A hepatite C é uma doença que leva à inflamação do fígado, sendo causada pelo vírus da hepatite C (VHC). Estima-se que existam 130 a 170 milhões de casos de infecção crônica pelo VHC por todo o mundo. Apesar do VHC ser principalmente hepatotrópico, foi evidenciada a detecção de RNA viral em células mononucleares do sangue periférico (PBMC). Evidências clínicas e experimentais têm demonstrado um importante tropismo do VHC por células do sistema imune, em especial por PBMCs. Apesar deste interessante achado, a importância da infecção do sistema imune na história natural da doença não é totalmente conhecida. É possível observar em alguns estudos que, em pacientes que desenvolvem infecção aguda, observa-se uma vigorosa resposta mediada por células T, específica ao VHC, mediada, por células T CD4+ e T CD8. Esta resposta é detectada na fase inicial da doença, e prolonga-se durante vários anos após a resolução do vírus. Pelo contrário, os pacientes que desenvolvem infecção crônica, apresentam, normalmente, respostas T fracas e / ou de curta duração, bem como defeitos nas funções efetoras de células T específicas. As respostas mediadas por células T com este perfil resultam, normalmente, em um baixo controlo da viremia e na sua persistência. Outra importante questão que permanece incerta até o presente momento é como a infecção das células imunes pelo VHC altera a sua função, principalmente no que concerne às PBMCs. Informações referentes às PBMCs e o VHC são pouco estudadas e referidas na literatura internacional, embora sejam de fundamental importância para a compreensão do seu peso na história natural da infecção. OBJETIVO: Baseado nessas incertezas o presente estudo tentará contribuir para a elucidação da influência do parasitismo por VHC na função de células TCD4+ e TCD8+ em pacientes naïve com indicação de tratamento com IFNpeg+RBV e comparar os dados obtidos antes, durante e pós-tratamento com os valores encontrados dos controles negativos, avaliando a influência do tratamento sobre a função dessas células. Verificar a presença do RNA-VHC nas PBMCs por RT-PCR em Tempo Real. Verificar a influência de fatores do vírus e do hospedeiro, tais como genótipo, carga viral, idade, gênero, raça, polimorfismo IL28B e biópsia hepática sobre as células parasitadas pelo VHC e sua produção de citocinas; Determinar a contagem e a função de linfócitos T CD4+ e CD8+ no sangue periférico desses pacientes; Determinar a influência do parasitismo de linfócitos pelo VHC sobre a função dos linfócitos TCD4+ e TCD8+. METODOLOGIA: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios, atendidos no Ambulatório de Hepatites da Divisão de Clínica de Doenças Infecciosas e Parasitárias do HC FMUSP. A contagem de células da subpopulação de linfócitos T CD4+ e CD8+ periféricas foram realizadas por citometria de fluxo FACSCanto II (BD) por meio do software MultiSet(BD). Foi analisada a presença/ausência do RNA-VHC em PBMC por PCR em tempo real no sistema TaqMan, no termociclador Applied Biosystems StepOne(TM). A função dos linfócitos TCD4+ e TCD8+ foram avaliadas através da técnica ELISPOT utilizanso o Kit Human IFN-gamma/IL-4 Dual-Color FluoroSpot. A contagem dos spots foi realizada em um leitor automatizado CTL-ImmunoSpot® S6 FluoroSpot Line. As PBMCs foram fracionadas e depletadas, utilizando-se o Kit Dynabeads FlowComp Human CD4 (Invitrogen Life Technologies) segundo as instruções do fabricante. RESULTADOS: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios. O genótipo 1 foi o mais prevalente 61,5%. O RNA do VHC foi detectado nas PBMCS em 88,4% dos pacientes. XVII Nossos resultados mostraram um aumento de células CD4 e CD8 parasitadas pelo VHC antes do tratamento, com valores estatisticamente significantes quando comparadas aos controles normais apenas no caso das CD4. Ocorreu, porém, um nítido prejuízo na produção de interleucinas por estas células parasitadas, particularmente a produção de IFN- y, com valores altamente significantes (0,009). Na semana 12, podemos ver o aumento de células CD4 no pré-tratamento, porém com diminuição na semana 12 e no follow up; entretanto a produção de IL-4 pelas células CD4 aumenta na semana 12 e cai novamente no seguimento; com as células CD8 ocorre leve queda na semana 12 porém uma tentativa de recuperação no follow up; sua produção de IFN-y cai na semana 12 e no follow up para números estatisticamente significantes. É a chamada \"exaustão\" de função destas células já descrita in vitro por alguns autores. Estes dados poderão ser muito úteis nas futuras observações desses pacientes que serão tratados com esquemas de DAAs sem interferon alfa. CONCLUSÃO: Em conclusão, os resultados desta pesquisa confirmam a importante influência do parasitismo das células CD4 e CD8 em suas funções / Hepatitis C is a disease that causes inflammation of the liver, caused by hepatitis C virus (HCV). It is estimated that there are 130 million to 170 million cases of chronic HCV infection worldwide. Although HCV is primarily hepatotropic was evidenced by detection of viral RNA in peripheral blood mononuclear cells (PBMC). Clinical and experimental evidence have demonstrated an important tropism of HCV by immune system cells, in particular by PBMC. Despite this interesting finding, the importance of infection of the immune system in the natural history of the disease is not fully known. It can be observed in some studies in patients who develop acute infection a vigorous response is observed mediated by T cells specific to HCV mediated by CD4 + and CD8. This response is detected at the initial stage of the disease, and extends for years after the resolution of the virus. Conversely, patients who develop chronic infection, are typically in the low T responses and / or short-term as well as defects in the effector functions of specific T cells. The responses mediated by T cells with the profile usually result in a low control of viremia and its persistence. Another important issue that remains unclear to date is how the infection of immune cells by HCV alters its function, especially with regard to the PBMCs. Information relating to the PBMCs and HCV are little studied and reported in the international literature, although they are of fundamental importance for the understanding of its weight in the natural history of infection. OBJECTIVE: Based on these uncertainties the present study attempts to contribute to the elucidation of the influence of parasitism by HCV on CD4 T cell function + and CD8 + in naïve patients with treatment indication with IFNpeg + RBV and compare the data obtained before, during and after treatment with the values found negative controls, assessing the influence of treatment on the function of these cells. Verify the presence of HCV-RNA in PBMC by RT-PCR in real time. Check the influence of virus and host factors such as genotype, viral load, age, gender, race, IL28B polymorphism and liver biopsy on the parasitized cells with HCV and their cytokine production; Determine count and function of lymphocytes T CD4 + and CD8 + peripheral blood of patients; To determine the influence of parasitism lymphocytes HCV on the function of CD4 + and CD8 + T lymphocytes. METHODS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls, treated at the Hepatitis Clinic of the Division of Clinical Infectious and Parasitic Diseases of the FMUSP. The lymphocyte subpopulation of T-cell count CD4 + and CD8 + peripheral been made by FACSCanto II flow cytometer (BD) through the multiset software (BD). the presence / absence of HCV-RNA in PBMC by real-time PCR was analyzed in the TaqMan system, Applied Biosystems thermal cycler StepOne (TM). The role of CD4 + T lymphocytes and CD8 + were assessed by ELISPOT technique utilizanso the Human Kit IFN-gamma / IL-4 Dual-Color FluoroSpot. The counting of the spots was carried out in an automated reader CTL-ImmunoSpot® S6 FluoroSpot Line. And fractionated PBMCs were depleted using Dynabeads kit is the Human CD4 FlowComp (Invitrogen Life Technologies) according to manufacturer\'s instructions. RESULTS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls. Genotype 1 was the most prevalent 61.5%. The HCV RNA was detected in PBMC in 88.4% of patients. Our results showed an increase of CD4 and CD8 cells parasitized with HCV prior to treatment, with statistically significant amounts compared to controls only if CD4. There was however, a marked impairment in production of interleukins for these parasitized cells, particularly the production of IFN- y, with highly significant values (0.009). At week 12, we can see the increase in CD4 cells before treatment, but with decreased at week 12 and at follow up; However IL-4 production by CD4 cells increased at week 12 and again falls in the following; with the CD8 cells is slightly lower at week 12 but a recovery attempt at follow up; their IFN-y production drops at week 12 and follow up to statistically significant numbers. It is called \"exhaust\" function of these cells in vitro been described by some authors. This data is very useful in future observations of these patients are treated with AADs regimens without interferon alfa. CONCLUSION: In conclusion, the results confirm the important influence of the parasitism of CD4 and CD8 cells in their functions

A hepatite C é uma doença que leva à inflamação do fígado, sendo causada pelo vírus da hepatite C (VHC). Estima-se que existam 130 a 170 milhões de casos de infecção crônica pelo VHC por todo o mundo. Apesar do VHC ser principalmente hepatotrópico, foi evidenciada a detecção de RNA viral em células mononucleares do sangue periférico (PBMC). Evidências clínicas e experimentais têm demonstrado um importante tropismo do VHC por células do sistema imune, em especial por PBMCs. Apesar deste interessante achado, a importância da infecção do sistema imune na história natural da doença não é totalmente conhecida. É possível observar em alguns estudos que, em pacientes que desenvolvem infecção aguda, observa-se uma vigorosa resposta mediada por células T, específica ao VHC, mediada, por células T CD4+ e T CD8. Esta resposta é detectada na fase inicial da doença, e prolonga-se durante vários anos após a resolução do vírus. Pelo contrário, os pacientes que desenvolvem infecção crônica, apresentam, normalmente, respostas T fracas e / ou de curta duração, bem como defeitos nas funções efetoras de células T específicas. As respostas mediadas por células T com este perfil resultam, normalmente, em um baixo controlo da viremia e na sua persistência. Outra importante questão que permanece incerta até o presente momento é como a infecção das células imunes pelo VHC altera a sua função, principalmente no que concerne às PBMCs. Informações referentes às PBMCs e o VHC são pouco estudadas e referidas na literatura internacional, embora sejam de fundamental importância para a compreensão do seu peso na história natural da infecção. OBJETIVO: Baseado nessas incertezas o presente estudo tentará contribuir para a elucidação da influência do parasitismo por VHC na função de células TCD4+ e TCD8+ em pacientes naïve com indicação de tratamento com IFNpeg+RBV e comparar os dados obtidos antes, durante e pós-tratamento com os valores encontrados dos controles negativos, avaliando a influência do tratamento sobre a função dessas células. Verificar a presença do RNA-VHC nas PBMCs por RT-PCR em Tempo Real. Verificar a influência de fatores do vírus e do hospedeiro, tais como genótipo, carga viral, idade, gênero, raça, polimorfismo IL28B e biópsia hepática sobre as células parasitadas pelo VHC e sua produção de citocinas; Determinar a contagem e a função de linfócitos T CD4+ e CD8+ no sangue periférico desses pacientes; Determinar a influência do parasitismo de linfócitos pelo VHC sobre a função dos linfócitos TCD4+ e TCD8+. METODOLOGIA: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios, atendidos no Ambulatório de Hepatites da Divisão de Clínica de Doenças Infecciosas e Parasitárias do HC FMUSP. A contagem de células da subpopulação de linfócitos T CD4+ e CD8+ periféricas foram realizadas por citometria de fluxo FACSCanto II (BD) por meio do software MultiSet(BD). Foi analisada a presença/ausência do RNA-VHC em PBMC por PCR em tempo real no sistema TaqMan, no termociclador Applied Biosystems StepOne(TM). A função dos linfócitos TCD4+ e TCD8+ foram avaliadas através da técnica ELISPOT utilizanso o Kit Human IFN-gamma/IL-4 Dual-Color FluoroSpot. A contagem dos spots foi realizada em um leitor automatizado CTL-ImmunoSpot® S6 FluoroSpot Line. As PBMCs foram fracionadas e depletadas, utilizando-se o Kit Dynabeads FlowComp Human CD4 (Invitrogen Life Technologies) segundo as instruções do fabricante. RESULTADOS: Foram estudados 52 pacientes com hepatite C crônica; destes, 17 pacientes foram tratados com IFNalfa+RBV; 10 controles sadios. O genótipo 1 foi o mais prevalente 61,5%. O RNA do VHC foi detectado nas PBMCS em 88,4% dos pacientes. XVII Nossos resultados mostraram um aumento de células CD4 e CD8 parasitadas pelo VHC antes do tratamento, com valores estatisticamente significantes quando comparadas aos controles normais apenas no caso das CD4. Ocorreu, porém, um nítido prejuízo na produção de interleucinas por estas células parasitadas, particularmente a produção de IFN- y, com valores altamente significantes (0,009). Na semana 12, podemos ver o aumento de células CD4 no pré-tratamento, porém com diminuição na semana 12 e no follow up; entretanto a produção de IL-4 pelas células CD4 aumenta na semana 12 e cai novamente no seguimento; com as células CD8 ocorre leve queda na semana 12 porém uma tentativa de recuperação no follow up; sua produção de IFN-y cai na semana 12 e no follow up para números estatisticamente significantes. É a chamada \"exaustão\" de função destas células já descrita in vitro por alguns autores. Estes dados poderão ser muito úteis nas futuras observações desses pacientes que serão tratados com esquemas de DAAs sem interferon alfa. CONCLUSÃO: Em conclusão, os resultados desta pesquisa confirmam a importante influência do parasitismo das células CD4 e CD8 em suas funções / Hepatitis C is a disease that causes inflammation of the liver, caused by hepatitis C virus (HCV). It is estimated that there are 130 million to 170 million cases of chronic HCV infection worldwide. Although HCV is primarily hepatotropic was evidenced by detection of viral RNA in peripheral blood mononuclear cells (PBMC). Clinical and experimental evidence have demonstrated an important tropism of HCV by immune system cells, in particular by PBMC. Despite this interesting finding, the importance of infection of the immune system in the natural history of the disease is not fully known. It can be observed in some studies in patients who develop acute infection a vigorous response is observed mediated by T cells specific to HCV mediated by CD4 + and CD8. This response is detected at the initial stage of the disease, and extends for years after the resolution of the virus. Conversely, patients who develop chronic infection, are typically in the low T responses and / or short-term as well as defects in the effector functions of specific T cells. The responses mediated by T cells with the profile usually result in a low control of viremia and its persistence. Another important issue that remains unclear to date is how the infection of immune cells by HCV alters its function, especially with regard to the PBMCs. Information relating to the PBMCs and HCV are little studied and reported in the international literature, although they are of fundamental importance for the understanding of its weight in the natural history of infection. OBJECTIVE: Based on these uncertainties the present study attempts to contribute to the elucidation of the influence of parasitism by HCV on CD4 T cell function + and CD8 + in naïve patients with treatment indication with IFNpeg + RBV and compare the data obtained before, during and after treatment with the values found negative controls, assessing the influence of treatment on the function of these cells. Verify the presence of HCV-RNA in PBMC by RT-PCR in real time. Check the influence of virus and host factors such as genotype, viral load, age, gender, race, IL28B polymorphism and liver biopsy on the parasitized cells with HCV and their cytokine production; Determine count and function of lymphocytes T CD4 + and CD8 + peripheral blood of patients; To determine the influence of parasitism lymphocytes HCV on the function of CD4 + and CD8 + T lymphocytes. METHODS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls, treated at the Hepatitis Clinic of the Division of Clinical Infectious and Parasitic Diseases of the FMUSP. The lymphocyte subpopulation of T-cell count CD4 + and CD8 + peripheral been made by FACSCanto II flow cytometer (BD) through the multiset software (BD). the presence / absence of HCV-RNA in PBMC by real-time PCR was analyzed in the TaqMan system, Applied Biosystems thermal cycler StepOne (TM). The role of CD4 + T lymphocytes and CD8 + were assessed by ELISPOT technique utilizanso the Human Kit IFN-gamma / IL-4 Dual-Color FluoroSpot. The counting of the spots was carried out in an automated reader CTL-ImmunoSpot® S6 FluoroSpot Line. And fractionated PBMCs were depleted using Dynabeads kit is the Human CD4 FlowComp (Invitrogen Life Technologies) according to manufacturer\'s instructions. RESULTS: We studied 52 patients with chronic hepatitis C; of these, 17 patients were treated with IFNalpha + RBV; 10 healthy controls. Genotype 1 was the most prevalent 61.5%. The HCV RNA was detected in PBMC in 88.4% of patients. Our results showed an increase of CD4 and CD8 cells parasitized with HCV prior to treatment, with statistically significant amounts compared to controls only if CD4. There was however, a marked impairment in production of interleukins for these parasitized cells, particularly the production of IFN- y, with highly significant values (0.009). At week 12, we can see the increase in CD4 cells before treatment, but with decreased at week 12 and at follow up; However IL-4 production by CD4 cells increased at week 12 and again falls in the following; with the CD8 cells is slightly lower at week 12 but a recovery attempt at follow up; their IFN-y production drops at week 12 and follow up to statistically significant numbers. It is called \"exhaust\" function of these cells in vitro been described by some authors. This data is very useful in future observations of these patients are treated with AADs regimens without interferon alfa. CONCLUSION: In conclusion, the results confirm the important influence of the parasitism of CD4 and CD8 cells in their functions

Die Virushepatitis gehört weltweit zu einer der häufigsten viralen Erkrankungen. Doch durch die Entwicklung von immer effizienteren Impfstoffen kann bei einer frühzeitigen Immunisierung eine Infektion verhindert werden. Ziel dieser Arbeit war es, ein Patientenkollektiv zu untersuchen, welches eine Impfung mit einem Hepatitis B-Impfstoff erhalten hatte, und dieses mit Probanden zu vergleichen, die nicht immunisiert waren. Diese Proben wurden auf ihre serologische und zelluläre Reaktivität mittels ELISA und ELISpot untersucht. Im ELISA zeigte sich bei 95 % der geimpften Personen eine positive Serokonversion nach zurückliegender Hepatitis B-Impfung. Um den Impfstatus genauer zu analysieren und bei seronegativen geimpften Probanden den zellulären Arm des Immun-systems zu verifizieren, wurde mittels ELISpot die IFN-γ-Sekretion von HBs-reaktiven T-Zellen untersucht und mit den Ergebnissen, welche in der Serologie gewonnen wurden, verglichen. Dabei zeigte sich, dass die zelluläre Untersuchung bei 43 von 94 untersuchten Patientenproben (46 %) ein positives Ergebnis aufwies. Zweiundzwanzig der 48 geimpften Patienten (46 %) hatten eine antigenspezifische IFN-γ-Sekretion und 21 der 46 Proben der aktuell nicht geimpften Patienten fielen im ELISpot positiv aus. Bei zwei seronegativ geimpften Patienten konnte jeweils ein positives Ergebnis im ELISpot gezeigt werden. Ein direkter Zusammenhang zwischen der Höhe des anti-HBs-Titers im ELISA und Anzahl der spot-bildenden Zellen im ELISpot konnte nicht gezeigt werden. Die Ergebnisse dieser Arbeit zeigen, dass die serologische Untersuchung mittels ELISA weiterhin als Goldstandard verwendet werden soll, um den aktuellen Schutz gegenüber einer Hepatitis B-Infektion zu verifizieren. Durch die zelluläre Untersuchung mit dem ELISpot-Verfahren kann bei weiterer Testoptimierung in Zukunft eine Nachweismethode für seronegative Geimpfte entwickelt werden. Vor allem sollten diese mit Hilfe des ELISpots genauer analysiert werden, um gegebenenfalls bei nicht vorhandener humoralen Immunität und einer ebenfalls fehlenden zellulären Immunität prophylaktische Maßnahmen einzuleiten.

Hintergrund: Um akute Rejektionen nach Nierentransplantation früh erkennen und behandeln zu können, gleichzeitig die Nebenwirkungen einer immunsuppressiven Therapie zu minimieren, wäre die Etablierung eines “Immunmonitorings”, welches zu jedem Zeitpunkt Hinweise auf die Aktivierung des Immunsystems des Empfängers gegen das Transplantat gibt, wünschenswert. Methodik: In dieser Studie wurden die Anzahl der spenderreaktiven, IFNgamma-produzierenden T-Zellen von 52 nierentransplantierten Patienten zu verschiedenen Zeitpunkten vor (prä-TX) und nach Transplantation (post-TX) im ELISpot-Assay gemessen und in Zusammenhang mit der klinischen Entwicklung gebracht. Außerdem wurde das Assay auf Reproduzierbarkeit untersucht und versucht zu optimieren. Ergebnisse: Eine stark erhöhte Anzahl spenderreaktiver Zellen prä-TX (>200 IFNgamma-spots/3*100000 PBMZ, n = 5) war immer mit einer akuten Rejektion des Transplantats assoziiert. Post-TX korrelierte die Anzahl der spenderreaktiven, IFNgamma-produzierenden Zellen mit der Nierenfunktion ein Jahr nach Transplantation. Diese Korrelation wurde in den Wochen 2 und 3 post-TX und bei Patienten ohne akute Rejektion, besonders deutlich. Hinsichtlich der methodischen Optimierung hat sich die magnetische Depletion CD2pos-Zellen als effektiv gezeigt, die IFNgamma-Sekretion von Stimulatorzellen zu unterbinden. Um die Reproduzierbarkeit des Assays zu verbessern sollten Stimulatorzellen im Überschuss und Empfänger-T-Zellen in einer konstanten Anzahl eingesetzt werden. Dabei sollte die Gesamtzellzahl über 1000000 Zellen/ml betragen. Conclusion: Das ELISpot-Assay ist zur Erkennung klinisch relevanter T-Zellsensibilisierungen vor und nach Transplantation geeignet. Vor einem Einsatz in der klinischen Routine sollten jedoch einige methodische Verbesserungen vorgenommen werden. / Background: In order to perform early diagnosis and treatment of acute rejections after renal transplantation while minimizing side effects of immunosuppression, an immune monitoring tool is needed, which gives information on the activation state of the immune system of the transplant recipient against the allograft at any given time. Methods: In this study, frequencies of donor-reactive, IFNgamma-producing T cells where measured in 52 renal transplant recipients at different time points before (pre-TX) and after transplantation (post-TX) using the ELISPOT-assay. The frequencies were correlated with clinical outcome. Also, the reproducibility of the assay and possibilities of optimization were tested. Results: Highly elevated frequencies of donor-reactive cells pre-TX (>200 IFNgamma-spots/3*100000 PBMC´s, n = 5) were always associated with acute rejection episodes after transplantation. Post-TX frequencies of donor-reactive, IFNgamma-producing cells correlated significantly with graft function one year post-TX. This correlation was strongest for frequencies in week 2 and 3 post-TX and in patients without acute rejection. Regarding the methodical optimization, magnetic CD2pos-cell depletion of donor leucocytes proved useful to inhibit IFNgamma secretion of stimulating cells. To improve reproducibility of the assay stimulating cells should be used as a surplus, a constant number of responding T cells should be chosen, and overall cell concentration should exceed 1000000 cells/ml. Conclusion: The ELISPOT-assay is a useful tool to detect clinically relevant T cell sensibilisation pre- and post-TX. Before it is routinely used some methodical alterations must be performed.

O tratamento de pacientes apresentando doenças inflamatórias imunomediadas com drogas anti-TNF-alfa aumenta o risco da reativação da tuberculose. Isso sugere que tais drogas possam afetar a imunidade celular destes. No entanto, há dados conflitantes sobre se esse tratamento suprime as respostas para o teste tuberculínico (TT) e os ensaios de liberação de interferon-gama (IGRAs) e poucos dados em pacientes com psoríase. O presente estudo avaliou pacientes com psoríase moderada a grave enfocando os efeitos do tratamento com infliximabe em suas respostas imunológicas celulares. Foram avaliadas as respostas imunes celulares de doze pacientes antes e durante o tratamento com infliximabe. As células mononucleares do sangue periférico (PBMC) foram estimuladas com a fito-hemaglutinina (PHA), o superantígeno enterotoxina B (SEB), um lisado de citomegalovírus (CMV), e antígenos de Mycobacterium tuberculosis, e a ativação de linfócitos foi avaliada por ELISPOT para enumerar células secretoras de IFN-y, por ELISA para detecção da secreção de IFN-y, e através da incorporação de[3H] timidina para medir a proliferação. O tratamento com infliximabe não levou à redução de INF-y e da resposta linfoproliferativa nos pacientes. Pelo contrário, aumentou a liberação desta citocina em culturas de PBMC estimulados com PHA e SEB por 12 h. Este efeito foi mais notado no pico do efeito clínico do agente anti-TNF (7 semanas de tratamento) e menos proeminente no seu nadir (logo antes da infusão da próxima dose). Reatividade imunitária ao CMV também não foi significativamente afetada, notando-se leve aumento pelo agente anti-TNF. É de se notar que secreção de IFN-y e resposta proliferativa a Mtb dos dois pacientes TT positivos foram, também, visivelmente aumentadas na semana 7, declinando quando infliximabe atingiu o seu nadir. Os efeitos deletérios do bloqueio do TNF em pacientes com psoríase grave, submetidos ao tratamento com infliximabe parecem ser atenuados, pelo menos parcialmente, por um efeito de aumento na imunidade mediada por células desses pacientes, possivelmente pela diminuição da ação imunossupressora decorrente do excesso de TNF / Treatment of patients with immune-mediated inflammatory diseases with anti-TNF agents increases the risk of tuberculosis reactivation, suggesting that it may affect their cellular immune response. However, there are conflicting data on whether anti-TNF treatment suppresses the responses to tuberculin skin test (TST) and interferon-y release assays and no information regarding psoriasis patients on anti-TNF treatment. The present study evaluated patients with moderate to severe psoriasis focusing on the effects of treatment with infliximab on their cellular immune responses. Cellular immune responses of twelve patients were evaluated before and during infliximab treatment. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohemaglutinin (PHA), the superantigen enterotoxin B (SEB), a cytomegalovirus lysate (CMV), and Mycobacterium tuberculosis antigens, and the lymphocyte activation was evaluated by ELISPOT for enumeration of IFN-y-secreting cells, ELISA for detection of secreted IFN-y, and by [3H]thymidine incorporation for proliferation measurement. Treatment with infliximab does not lead to reduction in the INF-y and lymphoproliferative responses of patients. It rather increased the overnight release of this cytokine in PBMC cultures stimulated with PHA and SEB. This effect was most noted at the peak of the clinical effect of the anti-TNF agent (week 7 of treatment) and less prominent at its nadir (just before infusion of the next dose). Immune reactivity to CMV was also either unaffected or slightly increased by the TNF blocking agent. Of note, the IFN-y and proliferative responses to Mtb from the two TST-responder patients were also remarkably increased at week 7, declining when infliximab reached its nadir. The deleterious consequences of TNF blockade in patients with severe psoriasis undergoing infliximab treatment may be in part attenuated by an enhancing effect on the cell mediated immunity of the patients, possibly due to the abbreviation of the immunosuppressive effect of TNF overexpression

O tratamento de pacientes apresentando doenças inflamatórias imunomediadas com drogas anti-TNF-alfa aumenta o risco da reativação da tuberculose. Isso sugere que tais drogas possam afetar a imunidade celular destes. No entanto, há dados conflitantes sobre se esse tratamento suprime as respostas para o teste tuberculínico (TT) e os ensaios de liberação de interferon-gama (IGRAs) e poucos dados em pacientes com psoríase. O presente estudo avaliou pacientes com psoríase moderada a grave enfocando os efeitos do tratamento com infliximabe em suas respostas imunológicas celulares. Foram avaliadas as respostas imunes celulares de doze pacientes antes e durante o tratamento com infliximabe. As células mononucleares do sangue periférico (PBMC) foram estimuladas com a fito-hemaglutinina (PHA), o superantígeno enterotoxina B (SEB), um lisado de citomegalovírus (CMV), e antígenos de Mycobacterium tuberculosis, e a ativação de linfócitos foi avaliada por ELISPOT para enumerar células secretoras de IFN-y, por ELISA para detecção da secreção de IFN-y, e através da incorporação de[3H] timidina para medir a proliferação. O tratamento com infliximabe não levou à redução de INF-y e da resposta linfoproliferativa nos pacientes. Pelo contrário, aumentou a liberação desta citocina em culturas de PBMC estimulados com PHA e SEB por 12 h. Este efeito foi mais notado no pico do efeito clínico do agente anti-TNF (7 semanas de tratamento) e menos proeminente no seu nadir (logo antes da infusão da próxima dose). Reatividade imunitária ao CMV também não foi significativamente afetada, notando-se leve aumento pelo agente anti-TNF. É de se notar que secreção de IFN-y e resposta proliferativa a Mtb dos dois pacientes TT positivos foram, também, visivelmente aumentadas na semana 7, declinando quando infliximabe atingiu o seu nadir. Os efeitos deletérios do bloqueio do TNF em pacientes com psoríase grave, submetidos ao tratamento com infliximabe parecem ser atenuados, pelo menos parcialmente, por um efeito de aumento na imunidade mediada por células desses pacientes, possivelmente pela diminuição da ação imunossupressora decorrente do excesso de TNF / Treatment of patients with immune-mediated inflammatory diseases with anti-TNF agents increases the risk of tuberculosis reactivation, suggesting that it may affect their cellular immune response. However, there are conflicting data on whether anti-TNF treatment suppresses the responses to tuberculin skin test (TST) and interferon-y release assays and no information regarding psoriasis patients on anti-TNF treatment. The present study evaluated patients with moderate to severe psoriasis focusing on the effects of treatment with infliximab on their cellular immune responses. Cellular immune responses of twelve patients were evaluated before and during infliximab treatment. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohemaglutinin (PHA), the superantigen enterotoxin B (SEB), a cytomegalovirus lysate (CMV), and Mycobacterium tuberculosis antigens, and the lymphocyte activation was evaluated by ELISPOT for enumeration of IFN-y-secreting cells, ELISA for detection of secreted IFN-y, and by [3H]thymidine incorporation for proliferation measurement. Treatment with infliximab does not lead to reduction in the INF-y and lymphoproliferative responses of patients. It rather increased the overnight release of this cytokine in PBMC cultures stimulated with PHA and SEB. This effect was most noted at the peak of the clinical effect of the anti-TNF agent (week 7 of treatment) and less prominent at its nadir (just before infusion of the next dose). Immune reactivity to CMV was also either unaffected or slightly increased by the TNF blocking agent. Of note, the IFN-y and proliferative responses to Mtb from the two TST-responder patients were also remarkably increased at week 7, declining when infliximab reached its nadir. The deleterious consequences of TNF blockade in patients with severe psoriasis undergoing infliximab treatment may be in part attenuated by an enhancing effect on the cell mediated immunity of the patients, possibly due to the abbreviation of the immunosuppressive effect of TNF overexpression

Die Virushepatitis gehört weltweit zu einer der häufigsten viralen Erkrankungen. Doch durch die Entwicklung von immer effizienteren Impfstoffen kann bei einer frühzeitigen Immunisierung eine Infektion verhindert werden. Ziel dieser Arbeit war es, ein Patientenkollektiv zu untersuchen, welches eine Impfung mit einem Hepatitis B-Impfstoff erhalten hatte, und dieses mit Probanden zu vergleichen, die nicht immunisiert waren. Diese Proben wurden auf ihre serologische und zelluläre Reaktivität mittels ELISA und ELISpot untersucht. Im ELISA zeigte sich bei 95 % der geimpften Personen eine positive Serokonversion nach zurückliegender Hepatitis B-Impfung. Um den Impfstatus genauer zu analysieren und bei seronegativen geimpften Probanden den zellulären Arm des Immun-systems zu verifizieren, wurde mittels ELISpot die IFN-γ-Sekretion von HBs-reaktiven T-Zellen untersucht und mit den Ergebnissen, welche in der Serologie gewonnen wurden, verglichen. Dabei zeigte sich, dass die zelluläre Untersuchung bei 43 von 94 untersuchten Patientenproben (46 %) ein positives Ergebnis aufwies. Zweiundzwanzig der 48 geimpften Patienten (46 %) hatten eine antigenspezifische IFN-γ-Sekretion und 21 der 46 Proben der aktuell nicht geimpften Patienten fielen im ELISpot positiv aus. Bei zwei seronegativ geimpften Patienten konnte jeweils ein positives Ergebnis im ELISpot gezeigt werden. Ein direkter Zusammenhang zwischen der Höhe des anti-HBs-Titers im ELISA und Anzahl der spot-bildenden Zellen im ELISpot konnte nicht gezeigt werden. Die Ergebnisse dieser Arbeit zeigen, dass die serologische Untersuchung mittels ELISA weiterhin als Goldstandard verwendet werden soll, um den aktuellen Schutz gegenüber einer Hepatitis B-Infektion zu verifizieren. Durch die zelluläre Untersuchung mit dem ELISpot-Verfahren kann bei weiterer Testoptimierung in Zukunft eine Nachweismethode für seronegative Geimpfte entwickelt werden. Vor allem sollten diese mit Hilfe des ELISpots genauer analysiert werden, um gegebenenfalls bei nicht vorhandener humoralen Immunität und einer ebenfalls fehlenden zellulären Immunität prophylaktische Maßnahmen einzuleiten.

<p>Transition metals such as nickel (Ni), cobalt (Co), palladium (Pd), chromium (Cr) and gold (Au) are widely used as alloys in jewelry and biomaterials such as orthodontic and orthopaedic appliances. These metals also cause cell-mediated allergic contact dermatitis (ACD) reactions in a significant proportion of the population upon prolonged direct exposure. The immune mechanisms underlying the response to these metals are not yet well defined. In the studies described in this thesis we therefore investigated the profile of cytokine responses to various metal ions <i>in vitro</i> and the relationship with the ACD reaction <i>in vivo</i>. In the first study, we investigated the relationship between the profile and magnitude of Ni²⁺-induced cytokine responses <i>in vitro</i> and the degree of <i>in vivo</i> reactivity to Ni²⁺. PBMC from Ni²⁺-reactive (ACD) and non-reactive control subjects were cultured with or without NiCl₂. The numbers of IL-4-, IL-5- and IL-13-producing cells and the concentrations of IFN-γ, IL-10 and IL-13 produced were analysed by ELISpot and ELISA respectively. Ni²⁺ elicited a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects with a positive correlation observed between the levels of the elicited cytokines and the degree of patch test reactivity. Hence, suggesting an involvement of both Th1- and Th2-type cytokines in ACD to Ni²⁺ and a direct association between the magnitude of the Ni²⁺-induced cytokine response overall and the <i>in vivo</i> reactivity to Ni²⁺. The impact of the regulatory cytokine IL-10 on Ni²⁺-induced Th1- and Th2-type cytokine responses in human PBMC was investigated in the next study. PBMC from blood donors with a history of Ni²⁺ reactivity and non-reactive control donors were stimulated with Ni²⁺ <i>ex vivo</i> with or without addition of human recombinant IL-10 (rIL-10) or neutralising mAb to IL-10. Depletion/enrichment experiments were performed to phenotype the Ni²⁺-specific cytokine producing cells. Exogenous rIL-10 significantly down-regulated the production of all cytokines but with a more pronounced effect on IFN-γ. IL-10 neutralisation, on the other hand, enhanced the levels of Ni²⁺-induced IFN-γ only. Ni²⁺-specific cytokine-producing cells in PBMC were found to be predominantly CD4⁺ T cells. Thus, IL-10 may play a regulatory role <i>in vivo</i> by counteracting the ACD reactions mediated by CD4⁺ T cells producing Th1-type cytokines. In the third study, we investigated the relationship between <i>in vivo</i> patch test reactivity to a number of metals (Ni, Co, Pd, Cr and Au) included in the standard and/or dental patch test series and <i>in vitro</i> responses to the metals in question. PBMC from metal patch test positive and negative control subjects were stimulated with a panel of eight metal salts and cytokine responses analysed by ELISpot and/or ELISA. A mixed Th1- (IL-2 and/or IFN-γ) and Th2-type (IL-4 and/or IL-13) cytokine profile was observed in PBMC from most metal allergic subjects upon <i>in vitro</i> stimulation with the metal(s) to which the subject was patch test positive. Our data suggest that other metals included in the standard and dental patch test series, just like Ni2⁺, induce a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects <i>in vitro</i>. We further developed a simplified ELISpot protocol utilising plates precoated with capture monoclonal antibodies (mAb) and subsequent detection in one step using enzyme-labelled mAb, for enumerating the frequency of allergen (Ni²⁺)-specific cytokine producing cells. This was compared with a regular ELISpot protocol, with an overnight incubation for capture mAb adsorbtion and detection with biotinylated mAb followed by enzyme-labelled streptavidin. PBMC from Ni²⁺-reactive and non-reactive subjects were incubated with or without NiCl₂ and the enumeration of cells producing IFN-γ, IL-4 or IL-13 by the two protocols were compared. PBMC from Ni²⁺-reactive subjects showed significantly higher Ni²⁺-induced IL-4 and IL-13 responses and the number of antigen-specific cytokine-producing cells determined by the two ELISpot protocols correlated well. In a nutshell, our data point to the potential use of <i>in vitro</i> cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.</p>

Transition metals such as nickel (Ni), cobalt (Co), palladium (Pd), chromium (Cr) and gold (Au) are widely used as alloys in jewelry and biomaterials such as orthodontic and orthopaedic appliances. These metals also cause cell-mediated allergic contact dermatitis (ACD) reactions in a significant proportion of the population upon prolonged direct exposure. The immune mechanisms underlying the response to these metals are not yet well defined. In the studies described in this thesis we therefore investigated the profile of cytokine responses to various metal ions in vitro and the relationship with the ACD reaction in vivo. In the first study, we investigated the relationship between the profile and magnitude of Ni2+-induced cytokine responses in vitro and the degree of in vivo reactivity to Ni2+. PBMC from Ni2+-reactive (ACD) and non-reactive control subjects were cultured with or without NiCl2. The numbers of IL-4-, IL-5- and IL-13-producing cells and the concentrations of IFN-γ, IL-10 and IL-13 produced were analysed by ELISpot and ELISA respectively. Ni2+ elicited a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects with a positive correlation observed between the levels of the elicited cytokines and the degree of patch test reactivity. Hence, suggesting an involvement of both Th1- and Th2-type cytokines in ACD to Ni2+ and a direct association between the magnitude of the Ni2+-induced cytokine response overall and the in vivo reactivity to Ni2+. The impact of the regulatory cytokine IL-10 on Ni2+-induced Th1- and Th2-type cytokine responses in human PBMC was investigated in the next study. PBMC from blood donors with a history of Ni2+ reactivity and non-reactive control donors were stimulated with Ni2+ ex vivo with or without addition of human recombinant IL-10 (rIL-10) or neutralising mAb to IL-10. Depletion/enrichment experiments were performed to phenotype the Ni2+-specific cytokine producing cells. Exogenous rIL-10 significantly down-regulated the production of all cytokines but with a more pronounced effect on IFN-γ. IL-10 neutralisation, on the other hand, enhanced the levels of Ni2+-induced IFN-γ only. Ni2+-specific cytokine-producing cells in PBMC were found to be predominantly CD4+ T cells. Thus, IL-10 may play a regulatory role in vivo by counteracting the ACD reactions mediated by CD4+ T cells producing Th1-type cytokines. In the third study, we investigated the relationship between in vivo patch test reactivity to a number of metals (Ni, Co, Pd, Cr and Au) included in the standard and/or dental patch test series and in vitro responses to the metals in question. PBMC from metal patch test positive and negative control subjects were stimulated with a panel of eight metal salts and cytokine responses analysed by ELISpot and/or ELISA. A mixed Th1- (IL-2 and/or IFN-γ) and Th2-type (IL-4 and/or IL-13) cytokine profile was observed in PBMC from most metal allergic subjects upon in vitro stimulation with the metal(s) to which the subject was patch test positive. Our data suggest that other metals included in the standard and dental patch test series, just like Ni2+, induce a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects in vitro. We further developed a simplified ELISpot protocol utilising plates precoated with capture monoclonal antibodies (mAb) and subsequent detection in one step using enzyme-labelled mAb, for enumerating the frequency of allergen (Ni2+)-specific cytokine producing cells. This was compared with a regular ELISpot protocol, with an overnight incubation for capture mAb adsorbtion and detection with biotinylated mAb followed by enzyme-labelled streptavidin. PBMC from Ni2+-reactive and non-reactive subjects were incubated with or without NiCl2 and the enumeration of cells producing IFN-γ, IL-4 or IL-13 by the two protocols were compared. PBMC from Ni2+-reactive subjects showed significantly higher Ni2+-induced IL-4 and IL-13 responses and the number of antigen-specific cytokine-producing cells determined by the two ELISpot protocols correlated well. In a nutshell, our data point to the potential use of in vitro cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.

Soluble amyloid-beta (Aβ) aggregates, including Aβ protofibrils, play a central role in Alzheimer’s disease (AD) and constitute a potential diagnostic biomarker and a therapeutic target. Aβ protofibrils promote synapse dysfunction and neurodegeneration, but the mechanisms behind these effects remain unclear. The aim of this thesis was to increase the knowledge of Aβ protofibrils in AD pathology. When measuring low abundant antigens, such as soluble Aβ aggregates, in plasma and CSF by immunoassays, there is a possibility of interference by heterophilic antibodies (HA). In paper I, we show that HA generate false positive signals, by cross-binding the assay antibodies, when plasma and CSF from AD patients and healthy controls were analyzed for soluble Aβ aggregates, using sandwich ELISAs. Natural anti-Aβ antibodies exist in AD patients and healthy individuals. Circulating Aβ and anti-Aβ antibodies may form immune complexes, masking epitopes on the anti-Aβ antibody, which makes the anti-Aβ antibody concentration difficult to measure. In paper II, the ELISpot technique enabled us to successfully measure B cell production of anti-Aβ antibodies. Our results show that anti-Aβ protofibril antibody production is present in both AD patients and healthy individuals, but is significantly higher in AD patients, indicating that the immune system attempt to eliminate the toxic Aβ species. Insufficient lysosomal degradation is proposed to cause sporadic AD. In paper III, we used a co-culture system of astrocytes, neurons and oligodendrocytes, to clarify the role of astrocytes in Aβ protofibril clearance. Astrocytes are the most prominent glial cell type in the brain, but their role in AD remains elusive. We found that astrocytes effectively engulf, but inefficiently degrade Aβprotofibrils. This result in a high intracellular load of toxic, partly N-terminally truncated Aβ and lysosomal dysfunction. Moreover, we found that secretion of microvesicles, containing N-terminally truncated Aβ, induce neuronal apoptosis. In paper IV, we show that treatment with the protofibril selective antibody mAb158 lead to enhanced Aβ clearance and thereby prevent Aβ neurotoxicity. Taken together, this thesis contributes with important knowledge on the role of Aβ protofibrils in AD pathogenesis and technical aspects that should be considered when measuring Aβ in human tissues.