A Cardiomiopatia Chagásica Crônica (CCC) é o elemento mórbido mais importante da Doença de Chagas e sua elucidação se tornou fundamental. Estudos da imunologia da CCC demonstram que o sistema imune desempenha um papel duplo no curso da doença, agindo de forma a controlar as formas parasitárias e ainda promovendo lesão tissular. Porém, pouco se sabe do papel das células estruturais, tais como os cardiomiócitos, no curso da doença. Sabe-se que, em outras patologias cardíacas, o IFN-γ, citocina produzida em abundância no coração dos pacientes com CCC, determina o priming de diversas populações celulares, modulando positivamente a sua resposta. Cardiomiócitos HL-1 e animais C3H/HePas foram primados com IFN-γ e desafiados com LPS para a dosagem de citocinas, simulando quadro agudo e crônico de infecção. Neste trabalho, determinamos que o IFN-γ modula positivamente a produção de diversas citocinas in vitro por células HL-1 (IP-10, MCP-1, G-CSF, RANTES, MIG, IL-6, MIF) e também in vivo no coração (IP-10, KC, G-CSF, LIF e IL-6). Além disso, in vitro, o IFN-γ foi capaz de diminuir a produção de VEGF e GM-CSF em relação aos grupos tratados apenas com LPS. Os dados corroboram a literatura e permitem concluir que os cardiomiócitos são capazes de participar ativamente da resposta inflamatória no coração e que são sensíveis aos produtos da mesma. O trabalho serve ainda de base para novos estudos sobre o perfil de citocinas expressas no coração no curso da infecção por T. cruzi e como os cardiomiócitos participam da resposta inflamatória em questão. / Chronic Chagasic Cardiomyopathy (CCC) is the most morbid element of Chagas Disease, the elucidation of its physiopathology being fundamental. However, little is known about the role of structural cells, such as cardiomyocytes, in the course of the disease. In other cardiac pathologies, it has been shown that IFN-γ determines the priming of several resident populations, positively modulating their response. In this work, HL-1 cardiomyocytes and C3H/HePas mice were primed with IFN-γ (in brief or extended protocols) and challenged with LPS, the cytokines produced being measured in the supernatants. We observed that IFN-γ positively modulates the in vitro production of many cytokines by HL-1 cells (IP-10, MCP-1, G- CSF, RANTES, MIG, IL-6, MIF) and also their in vivo production at the heart (IP-10, KC, G-CSF, LIF and IL-6). Besides, IFN-γ was able to decrease the LPS-induced production of VEGF and GM-CSF by HL-1 cells. Our data allow us to conclude that cardiomyocytes actively participate in the inflammatory response of the heart, being sensitive to products released by professional immune cells. This work may serve as a basis for further studies on the profile of the cytokines secreted in the heart tissue along the course of cardiac inflammation.

La SUMOylation est une modification post-traductionnelle qui gouverne divers processus cellulaires incluant immunité innée et défense antivirale. Des effecteurs de la synthèse d’IFN, de son signal de transduction ainsi que des facteurs de restriction sont modifiés par SUMO (Small Ubiquitin Modifier). Par ailleurs, certains virus exploitent la machinerie SUMO afin de contrecarrer les mécanismes de défense antivirale suggérant l’implication de SUMO dans l’interface virus et défense antivirale. A l’aide d’un modèle cellulaire exprimant les différents paralogues SUMO1, SUMO2 ou SUMO3 ou en diminuant l’expression de l’unique enzyme de conjugaison à SUMO, Ubc9, nous avons montré un effet différentiel de SUMO sur deux virus de la famille des Rhabdoviridae (virus de la stomatite vésiculaire (VSV) et le virus de la rage) et sur la réponse aux IFN alpha et IFN gamma. Le premier axe de recherche a permis de montrer que l’expression de SUMO inhibe la synthèse de l’IFN suite à l’infection par le VSV et le virus de la rage, rendant les cellules plus sensibles à l’infection par le virus de la rage. IRF3 est conjuguée à SUMO, ce qui corrèle avec l’inhibition de sa phosphorylation et l’inhibition de la synthèse d’IFN beta. En revanche, bien que la synthèse de l'IFN soit diminuée, l’expression de SUMO confère la résistance au VSV et inhibe sa transcription primaire. L’activité anti-VSV de SUMO est abolie par la déplétion de MxA. L’effet de SUMO est médié par sa capacité à augmenter l’oligomérisation et la stabilité de MxA. Par ailleurs, ce travail a permis d’identifier MxA comme nouvelle cible de la machinerie SUMO. MxA interagit avec SUMO de manière covalente sur la lysine K48 et de manière non covalente avec SUMO1. Le second axe de recherche a permis d’identifier SUMO comme un nouveau régulateur de la réponse aux IFN. La SUMOylation de STAT1 inhibe sa phosphorylation, régulant négativement le signal de transduction et par conséquent la transcription et la réponse biologique en réponse à l’IFN gamma. En revanche, l’expression de SUMO n’altère ni le signal de transduction ni la transcription en réponse à l’IFN alpha.Par ailleurs, dans les cellules exprimant SUMO3, l’IFN gamma et l’IFN alpha induisent la SUMOylation de PML par SUMO3 ce qui entraîne sa dégradation via le protéasome et inhibe les réponses biologiques médiées par PML. Ce travail a permis de montrer un rôle central de SUMO dans l’immunité intrinsèque et innée, médié par la SUMOylation de protéines cellulaires telles qu’IRF3, MxA, STAT1 ou PML. / SUMOylation modulates several cellular process including innate immunity and antiviral defense. Many key regulators involved in IFN induction, IFN signaling as well as various restriction factors are SUMOylated. Using cells stably expressing the different paralogs of SUMO; SUMO1, SUMO2 or SUMO3 and cells depleted of the only known SUMO conjugating enzyme, Ubc9, we show a differential effect on two viruses from Rhabdoviridae family (Vesicular Stomatitis Virus (VSV) and rabies virus) and on the response to IFN alpha and IFN gamma. First, we report that SUMO expression inhibits VSV- and rabies virus-induced IFN synthesis. Consequently, SUMO expression renders cells more sensitive to rabies virus infection. Overexpression of SUMO leads to IRF3 SUMOylation correlating rabies viral infection with both the inhibition of IRF3 activation and IFN beta synthesis. However, although SUMO inhibits VSV-induced IFN, SUMO confers resistance to VSV by inhibiting VSV primary transcription. Furthermore, the anti-VSV effect of SUMO is abolished in MxA depleted cells. Mechanistically, SUMO enhances MxA oligomerization resulting in the stabilization of the MxA protein. We also identified MxA as a new target of SUMO machinery. MxA interacts covalently with SUMO2/3 on lysine K48 and non-covalently with SUMO1. We then investigated the various roles of SUMO at different steps of the JAK/STAT pathway, including STAT activation, transcriptional response and IFN-induced biological effects, identifying SUMO as a new regulator of IFN response. The overexpression of SUMO leads to STAT1 SUMOylation and to a decrease in IFN-induced STAT1 phosphorylation resulting in an inhibition of IFN-gamma-induced transcription and biological responses. In contrast, SUMO expression does not alter IFN alpha signaling and transcriptional response. In addition, in SUMO3 expressing, IFN gamma;and IFN alpha induce SUMOylation of PML by SUMO3 inducing its degradation via the proteasome and inhibition of biological responses mediated by PML. Taken together our results show that SUMO plays a crucial role in innate and intrinsic immunity mediated by SUMOylated proteins such as IRF3, MxA, STAT1 or PML.

No description available.

The interferon (IFN) system is a powerful antiviral defense system. Host cell pattern recognition receptors (PRRs) recognise pathogen-associated molecule patterns (PAMPs) which when activated, lead to the transcription of the IFN-β gene. As a consequence IFN is secreted from the cell and activates the JAK-STAT pathway to up-regulate the transcription of IFN-stimulated genes (ISGs). The products of many ISGs inhibit viral replication and cell proliferation. Viruses encode IFN antagonists that dampen down the IFN response, making it less effective. However, within a virus population, there are always likely to be naturally occurring mutant viruses that have lost the ability to circumvent the host IFN response, and if isolated, these viruses would be unlikely to cause severe disease in the host and may therefore be developed as live attenuated virus vaccine candidates. To develop a methodology to rapidly isolate IFN-inducing mutant viruses, we generated an A549 reporter cell-line in which expression of GFP was driven by the IFN-β promoter. Using this cell-line, we show that the number of cells that became positive for GFP correlated with the amount of IFN secreted by the infected cells and the number of defective interfering (DI) particles within the virus preparations. However, we were unable to isolate IFN-inducing mutant viruses using the A549/pr(IFN-β).GFP cell-line(s). Possible reasons for this may be either that, in cells infected by IFN-inducing mutant viruses, an antiviral state was established independent of IFN that prevented virus replication in the reporter cells in which the IFN-β promoter was activated; or the viruses that activated the IFN-β promoter were DIs only which were not be able to replicate without non-defective helper viruses. A549/pr(IFN-β).GFP cells are also being used for high throughput assays to screen chemical libraries for compounds that block IFN induction. Such compounds may be potential candidates for anti-inflammatory drugs.

Mechanisms involved in the establishment of HCMV latency are poorly understood, however, work in our laboratory has demonstrated the ULb' encoded protein, pUL138, as the first viral determinant to function in the establishment of HCMV latency in CD34+ hematopoietic progenitor cells (HPCs). This work characterizes the transcripts that encode pUL138, identifies three novel ULb' proteins (pUL133, pUL135, and pUL136) and represents the first demonstration of an internal ribosome entry site (IRES) mediated expression of pUL138. pUL138 is encoded on three polycistronic transcripts of 3.6-, 2.7- and 1.4-kb in length. pUL133, pUL135 and truncated pUL136, are expressed on the 3.6-, 2.7- and 1.4-kb transcripts, respectively, in addition to pUL138. We demonstrate that pUL138 expression is inducible from the IRES on the 3.6- and 2.7-kb transcripts under conditions of cellular stress, whereas pUL138 expression from the 1.4-kb transcript is inhibited under these same conditions. Differential utilization of the UL138 transcripts and their respective encoded proteins may regulate the outcome of viral infection in a cell type or cell context dependent manner. The interaction of these proteins during HCMV latency is the focus of ongoing research. In addition, this work represents preliminary data regarding the type I interferon (IFN) response during HCMV during productive infection in MRC5 fibroblasts and during the establishment of HCMV latency in CD34+ HPCs.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Interferons (IFNs) comprise a group of antiviral cytokines; however, their expression at the porcine maternal-fetal interface and in fetal tissues has not previously been investigated. The purpose of this study was to analyze the expression of type I IFNs and their receptors in maternal and fetal tissues from sows infected with PRRSV. The approach was to use real-time RT-PCR to identify the expression of different subtypes of type I IFN genes. The results show that the constitutive gene expression of some subtypes including IFN-[alpha] and IFN-[alpha][omega] were detected in fetal lymphoid nodes (IFN-[alpha][omega]), placenta (several IFN-[alpha] subtypes and IFN-[omega]5) and particularly, thymus (multiple IFN-[alpha], IFN-[delta] and IFN-[omega]5). The results demonstrate that porcine type I IFNs are differentially expressed at the maternal-fetal interface and in fetal tissues in response to PRRSV infection.

Measures of temperament have been shown to influence physiological responses. Exit velocity (EV) has been identified as an objective, robust measure of temperament that can be used to predict subsequent performance of cattle. Additionally, previous studies from our lab indicate this measure of temperament may be related to production of interferon-γ (IFN-γ), a cytokine associated with cell-mediated immunity (CMI). Whereas research has investigated effects of EV upon immune responses, the overall goal of these studies was to examine this relationship under a variety of scenarios including human handling, transportation, and exposure to endophyte-infected tall fescue (E+) for determination of its ability to influence CMI in cattle. In each of 5 experiments, calves were classified as either high or low EV animals, based upon measurements obtained prior to initiation of experimental periods. The hypothesis for these studies was that calves with high exit velocities would have lower systemic immune responses to applied treatments. Two experiments were designed to examine the relationship between exit velocity and lymphocyte IFN-γ production during and following a period of exposure to E+ seed and increased temperature humidity index conditions. Preliminary measures of this cytokine indicated a positive relationship with EV. During application of heat and E+ treatment application, no differences in IFN-γ production were detected between EV or endophyte treatment groups. However, in both experiments, after temperatures were returned to thermoneutral and E+ heifers were placed on the endophyte-free treatment, the positive relationship between exit velocity and total lymphocyte production of IFN-γ observed in baseline samples was reestablished. Similarly, during an experiment examining IFN-γ production by lymphocytes in steers during the 4 weeks following a 10h, 805 km transport study, average lymphocyte production of IFN-γ was higher and lymphocyte proportions producing IFN-γ lower in low EV steers, but total lymphocyte production of this cytokine did not differ between exit velocity treatments. In a grazing and finishing study, cattle were placed on E+ or novel endophyte pastures, with balanced representation of low and high EV treatments within each pasture. During the subsequent finishing period, blood samples for lymphocyte IFN-γ production were collected from a single high EV calf from each pasture group. Neither endophyte nor exit velocity was detected to be related with lymphocyte production of IFN-γ. In an experiment examining changes in cytokine gene expression changes during acclimation to human handling, IFN-γ, Il-6, IL-10, and IL-12 were observed to increase linearly over the experimental period in all calves, irrespective of exit velocity designation. In the same experiment, whole period pro-inflammatory tumor necrosis factor-α expression was higher for high EV calves, but interferon-γ (IFN-γ) was lower in this same treatment group. These studies, cumulatively, indicate EV may be related to systemic production of IFN-γ, but abrupt changes to an animal’s environment may serve to mask this relationship.

diagnosticar precozmente y tratar a los individuos enfermos de forma apropiada. Por otro lado, el estudio de las personas infectadas permite aplicar, medidas de prevención y evitar que estas personas desarrollen la enfermedad. La prueba de la tuberculina (PT) ha sido utilizada durante los últimos 100 años como herramienta para el diagnóstico de la infección tuberculosa. Su principal inconveniente radica en que la mayoría de proteínas presentes en el PPD no son específicas de Mycobacterium tuberculosis. Esto provoca una disminución en la especificidad de la prueba, ya que individuos sensibilizados por exposición previa a micobacterias no tuberculosas (MNT) o vacunados con la BCG también responden inmunológicamente al PPD. Además, la PT presenta una baja sensibilidad en pacientes con alteraciones en la inmunidad celular. 70 La citoquina efectora clave en el control de la infección micobacteriana para la activación de los macrófagos y el desarrollo de la inmunidad protectora contra M. tuberculosis es el IFN-g. Por lo tanto, un método de inmunodiagnóstico basado en la cuantificación in vitro de la respuesta inmune celular puede ser una alternativa a la PT. En los últimos años, se han descrito dos antígenos específicos de M. tuberculosis: Early Secretory Antigen Target-6 (ESAT-6) y Culture Filtrate Protein 10 (CFP-10). Estos antígenos están ausentes en la cepa vacunal de la BCG y en la mayoría de MNT. También se ha estudiado un tercer antígeno llamado TB7.7. En este sentido han sido desarrollados diferentes métodos de cuantificación de la respuesta inmune celular utilizando estos antígenos específicos micobacterianos para la estimulación de las células T sensibilizadas y para la detección in vitro de IFN-g. En base a esta tecnología se han estandarizado dos técnicas comerciales: Quantiferon Gold In tube (QFN-G-IT) y T-SPOT.TB. QFN-G-IT estimula los linfocitos presentes en muestras de sangre total con los antígenos ESAT-6, CFP-10 y TB7.7 en un mismo tubo, y determina la producción de IFN-g mediante técnica de ELISA. Por otro lado, TSPOT. TB requiere una separación de células mononucleares para estimularlas con los antígenos ESAT-6 y CFP-10 por separado, y el IFN-g se detecta mediante ELISPOT. De esta forma, la Tesis se ha centrado en la estandarización y evaluación clínica de estas técnicas inmunológicas para el diagnóstico de la infección tuberculosa y TB activa, así como su aplicabilidad en la práctica clínica en pacientes adultos y pediátricos (inmunocompetentes e inmunodeprimidos), y el estudio del efecto de las MNT sobre la positividad de la PT. Finalmente, la Tesis se completa con la evaluación de nuevos marcadores y antígenos alternativos en el diagnóstico in vitro de la TB. En resumen, las técnicas basadas in vitro son más específicas que la PT, ya que están menos influenciadas por la vacuna de la BCG y la sensibilización a MNT. Por lo tanto, el uso de estas técnicas puede ayudar a reducir el número de quimioprofilaxis innecesarias en población adulta y pediátrica. La detección de IFN-g liberado por las células T sensibilizadas tras estimular con antígenos específicos de M. tuberculosis es una herramienta diagnóstica alternativa en el diagnóstico de la infección tuberculosa en población inmunocompetente e inmunodeprimida. Además, estas técnicas in vitro pueden ayudar en el inmunodiagnóstico de la TB activa. Por otro lado, el estudio de nuevos antígenos específicos y biomarcadores es una herramienta potencial para el estudio de la respuesta del huésped contra M. tuberculosis durante el tratamiento y la búsqueda de nuevos marcadores para el diagnóstico de la infección tuberculosa y TB activa. / The bases of tuberculosis (TB) control programs consist of a diagnosis and correct treatment of patients with active TB. An essential factor for controlling the spread of this disease is the ability to diagnose infected individuals in their early stages before they become infectious to others through the progression to active TB. The tuberculin skin test (TST) has been until now the only tool available for the diagnosis of latent tuberculosis infection (LTBI). Unfortunately, this test has some disadvantages because of its poor specificity that lead to false positive results by the BCG vaccine strain and nontuberculous mycobacteria (NTM) cross-reaction. Moreover, TST has a low sensibility in groups with impaired cellular immunity giving false negative results. CD4 T cells represent the major players in the protection against TB. Of greatest relevance are the T helper (Th) 1 cells, that produce IFN-g. The IFN-g cytokine activates macrophages for the development of a protective immunity against Mycobacterium tuberculosis. Therefore, an immunodiagnostic assay based on the in vitro quantification of this cellular immune response could be an alternative to the TST for the diagnosis of LTBI. In the last years, two specific M. tuberculosis antigens have been described. These antigens are early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), absent in BCG strain and in the majority of NTM. A new third M. tuberculosis antigen called TB7.7 has been also studied. In vitro assays for the diagnosis of LTBI, based on the detection of IFN-g secreted by effector T cells stimulated with these specific antigens, have been developed. There are two commercially available IFN-g T-cell based assays: QuantiFERON-TB Gold In-Tube (QFN-G-IT) and T-SPOT.TB, both approved by the U.S. Food and Drug Administration. QFN-G-IT test stimulates whole-blood with ESAT-6, CFP- 10 and TB7.7 in the same tube, and measures the concentration of IFN-g in supernatants with an ELISA assay. On the other hand, T-SPOT.TB assay stimulates isolated peripheral blood mononuclear cells with ESAT-6 and CFP-10 separately, and detects number of IFN-g producing T cells by means of an ELISPOT. In this sense, the main objectives of this Thesis are the standardization and clinical evaluation of these new immunological assays for the diagnosis of LTBI and active TB, their application in clinical practice in adult and pediatric immunocompetent and immunosuppressed population, and the evaluation of NTM effect in the LTBI diagnosis. Finally, we study and evaluate new antigens and potential biomarkers for the diagnosis of active TB and LTBI. In summary, IFN-g tests are more specific than TST because they are less affected by BCG vaccination and NTM sensitization. So, the utilization of these assays can help to reduce unnecessary LTBI treatment among adult and pediatric population. Therefore, detection of IFN-g produced by T cells after M. tuberculosis specific antigen stimulation is an alternative diagnostic tool for LTBI in immunocompetent and immunosuppressed patients. In addition, IFN-g assays could help in the immunodiagnosis of active TB. On the other hand, the study of new specific antigens and biomarkers is a potential tool for studying host immune response during anti-TB therapy and finding novel diagnostic markers for active TB and M. tuberculosis infection.

La caracterització genòmica del VHC al 1989, el desenvolupament de tècniques de detecció d’anticossos i, més recentment, la introducció de mètodes moleculars per a la identificació de l’ARN viral han permès pràcticament eliminar el risc de transmissió del VHC associat a la transfusió de productes sanguinis. No obstant, en la pràctica diària, les tècniques de cribatge d’anticossos i inclús els mètodes confirmatoris basats en immunoblots generen resultats indeterminats (presència d’anticossos enfront a un únic antigen viral en absència de virèmia), representant un problema pels Bancs de Sang a l’hora d’informar als donants sobre la causa de l’exclusió definitiva de la donació altruista. Per altra banda, tot i que l’eficàcia del tractament antiviral de la infecció persistent pel VHC ha millorat sensiblement en els últims anys, existeixen grups especials de pacients en els que la teràpia actualment disponible està contraindicada o és francament insuficient, pel que una millor comprensió dels mecanismes de persistència viral o resolució espontània de la infecció poden ser clínicament rellevants. La resposta immune cel·lular CD4+ i CD8+ tipus Th1 potent, multiespecífica i mantinguda és essencial per a la resolució espontània de la infecció, mentre que la infecció persistent es caracteritza per l’absència o la presència de cèl·lules específiques d’antigen funcionalment alterades. Estudis experimentals en ximpanzés han demostrat que l’absència de col·laboració per part dels limfòcits T CD4+ específics d’antigen condiciona la persistència viral, per la qual cosa és possible que la infecció crònica en l’ésser humà sigui deguda primàriament a una disfunció de la resposta CD4+. Les causes d’aquesta disfunció poden ser vàries, incloent la inducció d’anèrgia a través de lligands peptídics alterats tal com s’ha postulat en el cas de la tolerància a cèl·lules tumorals. En base a aquestes troballes, es van plantejar dos objectius principals de la tesi: (1) determinar el significat dels patrons d’immunoblot indeterminat en donants de sang i (2) avaluar la possible presència de limfòcits T CD4+ anèrgics en pacients amb infecció persistent, determinar la especificitat antigènica, i estudiar la possibilitat de restaurar la capacitat funcional de les cèl·lules T CD4+ específiques del VHC ex vivo. Els resultats dels estudis han permès demostrar en primer lloc que aproximadament la meitat dels donants amb patró d’immunoblot indeterminat representen curacions espontànies de la infecció, suggerint la utilitat de la tècnica d’ELISpot-IFN-γ per diferenciar donants falsos positius de les tècniques serològiques de cribatge d’aquells que presenten infeccions resoltes espontàniament. A més, es va determinar que la resposta cel·lular Th1 CD4+ enfront al domini helicasa de la proteïna NS3 és el factor discriminant de la resposta immune en infeccions resoltes. En el segon estudi es va demostrar presència de limfòcits T CD4+ específics de NS3 disfuncionals en la majoria dels pacients amb infecció persistent, independentment del seu perfil funcional, mitjançant l’activació transitòria antigen – específica del lligand del CD40 (CD154). També es va demostrar la possibilitat de revertir l’estat disfuncional de les cèl·lules T CD4+ específiques de NS3 mitjançant l’expansió in vitro en absència de l'estímul inductor d'anèrgia i en presència de citocines homeostàtiques (IL-7 i IL-15), proporcionant un nou instrument per entendre els mecanismes de persistència viral i investigar l'ús d'aquestes cèl·lules per a estratègies d'immunoteràpia adaptativa. / The HCV genome characterization in 1989, the development of antibodies detection techniques and, more recently, the introduction of molecular methods for identification of viral RNA have allowed to reduce the risk of HCV transmission associated with blood products transfusion. However, in current practice, the antibody screening techniques and the confirmatory methods based on immunoblot assays generate indeterminate results (antibodies against a single viral antigen in the absence of viremia), representing a problem for blood banks to inform donors about the cause of the definitive exclusion of altruistic donation. Moreover, although the effectiveness of antiviral treatment for persistent HCV infection has improved considerably in recent years, there are special groups of patients for whom currently available therapy is contraindicated or is frankly inadequate. A better understanding of the mechanisms of viral persistence or spontaneous clearance may be thus clinically relevant. Sustained persistence of powerful and multispecific CD4+ and CD8+ Th1 responses are essential for spontaneous HCV clearance, and persistent infection is characterized by the absence or the functional alteration of antigen-specific T cells. Experimental studies in chimpanzees have shown that the absence of HCV-specific CD4+ T lymphocytes cooperation can generate viral persistence, so it is possible that chronic infection in humans may be primarily due to a dysfunction of the CD4+ immune response. The causes of this dysfunction may be various, including the induction of anergy by altered peptide ligands, as has been postulated in the case of tolerance to tumour cells. Based on these findings, we raised two main objectives for this thesis: (1) to determine the significance of indeterminate immunoblot patterns in blood donors and (2) to evaluate the presence of CD4+ T lymphocytes with anergic phenotype in patients with persistent infection, to determine the antigenic specificity, and whether HCV-specific CD4+ T cells dysfunction can be reversed in vitro. Study results have allowed to demonstrate that approximately half of donors with indeterminate immunoblot pattern have resolved a previous HCV infection and suggested that ELISpot-IFN-γ might be a useful tool to distinguish donors with false positive serological techniques results from those with resolved spontaneous infections. In addition, we showed that the CD4+ Th1 immune response against NS3 helicase domain is the discriminate factor in the immune response in resolved infection. The second study showed that HCV-specific CD4+ T cells, although dysfunctional, are present in the peripheral blood of most patients with persistent HCV infection and can be easily detected, irrespectively of their functional profile, by the transient antigen - specific upregulation of CD40 ligand (CD154). We also demonstrated that it was possible to rescue NS3-specific CD4+ T cell response in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen and presence of homeostatic cytokines (IL-7 and IL-15), providing a new tool to understand the mechanisms of viral persistence and investigate the use of these cells for immunotherapy of adaptive strategies.

Aichi virus (AiV) is a small, nonenveloped RNA virus categorized to Picornaviridae. AiV infection causes mild gastroenteritis, but in neonates, AiV usually causes the risk of certain enterovirus-related clinical syndromes, such as fever, nausea, vomiting and diarrhea. The first case of AiV infection in Taiwan was diagnosed from a young patient with diarrhea in Kaohsiung Veterans General Hospital, and the AiV was successfully isolated. Antiviral innate immune system of our body plays the major role to defense virus invasion. Because AiV is an emerging picornavirus, the knowledge about its pathogenesis and the interaction with host innate immunity were totally absent. This study aims to investigate the mechanism of AiV regulating innate immune response. We first demonstrated that AiV is a type I IFN sensitive virus. IFN-£\2 treatment potently inhibited AiV replication. Real-time quantitative PCR data indicated that AiV induced only small amout of type I IFN gene expression, and the similar result was observed using IFN-£] luciferase reporter assay. In addition, the AiV triggered IFN-£] luciferase activity was progressively decreased in the late phase of infection. Immunoblotting assay showed that AiV evidently activated IRF-3 and IRF-7, the transcription factors of type I IFN induction. However, the retinoic acid inducible gene I (RIG-I) protein was cleavaged and its activity was downregulated by AiV. This data suggested that AiV triggered low level of type I IFN response may due to the negative feedback regulation of RIG-I activity. This immune evasion might be important for AiV replication in cells. Our study first reveals the status of innate immune response of AiV infection, and provides the basic virological theory for the development of anti-AiV drugs and vaccines in the future.