance and tolerance to host defense against infection can be distinguished in any host microbe system by examining the relationship between host health and pathogen levels. Using these parameters, and assuming health of the host when uninfected is equivalent between different host populations (vigor), a dose response curve can be generated to determine how host health changes as microbial levels change. Changes in health as microbe levels change would shift the host along the diagonal and indicate differences in resistance (Fig. 1). Changes in health without a change in microbe levels would shift the curve along the y-axis and would indicate differences in tolerance (Fig. 1). The more tolerant a host, the shallower the slope of the dose response curve would be. This method can be used to determine how different factors including environmental and genetic factors can influence host defenses. For example, mice infected with pathogen A that differ in the ability to thermally regulate their bodies may differ in how the shift within this health-bymicrobe space with respect to each other, revealing how temperature regulates resistance and tolerance in the context of pathogen A infection. Ayres and Schneider (2008, 2012) reported the point tolerance method, which uses microbial levels at a single defined time point to reveal how a single mutation in a component of the fly immune response can impact resistance and tolerance in response to different infection challenges (Fig. 1). Using a mouse malaria model, Raberg, Sim and Read (2007) reported that range tolerance, which measures host health at various pathogen doses, can reveal variations in resistance and tolerance (Fig. 1). Both models are based on the assumption that these relationships are linear and have been useful at identifying environmental, microbial and genetic factors that influence tolerance defenses. These relationships however are likely more complex and further experimentation and data points, for example, measuring pathogen burden over the course of the infection will reveal how differences in host populations influence resistance and tolerance at different stages of the infection.

CATABOLISM AND ENERGETIC COSTS OF THERMOREGULATION Fever responses caused by many forms of insult including sepsis induce a hypermetabolic state in the host (Frankenfield et al. 1997). Induction of a hypermetabolic state increases heat production to raise and maintain body temperature at the new

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to avoid severe hypoxia by reducing metabolic demands, have unraveled some mechanisms that may apply to other forms of hypothermia. Centrally, the glutamatergic action of the lateral POA of the hypothalamus has been implicated in the control of hypoxia-induced hypothermia in endotherms. This appears to be mediated by the inhibitory neurotransmitter gammaaminobutyric acid (GABA) and ionotropic GABA receptors in the rostral parapyramidal region of the medulla oblongata, as neurons directly project between these parts of the brain (Yoshida et al. 2009; Osaka 2014). Connecting the brainstem to the rest of the brain, the medulla oblongata contains respiratory, cardiac and vasomotor centers, positioning itself as a reasonable culprit of thermoregulatory action. While endogenous regulation of the hypothermic response involves the POA, the behavioral responses that mediate hypothermia appear to involve the neuronal bodies located in the dorsomedial nucleus and neural fibers passing through the paraventricular nucleus of the hypothalamus (Almeida et al. 2006a). In certain contexts, proinflammatory cytokines that typically act as pyrogens can also act as cryogens. For example, the proinflammatory cytokines IL-1β, IL-6 and TNF-α have been shown to act as cryogens in animal models of infection and systemic inflammation. In a mouse model of antibiotic-induced dysbiosis and intestinal injury, administration of broad spectrum antibiotics induced the expansion of a multi-antibiotic-resistant Escherichia coli strain. Upon disruption of the intestinal barrier with the sulfated polysaccharide dextran sulfate sodium (DSS), this E. coli translocated to extraintestinal tissues causing a sepsis-like disease characterized by multi-organ dysfunction and hypothermia. The authors found that this hypothermic response was dependent on activation of a component of the innate immune system called the NLRC4 inflammasome by this E. coli strain, which resulted in an overly exuberant response mediated by IL1β (Ayres, Trinidad and Vance 2012). The hypothermic response seen in this model and many other mouse infection models is likely due to the animals being thermally stressed. While pyrogenic infections cause fever at thermoneutrality and above, subneutral temperatures elicit a hypothermic response in response to similar agents in animals (Ivanov et al. 2003). Most animal vivariums that conduct research house their mice at subthermal temperatures (∼19◦ C–26◦ C; Speakman and Keijer 2012), despite knowledge that the rodent’s thermal neutral zone is ∼30◦ C, causing thermal stress (Watkinson and Gordon 1993; Swoap et al. 2008). Due to this, biological studies of infectious diseases and in general have been in the context of hypothermia. This hypothermic rather than hyperthermic response may result from various inflammatory stimuli including proinflammatory cytokines, differences in pathogen load, route/site of administration, ambient temperatures, circadian timing and other factors that influence host temperature differently when animals are under thermal neutral and thermal stressed conditions (Nomoto 1996; Szelenyi et al. 2004). With this in mind, it is important to recognize what we can learn from these ‘mistakes’ and use them as observations for relevant studies. Other cryogens potentially exist and take part in LPS-induced hypothermia, including the cytokines IL-10 and IFNγ (Leon 2004). 3-Iodothyronamine (T1 AM), an endogenous derivative of thyroid hormone, induces robust hypothermia in mice and rhesus monkeys, possibly related to hypothermic neuroprotective effects during stroke (Scanlan et al. 2004; Panas et al. 2010). Hypothalamic cysteinyl-LT (CysLT), an arachidonate metabolism product, 5-lipoxygenase and epoxygenase-derived eicosanoids have also been indicated as promoters of anapyrexia. This may suggest that LPS-induced hypothermia may be mediated by leukotrienes (Paul, Fraifeld and Kaplanski 1999; Kozak and

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References

elevated thermoregulatory set point. Studies in humans have suggested that the induction of fever causes a 30%–50% increase in metabolism and the average percent increase in metabolism for every 1◦ C of fever is ∼13% (Mackowiak and Plaisance 1998; Kluger et al. 1998; Kluger 2002). Consequences of the hypermetabolic response during fever are muscle proteolysis and a negative nitrogen balance in the host that results clinically in the wasting of energetic body tissues. While anabolic responses occur during infection, the magnitude of catabolic responses tends to be greater and results in the depletion of body tissues. The function of muscle wasting is not understood but possibly its function may serve to generate a source of amino acids that are mobilized to other parts of the body that are used for the anabolic requirements of the host response. Catabolism of body tissues does not become evident until the fever has been fully reached. Consistent with the notion that active thermoregulation during infection is beneficial for host defenses, muscle catabolism should also be beneficial for the host. Given that there are costs to mounting resistance responses (Iseri and Klasing 2014), the current hypothesis is that this muscle wasting occurs to fuel the resistance response. However, we propose that muscle catabolism is also necessary to fuel tolerance defense mechanisms that are induced by fever (Fig. 2). For example, defense responses that negatively regulate inflammatory responses and the induction of cytoprotective and tissue repair pathways are also energetically costly (Ayres and Schneider 2011).

The pathophysiology of muscle wasting is complex and incompletely understood. Factors including severity of the primary disease, proinflammatory cytokines such as TNFα, IL1β and IL-6, severity of the anorexic response, hormones, metabolism and pathogen factors are all believed to be the main drivers of skeletal muscle catabolism that can lead to the induction of atrophy-dependent programs in muscle (Beutler et al. 1985; Goodman 1991, 1994; Zamir et al. 1992; Costelli et al. 1993). The intestinal microbiota is also a critical regulator of skeletal muscle wasting in response to infectious and inflammatory diseases (Schieber et al. 2015). Colony born C57Bl/6 mice were protected from muscle wasting caused by DSS-induced intestinal injury compared to mice from Jackson Labs. Colony born mice harbored an Escherichia coli O21:H+ strain in the intestine that was absent in the mice from Jackson Labs. According to Koch’s postulates, administration of this commensal to Jackson mice resulted in protection from wasting induced by intestinal injury. This protection could be extended to oral infection with the pathogen Salmonella Typhimurium and lung infection caused by Burkholderia thailandensis. Escherichia coli O21:H+ mediates its protective effect by preventing infection/inflammation-induced systemic drop in circulating levels of IGF-1 in an NLC4 inflammasome-dependent manner without impacting pathogen infection levels indicating that this E. coli promotes tolerance. This protection was associated with E. coli O21:H+ occupying a new niche—white adipose tissue—during challenged states. In this case, muscle wasting

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Figure 1. Measuring the effects of thermoregulation on resistance and tolerance. (A) Host health and pathogen burdens of infected host populations that differ in their thermoregulatory responses are measured. Using these values, a health-by-pathogen plot is generated in (B) and (C) to determine how these host populations move in space with respect to each other. (B) Point tolerance. The relationship between host health and pathogen levels at a defined time point is examined. Shifts along the y-axis indicate health is changing as microbe levels are changing and indicate changes in resistance. Shifts along the y-axis indicate changes in tolerance. The steeper the slope the more tolerant the host. Adapted from Ayres and Schneider, (2008, 2012). (C) Range tolerance. The relationship between host health and different levels of pathogen are examined. Adapted from Raberg, Sim and Read (2007). (D) Wisteria rats were infected intraperitoneally with a clinical E. coli isolate and placed in either hyperthermic or hypothermic conditions. Morbidity and E. coli burdens in livers at 5 h post infection were measured. Shown are the data represented as point tolerance. Animals at 28◦ C have an increase in resistance. On the other hand, animals at ambient temperature that were allowed to develop hypothermia had lessened morbidity as characterized by decreased lung inflammation and neutrophil infiltration. Decreased tolerance is represented by a less steep slope. Data from Liu et al. (2012).

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appears to be maladaptive to the host by reducing host tolerance (Schieber et al. 2015). Clearly, this is inconsistent with the idea that wasting provides resources that enhance both resistance and tolerance mechanisms, as mentioned above. One possible explanation is that these studies were done in animals housed under thermally stressed conditions and that wasting in the absence of a febrile response is maladaptive. The effects of muscle wasting on tolerance defenses are likely to be complex and context dependent and will require further investigation.

REGULATION OF INFLAMMATION The most obvious way to promote tolerance would be to regulate the degree and duration of an inflammatory response during an infection. In doing so, the energetic costs associated with mounting an immune response would be minimized to avoid excessive and potentially pathogenic skeletal muscle catabolism. Furthermore, the potential tissue damage caused by the inflammatory response would be reduced. Elevated body temperatures induce the expression of heat shock factor 1 (HSF-1), which is involved in the acute heat response in mammals to activate expression of cytoprotective genes (Hasday and Singh 2000). HSF1 has been demonstrated to act as a negative transcriptional regulator of inflammatory responses. In LPS-treated human monocytes, HSF-1 inhibits transcription of genes encoding

proinflammatory cytokines (Cahill et al. 1996). Consistent with this, when hsf-1−/− mice were infected with Listeria monocytogenes, they exhibited significantly higher levels of serum TNFα and IFNγ and rapidly succumbed to infection without a significant difference in liver and spleen Listeria burdens (Murapa et al. 2011). Neutralization of TNFα promoted survival of infected animals deficient for HSF-1 demonstrating that inhibition of the inflammatory response by HSF-1 promotes tolerance (Murapa et al. 2011). Heat exposure appears to act as a negative regulator of inflammation in activated macrophages and this is dependent on HSF-1 transcriptional repressive behavior. Heat exposure also prevents transcriptional upregulation of proinflammatory cytokines by preventing the release of the damageassociated molecular pattern, HMGB1, reducing transcript stability and the recruitment of NF-kB to promoter regions of cytokine genes (Fiuza et al. 2003; Lee and Repasky 2012). In a mouse model of arthritis, mice exposed to higher temperatures had reduced joint damage associated with lower levels of circulating levels of TNFα and increased levels of the anti-inflammatory cytokine IL-10 in the joints (Lee et al. 2015). Downregulation of inflammation may also occur through the metabolic effects caused by the fever response. For example, IL-1β causes insulin resistance, and the resulting impairment in glucose uptake can limit further IL-1β transcription and reactive oxygen species (ROS) production (Jager et al. 2007; Wen et al. 2011; Benetti et al. 2013).

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Figure 2. Thermoregulatory induced tolerance mechanisms.

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Hypothermia can also reduce inflammation to promote tolerance. For example, Wistar rats infected with a clinical Escherichia coli isolate that were maintained under hypothermic conditions displayed less endotoxemia, organ dysfunction and lung neutrophil recruitment despite higher liver bacterial burden (Fig. 1) (Liu et al. 2012). Thus, shifts in the thermostatic set point of a host appear to provide a negative feedback mechanism that downregulates proinflammatory cytokine production to prevent excessive tissue damage.

SICKNESS-INDUCED BEHAVIORS

external temperatures regulate the circadian clocks (Garrity et al. 2010; Romeijn et al. 2012; Villamizar et al. 2012; Fan, Stuart-Fox and Cadena 2014). In endotherms, peripheral tissues have independent clocks that are regulated by the master clock in the brain called the suprachiasmatic nucleus (SCN) in the hypothalamus. The master clock is regulated by light-dark cycles and is resistant to temperature changes. The peripheral clocks respond to temperature changes (Reppert and Weaver 2002; Buhr, Yoo and Takahashi 2010). It appears that the SCN drives core body temperature rhythms that are sensed by the peripheral clocks. This suggests that fever and hypothermic responses may induce clock-dependent responses in peripheral tissues that influence host defenses. Reduced grooming and social withdrawal are additional sickness-associated behaviors in animals. Similar to sleep and changes in feeding, reduced grooming may serve as an energy conservation mechanism. Social withdrawal likely serves a beneficial function for a group rather than an individual. Workers of the ant Temnothorax unifasciatus infected with the fungal pathogen Metarhizium anisopliae leave their social network prior to death (Heinze and Walter 2010). In this context, social withdrawal would protect the group due to avoidance defenses. The exact function of social withdrawal and the extent to which it and other sickness-induced behaviors influences tolerance defenses and how this relates to temperature requires further exploration.

NEUTRALIZATION OF TOXINS Infection-induced tissue damage can release host-derived toxic compounds that must be dealt with to minimize further tissue damage. Mechanisms that do so would operate as tolerance mechanisms because they target the toxin rather than the pathogen (Fig. 2). For example, infection with Plasmodium, the causative agent of malaria, causes hemolysis freeing hostderived hemoglobin into circulation. Under inflammatory conditions, free hemoglobin is oxidized resulting in the release of its prosthetic heme groups into circulation, which is toxic to the host. Heat shock protein 32 (HSP-32, also known as heme oxygenase-1 HO-1) is induced during Plasmodium infection and in response to heat shock and catalyzes the degradation of free heme resulting in carbon monoxide, labile iron and biliverden (Seixas et al. 2009). Individuals hemizygous for the sickle cell mutation contain a point mutation in the beta chain of hemoglobin and have elevated basal levels of free heme and HSP32 expression and are less susceptible to Plasmodium infection (Ferreira et al. 2011). Using a transgenic mouse model hemizygous for the human sickle cell trait, HbS, investigators found that animals had increased circulating levels of free heme, which activated HSP32 expression via the Nrf2 transcription factor. Evidence suggests that the CO produced during the heme detoxification reaction by HSP32 prevented further release of cell-free hemoglobin into circulation during infection and the pathogenic effects of CD8+ T cells in cerebral malaria (Ferreira et al. 2011). This protection conferred by the HbS trait did so without influencing Plasmodium levels; thus, the detoxification mechanism of HSP32 promotes tolerance of malaria infection. In a mouse cecal ligation and puncture sepsis model, HSP32 was required for survival without an apparent difference in systemic bacterial levels (Larsen et al. 2010). A caveat of this model however is that the authors only measured culturable levels of bacteria which only represents a small fraction of the members of the intestinal microbiota.

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The fever response in animals and humans is accompanied by stereotypical behavioral modifications including anorexia, sleep disturbances, social withdrawal and grooming disturbances that are known as sickness-induced behaviors (Hart 1988) (Fig. 2). These behaviors appear to be highly conserved as they are found in both invertebrates and vertebrates. Similar to fever, the general assumption regarding these sickness-associated behaviors is that they are maladaptive consequences of the host response to the infection. However, because these behaviors are ubiquitous among animal species during infection it is likely that these are evolved behavioral adaptations to increase the chance of survival during infection (Hart 1988; Ayres and Schneider 2009, 2012; Ayres 2013). Hart (1988) proposed that the main purpose of these behaviors is to facilitate the febrile response to better promote resistance defenses. While there is evidence to suggest that resistance defenses are influenced by sickness-induced behaviors (Hart 1988; Morag et al. 1998; Ayres and Schneider 2009), we propose that fever induces tolerance defenses in part by promoting these behaviors. Given the energetic costs of fever, the induction of anorexia and the reduced consumption of food during infection seem counterintuitive. Foraging and capture of food is energetically demanding and a reduced motivation to eat may promote tolerance by conserving energy stores for host defense and minimize wasting pathology. Calorically restricted states induce stress responses in animals that may reduce the susceptibility of tissues to damage during infection (Koella and Sorense 2002; Partridge, Piper and Mair 2005; Burger et al. 2007; Kristan 2007; Libert et al. 2008; Mair and Dillin 2008; Ayres and Schneider 2009). Consistent with this, in Drosophila, mutations in the gustatory receptor Gr28b render flies constitutively anorexic. When infected with Salmonella Typhimurium, these flies have enhanced tolerance. Similarly, when flies are calorically restricted they have increased tolerance to infection compared to flies fed a standard diet (Ayres and Schneider 2009). These same parameters, however, rendered flies more susceptible to infection with Listeria monocytogenes due to resistance defects (Ayres, Freitag and Schneider 2008). Thus, the effects of sickness-induced anorexia on resistance and tolerance defenses will be context dependent. Infected animals typically exhibit sleepiness or fatigue and have a tendency to sleep during periods that they would otherwise be awake. Increasing sleep may be an additional means to conserve energy for the host defense response. Disruptions in sleep during infection lead to mortality in animal studies, for example, sleep deprived mice were more susceptible to infection with Plasmodium (Lungato et al. 2015). In humans, workers are more susceptible to infection (Pietroiusti et al. 2006). The circadian timing system in animals regulates the wakesleep cycle and synchronizes biological processes and is important for host defense against infections (Shirasu-Hiza et al. 2007; Stone et al. 2012; Allen et al. 2016). In ectotherms, both light and

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TISSUE SUSCEPTIBILITY TO DAMAGE Tissue susceptibility to damage during an infection is largely dependent on a cell’s ability to mount an appropriate compensatory stress response that will contribute to tolerance defenses (Fig. 2). In the absence of such responses, cells and tissues become vulnerable to host–pathogen interplay. For example, the host response to an infection places heavy demands on a cell’s protein synthesis machinery and can lead to the accumulation of damaged proteins as well as misfolded and unfolded proteins that leads to proteotoxicity. In Drosophila, pcmt, which encodes an L-isoaspartyl methyltransferase that is important for the damaged protein repair response, is required for tolerance of infection with the lethal intracellular bacterium Listeria monocytogenes (Ayres, Freitag and Schneider 2008). In Caenorhabditis elegans, the transcription factor xbp-1 that is a critical component of the unfolded protein response (UPR) promotes tolerance in worms challenged with the pathogen Pseudomonas, by limiting the accumulation of unfolded protein species (Richardson, Kooistra and Kim 2010). Changes in body temperature are sensed and trigger activation of stress response transcriptional programs, which include protection from proteotoxicity. In human cortical neurons, hypothermia induces the expression of cold shock proteins and endoplasmic reticulum stress leading to activation of the UPR. This can lead to cross-protection against oxidative stress in neurons (Rzechorzek et al. 2015). HSF-1 induces expression of a variety of genes including HSP-72, which inhibits proteotoxicity by facilitating the proper folding of newly translated or misfolded proteins (Mayer and Bukau 2005). In a variety of septic models, mice deficient for HSF-1 presented with impaired cardiac contraction and relaxation that was associated with increased production of immune effectors in cardiomyocytes, suggesting that HSF-1 may promote tolerance by maintaining cardiac function during sepsis (Robert et al. 2014). In a forward genetic ENU screen in mice, Kcnj8mydy/mydy mice have a null allele for Kir6.1 which encodes an ATP-sensitive potassium channel, and are hypersusceptible to challenges with LPS and MCMV infection without differences in viral titers compared to challenged wild-type mice. During infection, this channel maintains coronary homeostasis and prevents coronary artery vasoconstriction induced by the inflammatory response to maintain cardiac tonicity and prevent death (Croker et al. 2007). HSF-1 likely promotes tolerance via mechanisms independent of the protein stress response and downregulation of inflammation. In another study, T cells isolated from hsf-1−/− mice exhibit defective proliferation at febrile temperatures and this

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was due to an inability of HSF-1-deficient mice to restore ROS levels after heat challenge (Murapa et al. 2007). Thus, regulation of cellular stress responses by body temperature may represent a general tolerance mechanism that is protective against an array of infections. Cellular and tissue health is dependent on proper circulation of the body’s blood supply to provide oxygen and nutrients to the tissue. In the clinical setting of sepsis and in severe inflammatory disorders, dysregulated activation of the coagulation cascade and the inhibition of fibrinolysis can lead to disseminated intravascular coagulation that ultimately can cause multi-organ failure due to hypoxia caused by perturbations in the microcirculation (Levi, van der Poll and Schultz 2012). This causes ischemia of the tissue due to the limited amount of oxygen available for cellular aerobic respiration leading to a reduction in ATP and phosphocreatine levels and a switch in intracellular metabolism to anaerobic glycolysis. In anaerobic glycolysis, glucose is transformed into lactate generating 2 ATP molecules per glucose molecule. The resulting accumulation of hydrogen ions and lactate leads to the production of lactic acid and a drop in extracellular and intracellular pH, a condition called lactic acidosis. Patients who develop severe sepsis or septic shock have elevated circulating levels of lactic acid and lactic acidosis, and this is a marker of the severity of the infection. Early lactate normalization within the first six hours of resuscitation is a strong predictor of sepsis survival (Puskarich et al. 2013). In hibernating mammals, the internal thermostat is lowered and tissues function at lower temperatures. There is a shift from carbohydrate to lipid metabolism during hibernation limiting anaerobic metabolism and ischemic injury (Suozzi, Malatesta and Zancanaro 2009; Hindle et al. 2011; Xu et al. 2013; Quinones et al. 2014). In the hypothermic brain, metabolism also shifts from glucose to lipid utilization (Wong 1983; Drew et al. 2007; Darwazeh and Yan 2013). Thus, changing substrate utilization induced by temperature changes may represent a tolerance mechanism to prevent lactate build-up. An obvious mechanism to prevent tissue hypoxia in a septic patient would be to prevent dysregulation in the coagulation pathway to maintain blood flow to tissues. In a randomized controlled trial, mild hypothermia induction improved coagulopathy in septic patients (Johansen et al. 2015). In experimental studies, temperatures below 33◦ C reduce the synthesis and kinetics of clotting enzymes and plasminogen activator inhibitors as well as delay the initiation of thrombus formation and speed of clot formation (Valeri et al. 1987; Patt, McCroskey and Moore 1988; Michelson et al. 1994; Ruzicka et al. 2012), suggesting that cold temperatures may provide a defense strategy against severe coagulopathy. Once hypoxia occurs, however, the body must employ mechanisms to combat this stress. Increasing cardiac output and ventilation are possible defense strategies; however, these methods are energetically costly to the critically ill host. An alternative mechanism is to induce hypothermia to reduce the oxygen demand of tissues. Consistent with this, physiological mechanisms that promote hypothermia are induced in mammals during hypoxic conditions. Typically endotherms produce a thermogenic response when ambient temperatures are below thermoneutrality. However, in hypoxic rodents, the thermogenic response to cold temperatures is depressed (Tattersall and Milsom 2003; McAllen 2009). Furthermore, there is decreased heat production in hypoxic animals due to allocation of blood supply away from brown adipose tissue (McAllen 2009). Thus, hypoxic mammals appear to reduce the temperature threshold at which they begin their shivering response to generate heat for thermoneutrality. Both ectotherms and endotherms

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In addition to host-derived toxic compounds, mechanisms that neutralize microbial-derived toxins will promote tolerance of the host. Although we have evolved mechanisms to use LPS as an elicitor of tissue protective responses, systemic recognition of LPS by TLR4 leads to severe systemic inflammation. Alkaline phosphatases have been shown to modify LPS by dephosphorylating the lipid A moiety, which confers LPS toxicity (Koyama et al. 2002; Beumer et al. 2003; Tuin et al. 2006). Mild heat shock (39o C–41o C) has been shown to induce expression of alkaline phosphatase (Shui and Scutt 2001), as well as enhance enzymatic activity (Trieb, Blahovec and Kubista 2007). In a zebrafish model, intestinal alkaline phosphatase was required to prevent intestinal inflammation and pathology caused by the microbiota (Bates et al. 2007). Therefore, temperature regulation of alkaline phosphatase may represent a host-encoded strategy to promote tolerance by detoxification of microbial products.

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hibition of apoptosis by HO-1 promotes tolerance of malaria by preventing hepatic failure and death. In addition to controlling sensitivity to TNF, HSPs have been shown to influence downstream signaling upon activation of the death receptor (Meng et al. 1999; Hasday and Singh 2000; Johnson and Fleshner 2006). TNFα can also induce necrosis in some cell types (Morgan, Kim and Liu 2008). HSP90 has been reported to determine whether a cell will face an apoptotic or necrotic death fate (Fulda et al. 2010). HSPs also influence the intrinsic apoptosis pathway involving the release of cytochrome c from the mitochondria in response to cell death signals. Cytochrome c binds to Apaf-1, inducing oligomerization and recruitment of procaspase-9 resulting in the formation of the apoptosome and activation of caspase-9, which then activates caspase-3. This results in freeing of caspase-9 from the apoptosome and is then replaced with another caspase-9 molecule. HSP70 and HSP90 have also been observed to inhibit apoptosome formation (Beere et al. 2000; Pandey et al. 2000; Saleh et al. 2000). HSP-60 primarily resides in the mitochondrial matrix and has been shown to exert its antiapoptotic effects by influencing caspase-3 activation (Xanthoudakis et al. 1999). Various HSPs have also been demonstrated to block apoptosis by manipulating processes downstream of caspase activation (Creagh, Sheehan and Cotter 2000). During anaerobic glycolysis, the resulting acidosis leads to the influx of Ca2+ into cells and an inhibition of membrane-bound pumps and channels that are normally responsible for maintaining intracellular calcium homeostasis. Mitochondrial dysfunction may result from the excess intracellular Ca2+ leading to cell death via the intrinsic apoptotic pathway. Hypothermia has been shown to alleviate pump dysregulation and help maintain intracellular Ca2+ homeostasis and may prevent cell death (Siesjo et al. 1989; Hall 1997). This effect is likely indirect as studies have shown that, while not affecting apoptotic caspase activation, cold temperatures delay apoptosis in response to stress by delaying cytochrome c release (Goldstein et al. 2000). Heat and response proteins associated with heat have also been shown to promote various types of cell death. Permeabilization of the mitochondrial membrane is dependent on the activation and oligomerization of multidomain Bcl2-family proteins Bax and Bak. Heat can directly activate Bax and Bak to induce cytochrome c release (Pagliari et al. 2005). Necroptosis, a programmed form of necrosis type cell death, has important roles in host defense against viral infections and inflammation. Emerging evidence suggests that HSP90 is required for the induction of necroptosis (Jacobsen et al. 2016; Zhao et al. 2016). Thus, body temperature changes during infection likely directly or indirectly orchestrates the optimal balance of pro- and anticell death regulator mechanisms that likely influence host tolerance.

OPEN QUESTIONS AND FUTURE PERSPECTIVES It is clear from experimental and clinical studies that thermoregulation has broad beneficial effects for host defense against infectious diseases and beyond (Table 1, Fig. 2). However, we know very little about the mechanisms by which thermoregulation— both hypothermia and hypothermia influence host defenses. The field of thermoregulation in general and in the context of host defense needs to be reignited. We propose an integration of the conceptual framework of resistance and tolerance into these studies will shed light on our understanding of the

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including lizards, mice and rats chose to maintain lower body temperatures under hypoxic conditions through behavioral thermoregulation (Hicks and Wood 1985; Dupre and Owen 1992). The discovery that animals exhibit behavioral hypothermia in response to hypoxia provides support that hypothermia likely provides physiological protection under oxygen stressed conditions. Indeed, survival studies demonstrate that hypothermia induces a number of physiological benefits under oxygen limiting conditions. Survival of hypoxic mice was increased when animals were maintained at 35◦ C and reduced when housed at 40◦ C (Gordon 2001). Hypoxic lizards that were allowed to seek cold temperatures exhibited 100% survival. By contrast, 100% of hypoxic lizards that were not allowed to seek cold temperatures died (Hicks and Wood 1985). Rabbits that are anemic exhibited increased survival when maintained at hypothermic conditions presumably due to cold temperatures shifting the oxyhemoglobin dissociation curve to the left influencing oxygen accessibility (Gollan and Aono 1973). Thus, the induction of hypothermia in response to hypoxia may promote tolerance by reducing the oxygen demand by tissues, altering the affinity of hemoglobin for oxygen and avoiding the energetic costs that are associated with increasing cardiac output and ventilation. After ischemic episodes, restoration of circulation to tissues can be detrimental to tissue health and result in reperfusion injury. Cellular metabolites accumulate during ischemia that become oxidized upon the reintroduction of molecular oxygen to the tissues when blood flow is reestablished resulting in the accumulation of ROS. This activates an inflammatory response in the tissue that leads to tissue damage and death. Evidence from therapeutic hypothermia studies suggests that cold body temperatures can prevent reperfusion induced tissue injury. This protection is likely due to the fact that hypothermia reduces the levels of free radicals and stabilizes cell membranes, cellular swelling and edema (Ning et al. 2007; Lampe and Becker 2011). A wide variety of pathogens can cause tissue damage by induction of host cell death either by direct activation of host cell death machinery or indirectly through the host response to the infection. The dysregulated physiological responses that occur in severe systemic inflammatory conditions and sepsis, such as ischemia and reperfusion, cause cell death and lead to multiorgan failure. The cytokine TNFα is a central mediator of inflammation and induction of the febrile response (Jiang et al. 1999). TNFα has also cytolytic effects via the extrinsic apoptosis pathway involving activation of caspase-8 downstream of TNF receptor signaling (Micheau and Tschopp 2003; Walczak 2013). An important component of the heat shock response induced by fever is to promote stress responses that will enable a cell to survive during an inflammatory response. Janeway and colleagues showed that heat shock of fibrosacoma cells reduced sensitivity to TNFα-mediated lysis (Gromkowski, Yagi and Janeway 1989). Furthermore, incubation of TNFα secreting T lymphocytes at elevated temperatures reduced their secretion of TNFα and their cytolytic capabilities (Gromkowski, Yagi and Janeway 1989). Consistent with this, incubation of HeLa cells at a mild hyperthermic temperature of 40◦ C promoted thermotolerance and conferred protection from apoptosis induced by lethal hyperthermia (42◦ C –45◦ C). Protection was associated with the increased expression of various HSPs including HSP 27, 32, 60, 72 and 90 (Bettaieb and Averill-Bates 2008). In a mouse model of non-cerebral malaria, mice deficient for HSP32 (Hmox1−/− ) succumbed to infection due to hepatic failure despite having equivalent levels of plasmodium as infected wild-type mice. The heme detoxification conferred by HO-1 was required to prevent sensitization of hepatocytes to TNF-mediated apoptosis (Seixas et al. 2009). Thus, in-

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function of fever and hypothermic responses in surviving infectious diseases and beyond.

FUNDING This work was supported by NIH grant R01AI114929 (JSA), the NOMIS Foundation, the Searle Scholar Foundation (JSA), the Ray Thomas Edward Foundation (JSA). Conflict of interest. None declared.

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Vol. 43, No. 4

Antiviral Effect of Hyperthermic Treatment in Rhinovirus Infection C. CONTI,1 A. DE MARCO,2 P. MASTROMARINO,1 P. TOMAO,1

AND

M. G. SANTORO2,3*

Institute of Microbiology, School of Medicine, University “La Sapienza,” 00185, Rome,1 and Department of Biology, University of Rome Tor Vergata,2 and Institute of Experimental Medicine, CNR,3 00133 Rome, Italy Received 31 July 1998/Returned for modification 19 October 1998/Accepted 4 February 1999

Human rhinoviruses (HRV) are recognized as the major etiologic agents of the common cold. Starting from the observation that local hyperthermic treatment is beneficial in patients with natural and experimental common colds, we have studied the effect of brief hyperthermic treatment (HT) on HRV replication in HeLa cells. We report that a 20-min HT at 45°C is effective in suppressing HRV multiplication by more than 90% when applied at specific stages of the virus replication cycle. Synthesis of virus proteins is not affected by HT, indicating that the target for treatment is a posttranslational event. The antiviral effect is a transient cellmediated event and is associated with the synthesis of the 70-kDa heat shock protein hsp70. Unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat. The possibility that hsp70 could play a role in the control of rhinovirus replication is suggested by the fact that a different class of HSP inducers, the cyclopentenone prostaglandins PGA1 and 12-PGJ2, were also effective in inhibiting HRV replication in HeLa cells. Inhibition of hsp70 expression by actinomycin D prevented the antiviral activity of prostaglandins in HRV-infected cells. These results indicate that the beneficial effect of respiratory hyperthermia may be mediated by the induction of a cytoprotective heat shock response in rhinovirus-infected cells. Eukaryotic and prokaryotic cells respond to an increase in environmental temperature by expressing a specific set of cytoprotective proteins referred to as heat shock proteins (HSP) or stress proteins (13). HSP are utilized in the repair process following different types of injury to prevent damage resulting from the accumulation of nonnative proteins. In mammalian cells, HSP are induced in a variety of pathophysiological conditions, including fever, inflammation, oxidant injury, and virus infection (7). Induction requires the activation, translocation to the nucleus, and phosphorylation of a transregulatory protein, the heat shock transcription factor HSF (17). The 70-kDa heat shock proteins (hsp70) function as molecular chaperones and are encoded by a multigene family, including the constitutively expressed hsc70, the major inducible hsp70, the inducible hsp72, the glucose-regulated grp78/BiP, and the mithocondrial hsp75 (7, 13). A cytoprotective role of hsp70 in a variety of human diseases, including ischemia, inflammation, and infection, is widely recognized (7, 17). In the case of viral infection, evidence for the presence of HSP in intact virions or association of HSP with virus proteins during infection, as well as for the modulation of HSP synthesis by viruses, has been reported (23). However, the role of HSP in viral infection is still controversial. The possibility that elevated levels of hsp70 may interfere with viral replication has been suggested by a variety of studies describing the antiviral activity of cyclopentenone prostaglandins and other inducers of the heat shock response in negative-strand RNA viruses (reviewed in references 23 and 24). In the case of picornaviruses, induction of the heat shock response has been studied mainly during poliovirus infection. Constitutive hsp70 was shown to be associated with newly synthesized capsid precursor P1 of poliovirus, and the hsp70-P1 complex was found to be part of an assembly intermediate (14). On the other hand, poliovirus infection was shown to inhibit constitutive or heat shock-induced hsp70 synthesis starting 2 to 3 h after infection (14, 18). Infection with poliovirus type 2 was recently shown to prevent HSP induction also by cyclopentenone prostaglandins (4). Starting from the observation that HT is beneficial in patients with common colds, we have studied the induction of the

The human rhinoviruses (HRVs), members of the Picornaviridae family, are the major etiologic agents of the common cold (29). They include over 100 immunologically non-crossreactive serotypes, classified into a minor and a major group according to membrane receptor recognition (5, 32). HRVs contain four nonglycosylated structural proteins, VP1, VP2, VP3, and VP4, forming a capsid with icosahedral symmetry. Within the capsid lies a single-stranded RNA genome which serves as a monocistronic mRNA for the synthesis of the structural and nonstructural proteins of the virus. Upon entry into the host cell, the viral RNA is translated into a large polyprotein which is subsequently cleaved by virus-encoded proteases (19). Unlike other types of picornaviruses, the human rhinoviruses are adversely affected by acidic pH and replicate optimally at 33°C or colder. This may partly account for their predilection for the cooler environment of the nasal mucosa, limiting rhinoviruses to upper respiratory infections. Although there is an abundance of remedies for the common cold from nasal vasoconstrictors to vitamin C, no specific antiviral therapy has been found to be effective. Also, the large variety of immunologically non-cross-reactive rhinovirus serotypes and apparent antigenic drift in rhinoviral antigens cause major problems for the development of an effective vaccine (5). A different approach against rhinovirus infection was reported by Tyrrell et al., who demonstrated a beneficial role for local hyperthermia (20 to 30 min at 43°C) in improving the course of the disease in clinical trials in patients with natural and experimental common colds (31). In this case, brief hyperthermic treatment (HT) did not alter the frequency of antibody response in volunteers, suggesting either a direct effect of HT on rhinovirus replication or an inhibitory effect on inflammatory processes and the ensuing symptomatology. However, the mechanism of the anti-HRV activity of hyperthermia is not known.

* Corresponding author. Mailing address: Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy. Phone: 39-06-7259-4822. Fax: 39-06-7259-4821. E-mail: santoro@bio.uniroma2.it. 822

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INHIBITION OF RHINOVIRUS REPLICATION BY HYPERTHERMIA

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FIG. 1. Effect of HT during HRV replication in human cells. (A) HeLa cell monolayers were infected with HRV serotype 1B (10 PFU/cell) and subjected to HT (45°C, 20 min) soon after the adsorption period (time 0) or at 3, 6, or 9 h p.i. Virus titers were determined 12 h p.i. Bar C, untreated cells. Data represent the mean SD of duplicate samples of two independent experiments. (B) Temperature-dependent inhibition of HRV replication. HeLa cells were infected with HRV at 1 (F) or 10 (❍) PFU/cell and either kept at 33°C or subjected to 20 min of HT at different temperatures (37, 43, or 45°C) 6 h after infection. Virus titers were determined 12 h p.i. (C) HeLa cells were infected with HRV at 1 (F and Œ) or 10 (❍ and ‚) PFU/cell and either kept at 33°C (❍ and F) or subjected to 20 min of HT at 45°C (‚ and Œ) 6 h after infection. Virus titers were determined 12 or 24 h p.i. Data represent the mean SD of at least duplicate samples. , P 0.05.

heat shock response by hyperthermia and cyclopentenone prostaglandins during HRV infection in human cells. We provide evidence that, unlike poliovirus, rhinovirus infection does not inhibit the expression of hsp70 induced by heat or cyclopentenone prostaglandins and that both hyperthermia and prostaglandin treatment result in inhibition of HRV replication. The antiviral effect is a transient cell-mediated event, associated with hsp70 synthesis. MATERIALS AND METHODS Cell cultures. HeLa (Ohio) cells were grown at 37°C in a 5% CO2 atmosphere in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 IU of penicillin G per ml, and 100 g of streptomycin per ml. Virus infection and titration. Confluent HeLa cell monolayers were infected with HRV serotype 1B or 14 (3) for 1 h at 33°C at a multiplicity of infection (MOI) of 1, 5, or 10 PFU/cell. The viral inoculum was removed, and cell monolayers were washed three times with phosphate-buffered saline (PBS) and incubated with MEM containing 2% FCS at 33°C. Prostaglandin A1 (PGA1) and 9-deoxy- 9, 12-13,14-dihydro-prostaglandin D2 ( 12-PGJ2) (Cayman Chemical Co.) were stored in absolute ethanol and diluted to the appropriate concentration at the time of use. Control media contained the same concentration of ethanol diluent, which did not affect cell metabolism or virus replication. For the heating procedure, flasks were immersed in a temperature-controlled water bath (Grant Instruments) for 20 min at 45 0.01°C, unless specified otherwise. HT (45°C, 20 min) was not cytotoxic to uninfected HeLa cells as shown by the trypan blue exclusion technique 24 h after heat shock (data not shown). Virus production was determined by plaque assay as described previously (3). Briefly, after three cycles of freezing and thawing, serial 10-fold dilutions of HRV were prepared and inoculated on confluent HeLa cell monolayers in 35-mm-diameter plates. After 1 h at 33°C, the inoculum was removed, and cells were washed three times with PBS before the addition of MEM containing 2% FCS and 1% SeaPlaque agarose (Miles). After 3 days of incubation at 33°C in a 5% CO2 atmosphere, plaques were stained with 0.33% neutral red solution. For virus purification, HeLa cells infected with 10 PFU of HRV serotype 1B were labeled with [35S]methionine (25 Ci/ml/5 105 cells, 20-h pulse) in the presence of actinomycin D (0.5 g/ml) starting 5 h postinfection (p.i.). After three cycles of freezing-thawing and clarification at 6,000 g for 20 min at 4°C, the supernatants diluted in PBS were centrifuged at 12,000 g for 20 min and the virus was pelleted by centrifugation at 100,000 g for 4 h at 4°C. Unless otherwise specified, HRV serotype 1B was utilized for the experimental protocols. DNA, RNA, and protein synthesis. Confluent monolayers of uninfected or virus-infected HeLa cells (10 PFU/cell) were labeled for 12 h, starting soon after virus infection, with [3H]thymidine, [3H]uridine, or [35S]methionine (Amersham International) at a concentration of 5 Ci/5 105 cells for DNA, RNA, or protein synthesis, respectively, and the radioactivity incorporated into trichloroace-

tic acid (TCA)-soluble (uptake) and -insoluble (incorporation) material was determined as described previously (16). Protein synthesis and SDS-PAGE analysis. Confluent cell monolayers were labeled with [35S]methionine (1-h pulse, 5 Ci/ml/5 105 cells) at hourly intervals from 0 to 11 h p.i. in methionine-free medium containing 2% dialyzed FCS. Cells were usually preincubated for 15 min in methionine-free medium. After labeling, cells were washed and lysed in lysis buffer (2% sodium dodecyl sulfate [SDS], 10% glycerol, 0.001% bromophenol blue, 0.1 M dithiothreitol, 0.0625 M Tris HCl [pH 6.8]) and the radioactivity incorporated into TCA-insoluble material was determined. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a vertical slab gel apparatus (3% stacking gel, 10 or 12% resolving gel) and processed for autoradiography, as described previously (2). Autoradiograms were quantified densitometrically with a laser beam densitometer (Ultroscan XL; LKB) (2), and bands were expressed as relative peak areas. Virus proteins were identified on the basis of Mr and in relation to the position of viral marker proteins from [35S]methionine-labeled purified HRV serotype 1B. Immunoblot analysis. For immunoblot analysis, an equal amount of protein from each sample was separated by SDS-PAGE and blotted onto nitrocellulose, as described previously (27). After transfer, the filters were incubated with antihsp70 monoclonal antibodies (diluted 1:500) from HeLa cells (Amersham) in Ten-Tween 20 buffer (0.05 M Tris-HCl [pH 7.4], 5 mM EDTA, 0.15 M NaCl, 0.05% Tween 20), and the bound antibody was detected by using horseradish peroxidase-linked sheep antimouse antibody (Amersham International). Molecular weights were calculated by using Bio-Rad low-Mr markers. Statistical analysis. Statistical analyses were performed by using Student’s test for unpaired data. Data are expressed as the means standard deviations (SDs) of at least duplicate samples. P values of 0.05 were considered significant.

RESULTS Inhibition of HRV replication by brief HT in HeLa cells. The effect of brief HT on rhinovirus production in human cells was evaluated under one-step growth conditions. HeLa cells infected with HRV serotype 1B (10 PFU/cell) were subjected to a 45°C HT (20 min) soon after the adsorption period (time 0) or at 3, 6, and 9 h p.i. Cells were incubated at 33°C, and virus yield was quantitated by plaque assay at 12 h p.i. Figure 1A shows that HT, when applied in specific stages of the virus cycle, was strongly effective in inhibiting HRV replication for at least 12 h. The most dramatic effect was observed at 6 h p.i., with a reduction in virus yield of more than 99% relative to that of the control. HT applied at later times of the virus growth cycle (9 h p.i.) resulted in a decreased inhibitory effect (approximately 80% reduction relative to the control), whereas

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treatment soon after infection (time 0) had no significant effect on virus yield, indicating that inhibition of rhinovirus replication is not due to an aspecific cytotoxic effect of heat. To investigate whether this effect was temperature dependent and was influenced by the MOI of the virus, confluent HeLa monolayers were infected with HRV at an MOI of 1 or 10 PFU/cell for 1 h at 33°C and, at 6 h p.i., were subjected to HT at 37, 43, or 45°C for 20 min. Inhibition of virus replication, determined 12 h p.i. by plaque assay, was found to be temperature dependent, and a 2-log reduction in HRV yield was obtained after treatment at 45°C (Fig. 1B). Heat treatment at 43°C appeared to be less effective in cells infected with 10 PFU of HRV (approximately 40% reduction in virus yield relative to untreated control) than in cells infected with 1 PFU ( 70% reduction), whereas heat treatment at 45°C was similarly effective at low and high MOIs (Fig. 1B). Inhibition of virus replication by HT is transient. In fact, when virus yields from parallel cultures were measured at 24 h p.i., virus titers of heatstressed cells were comparable to that of untreated control cells (Fig. 1C), indicating that brief HT results in the delay, but not in the irreversible block, of HRV replication. The fact that inhibition of virus replication is transient further indicates that the reduction in virus yield is not caused by an aspecific irreversible cytotoxic effect of hyperthermia in HeLa cells. To investigate whether the antirhinoviral activity of hyperthermia was a general effect or was specific for serotype 1B which belongs to the HRV minor group according to membrane receptor recognition, the effect of HT was tested on HRV serotype 14, a representative member of the major HRV group (32). HeLa cells infected with HRV serotype 14 (1 PFU/ cell) were subjected to a 43°C HT (20 min) at 6 h p.i. Cells were incubated at 33°C, and virus yield was quantitated by plaque assay at 12 h p.i. HT was found to reduce the HRV serotype 14 yield by more than 90% at this time (control, 1.50 106 0.40 106 PFU/ml; HT, 1.36 105 0.50 105 PFU/ml). Effect of brief HT on cellular and viral protein synthesis. Inhibition of virus replication after HT has been previously associated with induction of HSP (6, 23). On the other hand, it has been shown that infection with different members of the Picornaviridae family, the polioviruses, prevents the expression of HSP stimulated by hyperthermia or chemical inducers of the heat shock response (4, 18). To investigate whether HRV infection could interfere with HSP expression and whether brief HT would affect HRV protein synthesis, HeLa cells infected with HRV (10 PFU/cell) were either kept at 33°C or subjected to HT (45°C, 20 min) at 6 h p.i. After a 1-h recovery period at 33°C, cells were labeled with [35S]methionine (1-h pulses at 33°C) at different times p.i. Uninfected cells were treated identically. Virus yield was quantitated by plaque assay at 12 h p.i. HT caused the expected reduction of virus yield at 12 h p.i. (Fig. 2D). As determined by [35S]methionine incorporation into TCA-insoluble material, heat stress was found to moderately ( 30%) inhibit protein synthesis in uninfected HeLa cells for a period of approximately 3 h (Fig. 2A). Under the conditions described, rhinovirus infection caused progressive inhibition of HeLa cell protein synthesis, and no difference between untreated and HT-treated cells was detected (Fig. 2A). Samples containing an equal amount of radioactivity were processed for SDS-PAGE analysis and autoradiography. As expected, in uninfected HeLa cells, HT induced the synthesis of a 72-kDa protein, identified as hsp70 by Western blot analysis with anti-hsp70 monoclonal antibodies (data not shown). hsp70 synthesis started 1 h after heat shock and continued at a lower level for the following 3 to 4 h (Fig. 2B and C). HRV infection did not induce HSP synthesis in HeLa cells. HRV-infected cells were able to respond to HT by synthesizing hsp70 in an

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amount comparable to that of uninfected cells (Fig. 2B and C), indicating that rhinoviruses, unlike polioviruses, do not interfere with HSP expression even in relatively late stages of infection in human cells. Levels of actin synthesis were instead decreased in HRV-infected cells (Fig. 2B). Finally, even though it greatly reduced virus yield, HT caused only a modest inhibition of HRV protein synthesis at 7 h p.i., whereas no difference in viral protein synthesis was detected between untreated and HTtreated cells at later times of infection (Fig. 2B and C), suggesting that HT could affect a posttranslational event in the replication cycle, possibly by interfering with the virus assembly process or with virus release from infected cells. In a parallel experiment, HeLa cells were infected with HRV at a low MOI (1 PFU/cell) and, after the 1-h adsorption period, were either kept at 33°C or subjected to HT (45°C, 20 min) at 6 h p.i. After a 1-h recovery period at 33°C, the cells were labeled with [35S]methionine for the following 2 h at 33°C. Uninfected cells were treated identically. Samples containing an equal amount of radioactivity were processed for SDS-PAGE analysis and autoradiography. As shown in Fig. 3A, the levels of hsp70 synthesis were comparable in uninfected and HRVinfected cells. For hsp70 identification, equal amounts of protein from each sample were separated by SDS-PAGE and processed for immunoblot analysis using anti-hsp70 monoclonal antibodies (Fig. 3B). Although virus proteins were not detectable by SDS-PAGE under these conditions, the synthesis of two polypeptides (indicated in Fig. 3A), whose identification is presently under investigation, was evident in untreated, but not in HT-treated, infected cells. Effect of PGA1 on rhinovirus infection. To investigate whether the antiviral effect of HT was a consequence of the induction of a heat shock response and HSP expression in the infected cell, we tested the effect of the cyclopentenone PGA1, which is a potent inducer of hsp70 synthesis (27), on HRV production after one cycle of virus growth. Confluent monolayers of HeLa cells were infected with HRV (10 PFU/cell) and, after the 1-h adsorption period, were treated with different concentrations of PGA1 or control diluent at 33°C. Virus yield was determined by plaque assay at 12 h p.i. PGA1 was found to reduce HRV production dose dependently, and an inhibition of more than 80% was observed at the concentration of 10 g of PGA1/ml (Fig. 4A). As shown above for HT, the antiviral effect of PGA1 was transient, and, at 24 h p.i., the virus yield in cells treated with 10 g of PGA1/ml (30 M) was equal to 70% of that of control cells. However, the readdition of PGA1 to infected cells at 12 h p.i. resulted in a virus yield reduction of more than 80% up to 24 h p.i. (data not shown). In a different type of experiment, HeLa cells were infected with HRV (100 PFU/dish) and, after the adsorption period (1 h, 33°C), were incubated with a semisolid medium containing agarose (1%) and PGA1 (10 g/ml) or control diluent. The number and the size of viral plaques were determined after neutral red staining at 48 and 72 h p.i. Under these conditions, PGA1 treatment inhibited HRV replication and caused a dramatic reduction in the number of plaques at 48 h p.i. (control cells, 98 12 plaques/dish; PGA1-treated cells, 8 3 plaques/ dish) (Fig. 4A, inset). At later times of infection (72 h p.i.), the number of plaques in PGA1-treated cells increased and was comparable to 60% of that in control cells; however, the plaque size was reduced by more than 50% in treated cells relative to that of the control. Under the conditions described above, PGA1 was shown not to be toxic to uninfected human cells, as determined by microscopic examination and vital dye uptake (Fig. 4A, inset). At the effective antiviral concentration, PGA1 also did not appear to affect nucleic acid or protein synthesis in HRV-infected cells at

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FIG. 2. Effect of HT on HRV protein synthesis. Cell monolayers infected with HRV (10 PFU/cell) were either kept at 33°C or subjected to HT (45°C, 20 min) 6 h after the adsorption period and labeled with [35S]methionine (1-h pulse) at 33°C at different times p.i. (A) Determination of radioactivity incorporated into TCA-insoluble material in uninfected (❍ and ‚) and infected (F and Œ) cells either untreated (❍ and F) or treated with HT (‚ and Œ). (B) Quantitative determination of viral proteins VP1 (‚ and Œ) and VP3 (❍ and F), as measured by densitometric analysis of autoradiographic patterns shown in panel C. Data are expressed as percentage of total proteins in untreated (❍ and ‚) or HT-treated (F and Œ) cells. Quantitative determination of hsp70 in uninfected ( ) and HRV-infected (■) cells is also shown. Levels of actin in uninfected (❍) and HRV-infected (F) untreated cells are shown as a control. (C) SDS-PAGE analysis and autoradiography of samples containing an equal amount of radioactivity from uninfected (U) and HRV-infected (HRV) HeLa cells, maintained at 33°C ( HS) or subjected to HT ( HS). hsp70 and HRV proteins VP1, VP2, and VP3 are indicated. (D) In the same experiment, virus titers were determined 12 h p.i. by plaque assay. Data represent the mean SD of duplicate samples. , P 0.05.

12 h p.i. (Fig. 5). Confluent monolayers of HeLa cells uninfected or infected with HRV (10 PFU/cell) were treated with PGA1 (10 g/ml) or with a different cyclopentenone prostaglandin, 12-PGJ2 (4 g/ml), 1 h after the adsorption period and then labeled with [3H]thymidine, [3H]uridine, or [35S]methionine for the following 12 h, as described in Materials and Methods. The results shown in Fig. 5 indicate that, at the doses tested, neither prostaglandin significantly affected either the uptake of [3H]thymidine or [35S]methionine or DNA or protein synthesis in uninfected and HRV-infected cells. Both prostaglandins caused a modest reduction in RNA synthesis in mock-infected cells, which did not appear to be due to a reduction in the uptake of precursors, since intracellular [3H] uridine levels were not decreased in prostaglandin-treated cells (Fig. 5C and D). Effect of PGA1 on host cell and HRV protein synthesis. To determine the effect of PGA1 treatment on the kinetics of HRV protein synthesis, HeLa cells were infected with HRV (5 PFU/cell) and treated with PGA1 (10 g/ml) or control diluent after the 1-h adsorption period. Cells were then labeled with [35S]methionine (1-h pulse) at different times p.i. In uninfected cells, as already described for the same cell line kept at 37°C (4), treatment with PGA1 at 33°C did not greatly affect the overall electrophoretic protein profile, but it induced the

synthesis of a 72-kDa cellular protein, which was identified as hsp70 by immunoblot analysis (data not shown). In HRVinfected cells, hsp70 synthesis started 4 h after PGA1 treatment and continued for up to 10 h (Fig. 4B), confirming that, unlike poliovirus, rhinovirus infection does not interfere with hsp70 expression. Under these conditions in control cells virus protein synthesis started 6 h after infection and several virus proteins were evident at 8 to 10 h p.i. PGA1 treatment was found to cause a delay in the synthesis of HRV proteins, which was not detected for up to 10 h p.i. Since the antiviral activity of cyclopentenone prostaglandins in negative-strand RNA virus models has been shown to be dependent on the induction of hsp70 synthesis (2, 20), we investigated the effect of actinomycin D, which is known to inhibit PGA1-induced HSP expression (2, 16), on HRV production. HeLa cells infected with HRV (10 PFU/cell) were treated with PGA1 (10 g/ml) or control diluent soon after the 1-h adsorption period in the presence or absence of actinomycin D (2 g/ml). Virus yields were determined at 12 h p.i. As shown in Fig. 6A, treatment with actinomycin D by itself did not affect HRV replication. Actinomycin D, however, completely prevented the inhibitory effect of PGA1, indicating that the antiviral activity is dependent on efficient cellular transcription and translation. In a parallel experiment, HRV-infected cells treat-

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FIG. 3. Effect of HT on hsp70 synthesis in HRV-infected cells. Cell monolayers either uninfected (lanes 1 and 3) or infected with HRV at a low MOI (1 PFU/cell) (lanes 2 and 4) were kept at 33°C (lanes 1 and 2; HS) or subjected to HT (45°C, 20 min) (lanes 3 and 4; HS) 6 h after the adsorption period and labeled with [35S]methionine (2-h pulse) at 33°C 1 h after heat treatment. (A) Samples containing an equal amount of radioactivity were separated by SDSPAGE analysis and processed for autoradiography. (B) Hsp70 was identified by immunoblot analysis using monoclonal anti-hsp70 antibodies.

ed with PGA1 or control diluent were labeled with [35S]methionine for 12 h in the presence or the absence of 2 g of actinomycin D/ml. SDS-PAGE analysis of [35S]methionine-labeled proteins showed that treatment with actinomycin D prevented PGA1-induced hsp70 synthesis in HeLa cells (Fig. 6B), suggesting that hsp70 could be involved in the control of HRV replication. Effect of 12-PGJ2 on HRV replication. To determine whether cyclopentenone prostaglandins other than PGA1 could inhibit rhinovirus replication, we investigated the effect of 12PGJ2 on HRV infection. HeLa cells infected with 10 PFU of HRV/cell were treated with different concentrations of 12PGJ2 or ethanol diluent after the 1-h adsorption period. Virus yield was determined by plaque assay at 12 h p.i. Treatment with 12-PGJ2 was found to reduce HRV production dose dependently, and an inhibition of approximately 90% was observed at the concentration of 4 g of 12-PGJ2/ml (12 M) (Fig. 7A). At this concentration, 12-PGJ2 was not toxic to uninfected HeLa cells and, as described above, did not significantly affect nucleic acid and protein synthesis in either uninfected or HRV-infected cells (Fig. 5). To investigate whether the antiviral activity of 12-PGJ2 was also dependent on cellular protein expression, HRV-infected HeLa cells (10 PFU/cell) were treated with 4 g of 12-PGJ2/ml in the presence or absence of actinomycin D (2 g/ml) for 12 h after infection. Treatment with actinomycin D completely prevented 12PGJ2-induced inhibition of rhinovirus replication, indicating that, as shown above for PGA1, the antiviral activity of 12PGJ2 is cell mediated (Fig. 7B). DISCUSSION The results described in the present report indicate that brief HT, when applied at specific stages of the virus cycle, is effective in blocking rhinovirus replication during primary infection of human cells. The inhibitory effect of HT on HRV replication is temperature dependent, and, under one-step multiplication conditions, a 20-min treatment at 45°C was found to

FIG. 4. Effect of PGA1 on HRV replication in HeLa cells. (A) Dose-dependent inhibition of HRV replication by PGA1. Confluent monolayers of HeLa cells were infected with HRV (10 PFU/cell) for 1 h at 33°C and treated with different doses of PGA1 or control diluent soon after the 1-h adsorption period. Virus yield was determined 12 h p.i. by plaque assay. Data represent the mean SD of duplicate samples of a representative experiment. , P 0.05. Each experiment was repeated three times with the same results. The effect of PGA1 (10 g/ml, 30 M) ( PGA1) or ethanol diluent ( PGA1) added directly to the agar overlay on the reduction of HRV plaque size and number is shown in the inset. Approximately 100 plaques were measured in triplicate cultures for each sample. (B) Confluent monolayers of HeLa cells, either uninfected (U) or infected with HRV (5 PFU/cell) for 1 h at 33°C (HRV), were treated with PGA1 (10 g/ml) ( PGA1) or control diluent ( PGA1) soon after the 1-h adsorption period and labeled with [35S]methionine (1-h pulse) at 33°C at different times p.i. Samples containing an equal amount of radioactivity were separated by SDSPAGE analysis and processed for autoradiography. HRV proteins VP1 and VP3 are indicated. hsp70 is indicated by the arrow.

be extremely effective, independently of the MOI. The most dramatic effect was observed when HT was applied at 6 h p.i., with a reduction in virus yield of more than 99% relative to that of the control. HT applied at later times of the virus growth cycle (9 h p.i.) resulted in a decreased inhibitory activity, whereas no significant effect on virus replication was found when heat shock was applied soon after virus entry into the cells. These results indicate that the antiviral effect is not due to a general change in membrane fluidity or cell metabolism. In fact, a brief exposure to high temperature (45°C for 20 min) did not damage HeLa cells and only moderately ( 30%) inhibited protein synthesis for a period of approximately 3 h in uninfected cells. HT also did not inhibit protein synthesis in HRV-infected HeLa cells (Fig. 2A). In spite of the dramatic reduction of HRV yield after one cycle of virus growth, HT of HeLa cells did not significantly

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FIG. 5. Effect of PGA1 and 12-PGJ2 on DNA, RNA, and protein synthesis in uninfected and HRV-infected HeLa cells. Confluent cell monolayers, either uninfected (U) or infected with HRV (10 PFU/cell) for 1 h at 33°C (HRV), were treated with PGA1 (10 g/ml, 30 M) (o), 12-PGJ2 (4 g/ml, 12 M) (■), or control diluent ( ) soon after the 1-h adsorption period and then labeled with [3H]thymidine (A and B), [3H]uridine (C and D), or [35S]methionine (E and F) for the following 12 h. (A, C, and E) Uptake of precursors by uninfected or HRV-infected cells. (B, D, and F) Incorporation of precursors into DNA, RNA, and proteins, respectively. Data represent the mean SD of duplicate samples.

alter the synthesis of viral proteins at 9 and 11 h p.i., indicating that the target for HRV inhibition could be a posttranslational event. This hypothesis is also supported by the finding that no antiviral effect is observed when HT is applied soon after virus entry into the host cell. A posttranslational event was previously suggested as the target for the antiviral activity of brief HT in vesicular stomatitis virus-infected monkey epithelial cells (6). However, the mechanism by which brief HT can control HRV replication at specific stages of the virus cycle remains to be established. In different types of models of acute RNA virus infection, the antiviral activity of hyperthermia has been associated with the induction of a protective heat shock response and the synthesis of HSP in the infected cell (reviewed in references 23 and 24). In the case of picornaviruses, increased levels of hsp70 have been detected in cultured neonatal myocardial cells from BALB/c mice after infection with two different picornaviruses, encephalomyocarditis virus and coxsackievirus B3 (11). Whether hsp70 is an unnecessary by-product of the viral infection or has a function in the viral life cycle is as yet unclear. hsp70 was also shown to be associated with newly synthesized capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells (14). The half-life of P1 was increased when bound to hsp70, and hsp70-P1 complexes were uncleavable by the viral protease. As anticipated in the introduction, infection with

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FIG. 6. Effect of PGA1 and actinomycin D on HRV replication. Confluent HeLa cell monolayers untreated (lanes 1 and 3) or pretreated with actinomycin D (2 g/ml) (lanes 2 and 4) for 1 h at 37°C were infected with HRV (10 PFU/cell) for 1 h at 33°C and treated with PGA1 (10 g/ml) (lanes 3 and 4) or control diluent (lanes 1 and 2). Actinomycin D was kept in the medium for the duration of the experiment. (A) Virus titers were determined 12 h p.i. by plaque assay. (B) In the same experiment, cells were labeled with [35S]methionine soon after PGA1 treatment for 12 h at 33°C. Samples containing an equal amount of radioactivity were separated by SDS-PAGE analysis and processed for autoradiography. A section of the fluorogram from native gels is shown. The position of hsp70 is indicated.

polioviruses, which are known to cause a dramatic shutoff of the host cell protein cap-dependent translation by proteolytically inactivating the cap-binding protein complex (22), inhibits constitutive as well as heat shock-induced hsp70 synthesis in human cells, even though to a minor extent in comparison to other cellular proteins (14, 18). We have recently shown that infection with poliovirus type 2 also prevents hsp70 synthesis after treatment with a different class of HSP inducers, the cyclopentenone prostaglandins (4). On the other hand, the translation of the glucose-regulated protein BiP was found to be increased in poliovirus-infected HeLa cells, at a time when cap-dependent translation of cellular mRNA is inhibited (28). We have now shown that, unlike polioviruses, rhinovirus infection does not induce the expression of glucose-regulated proteins and does not prevent heat shock-induced hsp70 synthesis in HeLa cells. Under the conditions described herein, comparable levels of this protein were detected in uninfected and HRV-infected cells. Based on the hsp70-P1 complex formation described in cells infected with other types of picornaviruses (14), it could be hypothesized that intracellular accumulation of high levels of hsp70 during specific stages of rhinovirus infection could impair virus maturation and/or release from the infected cells, possibly by hsp70 binding to viral polypeptides. Since HSP are known to be stable proteins (13), the relative time-specific effect of heat could be a consequence of the fact that hsp70 is utilized by the cell as a molecular chaperone as soon as it is synthesized. In this case, the availability of large amounts of newly synthesized protein could be

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FIG. 7. Effect of 12-PGJ2 on HRV replication. (A) Dose-dependent inhibition of HRV replication by 12-PGJ2. Confluent monolayers of HeLa cells were infected with HRV (10 PFU/cell) for 1 h at 33°C and treated with different doses of 12-PGJ2 or control diluent soon after the 1-h adsorption period. Virus yield was determined 12 h p.i. by plaque assay. (B) Confluent cell monolayers, either untreated (bars 1 and 3) or pretreated with actinomycin D (2 g/ml) for 1 h at 37°C (bars 2 and 4), were infected with HRV (10 PFU/cell) for 1 h at 33°C and treated with 12-PGJ2 (4 g/ml) (bars 3 and 4) or control diluent (bars 1 and 2). Actinomycin D was kept in the medium for the duration of the experiment. Virus titers were determined 12 h p.i. by plaque assay. Data represent the mean SD of duplicate samples of a representative experiment.

essential for interaction with virus proteins or viral components at different times of the virus cycle. It should be emphasized that inhibition of virus replication by HT is transient. If treatment is not repeated, HRV yields from HT-treated cells can reach the control level at later times of infection (24 to 48 h p.i.), confirming that the reduction in virus yield is not caused by an aspecific cytotoxic effect of hyperthermia in HeLa cells. The fact that hsp70 synthesis after heat shock is also transient, and it persists only for a period of 3 to 4 h after treatment, supports the possibility that high levels of hsp70 synthesis are necessary for the antiviral effect to persist. To investigate the possibility that hsp70 could play a role in the control of rhinovirus replication, we tested the effect of a different class of HSP inducers, the cyclopentenone prostaglandins, on HRV infection. Cyclopentenone prostaglandins of the A and J type (PGA and PGJ) are known to possess a potent antiviral activity against a wide variety of DNA and RNA viruses in different types of mammalian cells, as well as in animal models (12, 21, 24–26). The antiviral activity of these molecules has been associated with their ability to function as a signal for the induction of hsp70 synthesis via cycloheximide-sensitive activation of the transcription factor HSF1 (1, 24). Micromolar concentrations of PGA1, which did not inhibit cell metabolism, significantly reduced HRV yield after one cycle of virus growth while inducing hsp70 synthesis starting 4 h after the beginning of treatment. It is interesting to note that the amount of hsp70 induced by PGA1 at 33°C, the temperature used for HRV infection, was comparable to that at 37°C, as determined by immunoblot analysis (data not shown). Unlike poliovirus (4), HRV infection did not prevent hsp70 induction by PGA1. As shown previously in other cell types (2), PGA1 was a more effective inducer of hsp70 than heat, since synthesis of hsp70 persisted for at least 10 h after the addition of PGA1 as compared to 3 to 4 h after HT under the conditions used. PAGE analysis of HRV proteins showed that PGA1 caused a delay of virus protein synthesis, which did not start before 10 h p.i. This indicates that, as suggested in other virus-host cell models (23), induction of the heat shock response may affect rhinovirus

replication at more than one level. When HRV infection was allowed to proceed for up to 24 to 48 h p.i. without additional treatments, the virus yield progressively reached the level of mock-treated controls, indicating that the antiviral effect is reversible and not due to a cytotoxic effect of the drug. The possibility that hsp70 could be involved in the control of HRV replication is suggested by the fact that treatment with actinomycin D, which blocks hsp70 expression, prevented the inhibition of HRV production in PGA1-treated cells. Similar results were obtained with a different cyclopentenone prostaglandin, 12-PGJ2, a natural metabolite of PGD2, which presently occurs in human body fluids (10). Treatment with actinomycin D also prevented the antiviral effect of HT in HeLa cells (data not shown). As anticipated in the introduction, Tyrrell and coworkers have previously described that naturally acquired as well as experimental colds benefit from local HTs (43°C for 20 to 30 min) in randomized double-blind clinical trials in humans (30, 31). A significant reduction in the mean symptom scores was observed in treated patients. A transient reduction in virus shedding was reported only on the day of treatment, whereas the mean titers in convalescence and the frequencies of antibody response were not significantly different in treated and control groups. Regulation of heat shock gene expression was then hypothesized to be a possible target for the antiviral activity of respiratory hyperthermia (31). On the other hand, Hendley et al. have shown that two nasal treatments with steam had no effect on viral shedding in volunteers with experimental rhinovirus infections (9). No beneficial effects from steam inhalation on common cold symptoms were detected in other studies (8, 15). Differences in the techniques of administering therapy and in the strains of rhinoviruses were both hypothesized to be responsible for the discrepancy in these studies (15). Our results, which show that HT at 45°C is effective in inhibiting HRV replication in vitro when applied at specific stages of the virus cycle, point out that differences in the temperature utilized and in the time of treatment should be considered. Our results also suggest the possibility that HSP and hsp70 in particular could participate in an intracellular

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defense strategy during HRV infection. However, several questions on the mechanism by which hsp70 can interfere with rhinovirus replication during heat stress remain to be answered. A better understanding of the role of HSP in virus replication could be useful in the comprehension of the beneficial effect of fever or artificial pyrexia during virus infection and could indicate new strategies in the treatment of the common cold. ACKNOWLEDGMENTS We thank Carlotta Volpi and Alessandra Fiocchetti for excellent technical assistance. This work was supported by the Italian Ministry of Public Health, 1997 AIDS Research Project, and by the Italian National Research Council, P.F. “Biotechnology.” REFERENCES 1. Amici, C., L. Sistonen, M. G. Santoro, and R. I. Morimoto. 1992. Antiproliferative prostaglandins activate heat shock transcription factor. Proc. Natl. Acad. Sci. USA 89:6227–6231. 2. Amici, C., C. Giorgi, A. Rossi, and M. G. Santoro. 1994. Selective inhibition of virus protein synthesis by prostaglandin A1: a translational block associated with HSP70 synthesis. J. Virol. 68:6890–6899. 3. Conti, C., N. Orsi, and M. L. Stein. 1988. Effect of isoflavans and isoflavenes on rhinovirus 1B and its replication in HeLa cells. Antiviral Res. 10:117–127. 4. Conti, C., P. Mastromarino, P. Tomao, A. De Marco, F. Pica, and M. G. Santoro. 1996. Inhibition of poliovirus replication by prostaglandins A and J in human cells. Antimicrob. Agents Chemother. 40:367–372. 5. Couch, R. B. 1985. Rhinoviruses, p. 795–816. In B. N. Fields, D. M. Knife, J. L. Melnick, R. M. Chanock, B. Roizman, and R. E. Shope (ed.), Fields virology. Raven Press, New York, N.Y. 6. De Marco, A., and M. G. Santoro. 1993. Antiviral effect of short hyperthermic treatment at specific stages of vesicular stomatitis virus replication cycle. J. Gen. Virol. 74:1685–1690. 7. Feige, U., R. I. Morimoto, I. Yahara, and B. S. Polla. 1996. Stress-inducible cellular responses. Birkhauser Verlag, Basel, Switzerland. 8. Forstall, G. J., M. L. Macknin, B. R. Yen-Lieberman, and S. Medendrop. 1994. Effect of inhaling heated vapor on symptoms of the common cold. JAMA 271:1109–1111. 9. Hendley, J. O., R. D. Abbott, P. P. Beasley, and J. M. Gwaltney. 1994. Effect of inhalation of hot humidified air on experimental rhinovirus infection. JAMA 271:1112–1113. 10. Hirata, Y., H. Hayashi, S. Ito, Y. Kikawa, M. Ishibashi, M. Sudo, H. Miyazaki, M. Fukushima, S. Narumiya, and O. Hayaishi. 1988. Occurrence of 9-deoxy- 9, 12-13,14-dihydroprostaglandin D2 in human urine. J. Biol. Chem. 263:16619–16625. 11. Huber, S. A. 1992. Heat shock protein induction in adriamycin and picornavirus-infected cardiocytes. Lab. Investig. 67:218–224. 12. Hughes-Fulford, M., M. S. McGrath, D. Hanks, S. Erickson, and L. Pulliam. 1992. Effects of dimethyl prostaglandin A1 on herpes simplex virus and immunodeficiency virus replication. Antimicrob. Agents Chemother. 36: 2253–2258.

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13. Lindquist, S., and E. A. Craig. 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631–677. 14. Macejac, D. J., and P. Sarnow. 1992. Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. J. Virol. 66: 1520–1527. 15. Macknin, M. L., S. Mathew, and S. Medendrop. 1990. Effect of inhaling heated vapor on symptoms of the common cold. JAMA 264:989–991. 16. Mastromarino, P., C. Conti, R. Petruzziello, A. De Marco, F. Pica, and M. G. Santoro. 1993. Inhibition of Sindbis virus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat-shock protein synthesis. Antiviral Res. 20:209–222. 17. Morimoto, R. I., and M. G. Santoro. 1998. Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nat. Biotechnol. 16:833–838. 18. Muñoz, A., M. A. Alonso, and L. Carrasco. 1984. Synthesis of heat shock proteins in HeLa cells: inhibition by virus infection. Virology 137:150–159. 19. Palmenberg, A. C. 1987. Picornaviral processing: some new ideas. J. Cell. Biochem. 33:191–198. 20. Pica, F., A. De Marco, F. De Cesare, and M. G. Santoro. 1993. Inhibition of vesicular stomatitis virus replication by 12-prostaglandin J2 is regulated at two separate levels and is associated with induction of stress protein synthesis. Antiviral Res. 20:193–208. 21. Rozera, C., A. Carattoli, A. De Marco, C. Amici, C. Giorgi, and M. G. Santoro. 1996. Inhibition of HIV-1 replication by cyclopentenone prostaglandins in acutely infected human cells: evidence for a transcriptional block. J. Clin. Invest. 97:1795–1803. 22. Rueckert, R. R. 1990. Picornaviridae and their replication, p. 507–548. In B. N. Fields, D. M. Knipe, R. M. Chanock, J. L. Melnick, B. Roizman, and R. E. Shope (ed.), Virology, 2nd ed. Raven Press, New York, N.Y. 23. Santoro, M. G. 1996. Virus infection, p. 337–357. In U. Feige, R. I. Morimoto, I. Yahara, and B. S. Polla (ed.), Stress-inducible cellular responses. Birkhauser Verlag, Basel, Switzerland. 24. Santoro, M. G. 1997. Antiviral activity of cyclopentenone prostanoids. Trends Microbiol. 5:276–281. 25. Santoro, M. G., A. Benedetto, G. Carruba, E. Garaci, and B. M. Jaffe. 1980. Prostaglandin A compounds as antiviral agents. Science 209:1032–1034. 26. Santoro, M. G., C. Favalli, A. Mastino, B. M. Jaffe, M. Esteban, and E. Garaci. 1988. Antiviral activity of a synthetic analog of prostaglandin A in mice infected with influenza A virus. Arch. Virol. 99:89–100. 27. Santoro, M. G., E. Garaci, and C. Amici. 1989. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc. Natl. Acad. Sci. USA 86:8407–8411. 28. Sarnow, P. 1989. Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited. Proc. Natl. Acad. Sci. USA 86:5795–5799. 29. Sperber, S. J., and F. G. Hayden. 1988. Chemotherapy of rhinovirus colds. Antimicrob. Agents Chemother. 32:409–419. 30. Tyrrell, D. A. J. 1988. Hot news on the common cold. Annu. Rev. Microbiol. 42:35–47. 31. Tyrrell, D. A. J., I. Barrow, and J. Arthur. 1989. Local hyperthermia benefits natural and experimental common colds. Br. Med. J. 298:1280–1283. 32. Uncapher, C. R., C. M. DeWitt, and R. J. Colonno. 1991. The major and minor receptor families contain all but one human rhinovirus serotype. Virology 180:814–817.

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Reference 15 Kidney International, Vol. 59 (2001), pp. 1580–1589

HISTORICAL ARCHIVES

Substitution of renal function through skin catharsis: Evidence from the classical period to the Middle Ages ATHANASIOS A. DIAMANDOPOULOS and PAVLOS C. GOUDAS Department of Nephrology, “St. Andrew’s” State Hospital, Patras, Greece

Substitution of renal function through skin catharsis: Evidence from the classical period to the Middle Ages. The skin’s cleansing capacity has been known for centuries and has been used therapeutically and extensively for a great number of diseases. We studied the historical evolution of the methods used for catharsis through the skin, particularly for those in renal failure, by reviewing most of the existing ancient Greek and Byzantine codices dealing with the skin’s cleansing capacity. From the fragments cited in this article, it is evident that the ancient medical writers were well aware of the mechanism of perspiration, and through this process the excretion of several body toxins, they knew about renal failure as well as the influence of environmental temperature on blood purification via the skin. To validate their views, we reviewed the seasonal variation of the average values for blood urea, creatinine, and electrolytes for 161 regular dialysis treatment (RDT) patients in four dialysis units in southern Greece. The estimations were carried out during the winter/summer 1997, 1998, and 1999 terms and included three winter months and three summer months. We traced an unexpectedly large number of references in the ancient and medieval Greek medical literature concerning detoxification through the skin, mainly regarding patients in renal failure. This seasonal variation hypothesis is supported by the results of our retrospective study: there was a difference of 16 mg/dL in the average blood urea (mean winter urea 182 mg/dL, mean summer urea 166 mg/dL). We suggest that the ancients had a vivid idea about the substitution of renal function by the skin’s cleansing ability in renal failure. The previously mentioned phenomenon may be due to the elimination of blood urea through excessive perspiration. Our clinical results seem to verify their notions, and hence, the skin (like the peritoneum) may be considered a natural membrane for dialysis. We were unable to trace a similar report in the literature on the seasonal fluctuation of blood urea in dialysis patients.

For the past five years we have been interested in the study of the cathartic ability of various biological membranes [1]. More recently, we investigated in some Key words: renal failure, perspiration, ancient nephrology, Greek medical codices, Byzantine medical codices, detoxification using the skin, seasonal variation in detoxification. Received for publication February 24, 2000 and in revised form September 5, 2000 Accepted for publication October 6, 2000

 2001 by the International Society of Nephrology

detail the historical development of the use of the skin as an alternative route for catharsis in edematous and/or uremic humans. The reviewed literature was vast. Hence, this article presents only the first portion, extending from the classical period to the end of the Middle Ages. In a forthcoming report, we will present our findings from the historical literature from the Middle Ages up to the present time. Being enthralled by the ancients’ suggestion that the skin increases its cleaning ability in cases with renal failure, we attempted to test its validity on clinical grounds. Our findings show a significant decrease of the regular dialysis treatment (RDT) patients’ blood urea during the summer months. Although the idea of seasonal variation of biochemical and other parameters of humans and animals has not been ignored by the scientific community [2–4], we were unable to trace any similar report in the literature as far the mechanism that could explain this variation. We suggest that this decrease in our patients’ blood urea occurs because of its elimination through increased perspiration during the warmer period of the year. If this hypothesis is correct, then the ancients’ idea on the role of the skin as an alternative kidney is confirmed. It is interesting to note that the impact of the seasonal variations was very well known to the ancient Greeks as Hippocrates clearly stated in the following aphorism, “From all the weather conditions of the year the healthier and less deadly ones are the droughty and the rainless compared to the wet and rainy” [5]. Fourteen centuries later, Theophilus Protospatharius and Damascius commented on this aphorism and gave the following explanations: “Theophilus: . . . So, from all the seasons, as Hippocrates stated, the droughty are healthier and less deadly than the rainy ones. Because on the droughty seasons sweating eliminates the unwanted liquids, while on the rainy ones (the liquids) are collecting inside the body and rotten thus causing many problems.” Damascius concurred, “Because on the droughty seasons sweating eliminates the unwanted liquids, while on the rainy ones (the liquids) are collecting inside the body and rotten; except if one

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eliminates them every day by exercising or bathing or some other practice” [6]. Methods The historical materials of this research include practically all of the existing relevant Classical, Hellenistic, Roman, and Byzantine Greek codices, dating from the fifth century b.c. to the fifteenth century a.d. We read them in the various original dialects of the Greek language, and then translated them into English. CLASSICAL AGE: EMBEDOCLES, HIPPOCRATES, ARISTOTLE (500 TO 300 B.C.) Birth of physiology: The porosity of objects in nature The original observations of the various natural phenomena, whether wrongly or properly interpreted, led to the gradual development of theories pertaining to the interpretation of human’s physical mechanisms. The transfer of macrocosmic phenomena and their adaptation to the microcosm of humans and animals was the first step of this approach, and the observer had every reason to make this contrast. Menstruation, being adapted to the moon’s periodical appearance and disappearance, naturally did not go unnoticed. The earth itself had been personified and thus became a living organism that got warm, cold, or dry or was able to perspire. An essential parameter that applied to these observations was the awareness that all geologic and biological phenomena resting on mass or energy exchange were grounded on the existence of porous bodies. An old reference is given by Empedocles in his description for the receptivity of the senses: “Every body is affected seeing that it is penetrated through a number of pores by that substance which ultimately exercises its active influence and, thus, we see and hear and feel along with the other feelings (that exist). Moreover, things become visible despite the fact that air and water as well as other transparent bodies intervene due to the reason that these intervening bodies have pores which by virtue of their smallness are invisible; nevertheless, they are dense and they are arranged in series connection, and most of the transparent objects have more pores” [7]. An early reference of what centuries later will be termed an embryonic experimental proof of transcutaneous respiration is initially given, again by Empedocles, in his effort to explain the kinetics regarding the exchange of gases through the skin and combine it with the circulation of the blood. The idea was original in its conception: “Furthermore, all (animals) inhale and exhale in the following way. All animals have fleshy tubes that are void of blood and, in addition, these tubes are spread on the skin’s surface. Onto the orifices of these tubes the body’s uttermost surface has been cleaned through being furrowed

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with dense pores in such a way as to be able to contain the blood (in the body); a clean passage however, should be opened up for the air by means of the cuts and through the pores. Owing to this fact, when the thin blood rushes to an opposite direction in relation to these pores, the air dashes in them as an unrestrainable wave. When the air springs again through (the center of the body) to the surface, then the air is exhaled outwards. A similar event occurs when a young girl plays with a clepsydra made of glistening copper. When she supports the opening of the bottle’s neck with her lovely hand and sinks the clepsydra into the silver-colored water, the air can no more enter the vessel whereas the volume of air from within obstructs it as it falls onto the little pores until the girl (having withdrawn her hands) provides a free passage so that a dense stream of air comes out. Now then when the air has vacated the interior of the clepsydra, a proportional quantity of water comes into. The same occurs when the water has occupied the deep interior of the vessel, and through the human skin (that is, with the hand), the pore and the neck (of the vessel) as well as the outside air have been obstructed due to the fact that the air has been obsessed with the idea of penetrating into the vessel, holding the water at the neck’s outlet, producing a deep sound, maintaining under its possession the edge of the neck, until the girl provides a way out by removing her hand. Then, after this and exactly in the opposite direction, compared to what occurred before, the air falls inside and a proportional quantity of water is withdrawn. The same occurs with the blood that is moving with vehemence through the body’s parts. When it rushes inwards by returning backwards then the stream of air penetrates with swift undulation. When the blood rushes upwards from the body’s depth, the air is exhaled outwards in equal proportion(s).” [8]. The hydrological cycle regarding the earth as described by Aristotle was transferred to the human’s body in order to explain human physiology. With food the human received the nutritional substances necessary for life. These substances were digested and classified into the useful ones that remained in the body and the useless ones that were eliminated. The final carrier of the food, the undigested elements of food as well as the remnants of digestion, was the blood. The blood should ultimately undergo catharsis. A healthy body realized this catharsis through the intestinal tube (that is, in the form of feces), through the lungs (a reference to a statement attributed to Aristogenes was given by Aristotle [9]), through the kidneys by urine production, and through the skin by perspiration. According to Aristotle, some animals had no bladder because the nature of their bodies was not as warm as of other animals, and as such did not need to consume water and did not have liquid excrements. “Therefore not all animals have a bladder, as it seems that nature decided to give (a bladder) only to those which

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have a lung swarming with blood which is reasonable for them. Because in these that have such an organ, this nature (the warm) outweighs and are thirstier than the others and need not only dry food but more liquid and therefore their excretions are more than those their stomach can digest that have to be excreted. So there is a need for some organs to collect these excretions as well. So the ones (animals) that have such a lung also have a bladder, while from those that don’t, others drink less because their lung is spongy, and others, whatever liquid they need, they consume as food and not as a liquid like the insects and the fish, and similarly the alate and the squamate and the lepidoids, because of the small quantity of liquid they consume and because they convert to such (tissue) the extra excretion, none of them has a bladder, except the turtle from the squamates” [10]. Aristotle believed that “the blood-swarming animals have a warm nature.” The skin plays a significant role for the proper operation of the body, in that it constitutes a route for catharsis with regard to the substances that are to be eliminated. Aristotle, in his treatise titled Meteorologics, considered that there was a common reason for the creation of saline urine, of the even more saline respiration and of the salty sea: water passed through the soil, and the earth retained those elements that were useful, eliminating those that were salty and useless. Sweat was salty like seawater [11]. Moreover, Aristotle in his Problems, in the chapter titled “On Perspiration” questioned himself: “Why is perspiration salty? Because, since it is provoked by movement or heat, anything considered as unfamiliar to the blood or the fleshes is eliminated from the food. And soon this (alien substance) is separated and discharged. And it is salty considering that the body consumes whatever is sweet and rejects a substance that is alien and indigestible. And the latter when is discharged from below is called urine whereas when it is eliminated through the skin is called perspiration. Both are saline for the same reason” [12]. Aristotle’s observations on perspiration seem to have derived from relevant observations made on natural phenomena as described in Meteorologics [13]. Further than the Earth’s natural cathartic process, there was also the catharsis of the built or man-made environment. Hercules purified the Augea’s barns by turning the Alpheus river’s stream to run through them. The way that the rain fell and thus cleaned the city’s roads offered ideas for the purification of the body. Both the earth and the city were cleaned by the falling rain and the running water. Humans and animals were cleaned by the water they drank and the liquids they discharged [14]. Aristotle correlated the city and the environment with the good health of the body [15]. This correlation was developed and refined by the Byzantines, as discussed later in this article.

References

Hippocratic pathophysiology Hippocrates, the father of medicine, in his book, On Sufferings, referred to the cause of edemas and described them with exceptional detail: “An edema is mostly caused when catharsis does not occur, as in the case of a longstanding disease” . . . “When an edema is attributed to the absence of catharsis, then the abdomen is filled with water and the legs up to the shins are swollen while the shoulders, the chest and the thighs languish” [16]. According to Hippocrates, the humors and the fleshes were interchangeable both in health and in disease. The flesh could melt and become water and fill up the body’s cavities. Hippocrates identified four forms of renal diseases. In his work On the Inner Sufferings, he described them as follows, “Renal diseases are caused when the kidneys, having received the phlegm or choler or pus that is to be excreted, cannot eliminate them, resulting in its accumulation inside the kidneys and thus the appearance of the disease occurs.” To put it another way, this mechanism, which was actually suggested by Hippocrates, was identified with the reduction of the cathartic ability of the kidneys [17]. Therapeutics through the skin These conclusions of the views on pathophysiology also influenced the therapeutic approach to the issue. When the body malfunctioned and a therapeutic intervention was required, then catharsis was accomplished through either the intestinal tube with emetics, purgatives and enemas, or the skin. The most common method of catharsis through the skin was that of provoking perspiration. In ancient times, this was achieved with embrocations, cataplasms, sunbathing, and sand baths. A reference to the latter was not made by Aristotle nor does it exist in any written text of the period from 800 to 300 b.c. Later, however, Orivasios (fifth century a.d.) attributed such a reference to Herodotus (fifth century b.c.), and Antyllus (third century a.d.) did the same for Aetius (1st century a.d.). Nevertheless, the most widespread method was thermal baths and steam baths. In particular, “pyries” was a kind of a thermal bath that was accomplished by heating stones onto which water was thrown so that water vapors were produced, similar to modern saunas. The most significant reference to the use of thermal baths with regard to catharsis through the skin was given in Hippocrates’ work, On the Use of Water: “Warm water is employed for sprinkling(s) and steam baths that affect the entire body or some part of it as well as for the softening of rough skin, the relaxation of the tensed skin and the contracted nerves, the ecchymosis of fleshes and the excretion of sweat.” [18]. In On Diet of Acute Diseases, a work the authenticity of which was disputed by many, was written in the same period, and the author mentions, “The defeat of all diseases

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is accomplished either orally or from the abdomen or by the bladder or by some other organ. The cure provided by perspiration is a common one for all (diseases)” [19]. In the same book the author writes, “If we believe that we must make catharsis with the use of drugs, it is much safer to perform it from above (that is, from the mouth with emetics) with the use of hellebore. We should then abstain from the cathartic methods performed from below (that is, diuretic drugs and enemas). The best of all though is to provoke diuresis and perspiration and get the patient walking” [20]. In his book On Inner Sufferings, Hippocrates referred to the treatment of all four categories of renal diseases. For all of them, apart from prescribing diuretics and cathartic drugs, the treatment included hot compresses, thermal baths, and steam baths. A few references to skin catharsis were also made by Aristotle. Cataplasms had cathartic ability as well: “What is the efficiency of a cataplasm? It is to soothe and provoke perspiration and exhalation” [21]. In general, however, there was a lack of enthusiasm on behalf of the ancient Greeks regarding the experimental proof of their theories. This can be attributed to their general repulsion for manual work as well as to their philosophical stance that urged them to try to prove ceaselessly the grand principles of cosmology, biology, and politics on the basis of a rather latent theology or dialectical extremes, rather than being occupied with detailed work. In their discussions, they were pioneers in their tendency to place arguments above authoritative views. Their energy, however, was spent on argumentation against rival theories, acting in favor of the correctness of their own, without taking particular care in proving scientifically, and in detail, their theses [22]. They always tried to describe the forest without having previously perceived entirely what is a tree. Thus, they were opposed to the modern tendency of many researchers to produce a flood of observations with no final unified result, in effect describing so many trees that in the end the general view of the forest is lost. Aristotle, in the fourth century b.c., consciously tried to turn the philosophers’ interest to the common experiment, being, therefore, in contrast to the theory of Plato, his teacher. LATE ANTIQUITY: ERASISTRATUS, ACHIGENES, GALEN, ARETEUS, RUFUS (300 B.C. TO 300 A.D.) This section describes the views on catharsis during the classical age with the ideas of the poet and philosopher, Empedocles, and continue the corresponding description for later antiquity with other poets’ views. During the first century a.d., Ovid, in his poem “Metamorphoses,” described the ability of the humanized earth to absorb and re-excrete liquids when he told of the killing of Marsya

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and the shedding of tears for his death: “The fertile earth got soaked, and soaked it caught the tears and drank them deep into her veins. Transforming them into water, she sent them back out again to the open air” [23]. During the same period and in his poem “On the Nature of Objects” (50 b.c.), Lucretius presented a more altered version of the entire body’s pores, which corresponds to our topic more fully: “I will now try to remind (you) of how poriferous a body all things have, a fact which was also stated in my previous ode. Because, truly, although the fact that we realize this is important for many things, and at any event for those which I am going to straightforward speak, it is more than necessary to be certain that there is nothing more than (the truth) that a body is perforated by pores. One first such complex (gives evidence for this): in caves, the rocks above our heads discharge moisture and percolate muddy drippings. Likewise, sweat drips from our whole body” [24]. It is very important to realize that during these early phases of scientific thought, the concept of the communication of the body with the environment through the skin and the application of the four elements theory was implicitly understood and interlaced with all scientific applications, not only medicine. Birth of experimental physiology An impressive outcome of the methodology as initially introduced by Aristotle was a reference found in the papyrus “Anonymous Londiniensis,” probably written after the first half of the first century a.d. In it, a description of an experiment of Erasistratus of Cos (300 to 250 b.c.) was found: “. . . and Erasistratus performed the following experiment, he took an animal such as a hen or some other similar fowl and placed it into a caldron without providing it with food for quite some period of time. Then he weighed this fowl and its obvious excrements and he found them to be much less than the initial weight; thus he inferred that many discharges cannot be seen. This theory however, applies to man also. When men have drunk perfumes or have eaten garlic, this is made evident through their smell although is not otherwise sensible” [25]. A logical conclusion from the previously mentioned text was to assume that insensible transpiration was not only known to ancient Greeks, but it was also experimentally proven by them with a more or less quantitative measurement [26]. Galen’s natural and provoked skin catharsis Galen’s observations on physiology and on the function of various organs were unique. He identified the use of kidneys as a means of catharsis, which when they did not function properly, were substituted by other organs such as the gastrointestinal system and the skin. In particular, regarding the skin, he further developed

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References

Empedocle’s theory and claimed that the skin was full of pores resembling a sieve. He also maintained that under and onto the skin the arteries and the veins were anastomosed between them through minor pores (thus suggesting the function of the capillaries centuries before Harvey and Malpiggi). As a matter of fact, he concluded that aspiration and expiration were accomplished through the skin’s pores [27]. Galen expanded the theory of the four humors that he derived from Hippocrates. According to Galen, sudden edemas were attributed to the inefficiency of the kidneys to eliminate fluids, and he very elegantly differentiated between the anuria caused from kidney or bladder dysfunction [28]. Galen knew very well the cathartic ability of the skin. He was aware of the fact that the generation of perspiration purified the body and that this occurred both for those who suffer of a disease as well as for healthy people, since he wrote later: “Sweating then purifies the body. Indeed, similar to this (that is, perspiration) is that which is produced by low-effort exercise, baths and the summer heat.” It is exactly on the effect of the summer heat over the RDT patients’ blood urea that the last paragraphs are based.

appeared and prospered. Ruphus made an important reference in the section “on the sclerosis of kidneys” where he seemed to give a description of chronic renal failure: “Whenever scleroses develop in the kidneys they are painless and, as someone would expect, the loins are hanging and the hips are restricted in their movements and the legs are weak; they discharge a small quantity of urine resembling greatly the conditions affecting patients with edemas. And these patients of course, in the course of time, are filled up with water as the other viscera become sclerosed, too” [30]. In his works, Ruphus mentioned the same cathartic methods as the other physicians, namely venesection, enemas, diuretic drugs, embrocations, cupping, baths, and a careful diet. However, he added an interesting method for provoking perspiration in his work, On the Renal and Cystic Diseases, and in the paragraph on polyuria (urine diarrhea): “. . . because it is good for them to be able to perspire if diuresis stops. The best of all is a steam bath in a small vat with the head coming out from the top, so that, while the rest of the body is being heated, one can breathe cool air.”

Areteus’ opinions on the skin’s natural alternative cathartic ability Another significant representative of this period was Areteus from Cappadokia (second century a.d.). In his four preserved works, one can find many references to skin catharsis either through provocation or when the organism performs it on its own. In his book, On Causes and Signs of Chronic Sufferings: Book II, referring to dropsy, he wrote, “Dropsy is bad for any illness. However, from these (forms of dropsy) phlegmasia alba dolens (or milk-leg) is the most benign. This happens because, as it fortunately occurs in most cases, sweat, urine or diarrheas coexist and dropsy is solved.” Furthermore, in the same book, a little further on he wrote, “If in the case of dropsy, urine is much, dense and contains muddy materials, then there is some hope that dropsy will be solved. If, however, they are thin and few in quantity then this condition maintains the dropsy. If the disease changes course, as to its original form, and turns to the abdomen then by causing many and viscous watery evacuations the dropsy will be treated. Nevertheless, this therapy carries some risk due to the fact that many evacuations lead finally in the patient’s exhaustion or hemorrhage and death due to weakness. It is not dangerous if sweat solves (the dropsy), only when it is excreted in significant quantities. Because these (patients) do not sweat a lot” [29].

Archigenis’ nocturnal skin catharsis

Ruphus’ “chronic renal failure” Within this period, Ruphus from Efessus, whom the Byzantine doctor Oribasius called “a Great physician,”

A brief reference to catharsis through the skin also appeared in the few extant manuscripts by Archigenis, the physician who flourished in the emperor Trajan’s time (end of first and beginning of second century a.d.). He observed: “Common is the treatment of all dropsical patients. Their bed should be very soft, and especially for those showing anasarca edema, we should lay reed leaves under it and other drying herbs, like osier [agnus castus (or chaste tree)], kalaminth and the such. It is indeed wondrous the way in which the edema disappears during sleep so that some of those who were covered under piles of wheat, got up withered after their sleep. The covers should be rougher and the house temperature temperate according to the season of the year” [31]. Archigenis was probably the first physician in history who mentioned catharsis through the skin during sleep, something we try to achieve today by automated peritoneal dialysis during sleep. EARLY BYZANTIUM: ORIBASIUS, AETIUS, ALEXANDER OF TRALLES, PAUL OF AEGINA (300 TO 700 A.D.) Most physician writers of this period were involved in the study of ancient physicians. They repeated the theories and methods of the ancients, sometimes copying their texts exactly and other times combining various physicians’ methods, analyzing them, and often paraphrasing older views in order to present them as their

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own. Consequently, no rapid development in therapeutics occurred during this period. Oribasius’s sand baths According to researchers of Byzantine medical history, this period began with Oribasius from Pergamene (fourth to fifth century a.d.), who emphasized, “It is not useful to always cause perspiration in somebody who is bathing. Because we often take to a bath not to empty the body, but to moisten it all when very dry” [32]. The skin’s amphidromic permeability was obviously familiar to Oribasius. The therapeutic methodology’s objective did not change in Oribasius’ texts compared with more ancient writers. The body’s cathartic insufficiency constituted a significant cause of sickness, resulting in the accumulation of harmful substances. Chapter 8 of his tenth book was dedicated to the therapeutic use of sand baths. One of the indications, among others, was edema: “Heating through the sand is appropriate for asthmatic patients and those who have rheumatic diseases in the chest and abdominal diseases and gout and paralyzes and cachexia and edemas and any chronic painful illness. Suitable for therapy are all patients except for very young children.” Oribasius described this method in detail beginning from the way the sand was prepared: “You should therefore dig two or three deep holes of equal size at dawn and let them become overheated from the sun,” moving on to the positioning of the patient, depending on the illness, and extra care like covering the head so it didn’t get burned from the sun’s rays or the administration of fresh water if necessary. “As for the dropsical patients, the number of days that it (a sand bath) takes place should be proportional to the volume that must be removed. The benefit from this you should examine 21 days later and after a break of two or three days you should start again” [33]. The detailed description showed that the method was widely spread, and the instructions were derived from experience and observation, and not from untried theories. The time after Oribasius was rather poor in physician writers until the sixth century a.d. when two great personalities excelled in medicine. The first was Aetius Amidinus, and the second was Alexander from Tralles. Aetius wrote 16 books in which therapeutics dominated, combined with very few elements of anatomy and pathophysiology. Although they did not differ essentially from the writings by Orivasios and his predecessors, at certain points, he provided more information on his views. Aetius’s contribution to therapeutics In his third book and in the chapter on sand baths, Aetius mentioned that the objective of a sand bath as well as other methods of body heating was no other than

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the increase of insensible transpiration and perspiration: “the insensible transpiration it intensifies and the sweat it extracts.” The benefit of this increase of insensible transpiration and perspiration was evident “in dropsical and nephritic patients . . . and those who have developed a chronic disease of the cyst.” In Aetius’ work, phlebotomies were also mentioned, as well as thermal baths and cupping, and all other methods for catharsis referred to by previous physicians. Aetius also repeated his predecessor Archigenis’s opinion on the use of baths to provoke perspiration [34]. In the chapter “On Edemas,” he rendered his own pathophysiological interpretation and therapeutics for edemas, which were described in detail as edemas that leave a recess after exercising pressure and which should be treated with increased perspiration [35]. Alexander’s physical examination, diagnosis and treatment A generation after Aetius another famous physician appeared in Asia Minor, Alexander from Tralles of Lydia, (525 to 605 a.d.). He identified ascites from the “lurching as happens with a skinbag when one stirs the fluid that it contains,” tympanites that “when we beat it a sound is produced as occurs with a drum beating,” and anasarca edema from “the swelling of the entire body which when it is pressed with a finger a concavity is formed and when we stop pressing it the concavity does not immediately assume its previous form.” Alexander’s observations have great importance since he provided different treatments for each diagnosis, thus showing an understanding of the existence of different pathophysiological mechanisms. If a proper diagnosis was made, then “we treat ascites and tympanites with purgatives, whereas for the anasarca edema, we also employ venesection if it is needed” [36]. Paul’s therapeutic “safety rules” The earlier Byzantine age ended with Paul of Aegina (625 to 690 a.d.), who lived most of his life in Alexandria, Egypt. His auctorial work, Epitome, contained pharmaceutical and other treatments that in most cases were a mere copy of precedent authors’ views. Paul knew very well the consequences of a sudden loss of fluids and provided clear instructions with respect to the quantity that should be eliminated. In his description of abdominal catheterization for the relief of ascites, “Anyone interested in ensuring the patient’s safety must remove a small amount of fluid with the operation so that the patient is relieved of the force exerted by his excessive weight, and as for the remaining fluid this is eliminated with the use of medication that helps the body eliminate liquids, with sand-baths and sun-therapy as well as by

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recommending him to abstain from drinking liquid and by eating dry food.” The fluids that were accumulated in the suffering body could be eliminated either surgically as in the case of ascites or with classic methods such as sand bathing and enemas, or by combining both as it occured with cut cupping because, “not only blood comes out but fluids, too. And if it is necessary to remove a small quantity of liquids then a few or even one incision is enough; if however, the fluids are many then many incisions are also required” [37]. MIDDLE BYZANTINE AGE: STEFANUS FROM ALEXANDRIA, MELETIUS THE MONK (SEVENTH TO ELEVENTH CENTURY A.D.) After the seventh century a.d., Byzantium gradually lost its philosophical and scientific prestige. There were minor references in the works of a few scholars such as those of Stefanus from Alexandria and Meletius the Monk. Stephanus’s thoughts on the nature of perspiration Stefanus from Alexandria lived in Constantinople in the 7th century A.D. and taught in the university established by the Byzantine emperor Theodosius II. Very likely, Stefanus did not practice medicine himself; however he wrote many treatises, “memorandums” on Hippocrate’s, Galen’s, and Aristotle’s works, as well as various works on philosophy and astronomy. In his treatise on Hippocrate’s Prognosticon, he referred to the causes that produce perspiration. He also took up Erasistratus’ ideas inasmuch as he wrote: “The organ that produces sweat is the pores through which this sweat comes. And the raw material is all liquids, that is all juices that are in excess; this is proved by the color, the taste and the smell of perspiration in the baths as well as on the clothes of men who sweat, which have various colorings . . . various tastes . . . and various odors as they are generated by different fluids” [38]. Moreover, in his work Elaboration on Galen’s Therapeutics Dedicated to Glaphkon, he described baths and their uses [39]. Meletius’ physiology of digestion and perspiration and his microcosmos–macrocosmos perception Meletius the Monk, who lived approximately 850 a.d., was a conscientious compiler of famous ancient and Christian authors, as he himself clearly stated. In his reference on the physiology of digestion, he repeated Galen’s views and spoke of three kinds of digestion: that which occurred in the stomach, that of the liver, and finally, that of the rest of the body. In his “Essay on the Nature of Man,” he wrote: “The waste matter of the third digestion is derived from

References

the entire body and is called perspiration. It is purified through insensible pores. And all that takes place so, that this waste is not accumulated in the course of time and becomes decomposed into the intestines thus producing harm to the animal” [40]. In chapter 12 entitled “On the Skin and Hairs,” he wrote with respect to the usefulness of skin, “It eliminates from the body all that is redundant and for this reason it is entirely covered with holes so that respiration and sweat excretion are made possible.” He continued to perpetuate the age-old idea of the similitude between the microcosm and the macrocosm [41] when he wrote, “The creative or better yet, guardian nature . . . in caring for the animal, it created channeling pores through which the waste and muddy substances of the body are purified. Because as it knew that food is on one hand useful to the body but also has wasteful elements, for this reason it invented these (pores) just as they, who care for cities, build sewers and streams, so that whatever waste matter is collected it can be eliminated into lakes, rivers or seas.” LATER BYZANTINE: NICEPHORUS VLEMMYDES, NIKOLAUS MYREPSUS, IOANNIS AKTUARIUS (TWELTH TO FIFTEENTH CENTURY A.D.) In this period, Nicephorus Vlemmydes, Nikolaus Myrepsus, and Ioannis Aktuarius were distinguished physicians. One report on the medicine of this period was found in the poem “On Urines” by Nicephorus Vlemmydes, in which the methods for dealing with the disease did not change: enemas, baths, and embrocations [42]. Nikolaus Myrepsus demonstrated a method of spa therapy for very fat people, observing that a few days of increased perspiration, they thin and grew so much slimmer that “neither they who see them with their own eyes believe it” [43]. He, thus, calls to mind current advertisements of slimming centers, where by means of diuretics, massage, and saunas, they also promise tremendous weight loss. Ioannis Aktuarius, an eminent Byzantine doctor who lived during the fourteenth century, referred to the four digestions in his work, On Urines by the Wisest Aktuarius. The third digestion’s waste product was urine, and the fourth, which was the conversion of blood into flesh, produced a substance that was eliminated through the skin by insensible transpiration [44]. In his many works, there were also references to various methods of catharsis. In a chapter of his treatise on dropsical patients, Aktuarius described the following methods of treatment: diuretics, purgatives, enemas, emetics, perspiration, baths, scarifications, cataplasms, embrocations, and perforation of the abdominal cavity as Paul Aeginites described it. By the era of Late Byzantium, medical knowledge

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became more universal and not the exclusive asset of the Byzantines, the Arabs, and the Hebrews. The Latin West emerged from the Middle Ages to prepare for the early Renaissance. THE SKIN AS AN EXCRETORY ORGAN At this point, it is worth comparing some attributes between skin and kidneys. The blood supply of both kidneys ranges within 12 to 30% of the cardiac output, with the usual output being approximately 22% and is analogous to the skin’s. The kidneys’ ability to eliminate liquids rarely amounts a maximum of 10 L per day even in pathological cases of diabetes insipidus, while the skin can eliminate 12 L on a hot summer day [46]. Despite the fact that the average human skin surface is reported to be approximately 1.8 m2, in reality, this is much greater inasmuch as the skin has numerous folds as it occurs for example for a human weighing 70 kilos and 1.70 cm tall [(surface in m2) weight in kg0.425 height in cm0.725/ 139.315] [47]. It is also interesting to note that the surface of the peritoneum is also approximately 1.7 to 2 m2 [48]. The excretory ability of the skin is not limited to water, potassium, sodium, and urea. Creatinine, calcium, phosphorus, histamine, prostaglandins, amino acids, lactic acid, pyruvic acid, glucose, drug substances, and heavy metals are some of the substances that are found in the analysis of human perspiration [49]. Actually, the only restrictive element regarding the capacity of the skin to excrete substances is the aggregation of these substances. Finally, it is worth mentioning the excretion of bicarbonates in the excretory spiral, and their almost complete reabsorption in the excretory pore so that eventually the perspiration becomes acid (pH 5 to 6.5). The skin has approximately 2.5 million sweat glands, while each human kidney has approximately 1.2 million nephrons. In other words, the total number of a human’s nephrons amounts to the same number as that of the sweat glands. Both in the skin and the kidneys, as well as in many other tissues, the aquaporines seem to play the primary role for the elimination of water on a molecular level, as they are special protein channels on which the activity of the cellular membrane’s permeability to water depends. The role of the skin as an excretory organ is also demonstrated by the fact that in the sweat glands as well as in the kidneys receptors exist for aldosterone and antidiuretic hormone (ADH) [50]. The eliminated quantities of water and carbon dioxide through the skin are minimal in contrast to those eliminated by the lungs; nevertheless, they must be important for life, as shown by the following experiment. Fowls were placed in firmly closed boxes with their head protruding outwards. Although their respiration from the lungs was thus not obstructed, they died after a period of time because of the fact that their insensible transpiration was obstructed

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Table 1. Mean daily temperatures in central Greece in 1998 Mean temperature Month January February March April May June July August September October November December

Degrees Fahrenheit

Degrees Celsius

55 57 60 68 77 86 92 92 84 75 66 58

12.7 13.8 15.5 20 25 30 33.3 33.3 28.8 23.8 18.8 14.4

[51]. It is impressive that in the Hellenistic age, fowls were also closed in cages in order to prove experimentally the existence of insensible transpiration. (We describe this experiment later on.) The discussed limitations to the skin’s excretory capacity apply to humans and other mammals. In contrast, in organisms on a lower scale of evolution—such as the scyphozoans and similar creatures, which have a very thin cover for their bodies— the epidermis is their only respiratory organ since they entirely lack a respiratory system. In addition, the uricotelic animals such as reptiles eliminate large quantities of uric acid through their skin [52]. THE CLINICAL APPROACH To obtain an estimate of the validity of the antiquarian theory of the skin’s cleaning capacity, in a retrospective study design, we reviewed the average values for blood urea, creatinine, and electrolytes of all the patients in our dialysis unit in Patras, located in southwestern Greece, as well as those of three other dialysis units in Athens. The estimations were carried out during the winter/summer seasons of 1997, 1998, and 1999, and included three winter terms (that is, January, February, and March) and three summer terms (that is, July, August, and September). During these periods, the mean changes of temperature within the Patras’ and Athens’ area are very high (Table 1), as usually occurs in the Mediterranean countries. The total number included 161 patients on RDT. We compared 934 pairs of values for the same patients between winter and summer. Their dietary intake was unchanged during the period of the study. We found a significant difference of 16 mg/dL in the average blood urea between the winter and summer months (mean winter urea 182 mg/dL and mean summer urea 166 mg/dL; P 0005). There was no significant difference in the patients’ body weights between the winter and summer months [45]. Having reviewed the skin’s excretory mechanisms, we

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believe that the skin catharsis is a very attractive hypothesis to explain the seasonal variation findings on several grounds. First, a 10 C rise of the external temperature on the surface of the skin triples the sweating rate until the mechanism is saturated. Second, the mean temperatures between summer and winter in our country differ by 20 C. Third, the concentration of urea in human sweat is 1.5 to 2 times the blood urea concentration in both healthy humans and chronic renal failure patients. Finally, during summer days, the loss of water through the skin ranges from 1.5 to 2 L/24 hours. Therefore, we strongly suggest that the main reason for these results is the increased perspiration during summer days. It may not be an impressive increase, but the fact remains that the skin acts in this way operating at its minimum thermo-regulating capacity. CONCLUSIONS The hypothesis that the skin undertakes the role of a secondary kidney in renal failure is a very old one. The vast written material that survives from the classical and Byzantine periods shows the Greeks’ fascination with the concept and their goal to study it in detail. Its allotted role is augmented by an increase of the external temperature. We hope that our findings concerning a seasonal variation of the RDT patient’s blood urea reasonably support this antique idea. Epilogue The fifteenth century, in which this first part of our article stops, was not chosen arbitrarily. The period is considered the turning point between the Middle Ages and the Renaissance. During the fifteenth century a.d., four major evolutionary changes occurred: The fall of Byzantium to the Ottoman Turks, the expulsion of the Arabs from Spain, the invention of typography, and the discovery of America. Each one of these changes had a major impact on the evolution of scientific and, of course, medical knowledge. The impact of this evolution on the understanding of the mechanisms and applications of skin’s catharsis is presented in the second part of our study. ACKNOWLEDGMENTS The authors acknowledge the joined grants of the ISN and the “Greek Foundation for the study of the History of Nephrology,” without which this work would not have been possible. We also thank Professors A. Billes of Evangellismos Hospital, V. Xatziconstantinou of Amalia Fleming Hospital, and P. Ziroyannis of the General State Hospital for allowing us to include their patients’ data in this historical review. Reprint requests to Athanasios A. Diamandopoulos, M.D., Ph.D, Chorio Romanou, Patras, 26500, Greece. E-mail: goudasp@yahoo.com

References

REFERENCES 1. Diamandopoulos AA: History of peritoneal dialysis, in Proceedings of 2nd Symposium on Peritoneal Dialysis, Athens, Greek Nephrologic Society, 1995, pp 17–32 2. Tozawa M, Iseki K, Iseki C, et al: Seasonal blood pressure and body weight variation in patients on chronic hemodialysis. Am J Nephrol 19:660–667, 1999 3. Iseki K, Morita O, Fukiyama K: Seasonal variation in the incidence of end-stage renal disease. Am J Nephrol 16:375–381, 1996 4. Koch CD, Arnst E, Rommel K: Urea and creatinine levels and clearances: Observations in 25 healthy subjects for one year. J Clin Chem Clin Biochem 18:423–429, 1980 5. Hippocrates: Aphorisms, Hippocrate’s Complete Works (vol 91). Athens, Papyros, 1965, p 250 6. Theophilus Protospatharius: Commentarii in Hippocratis aphorismos, Scholia in Hippocratem et Galenum (vol 2), edited by Dietz FR, Amsterdam, Hakkert 1966, p 362 7. Aristotle: De Generatione et Corruptione. Edited by Mugler C, Paris, Les Belles Lettres, 1966, p 324 8. Aristotle: De Respiratione. Edited by Ross WD, Oxford, Clarendon Press, 1955, p 473 9. Aristotle: De Spiritu. Edited by Jaeger W, Leipzig, Teubner, 1913, pp 51–64 10. Aristotle: De Partibus Animalium, Aristotele Le Partie Des Animaux. Edited by Louis P, Paris, Les Belles Lettres,1956, p 670 11. Aristotle: Meteorologica, Aristotelis Meteorologicorum Libri Quattuor. Edited by Fobes FH, Hildesheim, Olms, 1967, pp 338–390 12. Aristotle: Problemata (sec 866). Edited by Bekker I, Berlin, De Gruyter, 1960 13. Aristotle: Meteorologica, Aristotelis Meteorologicorum Libri Quattuor. Edited by Fobes FH, Hildesheim, Olms, 1967, p 357 14. Aristotle: Meteorologica, Aristotelis Meteorologicorum Libri Quattuor. Edited by Fobes FH, Hildesheim, Olms, 1967, p 347 15. Aristotle: Problemata (sec 865), edited by Bekker I, Berlin, De Gruyter, 1960 16. Hippocrates: On Sufferings, the Greeks (vol 48). Athens, Kaktos 1992, p 56 17. Hippocrates: On Internal Sufferings: The Greeks. 1992, pp 137–144 18. Hippocrates: On the Use of Liquids: The Greeks. Athens, Kaktos, 1992, p 198 19. Hippocrates: On the Diet of Acute Diseases: The Greeks. Athens, Kaktos, 1992, p 125 20. Hippocrates: On the Diet of Acute Diseases: The Greeks. Athens, Kaktos, 1992, p 139 21. Aristotle: Problemata (sec 863), edited by Bekker I, Berlin, De Gruyter, 1960 22. Loyd G: Magic, Reason and Experience. Cambridge, Cambridge University Press, 1993, p 233 23. Ovidius: Metamorphoses, translated by Miller FJ, Cambridge, Loeb Classical Library, 1996, pp 6396–6398 24. Lucretius: On the Nature of Things, Great Works of Literature. Translated from Latin by Leonard WE, Focus Multimedia, 1995 25. Iatrica Anonymi Londiniensis Ex Aristotelis Iatricis Menoniis et Aliis Medicis Eclogae (sec 33, line 45). Edited by Diels H, Berlin, Reimer, 1893 26. Diamandopoulos AA, Goudas P: Nephrology, a newly rich specialty is looking for an illustrious ancestry: What about a famous grandfather? Am J Nephrol 20:163–165, 2000 27. Galen: De Simplicium Medicamentorum Temperamentis Ac Facultatibus Libri, Claudii Galeni Opera Omnia (11). Edited by Kuhn CG, Leipzig, Olms, 1965, p 402 28. Galen: De Symptomatum Causis Libri Iii, Claudi Galeni Opera Omnia (2). Edited by Kuhn CG, Leipzig, Olms, 1965, p 251 29. Areteus: On Causes and Symptoms of Chronic Sufferings B, the Greeks. Athens, Kaktos, 1992, pp 191–201 30. Rufus von Ephesus: De Renum et Vesicae Morbis, Ouevres de Rufus d’Ephese (sec 1). Edited by Daremberg C, Ruelle CE, Amsterdam, Hakkert, 1963 31. Archigenes: On Cachexia, Frammenti Inediti Di Archigene: Bolletino Del Comitato Per La Preparazione Della Edizione Nazionale Dei Classici Greci E Latini (vol 9). Edited by Calabro GL, 1961, p 71

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32. Oribasius: Collectiones Medicae, Oribasii Collectionum Medicarum Reliquiae (sec 19). Edited by Raeder J, Leipzig, Teubner, 1933 33. Oribasius: Collectiones Medicae, Oribasii Collectionum Medicarum Reliquiae. Edited by Raeder J, Leipzig, Teubner, 1933 34. Aetius Amidenus: Iatricorum Liber III, Aetii Amideni Libri Medicinales I-IV. Edited by Olivieri A, Leipzig, Teubner, 1935 35. Zervos S: Aetius Amidenus: Iatricorum Liber XV: On Oedemas. Athena, 21:7–8, 1909 36. von Tralles A: Therapeutica: On Oedema: On Ascites: On Therapy (vol 2). Edited by Puschmann T, Amsterdam, Hakkert, 1963, pp 439–443 37. Paulus Aeginites: Epitomae Medicae Libri Septem (vol 1). Edited by Heiberg JL, Leipzig, Teubner, 1921 38. Stefanus of Alexandria: Scholia in Hippocratis Prognosticon, Commentary in Hippocrates Prognosticon (sec 11). Edited by Duffy JM, 1975 39. Stefanus Atheniensis: Commentarii in Priorem Galeni Librum Therapeuticum Ad Glauconem. Scholia Hippocratem Galenum (vol 1). Edited by Dietz FR, Amsterdam, Hakkert, 1966, p 259 40. Meletius: De Natura Hominis, Anecdota Graeca E Coddici Manuscriptis Bibliothecarum Oxoniensium (vol 3). Edited by Cramer JA, Amsterdam, Hakkert, 1963, pp 107–108 41. Diamandopoulos AA: A history of natural membranes in dialysis. Am J Nephrol 17:304–314, 1997 42. Diamandopoulos AA: Musical Uroscopy. Edited by Ekdoseis A, Athens, Tehnogramma, 1996

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43. Eutihiades A: Introduction to Byzantine Medicine. Athens, Parisianos G, 1983, p 99 44. Dimitriadis K: Byzantinische Uroskopie. PhD Thesis, Bone, Ioannes Actuarius, 1971 45. Diamandopoulos AA, Goudas P, Hatzikonstantinou B, et al: Seasonal variations of the skin’s capacity as an alternative way for catharsis in patients under chronic dialysis, a multicentre study, in Proceedings of the 58th Scientific Meeting, Thessaloniki, Greek Nephrologic Society, 1999, pp 30–31 46. Ferry F: The Care of the Medical Patient. St. Louis, Mosby, 1998, p 336 47. Anonymous: Harrison’s Principals of Internal Medicine: Companion Handbook (13th ed). New York, Oxford University Press, 1994, p 842 48. Clinical Nephrology on CD-ROM. 1997 49. Benet L: Pharmacokinetics: The dynamics of drug absorption, distribution and elimination, in The Pharmacological Basis of Therapeutics (sec. 1), edited by Goodman Gilman A, Goodman L, New York, MacMillan, 1996, p 22 50. Kenzo S: Biology of the eccrine sweat gland, in Dermatology in General Medicine, edited by Fitzpatrick T, New York, McGraw Hill, 1993, p 237 51. Encyclopaedia Helios. Edited by Passas J, Athens, Helios, 1955 (in Greek) 52. Lehninger A, Biochemistry. New York, Worth Publishers, 1970, p 452

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from Hydrothermic Remedies... Water Temp : 39.5 - 43.5°C / 103-110°F

EFFECTS:Derivative (../Effects /Derivative.html) for:Congestions:

• Cerebral Congestion (../Problems /CerebralCongestion.html) • Passive Chest Congestion (../Problems /PassiveChestCongestion.html) • Pelvic Congestion (../Problems /PelvicCongestion.html) • Warming before Graduated Tonic Cold (GraduatedTonicCold.html) • Increase Immune Response for:Colds (../Diseases/Coryza.html) and Influenza (../Diseases/Influenza.html)

Definition A local immersion bath covering the feet and ankles at temperatures ranging from 39° - 43°C / 103-110°F.

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Heat As Medicine – Omicron / Flu Cure in 2 hours

Physiologic Effects

1. To provide local and reflex increase in blood flow through the feet and entire surface, producing decongestion in internal organs and brain (derivative effect). 1. to relieve congestive headache 2. to relieve chest congestion 3. to relieve pelvic congestion 2. To provide general warming of the body 1. to prepare patient for general application of heat 2. to prepare patient for tonic procedures 3. to produce sweating (when prolonged) 4. to help prevent or abort a cold 3. To aid relaxation and comfort 4. To provide a treatment for local inflammation of the feet 5. Increase white blood cell activity

Indications

1. Congestive Headache (../Problems /CongestiveHeadache.html) 2. Passive Chest Congestion (../Problems /PassiveChestCongestion.html) 3. Pelvic Congestion (../Problems /PelvicCongestion.html) including Prostatis (../Diseases /Prostatis.html), gall or renal Colic (../Problems/Colic.html), Nephritis (../Diseases /AcuteNephritis.html) and Toxaemia (../Problems /Toxaemia.html)

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References 4. Painful Abdominal Congestion (../Problems /VisceralCongestion.html) 5. Preparation for other, usually cold, treatments such as Graduated Tonic Cold (GraduatedTonicCold.html) 6. Warm the body 7. To stop Nosebleed (../Problems /Epistaxis.html) 8. Aid relaxation and comfort 9. Common Cold (../Diseases /Coryza.html) and Sore Throat (../Problems /ThroatInflammation.html) 10. Eye Pain (../Problems /ExternalEyeInflammations.html), Eye Inflammation (../Problems /EyeballInflammations.html), Earache (../Problems /MiddleEarInflammation.html)

Contraindications and Cautions

1. Obstructive circulatory disturbances 2. Diabetes (../Diseases /Diabetes.html) 3. Peripheral vascular diseases (../Problems /PeripheralVascularDisease.html) 4. Any condition where circulation in feet and legs is poor (like extreme vascular disease of feet and legs) or where there is a loss of sensation (../Problems /Paraesthesia.html) (feeling) in feet or legs.

Equipment

1. Foot tub or container large enough and deep enough; five gallon can or dish pan may be used but water should be at least 10 cm deep. 2. Thermometer, if available; if not,

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Heat As Medicine – Omicron / Flu Cure in 2 hours test water with elbow; temperature 39° - 43°C / 103-110°F. 3. Sheet or bath blanket 4. Turkish towel, cold compress if needed 5. Material for protection of bed (rubber sheet or plastic) 6. Pitcher or dipper to add hot water 7. Equipment for Fomentations (Fomentations.html) and Cold Mitten Friction (ColdMittenFriction.html) if needed

Procedure

Important Considerations

• Do not use hot foot bath in peripheral vascular disease unless specifically ordered by the physician; and, if used, maximum temperature is usually 39°C / 110°F. • Not recommended for frostbite • When adding hot water, be careful not to burn the patient

Preparation for Treatment

Explain procedure to patient Have room warm and free from draft Assemble materials Protect bedding If sweating is desired, have the patient drink warm or hot water before the bath • Patient may be lying or sitting, properly draped, i.e. covered with a sheet then a blanket, mitred at neck, with a towel around the neck • • • • •

Treatment

• The patient can be lying or sitting • Have the patient properly draped • Have water temperature 39° - 43°C / 103-110°F. and deep enough to cover a couple of inches above ankles. • Assist the patient to place his feet in the tub; place your hand under his feet

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References

• •

•

and into the water first to make sure the water is not too hot. Be sure the drape covers the tub Add hot water from time to time to increase the temperature gradually to 43°C / 110°F, of the hot foot bath; make sure you remove the feet first before adding the hot water or place one of your hands in the water between where you are pouring the water in and the patient's feet. Stir gently with your hand. Continue 10 - 30 minutes; check reaction for perspiration Use Cold Compress (ColdCompress.html) to head when sweating begins; renew often at least every 2 minutes and give water through a straw if sweating is continuing. When finished with treatment, pour cold or iced water over feet; remove from tub and rub and dry thoroughly If patient is perspiring, give an AlcoholSponge (AlcoholSponge.html) or other cooling treatment over the whole body and dry thoroughly when finished. The hot foot bath is commonly given in conjunction with other methods.

Completion of Treatment

• Give follow-up care as prescribed. • Be sure patient is warm and comfortable • Remove equipment. • Record treatment, temperature, time and reaction. • The patient should rest lightly covered following the treatment for 10 - 30 minutes so as to get rid of body heat and not perspire after getting dressed and leaving the building. (Thanks to Lesley)

To prevent or treat Colds (../Diseases/Coryza.html) and Influenza (../Diseases

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/Influenza.html)

• Drink hot water before the treatment • Use the lying treatment with Fomentations (Fomentations.html) • Treat until sweating freely for 5 minutes - add hot water frequently • Follow with Cold Mitten Friction (ColdMittenFriction.html)

For Congestive Headache (../Problems /CongestiveHeadache.html)

• Treat sitting in a comfortable chair. Aim for relaxation. • Start with hot water but don't add more • Continue for 20 minutes or until pain relief • Massage neck and shoulders afterwards

JHK

from Dr JH Kellogg's Hydriatic Techniques... 46, 40-50°C / 115, 104-120°F Begin at 40°C / 104°F and gradually increase over 4 min Derivative (../Effects/Derivative.html) Antiphlogistic (../Effects /Antiphlogistic.html) Revulsive (../Effects /Revulsives.html)

Derivative (../Effects /Derivative.html) 40-43°C / 104-110°F, 20 min - in

• Cerebral Congestion (../Problems /CerebralCongestion.html) • Pelvic Congestion (../Problems /PelvicCongestion.html) • Congestion (../Problems /Congestion.html) anywhere in upper body • to prevent Coryza (../Diseases /Coryza.html)

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References

Revulsives (../Effects /Revulsives.html) 46-52°C / 115-126°F 5 min followed by cold Pail Pour (PailPour.html) in

• Pelvic Atony (../Problems /PelvicAtony.html) • Pelvic Congestion (../Problems /PelvicCongestion.html) • Amenorrhoea (../Diseases /Amenorrhoea.html) • Indigestion (../Problems /Indigestion.html) Foot problems - 2-3x a day, 20-30min in

• acute Sprains (../Diseases /Sprains.html), • Anaesthesia (../Diseases /Anaesthesia.html) of the sole • Neuralgia (../Diseases/Neuralgia.html) of foot • Gout (../Diseases/Gout.html)

GKA

from Dr GK Abbott's Prescriptions... Derivative (../Effects/Derivative.html)

• Begin at 40°C / 104°F and increase to 48°C / 118°F • Finish with cold dash

Hydrotherapy Techniques:

Local Applications

 General Applications

 Compresses and Packs

(GeneralApplications.html)

(CompressesandPacks.html)

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 Local Applications (LocalApplications.html)

 Enemas (Enemas.html)

 Other Applications (OtherApplications.html)

 Hot and Cold Compresses

 Alternating Applications

(HotandColdCompresses.html)

(AlternatingApplications.html)

 Irrigations (Irrigations.html)

 Scotch Applications (ScotchApplications.html)

 Local Baths (LocalBaths.html)

 Temperature Table (TemperatureTable.html)

 Miscellaneous Local Treatments (MiscellaneousLocalTreatments.html)

 Special Compresses (SpecialCompresses.html)

Local Baths  Alternate Baths (AlternateBaths.html)  Alternate Foot Bath (AlternateFootBath.html)  Arm Bath (ArmBath.html)  Cold Foot Bath (ColdFootBath.html)  Cold Local Bath (ColdLocalBath.html)  Cold Rubbing Sitz (ColdRubbingSitz.html)  Cold Sitz Bath (ColdSitzBath.html)  Elbow Bath (ElbowBath.html)  Flowing Foot Bath (FlowingFootBath.html)  Hot Half Bath (HalfBath.html)  Hand Bath (HandBath.html)  Hot Foot Bath (HotFootBath.html)  Hot Local Bath (HotLocalBath.html)  Leg Bath (LegBath.html)  Neutral Sitz (NeutralSitz.html)  Partial Continuous Bath (PartialContinuousBath.html)

 Prolonged Cold Sitz (ProlongedColdSitz.html)

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References

 Revulsive Sitz (RevulsiveSitz.html)  Shallow Foot Bath (ShallowFootBath.html)  Sitz Bath (SitzBath.html)  Starch Bath (StarchBath.html)  Very Hot Sitz (VeryHotSitz.html)

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Reference 17 http:// ijp.mums.ac.ir Original Article (Pages: 4429-4437)

The Maternal Experiences of Child Care with Fever: a Qualitative Study Bahare Fallah Tafti1, *Naiire Salmani2, Sara Afshari azad31 MSc, Department of Meybod Nursing, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Assistant Professor, Faculty of Department of Meybod Nursing, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 3MSc, Faculty of Nursing and Midwifery, Tehran Medical Science, Islamic Azad University, Tehran, Iran. 1 2

Abstract Background One of the most common symptoms of diseases in infancy period is fever, and the concerns occurred could lead to encouraging parents to control fever as soon as possible. This study has been conducted to explore experiences of mothers caring children with fever. Materials and Methods This qualitative study was conducted using conventional content analysis. The data were collected through 14 unstructured individual interviews with a purposive sampling among the mothers having children with fever admitted to pediatric ward of Shahid Sadoughi hospital, Yazd-Iran. Data analysis was performed on a continuous and consistent comparisons basis. Results The mean and standard deviation of variables of mothers’ age (year), length of hospitalization of children (day), and age of children with fever (year) were 5.17 ± 28.25, 2.7 ± 4.2, and 2.3 ± 1.7, respectively. The experiences of participants were revealed in three themes of "concern

penetration", "in search of fever control", and "discomfort". Conclusion

Since the occurrence of fever is associated with concerns of parents and self-medication to control fever and discomfort of mothers, it is essential for the health care providers to design and implement the appropriate family-centered interventions to improve awareness and the performance of parents. Key Words: Child, Experience, Fever, Iran, Mother, Qualitative research.

*Please cite this article as: Fallah Tafti B, Salmani N, Afshari azad S. The Maternal Experiences of Child Care with Fever. Int J Pediatr 2017; 5(2): 4429-37. DOI: 10.22038/ijp.2017.21615.1807

*Corresponding Author: Salmani Naiire, PhD, Basij boulevard ,Imam Jafar Sadeq Hospital of Meybod ,Department of Meybod Nursing, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Email: n.salmani@ssu.ac.i Received date Dec.23, 2016; Accepted date: Jan.22, 2017

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and ibuprofen- induced kidney damage may occur (17). Another common method used to reduce body temperature is cooling the outer body done using a sponge and cold water bath, while there is little and insufficient evidence about the effects of cooling of the body on fever reduction (18). Furthermore, taking of the clothes and exposing the body of child with fever to open air, fanning children, preventing child form being in blanket (19), are of the other cares provided by mothers to reduce fever in children. How mothers manage fever in children is affected by ethnic and racial diversity (20), level of education, culture, socio-economic status and subjective norms (17). Although many studies have been done quantitatively on the performance of the parents caring children with fever, and extensive knowledge has been provided in relation to the performance level and method, deeper identification of care method needs further reflection, and the knowledge resulted from this deep reflection can help the health care providers to determine effective educational programs in order to reduce the negative performances and reinforce the positive ones (21), in a way that qualitative studies can be the provider of this knowledge, but there are few qualitative studies related to the mothers of children with fever and the affecting factors (22). Thus, the current study has been conducted to explore experiences of mothers caring children with fever.

1- INTRODUCTION Fever is one of the most common symptoms of diseases in infancy period, and it has been introduced as a reason for the increased referrals to hospitals. 30% of children who come to visit the pediatrics have fever (1), and about 40% of children under 6 months experience fever (2). Simultaneously with the fever, parents get anxious (3-5) and the concerns occurred lead to encouraging them to control fever as soon as possible, and this phenomenon is growing (6), so that fear and anxiety occur with fever in many mothers and it is called "fever phobia" (7). This fear does not stem from the fever itself, but from its possible side effects (8); and febrile convulsions and brain damage with it (9), dehydration and nausea (10) have been reported as causes of fear by most mothers. Therefore, the majority of mothers try to control their fever fast so that they can prevent higher fever (9). Many of the mothers start the treatment at home before visiting a physician (11), while fever is an indication of protecting performance of the body against invading pathogens (9), and fever is not a disease itself, it is a complex physiological response of the body against the disease (12). But parents mistakenly believe that higher fever is associated with the severity of disease, so they begin antipyretic therapy as soon as possible (13). Acetaminophen and Ibuprofen are of the most commonly used antipyretic medications (14); and mothers use these drugs more than enough, and incorrectly manage the fever (15). Most of the mothers stay up during the night and sometimes wake their children up to control the fever and prescribe more medicine. Doing so, they disrupt the child's rest in addition to their own rest, and increase their own fatigue. On the other hand, they expose their child to complications of high doses of the drug, so that acetaminophen-induced liver damage

2- MATERIALS AND METHODS This is a qualitative research with conventional content analysis approach. Participants in this study included 14 Iranian mothers, with the ability to speak Persian, interested in participating in research with the ability to communicate and transfer the experiences of care of children suffering from fever. Demographic characteristics of the participants are shown in Table.1. Research field was pediatric ward of

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Shahid Sadoughi hospital Yazd city of Yazd province, the Central of Iran. Being present in the ward and after primary communication, and introducing herself and explaining the purpose and importance of research for visitors, the researcher conducted the needed coordination for interviews with people who were willing to participate in research; oral and written informed consent were taken from all participants before the interview, and they were informed that they are free to get out of the research, and the researcher is obliged to observe all principles of research ethics, including confidentiality and anonymity of participants. Time and place of the interview were based on the willingness of participants, often after visiting hours and in the conference room or after child discharge and telephone coordination and determination of appointment so that the researcher can visit participants in their own house.

60 and 90 minutes. Sampling was done purposefully and continued to reach saturation. It was tried to collect data and select participants from mothers with the highest diversity of different economic, academic, and social levels, indigenous or non-indigenous, having children with different ages (infants, toddler, young, school age). A total of 14 unstructured individual in-depth interviews were conducted. Conventional qualitative content analysis was used for data analysis. This approach is used for subjective interpretation of text data; in this method, through systematic classification process, codes and themes are identified. Content analysis is beyond the objective content extraction from text data, and in this way, hidden themes and patterns can be revealed from within the data content of the study participants. Thus, concurrent with data collection, recorded interviews were transcribed line by line. It was read many times to understand the content of the statements of the participants, and then, the meaning units and primary codes were extracted. The codes were then classified based on similarities (23).

Data collection method was deep individual and unstructured interviews started with an open question, and then, the questions and answers were continued with regard to the objectives of the research based on the way of response by the participants. The interview was started by questions including "please tell me, how did you take care of your child when he had a fever?", "Why did you care for him/her?", then, based on the participants' answers, the interviewer used exploration questions such as "Would you please give me an example" or "please, explain more about the issue".

The proposed method was used to evaluate and increase the reliability and validity which are equivalent to the scientific strength of findings in the qualitative research of Guba and Lincoln (24). According to this method, four criteria of validity, transferability, reliability and verifiability were considered for assessment. For credibility and acceptability of data, the principal investigator tried to have continuous involvement with the data. To increase the transferability, findings were evaluated by two specialists in the field of qualitative studies that were outside of the research team. To verify the accuracy of the data and extracted concepts, the participants were reviewed.

All interviews were recorded by tape recorders with the permission of participants, and at the end of the interview, the participant was asked to state the additional content she has in the mind, and after thanking, the researcher also stated the possibility of subsequent interviews. Considering the interest of the participant, duration of an interview was an average of 40 minutes varied between Int J Pediatr, Vol.5, N.2 Serial No.38, Feb. 2017

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Table-1: The demographic characteristics of the participants Variables

Number (%)

Education

Job Native Gender (child)

High school

1(7.1)

Diploma

3(21.42)

College education

10(71.42)

Housewife

10(71.42)

Employee

4(28.57)

Yes

9(64.28)

5(35.71)

Girl Boy

8(57.14) 6(42.86)

The age average of mothers (year) 28.25_+5.17 The age average of children (year) 2.3+_ 1.7 The average length of hospitalization (day) 4.2_+ 2.7

issue that fear of the occurrence of complications from fever are the greatest reasons for concern and their main motive to act quickly to control fever. "I was so concerned when I saw my child with fever; he is a little kid, so many thoughts surrounded me: what should I do in case of seizure, because it may affect his brain. He is not old enough so that he can take drugs easily; I was always worry that I could not take care of him" (Interview 4, indigenous mothers, an 8-month child).

3- RESULTS Fourteen mothers of children with fever were interviewed in this study. Mean and standard deviation (SD) of age of mothers (year), length of hospital stay for children (day), and age of children with fever (year) were 28.25 ± 5.17, 4.2 ± 2.7, and 2.3 ± 1.7, respectively. In line with the question "how mothers explain care for children with fever?" the experiences of participants were revealed in three themes of "concern penetration", "in search of fever control" and "discomfort". Then, we discuss each of the themes.

In addition to concerns of increased risk of fever complications and increased fever, most of mothers were more concerned thinking about the possibility of child hospitalization, especially mothers, who had several children, were more concerned due to further responsibilities of parental role. "When the child got hot, I was worried about the possibility of brain damage on one hand, and that I could not take good care of my other child on the other hand. How can I say that when one’s child is sick and has high temperature, the

3-1. Concern penetration Onset of fever in children is associated with stress in mother. Increased fever, prediction of fever complications (seizures, mental retardation, and dehydration), prediction of hospitalization of children, concern of change in the parental roles, and inability to take care of a sick child were stated. Among the reasons cited, most parents agreed on the

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mother cannot think about anything else. She is always worried about the possibility of hospitalization, the mother has many responsibilities at home (Interview 7, nonindigenous mother, 1.5 year old child).

cold water and put his feet in the water, but he started shivering after a few minutes" (Interview 12 - indigenous mother - 2year-old child). "I had heard that salt water can stop fever, but it was ineffective" (Interview 10 – nonindigenous mother - 4-year-old child)/ Drug therapy was an action done by most mothers. Two common drugs used were Acetaminophen and Ibuprofen. For drug administration, a small number of mothers determined and prescribed the required dosage based on their previous experience and previous prescription. They were sure about the correctness of the prescribed dosage; and the majority of mothers prescribed the drug without calculating the required dose and without the knowledge of how to determine the dose. Some mothers were afraid of the side effects of the drug, so, they tried to prescribed the drug very little and frequently. Some other were afraid of seizures and mental retardation, so, they used Acetaminophen and Ibuprofen at a short distance from each other. The use of medicinal plants (Descurainia sophia, violet flower, hibiscus and jujube), was reported effective by mothers. They prescribed the decoctions or liquids from these plants based on their own experience. Taking off the child's clothes, fanning, and cooling the room air were introduced as care techniques by mothers. Most parents went to visit the physician after primary medical treatment actions at home and seeing their ineffectiveness.

3-2. In search of fever control Mothers were trying to control the child's fever; they used methods of fever measurement, self-medication (drug therapy, foot bath, taking off the child's clothes, fanning, cooling the room air, and medicinal plants), and referring to physician. Most mothers stated that they attempted to measure the child's fever at the beginning of fever, and the measurement was performed in different forms including touching the forehead and body of the child, and understanding the difference between the temperature of the child's body and her body, using forehead thermometer strips and axillary thermometers. Most mothers stated that they had no tools for measuring the temperature of the body at home, so, they have estimated the severity of fever simply touching the child, and some mothers did not know how to operate and read the temperature and were unable to use the thermometer despite having a thermometer at home. A mother of a 9-month nursing baby stated about measuring the child's body temperature that: "The body was very hot, I didn’t know how high the temperature was, I had a thermometer, but I didn’t know how to use it" (Interview 7 indigenous mother - 3-year-old child). When mothers diagnosed children with a fever, they started self-medication and mostly foot bath. Some used cold water for foot bath and stated that although they wanted to reduce fever with foot bath, the children started shivering and they stopped foot bath. Some added salt or alcohol to the water, but there was no reduction in the fever. Some mothers did foot bath with lukewarm water several times and had noticed a reduction in fever. "As soon as I felt my child’s fever, I brought a tub of

In fact, seeing the doctor immediately after the onset of fever was not their priority as a method of fever control. Mostly two days after the onset of fever and home care delivery and seeing ineffectiveness or increased fever, they had to see a doctor despite providing care at home. "I gave her Acetaminophen syrup several times, put his feet in the foot bath, no change occurred, so I took him to a doctor"

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(Interview 8 – non-indigenous mother – 3.7-year-old child).

the research method so that the reasons mentioned were resulted from the quantitative studies while the current study is qualitative, and parents stated the causes of concern during interview based on what they have experienced, but in quantitative studies, researchers included predicted and pre-defined causes in the questionnaire, and conducted a survey. After concerns occurred, mothers attempted to search for fever control, and used body temperature control, self-medication and doctor visit. Most mothers touched the child’s body to measure the temperature, some others did not how to apply and read thermometer despite having one at home. In literature, studies in various Asian, European and African countries reported different results in terms of how to control body temperature by mothers of children with fever, such that in the study done by Agrawal et al. (2013) in India, only 24 out of the 164 parents surveyed had used a thermometer to measure fever (30). In Turkey, among 816 mothers surveyed, 60% used the thermometer to determine the temperature of the child’s body at home (27). In this study conducted by Oshikoya et al. (2008) in Nigeria, 83.3% of mothers touched the child's body (forehead - chest - limbs) with back of the hand and examined the fever (31); and in Italy, from 388 parents, 302 parents used a thermometer to measure the temperature of their child's body (32).

3-3. Discomfort Mothers suffered from disorders in terms of sleep, rest and nutrition during care for children with fever, in a way that, frequent staying up during the time when the child had a fever caused fatigue. On the other hand, because of the stress caused by the disease and the imbalance in life, they did not get enough food. "I didn’t sleep well for three days. He had fever at night, I was worried, I couldn’t sleep at night, or woke up suddenly" (Interview 2 – indigenous mother – 1-year-old child). "When your child is sick, you cannot eat anything, because he/she cannot eat. I knew that my body had no energy and needed food, but everything was cluttered" (Interview 5 – non-indigenous mother – 2.8-year-old child). 4- DISCUSSION Statements made by participants showed that mothers' experiences in care of children with fever were concern penetration, in search of fever control and discomfort. Mothers’ concerns were caused by rising fever, fever complications, hospitalization of children, changing parental roles and inability to care for the child. These findings were also confirmed in many studies that the incidence of fever in children causes concern for parents (25), but the cause of concern for mothers is different in different cultures and countries, in a way that mothers’ lack of knowledge to control and manage fever, low age of the child with fever, low age of mother (26), being only one child (27), prediction of fever complications such as seizures and mental retardation (28), possibility of brain damage (29), are cited among the causes of concerns. These reasons are somehow different from the reasons identified in this study; this difference can be derived from

Differences in reported rates may be due to differences in the social, economic, cultural and educational status of mothers; because these factors are the most important determinants of the mothers’ level of knowledge and how they manage the fever (33). The self-medications mentioned by mothers included drug therapy, foot bath, taking off child's clothes, fanning, cooling the room air, and medicinal plants. The literature review showed that in different countries, parents have used various methods to reduce the

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child's fever. Rajput et al. (2014) in India stated that the most common fever control methods included acetaminophen prescription, body bath with sponge and lukewarm water, using honey, sugar water and medicinal plants (34). Oshikoya et al. (2008) in Nigeria showed that existing methods of mothers to control the fever includes taking off the child’s clothes and exposing the child's body to the air, bathing with lukewarm water, and cold water and fanning the child’s body (35).

needs are ignored (41). Following the failure to provide mothers’ needs, they suffer from change of mood and decline in performance and physical health (42).

Rekain et al. (2014) introduced acetaminophen and towel soaked with cold water as methods of reducing fever in Mokoro (an African country) (36). Pereira et al. (2012) in Brazil identified Acetaminophen and Diprofen as common drugs used by parents to lower their children’s fever (37). Through a review study on ways to manage fever in children by parents, Walsh and Edwards (2005) stated that the most common way is using antipyretics which are preferred by parents (38). In general, fever control by drug is of the conventional methods common in most countries surveyed, and other methods such as body bath, fanning, cooling and using medicinal plants are reported different depending on the type and context of studies. It can be interpreted that the social, economic, and educational levels, insurance status, and ethnicity of parents (39), are among the most important factors affecting the determination of the type of parents’ actions to manage fever and can explain the differences in performance.

The findings of the study indicated the fact that fever is a stressful event for mothers, and in most cases, the concern occurred is because of the risk of seizure with fever which is a driving factor for an immediate action to control fever. Most people often choose self-medication as a prior method to the doctor’s visit, so, they visit the physician in case of ineffectiveness of self-medications in controlling fever. In the meantime, they cannot meet the basic needs such as sleep, rest and eating, so, they experience discomfort. Therefore, focusing on family care and the important role of mothers in the care of children with fever, and according to the themes identified, it seems necessary to put the required educations about fever and the resulted seizures, and how to properly manage fever at home as the priorities of the care team interventional program. It must also be designed, implemented and assessed.

4-1. Limitations of the study This study was conducted in limited society and explained perspective of mothers with children hospitalized in teaching hospital. 5-CONCLUSION

6- CONFLICT OF INTEREST: None.

7- REFERENCES

Another finding of the study was mothers’ discomfort occurred as a result of fever in children and ongoing care of the child by mother. Conner, nelson (1999) the stated main needs of mothers with sick children to have a place to relax, bedding items, a quiet place and food. In fact, mothers need physical support and provision of food and sleep is very important (40), but usually during the child's illness, the mother's

1. Leocadio MC, Jabai AC, Rul JA, et al. Pagdikta (The dictation): the meanings inFilipino mothers’ experience of using herbal plants in the management of their children’s fever. Int J Public Health Res 2011:169-79. 2. Clarke P. Evidence-Based Management of Childhood Fever: What Pediatric Nurses Need to Know. J Pediatr Nurs 2014; 29(4):372–5.

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3. De S, Tong A, Isaacs D, Craig JC. Parental perspectives on evaluation and management of fever in young infants: an interview study. Arch Dis Child 2014; 99(8):717–23.

16. Crocetti M, Moghbeli N, Serwint J. Fever phobia revisited: Have parental misconceptions about fever changed in 20 years? Pediatrics 2001; 107:1241–46.

5. Richardson M, Purssell E. Who’s afraid of fever? Arch Dis Child 2015; 100(9):818–20.

17. Monsma J, Richerson J, Sloand E.Empowering parents for evidence-based fever management: An integrative review. Journal of the American Association of Nurse Practitioners 2015; 27:222–29.

6..Zyoud SH, Al-Jabi SW, Nabulsi MM, Tubaila MF, Sweileh WM, Awang R, et al. The validity and reliability of the parent fever management scale: a studyfrom Palestine. Matern Child Health J. 2015; 19(8):1890–97.

18. Meremikwu M, Oyo-Ita A. Paracetamol versus placebo or physical methods for treating fever in children. Cochrane Library 2009; 2:1–3.

7. Betz MG, Grunfeld AF. ’Fever phobia’ in the emergency department:a survey of children’s caregivers. Eur J Emerg Med 2006; 13(3):129–33.

19. Prakash Agrawal R, Singh Bhatia S, Kaushik A, Madhur Sharma Ch. Perception of fever and management practices by parents of pediatric patients. International Journal of Research in Medical Sciences2013; 1(4):397400.

8. Crocetti M, Moghbeli N, Serwint J. Fever phobia revisited:have parental misconceptions about fever changed in 20 years?. Pediatrics 2001; 107(6):1241–46.

20. Gasparini R, Marchisio P, Crovari P: Burden of influenza in healthy children and their households. Arch Dis Child 2004; 89(11):1002–7.

9. Chang LC, Liu CC, Huang MC. Parental knowledge, concerns, and management of childhood fever in Taiwan. J Nurs Res 2013; 21: 252e60.

21. Cohee LM, Crocetti MT, Serwint JR, Sabath B, Kapoor S. Ethnic differences in parental perceptions and management of childhood fever. Clin Pediatr 2011, 49:221–27.

10. Purssell E. Parental fever phobia and its evolutionary correlates.J Clin Nurs 2009; 18: 210e8. 11. Uday C. Rajput1, Srikanth Kulkarni, Sambhaji S. Wagh..Parental Knowledge, Attitude and Practices Regarding Fever in Their Children: A Hospital Based Observational Study. International Journal of Recent Trends in Science and Technology.2014; 10(3): 517-20.

22. Lagerlov P, Helseth S, Holager T. Childhood illnesses and the use of paracetamol (acetaminophen): a qualitative study of parents' management of common childhood illnesses. Family Practice 2003; 20(6), 717-23. 23. Hsieh H,Shannon S.Three approches to qualitative content analysis .Qualitative Health Research Journal 2005;15(9):1277-88.

12. Plaisance K, Mackowiak F. Antipyretic therapy. Archives of InternalMedicine, 2000; 160: 449–56.

24. Graneheim UH, Lundman B.Qualitative content analysis in nursing research:concepts,procedures and measures to achieve trustworthiness. Nurse Educ Today 2004; 24(2):105-112.

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35. Oshikoya KA, Senbanjo IO. Fever in Children: Mothers’ Perceptions and their Home Management. Iran J Pediatr Sep 2008; 18(3):229-36.

27. Gunduz S, Usak E, Koksal T, Canbal M. Why Fever Phobia Is Still Common? Iran Red Crescent Med J 2016; 18(8):e23827. 28. Youssef A, Abdullah M, Suleiman A, Mohammed A,Sameeh S, Amal H, et al. Parental perception of fever in children. Ann Saudi Med 2000; 20(3-4):202-5.

36. Rkain M. Rkain L, Safi M, Kabiri M, Ahid S , Benjelloun BDS. Knowledge and management of fever among Moroccan parents. Eastern Mediterranean Health Journal 2014; 20(6): 396-402.

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37. Pereira GL, Dagostini JM, Pizzol Tda S. Alternating antipyretics in the treatment of fever in children: a systematic review of randomized clinical trials. J Pediatr 2012; 88(4):289-96.

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https://onlinelibrary.wiley.com/doi/10.1111/j.1442-200X.1997.tb03584.x Reference 18

Evaluation of sponging and antipyretic medication to reduce body tempe...

Pediatrics International / Volume 39, Issue 2 / p. 215-217

Evaluation of sponging and antipyretic medication to reduce body temperature in febrile children SERAP AKSOYLAR, SADIK AKŞİT MD, SUAT ÇAǦLAYAN, IŞIN YAPRAK, RAHMI BAKILER, FUNDA CETIN First published: 19 January 2011 https://doi.org/10.1111/j.1442-200X.1997.tb03584.x Citations: 25

Abstract Two hundred and twenty-four children aged 6 months to 5 years, with rectal temperatures greater than or equal to 39°C (104°F), were randomly treated with sponging alone or with medication including a single oral dose of aspirin 15 mg/kg, or paracetamol 15 mg/kg, or ibuprofen 8 mg/kg. Twenty-three children were excluded from the �nal analysis because they did not complete the study. Demographic characteristics of the patients were found to be comparable in all groups. Rectal temperatures were recorded every 30 min for a 3 h period. During the �rst 30 min of intervention, sponging was found to be more e�ective than all of the three medications. After 60 min, the e�ects of each medication became superior to sponging with tepid water in reducing body temperature. Twenty-three children were excluded from the �nal analysis because they did not complete the study. Comparing the e�ect of the three di�erent medications, it was seen that the antipyretic e�cacy of aspirin and ibuprofen were signi�cantly more than paracetamol 3 h after intervention (P < 0.05). For the management of fever over 39°C, it is therefore recommended to give children an antipyretic drug, preferably ibuprofen, and at the same time to begin sponging to provide a rapid and sustained antipyresis.

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