Conscience & Art Núm. 4 by ConScienceandArt

VOLUMEN 2• NÚMERO 2 •ENE-ABR 2015

Inmunología

Microvesicles and the immunologic system

Sociología de la Ciencia Oncohematología Medicina Interna

Cancer Research & Bioinfomatics

Directorio Instituto Politécnico Nacional Enríque Fernández Fassnatch Director General Julio G. Mendoza Álvarez Secretario General Miguel Angel Álvarez Gómez Secretario Académico José Guadalupe Trujillo Ferrara Secretario de Investigación y Posgrado Jorge Edgar Puga Álvarez Coordinador Comunicación Social Mario González Pacheco y Morales Director interino de la ENCB Manuel Piñón López Subdirector Académico Griselda Ma Chávez Camarillo Jefa Interina de la SEPI

“La Técnica al Servicio de la Patria” www.ipn.mx

Editor Responsable Editor Dr. Luis Antonio Jiménez Zamudio Cuerpo Editorial Editorial Board Dr. Alejandro Francisco-Cruz M. en C. Félix Matadamas Martínez Dra. Georgina Filio Rodríguez Dra. Jacqueline Liszeth Oliva Ramírez Dr. Moisés Talavera Paulin M. en C. Pablo Núriban Valero Pacheco M. en C. Violeta Álvarez Jiménez M. en C. Violeta Castro Leyva M.C. José Pablo Romero López M.C. Martha Carnalla Cortés M.C. Nayar Alejandro Durán Hernández M. en C. Bibiana Ruíz Sánzhez Traducción Roger Milton Rubio Sánchez Consejo Editorial Editorial Advisers Dra. Isabel Wong Baeza ENCB-IPN, México Dra. Iris Estrada García ENCB-IPN, México Dra. Jeanet Serafín López ENCB-IPN, México Dr. Javier Sánchez García ENCB-IPN, México Dr. Rommel Chacón Salinas ENCB-IPN, México Dr. Rubén López Santiago ENCB-IPN, México Dra. Julieta Luna Herrera ENCB-IPN, México Dr. Jorge Barrios Payán INCMNSZ-SSA, México Dra. Dulce Adriana Mata Espinosa INCMNSZ-SSA, México Dr. Citlaltepetl Salinas Lara INNN-SSA, México Dra. Lourdes Arriaga Pizano CMN Siglo XXI, México Comité de Arbitraje Arbitration Commitee Dra. Esther Orozco Cinvestav-IPN, México Dr. Sergio Estrada Parra ENCB-IPN, México Dr. Germán Chamorro Cevallos ENCB-IPN, México Dr. Arturo Reyes-Sandoval Jenner Institute, University of Oxford, Reino Unido

Art Fortino Vázquez Arellano Dirección de Arte y Diseño Art and design director Nancy J. Tiol Colaboraciones Diseño Lizeth Arellano Aaron de la Vega Lara Publicación con arbitraje por pares ConScience &Art, beyond the method. Volumen 2, Número 1, enero-abril de 2015 es una publicación cuatrimestral editada por el Instituto Politécnico Nacional a través de la Escuela Nacional de Ciencias Biológicas: Prolongación de Carpio y Plan de Ayala SN, Colonia Santo Tomás, CP: 11340, Delegación Miguel Hidalgo, Distrito Federal, Tel: 57296000, Ext. 62367, http://conscienceandart.com, correo electrónico:conscienceandart@gmail.com. Editor responsable: Dr. Luis Antonio Jiménez Zamudio. Reservas de Derechos al Uso Exclusivo del número: 04-2014-071612423300203, ISSN: en trámite, ambos otorgados por el Instituto Nacional de Derechos de Autor. Responsable de la última actualización de este número: M.C. José Pablo Romero López, Departamento de Inmunología, ENCB-IPN, fecha de última actualización, 30 de abril de 2015. Las opiniones expresadas por los autores no necesariamente reflejo la postura del editor de la publicación. Queda estrictamente prohibida la reproducción total o parcial de los contenidos e imágenes de la publicación sin previa autorización del Instituto Politécnico Nacional. Todos los derechos reservados 2015 ConScience & Art Para mayor información y permisos conscienceandart@gmail.com Página de la revista: http://conscienceandart.wordpress.com/ http://conscienceandart.com

Índice 56

Microvesicles and the immune system.

Mycobacterium tuberculosis infection up-regulates mRNA expression for MICA and MICB in human macrophages.

Cancer research & bioinformatics

Medicina, enfermedad y arte

Revista cuatrimestral, sin fines de lucro. Todos los derechos reservados 2015 ConScience & Art. Para mayor información y permisos conscienceandart@gmail.com. Acceso a la revista e instrucciones para autores y contribuciones http://conscienceandart.wordpress.com/ http://conscienceandart.com/

Reseña de la portada

Congress itinerary

Prize itinerary

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 54

Editorial He tenido la fortuna de haber sido testigo del nacimiento de la revista ConScience & Art, y de valorar el esfuerzo y entusiasmo de un grupo de alumnos de posgrado de nuestra escuela, con la colaboración del Dr. Luis Antonio Jiménez Zamudio. Con éste, se alcanza ya el quinto número de la revista. ConScience & Art es un concepto que rompe todos los paradigmas, ya que se exponen los últimos hallazgos en diversas disciplinas de la ciencia, como la inmunología y la farmacología entre otros, creando una asociación muy interesante con el arte en sus diversas expresiones, brindando al lector un espacio de esparcimiento muy agradable, una forma muy particular de disfrutar de la ciencia sin sacrificar su rigor y formalidad. Mis más sinceras felicitaciones a todo el cuerpo editorial, que ha hecho posible esta revista y mis mejores deseos para que continúe con esta exitosa aventura.

I have had the fortune to have been witness of the birth of the ConScience & Arts magazine, and to value the effort and enthusiasm of a group of graduate students from this school, who in collaboration with Dr. Luis Antonio Jiménez Zamudio, have reached the publication of the fifth issue. ConScience & Art is ultimately a concept that breaks all established paradigms, as recent findings are reported in different scientific disciplines, such as immunology, pharmacology among others, while at the same time creating a very interesting association with art in its various expressions. The reader is thus allowed a pleasant recreational space and a very special way to enjoy science without compromising rigos and formality. My sincere congratulations to the entire editorial board of ConScience & Art, and my best wishes for the continued success of their aventure!

EDITORIAL

Rosalía Torres Bezaury Ex-Directora de la Escuela Nacional de ciencias biológicas del IPN.

Rosalía Torres Bezaury was, until December 2014, the Director of the Escuela Nacional de Ciencias Biológicas. The editorial board of ConScience & Arts wants to acknowledge her constant support and encouragement, and wishes her all kind of success in her new endeavours.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

INMUNOLOGÍA

Review

Microvesicles and the immune system.

Alvarez-Jiménez VD.1 Chacón-Salinas R.1 Estrada-García Iris.1 1

Departament of Immunology, National School of Biological Science, National Polytechnic Institute, Mexico City, Mexico. Cells of multicellular organisms communicate via many mechanisms that include not only direct cell-cell direct contact, but also by secreted molecules and vesicles that travel long distances in the body, which regulate different processes that modify diverse cellular functions. Evidence points to the existence of microvesicles (MVs) and their importance as mediators of intercellular communications. Because of their function, MVs are described as “functional signaling bags” because they contain a collection of multiple proteins, lipids and genetic material from the cells that originate them. These bags are transported to other places in the body, where they can alter the function and physiology of interacting cells. Because MVs are released by different physiological and chemical stimuli, modulating different biological processes, research is focused on elucidating their biogenesis, content and biological effect. They have been studied in such varied pathologies as cancer, neurological problems, autoimmune diseases, inflammatory processes, cardiovascular, infectious, metabolic, and allergic illnesses, as well as for their possible use in the development of new diagnostic techniques, therapies, vaccines, etc. In this review we will focus mainly on the characteristics and functions that different MVs have in the immune system as a main source of intercellular communication and modulation, and we will review their function in various pathologies with a view toward finding their possible treatment or prevention.

Intercellular communication and microvesicles. Membrane protein transference among cells of the immune system was described for the first time more than 30 years ago, in a study of bone marrow chimera, in which it was observed that donor thymocytes could acquire host-derived MHC molecules [1]. Since then, several reports have described the mechanisms involved in the transference of membrane fragments among cells. Such mechanisms include those involving direct cell-cell contact (through the proposed mechanisms of trogocytosis, nanotubes, trans-endocytosis and gap-junctions) or through the liberation of MVs. The functional consequences of this membrane transference involve the induction, amplification and/or modulation of the immune

response, as well as the acquisition of new functional properties in the host cells, such as chemotaxis, secretion of cytokines and expression of adhesion proteins. Moreover, recent reports showed the presence of ribonucleic acid messengers (mRNAs) and microRNAs inside these vesicles, suggesting the possibility that genetic material transference could affect host cell function. All of these results support the idea that transference of cellular membrane components is an effective and common way of intercellular communication [2]. Blistering is a physiologic mechanism generated during cellular development, protection and activation. An example of such CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 56

INMUNOLOGÍA

Formation of MVs is an important selfdefense mechanism, when cells (such as neutrophils, erythrocytes, oligondendrocytes, and platelets) protect themselves against attack by the complement system through exclusion of the attack complex membrane C5b-9 (MAC) of the cellular surface [4-9]. Since 2003, it is known that a great number of MVs are liberated in sick individuals when compared to healthy individuals in several pathologic conditions, for example, in inflammation, vascular dysfunction and cancer [10-11]. Oddly, the MV phenomenon is preserved throughout evolution, as it is evidenced by bacteria that release MVs to form biofilms or to establish a system of traffic signs in response to the external environment [12-13]. MVs are complex structures enclosed by a lipid bi-layer with trans-membrane proteins that contain hydrophilic soluble components derived from the cytosol of the donor cell. MVs usually measure from 50 to 1000 nm and are derived from the cellular membrane (ectosomes), or from multivesicular bodies (exosomes). Although MVs were first isolated from cellular cultures, their presence is evident in several biological fluids such as blood, plasma, urine, milk, synovial fluid, bronchoalveolar lavage, amniotic liquid and ascites [14]. It is therefore not surprising that MVs are proposed as an important factor in regulating diverse biological effects and pathologies where an altered immune response is involved. Extracellular vesicles biogenesis. At present, there is great confusion in the nomenclature of the MVs. Several investigators have tried to classify them on the basis of their structure, size and mechanism of formation. Several names have been used in an effort to include all these 57

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

particles, such as microparticles, vesicles, microvesicles, nanovesicles, exosomes, ectosomes, dexosomes and argosomes. However, all of these particles are similar in the composition of proteins and lipids of the cell that originated them, making their classification difficult only on that basis [15]. Up to now the best way to distinguish them is through their formation mechanism. As such, then, MVs are classified into exosomes, ectosomes and apoptotic bodies. Exosomes. Exosomes are the smallest vesicles. They measure 50-100 nm in diameter and are derived from multivesicular bodies that fuse with the plasmatic membrane, permitting their subsequent liberation toward the intercellular space [15-16]. Exosomes were observed for the first time more than 20 years ago and the term exosomes has been used since 1987 to refer to those inner vesicles of endosomal origin that are exocyted [17]. The exosomes are liberated by several cellular types, including reticulocytes [18], mast cells [19], dendritic cells (sometime called [22] [23] dexosomes) [20-21], small platelets , B lymphocytes , T [24] [25] [26] lymphocytes , epithelial and tumor cells . Exosome function depends mainly on the surface proteins and the type of cell from which they originated. The two most studied known functions of exosomes are the elimination of obsolete proteins during cellular maturation, and the intercellular communication by transference of genetic material among cells [28-29]. Exosomes are the vesicles most widely described in the immune system, where they have the capacity to present antigens to T lymphocytes and consequently are considered as stimulators of immune response [30-31], making them potential candidates for vaccines against cancer. At present there are several protocols and clinical studies that are evaluating these MVs as possible candidates for such possible therapy [32-34]. Interestingly, recent investigations have demonstrated that these MVs can also have suppressant effects on the immune response [35-37]. Ectosomes. The other large group of vesicles is the ectosomes, MVs that are derived from the cellular membrane through a process called ectocytosis [9, 38-40]. This vesicle group is the most heterogeneous due to its wide variation in size (200-1000 nm). They originate from the plasmatic membrane of neutrophils [12], DCs (dendritic cells) [22], platelets [23], and tumors [27]. These vesicles carry great amounts of membrane proteins from the originating cell. Several cells, including tumoral, release ectosomes spontaneously or in response to different stimuli. [41-44] In general,ectosomes are vesicles with cytosolic content that expose phosphatidylserine (PS) on the external face of its membrane [39]. Depending on their cellular origin, the ectosomes have been associated with a wide spectrum of biological activities, but more often with

communication is the mineralization of cartilage, bone and predentin. In this case the c a l c i f i c at i on is initiated by the liberation of MVs toward the extracellular matrix by chondrocytes, osteoblasts and odontoblasts [3].

INMUNOLOGÍA

proinflammatory and procoagulant factors. [41, 43, 45-48] Ectosomes have the capacity to deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions [49]. The ectosome formation depends on several mechanisms. It is known that each of the lipid layers of the membrane has a specific composition. The aminophospholipids: phosphatidylserine (PS) and phosphatidylethanolamine (PE) are oriented specifically toward the inner face of the membrane, whereas the phosphatidylcholine (PC) and the sphingomyelin (SP) are enriched in the external face. The lipids in the membrane are regulated mainly by three proteins: 1) translocase aminophospholipid to the inside, specific for PS and PE, known as flippase; 2) floppase to the exterior of the membrane, and 3) scramblase. Together, they promote the nonspecific bidirectional redistribution through the bilayer. The most remarkable change during the MV liberation is the

lipid distribution of the cellular membranes where there is a PS translocation to the surface, followed by the ectosome liberation because of the cytoskeleton fragmentation by intracellular Ca2+ [50]. Apoptotic vesicles. The vesicles related to apoptotic cells are different from other MVs, although their size varies between 50-500 nm. These vesicles are amorphous and they have a high content of histones. However, they can be confused with MVs of viable cells because of their size and the PS exposition on the external side [51]. The different types of MV described and their characteristics are shown in the following chart.

Feature

Exosomes

Ectosomes

Microvesicles

Apoptotic vesicles

Size Sucrose density Appearance by electron microscopy Sedimentation Lipid composition

50-10nm 1.13-1.19 g/mL Cup shape

50-200nm ND Bilamellar round structures

500->1000nm 1.16-1.28 g/mL Amorphous

100,000 g Enriched in cholesterol, sphingomyelin and ceramide; contain lipid rafts; expose phosphatidylserine

160-200,000 g Enriched in cholesterol and diacylglycerol; expose phosphatidylserine

100-1000nm ND Irregular shape and electron-dense 10,000 g Expose phosphatidylserine

Biogenesis

Internal compartments (endosomes)

Plasma membrane

Apoptotic cells

Microvesicle composition. The great amount of information that exists about the MV composition ref lects the great diversity of populations that have been studied by genomic and proteomic analysis. However,

1,200-10,000 g Expose phosphatidylserine

Table 1. Physical and chemical features of different microvesicles. The features described here are based on several observations of purified vesicles originating from different cells [51, 52] PS; fosfatidilserina, CD35; receptor complemento 1, TSG101; Gen 101 de susceptibilidad a tumor.

they have been difficult to classify, due to the fact that when a cell is stimulated it can release vesicles of the same type, but with different composition. It has been shown that the origin of the exosomes consists of diverse endosomal vesicles and multivesicular bodies with resulting heterogeneous exosomes in composition. An example of this diversity was shown when comparing the exosome proteins liberated from intestinal epithelial cells to the cellular line HT29-19A stimulated with IFN-g. The liberated exosomes were different in size and shape. Aside from their different protein composition, the exosomes liberated from the apical surface have molecules related to the formation of endosomes, such as syntaxin 3 and protein 2 joined to syntaxin, when compared with exosomes from the basolateral area , which have adhesion and costimulation proteins [53].

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 58

In general MVs contain several proteins, such as Alix/ sintenin, TSG101, HSP70, MFGE8, actylcholinesterase (AchE), annexin V, flotillin, transferrin receptor (TfR) and tetraspanins. They also contain lipids such as PS and ceramide. However, we must stress that MVs contains specific proteins depending on their cellular origin. For example, the T lymphocyte exosomes contain TCR and co-stimulation molecules (Figure 1), exosomes from tumor cells contain tumor antigens, those derived from platelets contain coagulation factors and DCs exosomes express MHC-II.Some reports indicate that this difference in MV composition can be achieved by inhibiting specific molecules that participate in MV genesis. For example, Rab27a inhibition in DCs decreases the secretion of exosomes that contain CD63, Hsp 70, TSG101 and Alix, but the secretion of MV that express CD9 and MFGE8 (lactadherin) is not affected. This opens the possibility of studying specific MV populations in order to elucidate their possible function [54].

Fig.1 Molecular composition of an ectosome originated from neutrophil.

Microvesicles functions in the immune system Due to their high content of active molecules, MVs can modulate both branches of the immune response. In innate immune cells, MVs have an important function. In mast cells MVs can transfer RNA from one cell to another, where the information is read and expressed in the protein with biological activity [55]. Some MVs derived from mast cells promote the maturation of DCs that acquire the capacity to present antigen to naïve T cells. [56] Regarding neutrophils, several reports indicate that they produce ectosomes. These MVs have enzymatic activity due to their myeloperoxidase and elastase content. Furthermore, the receptor for the C3b fraction of complement (CR1 or CD35) is expressed in the ectosome surface. That is why it was proposed that ectosomes function as ecto-organelles with antimicrobial activity in the extracellular environment. [38] Several studies showed that human neutrophils can release ectosomes spontaneously or in response to cellular activation (PMN-Ect), that differ in the components present in the MVs, such as the surface molecules LFA-1/CD11a, CD11b, FcgRIII/ CD16, L-selectin, HLA class I [39]; the enzymes MPO, elastase, 59

MMP-9, PR3; complement proteins CR1/CD35, MCP/CD46, CD59 and other molecules like CD66b, DAF/CD55 and CD59 [42] . PMN-Ect can have different functions depending on the target cell. For example, PMN-Ect has anti-inflammatory properties in vitro over human macrophages derived from monocytes [57], while immature DCs show modified morphology, reduced phagocytic capacity and increased TGF-b production. Furthermore, mature DCs have a low expression of maturity markers and a reduced capacity to induce proliferation in T cells when exposed to this kind of MV. These data are a clear evidence of how PMN-Ect have the capacity to modify the maturity and function of nearby cells during the inflammatory process and can be crucial during infection by several microorganisms [58]. González-Cano et al. demonstrated for the first time the liberation of PMN-Ect in response to the infection with M. tuberculosis (Mtb), Leishmania mexicana, Staphylococcus aureus, and Escherichia coli. They demonstrated the expression of CD35, PS, Rab5, Rab7 and gp91Phox, a sub-unity of cytochrome b555 in the membrane of the ectosomes, which

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

INMUNOLOGÍA

indicates that these MVs could have an important participation in infection with these microorganisms [59]. In this line, PMN-Ect induced by M. tuberculosis can interfere with mycobactericidal activity of infected macrophages [60]. MVs can also interfere with NK activity. For example, MV derived from Jurkat and Raji cellular lines express ligands of NKG2D that can be overexpressed under oxidative stress. These MVs have the ability of inhibiting the cytoxicity of NK cells [61] . Let us now examine how MVs produced by DCs regulate the immune response. Reports have shown that MV-DCs can induce tolerance, in a mechanism that is dependent on the state of cellular maturation. For example, MVs derived from immature DCs inhibit the alloreactive T cell response and extend the survival of allografts preventing the activation of NK cells, suggesting a powerful immunosuppressive effect of this kind of MV. Clinical studies with these vesicles and the immunosuppressant agent LF-15 demonstrated an induction of specific tolerance to allografts, that was characterized by a marked inhibition of proliferative response of alloreactive T cells [62, 63] The maturation state of the DCs with the efficient stimulation of the immune response by the MVs. In mature DCs the MVs contain more MHC II adhesion molecules such as ICAM-1 and costimulation molecules necessary for the efficient activation of T cells. These MVs, in contrast with those derived from immature DCs, can activate T and B naïve cells [64]. The maturation of the DCs is also related with the type of miRNA transferred to the MV [65]. MVs derived from DCs can be liberated spontaneously, but they can be increased by TLRs stimulation and by purinergic receptor rP2x7 [66-67]. MVs secreted from stimulated DCs with antigens in vitro induced the activation of specific antigen T CD4+ cells and CTLs. In other in vitro studies, it has been seen that this form of cellular activation occurs only in the presence of DCs CD8+promoting the proliferation and secretion of cytokines by T cells [68-69].

It has been demonstrated that MVs generated from DCs stimulated with OVA (ovalbumin) induced immunostimulant function T CD8+ memory cells [68]. In infections, the MVs can transport antigens of different pathogens and induce an effective specific immune response [70,71-72]. These studies suggest a high potential of these MVs for the development of vaccines, but further studies are needed to prove this. A regulating function has been seen in MVs from T cells. Their release is increased during early cellular activation because of intracellular Ca2+ liberation and result in different types of MVs according to their activation grade [44]. These MVs present biologically active molecules, such as those related with apoptosis, FASL and APO2 ligand, that promote death by apoptosis [73-74]. On the other hand, MVs of activated T CD8+ lymphocytes induce the activation of different pathways such as ERK and NF-kB in myeloma cells. This increases the expression of the metalloproteinase 9 (MMP-9) promoting the invasivity of carcinogenic cells in vitro, but without affecting the proliferation or apoptosis of carcinogenic cells, therefore suggesting that MVs of T lymphocytes can promote the progression of tumor cells [75]. Exosomes of T CD8 cells also contain granzymes and perforins, usually observed during the formation of the immunologic synapse, promoting the liberation of the MVs. They fuse with the target cell for their destruction [76]. MV liberation by B lymphocytes is promoted when there is specific stimulation with T CD4 lymphocytes and crosslinking of the BCR by antigens [77-79]. B cells can release MVs after infection with the Epstein-Barr virus and transfer viral proteins and RNA to B and DCs cells [80-81]. Most of the studies have been made in vitro. However, what are the physiological functions of the MVs in vivo? One of the most successful studies was observed in MVs derived from serum of women with full term delivery which expressed high levels of FASL and MHC II when compared with MVs from women with pre-term delivery. In vitro, these MVs inhibited T cell activation in a way dependent on FASL. Interestingly, this inhibition was higher in women that reached full term. CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 60

INMUNOLOGÍA

Therefore, it seems that this liberation of exosomes transitorily helps to control the tumor, but it is not known if this ultimately affects tumor progression [87]. Other interesting studies have demonstrated the effect of RNAs present in MVs. This genetic material is found mainly in exosomes. For instance MVs derived from serum samples of patients with cancer have the ability to modify physiological properties of tumor cells through miRNAs, such as metastasis [88-89], which suggests a possible MV tumor induction mechanism. This process might be dependent upon patient immunologic competence and the tumor state. Finally, DC derived MVs have activity in different mouse models that we can see in autoimmune gastritis [90] and in experimental models of infection in intestine with retrovirus [91], simplex herpes [92], and in lungs infected with the influenza virus [93]. In all these studies, the antigen was taken preferentially by DCs CD8+ that reside in lymph nodes by cross-presentation [94]. In tissue grafts, DCs from the transplanted tissue migrate to the spleen, where they transfer allopeptides captured in the graft to other DCs through the exosomes, producing a natural state of tolerance [95]. In another study, the in vivo transference of MHC-peptide was demonstrated among intestinal epithelial cells and bronchial cells to DCs in absence of stress signs. These points to a possible relevant mechanism for the development of tolerance induction to food or allergens [96].

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

Conclusions. MVs represent new line of study of intercellular communication. These structures are generated spontaneously and in cellular activation states. MVs have diverse and important functions in the immune cell response, all the way from mechanisms of presentation of antigen in promoting an efficient immune response, up to the induction of tolerance mechanisms. Several functions are still to be described that can potentiate their use in possible prophylactic or therapeutic treatment of disease where immune response is involved. The study of MVs promises to expand in the near future, as we continue to understand them more thoroughly.

These results indicate that MVs are produced in the placenta and block the activation of the immune response of the mother against the fetus [82]. Several studies have demonstrated that the secretion of the MVs of tumor cells inhibit the immune response [83-85] . In contrast with these studies, Thery et al. have demonstrated the opposite. They generated tumor cells that secret MVs with a single membrane antigen. These vesicles induce efficient activation of T CD8+ cells in vivo promoting elimination of the tumoral cells [86]. In patients with cancer, the quantity of exosomes in serum is increased with the progression of the tumor, which suggests that if the immune response is induced in vivo by exosomes derived from the tumor, this response is not enough to eliminate the tumor permanently.

INMUNOLOGÍA Si bien las microvesículas son pequeños cuerpos provenientes de una célula, cuyas funciones biológicas específicas hasta el momento no se han elucidado, estás estructuras han captado la atención de muchos investigadores los cuales las han descrito como una especie de contenedores de los desperdicios celulares (biomoleculas) hasta considerarlas tan importantes e indispensables en la biología de la célula debido a su función como mensajeros biológicos (llevando información clave a tejidos lejanos en el cuerpo, en los que pueden alterar su fisiología) hasta ser puentes de comunicación entre un mismo tipo de célula o entre diferentes tipos celulares. Es así como las microvesículas pueden representar para muchos la esperanza para entender fenómenos complejos y para entender enfermedades, sin embargo estas esferas de materia pueden ser un arma de doble filo acarreando y participando en procesos dañinos, al igual que una indefensa burbuja de jabón una vez estallada no hay vuelta atras.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 62

Reference.

1. Sharrow S. O., Mathieson, B. J. & Singer A. 1981. Cell surface appearance of unexpected host MHC determinants on thymocytes from radiation bone marrow chimeras. J. Immunol. 126, 1327–1335. 2. Davis D. M. 2007. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nature Rev. Immunol. 7, 238–243. 3. Anderson H. C. 2003. Matrix vesicles and calcification. Curr Rheumatol Rep. 5:222-226. 4. Campbell A. K. and Morgan B. P. 1985. Monoclonal antibodies demonstrate protection of polymorphonuclear leukocytes against complement attack. Nature 317:164-166. 5. Lida K., M. B. Whitlow M. B. and V. Nussenzweig. 1991. Membrane vesiculation protects erythrocytes from destruction by complement. J Immunol. 147:2638- 2642. 6. Morgan B. P., J. R. Dankert and Esser A. F. 1987. Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis. J Immunol. 138:246-253. 7. Scolding N. J., Morgan B. P., W. A. Houston, C. Linington, A. K. Campbell, and D. A. Compston. 1989. Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement. Nature. 339:620-622. 8. Sims P. J. and T. Wiedmer. 1986. Repolarization of the membrane potential of blood platelets after complement damage: evidence for a Ca++ dependent exocytotic elimination of C5b-9 pores. Blood 68:556-561. 9. Stein J. M. and J. P. Luzio. 1991. Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma membrane proteins and lipids into shed vesicles. Biochem J 274. (Pt 2):381-386. 10. Diamant M., Tushuizen M. E., Sturk A. and Nieuwland R. 2004. Cellular microparticles: new players in the field of vascular disease? Eur J Clin Invest. 34:392-401. 11. Freyssinet J. M. 2003. Cellular microparticles: what are they bad or good for? J Thromb Haemost 1:1655-1662. 12. Schooling S. R. and Beveridge T. J. 2006. Membrane vesicles: an overlooked component of the matrices of biofilms. J Bacteriol. 188:5945-5957. 13. Mashburn L. M. and M. Whiteley. 2005. Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature. 437:422-425. 14. Théry C., Ostrowski M and Segura E. 2009. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol. 9:581–593. 15. Théry C., L. Zitvogel and S. Amigorena. 2002. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2:569-579. 16. Coccuci E, Racchetti G, Meldolesi J. 2009. Shedding microvesicles: artefacts no more.Trends Cell Biol. 19:43-51. 17. Théry C. 2011. Exosomes: secreted vesicles and intercellular communications. F1000 Biology Reports. 3:15. 18. Johnstone R. M., M. Adam, J. R. Hammond, L. Orr, and C. Turbide. 1987. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 262:9412-9420. 19. Raposo G., Tenza D., S. Mecheri, C. Bonnerot, and C. Desaymard. 1997. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation. Mol Biol Cell 8:2631-2645. 20. Zitvogel, L., A. Regnault, A. Lozier, J. Wolfers, C. Flament, D. Tenza, P. RicciardiCastagnoli, G. Raposo, and S. Amigorena. 1998. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell derived exosomes. Nat Med. 4:594-600. 21. Le Pecq J. B. 2005. Dexosomes as a therapeutic cancer vaccine: from bench to bedside. Blood Cells Mol Dis. 35:129-135. 22. Heijnen, H. F., R. Fijnheer, H. J. Geuze, and J. J. Sixma. 1999. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood. 94:3791-3799. 23. Raposo G., H. W. Nijman, W. Stoorvogel, R. Liejendekker, C. V. Harding, C. J. Melief and H. J. Geuze. 1996. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 183:1161-1172. 24. Blanchard N., D. Lankar, F. Faure, A. Regnault, C. Dumont, G. Raposo and C. Hivroz. 2002. TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex. J Immunol. 168:3235-3241. 25. Barreto A, et al. 2010. Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function. Viral Immunol. 23:595–608. 26. Wolfers J., A. Lozier, G. Raposo, A. Regnault, C. Thery, C. Masurier, C. Flament, S. Pouzieux, F. Faure, T. Tursz, E. Angevin, S. Amigorena and L. Zitvogel. 2001. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med. 7:297-303. 27. Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL. 2010. Tumor-derived microvesicles induce, expand and up-regulate biological

activities of human regulatory T cells (Treg). PLoS ONE. 5:1169. 28. Chaput N., J. Taieb, N. Schartz, C. Flament, S. Novault, F. Andre, and L. Zitvogel. 2005. The potential of exosomes in immunotherapy of cancer. Blood Cells Mol. Dis 35:111-115. 29. Chaput N., C. Flament, S. Viaud, J. Taieb, S. Roux, A. Spatz, F. Andre, J. B. LePecq, M. Boussac, J. Garin, S. Amigorena, C. Thery, and L. Zitvogel. 2006. Dendritic cell derived-exosomes: biology and clinical implementations. J Leukoc Biol. 80:471-478. 30. Blanchard N, et al. 2002. TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex. J Immunol 168:3235–3241. 31. Van der Vlist EJ, Arkesteijn GJA, van der Lest CHA, Stoorvogel W, Nolte-’t Hoen EN, Wauben MH. 2012. CD4+ T cell activation promotes the differential release of distinct populations of nanosized vesicles. J Extracell Vesicles. 1:18364. 32. Li X., Z. Zhang, T. Beiter and H. J. Schluesener. 2005. Nanovesicular vaccines: exosomes. Arch Immunol Ther Exp (Warsz). 53:329-335. 33. Chaput N., J. Taieb, N. Schartz, C. Flament, S. Novault, F. Andre and L. Zitvogel. 2005. The potential of exosomes in immunotherapy of cancer. Blood Cells Mol Dis 35:111-115. 34. Chaput N., C. Flament, S. Viaud, J. Taieb, S. Roux, A. Spatz, F. Andre, J. B. LePecq, M. Boussac, J. Garin, S. Amigorena, C. Thery, and L. Zitvogel. 2006. Dendritic cell derived-exosomes: biology and clinical implementations. J Leukoc Biol. 80:471-478. 35. Valenti R., V. Huber, P. Filipazzi, L. Pilla, G. Sovena, A. Villa, A. Corbelli, S. Fais, G. Parmiani, and L. Rivoltini. 2006. Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes. Cancer Res. 66:9290-9298. 36. Li X. B., Z. R. Zhang, H. J. Schluesener and S. Q. Xu. 2006. Role of exosomes in immune regulation. J Cell Mol Med 10:364-375. 37. Clayton A, Al-Taei S, Webber J, Mason MD, Tabi Z. 2011. Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production. J Immunol.187:676–683. 38. Hess C., S. Sadallah, A. Hefti, R. Landmann and J. A. Schifferli. 1999. Ectosomes released by human neutrophils are specialized functional units. J Immunol. 163:4564-4573. 39. Gasser O., C. Hess, S. Miot, C. Deon, J. C. Sanchez and J. A. Schifferli. 2003. Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils. Exp Cell Res. 285:243-257. 40. Pilzer D., O. Gasser, O. Moskovich, J. A. Schifferli and Z. Fishelson. 2005. Emission of membrane vesicles: roles in complement resistance, immunity and cancer. Springer Semin Immunopathol. 27:375-387. 41. Nomura S., N. N. Tandon, T. Nakamura, J. Cone, S. Fukuhara and J. Kambayashi. 2001. High-shear-stress-induced activation of platelets and microparticles enhances expression of cell adhesion molecules in THP-1 and endothelial cells. Atherosclerosis 158:277-287. 42. Dolo V., A. Ginestra, D. Cassara, S. Violini, G. Lucania, M. R. Torrisi, H. Nagase, S. Canevari, A. Pavan and M. L. Vittorelli. 1998. Selective localization of matrix metalloproteinase 9, beta1 integrins, and human lymphocyte antigen class I molecules on membrane vesicles shed by 8701-BC breast carcinoma cells. Cancer Res. 58:4468-4474. 43. Satta N., F. Toti, O. Feugeas, A. Bohbot, J. Dachary-Prigent, V. Eschwege, H.Hedman and J. M. Freyssinet. 1994. Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. J Immunol. 153:32453255. 44. Wieckowski EU, Visus C, Szajnik M, Szczepanski MJ, Storkus WJ, Whiteside TL. 2009. Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes. J Immunol.183:3720–3730. 45. MacKenzie A., H. L. Wilson, E. Kiss-Toth, S. K. Dower, R. A. North and A. Surprenant. 2001. Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 15:825-835. 46. Mesri M. and D. C. Altieri. 1999. Leukocyte microparticles stimulate endothelial cell cytokine release and tissue factor induction in a JNK1 signaling pathway. J Biol Chem. 274:23111-23118. 47. Mesri M. and D. C. Altieri. 1998. Endothelial cell activation by leukocyte microparticles. J Immunol. 161:4382-4387. 48. Sabatier F., V. Roux, F. Anfosso, L. Camoin, J. Sampol and F. Dignat-George. 2002. Interaction of endothelial microparticles with monocytic cells in vitro induces tissue factor-dependent procoagulant activity. Blood. 99:3962-3970. 49. Dalli J, Montero-Melendez T, Norling LV, Yin X, Hinds C, Haskard D, Mayr M, Perretti M. 2013. Heterogeneity in neutrophil microparticles reveals distinct proteome and functional properties. Mol Cell Proteomics. Aug;12(8):2205-19.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

INMUNOLOGÍA

50. Bevers E. M., Comfurius P., Dekkers D. W. and Zwaal R. F. 1999. Lipid translocation across the plasma membrane of mammalian cells. Biochim Biophys Acta. 1439: 317–330. 51. Théry C., Ostrowski M. and Segura E. 2009. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9: 581-593. 52. Bobrie A, Colombo M, Krumerich S, Raposo G, Thery C. 2012. Diverse subpopulations of vesicles secreted by different intracellular mechanisims are present in exosome preparations obtained by differential ultracentrifugation. J Extracell Vesicles.1:18397. 53. Van Niel, G., G. Raposo, C. Boussac, R. Hershberg, N. Cerf-Bensussan and M. Heyman. 2001. Intestinal epithelial cells secrete exosome like vesicles. Gastroenterology 121:337-349. 54.Fernandez-Messina L, et al. 2010. Differential mechanisms of shedding of the glycosylphosphatidylinositol (GPI)-anchored NKG2D ligands. J Biol Chem. 285:8543–8551. 55. Valadi H. Ekström K, Bossios A, Sjöstrand M, Lee J. 2007.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature Cell Biol. 9:654–659. 56. Skokos D, et al. 2003. Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo. J Immunol. 170:3037–3045. 57. Gasser, O., J. A. Schifferli. 2004. Activated polymorphonuclear neutrophils disseminate anti-inflammatory microparticles by ectocytosis. Blood 104: 25432548. 58. Lee T. L., Y. C. Lin, K. Mochitate and F. Grinnell. 1993. Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and beta 1 integrin receptors. J Cell Sci 105. ( Pt 1):167-177. 59. Gonzáles-Cano P, Ricardo Mondragón-Flores, Luvia E. Sánchez-Torres, Sirenia Gonzáles-Pozos, Mayra Silva-Miranda, Amalia Monroy-Ostria, Sergio EstradaParra, Iris Estrada-García. 2010. Mycobacterium tuberculosis H37Rv induces ectosome release in human polymorphonuclear neutrophils. Tuberculosis 90: 125–134. 60. Azevedo Duarte T, Noronha-Dutra A. A, Joilda Silva Nery, Brum Ribeiro S, Thassila Nogueira Pitanga, José R. Lapa e Silva, Arruda S, Boéchat N. 2012. I-induced neutrophil ectosomes decrease macrophage Activation. Tuberculosis 92; 218- 225. 61. Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L. 2011. Thermal- and oxidative stress causes enhanced release of NKG2D ligandbearing immunosuppressive exosomes in leukemia/lymphoma T and B cells. PLoS ONE. 6:19. 62. Yang X, Meng S, Jiang H, Zhu C, Wu W. 2011. Exosomes derived from immature bone marrow dendritic cells induce tolerogenicity of intestinal transplantation in rats. J Surg Res.171:826–832. 63. Peche H, Heslan M, Usal C, Amigorena S, Cuturi MC. 2003. Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cellderived exosomes modulates allograft rejection. Transplantation. 76:1503–1510. 64. Laulagnier K, Motta C, Hamdi S, Roy S, Fauvelle F, Pageaux JF, Kobayashi T, Perret B, Bonnerot C, Record M. 2004. Mast cell- and dendritic cell derived exosomes display a specific lipid composition and an unusual membrane organization. Biochem J. 380:161–171. (64) Segura E, et al. 2005. ICAM-1 on exosomes from mature dendritic cells is critical for efficient naïve T-cell priming. Blood 106:216–223. 65. Montecalvo, A. et al. 2008. Exosomes as a short-range mechanism to spread alloantigen between dendritic cells during T cell allorecognition. J. Immunol. 180:3081–3090. 66. Obregon C, Rothen-Rutishauser B, Gerber P, Gehr P, Nicod LP. 2009. Active uptake of dendritic cell derived exovesicles by epithelial cells induces the release of inflammatory mediators through a TNF-alpha-mediated pathway. Am J Pathol. 175:696–705. 67. Skokos D, et al. 2003. Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo. J Immunol. 170:3037–3045. (67) Qu Y, Dubyak GR. 2009. P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways. Purinergic Signal. 5:163. 68. Hao S, Yuan J, Xiang J. 2007. Nonspecific CD4(+) T cells with uptake of antigenspecific dendritic cell-released exosomes stimulate antigen-specific CD8(+) CTL responses and long-term T cell memory. J Leukoc Biol. 82:829–838. 69. Admyre C, Johansson SM, Paulie S, Gabrielsson S. 2006. Direct exosome stimulation of peripheral human T cells detected by ELISPOT. Eur J Immunol. 36:1772–1781. 70. Colino J, Snapper CM. 2006. Exosomes from bone marrow dendritic cells pulsed

with diphtheria toxoid preferentially induce type 1 antigens pecific IgG responses in naive recipients in the absence of free antigen. J Immunol 177:3757–3762. 71. Aline F, Bout D, Amigorena S, Roingeard P, Dimier-Poisson I. 2004. Toxoplasma gondii antigenpulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection. Infect Immun. 72:4127–4137. 72. Colino J, Snapper CM. 2007. Dendritic cell-derived exosomes express a Streptococcus pneumoniae capsular polysaccharide type 14 cross-reactive antigen that induces protective immunoglobulin responses against pneumococcal infection in mice. Infect Immun. 75:220–230. 73. Martinez-Lorenzo MJ, et al. 1999. Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles. J Immunol. 163:1274–1281. 74. Monleon I, et al. 2001. Differential secretion of Fas ligand- or APO2 ligand/ TNF-related apoptosis inducing ligand-carrying microvesicles during activationinduced death of human T cells. J Immunol. 167:6736–6744. 75. Cai Z, et al. 2012. Activated T cell exosomes promote tumor invasion via Fas signaling pathway. J Immunol. 188:5954–5961. 76. Peters PJ, et al. 1989. Molecules relevant for T celltarget cell interaction are present in cytolytic granules of human T lymphocytes. Eur J Immunol. 19:1469– 1475. 77. Muntasell A, Berger AC, Roche PA. 2007. T cellinduced secretion of MHC class II-peptide complexes on B cell exosomes. EMBO J. 26:4263–4272. 78. Rialland P, Lankar D, Raposo G, Hubert P. 2006. BCR-bound antigen is targeted to exosomes in human follicular lymphoma B-cells. Biol Cell. 98:491–501. 79. McLellan A. D. 2009. Exosome release by primary B cells. Crit Rev Immunol. 29:203–217. 80. Vallhov H, et al. 2011. Exosomes containing glycoprotein 350 released by EBVtransformed B cells selectively target B cells through CD21 and block EBV infection in vitro. J Immunol. 186:73–82. 81. Pegtel DM, et al. 2010. Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci USA. 107:6328–6333. 82. Taylor, D. D., Akyol, S. & Gercel-Taylor, C. 2006. Pregnancy associated exosomes and their modulation of T cell signaling. J. Immunol. 176, 1534. 83. Taylor, D. D. & Gercel-Taylor, C. 2005. Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects. J. Cancer 92, 305–311. 84. Clayton A, Mitchell JP, Court J, Tabi Z. 2007. Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res. 67:7458– 7466. 85. Huber, V., Filipazzi, P., Iero, M., Fais, S. & Rivoltini, L. 2008. More insights into the immunosuppressive potential of tumor exosomes. J. Transl. Med. 6; 63 . 86. Zeelenberg, I. S. et al. 2008.Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Res. 68, 1228–1235. 87. Taylor, D. D. & Gercel-Taylor, C. 2008. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110, 13–21. 88. Skog, J. et al. 2008. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nature Cell Biol. 10, 1470–1476. 89. Scheinecker, C., McHugh, R., Shevach, E. M. & Germain, R. N. (2002). Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node. J. Exp. Med. 196, 1079–1090 90. Fleeton, M. N. et al. 2004. Peyer’s patch dendritic cells process viral antigen from apoptotic epithelial cells in the intestine of reovirus-infected mice. J. Exp. Med. 200, 235–245. 91. Allan, R. S. et al. 2006. Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming. Immunity. 25, 153–162. 92. Geurtsvan Kessel, C. H. et al. 2008. Clearance of influenza virus from the lung depends on migratory langerin+CD11b– but not plasmacytoid dendritic cells. J. Exp. Med. 205, 1621–1634. 93. Segura, E., Guerin, C., Hogg, N., Amigorena, S. & Thery, C. 2007. CD8+ dendritic cells use LFA-1 to capture MHC-peptide complexes from exosomes in vivo. J. Immunol. 179, 1489–1496. 94. Montecalvo, A. et al. 2008. Exosomes as a short-range mechanism to spread alloantigen between dendritic cells during T cell allorecognition. J. Immunol. 180:3081–3090. 95. Karlsson, M. et al. 2001. “Tolerosomes” are produced by intestinal epithelial cells. Eur. J. Immunol. 31:2892–2900. 96. Almqvist, N., Lonnqvist, A., Hultkrantz, S., Rask, C. & Telemo, E. 2008. Serumderived exosomes from antigen-fed mice prevent allergic sensitization in a model of allergic asthma. Immunology. 125:21–27.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 64

SOM

Original contribution

Mycobacterium tuberculosis infection up-regulates mRNA expression for MICA and MICB in human macrophages. Short communication Enrique Hernández-Martínez1, Erik Nieto-Patlán1, Jeanet Serafín-López1, Luz M. Aguilar- Anguiano2, Sergio Estrada-Parra1, Iris Estrada-García1 and Rommel Chacón-Salinas1*.

*Address all correspondence to: rommelchacons@yahoo.com.mx

INTRODUCTION Tuberculosis is one of the leading causes of death worldwide. This disease is caused by intracellular bacteria that belong to the Mycobacterium tuberculosis (Mtb) complex. The immune response against this pathogen plays a crucial role for bacterial containment and limiting damage in the host. The main cellular target of the infection is macrophages that usually are unable to eliminate ingested Mtb. This scenario changes until the induction of CD8+ and CD4+αβ T cells that activate infected macrophages after the recognition of mycobacterial antigens on classical MHC class I and class II molecules, respectively (Ernst, 2012). However the immune response has other strategies to recognize infected cells that do not imply direct recognition of antigens. Recognition of stressed by cells of the immune system is a strategy used by the immune response to eliminate cells that have suffered malignant transformation or that are target of an infectious agent (Hayday, 2009). Stressed cells usually induce the expression or upregultion of different molecules that are sensed by cells of the immune system. One group of this type of molecules is MHC class-I related molecules, which include MIC-A, -B and the UL-16 binding proteins or ULBP (Gonzalez et al., 2006). After stressing signals, these molecules are upregulated on the cell surface membrane where they can interact with their receptor NKG2D, a type II trans-

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

1. Department of Immunology, National School of Biological Sciences (ENCB), National Polytechnic Institute (IPN), Carpio y Plan de Ayala s/n Col. Santo Tomás, Mexico DF 11340, Mexico 2. Escuela Médico Militar y Hospital Central Militar, Secretaría de la Defensa Nacional (SEDENA), Blvd. Avila Camacho S/N, Lomas de Sotelo, Mexico DF, 11200, Mexico

SOM membrane protein that is expressed by NK cells, and subsets of NKT, γδ T cells, CD4+ and CD8+ αβ T cells (El-Gazzar et al., 2013). Engagement of NKG2D leads to cytokine production and cytotoxicity toward stressed cells either directly or through providing costimulation to effector cells (HuergoZapico et al., 2014). Some signals have been identified as inducers of cellular stress than induce an overexpression of NKG2D ligands, these include tumorigenesis, viral infections, injured tissue and inflammatory diseases (Raulet et al., 2013). However, scarce information exists about the role of bacterial infections and induction of NKG2D ligands. In this work we evaluated mRNA expression for MICA and MICB in human macrophages infected with the intracellular pathogen Mycobacterium tuberculosis.

with Mtb H37Rv reaching the maximum level of expression after 12 hpi and a gradual decrease at 24 and 48 hpi (Figure 1 a,b). To corroborate this finding the mRNA levels for MICA and MICB were compared between infected cells with Mtb and those stimulated with PMA, a well known inducers of their expression (Gregorie et al., 2009). We noticed that cells infected with Mtb expressed higher mRNA levels for MICA and MICB than those stimulated with PMA (Figure 1 c,d).

MATERIAL AND METHODS Mycobacterium tuberculosis H37Rv was grown in Middlebrook 7H9 medium (BD-Difco, USA) supplemented with 10% OADC (BD-Difco, USA) until they reached log-stationary phase. Bacteria was harvested, aliquoted and frozen at -70 oC until used. Human monocytic cell line THP-1 was obtained from ATCC and cultured at 37 ̊C with 5% CO2 in RPMI 1640 (Gibco USA) supplemented with 10% Fetal Bovine Serum (Gibco, USA). Macrophages were obtained as previously described (Park et al., 2007), briefly 1X106 cells were stimulated during 48 hours with 40 nM of PMA (Sigma), after this time adherent cells were washed and left unstimulated during 96 hours. After this time cells were infected during 2 hours with Mycobacterium tuberculosis H37RV at a ratio of 10 colony-forming units (CFU) per macrophage , or stimulated with 40 nM PMA. After different hours post-infection (hpi) cells were lysed with 1 ml of Trizol reagent (Invitrogen USA) and total RNA was extracted according to manufacturer instructions. cDNA was reverse transcribed from total RNA using M-MLVRT (Invitrogen USA). Twenty-five nanograms cDNA was subjected to q-RT PCR using a sequence detector (LightCycler 1.5 Roche) and target mixes for MICA, MICB and GAPDH (Taqman Gene Expression Assay, Applied Biosystems). Cycle threshold (CT) values for MICA and MICB were normalized to GAPDH as previously described (Reyes-Martinez et al., 2014).

RESULTS AND DISCUSSION Although THP-1 cells expressed basal mRNA levels for MICA and MICB these were increased after 6 hours post-infection CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 66

SOM

Our results show that infection of human macrophages with Mtb induces cellular stress that is reflected as an increase in mRNA levels for MICA and MICB. These results contrast with the observations made by Vankayalapati et al, who observed that human macrophages infected with Mtb only overexpressed ULBP1, but not MICA or MICB (Vankayalapati et al., 2005). These differences can be explained for several factors: Cells used by Vankayalapati et al were peripheral blood monocytes infected with the attenuated strain H37Ra and they evaluate the expression of proteins on the cell surface after 48 hours post- infection.

Figure 1. Mycobacterium tuberculosis infection of human macrophages induces overexpression of MICA and MICB mRNA. B) 10

MICB FOLD INCREASE

MICA FOLD INCREASE

8 6 4 2 0

60 40 20 0

HPI

D) 15 10

5 0

PMA

MICB FOLD INCREASE

C) MICA FOLD INCREASE

Mtb

40 30

20 10 0

PMA

Mtb

Macrophages derived for PMA activated THP-1 cells were infected for 2 hours with Mtb at a MOI of 10. After different hours post- infection mRNA levels for MICA (a) and MICB (b) were evaluated. Macrophages were stimulated with either PMA or infected with Mtb, mRNA levels for MICA (c) and MICB (d) were assessed by real time PCR after 12 hours. *p<0.05 one way ANOVA (Bonferroni test) compared to not-stimulated macrophages.

CONS ART * VOLUMEN 2 * NĂ&#x161;MERO 2 * ENERO-ABRIL 2015

Although we did not evaluate protein levels on the cell surface of macrophages, our results shows that the cellular stress induced by infection with virulent Mtb promote an early and transient expression of MICA and MICB genes, that is turned off at 48 hpi that could explain the observation made by Vankayalapati et al. Whether this increase in mRNA expression for MICA and MICB is

SOM

reflected in an increase in proteins levels on the cell surface needs to be pursued. Although the first step in the regulation for these genes is their transcription, further regulatory steps are known, like RNA stabilization, protein stabilization and cleavage from the cell membrane (Raulet et al., 2013). How Mtb induces stress in infected macrophages that lead to an increase in mRNA for MICA and MICB is unknown. Previous reports indicate that TLR signaling can lead to an in ligands for NKG2D (Hamerman et al., 2004), and is well known that Mtb presents several ligands capable to active different TLR (Mortaz et al., 2014). A second pathway is related with oxidative stress that also induce an increase in MICA and MICB (Yamamoto et al., 2001); Mtb is also known to promote high levels of reactive oxygen species through activation of macrophage NADPH oxidase (Deffert et al., 2014).

This cellular stress induced by Mtb can have a relevant role in the immune response to this pathogen. Previous reports indicated that the increase in ULBP1 facilitates lysis of infected cells by NK cells (Vankayalapati et al., 2005), however we hypothesize that other cell populations can also be activated by cellular stress signals induced by Mtb like γδ, CD4 and CD8 T cells, whose participation is critical to contain Mtb infection (Ernst, 2012). In conclusion, our results show that infection of human macrophages with Mycobacterium tuberculosis induce cellular stress that is reflected as an increase in mRNA levels for MICA and MICB. Acknowledgments We thank Claudia Iturbe-Haro and Jessica Castañeda-Casimiro for technical help. This work was funded by by SIP-IPN and by SEDENA A022 2013.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 68

REFERENCES Deffert, C., Cachat, J., and Krause, K.H. (2014). Phagocyte NADPH oxidase, chronic granulomatous disease and mycobacterial infections. Cell Microbiol 16, 1168-1178. El-Gazzar, A., Groh, V., and Spies, T. (2013). Immunobiology and conflicting roles of the human NKG2D lymphocyte receptor and its ligands in cancer. J Immunol 191, 1509- 1515. Ernst, J.D. (2012). The immunological life cycle of tuberculosis. Nat Rev Immunol 12, 581-591. Gonzalez, S., Groh, V., and Spies, T. (2006). Immunobiology of human NKG2D and its ligands. Curr Top Microbiol Immunol 298, 121-138. Gregorie, C.J., Wiesen, J.L., Magner, W.J., Lin, A.W., and Tomasi, T.B. (2009). Restoration of immune response gene induction in trophoblast tumor cells associated with cellular senescence. J Reprod Immunol 81, 25-33. Hamerman, J.A., Ogasawara, K., and Lanier, L.L. (2004). Cutting edge: Tolllike receptor signaling in macrophages induces ligands for the NKG2D receptor. J Immunol 172, 2001-2005. Hayday, A.C. (2009). Gammadelta T cells and the lymphoid stress-surveillance response. Immunity 31, 184-196. Huergo-Zapico, L., Acebes-Huerta, A., Lopez-Soto, A., Villa-Alvarez, M., Gonzalez- Rodriguez, A.P., and Gonzalez, S. (2014). Molecular Bases for the Regulation of NKG2D Ligands in Cancer. Front Immunol 5, 106. Mortaz, E., Adcock, I.M., Tabarsi, P., Masjedi, M.R., Mansouri, D., Velayati, A.A., Casanova, J.L., and Barnes, P.J. (2014). Interaction of Pattern Recognition Receptors with Mycobacterium Tuberculosis. J Clin Immunol. Park, E.K., Jung, H.S., Yang, H.I., Yoo, M.C., Kim, C., and Kim, K.S. (2007). Optimized THP- 1 differentiation is required for the detection of responses to weak stimuli. Inflamm Res 56, 45-50. Raulet, D.H., Gasser, S., Gowen, B.G., Deng, W., and Jung, H. (2013). Regulation of ligands for the NKG2D activating receptor. Annu Rev Immunol 31, 413-441. Reyes-Martinez, J.E., Nieto-Patlan, E., Nieto-Patlan, A., Gonzaga-Bernachi, J., Santos- Mendoza, T., Serafin-Lopez, J., Chavez-Blanco, A., SandovalMontes, C., Flores-Romo, L., Estrada-Parra, S., et al. (2014). Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype. Immunol Invest 43, 436-446. Vankayalapati, R., Garg, A., Porgador, A., Griffith, D.E., Klucar, P., Safi, H., Girard, W.M., Cosman, D., Spies, T., and Barnes, P.F. (2005). Role of NK cellactivating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium. J Immunol 175, 4611-4617. Yamamoto, K., Fujiyama, Y., Andoh, A., Bamba, T., and Okabe, H. (2001). Oxidative stress increases MICA and MICB gene expression in the human colon carcinoma cell line (CaCo-2). Biochim Biophys Acta 1526, 10-12.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

This paper shows us how a cell marks itself for sacrifice for the better good of the community, which might remind us to some famous stories of self-sacrifice, for example the crucifixion of Jesus Christ or Gilda’s act of love in the Verd’is opera ‘Rigoletto’. We can think on Scheherazade from the tale of the Arabian Nights who is offered as bait and favored at the end of the story.

SOM

Review

Cancer Research Bioinformatics

Alejandra Cervera

Systems Biology Laboratory, Research Programs Unit, Genome-Scale Biology and Institute of Biomedicine, University of Helsinki, Finland. alejandra.cervera@helsinki.fi

â&#x20AC;&#x153;Our cells resemble little factories that operate with clockwork precision. Proteins are the workforces in charge of most of the cells functionsâ&#x20AC;?

ancer is the second largest cause of death in developed countries and its incidence around the world is on rise. According to World Health Organization (WHO) around 7.6 million people died of cancer in 2008, however, by 2030 it could be increase to 13.1 million. Millions of dollars are invested annually on the search of new treatments and prevention methods. The term cancer is used to refer a large group of diseases that can affect any organ in the body. These disorders are genetic in nature, caused by abnormalities in the genome that may be inherited, but in most cases are acquired with time. In contrast to the Mendelian type of genetic disorders, where a single gene is affected, cancer is a multifactorial complex disease. The main characteristic of multifactorial or complex diseases is that several genes are usually involved in the progression of the disease, as well as, environmental or lifestyle factors. Despite the fact that cancer is so thoroughly studied, the mechanisms behind its development and progression are still not completely understood.

lated into different amino acids to conform proteins. The specific sequence of nucleotides that codify a protein is a gene. Since all cells in a single organism have exactly the same DNA, the cells decide which proteins will be synthesized according to its levels of existing proteins and the attached markers of DNA that promote or inhibit the gene expression. The cell depends on obtaining the precise instructions to synthesize proteins from the DNA. Unfortunately, there are many ways in which the information flow between genes and proteins can be disrupted causing an inadequate cell function. For example, cell exposure to radiation is known to cause mutations into its DNA. When a nucleotide inside a gene is replaced by one of the other three types of nucleotides, the protein blueprint is corrupted. An error in the sequence cannot affect the protein product or it can yield a defective protein or no protein at all. If we talk about mutations caused by radiation, we can highlight that carcinogens can affect the protein production due to their tendency to bind to the DNA interfering with the gene expression.

One of the aims of medical research is to elucidate how normal cells become cancerous. Our cells resemble little factories that operate with clockwork precision. Proteins are the workforces in charge of most of the cells functions. Different proteins perform different tasks, so specific proteins need to be available in the cell at different time. The blueprints for producing the proteins that cells need are found in the DNA. The DNA is a molecule of two chains with smaller subunits called nucleotides (adenine, thymine, guanine and cytosine). The order in which the nucleotides appear serves as a code that can be trans-

Cells have many mechanisms that keep the DNA intact, but it is often the case that the genes in charge of protecting the DNA from alterations are precisely the ones under attack. The main function of TP53 gene is to protect the cells from DNA damage, but frequently it is mutated in many cancer cells. When mutations start to accumulate into the DNA, the cell is compromised and its behavior changes completely. The new acquired mutations that compromise many basic cell functions promote tumor formation or cell death, due to the loss of cell ability to regulate its replication. CONS ART * VOLUMEN 2 * NĂ&#x161;MERO 2 * ENERO-ABRIL 2015 70

Bioinformatics As random as the accumulation of mutations may seem, it has been observed that different cancer types favor specific mutations. The most well known examples are the mutations in BRCA1 and BRCA2 genes that are common in breast and ovarian cancers. Similarly to TP53, BRCA1 and BRCA2 are also tumor suppressor genes that help to repair the DNA damage or promote the cell death, when the corruption of the sequence is too extensive. Mutations in BRCA1 for example leads to 60 to 90% of probability to develop breast cancer and 40 to 60% of probability to develop ovarian cancer. RB1 is also a tumor suppressor gene that is usually mutated in most cancers. A single mutation in each of the two copies of RB1 is the cause of retinoblastoma, a cancer of the eye retina. However, many other cancers need different genomic modifications to progress. Identify the specific mutations that lead to cancer is not quite simple since cancer cells tend to accumulate a great number of alterations, usually referred as passenger mutations (mutations that are just there that occur incidentally because of mutational processes without being beneficial for the cancer in any way). Cancer researchers try to understand the mechanisms of the disease progression by identifying the genes that differ between normal and cancer cells. One way of identifying these genes is analyzing the DNA of the cells inside the tumor. For this purpose, a biopsy from the tumor is taken from the patient. Then, DNA is extracted from a sample of tumor containing thousands of cells that can include normal tissue, immune system cells, and cancer cells that have accumulated different sets of mutations. The combined DNA extracted from the tumor is sheared into million of pieces and then sequenced to know the exact code for each gene. 71

Current sequencing technologies produce millions of scrambled reads only tens of bases long that have to be pieced together to reconstruct the genome of study. The human genome is 3 billion of nucleotides long and contains about 20,000 different protein-coding genes. Reassembling the short reads obtained from sequencing machines into complete genomes is the first of many challenges that Bioinformaticians tackle prior to identifying mutations. Bioinformatics is an interdisciplinary scientific field that makes use of tools and methods from computer science, mathematics, genetic engineering and statistics to study and process biological data. The step of reconstructing the genome is usually referred as assembly or alignment and constitutes one still active area of algorithm development in bioinformatics. Thanks to the Human Genome Project a reference human DNA sequence is available on the net and the short reads can be directly aligned to it. When a reference genome is not used, then the process of joining together the short sequences into a whole genome is called assembly. Neither alignments nor assemblies can be perfect, since the human genome has many repetitive regions that so far have not been inferred in their totality. Nowadays is possible to identify the majority of differences in the gene sequences between healthy and cancer genome. Cancer cells present common aberrations into their DNA that include: single nucleotide variations and several base deletions (bases missing), insertions (extra bases), amplifications (bases inserted several times in a row), and translocations (DNA fragments that are copied or cut from one place and inserted somewhere and possibly in a reverse order). Since some of these alterations are usually found in a single tumor, scientists compare the DNAs of several patients with the same type of cancer, and preferably also same subtype, with the aim to identify the possible cause of the uncontrolled cell proliferation. Examples of known gene alterations that lead to cancer include: mutations that activate the KIT gene causing gastrointestinal tumors, mutations in MET that lead to renal cancer and the formation of the BCR-ABL fused protein responsible for myeloid leukemia. The tumor genome study is quite useful to understand the involvement of different genes in cancer progression, but is usually not enough, since actual gene expression cannot be directly assessed. If we want to know what is really going on, which proteins are being produced in the cell, then we need to look at RNA level. When a protein is needed in the cell, its blueprint has to be copied from the DNA into a mRNA (messenger RNA), molecule that can

CONS ART * VOLUMEN 2 * NĂ&#x161;MERO 2 * ENERO-ABRIL 2015

SOM

SOM be translated into an amino acid chain by the ribosome. Aminoacids are the subunits that form the proteins. Similar to the DNA, the mRNA is also extracted from the tumor cells, is sheared and sequenced. The mRNA fragments also have to be pieced together, but this time with the aim of reconstructing individual gene sequences and estimating their abundance.

Despite the fact that genes are several orders of magnitude shorter than the DNA, the assembly or alignment step is not straightforward. At DNA level, the genes are formed by successions of exons and introns. Exons are blocks of DNA that will be translated into amino acids, while introns are usually discarded. To complicate things further, varying combinations of exons can be used to create different proteins from the same gene. This process of including some exons while not others is called alternative splicing. In some cases, the proteins variants perform similar functions, in others; they can perform complete different functions that could block their own expression. The splice variants of a gene may be expressed at different stages of the cell cycle, but it has also been showed that some isoforms are only expressed in cancer cells, as it is the case with the CD44 gene. Assembling the short-reads sequences into the right splice variant of the gene is still an open bioinformatic problem. Since different isoforms share some of exons, and the short-reads rarely overlap more than one exon junction (places where consecutive exons are joined together, i.e where an intron has been spliced out), we need to use a statistical model of read coverage evenness to accept or reject these predicted isoform and the unambiguously decision. Despite the complexities, algorithms are capable of mapping most of the reads of individual genes allowing us to quantify their expression by counting how many reads have been assigned to each gene. The more copies of the same gene are found in the cell means that this gene is more active. When control samples are available, the gene expression estimates between cancer and normal cells can be compared. The differential expression analysis is often used to identify genes that are changing on the processes occurring in cancer and that are not affected in healthy tissues, but unfortunately the normal biological variation between cells can complicate the identification of the real drivers of the disease. Gene expression can also be used to corroborate the relevance of aberrations found at DNA level. Amplifications, deletions, and some

single nucleotide variations alter the gene expression patterns located in the affected region of the DNA. Even though quantifying the mRNA is a good measure of identifying expressed genes it comes with some caveats. Exist small species of RNA,, such as microRNAs (miRNAs) and silencing RNAs (siRNAs) that are known to interfere with the translation of mRNA into proteins. MicroRNAs are usually 21-25 nucleotides non-coding RNAs, short sequences that bind to specific mRNAs promoting their early degradation or inhibiting their translation. miRNAs have been revealed to play important roles in many biological systems, ranging from the development and differentiation of cells to tumors, including those in the mammalian immune system. Dysregulation of miRNAs has been associated with disease, but it still unknown about them and their role in cancer, and therefore continue to be an active field of study. In that sense, in the last 5 years various miRNAs have associated with cell proliferation, resistance to apoptosis, differentiation, immunity and cancer initiation, progression and metastasis. Thanks to their size, miRNA do not need to be sheared prior to sequencing, and therefore do not need to be reassembled. The catch with miRNAs comes when we are trying to find the target genes of each miRNA. On that sense several databases or algorithms for miRNA target prediction have been created and are widely available, wich can predict miRNA targets across different species. The most established and widely used miRNA target prediction algorithms are TargetScan, Miranda and PicTar, however, the evaluation of their utility of each prediction algorithm is difficult as there is no comprehensive set of experimentally validated targets. Furthermore, it is on depending on the cell cycle, the cell type, as well as, tools for predicting targets for novel miRNAs, but still heaps of work are still needed to fully characterize them. Alternatively, the 3D structure of the DNA can also be disrupted, adding an additional layer of complexity to gene regulation. Chemical changes that do not cause a nucleotide substitution, but that alter the DNA structure making it more or less accessible to transcription are called epigenetic modifications. Epigenetic marks play an important role in gene regulation in our cells and their configurations are cell type specific. The best studied of the epigenetic marks is the methylation of the DNA. Methylation occurs in parts of ge-

CONS ART * VOLUMEN 2 * NĂ&#x161;MERO 2 * ENERO-ABRIL 2015 72

SOM nome called CpG sites, a cytosine followed directly by a guanine where, a methyl group is added to the cytosine along the genome, leading to a tighter DNA 3D structure (heterochromatin). If the promoter region of a gene (the upstream sequence of a gene where the transcription begins) is methylated it results in the silencing of a gene. In fact, epigenetic modifications have been observed in several diseases including cancer.

Recommended bibliography [1]. References Garber M, Grabherr MG, Guttman M, Trapnell C. (2011). Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods 8(6):469-77. Mardis ER (2008). Next-generation DNA sequencing

Tumor suppressor genes, like TP53, can be inactivated by methylation of its promoter region. In a similar way, loss of methylation leads to an unstable genome where further DNA damage may result in the expression of cancer promoting genes (oncogenes). It comes as no surprise that in general cancer genomes have been found to be hypomethylated compared to normal genomes, except for promoter regions of tumor suppressor genes where the opposite occurs.

methods. Annu Rev Genomics Hum Genet. 9:387-402. Martinez NRT, Louafi F, Snchez ET. (2011). The Interleukin 13 (IL-13) Pathway in Human Macrophages Is Modulated by MicroRNA-155 via Direct Targeting of Interleukin 13 Receptor a1 (IL13Ra1).J Biol Chem 286 (3): 1786-1794. Morozova O, Marra MA. (2008). Applications of nextgeneration sequencing technologies in functional genomics. Genomics. 92(5):255-64. Kristensen VN, Lingjærde OC, Vollan HKM, Frigessi

The sequencing cost has been in constant decline during the past years allowing us to study cancer tumors in great detail and numbers. Nevertheless, the promise of personalized medicine has not fully arrived to the clinic. Tailored treatments to patients with specific genomic alterations do exist, but not as many as we would like to have by now. Despite the fact that hundreds of algorithms and tools for analyzing our genomes have been published, nowadays exist few golden standards for processing the data. The combination of different levels of information to find a specific cancer treatment remains as an important challenge to be resolved. The role of bioinformatics has progressively become more and more important in our quest to find new and more efficient cancer treatments; a trend that undoubtedly will certainly continue as long as new technologies become available as new algorithms are required.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

A, Børresen-Dale A-L. (2014). Principles and methods of integrative genomic analyses in cancer. Nature Reviews Cancer 14: 299-313 Ziller MJ, Gu H, Müller F, Donaghey J, Tsai LT, Kohlbacher O, De Jager PL, Rosen ED, Bennett DA, Bernstein BE, Gnirke A, Meissner A. (2013). Charting a dynamic DNA methylation landscape of the human genome. Nature 500:477-81.

Resembling the Sherlock Holmes cleverness, necessary to uncover clues unseen and solve the unsolvable and strange case described in “The adventure of the red circle”, in cancer research, we need the same qualities to uncover its mysteries. As well as the shrewdness of Holmes was essential to discern between the suspects and find out the real guilty, making successful arrangements on bioinformatic algorithms could be essential to discern between normal and cancerous cells. As Holmes, in that short story, discovered a kind of secret communication between the main suspects to be safe and stay alive, the bioinformatics could be our own “Sherlock” to uncover the hidden alterations inside of affected cells.

One method to identify the methylation patterns is the transformation of the modified cytosines into uracil (a type of nucleotide usually only present in RNA) and then sequencing the DNA. Uracils in the sequence will point to the CpG sites where the cytosine has been methylated. Restoring the original epigenetic patterns in cells is believed to be possible and new generation of cancer drugs are being created with this aim. Among them, several DNA methylation inhibitors have been proved to remove the silencing mark from tumor suppressor genes and are currently under clinical trial.

SOM

Enfermedad y arte,

Medicina

colaboraciones

no intencionales

Ceja-Reyes Ana Isabela, Romero-López José Pabloa,b,c

Ilustration by Kat Powell

(a) Médico Cirujano egresado de la FES Iztacala, UNAM, (b) Estudiante de Maestría en Ciencias Quimicobiológicas, Laboratorio de Inmunoquímica I, Escuela Nacional de Ciencias Biológicas, IPN. (c) Programa Interdisciplinario de Investigación en Medicina (PROINMED) FES Iztacala, UNAM.

Como resultado de la obra de los seres humanos, el arte es susceptible de ser afectado por aquello que aqueja a su creador y parte de éstos elementos que pueden modificar la producción de un artista, reside en su estado de salud. Al imprimir una idea o una imagen en una obra de arte, encontramos ésta magnífica colaboración no intencional; los efectos de los avances científicos de la época se ven claramente reflejados a un lado de la historia del arte; las enfermedades de las personas retratadas que pudieron pasar ocultas por tratarse de entidades patológicas desconocidas, o simplemente ignoradas, han sido descubiertas mucho tiempo después por el estudio de las obras. En ésta revisión se pretende hacer una brevísima reseña de la estrecha relación que existe entre la medicina, la enfermedad y el arte, para rescatar la estrecha unión artístico-científica que tiñe de interés ambas partes. La anatomía como disciplina científica, ha intervenido directamente en aquellas expresiones artísticas que requieran este conocimiento para describir la apariencia de un animal o del hombre. Remontándonos al renacimiento, tenemos la obligación de destacar a dos de los grandes de la época, Leonardo da Vinci y Miguel Ángel Buonarroti quienes al no conformarse con una rama del saber, decidieron realizar estudios anatómicos de forma clandestina, deseosos de realizar sus obras más apegadas a la realidad. Éstos estudios ejercieron gran influencia en su trabajo, influencia reflejada en el famoso dibujo del “Hombre de Vitrubio” de Da Vinci, realizado a partir de los textos del arquitecto de la antigua Roma Vitruvio –de quien el dibujo toma su nombre, donde se reflejan las proporciones del cuerpo humano, explicándonos la perfecta armonía de la anatomía humana. La descripción de esta imagen (Fig. 1) describe que, si se centra un compás en el ombligo de un hombre colocado sobre la espalda, con las piernas suficientemente extendidas para que su altura disminuya 1/14 del total y los hombros elevados hasta que los dedos estén al nivel del borde superior de la cabeza, se forma una circunferencia donde los dedos de ambas manos y pies tocan el perímetro del círculo así trazado, además de que se puede formar un cuadrado con los bordes de la imagen1. CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 74

SOM Fig.

Da Vinci L, Hombre de Vitruvio. 1492; pluma y tinta en papel, Galería de la Academia, Venecia, Italia

Fig.

Buonarroti MA, David. 1501-1504; mármol de Carrara, Galería de la Academia, Florencia, Italia

Otro resultado de esta obra tan importante, es el conocimiento sobre las proporciones, de tal manera que encontramos que “la longitud de los brazos extendidos de un hombre es igual a su altura”, “Un pie es la anchura de cuatro palmas” y “La altura de un hombre son cuatro antebrazos, entre otras. 1 Un ejemplo en el que se ve plasmada la maestría de los estudios anatómicos de la época y su influencia en el arte es el “David” de Miguel Ángel (Fig. 2), escultura en la que se representa cada músculo visible a través de la piel, marcado perfectamente de acuerdo con la posición que tiene David, con la cara vuelta hacia un extremo (mirando a su adversario), con una mirada calculadora, frunciendo el entrecejo como midiendo a su contrincante.2 Podemos observar los mínimos detalles, todos los miembros manifiestan un reposo tenso, marcándose tendones, músculos y realzándose las venas (Fig 3) es la captación del momento anterior a la acción, la máxima concentración antes del acontecimiento de defender a un pueblo de un gigante. 75

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

SOM Fig.

Da Vinci L, La Gioconda. 1503; óleo sobre tabla de álamo, Museo del Louvre, París, Francia

Fig.

de mama. Ésta obra llamada “Betsabé con la carta del Rey David” (Fig. 5), es una escena extraída del relato bíblico en el que la protagonista, Betsabé, recibe una carta del Rey David en la que la invita a su palacio, se encuentra consternada, reflejando una expresión de angustia por tener que tomar la decisión entre permanecer fiel a su marido u obedecer al rey. En la pintura se observa que la modelo presenta tumoraciones axilares y retracción cutánea del cuadrante inferior externo de la mama izquierda 4, signos que hacen sospechar de cáncer de mama–Situación que la aquejaría más que la mala noticia de la carta-.

La pintura es quizá la actividad artística que ha dejado los testimonios más tangibles de la huella de diversas enfermedades que aquejaban a los protagonistas y a sus creadores. El retrato más famoso del mundo, “La Gioconda” de Leonardo Da Vinci (Fig. 4) ha suscitado desde hace más de un siglo diferentes hipótesis, rumores y sospechas en el ambiente médico, llegando a ser considerada por algunos como “Un compendio de medicina interna”3, debido a las múltiples enfermedades o situaciones que le han sido adjudicadas. Resumiendo, a ésta mujer de sonrisa enigmática, se le ha diagnosticado embarazo, enfermedades neurológicas como parálisis facial periférica, estrabismo, hipoacusia, parkinsonismo, alteraciones periorales como traumatismos bucales, bruxismo, tics distónicos, esclerodermia, sífilis, alopecia universal, hipercolesterolemia familiar y tricotilomanía con depresión y ansiedad3. Actualmente, gracias a las nuevas tecnólogias aplicadas al estudio del arte, tenemos otra mirada sobre la salud de la Gioconda, se han descartado las condiciones patológicas ya mencionadas y se han atribuido a imperfecciones del barniz y limpieza errónea de la pintura, siendo su única afección la presencia de un lipoma o fibrolipoma en el dorso de la mano derecha3.

Pasando a una de las pinturas más emblemáticas del arte occidental, “Las Meninas” de Velázquez (Fig.6), encontramos representada una condición corporal frecuentemente usado en la pintura barroca, el enanismo (acondroplasia), condición que no era plasmada tan frecuentemente por tener una alta incidencia en la población, sino porque muchos bufones de la corte eran enanos, ya que por su baja estatura con frecuencia eran destinados a acompañar a los niños5. La obra de “Las Meninas” parece situarse en la infanta Margarita, de cinco años, quién está rodeada por sus damas de honor y dos enanos a su izquierda. Se logran apreciar características propias de la facie del acondroplásico como la naríz chata, frente ancha y prominente y obviamente la baja estatura5.

Fig. Rembrandt H, Betsabé con la carta del rey David.1654; óleo sobre lienzo, Museo del Louvre, París, Francia

Hace más de trecientos años, Rembrandt plasmó otro ejemplo en el que se observa una de las enfermedades con más prevalencia en nuestro medio, el cáncer

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 76

SOM Fig.

Velázquez D, Las Meninas. 1656, óleo sobre lienzo, Museo del Prado, Madrid, España

Cualquier enfermedad, por insignificante que llegue a ser, deja alguna huella en el organismo al que ataca. En el caso de los artistas, estas huellas quedan marcadas por la eternidad en sus obras, por lo que se mencionarán algunos ejemplos. En primer lugar, hablaremos sobre Claude Monet, figura clave del movimiento impresionista. Hablar de Monet es hablar de luz, sobras, modernidad, abstracción y adaptación a la enfermedad; su sintomatología inició a la edad de 72 años cuando éste famosísimo pintor detectó cierto grado de deterioro de la visión, además de percibir que los colores habían cambiado, esto coincide con un viaje a Venecia, donde realizó algunas series de cuadros venecianos en los que se percibe un predominio de colores ocres; él mismo refiere pintar de memoria sin poder ver los colores fríos, azules o violetas. En 1918 escribe lo siguiente: “No logro percibir los colores con la misma intensidad que antes. Los rojos parecen como lodosos, mi pintura se vuelve mas y mas obscura… por un lado confío en las etiquetas de los tubos de pintura y por el otro confío en mi memoria”6. Fue hasta 1922 que tras ser examinado por diferentes oftalmólogos se le diagnostican cataratas, éstas modifican progresivamente la sensibilidad al contraste, actuando como un filtro amarillo que aumenta la visión de los colores cálidos como los ocres y los marrones y que impide la visualización de los colores fríos, los azules y los violetas. Por

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

Fig.

SOM

ello a la edad de 83 años se le realizó una iridectomía y en una segunda sesión, extracción extracapsular con aspiración de masas que se complicó, requiriendo una tercera operación para la sección de una membrana del ojo derecho. El pintor tardó tiempo en percibir bien los colores y nunca aceptó operar el ojo izquierdo. En aquel tiempo removían el cristalino, sin trasplantar uno nuevo, por lo que su visión quedó realmente deteriorada, necesitando así gafas afaquicas7.

Monet C, El puente Japonés. 1899; óleo sobre tela, Galeria Nacional, Londres, Inglaterra

Fig.

Podemos ser testigos de su enfermedad, ya que le gustaba realizar series de cuadros, aquí observamos “El puente japonés”(Fig. 7), pintado a los 59 años, cuando aún no presentaba ninguna sintomatología, observamos el espectro de luz y sombras, un balance de colores y detalles. Comparamos una segunda versión realizada en 1922 (Fig.8) en la que la catarata ya había modificado su percepción espacial y la visión de los detalles, observamos el tinte ocre y predominio de los marrones. Después, una tercera versión de (Fig.9 ), posterior su cirugía, en la que la afaquia potencia el espectro azul produciendo una percepción azulada llamada cianopsia.6,7 Monet adaptó el arte a su enfermedad, pintaba a determinada hora del día, prefería trabajar con la luz tenue del crepúsculo para mantener cierto sentido del color, esta situación no lo detuvo a seguir pintando convirtiéndose así, en uno de los exponentes más importantes del impresionismo.

Monet C, El puente Japonés. 1922; óleo sobre tela, Museo Marmottan, París, Francia

Fig.

Monet C, El puente Japonés. 1926; óleo sobre tela, Museo Marmottan, París, Francia

Otro claro ejemplo de la influencia del estado de salud en la producción artística es el caso del famoso pintor Paul Klee (18791940) En 1933, Klee, que hasta entonces trabajaba como profesor en el colegio de Düseldorf, fue despedido y estigmatizado como un “artista degenerado”. Al regresar a su pueblo natal tuvo exhibiciones de arte sin éxito y llegó al grado de ser juzgado como esquizofrénico, lo que lo llevó a un estado de aislamiento total. En 1935, Klee sufrió afecciones respiratorias que fueron diagnosticadas como bronquitis y neumonía, a esto se agregaron anemia y debilidad, con el tiempo la piel de su cara y cuello se engrosó y endureció a tal grado que no podía abrir la boca con facilidad. En sus obras artísticas se puede apreciar el cambio evidente de su transformación causada por la enfermedad. Klee falleció en 1940 debido a miocarditis sin contar con ningún diagnóstico etiológico. Hoy en día se sugiere que el motivo de todos los síntomas y signos de éste pintor fue esclerosis sistémica difusa, una de las enfermedades autoinmunes más severas, recientemente estudiada que en los años de Klee era prácticamente desconocida o incluida dentro del espectro clínico de la esclerodermia8. En las ilustraciones se aprecia la comparación de la producción artística de éste importante pintor antes (Fig. 10)y después (Fig. 11) de sufrir la devastación resultante de la autoinmunidad que lo llevó a la tumba, no es dificil encontrar el lado pesimista del pintor al ilustrar la muerte de la manera en la que lo hace ya que en ésta y otras obras

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 78

SOM

Fig.

11 10

Klee P. Muerte y fuego. 1940; óleo sobre yute, Museo Paul Klee, Berna, Suiza

demuestra el sentimiento de estar atrapado en un cuerpo acartonado en el precipicio hacia el fin de la vida. Tal como estas obras, se encuentran muchas otras, en las que el talento, la formación y un ojo perspicaz han dejado como legado universal obras opulentas, que entre muchos otros mensajes, nos concede admirar el arte desde otro punto de vista, uno en el que la adjunción del arte y ciencia recrea, interpreta y articula ambas actividades. De igual forma sería una estimulante opción que diversas profesiones examinen estas obras de arte, para que desde su original interpretación, nos revele puntos de vista no explorados.

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

Referencias 1. Panero J, Zelnik M. Las dimensiones humanas en los espacios interiores. Ediciones G. Gill. 1987:15-17 2. Stone I. La agonía y el Éxtasis. Vida de Miguel Ángel. Emece 2003:287-355. 3. Barbado Hernández FJ. Otra mirada médica a La Gioconda. Rev Clin Esp. 2012;212(11):549-550. 4. Vaidya JS. Breast cancer: an artistic view. The Lancet Oncology. 2007;8:583585. 5. Sierra Valentí X. Medicina y enfermedad en el arte barroco. Actas Dermosifiliogr. 2007;98:570-4. 6. Marmor MF. Ophtalmology and Art: Simulation of Monet´s Cataracts and Dega´s Retinal Disease. Arch Ophthalmol. 2006;124:1764-1769 7. Álvarez-Suárez ML. Las Cataratas de Monet. Arch Soc Esp Oftalmol. 2005;8 8. Suter H. Case report on the illness of Paul Klee (1978-1940). Case Rep Dermatol. 2014; 6:108-113.

Fig.

Klee P. Crystal Gradation. 1921; acuarela. Kunstmuseum Basel, Basel, Suiza

SOM

Reseña del artista / Artist review

https://www.behance.net/katpowell

My artwork is a showcase of the creative mind teetering between the nature flow of paint and the nurturing skill of my hands and mind. To push this idea of attempting to understand which side is dominating I choose to draw skeletal creatures in my artwork. I create images of anatomy and skeletons because, out of all the things in the world, your own bones and muscles are the closest thing to you and yet, even though they have been close to you since birth, you still don’t fully understand them. All of these fascinating structures, mechanics, and self-regulating organs are barely hidden under a small barrier that constantly drives us to discover how life can be sustained. That is the purpose behind the image “Rod of Asclepius”.

El trabajo de esta artista representa claramente el nexo difuso pero fuerte que se mantiene entre las ciencias de la salud y el arte, representando la unión entre lo palpable (anatomía) y lo creado (arte), el trabajo de Kat Powell ejemplifica la estrecha relación entre “El arte y la medicina”

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015 80

Rese単a de la portada

Interview Where were you born and where do you live? I was born in Milwaukee, WI, USA and have lived here all my life.

What is your professional formation and where did you study?

I attended the Milwaukee Institute of Art and Design, but only for one semester. I consider myself a self-taught artist.

How can you describe your artistic work?

It’s a blending of joy and sorrow. I try to bring a balance of the positive and negative in each work. I feel life is a constant balancing act of both good and bad, and you can have an appreciation of both. Even in the ugly there is beauty.

Have you ever been related with anatomy or any other branch of the science? I have, actually. When I’m not creating art, I work as a registered nurse. Not passionately, but it pays the bills.

What do you think about scientists who get involved with art or vice versa, artists that touch science on their work?

That maybe reaching into a realm that is structurally different than what you’re used to can open doors in the mind and spark ideas that may not have been originally thought of without stepping out of our comfort zone.

What do you think about our effort to demonstrate the link between this two faces of life(science and art)?

It’s very interesting. I like the blending of images and theories. Fresh and stimulating.

Could you tell us something about the creation of these collages? What was your inspiration to do them? Well, mostly they were a sort of creative masturbation. I love to paint surrealistic images but I prefer to actually look at a subject to draw vs drawing straight out of my head. Digital collage was a solution to that dilemma. Some of the collages I never get to painting and I just use them as limited edition prints.

The personality of an artist is definitively its most valuable piece of art. How do you believe that your personality have influenced your art? I feel my personality is directly transferred into my work. Some pieces are things I fantasize about, some are pulled from my childhood and others are my translation of the world around me. I have been told that making art is very egocentric. I agree...how can you separate yourself from something you’re creating. Thank you so much.

Entrevista ¿Dónde naciste? y ¿Dónde vives? Nací en Milwaukee WI, EUA y he vivido aqui toda mi vida

¿Qué formación profesional tienes? y ¿Dónde estudiaste?

Asistí al Instituti de Arte y Diseño de Milwaukee, pero solo por un semestre. Me considero una artista auto-educada.

¿Cómo podrías describir tu trabajo artístico?

Es una mezcla de alegría y tristeza. Trato de proporcionar un balance de lo positivo y negativo a cada trabajo. Siento que la vida es un acto de balance constante entre el bien y el mal, y puedes tener una apreciación de ambos. Incluso en la fealdad hay belleza.

¿Alguna vez has estado relacionada con la anatomía o cualquier otra rama de la ciencia?

De hecho si. Cuando no estoy creando obras de arte, trabajo como una enfermera registrada, no muy apasionadamente, pero ese trabajo paga las cuentas.

¿Qué opinas a cerca de los científicos que se han involucrado con el arte, o viceversa, artistas que de alguna forma han tocado la ciencia en su trabajo?

Pienso que tal vez, alcanzando un reino que es estructuralmente diferente de aquel al que estas acostumbrado puede abrir puertas en la mente y generar ideas que no habrían sido pensadas sin dar un paso fuera de tu área de comfort.

¿Qué opinas sobre nuestro esfuerzo por demostrar el enlace enre éstas dos caras de la vida (arte y ciencia)? Es muy interesante. Me gusta la mezcla entre imagenes y teorías. Es fresco y estimulante.

¿Nos podrías decir algo sobre la creación de éstos collages?¿Cuál fué tu inspiración para hacerlos? .

Bueno, en su mayoría, fueron un tipo de masturbación creativa. Me encanta pintar imágenes surrealistas aunque prefiero ver realmente a un sujeto que dibujar que dibujar algo salido directamente de mi mente. Los colages digitales fueron una solución a ese dilema. Nunca llegué a pintar algunos de los collages, y solo los usé como impresiones de edición limitada.

Definitivamente, la personalidad de un artista es su más valiosa obra de arte. ¿Cómo crees que tu personalidad ha influido tu trabajo? Siento que mi personalidad está directamente transmitida a mi trabajo. Algunas obras son cosas en las que fantaseo, algunas son tomadas de mi infancia y otras son traducciones mías del mundo que me rodea. Me han dicho que hacer arte es algo muy egocentrico. Estoy de acuerdo...¿cómo podrías separarte de algo que estás creando?. Muchas gracias.

INMUNOLOGÍA

XVIII Congreso Latinoamericano de Alergia, Asma e Inmunología del 14 al 16 de marzo de 2015 Buenos Aires, Argentina 4th European Congress of Immunology, Vienna, September 6-9, 2015.

CONGRESS

itinerary

Annual International Conference on Pharmacology and Pharmaceutical Sciences (PHARMA) 26-27 October Bangkok Thailand V Congreso de la rama de Especies Reactivas del Oxígeno en Biología y Medicina de la Sociedad Mexicana de Bioquímica y el VI Taller Internacional de Aspectos Comparativos del Estrés Oxidativo en Sistemas Biológicos del 18 – 21 de marzo del 2015 Morelos, México.

International Conference on Immunology September 25 - 26, 2015 London, United Kingdom

XI Congreso de la Asociación Latinoamericana de Inmunología - ALAI X Congreso Colombiano de Alergia Asma e Inmunología - ACAAI Octubre 13-16, 2015 Medellín, Colombia

56 CONGRESO NACIONAL AMEH 01 de mayo del 2015 Acapulco, México Metabolism and Cancer del 07 al 10 de junio de 2015 Bellevue, USA Congreso Centroamericano y del caribe de Parasitología y medicina tropical del 12 al 13 de junio de 2015 Bávaro, Punta Cana, República Dominicana 3rd Annual International Conference on Advances in Medical Research (CAMR 2015) del 17 al 18 de agosto de 2015 Singapore, Singapur

CONS ART * VOLUMEN 2 * NÚMERO 2 * ENERO-ABRIL 2015

50° Congreso Mexicano de Química del 07 al 10 de octubre Querétaro, México

PRIZE

itinerary • Call Mobility Grants AMC International and Advanced Fellowships CONACYT

• La Academia Mexicana de Ciencias, el Consejo Nacional de Ciencia y Tecnología y el Consejo Consultivo de Ciencias de la Presidencia de la República, abren a concurso las “Becas para Mujeres en las Humanidades y las Ciencias Sociales“

• ESTANCIAS DE VERANO EN EE.UU. PARA INVESTIGADORES JÓVENES, AMC-FUMEC 2015 • UC MEXUS-CONACYT Postdoctoral Fellowship Program 2015-16

http:// www.conscienceandart.com http://conscienceandart.wordpress.com

/ConScienceArt @ConScience_Art