AABS 2.3 (2015)

ISSN: 2349-6991

AABS Annals of Applied Bio-Sciences 2015: Vol. 2; Issue 3

Contents Sr. No. 1.

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Title of The Article

Type of The Article

Authors

A Review on Major Bioactivities of Bacopamonnieri.

Review Article

Gayatridevi Pushkar, Bhupendra Pushkar, Rohini Sivabalan1

Aqueous Phytal extracts as source for staining in gel based protein separation techniques

Original Article

Isolation and characterization of potential microbe for bio-remediating heavy metal from Mithi river

Original Article

Effect of temperature variation and determination of optimum temperature of alpha amylase activity in Telescopium telescopium.

Original Article

Histopathological lesions of nasal cavity, paranasal sinuses and nasopharynx

Clinical and haematological characteristics of autoimmune haemolytic anemia: retrospective analysis of 10 cases Anaplastic Thyroid carcinoma: A Rare Case

Persistent m端llerian duct syndrome: a case report

Original Article Original Article

Case Report Case Report

Bhupendra K. Pushkar, Pooja I. Sevak, Anamika Singh

Page No. R1-R11 A20-A27

JayaPrada Rao Chunduri, Harsha Mota

A28-A35

Janice Jaison, Deepa Topandas Tekwani

A40-A46

Sharvari Kudtarkar, Prakash V. Desai

Maryann Margaret Bukelo, Srikanth Umakanthan, Sharada Rai1

Mangala R Nagare, Joshi Sneha R, Ashwini Karwande, Smita Pathak, Tekwani Deepa T, Janice Jaison Chandrahas R. Godbole, Sneha Joshi, Janice Jaison

A37-A39

A47-A50 C6-C9 C10-C13

Isolated cysticercus cellulosae of medial rectus muscle presenting with a mass over the inner region of the eye: a rare case report and treatment review

Case Report

Jagriti Rana1, S. P. Singh, Vijay Kumar Jha, S. Khanduja

C14-C17

Case Report

C18-C21

Hemoglobin H Disease: A rare case report and its diagnostic challenge

Case Report

Rajeshwari K, N.V. Dravid1, Karibasappa GN, Akshay Surana

Acute Eosinophilic Appendicitis: Case report of three cases with brief review of literature

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Editorial Team Editor-in-Chief Dr Shelly Sehgal, Consultant Pathologist, Delhi, India Dr Dipti Agrawal, Delhi, India

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Dr Bhupendra Pushkar, Asst. Professor, Dept. of Biotechnology, University of Mumbai, India

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Dr Chandrassegar Saravanan, Novartis Institutes for Biomedical Research, Technology Square, Cambridge, MA Dr Nandhakumar Balakrishnan, College of Veterinary Medicine North Carolina State University, Raleigh

Prof. M H Fulekar, Professor & Dean, School of Environment and Sustainable Development, Central University Gujarat,, India Dr Igor Iuco Castro-Silva, Professor, Clinica Odontologica, Faculdade de Ciencias do Tocantins, Brazil Kapil Agarwal, Engineer, Nagoya City, Aichi, Japan

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Dr D A Bhiwgade, Dept. of Biotechnology & Bioinformatics, Padmashree Dr. D. Y. Patil University, Navi Mumbai,, India Dr Radhika P Kamdar, Emory University School of Medicine, Georgia,, United States Dr Saba Hasan, Asst. Prof. Amity University, Lucknow, India

Dr Manav Kapoor, Assistant Professor Neuroscience, Icahn School of Medicine, Mount Sinai, New York, NY, 10029

Managing Editor

Dr Prashant Goyal, Director-Laboratory & Quality Manager, Delhi, India

Review Article

A Review on Major Bioactivities of Bacopamonnieri.

Gayatridevi Pushkar*1, Bhupendra Pushkar2, Rohini Sivabalan1 1 2

Department of Zoology, Ramnarain Ruia College, Matunga, Mumbai. India Department of Biotechnology, University of Mumbai, Kalina campus, Santacruz (east),Mumbai, India.

Keywords: #########

Abstract

Bacopa monnieri (family: Scrophulariaceae) is a reputed drug of Ayurveda. It is used in traditional medicine to treat various nervous disorders and for promoting memory and intellect. This medicinal plant is locally known as Brahmi. It is known as a memory enhancer and many preparations of brahmi are now commercially available in the market. Herbal medicines are gaining importance hence B. monnieri has been studied extensively for its chemical constituents, constituents responsible for memory enhancing effect and also its various other useful effects. As now a days natural products are much preferred so the possible use of brahmi as a neuropsycotropic drug is also gaining importance. Some of its effects has been established in several in vivo and in vitro models. This article reviews the useful effects of this plant.

*Corresponding author: Gayatridevi Pushkar, Department of Zoology, Ramnarain Ruia College, Matunga, Mumbai-400 019, INDIA. Email: pushkargayatri16@gmail.com.

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Review article

Introduction The interest in the use of herbal medicines are gaining importance all around the world. The World Health Organization (WHO) estimates that 80% of the world‟s population presently uses herbal medicine for some aspects of primary health care [1].Similarly the use of natural productfor neuropsycotropic drugs are alsogaining importance.

In this contextBacopamonnieri can be useful herbal drug option for neuropsycotropic disorders.Since it is extensively used as a nerve tonic and thought to improve memory in ayurvedic medicines[2].Though Brahmi is now promoted as the „brain booster‟ of the new millennium, Ayurvedic medicine has known this for centuries. It is highly valued in conditions affecting the nervous system and brain. This review summarizes our current knowledge of the major bioactivities of Bacopamonnieri.

Description of plant Bacopamonnieri(BM) or Brahmi, a plant in the family Scrophulariaceae, has been used in the Ayurvedic system of medicine for centuries. It has been claimed as a nerve tonic and extensively used for treatment of various neurological and neuropsychiatric diseases[2].BM locally known as brahmi in India, has been used for centuries in the Ayurveda.

The herb has been mentioned in several ancient Ayurvedic treatises including the 'CharakaSamhita' since sixth century AD, in which it is recommended in formulations for the management of a range of mental conditions including anxiety, poor cognition and lack of concentration, as a diuretic and as an energizer for the nervous system and the heart[3][2]. Specific uses include the treatment of asthma, insanity and epilepsy[4][2]. The plant has been utilized extensively as a nootropic, digestive aid and to improve learning, memory and respiratory function[5],[6]. The herb is a small creeping herb with numerous branches, small oblong leaves and light purple or small and white flowers, with four or five petals . It is found in wetlands throughout the Indian subcontinent in damp and marshy or sandy areas near streams in tropical regions. The genus Bacopa includes over 100 species of aquatic herbs distributed throughout the warmer regions of the world [7]. The entire plant is used medicinally[8].

Chemical composition The pharmacological effects of Bacopamonnieri are attributed to the presence of a number of biologically active compounds, including alkaloids, saponins and sterols[9]. The compounds responsible for the memory enhancing effects of Bacopamonnieri are triterpenoidsaponins called "bacosides" [12]. The major chemical entity shown to be responsible for neuropharmacological effect of BM is bacoside A, assigned as 3-(a-L-arabinopyranosyl)-O-b-D-glucopyranoside-10, 20--dihydroxy-16-keto-dammar-24-ene[12].Bacoside A usually co-occurs with bacoside B; the latter differing only in optical rotation[13].The isolation of D-mannitol and a saponin, hersaponin and potassium salts was also reported [2][11]. On acid hydrolysis, bacosides yield a mixture of aglycones, bacogenin A1, A2, A3, [14],[15],[16] which are artefacts, and two genuine sapogenins, jujubogenin andpseudojujubogenin and bacogenin, A4, identified as ebelin lactone pseudojujubogenin, were isolated[17].Successively, a minor saponinbacoside A1 and a new triperpenoidsaponin, bacoside A3, were isolated[2][17].Later, three new dammarane-type triterpenoidsaponins of biological interest, bacopasaponins A, B and C, isolated and identified as 3-O-α-L-arabinopyranosyl- 20-O-α-Larabinopyrasonyl-jujubogenin, 3-O-[α-Larabinofuranosyl-(1→2)- α-L-arabinopyranosyl] pseudojujubogenin and 3-O-β- D- glucopyranosyl (1→3)-{α-L-arabinofuranosyl-(1→2)}-α-L arabinopyrasonyl] pseudojujubogenin and also a new dammarane-type pseudojujubogenin glycoside, bacopasaponin D, 3-O-[α-L-arabinofuranosyl- (1→2)-β-D- glucopiranosyl] pseudojujubogenin were identified by spectroscopic and chemical transformation methods[9][18].

In view of the increasing interest in this herbal plant, yet two new pseudojujubogenin glycosides designated as bacopaside I and II were isolated from glycosidic fraction of the methanol[19]. Subsequently, three new saponins from BM, designated as bacopasides III, IV and V with structures 3-O α-L- arabinofuranosyl-(1→2)-β-D-glucopyranosyljujubogenin, 3- O-β-D-glucopyranosyl- (1→3)-α-L- arabinopyranosyljujubogenin, 3-O-β-D-glucopyranosyl-(1→3)- α-Larabinofuranosyl pseudojujubogenin were isolated[20]. In addition, the isolation of three new phenylethnoid glycosides, viz. monnierasides I-III along with the known analogue plantainoside B was reported from the glycosidic fraction of BM[21][28]. Moreover, an isolation of a new saponin, a juju-

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R-3 AABS. 2(2) 2015 bogenin, named bacopasaponin G, and a new glycoside, phenylethyl alcohol was also reported [22][28] . The drug is characteristically designated on the basis of its total bacosides content which are tetra cyclic triterpenoidsaponins.

These steroidal saponins called Bacoside A &Bacoside B are considered Bacopaâ€&#x;s most therapeutic constituents.The chemical structures of saponinsisolated from BM are shown In [Figure 1a], bacoside A levorotatory, and [Figure 1b], bacoside B dextrorotatory, [Figure 2]triterpinoidsaponin and bacogenin A4.

Fig 1a: BACOSIDE A (Levorotatory)

Fig 1b: BACOSIDE B (Dextrorotatory)

Fig 2: TRITERPENOID SAPONIN and BACOGENIN A4

The possible mode of action of brahmi on brain The BM extracts and isolated bacosides have been extensively investigated for their neuropharmacological effects. The triterpenoidsaponins and their bacosides are said to be responsible for BM's ability to enhance nerve impulse transmission [28]. It was suggested that bacosides induce membrane dephosphorylation, with a concomitant increase in protein and RNA turnover in specific brain areas[24]. The other proposal that was put forward was that BM enhances protein kinase activity in the hippocampus which may also contribute to its nootropic action and thus it would aids in repair of damaged neurons by enhancing kinase activity, neuronal synthesis and restoration Annals of Applied Bio-Sciences, Vol. 2; Issue 2: 2014

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R -4 of synaptic activity and ultimately nerve impulse transmission[25].It is clear that brahmi exhibit useful effects on brain and required further research so that its mode of action can be identified. However some of these useful effect which have been studiedare discussed below.

Review article

Antidepressant and Antianxiety Effects Currently available treatment of depression is often associated with several undesirable side effects and it is effective only in a certain portion of the patients [27]. Hence the search for novel pharmacotherapy from medicinal plants for psychiatric illnesses has progressed significantly. From ancient timebrahmi have been used to treat psychiatric disorders and can be potential candidate for neuropsycotropic drug.According to one study, the BM extract in the dose range of 20-40 mg/kg was found comparable to standard anti-depressant drug imipramine in anti-depressant activity in rodent animals[29] The same study has postulated the role of serotonin and GABA (gamma amino butyric acid) in the mechanism of action attributed for its antidepressant action along with its anxiolytic potential , based on the compelling evidence that the symptoms of anxiety and depression overlap each other[29].

The traditional use of BM as an anti-anxiety remedy in Ayurvedic medicine is supported by both animal and clinical research[28].One of the study with 35 patients with diagnosed anxiety neurosis demonstrated that administration of brahmi syrup (30 mL daily in two divided doses, equivalent to 12 g dry crude extract of bacopa) resulted in a significant decrease in anxiety symptoms, level of anxiety, level of disability and mental fatigue and an increase in immediate memory span[30].In another study, effects of a standardized BM (300 mg/day) on cognitive performance, anxiety and depression in the elderly was evaluated[31]. The study provided further evidence that BM has a good potential for safely enhancing cognitive performance in the ageing. [31] Anti-Epileptic Effects The use of Bacopamonnieri for treatment of epilepsy has been studied ,since it exhibit various effects on brain.

According to one of the study on the neuroprotective role of B. monnieri extract in alteration of glutamate receptor binding and gene expression of (N-methyl-D-aspartate) NMDA R1 in hippocampus of temporal lobe of epileptic rats along with pilocarpine-induced epilepsy, there was significant down regulation of NMDA R1 gene expression and glutamate receptor binding without any change in its affinity. B. monnieri treatment of epileptic rats significantly reversed the expression of NMDA R1 and glutamate receptor binding alterations to near-control levels.

This study also showed a significant increase in the activity of glutamate dehydrogenase, which neared the control level after B.monnieri treatment [32]. This indicate the neuroprotective role of B. monnieri extract in glutamate-mediated excitotoxicity during seizures and cognitive damage occurring in association with pilocarpine-induced epilepsy[32] One of the research demonstrated that hersaponin (an active constituent) exhibited protection against seizures in mice and mentioned the possibility of its use as an adjuvant in treatment of epilepsy[33].

One of the study examined the anticonvulsant properties of BM extracts in mice and rats[34].When administered acutely at lower doses anticonvulsant activity was not observed. But intraperitoneal injections of high doses of BM extract given for 15 days demonsterated anticonvulsant activity[34]. It is postulated that the anti-convulsive effects could be mediated through GABA which is involved in neural impulse transmission, because substances which stimulate GABA are known to possess anticonvulsant, pain relieving and sedative activities [34].The glutamate gene expression during epilepsy in adult and hypoxic insult to brain during the neonatal period and the therapeutic role of neuroprotective supplement were studied and also the role of metabotropic glutamate-8 receptor (mGluR8) gene expression in cerebellum during epilepsy and neuroprotective role of Bacopamonnieri extract in epilepsy were evaluated[35].During epilepsy a significant down-regulation of mGluR8 gene expression was observed which was up-regulated near control level after B. monnieri treatment. This showed that B. monnieri treatment to epileptic rats significantly brought the reversal of the down-regulated mgluR8 gene expression toward control level[35].

Antioxidant and Adaptogenic Properties As stress is linked to many diseases, research on an effective antistress agent (adaptogen) from plants has gained importance. In one of the study the adaptogenic property of a standardized extract of Bacopamonnieri against acute stress (AS) and chronic stress (CS) models in rats was studied[36].The Pretreatment with B. monnieri at 40 mg/kg significantly reduced the AS-induced increase in the ulcer , adrenal gland weight, plasmaglucose,( asparhttp://www.pacificejournals.com/aabs

R-5 AABS. 2(2) 2015 tate aminotransferase) AST, and(creatine kinase) CK. A dose of 80 mg/kg significantly reversed the AS-induced changes in adrenal gland weight, spleen weight, plasma glucose, alanine aminotransferase (ALT), and AST[36].

BM extract or bacosides have shown an antioxidant activity [37],[38],[39],[40],[41],[42] and antistress[43]. A previous study suggests an involvement of the GABA-ergic system in the mediation of these effects of BM[44].Based on animal study results, bacosides were shown to have antioxidant activity in the hippocampus, frontal cortex and striatum[45].One of the study has shown that the BM extracts modulate the expression of certain enzymes involved in generation and scavenging of reactive oxygen species in the brain[46].

In one of the study on rats BM showed the potential to be effective in stress and can be beneficial in the management of stress related conditions[47].BM was found to induce the constitute expression of heat-shock protein (HSP 70), and also induced the(cytochrome P450) CYP 450 enzymes in all regions of brain[47].The level of Hsp70 was found to be increased in brain as a response to stress. An increase in the activity of CYP 450-dependent enzymes 7-pentoxyresorufin-odealkkylase (PROD) and 7-ethoxyresorufin-o-deethylase (EROD) was observed in all the brain regions after exposure to stress alone and with both doses of BM although the magnitude of induction observed was less with a higher dose of the same[47].Thus, it was suggested that the BM primed the brain for stress by stockpiling these useful enzymes even before stressful conditions and that our susceptibility to stress could be lowered by using this medicinal herb[47]. It was sugessted that this induction may be an adaptive response to the stress which needs further investigation. The level of super oxide dismutase (SOD) was also increased when pre-treated with BM. This data indicates that BM has a potential to modulate the activities of HSP 70, CYP 450 and SOD and thereby possibly allowing the brain to be prepared to act under adverse condition like stress[47].

One of the study concluded that BM helps in coping with combined hypoxic, hypothermic and immobilization stress that could lead to onslaught of 'free radicals'[48].The results also indicated that this extract exhibits interesting antioxidant properties, expressed by its capacity to scavenge superoxide anion and hydroxyl radical, and to reduce H 2 O 2 -induced cytotoxicity and DNA damage in human fibroblast cells[41],[49],[50]. BM extract has also shown neuroprotective effect against aluminium-induced oxidative stress in the hippocampus of rat brain[51].According to one study aqueous extract of BM reduced nicotine-induced lipid peroxidation (LPO) and conferred geno protection in Swiss mice[52].One of the recent study has shown the protective role of bacoside A against chronic cigarette smoking-induced oxidative damage in rat brain[54]. This antioxidant activity of BM is able to explain, at least in part, the reported antistress, cognition-facilitating and antiageing effects of BM[55]. and may justify further investigation of its other beneficial biological properties and the potential antistress agent.

Alzheimer’s disease Since it has been claimed that B. monnieri is efficacious in the treatment of memory loss, it can bepotentialiy beneficial in the treatment of Alzheimer‟s[26]. According to one study involving short- and long-term treatment with B. monnieri extract showed reduction in brain Aβ (amyloid beta) levels in the cortex and reversed the behavioural deficits in mice[53]. The effect of subchronic (14 days) administration of a standardized extract of Bacopamonnieri (bacoside A content 82%) was evaluated on two animal models of Alzheimer's disease induced by intracerebroventricular administration of colchicines and by lesioning of the nucleus basalismagnocellularis (nbm) with ibotenicacid[69]. Subchronic administration of Bacopamonnieri at a dose of 10mg/kg significantly reduced the magnitude of memory deficits induced by both compounds as observed on days 7 and 14, while the effects were evident with the lower dose only on day 14 [69]. In the same study, BM was also shown to reverse the depletion of acetylcholine, the reduction in choline acetylase activity and a decrease in muscarinic cholinergic receptor binding in the frontal cortex and hippocampus[69]. The study indicates the potential of B. monnieri as a therapeutic agent for treatment of alzheimer‟s disease further research in this area.

Gastrointestinal Effects Even while Brahmi is widely used to improve intellectual functions, a study was done on the prophylactic and curative effects of standardized extract of Brahmi in various gastric ulcer models [56]. And the study also included evaluation of standardized Brahmi extract on other contributing factors towards ulcerogenesis. And it was found that the effect was due to augmentation of the defensive mucosal factors like increase in mucin secretion, life span of mucosal cells and gastric antioxidant effect rather than on the offensive acid-pepsin secretion[56]. Animal and in vitro studies suggested that BM may have a protective and curative effect on gastric ulcers, and studies were reported for its antiulcerogenic activity[57],[58],[59],[60],[61]. A recent in vitro study also demonstrated its specific anAnnals of Applied Bio-Sciences, Vol. 2; Issue 2: 2014

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R -6 ti-microbial activity against Helicobacter pylori, a bacterium associated with chronic gastric ulcers. When the extract was incubated with human colonic muscosal cells and H. pylori, it resulted in the accumulation of prostaglandin E and prostacycline, prostaglandins known to be protective for gastric mucosa[62]. Further study is required to find the exact mechanism of this effect and to correlate its use in gastric related diseases.

Review article

Sedative and Tranquillizing Properties Bacopamonnieri as a sedative and tranquillizing properties was also studied[28]. According to one study sedative effect was due to glycosides named hersaponins [63]. And subsequent studies has found that the alcoholic extract, and to a lesser extent the aqueous extract of the whole plant exhibited tranquilizing effects on albino rats and dogs[64]. It has also been found that the alcoholic extract of the plant and chlorpromazine improved the performance of rats in motor learning[65]. Another study has reported that a single dose of the glycoside hersaponin is better than pentobarbitone in facilitating acquisition[66].

Cognition Cognition is the mental processing that includes the attention of working memory, comprehending and producing language, calculating, reasoning, problem solving, and decision making. It is the process by which the sensory input is transformed, reduced, elaborated, stored, recovered, and used. In general cognition refers to an information processing view of an individual[67]. B. monnieri demonstrated enhanced attention and cognitive processing capability together with enhanced working memory[68]. According to one studystandardized bacosides-rich extract of BM, reversed the cognitive deficits induced by intracerebroventricularly administered colchicines and injection of ibotenic acid into the nucleus Basalismagnocellularis [69]. Also BM was shown to reverse the depletion of acetylcholine, the reduction in choline acetylase activity and a decrease in muscarinic cholinergic receptor binding in the frontal cortex and hippocampus[69]. The cognition facilitating activity of the BM extract is attributed to the saponins, Bacoside A and Bacoside B, which are effective in much lower doses[70],[71].

Numerous clinical studies have been carried out to date to establish the efficacy of BM in memory and attention disorders and to study its acute and chronic effects clinically on cognitive function[28].One of the study was done on Seventy-six adult volunteers aged between 40 and 65 years. The results showed a significant effect of BM on the test for the retention of new information. In the follow-up tests, it was found that the BM decreases the rate of forgetting of newly acquired information. In adults, only chronic administration was shown to enhance cognitive effects[73]. In another study on 38 healthy volunteers (ages 18-60), subjects were given a single dose of 300 mgBM[73][74]. These results were attributed to BM's antioxidant properties and/or its effect on the cholinergic system[75] [76].

In one of the study conducted on 36 children diagnosed with attention deficit/hyperactivity[77].A significant benefit was observed in BM-treated subjects as evidenced by improvement on sentence repetition, logical memory and paired associated learning tasks[77]. The efficacy of standardized BM in subjects with age-associated memory impairment (AAMI) without any evidence of dementia or psychiatric disorder was evaluated in one of the study. BM produced significant improvement in mental control, logical memory and paired associated learning during the 12-week drug therapy.BM was found to be efficacious in subjects with age-associated memory impairment[78].

Side Effects and Toxicity BM has a record of several hundred years of safe therapeutic use in Ayurvedic medicine. In one of the study the aqueous extract given orally at a dose of 5 g/kg and the alcoholic extract given orally at a dose of 17 g/kg and both extracts did not produce any gross behavioural changes at these levels [28]. According to one study concentrated bacosides given in single (20-30 mg) and multiple (100-200 mg) daily doses were well tolerated and without adverse effects[79].

Other useful effect Although BM is usually more known for its effect on brain and memory, it exhibit various other effects also. According to one study BM has shown protective effect against DNA damage in astrocytes [81] and human fibroblasts[82]. In another study anti-cancer effect of BMwas studied. And suggested that the anticancer effect of BM extracts is possibly due to inhibition of DNA replication in cancer cell lines[83]. Effect of brahmi on thyroid hormones was also studied. And it was found that high doses (200 mg/kg) of BM increased the thyroid hormone, T4, http://www.pacificejournals.com/aabs

R-7 AABS. 2(2) 2015 by 41% when given orally. T3 was not stimulated, suggesting that the extract may directly stimulate synthesis and/or release of T4 at the glandular level, while not affecting conversion of T4 to T3. This study indicated that BM extract did have a stimulatory effect on thyroid function, but on very high doses[84].

BM was also reported to possess anti-inflammatory activity via inhibition of prostaglandin synthesis and lysosomal membrane stabilization[85],[86]. In vitro study using rabbit aorta and pulmonary artery has demonstrated that BM exerts a vasodilatory effect on calcium chloride-induced contraction in both tissues. It is believed to exert this effect via interference with calcium channel flux in tissue cells[87]. Animal studies have demonstrated that BM have a relaxant effect on chemically-induced bronchoconstriction, probably via inhibition of calcium influx into cell membranes[28]. A subsequent study on rats in which methanol sub-fractions of BM were given to anesthetized rats prior to induction of bronchoconstriction with carbachol, an acetylcholine analogue. Nearly all of the BMsubfractions inhibited carbachol-induced bronchoconstriction, hypotension and bradycardia in this animal model[88].

An invitro study also demonstrated that a methanol extract of BM possessed potent mast cell stabilizing activity comparable to disodium cromoglycate, a commonly used allergy medication[90]. These studies indicated the potential usefulness of BM extracts in bronchoconstrictive and allergic conditions[28]. In vitro and animal studies have demonstrated that the BM might potentiate the effect when taken with some synthetic drugs or it might have a protective effect against certain drugs and their negative side effects [28]. BM has been noted in animal models to decrease the toxicity of morphine and phenytoin (PHT). Administration of BM with morphine significantly decreased lipid peroxidation (LPO) and increased levels of antioxidant enzymes and glutathione in rat hepatic tissue, when compared to morphine alone. These results suggested a protective effect for BM on the hepatic antioxidant status in morphine-treated rats[91].

In mice, BM administration with phenytoin (PHT) significantly reversed phenytoin-induced cognitive impairment, as noted by improved acquisition and retention of memory[92]. Both acquisition and retention of memory were improved without affecting the anti-convulsant activity of PHT. These results suggested a potential corrective effect of BM in phenytoin-induced cognitive deficit[93]. An animal study found that the effects of chlorpromazine, a drug similar to (perphenazine, prochlorperazine, thioridazine), were enhanced when a BM was given along with it[94],[95]. More study is required in this area.

Conclusion Bacopamonnierihas been used in traditional Ayurvedic medicine for various indications including memory decline, inflammation, pain, pyrexia, epilepsy and as a sedative [7]. Accumulative lines of evidence have demonstrated that Bacopamonnierihas the wide variety of neuropharmacological actions. Further research are required to ascertain the findings mentioned in this review. The activity of BM both as an anxiolytic and anti-depressant needs further evaluation, its potential as an anti-epileptic treatment and as a treatment to correct side effects of anti-epileptic drugs is another area to be studied in future. The antioxidant activity of brahmi may be useful in the treatment of human pathologies in which free radical production plays a key role which requires further study.

Futher studies are required to establish the effect of Bacopamonnieri on thyroid hormones, astrocytes cells, bronco-constriction and also the possible mechanism by which it exibit this effects. Which opens up interesting avenues for further research and offers new perspectives in the treatment of these diseases. Hence Bacopamonnieri has a promising future prospect.

Abbrevations: BM :Bacopamonnieria.

GABA : gamma amino butyric acid.

NMDA:N-methyl-D-aspartate . mGluR8 :metabotropic glutamate-8 receptor.

AST : aspartate aminotransferase.

AS : acute stress.

CS : chronic stress.

CK :creatine kinase.

ALT : alanine aminotransferase.

HSP : heat-shock protein.

CYP450 :cytochrome P450.

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Review article

PROD :pentoxyresorufin-odealkkylase .

EROD :ethoxyresorufin-o-deethylase.

SOD : super oxide dismutase.

LPO :lipid peroxidation.

Aβ : amyloid beta.

AAMI :age-associated memory impairment.

PHT : phenytoin.

Acknowledgements None.

Funding None.

Competing Interests None declared.

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67. Bhattacharya SK, Kumar A, Ghosal S. Effect of Bacopamonniera on animal models of Alzheimer's disease and perturbed central cholinergic markers of cognition in rats. In: DV Siva Sankar, editors. Molecular Aspects of Asian Medicines. New York: PJD Publications; 1999. p. 27-58.

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70. Sairam K, Rao CV, Babu MD, Goel RK. Prophylactic and curative effects of Bacopamonniera in gastric ulcer models.Phytomed 2001;8:423-30.

71. Roodenrys A, Booth D, Bulzomi A, Phipps A, Micallef C, Smoker J. Chronic effects of Brahmi (Bacopamonnieri) on human memory. Neuropsychopharmacol 2002;27:279-81.

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(Brahmi).Ind J Pharmacol 1997;29:S359-S65.

78. Martis G, Rao A, Karanth KS. Neuropharmacological activity of Herpestismonniera. Fitoterapia 1992;63:399-404.

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84. Jain P, Khanna NK, Trehan T, Pendse VK, Godhwani JL. Anti-inflammatory effects of an Ayurvedic preparation, BrahmiRasayan, in rodents.Ind J ExpBiol 1994;32:633-6.

85. Dar A, Channa S. Calcium antagonistic activity of Bacopamonniera on vascular and intestinal smooth muscles of rabbit and guinea-pig. J Ethnopharmacol 1999;66:167-74.

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Original Article

Isolation and characterization of potential microbe for bio-remediating heavy metal from Mithi river Bhupendra K. Pushkar*, Pooja I. Sevak, Anamika Singh Department of Biotechnology, University of Mumbai, Kalina, Santacruz (E), Mumbai, Maharashtra, India Keywords: Isolation, Characterization, Bioremediation, Heavy Metal, Pollutants.

Abstract

Background: Heavy metals contamination is common problem in the current time in river water and other natural resources. Mithi River receives the different kind of industrial and domestic wastes, which also includes toxic heavy metal pollutants. Bioremediation of the heavy metal pollutants can be a promising option for their removal.

Methods: Isolation and characterization of the heavy metal resistant microorganisms obtained from the Mithi River was performed using basic biochemical test and confirmed by 16S rDNA technique. SEM analysis, MIC and flocculation index were carried out to detect the efficiency of bacteria for bioremediation.

Results: Significant populations of heavy metal resistant microorganisms were found in Mithi River. Microorganisms of Mithi River studied in the current research were gram negative as well as gram positive.

Conclusion: Microorganisms having the potential of bioremediation of heavy metals were isolated successfully from Mithi River and their identity was confirmed. The potential of the microorganism for their capacity of bioremediation of heavy metals was also confirmed.

*Corresponding author: Dr Bhupendra K. Pushkar, Department of Biotechnology, University of Mumbai, Kalina, Santacruz (E), Mumbai, 400098, Maharashtra, India, Email: bhupendrapushkar@gmail.com , phone +919821644722,

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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1. Introduction Environmental contamination owing to the anthropogenic activities and the natural resources is increasing progressively on account of an unabated increment in population, industrialization and urbanization (1). Heavy metal(s) are widespread pollutants of great concern as they are non-degradable and thus persistent (2). Extensive industrialization and improper disposal are attributed to be a prime factor responsible for the release of heavy metals into the ecosystems (3). “Heavy metal� is a general collective term, which applies to the group of metals and metalloids with atomic density greater than 4000 kg m-3, or 5 times more than water (4, 5). The different sources of heavy metals like Pb, Cd, Hg, Cr, As, Cu and Ni are lead acid batteries, paints, E-waste, Smelting operations, coal- based thermal power plants, ceramics, bangle industry, chlorine-alkali plants, electroplating and thermal power plants etc. (6).

Some of these metals are useful to the body in low concentration like arsenic, copper, iron, nickel, etc. but are toxic at high concentration (7). Heavy metals contamination impose various health problems like headache, irritability, abdominal pain and various symptoms related to the nervous system, anxiety, bladder and kidney cancer (8) either by displacing the vital nutritional minerals from their original place, thereby, hindering their biological function or accumulating, thereby disrupt function in vital organs and glands such as the heart, brain, kidneys, bone, liver, etc. (9).

Conventionally, heavy metals remediation can be classified under three categories. Chemical, Physico-Chemical and Biological/Biochemical/Biosorptive Technologies (5).Bioremediation is superior to other remediation processes as these processes generate additional pollution, expensive and often do not permanently alleviate the pollution hazard (10, 11, 12).

Bioremediation is a collective term used to describe the use of biological systems such as microorganisms to decontaminate polluted soil, water or air. Bioremediation is the process of breaking down or transforming hazardous materials into simple nontoxic substances by biological treatments (13). Application of microorganisms for heavy metals remediation is considered as a natural, stable and economical solution (14).

Generally, the higher concentration of these metals above threshold levels has deleterious impact on the functional activities of microbial communities. Microorganisms inhabiting in metal polluted areas have evolved various mechanisms to resist themselves against metal stress when exposed to higher concentration of toxic waste (15). These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell, and reduction of the heavy metal ions to a less toxic state (16) Various microorganisms such as Pseudomonas Spp., Closteridium Spp. Bacillus Spp. Escherichia Spp. etc. have been reported as efficient candidates for bioremediation of heavy metals ( 17, 18 ).Microorganisms and microbial products can be highly efficient bioaccumulators of soluble and particulate forms of metal especially dilute external solutions (19).

Mithi River home of diverse micro flora originates at Powai and meets Arabian Sea at Mahim Creek flowing through residential and industrial complexes of Powai, Saki Naka, Kurla and Mahim over a distance of about 15 km (20). The river receives raw sewage, industrial waste and garbage of all types.

There are many illegal industrial activities along the bank of the river (21). Themicroflora of Mithi River can be used for bioremediation purpose of heavy metals as the contamination level of the river make the flora of the river resistance to the heavy metals.

The purpose of present study is to isolate and characterize the microbes from Mithi River which can be further used as a potential tool for bioremediation and removal to toxic metals from the heavy metal polluted areas.

2. Materials and Methods

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Original Article

2.1 Microbial load of the Mithi River- Microbial load of water is also a very important factor in the quality of water. The number of bacteria present in the Mithi River is determined by diluting the water sample (Mithi River) till 10-5 and plating dilutions on the nutrient agar containing 0.5% peptic digest, 0.5% sodium chloride, 0.15% beef extract, 0.15% yeast extract and 1.5% agar. The total CFU/ml was calculated by using the number of colonies observed on nutrient agar plates.

2.2 Isolation and purification of heavy metals resistant microorganisms from Mithi riverBacteria resistant against lead and mercury were isolated from water sample collected from Mithi River at different sampling points (Kalanagar, BKC area).Water sample was plated nutrient agar medium containing 10 ppm ofPb and Hg as salt of Pb(NO2)2 and HgCl2 (Pb and Hg added to separate plates). After incubation at 37°C for overnight, isolated colonies were re-streaked again and again till single type of cells are observed in gram staining. Glycerol stock of purified culture were prepared in triplicate and stored at -20°C for further study.

2.3 Characterization and Identification- Isolated bacterial cultures were characterized by biochemical analysis. Bacterial cultures were analysed by indol, methyle red, Voges–Proskauer, citrate utilization, triple sugar iron, catalase, motility, gelatinase test and sugars like glucose, lactose, sucrose, maltose, mannitol and xylose.

2.4 Molecular identification by 16S rRNA PCR-The genomic DNA were extracted from cells by heating the single colony in 100 µl of sterile distilled water for 10 minutes in boiling water bath. The supernatant was collected and used as template DNA. 27F 5’ AGA GTT TAG TCCTGG CTC AG 3’ and 1492R 5’ GGTTAC CTTGTTACGACT T 3’ (Merck Millipore, India) were used as forward and reverse primers. Reaction mixture of 40 µl was prepared by 2x of master mix (Genei), nuclease free water, and 20pmol/µl of forward and reverse primer. PCR amplification was carried out on thermocycler (Applied biosystems). PCR cycle was as follow initial denaturation was at 94˚C for 7 minutes, 38 cycles of denaturation at 94˚C for 30 seconds, primer annealing at 54˚C for 35 seconds, DNA extension at 72˚C for 45 seconds and final extension was at 72˚C for 10 minutes. The amplified PCR product was checked on agarose gel electrophoresis and given for sequencing toxcelris Labs Ltd., Ahmedabad. DNA sequences were compared with already submitted sequence in database using BLAST software.

2.5 Determination of heavy metal tolerance of resistant isolate- Maximum tolerance limit of isolated bacteria against Lead was determined. The isolated bacteria were grown in nutrient broth in presence of lead at concentration of 10, 30, 50, 100, 200, 300, 400, 500, 700 ppm. The tube were incubated at 37°C for 24 hours.

2.6 SEM analysis of heavy metal resistant isolates- In order to study the effect of heavy metals of bacterial surface, isolated bacteria were subjected to scanning electron microscopy (FEI quanta FEG 250). Culture without metals was used as control and culture with metals was taken as test. Cells were centrifuged at 10,000 rpm for 5 minutes. The pellet was washed with sterile PBS and re-centrifuged at 10,000 rpm for 5 minutes. This step is repeated two times to remove the traces of media. Re-suspend the pellet in 500 µl of PBS. Mix the pellet homogeneously with PBS than load 50 µl of mixed culture on glass coverslip. Fix the culture by putting 3.7% formaldehyde on coverslip. Mix it by gently moving coverslip and keep it for 5 minutes. Dehydrate the culture in oven at 80°C for one hour. The bacteria was viewed at low vacuum.

2.7 Flocculation index of the isolate- Flocculation index in absence and presence of lead was determined for isolated bacteria.One was control without any lead and other media tubes containing 20, 40, 80 ppm of lead. 1 x 10-7 cells/ ml was inoculated in each nutrient broth tube. The tubes were incubated at 37°C for 24 hours. After 24 hours OD600 was determined after mixing the media (F0).

The tubes were kept undisturbed at 37°C for 2 hours to let the cells settle down. Supernatant were taken into new tubes without shaking the media and OD600was taken of each tubes (Ft). The flochttp://www.pacificejournals.com/aabs

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culation index was determined using the formula given below (Sannasi P. et. al.2009);% Flocculation index = F0-FtX 100 F0 3. Results and Discussion 3.1 Microbial load of the riverwater sample of obtained from Mithi River was cultured on culture media for isolation. The number of colonies grown on the plates is given in table 1. The CFU count of the river was found to be 4.2x105 cells/ml and the CFU count of river water in presence of 10 ppm of lead was 1.7x105 cells/ml. Decrease in CFU of river water in presence of lead indicate that not all the microbes of the river are able survive at 10 ppm of lead. 40.48% of the microbe from water sample were found to grow in the presence of lead. This indicates that the significant population of micro flora of the river is acclimatized to the stress condition exerted by the metal present in the river on the bacterial cells. Further, the microorganisms isolated from the Mithi River could be used as the potential candidate for the bioremediation of heavy metals.

Table 1: Number of colonies observed after plating the dilutions on the nutrient agar plates without any lead and in presence of 10 ppm of lead.

Dilution Neat 10-1 10-2 10-3 10-4 10-5 3 1 CFU (without lead) Confluent Confluent 148 26 Confluent Confluent 113 22 2 0 CFU (with lead) 3.2 Isolation of heavy metals resistant microorganisms from Mithi River-Organisms was carried out in the culture media containing lead. Five organisms which had shownthe significant resistance towards lead were isolated successfully and purified. Thesemicroorganisms were selected on the basis of their differential colony characteristics and were given a code of WPb-1, WPb-2, WPb-3, WPb-4 and WPb-5. Majority of the lead resistant microorganisms that were isolated showed the gram negative character.Various investigations have reported the gram negative character of the lead resistant microorganisms which corroborates with the current investigation (22).

Table 2: Colony characteristics of isolates.

Sr. No.

Media used

Characters

WPb-1

WPb-2

WPb-3

WPb-3

WPb-5

1 2 3 4 5 6 7 8

NA NA NA NA NA NA NA NA

Size(mm) Shape Color Margin Opacity Elevation Consistency Gram’s Nature

1 Circular White Entire Translucent Convex Smooth Gram + Bacilli

3 Circular White Entire Opaque Convex Mucoid Gram –ve coccobacilli

2.5 Circular Creamish Entire Translucent Raised Smooth Gram –ve coccobacilli

3 Circular Creamish Entire Opaque Convex butyrous Gram –ve coccobacilli

2 Circular Creamish Entire Translucent Convex Mucoid Gram –ve coccobacilli

NA-Nutrient agar 3.3 Characterization and Identification- Different colony characteristics were observed for each isolate.

Colony characteristic are mentioned in the table no. 2. The biochemical analysis of five isolate are presented in Table 3.

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Table 3: Biochemical test of five isolated bacteria.

Test Indol test Methyl red

WPb-1 Negative Positive

WPb-2 Negative Negative

WPb-3 Negative Positive

WPb-3 Negative Positive

WPb-5 Negative Negative

Voges–Proskauer

Negative

Negative

Negative

Negative

Negative

Citrate utilization

Negative

Positive

Negative

Negative

Positive

Triple sugar iron

Red/Red

Red/yellow, gas production

Yellow/Yellow

Yellow/Yellow

Red/Red

Catalase

Negative

Negative

Negative

Negative

Negative

Motility

Non-motile

Non-motile

Non-motile

Non-motile

Non-motile

Gelatinase

Negative

Negative

Negative

Negative

Negative

Glucose Sucrose Lactose Mannitol Maltose Xylose

Negative Negative G Negative Negative Negative

FG FG F FG FG G

Negative F Negative F FG Negative

F F Negative Negative F Negative

Negative Negative Negative Negative Negative Negative

F:Sugar fermented, G: gas production 3.4 Molecular identification by 16S rRNA- PCR amplification of 16S rDNA of all the isolates produced 1.5 Kb products. The distinct bands were seen adjacent to 1.5 kb band of DNA ladder on electrophoresing the DNA on 1.5% agarose (fig. 1). The bacteria identified were Bacillus thuringenesis strain KPWP-1, Enterobacter cloacae, Acinetobacter barumanii strain AIEB-2, Pseudomonas fluorescens and Acinetobacter johnsonii. Bacillus sp. and Pseudomonas sp. are also commonly found in industrial effluent and many researchers have isolated and studied heavy metal bio sorption competence of this group of microorganisms (23). This indicates that the microorganisms isolated and selected in the current investigation have the potential for the bioremediation of heavy metal which is also supported by their growth in presence of heavy metals.

Figure 1: AGE of 16S rDNA PCR product. Band of 1.5 kb were observed for WPb-1, WPb-2, WPb-3, WPb-4 and WPb-5 isolates.

3.5 Determination of heavy metal tolerance of resistant bacterialisolate- Metal tolerance of all the bacterial isolates was found to be very high. The growth of four bacterial isolates out of five was seen till 500 ppm which indicates that the isolated bacteria can survive in the high concentration of metal and can be used as the potential candidate for the bioremediation. The growth of bacteria at different metal concentration was shown in the table 4. The MIC of WPb-1, WPb-2, WPb-3, WPb-4 and WPb-5 for lead was found to be 500 ppm, 600 ppm 600 ppm, 600 ppm and 600 ppm respectively.

Four out of five isolate showing MIC of 600 ppm of lead. High tolerance of the lead by the bacterial isolates of the current investigation indicates their potential for the bioremediation of heavy metals.

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Table 4: Tolerance limit of the isolated bacteria.

Sr. No. Concentration of lead (ppm) WPb-1 WPb-2 WPb-3 WPb-4 WPb-5 10 + ++ ++ ++ ++ 1 25 + ++ ++ ++ ++ 2 50 + ++ ++ ++ ++ 3 100 + ++ ++ ++ ++ 4 200 + + ++ ++ + 5 300 + + ++ ++ + 6 400 + + + ++ + 7 500 + + ++ + 8 600 9 + Sign indicates growth and ++ sign indicates dense growth & - no growth.

3.6 SEM analysis of heavy metal resistant bacterial isolates- Difference in the image of the WPb-2 was seen after SEM analysis. The surface of bacteria shown accumulation of lead when grown in the presence of lead. The SEM analysis indicates that the bacteria inhibit influx of metal in the bacterial cell. This mechanism may be used by the microorganism at the initial stages when exposed to the metal. Some morphological and physiological changes in bacteria have been observed when exposed to metals. The production of exopolymers or biopolymers is sometimes related to the cell’s defense mechanisms as it immobilizes toxic heavy metal ions thus inhibiting them from entering the cell as reported by Sannasi P. (24).

Figure 2:SEM images of the WPb-2. (a)SEM image of WPb-2 without lead and (b)SEM image of WPb-2 in presence of lead magnified to 10,000X. Arrow showing the accumulated lead on the surface of WPb-2 isolate.

3.7 Flocculation index of the isolate- The flocculation index of the bacterial isolates in the current investigation increased with the increase of the lead concentration. Control group of bacteria showed low flocculation index as compared to the bacterial isolates growing in presence of heavy metals.

This may be due to the non-availability of the metal ions on the surface of the bacterial isolates containing tube as there is no metal ions to bind on the surface on bacteria make floccules of the cells which settle down faster than unfloculated cells. This result confirms the binding and accumulation lead on the surface of the bacteria.

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Table 5: The flocculation index of WPb-2 isolate at different concentration of lead Growth tube Flocculation index (%) 5.1 Control tube 6.0 Metal tube (40ppm) 6.7 Metal tube (80ppm) 7.0 Metal tube (100ppm) Conclusion: Heavy metal resistant microorganisms were isolated from the Mithi River. Both gram negative and positive microorganisms studied in the current investigation have shown the capability of bioremediation of heavy metals. Microorganisms, viz Bacillus thuringenesis strain KPWP-1 and Pseudomonas fluorescens, isolated and studied under the current investigation have been earlier reported for their bioremediation characteristics. While the microorganisms such as Enterobacter cloacae, Acinetobacter barumanii strain AIEB-2 and Acinetobacter johnsonii have not been reported so far for their bioremediation characteristics. Some new species was also identified that are not extensively found in bioremediation process. On the basis of results of MIC, SEM and flocculation, it has been observed in the current investigation that these microorganisms have the potential to grow in the natural sites containing the toxic heavy metal. Further, these organisms may possess the mechanism for the bioremediation of heavy metals. Exopolymer secretion by the microorganisms studied in the current investigation may be one of the mechanisms used by them to survive in the metal contaminated sites and remediate the heavy metals. The isolated microorganisms of the present study can be used for bioremediation of heavy metal.

Acknowledgement: The authors are thankful to Nation Centre for Nanotechnology and Nonosciences for technical support.

Funding: UGC under Innovative Research Activities, XII plan scheme. Publication of this article was co-sponsored by the management of Annals of Applied Bio-Sciences.

Competing Interests: None declared References: 1.

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J.P. Rupareliaa, S.P. Duttaguptab, A.K. Chatterjeec and S. Mukherjia. Potential of carbon nanomaterials for removal of heavy metalsfrom water. Volume 232, Issues 1–3, 30 November 2008, Pages 145–156.

MeghrajHookoom and DaneshwarPuchooa.Isolation and Identification of Heavy Metals Tolerant Bacteria from Industrial and Agricultural Areas in Mauritius. Current Research in Microbiology and Biotechnology, Vol. 1, No. 3 (2013): 119-123.

M.A. Hashim, Soumyadeep Mukhopadhyay, Jaya Narayan SahuandBhaskarSengupta. Remediation technologies for heavy metal contaminated groundwater. Journal of Environmental Management 92 (2011) 2355e2388.

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Reena Singh, NeetuGautam, Anurag Mishra, and Rajiv Gupta. Heavy metals and living systems: An overview. Indian J Pharmacol. 2011 May-Jun; 43(3): 246–253.

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Shailendra Singh, Seung Hyun Kang, Ashok Mulchandani and Wilfred Chen. Bioremediation: environmental clean-up through pathway engineering. Current Opinion in Biotechnology, 19:437–444, 2008.

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Shruti Murthy, Geetha Bali and S. K. Sarangi. Biosorption of Lead by Bacillus cereus Isolated from Industrial Effluents. British Biotechnology Journal 2(2): 73-84, 2012.

Hadis Ghodsi, MehranHoodaji, ArezooTahmourespour and Mohammad Mehdi Gheisari. Investigation of bioremediation of arsenic by bacteria isolated from contaminated soil. African Journal of Microbiology Research Vol. 5(32), pp. 5889-5895, 30 December, 2011.

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Original Article

Aqueous Phytal extracts as source for staining in gel based protein separation techniques JayaPrada Rao Chunduri*, Harsha Mota Department of Biotechnology, Mithibai College, Bhakti Vedanta Marg, Vile Parle (w), Mumbai, India Keywords: PAGE, Natural İngredients, Staining Solutions

Abstract

PAGE (Polyacrylamide gel electrophoresis) has been considered as best and easy gel based protein separation technique among rapidly growing array of proteomic technologies. The post electrophoresis procedures usually comprise staining techniques to identify the separated proteins using CBB, Silver nitrate, fluorescent stains etc. Chemical stains used in staining methods are non eco-friendly and non-degradable. Natural plant extracts used as stains for the development and identification of protein bands is a novel idea. Current study focuses on the development of new staining techniques using natural ingredients like tea, coffee, henna and beetroot which are easily available. The results found to be 100% reliable, more ecofriendly, cost effective. Application of natural stains in the routine proteomic studies could be a step towards ecofriendly approaches in science.

*Corresponding author: Dr. JayaPrada Rao Chunduri, 1204, G-wing, Raheja Vistas, Raheja Vihar, Chandivali Farm Road, Powai, Andheri-E, Mumbai-72, India Email: jayapradachunduri@gmail.com

This work is licensed under the Creative Commons Attribution 4.0 License. Published by Pacific Group of e-Journals (PaGe)

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Introduction Natural colorants have been in use since ancient times for dying and coloring purpose for eg., wall painting of Ajanta, Ellora. The leaves of Lawsonia inermis has been well known example in the Indian subcontinent for decorating hands, soles, and dyeing beard and hair to impart beautiful shades of dark red color. Dyes are also obtained from beetroot, cranberry; milkwort flowers (yellow dyes), stigmas of saffron flowers (orange dyes) and indigo (Blue).

Staining procedures play very important roles in bio-molecular studies. It has been used to stain cell nucleus, cell membrane, protein, DNA, etc. The use of stains has been very predominant in molecular biology studies such as in proteomics for identification, characterization and further processing of bio-molecules.

Proteins are the primary functional agents of biological system and regulate metabolic processes, signal transduction, small molecule/ion transport, cell replication, and apoptosis. Proteome examination is a critical way to analyze how a cell responds to its environment. Polyacrylamide gel electrophoresis (PAGE) remains the well accepted, widespread, and successfully implemented technique for the assessment, high-resolution separation, and characterization of these critical molecules1. Protein separation by one or two dimensional electrophoresis (1D/2D) is largely used in proteomic approaches because of both high resolution and the availability of powerful image analysis software for gel comparison and compatibility with subsequent protein characterization by mass spectrometry2. For these various aspects, the selection of the protein staining procedure is of major importance 3. Coomassie blue has been the most widely used non-covalent dye for post-electrophoretic protein staining4. However, it suffers from a low sensitivity in protein detection, including in the improved colloidal version 5. In contrast, the other classical protein stain, silver nitrate, displays an excellent sensitivity but could interfere with protein analysis by mass spectrometry6. Different fluorescent dyes have been introduced 7 recently, for eg. Sypro Ruby8, and Ruthenium red-based dyes9. However, their use remains relatively limited, probably due to their cost and/or technical difficulties. These stains are neither easily biodegradable nor environment-friendly. CBB is toxic and its use necessitates specialized disposal efforts.

Current day’s need is to develop a new stain which is natural, efficient, effective and sharp in characterizing the bio-molecules without harming the environment. Use of aqueous extracts natural phytal origin for the development and identification of protein bands is a novel idea. Phytal extracts can excel over standard stains by simplicity and non toxicity.

Earlier studies indicated that for the preparation of Henna (Lawsonia inermis) extractions, chemical based combinations of CaOH and 50% ethanol10 or acidic aqueous 11 were used as extraction media. These henna extractions were used as stains in protein staining techniques. Aqueous henna leaves extract (cold or hot) oxidized with potassium permanganate was used as a substitute to the usual counter stains used in Gram staining12 During the current research an attempt was made to develop stain by using henna in combinations with 1.beetroot and 2. coffee and tea and its application in the post –elelctrophoretic staining.

Materials and Methods Sample: Blood Serum and Bovine Serum Albumin were considered. Each was mixed with appropriate quantity of gel loading dye.

Reagents: 1.5 M Tris-HCl ( pH 8.6), 10% APS , TEMED, Tris glycine buffer, Gel loading dye Gel electrophoresis: The gel used was 10% polyacrylamide. Standard Protocols were followed for the preparation of PAGE gel using acrylamide:bisacrylamide ( ratio 29:1) solution, 10%APS, Tris HCl buffer (pH 8.6), and TEMED,. The Tris Glycine buffer (pH8.3) was used as tank buffer ( 25mM Tris base, 0.2 M Glycine, pH 8.6). Prior to electrophoresis, the samples were heated in the presence of sample buffer (70 mM Tris–HCl (pH 6.8), 11.4% glycerol and 0.01% bromphenol blue) at 100°C for 30 seconds in a boiling water bath. Protein samples were loaded into the individual wells, and electrophoresis processed using 100 Volt of current 13.

Preparation of staining solutions:  Preparation of Standard Staining solution (CBB R 250): CBB R-250 (0.25 g) was dissolved in mixture of methanol: D/W: Glacial acetic acid in ratio of 45:45:10.

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 Preparation of phytal stains: The plant leaves of henna (Lawsonia inermi, Family: Lythraceae ), root of Beetroot (Beta vulgaris, Family: Amaranthaceae), dried leaves of tea (Camellia sinensis, Family: Theaceae) and dried seed  Powder of coffee (Coffeea Arabica, Family: Rubiaceae) were considered for the preparation of herbal stains.

Henna had been considered as main ingredient along with carrot and beetroot or coffee and tea.  Combination 1: Equal quantity of 5gm of tea and 5gm of coffee powder were boiled in 50 ml of distilled water and 4% henna extraction was made in decoction and filtered. This was considered for staining purpose  Combination 2: A 4% decoction was prepared in D/W of equal quantity of henna powder and beetroot. It was then filtered and used for staining purpose.  A 4% decoction was prepared in D/W of equal quantity of henna powder and beetroot. It was then filtered. Used for staining purpose Protein staining: Standard solution: CBB stain was used as control: After separation, the gels were carefully transferred to a steel tray filled with distilled water. The staining solution was added to the gel and kept at room temperature for 1 hour. The gel was washed with D/W and destaining was carried out at room temperature for 2 hours using destaining solution.

Test solution: After separation, the gels were carefully transferred to a steel tray filled with distilled water. Gels were stained with individual stains (HB and HCT) immediately after removing distilled water considering different temperature conditions (Room temperature, 37°C and 120°C) and time variations (30/60/90min). Destaining was carried out for 1 hour using 6% acetic acid/Vinegar without the need of fixation step.

Phytochemical Screening: The preliminary phytochemical tests were performed for testing different chemical groups present in extracts.

Alkaloids: 100 mg of extract was added to small quantity of Wagner's reagent [Solution of iodine in potassium iodide]. The presence of reddish brown precipitate indicates the presence of alkaloids14. 100 mg of extract was added with small quantity of Hager's reagent [saturated solution of Picric acid]. The presence of alkaloids is indicated by yellow precipitate14,15 Glycosides: 2 mL of extract was treated with 0.4 ml of glacial acetic acid containing a trace amount of FeCl3. 0.5 ml of concentrated H2S04 was also added by the side of the test tube. Persistent blue color appeared in the acetic acid layer indicates the presence of cardiac glycosides.14 Tannins: 5 ml of extract was added with few drops of 5% FeCl3. Deep blue black colour indicates the presence of tannins14.

Flavonoids: 5 ml of extract was added with few drops of NaOH solution. An intense yellow color, which turns to colorless on addition of few drops of dil.H 2SO4 indicates the presence of flavonoids11.

Terpenoids: 5 ml Extract was treated with 5 ml CHCl3 and few drops of conc. H2SO4. Shake well and allow it to stand for some time. Formation of yellow colored lower layer indicates the presence of terpenoids 14,15 Phenols: A few drops of extract was taken in a test tube and added with few drops of 1% FeCl3. The formation of a red, blue, green, or purple coloration indicates the presence of phenols14.

St. Sabouraud’s agar containing dextrose and peptone was used to isolate the fungus grown on the phytal extract as the high concentration of dextrose and acidic pH (pH 5.6) make this medium selective for fungi.

Gel Analysis techniques: MyImage Analysis software of Thermo Scientific was used to analyse the gel characteristics with respect to Rf value of bands, relative quantity and purity of the bands. The data was used to assess the similarity between the world wide used CBB and the current phytal aquatic staining techniques.

The statistical analytical methods such as correlation and student t test were performed to assess the significant similarity between the different staining procedures and their protein binding expressions.

Result The study has revealed the presence of phytochemical constituents. Important phytochemicals such as terpenoids, flavonoids, alkaloids, phenols, glycosides, tannins and steroids were present in the phytal extracts. The phytal extract of henna and beetroot contained steroids, terpenes, glycosides, tannins, phenol, flavonoids, and alkaloids http://www.pacificejournals.com/aabs

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whereas phytal extract of henna showed absence of steroids, terpenes, and flavonoids. Tannins were absent in beetroot. Similarly, the phytal extract of tea and coffee contained steroids, terpenes, glycosides, tannins, phenol, flavonoids, and alkaloids whereas phytal extract of coffee showed absence of terpenes. Thus, the combinatorial effect may have shown the staining ability. The phytochemical constituents of the phytal extracts found to be different at their individual levels to that in combination (Table-1). Analysis of plant extracts revealed the presence of flavonoids, glycosides, phenols, saponins, steroids and tannins in most of the selected plants which could be responsible for the observed binding property with the protein and staining property.

Table 1: Phytochemical analysis of phytal extracts of Tea, Coffee, Henna, Beetroot and in combinations of Tea and Coffee, Henna and beetroot, henna.

Stains Flavonoids Steroids Alkaloids Phenols Tannins Glycosides Terpenes Tea + + + + + + + Coffee + + + + + + Henna+ + + + + + + + Tea+Coffee Henna + + + + Beetroot + + + + + + Henna+Beetroot + + + + + + +

Phytal stains and PAGE Gel: Staining and destaining methods of standard CBB R 250 and phytal extract showed remarkable results. In our effort to develop and optimize a sensitive protein detection procedure using henna, beetroot, tea and coffee aqueous extract, a number of conditions for staining process were tested.

Temperature: Temperature is important factor for gel staining protocol and expression of protein. Staining at 37°C for 60 minutes was found to be enough to obtain a clear band for both phytal extracts of tea and coffee. Room temperature showed equal efficacy as that of 37°C of HCT. On the contrary, staining at 120°C for 60 minutes indicated no clear bands (Fig.1).

Fig.1: Efficiency of Natural stains’ (Henna Beetroot (HB) and Henna, Coffee and Tea (HCT)) staining with respect to temperature on Protein band expression. Serum was separated using PAGE and stained with staining solutions at RT (1), 37°C (2), and 120°C (3), and CBB stain (4) for 60 minutes.

Time variation: Time is a very important factor for protein detection in protocol. Gel staining under different staining times at 37°C indicated the impact on binding efficacy of the phytal stains. Visual observations of 60 min time variations indicated similar results in the case of both types of aqueous stains.

Staining for 60 minutes was found to be enough to obtain a clear band. However, staining for 30 minutes was less effective, and no clear bands were obtained. The color of the bands and backgrounds became more intense and unclear as the staining time increased. for both types of aqueous stains (Fig.2)

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Fig 2: Efficiency of Natural stains’ (Henna Beetroot (HB) and Henna, Coffee and Tea(HCT)) staining time and Protein band expression. Serum was separated using PAGE and stained with staining solutions for 30 minutes (1), 60 minutes (2), and 90 minutes ( 3).

Staining protocol for both phytal solutions was standardized. Best results obtained when the PAGE gels were stained at 37°C for 1 hour followed by destaining with 6% Acetic acid for 1 hour. Protein bands of BSA and blood serum were observed after staining the gel with aqueous extract of beetroot and henna (Fig.3). Similarly, separated protein bands of blood serum and BSA samples were observed after staining of gel with phytal extract of HCT (Fig.4).

Fig. 3: Gels with BSA(1) / serum (2)sample stained by Henna and beetroot (HB) phytal extract.

Fig. 4: Gels with Serum(1) / BSA sample (2) stained with henna, coffee and tea phytal stain (1:stains used 1-6, 7- CBB and, 2 : 1-4 fresh stain; 5,6 - 1 month old stain).

MyImage Analysis software of Thermo Scientific was used to assess expression of the intensity levels and binding characterization of protein bands by natural stains and the standard CBB. The photographs of gels stained with both stains were used for analysis. The Rf value, relative quantity and percentage purity of each band of protein was assessed using this computer software. (Fig.5 and 6)

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It was observed that percentage purity of the protein band stained with standard CBB stain and test stain i.e. phytal extract of beetroot and henna; and CBB stain and test stain i.e. phytal extract of henna, coffee and tea showed almost similar results (Table-2). Similar trends were observed for relative quantity of each protein band as well. However, relative quantity and percentage purity of protein bands visualized by HCT stain were relatively higher compared to that of henna and beetroot stained protein bands indicating the efficiency of HCT stain better than that of beetroot and henna stain.

Fig.5. Rf value of bands of BSA and serum samples stained with HB stain.

Fig.6: Rf value of bands of BSA and serum samples stained with HCT stain Table 2: Percentage purity of each protein band was analysed. Proteins were stained with CBB, Henna + beetroot, and Tea+ coffee.

Stains Sample Percentage purity Band 1 Band 2 Band 3 Band 4 BSA 7.82±0.50 8.64±1.00 13.15±0.99 28.39±11.56 Henna+ Beetroot CBB

BSA

8.01

8.19

13.37

14.77

Henna + Beet root

Blood Serum Blood Serum BSA

3.48±0.06

2.48±0.40

6.33±0.36

6.48±0.41

3.93

1.98

8.82

5.66

6.4±0.14

10.33±0.91

15.25±0.71

23.14±9.06

Blood Serum Blood Serum

6.66±1.33

7.79±1.09

11.77±2.79

6.21

8.25

13

CBB Henna+ Tea + Coffee Henna + Tea + Coffee CBB

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Statistical analysis of the results of two phytal stains and CBB staining techniques with respect to the human serum sample and Bovine albumin serum samples separated by Polyacrylamide gel electrophoresis was performed. A positive correlation between Rf values of bands, relative quantity and purity of the bands was observed. The significant differences with respect to RF values, purity of the band observed and relative quantity expressed after staining were assessed with student T test. The test results between HB staining, HTC staining and standard CBB have not shown any significant differences with respect to different parameters. These results were observed irrespective of the protein sample such as serum and BSA. (t cal values <2.42 at 0.05 significance)

Discussion 1DE is the most sought after procedure due to its high resolving power, large sample loading capacity and display of several proteins on a single gel. This gives an ample scope of producing a direct and global view of a sample proteome at a given time. Several dyes are available to label proteins, either before or after electrophoresis. These include quite expensive (Sypro ruby) or economical (colloidal Coomassie blue, silver nitrate) and affordable stains, which may not cause any obstacles in protein identification by mass spectrometry (CBB). Despite a moderate amount of success in detecting and quantifying proteins in-gel, each staining procedure has its own limitations. All the traditional staining solutions contain methanol, acetic acid, or phosphoric acid which not only produce unpleasant smell but also cause environmental pollution.

The two staining solutions developed along with their staining procedures largely depend on water and no acids are needed for the staining solution preparation. The phytal extracts used for staining process have promising future in terms of staining the protein as efficiently as that of widely used CBB. The current staining procedure does not require washing and fixing steps but requires only the staining step. However, the protocol requires 6% acetic acid for destaining.

Conclusion Post-electrophoretic protein staining procedures consider most widely used non covalent dye Coomassie blue. The other classical protein stains include silver nitrate, different fluorescent dyes such as Sypro Ruby, and Ruthenium red-based dyes that have limited usage probably due to their cost and/or technical difficulties 16. In comparison, the current aqueous based phytal stains have advantages such as simplicity, low cost, environmental friendliness; do not require many reagents (except Vinegar or 6% acetic acid). Unlike the other chemical based extractions of henna, the current procedure involves extraction of stain from the leaves of henna and in combination of beetroot or tea and coffees using only the distilled water as extraction source. The protocol includes only staining and quick destaining steps. Stains are reusable up to several times, provided properly refrigerated but indicate a minimal intensity differences in the gel13.

Acknowledgements We thank Dr. D.V. Kamat, Co-Ordinator (Biotechnology Dept) and I/C Principal for his constant encouragement and SVKM management for the facilities provided during this research project.

Funding None.

Competing Interests None declared.

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AABS; 2(2): 2015 Neuhoff V, Arold N, Taube D and Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9, 255-262.

Mortz E, Krogh TN, Vorum H and Gorg A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 2001, 1, 1359-1363.

Westermeier R, Marouga R. Protein detection methods in proteomics research. Bioscience Rep. 2005, 25, 19-32.

Berggren K, Chernokalskaya E, Steinberg TH, et al. Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis 2000, 21, 2509-2521.

Rabilloud T, Strub JM, Luche S, van Dorsselaer A and Lunardi J, Comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.

Proteomics 2001, 1, 699-704.

Ali R, Sayeed SA, A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis. Electrophoresis, 1990, 11(4),343-344 Tyagi R. K., Sagar R. and Datta K., A sensitive method for detection of proteins in polyacrylamide gels and on protein blots using acidic henna leaf extract, Biotechnology Techniques, 1994, 8(8), 583-588.

Chukwu OC, Odu C E, Chukwu D I, Hafiz N, Chidozie V N and Onyimba I A, Application of extracts of Henna (Lawsonia inamis) leaves as a counter stain, African Journal of Microbiology Research,2011, 5(21), 3351-3356.

Dong WH, Wang TY, Wang F, Zhang JH , Simple, Time-Saving Dye Staining of Proteins for Sodium Dodecyl Sulfateâ&#x20AC;&#x201C;Polyacrylamide Gel Electrophoresis Using Coomassie Blue. PLoS ONE,2011, 6(8): e22394.

Thakur N, Vikrant A, Preliminary Phytochemical Analysis of the Extracts of Psidium Leaves, Journal of Pharmacognosy and Phytochemistry,Middle-East Journal of Scientific Research,2014, 19 (11), 1421-1424.

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François Chevalier, Standard Dyes for Total Protein Staining in Gel-Based Proteomic Analysis, Materials 2010, 3, 4784-4792.

Annals of Applied Bio-Sciences, Vol. 2; Issue 2; 2015

ISSN: 2349-6991

Original Article

Effect of temperature variation and determination of optimum temperature of alpha amylase activity in Telescopium telescopium.

Sharvari Kudtarkar*, Prakash V. Desai Department of Zoology, Goa University, Taleigao Plateau, Goa, India.

Keywords: Alpha amylase, Telescopium telescopium, Optimum temperature

Abstract

The changes in enzyme activity of α- amylase from hepatopancreas of Telescopium telescopium from the Chorao Island of Goa State were investigated during year 2004 to 2006. The enzyme activity was observed highest at 20 minutes of incubation time and was 30.0 ± 10.83 µ mols/mg protein/min. The lowest enzyme activity was observed at zero minute of incubation and was 19.17 ± 1.23 µ mols/mg protein/min. At 40 0C of incubation temperature the highest enzymatic activity was observed and was 32.05 ± 3.307 µ mols/mg protein/min which is recorded as the optimum temperature.

The enzyme activities were also noted for one week starved Telescopium telescopium in laboratory.

*Corresponding author: Mrs. Sharvari Kudtarkar. Zoology Department, Goa University. Taleigao Plateau, Goa- 403206.

Mobile - 8080672010

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Introduction Telescopium telescopium (Linnaeus 1758) is herbivorous and detritus feeder. This species is the last remaining survivor of a genus that had several fossil records. It occurs in different size ranges during their development and growth. Maximum size recorded was 110 mm in length. It has tolerance to wide range of salinities and temperature as it is an estuarine gastropod and it remain exposed to sun during low tides for several hours. Large variations in salinity and temperature occur in the intertidal region[1],Telescopium telescopium must be highly adapted to avoid being dried out. At the optimum temperature range the rate of reaction rises because of the increase in the number of molecules with the required energy of activation. After exceeding the optimum temperature range in a specific set of conditions the rate of reaction decreases constantly and with further temperature rise it falls off to zero.

Materials and Methods Specimens of Telescopium telescopium of mean shell length of 9.1 cm (range 8.9 to 11.1 cm) were obtained from Mandovi estuary, Chorao islands, Goa. The gastropod was carefully opened with the help of bone cutter. The intestinal region was located and hepatopancrea was isolated aseptically. The epithelial sheath that covers the tissue was carefully removed with the help of forceps. Wet weight of each animal was measured and recorded. Samples were collected with the same procedure after one week of starvation. The samples were homogenized in chilled mortar and pestle in 10 ml phosphate buffer of pH 7 and centrifuged in a cooling centrifuge at 2000RPM for 20 mins. Supernatant was collected and stored at 4 0C and further used for the enzyme extraction, purification and assay. Purified enzyme solution obtained from purification steps is used to determine the amylase enzyme activity. Ammonium sulphate precipitations were performed as described by Deutscher.[2] Supernatant produced through crude homogenate centrifugation was brought to 25% saturation with ammonium sulphate and subsequently centrifuged at 20,000 rpm for 30 mins at 0 0C. The resulting pellets were pooled and stored at 4 0C. Each pellet was resuspended in deionised water; subsequently the process was repeated to obtained pellet with 50% and 75% ammonium sulphate saturation. For the removal of salts from protein solution, dialysis was performed. Regenerated cellulose dialysis tubing 3500 MWCO was used for dialysis. The ammonium sulphate precipitated fractions including all supernatants and pellet solutions were dialysed twice for 24 hours dialysis against distilled water at 4 0C. The dialyzed solution was pipette out of the tubing and stored. The volume of each fraction was measured and recorded. Fractions of pellet solutions and supernatant solutions were collected separately and tested separately for alpha amylase activity. The fifth to seventh fraction of pellet solution exhibited maximum enzyme activity (90-95 %) and these fractions were used subsequently for column chromatography.

Column chromatography was performed.[3] Amylase from crude extract dialysate was purified using DEAE cellulose. Flow rate was 1ml/min. All fractions were 1ml in volume and collected in size exclusion chromatography fraction tube. Using peristaltic pump the six times diluted solution with binding and washing buffer of fraction was loaded on to Hi Trap Benzamidine column. Finally 10 fractions were collected and tested for enzymatic activity and were then pooled for use in subsequent analysis.[4] The specific activity was determined by estimating protein from the tissue extract following the method of Lowry et al [5]and was expressed as units per mg soluble protein(Âľ mg -1) For the appropriate incubation time for amylase activity of hepatopancreas of Telescopiumtelescopium the reaction mixture was incubated at different time intervals starting from zero minute to 5,10,15,20,25,30 and 40 mins. Tubes were setup containing reaction mixture of (0.5%) starch, phosphate buffer (pH 7) and sodium chloride (1.0%). Tubes were placed in water bath at 37 0C and enzyme solution equilibrated at same temperature was added. Tubes then incubated at different time intervals as mentioned above. Reaction was stopped using 2M NaOH. Tubes then boiled for 5mins. Extinction was read at 540 nm against blank in spectrophotometer and enzyme activity was calculated using formulaMass of Maltose in mg/gm protein =

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The optimum temperature for maximum enzyme activity as determined by varying incubation temperature of the reaction mixture as 10, 20, 30, 40, 50, 60, 70 0C. All enzymes start to denature at temperatures above their optimum temperature. The reaction mixture was same. The tubes were incubated for 20 mins in a water bath at the desired Annals of Applied Bio-Sciences, Vol. 2; Issue 2; 2015

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temperature regimen. The enzyme activity was calculated as described earlier.

Result Graph no. 1 shows the enzyme activities at various incubation times. The amylase activity was minimum at zero min of incubation, later the enzyme activity elevated with increase in incubation time to 20 mins. The highest enzyme activity was observed at 20 mins of incubation time and it was 30.0 ±10.83 µ mols/mgprotein/min. The lowest enzyme activity was for zero minute of incubation and was 19.17 ± 1.23 µ mols/mgprotein/min. Effect of variable incubation temperature on the amylase enzyme activity can be revealed from graph no. 2. The amylase activity at 10 0C was 13.75 ±1.25 µ mols/mgprotein/min. Later the activity showed elevation with increase in temperature (32.05 ± 3.307 µ mols/mgprotein/min). After this enzyme activity showed decrements. The lowest enzyme activity was observed for 70 0C of incubation temperature ( 9.58 ± 5.636 µ mols/mgprotein/min). Subsequently the amylase activity can be observed in one week starved animals.

35

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0 0

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Effect of temperature variation mean fresh mean 1wk

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20 oC

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Discussion

Many researchers have exposed the molluscan amylases to variety of substrates.[6], [7]These researchers used glycogen, amylase, amylopecten, agar, meltotriose, maltotetraose, etc. They have found that amylase I and II could digest amylopectin to maltotraose and maltopentose.Broekhuysen (1941) determined the lethal temperatures and the survival timesat high temperatures in some intertidal marine gastropods.[8] Evans (1948) investigated the mean lethal temperature varied from 46.3 0C to 36.2 0C for some littoral gastropods.[9] The starch used in the present study was a combination of amylose and amylopecten. Besides α- amylases of Abalone is known to digest glycogen particularly amylase II. In the present study effect of incubation temperature variation was observed. Since temperature is a measure of molecular agitation it controls the rate of chemical reaction and is one factor limiting energy liberation and organismic growth. Present investigation shows the Optimum temperature for amylase activity in hepatopancreas of Telescopiumtelescopiumis 40 0C. The Maximum activity observed at 20 mins of incubation time. In nature the chances of telescopium reaching a temperature of 40 0C are rare however; the body temperature on mudflats could reach to 34 to 36 0C during summer. Thus in natural conditions the amylase activity though may not reach to a peak level, it still would be substantially high enough to promote carbohydrate digestion at a relatively faster rate.

Acknowledgements I take this opportunity to express my profound gratitude to my guide Prof.Dr. P.V. Desai. For his guidance and unfailing patience and without whose support and encouragement, this wok would not have been completed.

I equally thankful to Dr. Shanti Desai for providing valuable assistance in my work. I am also thankful to teaching and non-teaching staff of the Zoology Dept. Goa University.

Funding None.

Competing Interests None declared.

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

Joshi GV, Jamale BB (1975) In” Ecological studies in mangroves of Terekhol and Vashisti rivers”, p.

751-760. Bull Dept. Mar. Sci. University Cochin-7, vol-4.

Deutscher M.P.(1990), Methods in enzymology” Vol.182.(Guide to protein purification) Academic Press New York. Pp 380- 392.

Fagan J.M. (1986), “Demonstration of two distinct high molecular weight proteases in rabbit reticulocytes”.

Jr. Biological Chemistry Vol. 262.No 6,pp 2451- 2457.

Alexander P. and Block R.J.A. (1961). In “Laboratory manual of Analytical methods of Protein Chemistry”, Vol.1-3, p.160-161.: Pergamon Press, Oxford.

Lowry OH, et al. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951:193:265-75.

Bertrand Tatoinkoufossi, Frederic tavea, (2004).“Production and partial characterization of a thermostable amylase from ascomycetes yeast strain isolated from starchy soil”; African Jr. of Biotech. 2004;4(1):14-18.

Ching Yu Tsao,Yun-zu Pan, et al. Purification and characterization of Amylases from small Abalone. Jr. Agric Food Chem. 2003;51:1064- 1071.

Broekhuysen G. J. “A preliminary investigation of the importance of desiccation, temperature and salinity as factors controlling the vertical distribution of certain intertidal marine gastropods in False Bay, South Africa.”

Trans RoySoc .S. Afr, 1941;28:255 Evans R.G. (1948) “The lethal temperatures of some common british littoral molluscs” Jr.Anim. Ecology,17,165.

Annals of Applied Bio-Sciences, Vol. 2; Issue 2; 2015

ISSN: 2349-6991

Original Article

Histopathological lesions of nasal cavity, paranasal sinuses and nasopharynx Janice Jaison*, Deepa Topandas Tekwani Department of Pathology, MIMER Medical College, Talegaon (D) Pune, India Keywords: Polypoid lesions, nasal cavity, paranasal sinuses, nasopharynx

Abstract Background: The parts of the upper respiratory tract included in the present study are- Nasal Cavity, Paranasal sinuses and Nasopharynx. Majority of the lesions at these sites are polypoid. It is difficult to comment upon the nature of the lesion- whether neoplastic or non-neoplastic. Hence histopathological examination is essential for both ENT surgeons as well as pathologists.

Methods: The study involved an analysis of specimens received in the surgical pathology laboratory from the department of ENT over a period 5 years.

Result: A study of 104 lesions was undertaken. They constituted 3.06% of the total histopathological work load at our institution. Inflammatory and tumor like lesions constituted 74.04% followed by malignant tumors constituting 13.46% and benign tumors constituting 12.50% of the total number of cases.

Conclusion: The age group in this study ranged from 7 to 75 years. Inflammatory and tumor like lesions were seen in all age groups, while benign tumors showed a peak incidence in the 1st and 2nd decade and malignant tumors were common in the 4th to 6th decade. Males were more commonly affected by the various lesions as compared to females. Inflammatory and tumor like lesions were the most common lesions occurring at these sites, followed by malignant tumors and benign tumors respectively.

*Corresponding author: Dr. Janice Jaison, Flat no 2, Jai Apts, Plot no 121, Sect-26, Pradhikaran, Nigdi, Pune-44, India

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Introduction The parts of the upper respiratory tract included in our study are: Nasal cavity, Paranasal sinuses and Nasopharynx. The upper respiratory tract gets exposed to a variety of infections and many other influences in the environment. Due to the effect of these factors, various infections, tumor like lesions and true neoplasms occur in the upper respiratory tract. Fortunately, lesions of the upper respiratory tract though are most common afflictions of humans; the overwhelming majority are more nuisances than threats. The main lesions of this region are inflammations- both acute and chronic. Majority of these lesions of the nose and paranasal sinuses are polypoid. From the clinical examination of the patient or from the macroscopic examination of the specimen it is difficult to comment upon the nature of the lesion-whether neoplastic or non-neoplastic. Hence histopathological examination is essential for both ENT surgeons as well as pathologists.

Materials and Methods The study involved analysis of specimens received in the surgical pathology laboratory from the Department of ENT over a period of 5 years. The personal details of the patient were noted along with the presenting complaints.

A detailed clinical examination with special reference to ear, nose and throat examination was performed. Routine blood investigations were done in all patients and radiological investigations like X-ray, CT Scan and MRI were also done wherever relevant. A Final diagnosis was offered after Histopathological examination. The specimens received and studied were the result of the following operative procedures 1. Nose â&#x20AC;&#x201C; Excision biopsy, polypectomy 2. Paranasal Sinuses- Ethmoidectomy: Caldwell-Luc operation, Total Maxillectomy/Extended maxillectomy/lateral Rhinotomy/neck dissection 3. Nasopharynx- Adenoidectomy All of the above specimens on being received in the surgical pathology laboratory were fixed in 10% formalin overnight and a detailed gross examination was done the subsequent day. All tissues submitted were processed according to routine histological techniques and paraffin sections were obtained. All sections were stained by haematoxylin and eosin (H&E) method and examined microscopically.

Special stains were performed wherever necessary. These included- Reticulin, Mucicarmine, PAS, Acid fast stain, Grocottâ&#x20AC;&#x2122;s methanamine silver and Giemsa

Result The present study includes the lesions of the nasal cavity, paranasal sinuses and nasopharynx. These lesions were studied over a period of five years. During the course of this study, a total of 3393 specimens were received in the surgical pathology department. Of these, 104(3.06%) constituted the specimens of nose, paranasal sinuses and nasopharynx (Fig 1). The lesions were grouped into 3 categories: Inflammatory and tumor like, benign tumors and malignant tumors. Inflammatory and tumor like lesions were the commonest constituting 74.04% of the total cases, followed by malignant tumors constituting 13.46% and benign tumor constituting 12.5% of the cases (Fig. 2).

3.06% Fig 1- Comparison of cases studied with the total workload of surgical pathology department total workload cases studied

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13.46%

Inflammatory and tumor like lesions

Fig 2: Comparison between % of inflammatory and tumor like lesions, Benign and Malignant tumors

Benign tumors

12.5%,

Malignant tumors

74.04%,

Fig 3: Photomicrograph of Inflammatory polyp, showing lining respiratory epithelium beneath which is seen a predominantly lymphocytic inflammatory infiltrate with a few plasma cells and neutrophils (H& E, X 40) Fig 4: Photomicrograph of Allergic polyp showing a predominantly eosinophilic inflammatory infiltrate with a few plasma cells and lymphocytes. (H&E, X40) In the inflammatory and tumor like conditions, Inflammatory polyps were the commonest lesions. Out of the 77 cases of inflammatory and tumor like lesions, there were 28 cases of Inflammatory polyps constituting 26.93%.

This was followed by Allergic polyps, of which there were 22 cases (21.16%), 14 cases of Rhinoscleroma (13.46%), 4 cases of Rhinosporidiosis (3.85%), 3 cases of Atrophic Rhinitis (2.89%) and 2 cases each of Rhinophyma, cholesterol granuloma and Adenoids (1.92% each).(Fig.3-7) Out of the total of 104 cases, there were 13 cases of benign tumors. There were 5 cases each of Nasopharyngeal angiofibroma and Inverted Papilloma constituting 4.81% each followed by 3 cases of Capillary haemangioma constituting 2.88%. (Fig 8-9) 14 malignant tumors were studied. Well differentiated squamous cell carcinoma of the maxillary sinus was the commonest lesion in this group. There were 4 cases constituting 3.85% followed by 2 cases each of Adenoid cystic carcinoma, Olfactory neuroblastoma and Well differentiated squamous cell carcinoma of nasopharynx. (1.92% each).( Fig 10)

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Fig 5: Photomicrograph of Rhinoscleroma showing stratified squamous epithelium, beneath which are seen plenty of foamy macrophages. (H&E,X4) Fig 6: Photomicrograph of Rhinoscleroma showing a naked eosinophilic hyaline mass- Russell body seen in a background of chronic inflammatory cells (H&E,X40)

Fig 7: Photomicrograph of Rhinosporidiosis showing an ulcerated respiratory epithelium. Beneath is seen a vascular inflammatory granulation tissue, containing sporangia in varying stages of maturity enclosing spores(H&E, X4) Fig 8: Photomicrograph of Inverted Papilloma showing epithelial proliferation accommodated by infolding into the edematous stroma (H&E,X10)

The remaining malignant lesions were Poorly differentiated carcinoma of nose, Mesenchymal chondrosarcoma and Well differentiated carcinoma of ethmoidal sinus of which single cases were studied (0.96% each). (Table 1) Inflammatory and tumor like lesions were common in the 1 st to 3rd decade, benign tumors were commonest in the 1st and 2nd decade, while malignant tumors were common in the 4 th to 6th decade. Inflammatory and tumor like lesions and benign tumors were more common in males, while malignant tumors were common in females.

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Fig 9: Photomicrograph of Juvenile Nasopharyngeal Angiofibroma showing stratified squamous epithelium beneath which are seen plenty of capillary sized blood vessels. ( H&E, X4) Fig 10: Photomicrograph of Adenoid Cystic Carcinoma showing classical cribriform pattern (H&E, X10) Table 1. Incidence of various lesions Lesions Allergic polyp Inflammatory polyp Rhinoscleroma Rhinosporidiosis Rhinophyma Atrophic Rhinitis Cholesterol Granuloma Adenoids Nasopharyngeal angiofibroma Inverted Papilloma Capillary Haemangioma Poorly differentiated Ca of nose Adenoid Cystic Ca of ethmoidal sinus Adenoid Cystic Ca of maxillary sinus Well differentiated SCC of maxillary sinus Well differentiated SCC of ethmoidal sinus Mesenchymal Chondrosarcoma Olfactory neuroblastoma Well differentiated SCC of nasopharynx

Number of cases 22 28 14 4 2 3 2 2 5 5 3 1 2 1 4 1 1 2 2

% of cases 21.16 26.93 13.46 3.85 1.92 2.89 1.92 1.92 4.81 4.81 2.88 0.96 1.92 0.96 3.85 0.96 0.96 1.92 1.92

Discussion The present study of histopathology of lesions of the nasal cavity, paranasal sinus and nasopharynx included 104 lesions encountered over a period of 5 years.

Out of these 104 cases, 27 cases were Neoplastic (25.96%) and 77 (74.04%) were inflammatory and tumor like lesions. Of the neoplastic lesions 13(48.15%) were benign and 14 (51.85%) were malignant tumors. This is in agreement with Ash1, Buchnan and Salvin2, Ghosh and Bhattacharya3. But Tandon4 reported incidence of benign tumors as 73.3% and malignant tumors as 26.5% in their study.

In the present study the ratio of benign to malignant tumors in the nose was 1.3:1 and in the paranasal sinuses, only malignant tumors were found. This is in accordance with Friedman and Osborn 5 who found the ratio of behttp://www.pacificejournals.com/aabs

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nign to malignant tumors in the nasal cavity about 6:1, while in the paranasal sinuses the ratio was reverse, there being and increase in the number of malignant tumors.

Rhinoscleroma: in the present study there were 14 cases of Rhinoscleroma. There was a male predoinance with male to female ratio of 6: 1, which is in agreement with Kakar and Sinha 6. . No particular age group predominance was seen. Rhinoscleroma was common in the 1st to 4th decade. The findings of the present study coincide with studies carried out by Kakar and Sinha6. All the patients were from poor socio-economic strata.

Rhinosporidiosis: in the present study, there were four cases of Rhinosporidiosis. All of them were male patients.

Many Indian workers- Ratnakar7 , Makannavar8 and Ahluwalia H9 have reported cases of rhinosporidiosis collected over years. Rhinosporidiosis is commonly encountered in the 1 st to 3rd decade. In our study, out of the 4 cases, 2 cases were encountered in the 3rd to 4th decade, 1 case in the 2nd to 3rd decade and 1 case in the 4th to 5th decade. All the 4 cases studied occurred in male patients, which corresponds to the studies carried out by various Indian authors indicating that males are predominantly affected in Rhinosporidiosis.

Nasal Polyps: These were the commonest lesions found in our study of 104 cases. Out of 77 non neoplastic lesions included in our study, 50 cases were of inflammatory and allergic polyp. Antrochoanal polyps mainly presented in the second and third decade but no such age distribution was observed in ethmoidal polyps. Female predominance was seen in our series of cases. The incidence of nasal polyps in our studies corresponds to the incidence of nasal polyps in studies carried out by Tandon4. Allergic polyps were common as compared to inflammatory polyps in studies carried out by Tandon4. In our study, however, inflammatory polyps were common as compared to allergic polyps and constituted 56%.

Atrophic Rhinitis: Three cases of atrophic rhinitis were studied. 2 cases in females and 1 in a male patient. The epithelial changes varied from partial squamous metaplasia to total squamous metaplasia with keratinization. The lamina propria showed chronic inflammatory infiltrate, granulation tissue and fibrosis. These findings correspond to the earlier studies carried out by Anand and Agarwal 10, Sinha 11 and Ishwar Singh 12.

Cholesterol Granuloma: 2 cases of cholesterol granuloma were recorded. These were similar to the cholesterol granuloma of the middle ear. They are said to occur as a result of extensive degeneration within an inflammatory polyp.

Rhinophyma: 2 cases of rhinophyma were studied. They showed classical histological features of hyperkeratosis of the epidermis and hypertrophy and hyperplasia of sebaceous glands.

Adenoids: 2 male patients presented with nasal obstruction and difficulty in breathing due to enlarged adenoids.

Benign lesions Nasopharyngeal angiofibroma: in the present study, there were 5 cases of angiofibroma. Of the 5 cases, 4 cases were males and one case was a female. The age group was 1 st and 2nd decade of life. This is in agreement with English 13 and Biller 14.

Inverted Papilloma: in the present study, there were 5 cases of inverted papilloma, 4 male patients and one female. Male predominance was seen in our study with majority of cases occurring in the 5 th to 6th decade. This is in accordance with studies carried out by Lawson and Billen. 15 Capillary Haemangioma: in the present study, there were 3 cases of capillary haemangioma. All of the 3 cases were male patients. All patients presented with epistaxis and nasal obstruction.

Malignant lesions In the present study of 104 cases, there were 14 cases of malignant neoplasms. Out of the 14 cases, 2 cases were malignancies of nasopharynx, 8 cases of malignancies of paranasal sinuses and 4 cases of malignancies of the nasal cavity. Maxillary sinus was most commonly involved (35.71%). This is in comparision with Frazell and Lewis 16 Acheson 17, Lewis and Castro 18 and Wang 19 In the present study, squamous cell carcinoma was the commonest of the malignant tumors encountered in the paranasal sinuses comprising 62.5%.

Conclusion The age group in this study ranged from 7 years to 75 years. For benign tumors peak incidence was in the 1 st and 2nd decade and for the malignant tumors in the 4 th to 6th decade of life. In tumor like and inflammatory lesions male to female ratio was 1.2 :1, while in neoplastic cases, males were affected more as compared to females and ratio of male to female cases was 1.07: 1.

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In the tumor like and inflammatory lesions simple nasal polyps were the commonest (64.93%).Inflammatory nasal polyps were common as compared to allergic nasal polyps and constituted 56%.The malignant tumors (51.85%) were common as compared to the benign lesions (48.15%).The maxillary sinus was commonly affected (35.71%) in all malignant neoplasms. The most common histological type of malignancy encountered was squamous cell carcinoma (57.14%) As most of the lesions of nasal cavity, paranasal sinus and nasopharynx can present as polyp like lesions, their histopathological examination can help the surgeons in diagnosis of a disease and choosing the right modality of treatment for the patient.

Acknowledgements My most heartfelt and sincere gratitude to my guide Brig. (Dr) S. K. Basu, for his steadfast yet patient guidance without which this study would never have been completed. My sincere thanks to Dr. Neeta Kulkarni, who provided me with the clinical material and subsequent exchange of notes. I am grateful to Dr. Smita Pathak, for the invaluable assistance graciously rendered to me during this study.

Funding None.

Competing Interests None declared.

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1966; 47:14 Tondon P. L., Gulati J. and Mehta N., Histopathological study of polypoidal lesions in the nasal cavity. Indian Journal of Otolaryngol, 1971; 13:3 Friedman I, Osborn DA. Pathology of Granulomas and Neoplasms of the Nose and Paranasal Sinuses. Churchill Livingstone, Edinburgh London Melbourne and New York 1982 Kakar P., Sinha A., The problems of scleroma in India. Indian Journal toof Olaryngol, 1964; 16:164 C Ratnakar, M Madhavan, Vanaja Sankaran, Andrew J Veliath, N K Majumdaar, Vasudev Anand Rao. Rhinosporidiosis in Pondicherry. Journal of Tropical Medicine and Hygiene, 1992; 95 (4):280-283.

J. H. Makannavar, S. Sateesh Chavan: Rhinosporidiosis- A clinicopathological study of 34 cases. Indian J Pathol Microbiol 2001; 44:17-21 Ahluwalia H. K. B., New interpretation of Rhinosporidiosis. J Submicrosc Cytol Pathol. 1992; 24: 109-11 Anand CS, Agarwal SR. A histopathological study in Atrophic Rhinitis. JIMA, 1972; 59:278-281 Sinha S. N., Samuel K.C., Juneja HIS, Mittal D.P: A study of exfoliative cytology of atrophic rhinitis. J Laryngol Otol, 1975; 89:1027-1041 Ishwar Singh, R. M. Raizada, V. N. Chaturvedi, S. K. T. Jain, S.N. Ingole Study of histopathological changes in Atrophic Rhinitis, Indian Journal of Otolaryngology and Head & Neck Surgery, 1999; Vol 51, No.1: 21-24 English GM, Hemenway WG, Cundy RL: Surgical treatment of angiofibroma. Arch Otolaryngol, 1972; 96:312 Biller HF, Session OG, Ogura JH: Angiofibroma- A treatment approach. Laryngoscope, 84; 695-705 Lawson W, Billen H. F., Jacobson A, Som P, The role of conservative surgery in the management of inverted papilloma.

Laryngoscoy, 1983 (2); 93:148-155.

Frazell EL, Lewis JJ: Cancer of nasal cavity and accessory sinuses. Report of management of 416 patients. Caner, 1963; 16:1263 Acheson E. D., Cowdell R. H., Jolles B., Nasal cancer in the Northampshire Boot and Shoe Industry. Br. Med. Journal 1970; 1: 385-93 Lewis J. S., Castro E. B., Cancer of the nasal cavity and paranasal sinuses. J Laryngol Otol, 1972; 86: 255-262 Cheng VST, Wang CC: Carcinoma of the paranasal sinuses. A study of 66 casees. Cancer, 1977; 40 (6):3038-3041.

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Original Article

Clinical and haematological characteristics of autoimmune haemolytic anemia: retrospective analysis of 10 cases Maryann Margaret Bukelo1, Srikanth Umakanthan*2, Sharada Rai1 1 2

Department of Pathology, Kasturba Medical College, Mangalore, India Department of Paraclinical Sciences, The University of the West Indies, Trinidad Keywords: Autoimmune haemolytic anemia, cold agglutinin disease, direct antiglobin test,

Abstract Background: Autoimmune haemolytic anemia (AIHA) is a disease of red cell destruction due to different antibody types. They are of two types: warm antibody AIHA (IgG type) and cold agglutinin disease (IgM type). Direct Coombs test is the cornerstone for diagnosis of AIHA. Methods and results: A retrospective analysis of ten cases diagnosed with DAT were included. The cases showed female predilection (M: F = 1:9). Fatigue and breathlessness was the commonest presentation (90%) and Pallor was the most common (90%) physical examination finding. MCV (>100 fl) was seen in 9 cases (90%). Direct antiglobulin test (DAT) was positive in all ten cases (100%).

Conclusion: AIHA is common in females and patients mainly present with anemia. Secondary cold AIHA was more common than primary and warm type of AIHA. Secondary AIHA was associated with connective tissue disorders.

*Corresponding author: Dr Srikanth Umakanthan, Department of Paraclinical Sciences, Pathology Unit, Building 5, Room 30, The University of the West Indies, Trinidad. email: dr.u.srikanth@gmail.com, phone: 1(868)- 473-0728

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Introduction Autoimmune haemolytic anemia (AIHA) is one of the most common causes of acquired haemolytic anemia caused by circulating antibodies against antigens on the red cell membrane. 1 Depending on the nature of the antibodies they are of two major types: warm antibody AIHA (IgG type) and cold agglutinin disease (IgM type). 2 Direct Coombs test is the cornerstone for diagnosis of AIHA. Clinical presentation is mainly attributed to the rate of hemolysis thereby, manifesting anemia like fatigue, breathlessness and also systemic symptoms like fever, joint pains and bleeding. 1 With this background, a clinico â&#x20AC;&#x201C; haematological profile of patients with AIHA were assessed using a retrospective study of 10 patients.

Materials and Methods A retrospective analysis was performed on patients diagnosed with AIHA by direct antibody test (DAT) from December 2012 to February 2013. The details pertaining to the disease status which included presenting complaints and physical examination findings were retrieved. The laboratory parameters including haematological, serological and immunological findings were then correlated. Other tests including bone marrow aspiration biopsy, lymph node biopsy and ANA profile was performed wherever necessary.

Result A total of 10 cases were evaluated. The age at presentation ranged from 26 to 80 years and there was a female predilection (M: F = 1:9). Fatigue and breathlessness was the commonest presentation (90%) in our patients. [Fig 1]Pallor was the most common (90%) physical examination finding. Organomegaly (splenomegaly, hepatomegaly and hepatosplenomegaly) was seen only in AIHA â&#x20AC;&#x201C; cold type. [Fig 2]

Fig 1: Modes of clinical presentation

Fig 2: Physical examination findings

Laboratory findings was correlated and in our study moderate anemia (Hb 6-10 gm%), raised indirect bilirubin (>2mg/dl), raised Lactate dehydrogenase (>450 Units) was observed in all ten cases. Raised MCV (>100 fl) was seen in 9 cases (90%) and raised ESR (>50mm/hr) was seen in 6 cases (60%). [Table 1]

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Figure 1: (A) Microscopy Low Power: RBC agglutinates seen. (B) High power : RBC agglutinates; (C) Microscopy peripheral smear: Spherocytes & Polychromatophils; (D). High power (40x): Leukoagglutinates seen (E). High power (40x): Nucleated RBC seen. (F).

Case of plasma cell dyscrasia: Bone marrow biopsy, Plasma cells and scattered hemosiderin laden macrophages seen

Morphological features on peripheral smear examination revealed RBC agglutinates, spherocytes and polychromasia.

Nucleated RBC and leukoagglutinates were also seen. [Figure 3: A-E] Serologically, Direct antiglobulin test (DAT) was positive in all ten cases (100%). Other tests included four cases (40%) of ANA positive, two cases (20%) of AntidsDNA positive and bone marrow biopsy in a case of plasma cell dyscrasia revealed abundant plasma cells.

This clinico-hematological correlation revealed that nine out of ten cases were of cold AIHA and one was of warm AIHA. Four out of ten cases are secondary AIHA associated with connective tissue disorder (two cases confirmed SLE).

Other cases were plasma cell dyscriasis with AIHA on bone marrow biopsy (1/10), [Figure 3: F] primary AIHA (1/10), essential thrombocytosis (1/10), malaria (1/10), Mycoplasma pneumonia (1/10) and hypothyroidism (1/10).

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Discussion Cold agglutinins are polysaccharide antibodies that react with the red cells at lower temperature (<37 0 C) and are almost always IgM type. This type of AIHA requires accurate diagnosis as it heralds a therapeutic challenge. Hence understanding its clinical manifestations and laboratory diagnosis is very much essential for accurate therapeutic options.

Few vital studies have been undertaken in Indian population on AIHA. Alwar et al, studied 175 cases, the commonest presentation was fatigue and breathlessness attributed to anemia (36%). Pallor (98%) was a universal finding. Serologically, 45% of primary AIHA cases showed both DAT and IAT positivity. Cold agglutinin titer was significantly raised in two cases.1 Choudhry et al, studied 21 cases, in which pallor (89%) was the most common feature, splenomegaly (81%) was higher than hepatomegaly (76%). DAT positivity was seen in 19 cases, while IAT was positive in 7 cases. 3 In a study by Baek et al, Coomb’s test was positive in 96% of the cases. Out of 32 cases, 32 were warm and 1 case was cold antibody type.

MCV was raised with range of 107 +/- 22. Thus MCV is considered an important index for AIHA. 4 The diagnosis of CAD mainly depends on typical DAT findings. Specific DAT for IgG is usually, but not always, negative.5,6 Other investigations include complement assessments (C3, C4 and CH50), electrophoresis with immunofixation, trephine biopsy including immunohistochemistry, flow cytometry of aspirate, chest X ray and abdominal ultrasonography.7,8 The cornerstone for CAD management is nonpharmacological measures which include avoiding cold exposure and increasing warm clothing. Pharmacological therapies are mainly aimed to suppress aberrant IgM protein production and mainly include corticosteroid therapy.9 Drugs such as Cladribine, Rituximab, Fludarabine and Eculizumab are all under evaluation. It is also important to note that splenectomy is contraindicated in CAD as hemolysis occurs outside the spleen.10 Conclusion In a retrospective analysis performed on 10 cases, AIHA was common in females and patients mainly presented with anemia. An increased MCV in a background of a haemolytic blood picture should raise a suspicion of cold AIHA. Secondary cold AIHA was more common than primary and warm type of AIHA. Secondary AIHA was associated with connective tissue disorders.

Acknowledgements: None.

Funding: None.

Competing Interests: None declared.

References 1.

Alwar V, Shanthala DAM, Sitalakshmi S, Karuna RK. Clinical patterns and haematological spectrum in autoimmune haemolytic anemia. Journal of Laboratory Physicians.2010;2(1):17-20 2. Berentsen S, Sundic T. Red Blood Cell Destruction in Autoimmune Hemolytic Anemia: Role of Complement and Potential New Targets for Therapy.Biomed Res Int. 2015;2015:363278. Epub 2015 Jan 29.

3. Choudhry VP, Passi GR, Pati HP. Clinico-hematological spectrum of auto-immune hemolytic anemia: an Indian experience. J Assoc Physicians India. 1996 Feb;44(2):112-4.

4. Baek SW, Lee MW, Ryu HW, Lee KS, Song IC, Lee HJ, Yun HJ, Kim S, Jo DY. Clinical features and outcomes of autoimmune hemolytic anemia: a retrospective analysis of 32 cases.Korean J Hematol. 2011 Jun;46(2):111-7.

5. Berentsen S1, Ulvestad E, Langholm R, Beiske K, Hjorth-Hansen H, Ghanima W, Sørbø JH, Tjønnfjord GE. Primary chronic cold agglutinin disease: a population based clinical study of 86 patients. Haematologica. 2006 Apr;91(4):460-6.

6. Genty I, Michel M, Hermine O, Schaeffer A, Godeau B, Rochant H. Characteristics of autoimmune haemolytic anemia in adults: Retrospective analysis of 83 cases. Rev Med Interne 2002;23:901-9.

7. Gertz MA. Cold agglutinin disease. Haematologica 2006;91(4):439-441.

8. Siberstein LE, Berkman EM, Schreiber AD. Cold hemagglutinin disease associated with IgG cold reactive antibody.

Ann Intern Med 1987;106:238 9. Berentsen S. How I manage cold agglutinin disease. Br J Haematol 2011;153(3):309-317 10. Berensten S. Complement, cold agglutinins, and therapy. Blood 2014:123:4010

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Case Report

Anaplastic Thyroid carcinoma: A Rare Case Mangala R Nagare*, Joshi Sneha R, Ashwini Karwande, Smita Pathak, Tekwani Deepa T, Janice Jaison Department of Pathology, MIMER Medical College, Talegaon (D), Pune, India Keywords: Anaplastic carcinoma, Goiter, Thyroid, Pathology

Abstract

Anaplastic carcinoma of thyroid is one of the most lethal tumours in humans. They are rare and usually occur in cases of long standing thyroid disease and other thyroid tumours. Prognosis is poor with a mean survival rate of about 6 months after diagnosis. However patients with small foci of anaplastic carcinoma, in well-differentiated thyroid tumours, that are excised completely, are reported to have better survival rates. Death is usually due to local recurrence or distant metastasis to lungs, bone or brain.

We report an interesting case of anaplastic thyroid carcinoma, in a 47 year old lady with longstanding co-existing goiter.

*Corresponding author: Dr Mangala R Nagare, C/502, Tejowalaya, Near Cipla Foundation, Warje, Pune-52, India e-mail: manalnagare@yahoo.co.in; Mobile: +91 9881099589,

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Introduction Anaplastic thyroid carcinomas (ATC) are the most lethal tumors in humans 1. Anaplastic thyroid carcinoma (ATC) is a rare malignancy, accounting for only 1to 2% of all thyroid cancers 2. It usually occurs in a case of long standing thyroid disease and other thyroid tumors3, 4. Prognosis is poor with mean survival rate of about 6 months after diagnosis4, 5. Here we report a case of anaplastic carcinoma of thyroid in a middle aged female with co-existing goiter.

Case Report A 47 yrs old female presented with a large midline neck swelling 5cm X 4cm moving freely upwards with deglutition since last 4yrs. Patient also complained of mild dysphagia. There was no history of pain, fever, altered voice or breathlessness. No history of sudden increase in size. No palpable cervical lymph nodes were noted. Patient was euthyroid clinically and thyroid function tests were within normal limits.

X ray neck showed an ill-defined soft tissue mass in pre tracheal space with multiple foci of calcification suggestive of goiter with minimal deviation of trachea. X ray chest was within the normal limits.

A blind fine needle aspiration cytology (FNAC) was done, 4cc of frank colloid was aspirated and diagnosis of colloid goiter with cystic change was given. However correlation with ultrasonography was advised to rule out any solid area but unfortunately it was not done.

A sub total thyroidectomy was performed and the specimen was sent for histopathological examination. Grossly the specimen was measuring 7x4x3 cm. and was externally unremarkable. On cut section one lobe was completely cystic containing colloid and other lobe showed well circumscribed encapsulated grayish white solid tumor surrounded by thyroid tissue (Fig 1, 2, 3). On microscopy sections from solid tumor mass showed tumor composed of large pleomorphic cells, few were spindle shaped arranged in groups, clusters and solid sheets and interlacing fascicles and showed atypical mitotic figures. Also seen were tumor giant cells, capsular invasion and vascular emboli. The tumor was surrounded by thyroid tissue showing changes of goiter (Fig 4 & 5). From the histopathological features the diagnosis of Anaplastic thyroid carcinoma was made.

Fig 1 Showing Specimen of subtotal thyroidectomy and Fig 2 Showing Specimen of thyroidectomy with one completely solid and other completely cystic lobe Fig 3 Tumor mass along with normal thyroid

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Fig 4 Photomicrograph showing anaplastic areas. Inset shows tumor giant cells.

Fig 5 Photomicrograph Showing Capsular invasion and inset shows vascular emboli The patient was lost to follow up for 4 months. After that patient came with complaints of right sided neck swelling and PET scan reports. The fluorin -18-FDG-(fluorodeoxyglucose)-positron emission tomography (18 F-FDG PET) scan showed small FDG avid right level II cervical lymph node. No evidence of any other FDG avid distant metastasis was present on whole body survey. Hence, a left hemithyroidectomy with right sided modified radical neck dissection was done & the specimen was sent for histopathology.

Gross: left hemithyroidectomy specimen measuring 4x3x0.5cm was received with two small lymph nodes. No obvious tumor was seen on grossly. Microscopy show colloid goiter and lymph nodes showed no evidence of tumor mass. Right sided radical neck dissection showed tumor mass measuring 4.5x4x2cm with surrounding muscles showing infiltration of the same tumor. On cut section the mass was friable, soft, grayish white with areas of necrosis (fig 3). On microscopy no lymph node tissue was identified. The tumor mass showed histological features of anaplastic thyroid carcinoma invading the muscles. So this whole tumor mass is the metastasis of Anaplastic Thyroid Carcinoma (ATC), which was diagnosed previously.

Discussion Anaplastic thyroid carcinoma usually presents in elderly patients as rapidly growing mass associated with hoarseness, dysphagia and dyspnoea5, 6. The sex distribution shows that females are affected more frequently than males with ratio of 1.5:16. In our case, the patient was a middle aged female with complaints of dysphagia.

Etiological factors including iodine deficiency, radiation exposure, preexisting thyroid disease, long standing goiter is a well-known risk factor for ATC3.In our case, the patient had history of goiter since more than 4 yrs. The tumor has higher incidence in regions of endemic goiter and history of goiter is reported in over 80% cases4.

The FNAC is reported to be 90% accurate in diagnosing ATC2. But in our case the swelling was diffuse and cystic and initial aspiration showed 4 cc frank colloid hence diagnosis was colloid goiter was made, which was misleading. Hence, even though on blind FNAC we get ample cellularity, a USG correlation and USG guided FNAC from solid areas should be mandatory in cases of solid-cystic lesions of thyroid.

ATC is an extremely lethal tumor seen in usually elderly but should be considered in any age group as in our case patient was a middle aged female. A close follow up of such patients is extremely mandatory as the tumor is

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highly aggressive and death is usually due to distant metastasis. In our case, the patient was young at the time of diagnosis, but showed the presence of metastasis in right sided radical neck dissection.

Grossly, ATC is usually fleshy and tan â&#x20AC;&#x201C; white with areas of hemorrhage and necrosis.

Microscopically, it is typically composed of a variable admixture of spindle cells, epithelioid cells, and giant cells.

Necrosis, vascular invasion and mitoses are quite prominent 4, 5. Similar findings were noted in our patient also.

Conclusion ATC is rare malignancy.2Imaging techniques and USG-guided FNACs will be helpful in diagnosis of thyroid lesions. A close follow up of such patients is extremely mandatory as the tumor is highly aggressive and death is usually due to distant metastasis.

Acknowledgements None.

Funding None.

Competing Interests None declared.

References 1.

N Bhat, Subramaniam Vinayak E. Anaplastic thyroid carcinoma with paraneoplastic syndrome. Bombay hospital Journal, 2006;48:655-658.

2.

Sze-How NG, Imisairi Ab Hadi. The aggressive anaplastic thyroid carcinomas: A case report and institutional review. World Journal of Endocrine surgery, Jan-April 2012; 4:20-22

3.

Peter Humphrey, Louis Dehner, John Pfeifer. Chapter 24, Thyroid, The Washington Manual Of Surgical Pathology. Edition 1st.415-416.

4.

Scott L Boerner, Sylvia L. Asa. Chapter 12 Squamoid, Spindle and giant cell lesions. Biopsy interpretations of thyroid. 2010, 227-252.

5.

Pathology of thyroid and parathyroid diseases. Sternbergâ&#x20AC;&#x2122;s Diagnostic Surgical Pathology. 5th ed; 518-519.

6.

N Ordonez, Z. Baloch et al. Pathology and genetics of Tumors of thyroid and Parathyroid, Undifferentiated (anaplastic) carcinoma. WHO 8; Tumors of endocrine organs.77-79 .

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Case Report

Persistent m端llerian duct syndrome: a case report Chandrahas R. Godbole*, Sneha Joshi, Janice Jaison Department of Pathology, MIMER Medical College, Talegaon Dabhade, Pune Keywords: Hernia uteri inguinale, Mullerian inhibiting factor, Persistent mullerian duct syndrome

Abstract

Persistent mullerian duct syndrome (PMDS) is usually an accidental finding either during orchipexy or during routine inguinal hernia repair in male patients presenting with maldescended or cryptorchid testes. It is caused by a defect in the mullerian inhibiting factor. Intraoperatively, mullerian remnants consisting of an infantile uterus and fallopian tubes are usually found. Familiarity with PMDS is necessary to diagnose the condition. We report a case of PMDS in a 45-year-old male presenting with right inguinal hernia.

*Corresponding author: Dr Chandrahas Godbole, Flat 1, Sukhada Society, Mahaganesh colony, Paud Road, Pune 411038 India email -chandrahasgod@yahoo.com; Mobile: +91-9850029749

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Introduction Persistent mullerian duct syndrome (PMDS) is a rare form of male pseudohermaphroditism, characterized by the presence of a uterus and fallopian tubesin genotypically and phenotypically normal males owingto failure of mullerian duct regression.[1] The syndrome is caused either by an insufficient amount of mullerian inhibiting factor (MIF) or due to the insensitivity of the target organ to the MIF. The diagnosis of PMDS is often established during operative treatment of associated abnormalities such as inguinal hernia and undescended testis in a genotypically and phenotypically normal male.[2]

Case Report A 45 year old male patient came with complaints of right inguinal hernia since 4 months. Hernia was repaired and inguinal canal contents were removed.

On dissection, uterus like structure along with a fallopian tube and mass resembling undescended testis were separated and were submitted for further histopathological evaluation. Grossly, Theuterus measures about 4.5 x 4 x2 cm in size and fallopian tube measures 7 cm. The testis measured 2.5x2x1 cm (Fig. 1 and 2)

Fig 1-Inguinal hernia contents- Uterus and testis: Fig 2-Inguinal hernia contents- Fallopian tube and testis.

Fig 3- Histopathology- Atrophic endometrium and myometrium; Fig 4. Histopathology â&#x20AC;&#x201C; undescended testis

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The section from the uterus showed atrophic endometrium which measured 0.1 cm in thickness. The myometrium was unremarkable (Fig. 3). The fallopian tube showed normal tubal histology. No ovarian tissue was found. Undescended testis was atrophic with tubular basement membrane thickening and Leydig cell hyperplasia (Fig 4)

Discussion PMDS is a rare form of male pseudo-hermaphroditism, characterized by the presence of a uterus and fallopian tubes owing to failure of mullerian duct regression in genotypically normal males. [1] Nilson described the condition in 1939 and termed it as hernia uteri inguinale .[3] American NationalInstitute of Health estimates that there are less than 200000 cases of PMDS in US.Exact incidence in India is not known.[3] In a human foetus, both mullerian and wolffian ducts, the anlagen of the female and male reproductive tracts, respectively, are present at 7-week gestation.

The normal sex differentiation in males is controlled by testosterone and MIF. Testosterone has a direct local effect on the wolffian ducts, including differentiation into the epididymides, vas deferens, and seminal vesicles. Also the formation of the urogenital sinus and male external genitalia requires in situ conversion of testosterone into dihydrotestosterone. Despite the normal male genotype and the subsequent normal development of foetal testis, if there is a failure in production of MIF or insensitivity of the target organ to MIF, Mullerian structures do not regress.

Since the secretion and action of testosterone is not affected, the Wolffian (mesonephric) duct derivatives and the external genitalia of the foetus progress in the normal male direction. An intersex condition is therefore not usually suspected. But the malformation is incidentally detected during operative treatment of associated abnormalities such as an inguinal hernia or an undescended testis, generally in the first year of life. Henceforth, the diagnosis of PMDS is often established when a uterus and/or fallopian tube is found along with undescended testis in a genotypically and phenotypically normal male child.

In PMDS, the testes are usually histologically normal, apart from lesions due to longstanding cryptorchidism. The overall incidence of malignant transformation in these testes is 18%, similar to the rate in abdominal testes in otherwise healthy men.[4] Clinically, PMDS cases are divided into three categories: 1. Majority (60–70%) with bilateral intra-abdominal testisin a position analogous to ovaries.

2.Smaller group (20–30%) with one testis in thescrotum, associated with contralateral inguinal herniawhose contents are testis, uterus and tubes (classicalpresentation of hernia uteri inguinale).

3. Smallest group (10%) where both the testes are located inthe same hernial sac along with the müllerian structures(transverse testicular ectopia - TTE). PMDS accountsfor 30–50% of all cases of TTE.[3] Our case is a classical presentation of PMDS- hernia uteri inguinalewith one testis in thescrotum, associated with contralateral inguinal herniawhose contents are testis, uterus and fallopian tube.

Conclusion In cases of unilateral or bilateral cryptorchidism associated with inguinal hernia, as in our patient's case, the possibility of persistent Mullerian duct syndrome should be kept in mind in order to prevent further complications such as infertility and malignant change.

Acknowledgements None.

Funding None.

Competing Interests http://www.pacificejournals.com/aabs

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None declared.

References 1.

2.

3.

4.

Nayak VJ, Kamath AS, Krishnappa MH, Bylappa SK. Persistent mullerian duct syndrome: A case report and review of the literature. Int J App Basic Med Res 2014 ;4:125-7 Nishikant N Gujar, Ravikumar K Choudhari, Geeta R Choudhari, Nasheen M Bagali, Harish S Mane, Jilani S Awati and VipinBalachandran.Journal of Medical Case Reports 2011; 5:586 Manjunath BG,Vasanth G. Shenoy , Preetham Raj. Persistent müllerian duct syndrome: How to deal with themüllerian duct remnants - a review. Indian J Surg 2010; 72:16–19 Shrinivasan Chamrajan, Nidhi H Vala, Jatin R Desai, Niraj N Bhatt. Persistent mullerian duct syndrome in a patient with bilateral cryptorchid testes with seminoma. Journal of Human Reproductive sciences. 2012 ; 52:215-217.

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Case Report

Isolated cysticercus cellulosae of medial rectus muscle presenting with a mass over the inner region of the eye: a rare case report and treatment review Jagriti Rana1, S. P. Singh1, Vijay Kumar Jha*2, S. Khanduja2 1

Department of Ophthalmology, Moti Lal Neharu Medical College, Allahabad, India Department of Pathology, Institute Of Medical Sciences, Banaras Hindu University, Varanasi, India 3 Department of Radiodiagnosis, Moti Lal Neharu Medical College, Allahabad, India 2

Keywords: Cysticercosis, Extraocular muscle, Ultrasound biomicroscopy

Abstract

Ocular involvement with or without brain involvement is common in Cysticercosis but isolated Cysticercus cellulosae of medial rectus muscle is very rare. Herein we are presenting a case of isolated cysticercosis of medial rectus in young male which was diagnosed on radiology and responded well to albendazole and predinisolone.

*Corresponding author: Dr. Vijay Kumar Jha, Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, INDIA Email: vj.bhu07@gmail.com

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Introduction Cysticercosis is caused by acquiring infection with the larval stage - cysticercus cellulosae of Taenia solium. Cysticercosis is prevalent in developing countries of Latin America, Indian subcontinent, Africa and China especially in areas of poor sanitation1. Ingestion of contaminated food especially undercooked pork, contaminated water and infected vegetables are sources of infection. Ocular involvement occurs in 1.8 to 4.5% of the cases2. Ocular adnexal involvement is exceptionally rare3. Orbital neurocysticercosis shows extremely varied clinical presentations like headache, diplopia, restriction of movements, mass, ophthalmic migraine and rednees4. We, hereby present a case of 20 year old male from eastern UP who presented with an adnexal mass over the inner region of the right eye.

Case Report A young male of 20 years presented in the out patients department of ophthalmology in SRN Hospital, Allahabad with the chief complaint of red mass over the right eye for 3 months which was gradually increasing in size with foreign body sensation. The swelling was not associated with protrusion of the eyeball, diminition of vision, diplopia, pain, seizures, nausea, vomiting and dizziness. There was no history of antecedent trauma to the right eye.

On examination no proptosis was appreciated. The ocular movements were within normal limits except slight restriction of adduction in right eye. The visual acuity and the intraocular pressure in both the eyes were normal. There was a well circumscribed reddish nodular mass near the medial canthus of the right eye which was 8 x 12 mm in size. Conjunctiva over the mass was congested. Fundus examination of both the eyes was normal.

Investigations: All routine investigations including haemoglobin, ESR, blood sugar, blood urea and serum creatinine were within normal limits. Total leucocyte count was high (12,300/µl) with neutrophilia( polymorphs – 83%). Eosinophil count was within normal range (2%). Stool examination did not show any ova or cyst. Central nervous system examination revealed no abnormality.

Imaging studies: Ultrasonography – B - scan of right eye showed a cystic lesion measuring around 10 x 9 mm in size with hyperechoeic speck (scolex) within it, over the medial rectus muscle which appeared as hyperechoeic thickening.

Rest of the vitreous, retina, the optic disc and other extra ocular structures were within normal limits.

Ultrasound biomicroscopy (UBM) of right eye revealed a cyst of dimension 12.73 x 9.28 mm. There was a hyperechoeic mass inside the inner wall of the cyst measuring 5.46 x 8.26 mm. Hyperechoeic mass showed hyperechoeic speck ( scolex ) of dimension 2.44 x 5.93 mm. CT scan of the brain and right eye showed a cystic lesion of about 8 x 13 mm in size with the thickening of the medial rectus muscle. Brain parenchyma did not show any cyst or features of neurocysticercosis.

Treatment Review: The patient was kept on Albendazole 400 mg daily along with oral prednisolone 10 mg per day for four weeks. Patient improved gradually and the mass resolved. A repeat B – scan and UBM done after four weeks showed loss of the scolex, collapse of the cyst wall and regression in cyst size with return of ocular motility to normal.

Fig -1:- Ultrasound biomicroscopy of right eye showed a cystic mass in the rectus muscle.

Fig-2:- Right orbital ultrasound biomicroscopy (A & B scan) showed a well defined cystic lesion with eccentric hyperechoic area suggesting scolex (Cyticercus).

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Discussion Cysticercosis is a common problem in developing countries of South East Asia, Africa, South America and Eastern Europe1. Despite the high incidence of brain and ocular involvement in cysticercosis, extraocular muscle cysticercosis is very rare5. It commonly affects children and young adults 6. Medial rectus is the most commonly involved extra ocular muscle7, however in another study inferior rectus was the commonest muscle involved 1. In our case medial rectus muscle was involved. Common clinical presentataions include – diplopia with motility restriction, proptosis, orbital cellulitis, ptosis and pseudotumor formation.

Eosinophilia, inflammatory markers and ELISA for cysticercus specific antibodies are insensitive markers1,7. Orbital imaging is the key for diagnosing myocysticercosis. The presence of high amplitude spikes corresponding to the cyst wall and scolex on A – scan ultrasonography or an intracystic scolex on B – scan ultrasonography or computed tomography scan is diagnostic1,7.

We chose orbital sonography (B - scan) as both diagnostic modality and for assessing the cyst on follow - up visits as it was most cost effective. Ultrasound and CT both are comparable in ability to detect scolices 1. The CT scan of orbit and head was undertaken to substantiate the initial diagnosis. However CT may be preferable because intracranial CT can identify cerebral cysticercosis, the incidence of which is as high as 16.7% in a case series of myocysticercosis 8.

Early treatment with Albendazole and adjunctive systemic steroid is highly effective for treatment of myocysticercosis.1,6,8,9.

Death of parasite evokes strong inflammatory response secondary to release of toxins which is effectively suppressed by systemic steroids5. Some trials evaluating the clinical efficacy of Albendazole on extraocular motility outcome have demonstrated cyst elimination rate of more than 90% and the time to recovery of ocular motility ranging from 3 to 35 months7,9.

There are some contrasting reports describing limitations of ocular motility after albendazole therapy1,10 but failure to administer adjunctive steroid may explain the poorer outcome of these studies. A shorter duration of follow up could also be one of the reasons why a favourable outcome and extraocular motility was not observed 10.

Surgical excision of an extraarticular muscle cyst has been described 5. However, the potential role of damage to adjacent tissue and adhesion from surgical exploration should not be taken lightly particularly when effective medical therapy is available. Our patient responded well to albendazole with significant improvement of ocular movement in four weeks.

Conclusion Thus our study concludes that diverse clinical presentations of myocysticercosis mandates a high degree of clinical suspicion especially in a patient coming from endemic areas. Ultrasonography, CT scan and MRI help to substantiate the diagnosis. Stool examination should be recommended for all members of the family to detect asymptomatic carriers so that treatment with albendazole could be started.

Acknowledgements None.

Funding None.

Competing Interests None declared.

References 1.

2.

3.

4.

5.

6.

Sekhar GC, Honavar SG. Myocysticercosis: experience with imaging and therapy. Ophthalmology 1999; 106:2336 – 2340.

Wadia NH. Neurocysticercosis. Neurological practice in Indian prospective 2005;215-51.

Chandrashekhar G, Lemke BN. Orbital cysticercosis. Ophthalmology 1997;104:1599-604 Malik SRK, Gupta AK, Choudhary S. Ocular cysticercosis: Am J of Ophthalmol. 1968; 66:1168 – 71.

Shadangi P, Saggar V, Mittal SR, Singla R, Kumar R. Isolated Cysticercosis of inferior rectus muscle presenting with eccentric proptosis – a rare case report and treatment review. Journal of clinical and diagnostic research 2010; 4:2536-2539.

Puri P, Grover AK. Medical management of orbital myocysticercosis: a pilot study. Eye 1998; 12:795 -9.

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7.

Pushker N, Bajaj MS, Chandra M, Neena. Ocular and orbital cysticercosis. Acta Ophthalmol Scand 2001;79:408-13.

8. Sihota R, Honavar SJ. Oral albendazole in management of extraocular cysticercosis. British Journal of Ophthalmology. 1994, 78: 621-23.

9. Pandey PK, Chaudhari Z, Sharma P, Bhomaj S. Extraocular muscle cysticercosis masquerade. J pediatr Ophthalmol strabismus 2000; 37:273-8.

10. Kumar M, Saroha V, Sharma A, Pandav S, Singh U. Extraocular muscle cysticercosis: clinical presesntation and outcome of treatment. J Pediatr Ophthalmol Strabismus 2005; 42: 28-33

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Case Report

Acute Eosinophilic Appendicitis: Case report of three cases with brief review of literature Rajeshwari K*1, N.V. Dravid1, Karibasappa GN2 , Akshay Surana1 1

Department of Pathology ACPM Medical College, Dhule, Maharashtra, India Department of Public Health Dentistry, ACPM Dental College and Hospital, Dhule, Maharashtra, India.

2

Keywords: Eosinophils, Appendix, Eosinophilic appendicitis.

Abstract

Acute eosinophilic appendicitis (AEA) is a rare variant of appendix inflammation. The histologic hallmark of this entity is eosinophilic infiltration of the musularis layer with accompanying oedema separating the muscle fibres with out neutrophilic infiltration.

To the best of our knowledge there are very few cases of eosinophilic appendicitis (EA) in the absence of any other abnormality reported in the literature. Hence, we made an attempt to stud y the cases of AEA and to draw the relevant conclusion about the disease pathogenesis. Out of total 159 appendectomy cases, three cases were found to have eosinophilic appendicitis (EA) and these cases were studied for clinical and pathological findings. Incidence of 0.02% (3/15) with male preponderance was found. We herein, present the cases of AEA which is rare variant and less understood entity.

*Corresponding author: Dr. Rajeshwari K, Assistant Professor, Department of Pathology ACPM Medical College, Dhule, Maharashtra, India Email:ujwalgk@gmail.com; Mob : 91750768899

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Introduction Acute eosinophilic appendicitis (AEA) is a rare clinical entity. It is characterized by acute presentation and grossly inflamed appendix with absence of neutrophils in the muscle layer. The histologic hallmark of AEA is eosinophilic infiltration of the muscularis propria with accompanying oedema separating the muscle fibres. [1] Eosinophilic appendicitis is less well understood disease entity which needs detailed study of reported cases and clear cut defining criteria.

There are very few studies with respect to observation of eosinophilic infiltration of the muscularis propria in the absence of any other abnormality. [2,3] Hence in the present study an attempt was done to study the cases of AEA

Case Report A total of 159 appendectomy specimens were received in the department of pathology during the study period of June 2013- January 2015. All the cases of appendicitis were screened and those cases fulfilling the criteria of eosinophilic appendicitis were included in the study. Criteria to diagnose EA used were transmural eosinophilic infiltrate in the wall of appendix, more than 25 eosinophils per high power field in muscularis mucosa, absence of polymorphs or any other pathology in the wall. [4] Routine investigation like complete blood count (CBC), peripheral blood smear,urine and stool examination was carried to rule out peripheral blood eosinophilia and parasitic infestation. [5] Out of total 159 appendectomy specimens.

Three cases of AEA were found accounting for an incidence of 0.02% (3/159). All the three patients were extensively studied by taking detailed clinical clinical history, physical examination and relevant investigations to rule out any allergic pathology and worm infestation.

Case 1: Twenty five years female complained of recurrent pain abdomen in the right iliac region just below the umbilicus since 6weeks. Clinically the case was diagnosed as sub-acute appendicitis. Grossly the appendix was enlarged, oedematous and congested. Cut section revealed patent lumen. On microscopic examination the case was categorized as AEA. Case 2: Fourty yearâ&#x20AC;&#x2122;s male presented to surgical outpatient department with recurrent pain abdomen and vomiting since two weeks. Patient was evaluated for intestinal obstruction. Radiological examination showed signs of small bowel obstruction. Laprotomy was done with resection and anastomosis and also found that the appendix was inflamed, congested and edematous insitu, hence appendectomy was done.

Grossly the resected appendix was enlarged, congested and on cut section showed lumen obliteration. On histopathology the case fulfilled the criteria of AEA. Case 3: Sixty seven years male patient presented with acute abdominal pain, vomiting and abdominal distention since two days. On admission to the casualty the patient condition started deteriorating and was haemodinamically unstable. Radiological evaluation showed features of pneumatosis intestinalis with haemoperitonium. Clinical diagnosis of intestinal perforation with signs of peritonitis was made. Emergency exploratory laprotomy was performed and revealed enlarged, oedematous and perforated appendix with haemoperitonium.

Grossly the resected appendix was oedematous, congested and enlarged measuring 5cm in length with perforation in the wall of the appendix measuring 0.1x0.2 cm. Cut section showed obliteration with mucous secretion (figure 1). On histopathology showed features of AEA (figure 2 and 3) with signs of peritonitis.

Post operatively all the three patients were advised follow up CBC with absolute eosinophil count, upper gastro- endoscopy of the gastrointestinal tract (GIT) for tissue eosinophilic infiltration and stool examination to rule out parasitic infestations. During the follow up period of one year, upper GI endoscopy and biopsy was performed which showed no eosinophilic infiltration in the stomach.

Age range of the patients was between 25-67years (table 1). Mean age of presentation was 44years. None of the case had history of allergic disorder or peripheral smear eosinophilia/tissue eosinophilic infiltration. In all the three cases stool examination for parasite was negative.

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Figure 1: Gross photograph of appendectomy specimen showing enlarged, oedematous and obliterated lumen with mucous secretions.

Figure 2 â&#x20AC;&#x201C; Microphotograph of appendicular mucosa showing ulceration with dense and diffuse infiltration of eosinophils in all coats (H and E, x100).

Figure 3 - Microphotograph of muscularis propria showing dense and diffuse infiltration of eosinophils (H and E, x400).

Table 1 Clinical summary of three cases in acute eosinophilic appendicitis.

Case

Age/Sex

Clinical diagnosis

Peripheral blood eosinophilia

Stool Examination

Gross features

1.

25/female

Sub-acute intestinal obstruction

Absent

Negative

Oedema and congestion

2.

40/male

Intestinal obstruction

Absent

Negative

Oedema and congestion

3

67/male

Intestinal perforation with signs of peritonitis

Absent

Negative

Oedema congestion and perforation in the wall of appendix

Discussion AEA was first proposed by aravindan in 1997 [6] and defined by aravindan et al in 2010. [1] He proposed that eosinophilic infiltration is an early event of appendicitis and represents the part of a type 1 hypersensitivity reaction to an allergen and primary pathologic changes characterized by eosinophilic oedematous lesion in the appendix. [1] These observations are novel and hypothesis requires further testing.

If the lesion is infected by bacteria, acute suppurative appendicitis occurs and if there is no infection, AEA occurs. The blood eosinophil count first increases and then decreases over time in cases with acute suppurative and eosinophilic appendicitis. [1] On the contrary, eosinophilia persists and does not resolve over time in eosinophilic gastroenteritis cases.[5] Thus, AEA should be evaluated as a variant of acute appendicitis rather than an extension of eosinophilic ga-

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stroenteritis. [1, 6] Norman J Carr suggests that an eosinophil count in excess of 10 per mm2 (25 per 10 HPF) could be abnormal. He also states differential diagnosis for this eosinophilic infiltrate as eosinophilic enteritis and infestation by parasites. [3] In eosinophilic enteritis depending upon the involvement of different layers of intestinal wall, symptoms may vary.

The mucosal form of eosinophilic enteritis (most common variant) is characterized by vomiting, abdominal pain, and diarrhea, blood loss in stools, iron deficiency anemia, malabsorption and protein loosing enteropathy. The muscularis form is characterized by infiltration of eosinophils predominantly in muscle layer leading to intermittent obstructive symptoms and with complications like aspiration and perforation as in one of our case of eosinophilic appendicitis.

Serosal form is characterized by exudative ascitis with intense peripheral eosinophilia. [7] Because the pathogenesis and etiology of the disease is not well understood, no standard for the diagnosis of eosinophilic enteritis exists. Tally et al [5] have identified three main diagnostic criteria: i. Presence of gastrointestinal symptoms. ii. Biopsies demonstrating eosinophilic infiltration of one or more areas of gastrointestinal tract (GIT). iii. No evidence of parasitic/extrinsic disease.

Peripheral eosinophilia has been reported in upto 80% of the cases by Tally et al. [5] However, the definite diagnosis of eosinophilic enteritis requires histological evidence of eosinophilic infiltration. [7] Steroid is the mainstay of treatment of eosinophilic enteritis and sodium chromoglycate, catotifen, montelucast may be tried. Complicated case with obstruction and perforation requires surgical intervention. Otherwise surgeon should avoid unnecessary surgical intervention in case of eosinophilic enteritis. In case of parasitic infestation it results in tissue injury and local irritation in the gastrointestinal tract and cure is possible with medical therapy. Hence once eosinophilic appendicitis is being diagnosed, the patient should be completely evaluated for eosinophilic enteritis and parasitic infestations.

In our study all the three cases showed eosinophils in all the layers including muscularis propria leading to obstruction and perforation. None of the case revealed peripheral blood eosinophilia/tissue eosinophilic infiltration of >1 site of GIT which was ruled out by history, peripheral blood smear examination and by upper GI endoscopy. The cases also showed negative for parasite in stool examination. During the follow up period upper gastrointestinal endoscopy with biopsy from two to three areas, were performed which showed no eosinophilic infiltration.

Conclusion Acute eosinophilic appendicitis is a rare event and less well understood entity and an early marker of acute appendicitis.

Hence if patient undergoes laprotomy for various etiologies and if appendix found congested should be removed.

Adopting such therapeutic modality can prevent future occurance of acute appendicitis and hence need for appendectomy later in life. It is important to study AEA cases in detail for better understanding of disease pathogenesis and its significant role in patient management.

Acknowledgements: None.

Funding: None.

Competing Interests: None declared.

References 1.

2.

3.

4.

5.

6.

7.

Aravindan KP, Vijayavaghvan D, Manipadam MT. Acute eosinophilic appendicitis and significance of eosinophils- edema lesion. IJPM.2010; 53(2): 258-261.

Norman J, Leslie H. Appendix. In: Weidner N, Cote R, Suster S, Weiss LM . Modern surgical pathology. 2nd ed.

Saunders Elsevier. 2009 vol.1. pp. 837-852.

Carr NJ. The pathology of acute appendicitis. Ann Diag Pathol 2000;4: 46-58.

Gaurav Jain, Sujata R Kanetkar. Eosinophilic appendicitis: Case report of five cases & review of literature. Journal of medical research and practice 2013; 2(8):208-211.

Tally NJ, Shroter RG, Philips SF, Zinsmeister AR. Eosinophilic gastroenteritis: a clinicopathological study of patients with disease of the mucosa, muscle layer and the subserosal tissue. Gut.1990:31:54-58.

Aravindan KP. Eosinophils in acute appendicitis: Possible significance. IJPM.1997; 40(4): 491-498.

Rafiq A. Sheik, Thomas Prindiville, Shagufta Yasmeen, Boris H. Ruebner. Eosinophilic Gastroenteritis. JK- Practitioner 2003; 10(1):1-3.

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Case Report

Hemoglobin H Disease: A rare case report and its diagnostic challenge R. Sindhu*, S.K. Behera, D.P. Mishra Department of pathology, MKCG Medical College, Berhampur, Odisha, INDIA. Keywords: Pregnancy, HbH disease. Supravital staining. Thalassemia, Hemoglobin

Abstract Thalassemia the most common monogenic gene disorder in the world is especially frequent in Mediterranean countries. Hemoglobin H (Hb H) disease is a special variant of Îą-thalassemia presenting as microcytic hypochromic anemia. The clinical phenotypes of most individuals remain unnoticed unless there occurs an acute hemolytic crisis and they are most often under-diagnosed or misdiagnosed as iron deficiency anemia. Here we report a case of 34 year old pregnant women who presented with pallor and mild splenomegaly.

Complete blood count (CBC) showed decrease in Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) and increase in Red Cell Distribution Width (RDW) and reticulocyte percent. Peripheral smear showed microcytic hypochromic Red Blood Corpuscles (RBCs) with severe anisopoikilocytosis. Supravital staining of peripheral blood showed Hb H inclusions in RBCs. The increase in reticulocyte percent here is due to mature RBCâ&#x20AC;&#x2122;s having Hb H inclusions that are misinterpreted as reticulocytes by automated cell counters. Hence, in a background of variable clinical presentation of this disease, we consider this case to highlight the importance of simple peripheral smear examination and supravital staining of peripheral blood in the diagnosis of Hb H disease which are often misdiagnosed as iron deficiency anemia by seeing CBC results. It is also important to emphasize the importance of early diagnosis of these cases to facilitate implementation of proper preventive health care measure, ensure fetal well being and prompt treatment of potentially serious hemolytic crisis that can occur during pregnancy.

*Corresponding author: Dr. R. Sindhu MBBS, Post graduate in Department of pathology, MKCG Medical College, Berhampur, Odisha, 760004, INDIA. Email Id: sindhushankar51@gmail.com, Ph: 07854831852

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Introduction Hb H disease is a special variant of α-thalassemia that is more prevalent in South East Asia, Middle East and Mediterranean and in Indian subcontinentbecause of high carrier frequency of (--SEA) type of α-thalassemia deletion gene [1]. The clinical phenotypes of most individual with Hb H disease are very mild and may not be noticed during his/her entire life unless a routine full blood count is done or when there is an acute hemolytic crisis in conditions like infections, pregnancy like in our case where anemia is not improved even after iron/folic acid therapy. It is virtually important to carry out prospective and in depth studies of pregnancy in women with Hb H disease to define the risk and criteria for treatment. Another important issue is related to testing the women’s partner and genetic counselling [2].

Case Report A 34 year-old pregnant women came for her general checkup to our hospital. She had a previous history of cesarean section and 3 units of blood transfusion during her last pregnancy. During the present pregnancy she presented with pallor and mild splenomegaly. Her Hb started decreasing in-spite of taking regular oral iron/folic acid therapy. She has been treated with repeated blood transfusions. In between her pregnancies she was absolutely asymptomatic.There was no family history and her first child was apparently normal.Aroutine complete blood analysis was done using automated cell counterand report as follows: Hb- 8.4gm%, MCV-57.5fl, MCH-17.8pg, MCHC-31.0g/dl, Total RBC count -4.71×109/cu.mm, RDW-28.2%. Total Leucocyte Count -11,700/cu.mm and platelet count-1.6lacs/cu.mm, Reticulocyte count-6%.Peripheral smear showed microcytic hypochromic RBC’s with severe anisopoikilocytosis. Good number ofteardropcells, elongated cells and target cells were seen (Figure-1). Few cells show basophilic stippling.WBC showed neutrophilia with toxic granules. Supravital staining with 1% brilliant cresyl blue of peripheral blood showed Hb H inclusions (Figure-2). Hb electrophoresis by agarose gel showed thick band in “A” region. High Pressure Liquid Chromatography (HPLC) showed presence of Hb H. With these findings a diagnosis of α- thalassemia (Hb H disease) was made.

FIGURE 1: Microcytic and hypochromic RBCs with anisopoikilocytes- tear drop cells, target cells.(Leishman Stain, 400X) FIGURE 2: Cells showing golf like inclusion bodies in new methylene blue stain (x400X).

Discussion Thalassemia is perhaps the most common single-gene disorder in the world. More than 50% of the population appears to have a clinically silent form of α-thalassemia. HbH disease is recognized with increasing frequency in the eastern oasis of Saudi Arabia[3]. Around 300,000-400,000 severely affected infants are born every year, more than 95%, of who are in Asia, India, or the Middle East. Hb H disease results from the presence of only one functional α gene, usually as a consequence of the compound heterozygous state for α 0-thalassemia/α+-thalassemia (–/-α or –/αTα).

It occurs due to deletion or non-deletion mutations in α-gene on chromosome 16.

The disease presents with different phenotype ranging from asymptomatic to need for periodic transfusions, and fatal fetal hydropsfetalis in utero [4].

The severity is high in case of non-deletion mutation, constant spring mutation and if α 2 gene is affected [5]. When the level of α globulin gene synthesis falls below 70% of normal in adult life the excess βglobulin chains form tetramers of Hb H in the cell.The typical inclusion-body cells have a golf-ball like appearance with stippling regularly distributed over a blue stained background.

HbH has a high affinity for oxygen. These Hb H inclusions bind with the band 3 protein in RBC cell membrane and causes damage mostlyto mature red cells and a lesser extent to erythroid precursors, leading mainly to hemolysis and minimally to ineffective erythropoiesis. Certain conditions like infections, pregnancy and intake of certain oxidant drugs increases the Hb H inclusions leading to acute hemolytic crisis and precipitous drop in Hb. All affected individuals have a variable degree of anemia, decreased MCV and MCH and normal or slightly reduced level of Hb A 2.

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The amount of Hb H may be variable ranging from 1-40%. This HbH (fastest Hb) may escape from detection because of its high instability. Molecular analysis is required to confirm the hematological observation.

The important differential diagnosis that to be considered is iron deficiency anemia the most common anemia in pregnancy in India which also presents with decreased MCV, MCH, MCHC and increase in RDW and reticulocyte percent when on treatment. Since the Hb of the patient drops down when she was on iron therapy, the increase in reticulocyte percent couldnot is due to iron therapy. It is due to mature RBCs containing Hb H inclusions that are regarded as reticulocytes by cell counters. This entitles the importance of detailed peripheral smear examination along with a supra-vital staining showing Hb H inclusions in the diagnosis of Hb H disease and provides a way for other investigations like genetic studies for further evaluation. If diagnosed early it prevents from other unnecessary tests and false treatment to the patient with iron therapy. Other conditions where we could find Hb H inclusions are erythroleukemia and myelodysplasia. But the clinical condition and investigations done does not correlate with the above conditions.

It is also important to identify couples at the earliest who are at risk of conceiving fetus with Hb H disease. When one parent carries αo thalassaemia (--/αα) and the other carries an α+ thalassaemia (-α/αα) the risk of their offspring having HbH disease is 1:4 (25%). If the carrier of α+ thalassaemia is a homozygote clearly the risk of HbH disease is 1:2 (50%) [6]. Therefore, in these cases prenatal diagnosis should be done. It can be made by fetal DNA analysis at the earliest by 9-12 weeks by Chorionic Villous Sampling. DNA based diagnosis are highly accurate and specific.

HbH disease places fetuses at significant risk for growth restriction, preterm birth, and low birth weight, resulting in increased perinatal mortality [7]. Since both pregnancy and thalassemia are associated with a higher risk of thrombosis due to a hypercoagulable state, pregnant women with HbH disease will theoretically be at higher risk for thromboembolism [8]. Common obstetric complications, such pre-eclampsia, antepartum hemorrhage, and postpartum hemorrhage have been noted in some studies.

Conclusion HbH disease is an under-diagnosed entity in the Indian subcontinent. We feel that a careful evaluation for Hb H inclusions on reticulocyte preparation would help in diagnosing these cases. The early diagnosis of these cases will also facilitate implementation of proper preventive health care measure, ensure fetal well being and prompt treatment of potentially serious hemolytic crisis and infection and also to heighten the awareness of other devastating α and β thalassemia syndromes in the community.

Acknowledgements None.

Funding None.

Competing Interests None declared.

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Chui DHK, Waye JS. Hydropsfetalis caused by α-thalassemia: an emerging health care problem. Blood. 1998; 91:2213-2222.

David H. K. Chui, SuthatFucharoen, and Vivian Chan. Hb H disease: not necessarily a benign disorder blood, 2003 volume 101, number 3.

Munchi N, De Silva V, While IM. The frequencies of HbS α and β-thalassemia in Saudi Arabia Preliminary national values. Saudi Med J 1989; 10:62–65.

Mirabile E, Samperi P, Di Cataldo A, Poli A, La Spina M, Schilirb G. Phenotype-genotype correlation in Sicilian patients with Hb H. Eur J Haematol. 2000; 65:306-309.

Fucharoen S, Winichagoon P, Pootrakul P, et al. Differences between two types of Hb H disease, alpha-thalassemia 1/alpha-thalassemia 2 and alpha-thalassemia 1/Hb constant spring. Birth Defects Orig Artic Ser 1987;23: 309–315.

Cornelis L Harteveld, Douglas R Higgs. α- thalassemia Harteveld and Higgs Orphanet Journal of Rare Diseases 2010, 5:13.

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