Magister Scientiae Dentium - MSc(Dent) / The initiation of highly active antiretroviral therapy has shown to result in successful suppression of viral replications followed by an increase in CD4 lymphocytes, a partial recovery of T-cell specific immune responses and decrease susceptibility to opportunistic pathogens. Aim: The aim of the present study was to determine the prevalence of oral lesions in patients before and after undergoing HAART. Methods: The study design was longitudinal and descriptive, investigating the prevalence of oral lesions presenting in HIV/AIDS patients at baseline, 3 and 6 months after taking HAART. A convenience sample size of 200 participants was targeted. Results: 210 HIV positive patients participated at baseline. At 3 months, 96 (46%) and at 6 months, 52 (25%) were available for review respectively. At baseline 210 HIV positive patients were recruited into the study from three hospitals. Two infectious disease hospitals belonged to the City of Harare and the other is a government hospital. Just over two thirds were female (64.3%) and the age ranged as follows: 21-30 (17%); 31-40 (44%); 41-50 (26% and 51-60 (9%).Discussion: HAART appears to be effective in reducing the prevalence of oral lesions in persons with AIDS likely due to the immunological reconstitution. Oral candidiasis remains the most prevalent oral opportunistic infection in immuno-suppressed individuals and hence its important predictive value for immuno-suppression defined as CD4-cell count level <200/mL of blood. All oral lesions strongly associated with HIV infection with the exception of non-Hodgkin’s lymphoma were diagnosed at baseline. CD4 cell count level increased after initiation of HAART. T-lymphocytes that are formed after the introduction of HAART may not provide sufficient protection against some lesions like parotid gland disease and HPV conditions (planar warts). HAART failure was detected in some patients who had negative CD4-cell count at 6 months compared to the baseline parameters. Conclusions: HIV-positive patients experience oral pain during the course of their disease, eating, drinking and swallowing. Further longitudinal studies are required in order to ascertain the prevalence of these lesions at three and six months and the effect of HAART.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-05-22T14:08:18Z No. of bitstreams: 1 Raimundo Coutinho Junior. Perfil de ativação... 2014 .pdf: 1722380 bytes, checksum: 909bc68a97d8e0a06206d205cd3557f7 (MD5) / Made available in DSpace on 2014-05-22T14:08:18Z (GMT). No. of bitstreams: 1 Raimundo Coutinho Junior. Perfil de ativação... 2014 .pdf: 1722380 bytes, checksum: 909bc68a97d8e0a06206d205cd3557f7 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / O vírus linfotrópico das células T humanas do tipo 1 (HTLV-1) é o agente etiológico da mielopatia associada ao HTLV / paraparesia espástica tropical (HAM / TSP ), que ocorre em menos de 5 % dos indivíduos infectados. A resposta imune controla parcialmente a infecção, porém pode estar ligada a patogênese da doença. O objetivo deste estudo foi caracterizar fenotipicamente as subpopulações de linfócitos T, em pacientes assintomáticos e com diagnóstico de HAM/TSP. Foram avaliados 103 pacientes acompanhados no Centro de HTLV da Escola Bahiana de Medicina e Saúde Pública (EBMSP) e 19 controles não infectados. Os pacientes foram categorizados de acordo com o grau de certeza do diagnóstico de HAM/TSP: possível (Ps), provável (Pb) e definido (D), além de pacientes assintomáticos (ASS). O perfil fenotípico (CD25, CD45RA, CD45RO, HLA-DR, CD25, CCR-7, CD62L) das subpopulações de linfócitos T CD4+ e T CD8+ e a expressão da proteína Tax de FoxP3 na subpopulação T CD4+ foram avaliados por citometria de fluxo. A carga proviral do HTLV foi quantificada por reação em cadeia da polimerase (PCR) em tempo real. As proporções dos linfócitos TCD4+HLA-DR+ e TCD8+HLA-DR foram significativamente maiores nos pacientes com HAM/TSP Ps, Pb e D comparados aos controles não infectados (p=0,003). Comparando-se o grupo de infectados, as proporções de linfócitos TCD4+ e TCD8+ expressando HLA-DR foram significantemente maiores em pacientes com HAM/TSP-Ps e D comparados a pacientes assintomáticos (p<0,0001). Além disso, houve uma correlação positiva entre porcentagem de linfócitos T CD4+ HLA-DR+ e a carga proviral (r=0,5, p=0,0003). As proporções de linfócitos T CD4+CD25+ foram maiores nos pacientes com HAM/TSP D comparados aos controles não infectados. Observou-se um aumento na proporção de células reguladoras (T CD4+FOXP3+) nos indivíduos assintomáticos e nos pacientes com HAM/TSP-D comparados a indivíduos não infectados (p=0,04). A carga proviral HTLV-1 foi maior no grupo de pacientes HAM/TSP-D do que em pacientes assintomáticos (p=0,0001). Observa-se uma diferença estatística entre a proporção de células CD4+TAX+ dos indivíduos assintomáticos (0,26%) e os pacientes HAM/TSP-Ps (5,26%) (p=0,04). Em conclusão, os pacientes com HAM/TSP Ps, Pb e D apresentam um perfil fenotípico de ativação celular, comparados aos controles não infectados. Além disso, os pacientes com HAM/TSP Pb e D apresentam maior expressão de HLA-DR nas subpopulações de linfócitos T CD4+ e T CD8+, comparados aos assintomáticos, indicando um perfil imunológico semelhante nestes dois subgrupos de pacientes. / The human T-cell lymphotropic vírus type 1(HTLV-1) is the etiological agent of HTLV- associated myelopathy/ Tropical spastic paraparesis(HAM/TSP), wich occurs in less then 5% of the infected individuals. The immune response partially controls the infection, but may be linked to the pathogenesis of disease. The aim of this study was to characterize phenotipically T lymphocyte subpopulations in asymptomatic and in patients diagnosed with HAM/TSP. We evaluated 103 patients treated at the center for HTLV of Bahia School of Medicine and Public Health (EBMSP) and 19 uninfected controls. Patients were categorized as asymptomatic and according to the degree of certainty of the diagnosis of HAM/TSP: Possible(Ps), Probable(Pb) and Definite(D). The phenotypic profile (CD25, CD45RA, CD45RO, HLA-DR, CCR-7, CD62L) subpopulation of CD4+ and CD8+ T Cells and expression of the protein Tax and Foxp3 in the subpopulation CD4+ T cells were evaluated by flow cytometry. The HTLV proviral load was quantified by polymerase chain reaction (PCR) in real time. The proportions of CD4+ and CD8+ expressing HLA-DR+ were significantly higher in patients (p<0.0001). In addition there were a correlation between CD4+ HLA-DR+ or CD8+ HLA-DR+ and the proviral load(R=0,5;p<0,0001). The proportions of CD4+CD25+ were higher in patients with HAM/TSP D compared to infected controls. There was an increased proportion of regulatory cells (CD4+Foxp3+) of asymptomatic individuals and patients HAM/TSP D, compared to the uninfected individuals (p=0.04). . There is a statistical difference between the proportion of CD4+TAX+ asymptomatic individuals (0.26%) patients and HAM/TSP-Ps (5.26%) (p = 0.04 ) . In conclusion, patients with HAM/TSP Ps, Pb and D exhibit a phenotypic profile of cell activation, compared to uninfected controls. In addition, patients with HAM/TSP Pb and D show higher expression of HLA -DR in subpopulations of T lymphocytes CD4+ and CD8+ T cells, compared to asymptomatic, indicating an immunological profile similar in both groups of patients

A doença de Chagas, moléstia causada pelo protozoário Trypanosoma cruzi, resulta freqüentemente em um quadro de miocardite crônica que pode levar à morte do paciente. As causas que determinam a afetação cardíaca não estão totalmente esclarecidas. A nossa hipótese de trabalho é que o desafio poderia aumentar a ação efetora do sistema imune frente ao T. cruzi, resultando em redução da carga parasitária sistêmica e local, e, em longo prazo, em diminuição da patologia. A análise histopatológica mostrou que a manutenção prolongada do desafio foi capaz de reduzir moderadamente mas significantemente a patologia no coração. Estas experiências podem auxiliar a esclarecer a etiopatogenia do processo patológico cardíaco, confirmando que esta seja devida à reação frente aos parasitas remanescentes. Mais importante, os nossos resultados podem abrir uma esperançosa perspectiva de tratamento aos indivíduos de fase crônica. / Chagas disease caused by Trypanosoma cruzi frequently results in chronic myocarditis that may result in death. The mechanisms underlying cardiac pathology are not totally clear. Our working hypothesis was that challenge could increase the immune systems effector activity against T. cruzi, leading to reduction in systemic and local parasite loads, which in turn could result, at the long run, in pathology diminution. The hystopathological analysis revealed that sustained challenge led to a small, but significant, decrease in heart pathology. These results could help to elucidate the etiopathogeny of chagasic heart lesions, inasmuch as they support the hypothesis of reactivity towards locally-persisting parasites. More important, our results open a hopeful perspective for the treatment of patients at the chronic phase.

Submitted by ACACIO DUARTE PACHECO (acacio@fmva.unesp.br) on 2016-09-01T16:16:58Z No. of bitstreams: 1 Acácio Impressão Tese Final.pdf: 1381975 bytes, checksum: 5982f80f260a714c02111156f309cccb (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-09-02T18:54:19Z (GMT) No. of bitstreams: 1 pacheco_ad_dr_araca.pdf: 1381975 bytes, checksum: 5982f80f260a714c02111156f309cccb (MD5) / Made available in DSpace on 2016-09-02T18:54:19Z (GMT). No. of bitstreams: 1 pacheco_ad_dr_araca.pdf: 1381975 bytes, checksum: 5982f80f260a714c02111156f309cccb (MD5) Previous issue date: 2016-07-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A ocorrência de miocardite em cães naturalmente acometidos por Leishmania infantum chagasi tem sido demonstrada em estudos recentes, contudo o tipo de resposta imune incitada no tecido cardíaco de cães com leishmaniose visceral (LV) ainda não foi determinada. Sendo assim, foram avaliados fragmentos da: parede livre de átrio direito (AD), parede livre de ventrículo direito (VD), parede livre de ventrículo esquerdo (VE) e septo interventricular (SIV) de 20 cães naturalmente acometidos por LV para determinação das alterações histopatológicas e da resposta imune no tecido cardíaco. A presença de miocardite foi observada em 75% dos animais avaliados, sendo o infiltrado inflamatório do tipo linfoplasmocitário a alteração mais comum nesses fragmentos. A imunomarcação de Leishmania spp. foi observada em 60% dos animais. Não houve correlação entre a intensidade do infiltrado inflamatório e a presença de Leishmania sp. no miocárdio (p=0,0682). A imunomarcação do parasito apresentou correlação com a presença de linfócitos T CD4+ (p<0,0001) e T CD8+ (p=0,0002), linfócitos B CD79+ (p=0,046) e IgG (p=0,040). Os níveis de IL-4 (p=0,030), IL-12 (p=0,002), IFN- (p<0,0001) foram menores em cães infectados em relação ao grupo controle, independente da presença ou não do parasito no miocárdio (p>0,05). No presente estudo, a presença de alterações miocárdicas, associadas a resposta imunológica mista foram comuns em cães com LV. Novos estudos avaliando paralelamente, a presença de inflamação miocárdica e o perfil de citocinas no tecido cardíaco, à resposta imunológica sistêmica são indicados para confirmação da resposta imunológica presente no miocárdio de cães acometidos por LV. / Recent studies have demonstrated the occurrence of myocarditis in dogs naturally infected with Leishmania infantum chagasi, however the type of immune response in cardiac tissue of dogs with visceral leishmaniasis (VL) has not been determined. For this purpose, fragments from right atrial free wall (AD), right ventricular free wall (RV), left ventricular free wall (LV) and interventricular septum (IVS) of 20 dogs naturally affected by VL were evaluated to determine the histopathological changes and the immune response in heart tissue. The presence of myocarditis has been reported in 75% of the evaluated animals, and an lymphoplasmocytic inflammatory infiltrate was the most common change in these fragments. The immunostaining of Leishmania sp. was observed in 60% of the evaluated animals. There was no correlation between the intensity of the inflammatory infiltrate and the presence of Leishmania sp. (p = 0.0682). The immunostaining of the parasite correlated with the presence of CD4 + (p <0.0001) and CD8 + T lymphocytes (p = 0.0002), CD79 + B lymphocytes (p = 0.046) and IgG (p = 0.040). IL-4 (p = 0.030), IL-12 (p = 0.002) and IFN- levels (p <0.0001) were lower in the infected dogs when compared to control group, regardless of the presence or absence parasite in the myocardium (p> 0.05). The results obtained in this study suggest that the presence of myocardial changes is common in dogs with VL, associated with a mixed immune response in heart tissue. However, the local inflammatory process seems not enough to trigger the production of cytokines normally involved in VL, possibly due to a low parasite load in dogs myocardium, especially when compared to other organs such as spleen, liver and bone marrow. Further studies evaluating at the same time the presence of myocardial inflammation and the behavior of these circulating cytokines in target organs with immune activity are indicated to confirm the immune response present in the myocardium of dogs affected by VL. / FAPESP: 2013/12129-8 / CNPq: 141536/2013-6

Orientador: Mary Marcondes / Banca: Wagner Luis Ferreira / Banca: Katia denise Saraiva Bresciani / Banca: Heitor Flavio ferrari / Banca:fernanda Muller de Oliveira Ravai / Resumo: A ocorrência de miocardite em cães naturalmente acometidos por Leishmania infantum chagasi tem sido demonstrada em estudos recentes, contudo o tipo de resposta imune incitada no tecido cardíaco de cães com leishmaniose visceral (LV) ainda não foi determinada. Sendo assim, foram avaliados fragmentos da: parede livre de átrio direito (AD), parede livre de ventrículo direito (VD), parede livre de ventrículo esquerdo (VE) e septo interventricular (SIV) de 20 cães naturalmente acometidos por LV para determinação das alterações histopatológicas e da resposta imune no tecido cardíaco. A presença de miocardite foi observada em 75% dos animais avaliados, sendo o infiltrado inflamatório do tipo linfoplasmocitário a alteração mais comum nesses fragmentos. A imunomarcação de Leishmania spp. foi observada em 60% dos animais. Não houve correlação entre a intensidade do infiltrado inflamatório e a presença de Leishmania sp. no miocárdio (p=0,0682). A imunomarcação do parasito apresentou correlação com a presença de linfócitos T CD4+ (p<0,0001) e T CD8+ (p=0,0002), linfócitos B CD79+ (p=0,046) e IgG (p=0,040). Os níveis de IL-4 (p=0,030), IL-12 (p=0,002), IFN- (p<0,0001) foram menores em cães infectados em relação ao grupo controle, independente da presença ou não do parasito no miocárdio (p>0,05). No presente estudo, a presença de alterações miocárdicas, associadas a resposta imunológica mista foram comuns em cães com LV. Novos estudos avaliando paralelamente, a presença de inflamação miocá... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Recent studies have demonstrated the occurrence of myocarditis in dogs naturally infected with Leishmania infantum chagasi, however the type of immune response in cardiac tissue of dogs with visceral leishmaniasis (VL) has not been determined. For this purpose, fragments from right atrial free wall (AD), right ventricular free wall (RV), left ventricular free wall (LV) and interventricular septum (IVS) of 20 dogs naturally affected by VL were evaluated to determine the histopathological changes and the immune response in heart tissue. The presence of myocarditis has been reported in 75% of the evaluated animals, and an lymphoplasmocytic inflammatory infiltrate was the most common change in these fragments. The immunostaining of Leishmania sp. was observed in 60% of the evaluated animals. There was no correlation between the intensity of the inflammatory infiltrate and the presence of Leishmania sp. (p = 0.0682). The immunostaining of the parasite correlated with the presence of CD4 + (p <0.0001) and CD8 + T lymphocytes (p = 0.0002), CD79 + B lymphocytes (p = 0.046) and IgG (p = 0.040). IL-4 (p = 0.030), IL-12 (p = 0.002) and IFN- levels (p <0.0001) were lower in the infected dogs when compared to control group, regardless of the presence or absence parasite in the myocardium (p> 0.05). The results obtained in this study suggest that the presence of myocardial changes is common in dogs with VL, associated with a mixed immune response in heart tissue. However, the local inflam... (Complete abstract click electronic access below) / Doutor

A doença de Chagas, moléstia causada pelo protozoário Trypanosoma cruzi, resulta freqüentemente em um quadro de miocardite crônica que pode levar à morte do paciente. As causas que determinam a afetação cardíaca não estão totalmente esclarecidas. A nossa hipótese de trabalho é que o desafio poderia aumentar a ação efetora do sistema imune frente ao T. cruzi, resultando em redução da carga parasitária sistêmica e local, e, em longo prazo, em diminuição da patologia. A análise histopatológica mostrou que a manutenção prolongada do desafio foi capaz de reduzir moderadamente mas significantemente a patologia no coração. Estas experiências podem auxiliar a esclarecer a etiopatogenia do processo patológico cardíaco, confirmando que esta seja devida à reação frente aos parasitas remanescentes. Mais importante, os nossos resultados podem abrir uma esperançosa perspectiva de tratamento aos indivíduos de fase crônica. / Chagas disease caused by Trypanosoma cruzi frequently results in chronic myocarditis that may result in death. The mechanisms underlying cardiac pathology are not totally clear. Our working hypothesis was that challenge could increase the immune systems effector activity against T. cruzi, leading to reduction in systemic and local parasite loads, which in turn could result, at the long run, in pathology diminution. The hystopathological analysis revealed that sustained challenge led to a small, but significant, decrease in heart pathology. These results could help to elucidate the etiopathogeny of chagasic heart lesions, inasmuch as they support the hypothesis of reactivity towards locally-persisting parasites. More important, our results open a hopeful perspective for the treatment of patients at the chronic phase.

Le VIH-1 manipule les cellules présentatrices d’antigènes (APC) qui orchestrent les réponses immunes innées et adaptatrices, pour se propager dans l’hôte et établir le réservoir viral. Au laboratoire, nous étudions le rôle de l’autophagie dans les interactions entre les cellules dendritiques (DC) et le VIH-1 et la présentation des antigènes viraux. Dans divers modèles, la macroautophagie et l’autophagie médiée par les chaperonnes (CMA) semblent en effet être impliquées dans l’apprêtement d’antigènes sur les molécules du CMH. Ainsi, nous avons montré, dans une étude précédente, que la macroautophagie participait à la dégradation du VIH entrant dans les DC, conduisant à l’activation de lymphocytes T (LT) CD4+ spécifiques du VIH-1.Bien que sa réplication y soit limitée, le VIH-1 peut également infecter productivement les DC. J’ai donc voulu vérifier si les protéines virales néosynthétisées du virus peuvent constituer une source additionnelle d’antigènes. J’ai montré que, de façon remarquable, dans les DC infectées, des antigènes endogènes du VIH-1 peuvent être présentés par les molécules du CMH-II aux LT CD4 spécifiques. En utilisant différents outils, comme des inhibiteurs de l’autophagie ou des shRNA, j’ai montré que ni la macroautophagie ni la CMA ne contribuent significativement à l’apprêtement d’épitopes de la protéine virale Gag néosynthétisée sur les molécules du CMH-II. En parallèle, j’ai utilisé une protéine de fusion, Gag-LC3, pour acheminer spécifiquement Gag dans les autophagosomes (LC3+) des DC. Dans ce contexte, les drogues qui inhibent la macroautophagie réduisent drastiquement la présentation d’épitopes de Gag aux LT CD4. De façon remarquable, la présence de Gag dans les autophagosomes conduit à la génération d’épitopes antigéniques qui, dans le contexte infectieux, ne sont pas apprêtés sur les molécules de CMH-II par la voie endogène. Ainsi, diriger des protéines du VIH dans les autophagosomes conduirait à des variations dans le répertoire des antigènes endogènes présentés sur les molécules de CMH-II. Pour évaluer l’impact de l’autophagie sur la réplication du VIH dans les DC, j’ai ensuite analysé si la protéine Gag néosynthétisée pouvait être dégradée dans les autophagosomes. Dans les DC infectées, contrairement aux observations déjà décrites dans les macrophages, Gag ne colocalise ni avec les vésicules autophagiques LC3+, ni avec p62, une protéines adaptatrice impliquée dans le ciblage des protéines dans les autophagosomes. Ces résultats suggèrent que, dans ce contexte, les virions nouvellement produits ne sont pas acheminés et dégradés dans les autophagosomes. La protéine de fusion Gag-LC3 est utilisée dans ces expériences comme contrôle positif de colocalisation. Pour déterminer si mes observations pouvaient révéler un mécanisme d’échappement développé par VIH-1, j’ai utilisé différentes souches virales mutantes, modulé le flux autophagique avec des drogues et des ligands TLR, et exprimé Gag dans les DC en l’absence d’autres protéines virales. Dans l'ensemble, mon travail suggère que le VIH-1 ne manipule pas la macroautophagie dans les DC productivement infectées. En outre, la modulation de l’autophagie dans les DC (à l'aide de shRNA) n'a aucune incidence sur la réplication du VIH-1 et sur sa propagation.Mes travaux mettent en lumière la complexité des interactions entre l’autophagie et le VIH-1 dans les DC. Contrairement à ce qui a été observé lors des étapes d’entrée du virus, le virus ne semble pas être acheminé dans les autophagosomes une fois les DC infectées, et l’autophagie ne participe pas à l’apprêtement des antigènes néosynthétisés sur les molécules de CMH II. Cependant, les DC infectées activent de façon efficace les LT CD4 spécifiques du virus. Forcer l’acheminement d’antigènes du VIH dans les autophagosomes augmente fortement cette activation, et semble conduire à une diversification du répertoire des épitopes présentés sur les molécules de CMH-II par la voie endogène. / HIV-1 manipulates antigen-presenting cells (APC) such as dendritic cells (DC), witch orchestrate innate and adaptive immune responses, in order to propagate in the host and to establish viral reservoirs. We are studying the role of autophagic processes in DC/HIV-1 interactions with a focus on antigen presentation. We have previously shown that macroautophagy in DC participates in the degradation of incoming HIV-1 particles leading to activation of HIV-1-specific (HS) CD4 T cells. HIV-1 can also productively infect DC. I thus first asked whether neo-synthetized viral proteins might represent an additional source of HIV-1 antigens. Remarkably, I have shown using infected monocyte derived DC that de novo expression of Gag leads to the activation of HS CD4 T cells, highlighting that this antigen is endogenously processed in order to be presented into MHC-II molecules. Since macroautophagy and chaperon-mediated autophagy (CMA) are known to be involved in this process for other viral antigens and model antigens, I then dissected the role of these two pathways. Using several tools including inhibitors and shRNA, I demonstrated that in HIV-1-infected DC neither macroautophagy nor CMA contribute significantly to the processing of HIV-1-Gag epitopes into MHC-II molecules. I also used a Gag-LC3 fusion protein to specifically channel Gag into LC3+ autophagic vesicles in DC. In this context, inhibiting autophagy dramatically reduced the presentation of HIV-1-Gag epitopes to CD4+ T cells. Strikingly, channelling Gag into autophagosomes generated epitopes that were not processed endogenously in the context of HIV-1 infection. Thus specifically directing HIV-1 proteins toward autophagosomes might influence the repertoire of MHC II-restricted HIV-1 antigens. I further analyzed whether autophagy could affect HIV-1 replication in infected DC. In these cells, in contrast to what has been described in macrophages, Gag did not colocalize with LC3 or with the autophagic adaptor p62, suggesting that newly-produced HIV-1 particles are not sequestrated into autophagosomes. The Gag-LC3 fusion protein was used here as a positive control of colocalization. To determine whether my findings might reveal a DC-specific escape mechanism developed by HIV-1, I used various HIV-1 mutants, enhanced autophagic flux using drugs or TLR ligands, and expressed Gag in the absence of other HIV-1 proteins. Overall, my work suggests that HIV-1 does not manipulate autophagy in productively-infected DC. Moreover, modulating autophagy in DC (using shRNA) does not impact HIV-1 replication and propagation. Finally, my work highlights the complexity of the interactions between the autophagic process and HIV-1 replication in DC. Unlike during viral entry, HIV-1 does not seem to be targeted into autophagosomes after viral replication in infected DC, and autophagy does not contribute significantly to the processing of endogenous viral antigens. Nonetheless HIV-1-infected DC efficiently activates HS CD4 T cells, and targeting HIV antigens into autophagosomes greatly enhances this activation and might broaden the repertoire of MHC-II-restricted antigen. Further dissection of the various routes of endogenous HIV antigen processing would aid in the development of innovative vaccines.

Hintergrund: Die Versorgung von Immunzellen mit Energie in Form von ATP ist Grundlage eines funktionstüchtigen Immunsystems. Diese wird durch die mitochondriale OXPHOS oder durch die zytosolische Glykolyse gewährleistet. Sauerstoff und Glukose stellen die Hauptsubstrate dieser Stoffwechselprozesse dar. Fragestellung: Unter pathologischen Bedingungen wie sie in Entzündungsgebieten herrschen, konnte ein relativer Sauerstoffmangel experimentell nachgewiesen werden. Ziel dieser Arbeit war es herauszufinden, in welcher Weise die Funktionen einer definierten Lymphozytenpopulation (CD4+) durch Sauerstoffmangel beeinflusst werden. Methoden: Nach Isolation von CD4+ Zellen aus peripherem Blut gesunder Spender, wurden definierte Zellmengen stimuliert und in einem mit einer Sauerstoffelektrode ausgestatteten Gefäß unter Luftabschluß inkubiert. Zu definierten Zeitpunkten wurden Proben zur ATP-Messung entnommen, sowie Protein- und RNA-Lysate hergestellt. Die Vitalität zu Anfang und zum Ende der Inkubation wurde mittels Propidium-Jodid-Färbung im FACS bestimmt. Aus gesammelten Überständen wurden mittels Multiplex-ELISA die Konzentrationen von IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha und MCAF gemessen. Als Kontrollen dienten unter Normoxie inkubierte Aliquots der Zellsuspensionen. HIF-1alpha wurde mit Immunoblotting nachgewiesen. Transkriptionsänderungen von SOD1 und HK1 wurden durch SYBR-Green Real-Time-PCR quantifiziert. Ergebnisse: Stimulierte CD4+-Zellen von Normalspendern schütten unter dem Einfluss von Hypoxie vermehrt proinflammatorische und chemotaktisch wirksame Zytokine, sowie zur Differenzierung notwendige antiinflammatorische Zytokine aus. Die Verfügbarkeit von Glukose hat hierauf einen verstärkenden Effekt. Eine hypoxische Umgebung sorgt in Abhängigkeit von der Versorgung mit Glukose für eine Anpassung der zellulären Atmungsrate. Glukose ist für die Aufrechterhaltung eines konstanten ATP-Levels verantwortlich. Die glykolytische Energiegewinnung unter Hypoxie kompensiert den Ausfall der OXPHOS. Hypoxie führt bei stimulierten CD4+-Zellen bei freier Glukoseverfügbarkeit zu einer vermehrten Transkription des Hexokinase1-Gens. Glukosemangel bewirkt dagegen in hypoxischer Umgebung eine Transkriptionssteigerung des SOD1-Gens. / Background: The energy supply of immune cells in form of ATP is the cornerstone of a functional immune system. This supply is realized by either mitochondrial OXPHOS or cytosolic glycolysis. Oxygen and glucose present the main substrates in these metabolic processes. Objective: Relative shortness of oxygen could be determined experimentally under pathological conditions present in inflamed tissues. The aim of this study was to determine the extent of hypoxic influence on the cellular function of CD4+ lymphocytes. Methods: Human CD4+ cells were isolated from peripheral blood of healthy blood donors by MACS sorting. Following a defined protocol cells were stimulated and incubated in a sealed container with a Clark type electrode. Samples were taken for measurements of ATP content. RNA- and Protein lysates were made to quantify the transcription of SOD1 and HK1 by SYBR green RT-PCR and look for the presence of HIF-1alpha by immunoblot analysis respectively. Supernatants were used to measure the expression of IL-1beta, IL-2, IL-6, IL-8, IL-10, TNF-alpha and MCAF using a multiplex ELISA assay. Aliquots of cell supspensions incubated under normoxic conditions served as controls. Results / Conclusion: Under the influence of hypoxia stimulated CD4+ lymphocytes of healthy blood donors express proinflammatory and chemotactically active as well as anti-inflammatory cytokines important for cell differentiation. The availability of glucose leads to an increase of this effect. An hypoxic environment dependant on the availability of glucose leads to an adaptation of cellular respiration. Glucose deficiency provokes an increase in cellular oxygen utilization. The availability of glucose is responsible for a constant intracellular ATP level. This proves that in CD4+ lymphocytes glycolysis is capable of compensating for hypoxically impaired oxidative phosphorylation thus providing enough ATP to enable cellular function. Hypoxia under glucose provision leads to an increase in mRNA expression for HK1, a key enzyme of glycolysis. Lack of glucose under hypoxic conditions results in an increase in mRNA expression for SOD1. Glucose therefore serves in CD4+ cells as an agent of constant energy supply that leads to cell survival and an upkeep of a proinflammatory environment through cytokine expression.