Herpesviruses have co-speciated with several vertebrate and invertebrate animals throughout the history of evolution. In the immunocompetent human host, primary infection is usually benign, whereafter the virus is brought into life-long latency. Viral reactivation can however cause severe disease in immunocompromised, and rarely also in immunocompetent, patients. The overall aim of this thesis was to study the immunologic effects of cytomegalovirus (CMV) and herpes simplex type 1 (HSV-1) infection in neurocognitive disorders. CMV is known to promote T-cell differentiation towards a more effector-oriented phenotype, similar to what is seen in the elderly. We have addressed the frequency of CMV-specific CD8+ T-cells in Alzheimer's disease (AD). Furthermore, we have investigated whether AD patients present with a different CMV-specific immune profile, overall CD8 phenotype or inflammatory cytokine response to anti-CD3/CD28 beads, CMV pp65 and amyloid beta. Subjects with AD presented with a lower proportion of CMV-specific CD8+ T-cells compared to non-demented (ND) controls, but no differences in overall CD8 differentiation were seen. Overall, AD subjects presented with a more pro-inflammatory peripheral blood mononuclear cell (PBMC) phenotype. When PBMCs were challenged with CD3/CD28-stimulation, CMV seropositive AD subjects presented with more IFN-γ release than both CMV seronegative AD subjects and CMV seropositive ND controls. For effective screening of humoral herpesvirus immunity, both in research and in clinical practice, efficient immunoassays are needed. We have addressed the methodology of multiplex herpesvirus immunoassays and related bioinformatics and investigated antibody levels in AD patients and ND controls. Subjects with AD presented with lower levels of human herpesvirus 6 (HHV-6) IgG. However, there was no difference in HHV-6 DNA levels in PBMCs between the groups. Herpes simplex encephalitis (HSE) is a devastating disease, where antiviral treatment has greatly decreased mortality but not eliminated the associated long-term neurocognitive morbidity. We have investigated the correlation between N-Methyl-D-Aspartate Receptor (NMDAR) autoimmunity and recovery of neurocognitive functions after HSE. Approximately one quarter of all HSE cases developed NMDAR autoantibodies within 3 months after onset of disease. Antibody development was associated with an impaired neurocognitive recovery during the two year follow-up and could become an important therapy guiding factor in the future.

S'ha establert un sistema experimental autòleg per a poder estudiar la resposta de les cèl.lules Natural Killer (NK) contra les cèl.lules dendrítiques derivades de monòcits (moDC), infectades pel Cytomegalovirus humà (HCMV). Els nostres resultats mostren que les cèl.lules NK responen contra les moDC infectades per HCMV, que presenten una expressió de les molècules MHC de classe I a superficie reduïda. Específicament, demostrem que la infecció per HCMV disminueix l'expressió en superficie d'HLA-E en les moDC, alliberant així la inhibició de les cèl.lules NK NKG2A+. Mostrem que els NKR anomenats NKp46 i DNAM-1 tenen un paper dominant en el reconeixement de les moDC infectades per HCMV i evidenciem la importància de la dinàmica dels mecanismes d'immunoevassió en la susceptibilitat a la resposta NK. Finalment, trobem que els interferons de tipus I i la IL-12 secretats en resposta a la infecció per HCMV, a més de participar en l'activació de la cèl.lula NK i en la secreció d'IFN-, inhibeixen l'expressió i la funció de NKG2D en les cèl.lules NK, com un mecanisme de regulació potencial per prevenir la reactivitat NK contra cèl.lules veïnes sanes. / Suitable experimental conditions have been established to dissect the role of NK cell receptors (NKR) and cytokines in the NK cell response against autologous human cytomegalovirus (HCMV) infected monocyte derived dendritic cells (moDC). Our results reveal that NK cells are capable of responding to HCMV infected moDC that have down-regulated surface MHC class I molecules. In particular, we prove that HCMV infection decreases surface HLA-E expression on moDC, thus releasing NKG2A+ NK cells from inhibition. We show that NKp46 and DNAM-1 NKR play a dominant role in the recognition of HCMV infected moDC and we provide evidences stressing the importance of the dynamics of viral immune evasion mechanisms in NK cell susceptibility. Finally, we find that type I interferons and IL-12 secreted in response to HCMV infection, beyond their participation in NK cell activation and IFN- secretion, transiently inhibit the expression and function of NKG2D in NK cells, thus providing a potential regulatory feedback mechanism to prevent NK cell reactivity against bystander healthy cells.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T12:05:48Z (GMT). No. of bitstreams: 1 Parola_DanielaCorte_M.pdf: 2987280 bytes, checksum: 06dcaf150379c56fac854a42d4bc3453 (MD5) Previous issue date: 2007 / Resumo: O membro protótipo da subfamília dos betaherpesvírus, o citomegalovírus humano (HCMV), é o patógeno mais importante em pacientes transplantados, incluindo aqueles que receberam células de medula óssea ou enxerto de células tronco. Testes diagnósticos para identificar precocemente a infecção ativa pelo HCMV e uma terapia antiviral pré-clínica são medidas significantes para o controle desse vírus. Dois betaherpesvírus descobertos recentemente, o herpesvírus humano 6 (HHV-6) e o herpesvírus humano 7 (HHV-7) são geneticamente mais próximos um ao outro do que ao HCMV. Ambos têm alta prevalência na população em geral. Esses vírus não são tão patogênicos quanto o HCMV, porém o HHV-6 pode causar doenças como encefalite, hepatite e supressão da medula óssea. O objetivo do presente estudo foi avaliar o impacto clínico desses três vírus em transplantados de medula óssea. A monitorização pela N-PCR é importante método precoce para o controle da infecção ativa ou da doença pelo HCMV. A reação em cadeia da polimerase tipo nested (N-PCR) foi utilizada prospectivamente na monitorização de 43 pacientes para identificar as infecções ativas e a doença causada pelo HCMV, HHV-6 e HHV-7 por um período superior a 150 dias pós-transplante. Quarenta e um pacientes receptores adultos de células de medula óssea ou células tronco, com doença maligna e dois pacientes com doença não-maligna foram incluídos nesse estudo. Aciclovir foi administrado em baixas doses, como terapia profilática para infecção por herpesvírus tipo 1. Pacientes com infecção ativa por HCMV receberam terapia pré-clínica com ganciclovir. As incidências de positividade para infecção ativa por HCMV no sangue periférico, HHV-6 e HHV-7 no soro detectadas por N-PCR foram de 72%, 4,6% e 13,9%, respectivamente. A doença por HCMV ocorreu em 8 dos 43 pacientes (18,9%), no trato gastrintestinal e todos apresentaram N-PCR positiva para infecção por HCMV e um apresentou N-PCR positiva para HHV-6 no soro. Nenhum dos pacientes com doença por HCMV apresentou infecção ativa por HHV-7. A infecção ativa foi por HHV-6 e HHV-7 foi baixa em nossa casuística. A doença por HCMV permanece a mais a causa mais importante de impacto clínico após o transplante de medula óssea. Estudos adicionais necessitam serem feitos para uma melhor compreensão da relação entre os herpesvírus HCMV, HHV-6 e HHV-7 nos pacientes transplantados de medula óssea e células tronco periféricas / Abstract: The prototype member of the Betaherpesvirus subfamily, human cytomegalovirus (HCMV), is the most important infectious pathogen in transplant recipients, including those receiving bone marrow (BM) or stem cell (SC) grafts. Rapid diagnostic tests to identify active CMV infection, and pre-emptive therapy are significant improvements in the management of CMV. Two newly identified betaherpesviruses, human herpesvirus-6 (HHV-6) and human betaherpesvirus-7 (HHV-7), are genetically more closely related to each other than to CMV. Both are highly prevalent in the general population. These viruses are not as pathogenic as CMV but HHV-6 can cause disease such as encephalitis, hepatitis and bone marrow suppression. The aim of this study was to evaluate the clinical impact of these three viruses in bone marrow and stem cell transplantation patients. Monitorization with Nested-PCR is important in the control of active CMV infection or disease. Nested polymerase chain reaction (N-PCR) was used prospectively to monitor 43 patients for evidence of active infections and diseases caused by HCMV, HHV-6 and HHV-7 for up to 150 days after transplant. Forty-one adult recipients of BM or SC graft with malignant diseases and two patients with non-malignant diseases, and with a risk for CMV disease (D+/R+; D+/R-) were enrolled in this study. Aciclovir was used before the transplant at low doses, as prophylactic therapy for HHV-1. Patients with active CMV infections received pre-emptive therapy with ganciclovir. The incidence of positive active HCMV in blood, HHV-6 and HHV-7 in serum detected by Nested-PCR was 72%, 4.6% and 13.9%, respectively. HCMV disease occurred in 8/43 patients (18.6%), in the gastrointestinal tract and all presented positive N-PCR for active HCMV infection and one presented active infection by HHV-6 detected in serum. None of the patients presented active HHV-7 infection. Co-infection by HHV-6 and HHV-7 was low. CMV disease remains the most important disease after BMT. Future studies can be made to a better understanding in relationship between HCMV, HHV-6 and HHV-7 in BM or SC transplantation recipients / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Introdução: Citomegalovírus (CMV) é um DNA vírus de alta prevalência, e tem uma capacidade peculiar de infectar e permanecer integrado ao DNA das células do hospedeiro, mantendo-se na forma de infecção latente. O vírus também pode ocasionar doença, o que normalmente ocorre em pacientes imunocomprometidos, promovendo o aumento da morbidade e mortalidade nestes pacientes. As doenças inflamatórias intestinais (DII), doença de Crohn (DC) e retocolite ulcerativa (RCU), são enfermidades crônicas que afetam o trato gastrointestinal. A fisiopatologia e o tratamento destas doenças, muitas vezes, pode induzir um estado de imunossupressão. Isso incitou a ideia de que os pacientes com DII são mais susceptíveis à infecção e doença por CMV. Ainda há dúvidas e controvérsias sobre a relação entre a doença inflamatória intestinal e o CMV. Objetivos: Avaliar a frequência de infecção por CMV em pacientes com doença inflamatória intestinal, e se existe associação entre replicação viral do CMV com a atividade da DII, mediante índices clínicos e laboratoriais. Metodologia: Pacientes com DII previamente diagnosticada foram submetidos à entrevista, revisão de registros e coleta de amostras de sangue e fezes. Foram realizados os seguintes exames: pesquisa de citomegalovírus por IgG e IgM no sangue, pela técnica de reação em cadeia por polimerase (PCR) em tempo real no sangue e pela técnica de PCR qualitativa nas fezes. Estes resultados foram correlacionados com os valores de hemoglobina, proteína C-reativa, velocidade de hemossedimentação, calprotectina fecal e índices clínicos. Resultados: Quatrocentos pacientes foram elegíveis, sendo 249 com DC e 151 com RCU. No grupo de pacientes com DC, 67 apresentavam doença moderada ou grave pelo índice clínico, porém 126 se mostravam com doença ativa mediante a avaliação da calprotectina fecal. No grupo de pacientes com RCU, 21 exibiam doença moderada pelo índice clínico, mas 76 se encontravam com doença ativa, mediante a avaliação da calprotectina fecal. Drogas imunossupressoras foram amplamente utilizadas pelos pacientes, 143 pacientes com DC faziam uso de azatioprina e, destes, 48 usavam terapia combinada (anti TNF-alfa + azatioprina). Na RCU, a azatioprina foi usada por 41 pacientes e, destes, sete faziam uso de terapia combinada. Avaliando os dois grupos, 90,9% dos pacientes apresentaram anticorpos IgM contra o CMV no sangue e dez pacientes também exibiram IgG. A detecção do DNA CMV PCR em tempo real no sangue apresentou valores abaixo do limite inferior (150 cópias/mL) em todos os 400 pacientes. Enquanto isso, o DNA CMV PCR qualitativo, realizado na amostra fecal, indicou nove pacientes expressando valores positivos. Com efeito, nos 400 pacientes, identificaram-se 332 infectados sem replicação viral, 19 pacientes com replicação viral e 24 não infectados. Os pacientes com DII em uso de terapia combinada apresentaram uma chance maior de replicação viral 3,63 vezes em comparação aos pacientes que não fizeram uso deste tratamento. Conclusão: A infecção latente pelo CMV foi bastante prevalente, mas a infecção ativa foi rara. A utilização de terapia combinada, entretanto, em doentes com DII, tem associação com a replicação viral do CMV, mas sem indicar relação com a atividade inflamatória da DII / Background: Cytomegalovirus (CMV) is a highly prevalent DNA virus that has a peculiar ability to infect the host and remains integrated to his DNA as a latent infection. The virus can also appear in the form of disease, which most commonly occurs in immunocompromised patients, increasing their morbidity and mortality. Inflammatory bowel diseases (IBD), Crohn\'s disease (CD) and ulcerative colitis (UC) are chronic diseases that affect the gastrointestinal tract. The pathophysiology and treatment of these diseases often induce a state of immunosuppression, hence the assumption that patients with inflammatory bowel disease may be at greater risk for cytomegalovirus disease. However, there are still doubts and controversies about the relationship between IBD and CMV. Aim: Evaluate the frequency of CMV infection in patients with IBD correlating it with clinical and laboratorial activity indices of IBD. Methods: Patients with a previous diagnosis of IBD underwent interviews, a medical record review and collection of blood and fecal samples. The search of CMV was performed by IgG and IgM blood serology, real-time PCR in blood and qualitative PCR in feces. These results were correlated with red blood cell levels, Creactive protein, erythrocyte sedimentation rate and fecal calprotectin. Patients with CD were evaluated by Crohn\'s disease activity index and UC patients, by Truelove & Witts index. Results: Four hundred patients were eligible: 249 patients with CD and 151 with UC. In the CD group, using clinical index, 67 patients had moderate or severe disease, but 126 patients presented with active disease by evaluating fecal calprotectin. In patients with UC, 21 exhibited moderate disease by clinical index, but 76 patients presented with active disease by evaluating fecal calprotectin. Immunosuppressive drugs were widely used by patients. On CD, 143 patients of them were using azathioprine, and of these, 48 were using combo therapy (anti TNFalpha + azathioprine). On the UC, azathioprine was used in 41 patients, and seven of these were taking combo therapy. The great majority of patients (90,9%) had positive CMV IgG, ten patients had positive CMV IgM, nine patients had positive qualitative detection of CMV DNA by PCR in faeces, and in all 400 patients quantitative detection of CMV DNA by real-time PCR in blood was negative. In the 400 patients, we identified 332 CMV infected without viral replication, 19 patients CMV infected with viral replication (active infection) and 24 non-infected CMV patients. Analyzing the 19 patients with active infection, we only found an association with the use of combo therapy (anti TNF-alpha + azathioprine), and patients on combo therapy have a viral replication chance 3.63 times compared to patients who do not use this treatment. Conclusion: Latent cytomegalovirus infection is extremely frequent in the inflammatory bowel disease population, but the active cytomegalovirus infection is rare; and the use of combination therapy in patients with IBD is associated with viral replication of CMV, but without presenting relation to inflammatory activity of IBD

Orientadores: Laura Sterian Ward, Alfio José Tincani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T20:17:52Z (GMT). No. of bitstreams: 1 Almeida_JacquelineFatimaMartins_M.pdf: 2198135 bytes, checksum: 94a76c347fc27e9b1fc05d077aa728a0 (MD5) Previous issue date: 2014 / Resumo: Agentes biológicos causadores de neoplasias humanas têm sido alvo de pesquisas científicas nos últimos anos, especialmente os vírus, considerados responsáveis pela causa de cerca de 20% de todos os tipos de cânceres em geral. No entanto os mecanismos pelos quais estes vírus causam tumores variam dependendo do tipo de sua espécie, da célula alvo e de fatores que concernem ao hospedeiro. Os herpes vírus, como o Herpes Simplex Virus tipo 2 (HSV-2), Epstein-Barr vírus (EBV), Citomegalovirus (CMV) e Herpesvirus Humano tipo 8 (HHV-8) têm sido associados com diversas neoplasias malignas e também com doenças autoimunes tireoidianas. O objetivo do nosso trabalho foi verificar a presença da infecção destes herpes vírus em nódulos tireoidianos benignos e malignos procurando indícios de uma possível associação com tumores tireoidianos. Foram coletadas amostras de tecido tumoral tireoidiano e soro de 153 portadores de nódulos de tireoide (136 mulheres e 17 homens, 46±15 anos) incluindo 65 tumores benignos (48 bócios, 17 adenomas foliculares e 09 portadores de doença tireoidiana autoimunes sendo 02 doenças de Graves e 07 Hashimotos) e 79 tumores malignos (78 carcinomas papilíferos e 01 carcinoma folicular). Também obtivemos 75 tecidos tireoidianos normais extraídos do lobo contralateral da lesão em 75 tumores. Foi realizada análise sorológica por ELISA para detecção de anticorpos dos herpes vírus e foi realizada análise da Carga Viral por PCR em Tempo Real. Quarenta e cinco (29,4%) pacientes (45/153) possuíam anticorpos anti-HSV2; 83,7% anti-CMV (128/153); 98% anti-EBV (150/153) e nenhum paciente possuía anticorpos anti-HHV8, o que é compatível com as estimativas esperadas na população brasileira. As concentrações de anticorpos anti-EBV foi, em média maior do que o do HSV-2 e do que o do CMV (p<0,0001). Os herpes vírus HSV-2, CMV e HHV-8 não foram encontrados em nenhum tecido tireoidiano da população estudada. No entanto, encontramos sequências de DNA do EBV em 20 amostras de tecidos tireoidianos (04 bócios, 03 DAIT e 13 CP). Embora a carga viral média fosse de 1068 cópias/µg nos casos malignos e de 374 cópias/µg nos casos benignos, esta diferença não se mostrou estatisticamente significante. Foram encontrados também 6/75 (8%) casos positivos para a infecção por EBV nos tecidos normais. A carga viral foi, em média, maior nos tumores (126,5 cópias/µg) do que em seus respectivos tecidos normais (93 cópias/µg; p=0,0207). Não houve correlação entre a presença de EBV e as características clínicas ou de evolução dos pacientes. Embora, para melhor compreensão de seus mecanismos de ação, sejam necessários novos estudos moleculares e microbiológicos, a presença da alta carga viral do EBV nos tecidos tumorais tireoidianos em relação aos tecidos normais correspondentes sugere que este vírus pode exercer um papel no desenvolvimento de nódulos tireoidianos / Abstract: Biological agents that cause human cancers have been the subject of scientific research in recent years, especially viruses considered to be responsible for the cause of about 20% of all cancers in general. Although the mechanisms of latent infection and carcinogenesis vary depending on the particular virus, target cells, and host factors. Herpesviruses, such as Human Simplex Virus type 2 (HSV-2), Epstein - Barr virus (EBV), Cytomegalovirus (CMV) and Human Herpes Virus type 8 (HHV-8) have been associated with human malignancies and also with thyroid autoimmunity. We aimed to analyze the presence of these viruses in benign and malignant thyroid nodules looking for evidence of a possible association with thyroid tumors. Serum and thyroid specimens were prospectively collected from 153 thyroid nodule patients (136 females and 17 males, aged 46±15 years) including 78 papillary thyroid cancers (PTC), 01 follicular thyroid cancer (FTC), 17 follicular adenomas and 48 goiters. We used ELISA to screen all patients for the presence of the viruses and a real-time quantitative PCR (qPCR) technique to analyze thyroid tissues viral load on antibody-positive patients. Forty-five (29.4%) patients (45/153) presented anti-HSV2 antibodies; 83.7% anti-CMV (128/153); 98% ani-EBV (150/153) e no patient presented antibodies anti-HHV8, which would be expected to be found in Brazilian population in general. In both malignant and benign groups the medians of antibodies anti-EBV were higher than HSV-2 and CMV medians (p<0,0001). There was no evidence of the presence of HSV-2, CMV and HHV-8 in the population studied. Nevertheless we found EBV¿s DNA sequences in 20 thyroid tissue samples. Even though the average malignant viral load was 1068 copies/µg in 13 cases and 374 copies/µg in 7 benign cases, there was no statistically significant difference between groups. We also found 6 (8%) positive cases for EBV viral load out of 75 normal tissues. In paired test the viral load median was higher in tumors (126.5 copies/µg) than in their respective normal tissues (93 copies/µg; p=0.0207). We did not find any association among the presence of EBV and/or its viral load and any clinical or pathological feature. Althought molecular studies are needed to a better comprehension of the mechanisms underlying the relation between EBV and thyroid nodules, the presence of high EBV copy numbers in thyroid tumors, especially in PTC cases, in comparison with normal tissues, suggests that this virus may play a role in the development of thyroid nodules / Mestrado / Clinica Medica / Mestra em Clínica Médica

Orientador: Sandra Cecília Botelho Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T18:24:50Z (GMT). No. of bitstreams: 1 Peres_RenataMariaBorges_D.pdf: 6331424 bytes, checksum: 507ca7bba502f408e486703c76d9ef20 (MD5) Previous issue date: 2014 / Resumo: Introdução: Considerando-se que o transplante de células-tronco hematopoiéticas (TCTH) está associado à significativa morbidade e mortalidade devido à toxicidade do regime de condicionamento, sepse, doença do enxerto-contra-hospedeiro (DECH), entre outras complicações, sua indicação requer uma avaliação cuidadosa do risco. As infecções estão associadas a elevados índices de óbitos no pós-transplante, e seu aparecimento pode estar associado a fatores como o regime de condicionamento, imunodepressão para a prevenção da DECH, a própria DECH e a terapia adicional para a doença de base após o transplante. Dentre as infecções virais, o citomegalovírus humano (HCMV) é o patógeno mais importante no pós-TCTH pela morbidade e mortalidade associadas a ele. Por este motivo os pacientes submetidos ao TCTH são monitorados por testes laboratoriais sensíveis e específicos para o diagnóstico precoce da infecção ativa causada pelo HCMV, permitindo assim, a administração imediata da terapia antiviral. A antigenemia tem sido indicada como "padrão ouro" para guiar o tratamento precoce desta infecção. Embora a PCR em tempo real seja outra técnica muito utilizada para este fim, esta pode ser demasiadamente sensível para uso clínico, podendo haver diagnósticos positivos em pacientes que não apresentam risco de desenvolvimento da doença causada pelo HCMV. Objetivos: Determinar um valor de cutoff para diferenciar infecção latente de infecção ativa, além do risco do desenvolvimento da doença causada pelo HCMV. Métodos: Neste trabalho, 49 pacientes submetidos ao TCTH do tipo alogênico foram monitorados desde o dia do transplante até o dia +150 pelas técnicas de antigenemia e pela PCR em tempo real. Resultados: Após a construção da curva ROC o cutoff encontrado para indicar a reativação do HCMV foi 116 cópias. Baseado nesta determinação, os resultados positivos da PCR em tempo real (?116 cópias) foram comparados com resultados positivos da antigenemia (? 3 células pp65 positivas), observando associação estatisticamente significante entre eles. Sendo assim, a PCR em tempo real com 116 cópias como cutoff foi adotada como o único critério para definir a infecção ativa causada pelo HCMV nos pacientes incluídos no estudo. Vinte (40,8%) dos 49 pacientes tiveram infecção ativa causada pelo HCMV durante o monitoramento, ocorrendo com maior frequência entre os dias +41 e +50 pós-TCTH. Dezoito (90%) desses 20 pacientes foram tratados com ganciclovir e 16 deles atingiram a negativação da PCR em tempo real numa mediana de 6 dias após o início do tratamento. Quatro (8,2%) pacientes tiveram doença causada pelo HCMV comprovada por biópsia do trato gastrointestinal, 1 deles sem diagnóstico da viremia. Dois (50%) dos 4 pacientes que tiveram doença causada pelo HCMV não responderam bem ao tratamento antiviral, evoluindo para o óbito por esta causa numa mediana de 6,5 dias após o início do tratamento com ganciclovir. Outros 15 pacientes morreram por infecções fúngicas e bacterianas, DECH, recidiva da doença de base ou doença veno-oclusiva hepática. Conclusões: Pacientes submetidos ao TCTH não aparentado apresentam maior risco de infecção ativa causada pelo HCMV, o aumento repentino da carga viral parece ter relação com o aparecimento da doença causada pelo HCMV, a gravidade desta doença pode estar associada com o tempo de resposta ao tratamento antiviral e a manifestação de doença causada pelo HCMV e provável doença causada pelo HCMV aumentam o risco de óbito no pós-TCTH / Abstract: Considering that allogeneic hematopoietic stem cell transplantation (HSCT) is associated with significant morbidity and mortality due to toxicity of the conditioning regimen, sepsis onset, graft versus host disease (GVHD) manifestation, among other complications, its indication requires careful risk assessment. Infections are associated with high rates of deaths after transplantation, and their appearance can be associated with factors such as conditioning regimen, immunosuppression to prevent GVHD, GVHD itself and additional therapy for the underlying disease after transplantation. Among the viral infections, human cytomegalovirus (HCMV) is the most significant pathogen after HSCT, considering morbidity and mortality associated with it. For this reason, patients submitted to HSCT are monitored by sensitive and specific laboratory tests for early diagnosis of HCMV reactivation, thus allowing immediate administration of antiviral therapy. The antigenemia method has been indicated as the gold standard for guiding the early treatment of this infection. Although real-time PCR is another technique widely used for this purpose, this may be too sensitive for clinical use, there may be positive diagnoses in patients without risk of developing HCMV disease. Objectives: Determining a cutoff value to distinguish latent infection from active infection and the risk of developing HCMV disease. Methods: In this study, 49 patients undergoing allogeneic HSCT were monitored from the day of transplantation until day +150 by antigenemia and real-time PCR. Results: After constructing the ROC curve, the cutoff found for HCMV reactivation was 116 copies. Based on this determination, the positive results of real-time PCR (? 116 copies) were compared with results of positive antigenemia (pp65 positive cells ? 3), observing statistically significant association between them. Thus, the real-time PCR with 116 copies as cutoff was adopted as the sole criterion to define the active infection in the patients included in the study. Twenty (40.8%) of the 49 patients had HCMV reactivation during monitoring, occurring most frequently between days +41 and +50 after HSCT. Eighteen (90%) of these 20 patients were treated with ganciclovir and 16 have reached their negative real-time PCR in a median of six days after the beginning of treatment. Four (8.2%) patients had positive gastrointestinal tract biopsy for HCMV disease, one of them with no diagnosis of viremia. Two (50%) of four patients who had HCMV disease have not responded well to antiviral therapy, progressing to death by HCMV disease at a median of 6.5 days after starting treatment with ganciclovir. Another 15 patients died from fungal bacterial infection, GVHD, underlying disease relapse or veno-occlusive hepatic disease. Conclusions: Patients undergoing unrelated HSCT had higher risk of HCMV reactivation; the sudden increase in viral load seems to be related to the onset of HCMV disease; the severity of HCMV disease may be associated with the response time to antiviral therapy; and HCMV disease manifestation and probable HCMV disease increase the risk of death after HSCT / Doutorado / Clinica Medica / Doutora em Clínica Médica

Les monocytes classiques (cMo) sont des phagocytes mononucléés circulant dans le sang et capables de migrer vers les tissus enflammés pour s’y différencier en monocytes inflammatoires, cellules dendritiques dérivées de monocytes (MoDC), macrophages (MoM) ou cellules myéloïdes suppressives. Selon le contexte physiopathologique, les cellules dérivées de cMo peuvent être bénéfiques ou néfastes. Dans l’infection par le cytomégalovirus murin (MCMV) leur rôle est controversé. Les divergences apparentes dans la littérature pourraient s’expliquer par l’utilisation de souches distinctes de souris ou de virus, l’étude d’organes différents, et la confusion existante sur l’identité et la plasticité de différents sous-types de cellules dérivées de cMo. Par des analyses transcriptionnelles, morphologiques et fonctionnelles, mon travail de thèse montre que, dans la rate de souris infectées par MCMV, les cMo se différencient simultanément en monocytes inflammatoires, MoDC et MoM. Cette différenciation est abrogée lorsque les cMo sont incapables de répondre aux interférons de type I (IFN-I), massivement produits dans les infections virales, qui boostent l’immunité intrinsèque antivirale et promeuvent l’activation des cellules immunitaires innées et adaptatives. La déplétion des cMo compromet le contrôle de l’infection et les réponses des cellules Natural Killer et des lymphocytes T CD8+. Mon travail montre que, dans les souris infectées par MCMV, les cMo se différencient, de manière dépendante de l’IFN-I, en trois sous-types cellulaires distincts qui contribuent à la fois au contrôle de la réplication virale et à la promotion de réponses immunitaires innées et adaptatives protectrices. / Classical monocytes (cMo) are mononuclear phagocytes mainly localized in the blood at steady state. Upon inflammation cMo migrate into inflamed tissues where they can differentiate in inflammatory monocytes, monocyte-derived dendritic cells (MoDC), monocyte-derived macrophages (MoM) or myeloid derived suppressor cells (MDSC). Depending on the physiopathological context, cMo-derived cells can be beneficial or detrimental. There are major discrepancies between published reports on the role of cMo during MCMV infection. This may be due to the use of distinct strains of mice or of virus, to the study of different organs, or to the confusion existing in the field regarding the identity and the plasticity of the different types of cMo-derived cells. During my PhD, by combining gene expression profiling, morphological, phenotypical and functional studies, I have shown that splenic cMo in MCMV-infected mice encompass cells that had simultaneously differentiated in vivo into either inflammatory monocytes, MoDC or MoM. This cMo differentiation is abrogated in the absence of responsiveness to type I interferons (IFN-I), which are highly produced during viral infections and boosting cell-intrinsic anti-viral immunity as well as promoting the activation of innate and adaptive immune responses. cMo depletion compromises the control of MCMV replication and the antiviral responses of Natural Killer cells and CD8+ T lymphocytes. My PhD work demonstrates that, in MCMV-infected mice, cMo differentiate, via an IFN-I-dependent pathway, into three distinct cell subtypes that are involved both in the control of MCMV replication and in the induction of protective innate and adaptive immunity.

Natural Killer (NK) cells, members of the innate lymphoid cells (ILCs), are known to play an important role in the defense against foreign cells and abnormal host cells that have arisen due to viral infection or cancer inducing mutations. The typical immune response of NK cells involves the release of cytotoxic granules containing perforin and granzyme, and the secretion of immune-regulatory cytokines such as interferon gamma (IFN-γ). Unlike the adaptive lymphocytes such as T cells and B cells, NK cells do not require prior sensitization, enabling them to initiate an immune response much faster. This unique feature of NK cells is made possible by the utilization of an array of germline encoded receptors; but on the other hand, it limits NK cells ability to respond against rapidly evolving pathogens. NK cells overcome this shortcoming with an antibody-assisted process called antibody dependent cellular cytotoxicity (ADCC). A novel subset of human NK cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently discovered in cytomegalovirus positive (CMV+) individuals. This NK cell subset, called g-NK cell, was characterized by a deficiency in the expression of FcεRIγ, an adaptor protein that associates with CD16 which enables ADCC. Surprisingly, despite this deficiency, g-NK cells displayed an enhanced ADCC as compared to their conventional counterparts. Furthermore, having a long-lasting memory-like NK-cell phenotype suggests a role for g-NK cells in chronic infections. This study investigates the importance of g-NK-cells in clinical settings, first by investigating whether the presence of g-NK cells is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK cell proportions and function in the peripheral blood mononuclear cells (PBMCs) of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of g-NK cells, while having similarly enhanced ADCC responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56neg NK cell population often found in chronic HCV patients, suggesting their involvement in the immune response against HCV. Rituximab is a chimeric anti-CD20 antibody used to treat B cell lymphoma patients; and studies have suggested that its efficacy is associated with the ADCC potency and CD16 affinity. Since g-NK cells are characterized by their superior ADCC compared to their conventional counterpart, I decided to investigate whether the presence of g-NK cells can improve the effectiveness of rituximab against malignant B cells in the context of lymphoma and leukemia. The analysis of g-NK cells’ ADCC response against rituximab-coated lymphoma cell lines and B cells from a CLL patient indicated a superior ADCC by g-NK cells compared to their conventional NK cell counterparts. Taken together, for the first time, my findings indicate that the presence of g-NK cells in CMV+ individuals is associated with milder liver disease in chronic HCV infection. In addition, an enhanced ADCC response by g-NK cells upon encountering rituximab coated target cells suggests the beneficial roles of g-NK cells, and opens an avenue for novel therapeutic approaches where g-NK cells can be utilized to treat persistent diseases such as chronic viral infection and cancer.

Human cytomegalovirus (HCMV) is a ubiquitous virus of the herpes type that infects a high percentage of some populations. One of the most researched genes expressed by HCMV with close homology to human chemokine receptors is the US28 G protein-coupled receptor. Study design: This study was initiated to elucidate the intracellular signalling pathways of an inflammatory factor (IL-6) and an angiogenic factor (STAT3) triggered by the viral US28 oncogene and the presence of US28 in the HCMV viral particle. These pathways were observed by introducing the US28 gene into two human cell lines by infection with a HCMV strain that expresses the US28 gene (wild type), and two HCMV strains where the US28 gene was deleted (ÄUS28 and ÄUS28/UL33). Special attention was directed at the expression of IL-6 after promotion of the US28 gene and subsequent phosphorolation of STAT3. A new US28 antibody was validated and a method developed in an attempt to determine US28 on the viral particle. The following techniques were applied: Cell culture work, two mammalian cell lines were used, HFF’s and U373 MG. Virus stock titre determination to determine the multiplicity of infection. Protein quantitation to determine very small quantities of protein for Western blot analysis. ELISA for the quantitative determination of IL-6. Western blotting for phospho- STAT3 determination and validation of the US28 antibody. Immunocytochemistry was used for back titrations of virally infected cells. Immunofluorescence assay and use of confocal microscopic techniques was used for the location of the US28 gene in the virion and for tSTAT3 translocation to the nucleus. Conclusion: A clear increase in IL-6 secretion (495% ± 1%) was seen, and this was after only an hour in HCMV WT infected cells. From the increase in IL-6 secretion a subsequent increase in STAT3 phosphorylation was detected in the same samples. A clear link has been established between IL-6 and STAT3. A method to determine whether US28 was present in the HCMV viral particle was designed and preliminary results obtained. The results were inclusive. / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

Orientadores: Sandra Cecília Botelho Costa, Sandra Helena Alves Bonon / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T11:43:36Z (GMT). No. of bitstreams: 1 Peigo_MurilodeFreitas_M.pdf: 1466925 bytes, checksum: 094e2cd00645843fd5fa2c56ca66c2d5 (MD5) Previous issue date: 2012 / Resumo: O Lúpus Eritematoso Sistêmico (LES) é uma patologia sistêmica do tecido conjuntivo, que se apresenta de maneira variada na dependência do órgão afetado, da gravidade de seu acometimento e da idade do paciente, tendo influência de fatores raciais, de padrões imunológicos e ambientais. Pacientes lúpicos têm grande predisposição para desenvolver infecções graças à imunossupressão induzida pela própria doença como pelo uso de vários medicamentos em seu tratamento. Infecções causadas por herpesvírus, principalmente o Citomegalovírus Humano (CMV) e o Epstein-Barr (EBV), têm sido implicadas em várias doenças autoimunes graves, incluindo o LES. A reativação dos herpesvírus 6 e 7 (HHV-6 e HHV-7) geralmente ocorre em pacientes imunodeprimidos, mas seus papéis ainda são pouco estudados. As infecções por herpesvírus têm influência tanto no início do processo autoimune quanto na exacerbação da progressão da doença. A identificação de pacientes com alto risco de desenvolver doença pelos herpesvírus pode ser realizada utilizando técnicas de detecção de infecção ativa, como a Reação em Cadeia da Polimerase (Nested-PCR) e a detecção do antígeno pp65 do CMV (Antigenemia). Dependendo do caso, estes pacientes podem receber tratamento com antivirais. Diante do exposto, os objetivos deste estudo foram: monitorizar os pacientes com LES em relação à infecção ativa por CMV, EBV, HHV-6 e HHV-7, utilizando as técnicas de Nested-PCR e de antigenemia, bem como avaliar o impacto clínico dessas infecções. Foram incluídos neste trabalho, amostras de sangue de 71 pacientes em seguimento no Departamento de Reumatologia da Faculdade de Ciências Médicas ¿ UNICAMP, com diagnóstico de LES confirmado, sendo que 20/71 (28%) estavam com o lúpus ativo (SLEDAI ? 8) e 51/71 (72%) dos pacientes não tinham atividade lúpica (SLEDAI < 8). Das amostras de sangue pesquisadas, 10/71 (14%) foram positivas para os herpesvírus estudados, sendo que 90% destes pacientes com infecção ativa apresentavam o lúpus em atividade (p?0,006). Infecção ativa pelo CMV ocorreu em 4 pacientes (5,6%). HHV-7 foi detectado em 4 amostras (5,6%). Dois outros pacientes apresentaram dupla infecção por CMV e HHV-7 (2,8%). Infecção ativa pelo EBV e HHV-6 não foi detectada em nenhuma das amostras analisadas. Dois pacientes foram a óbito, sendo que um deles evoluiu com sepse de foco pulmonar (provável doença por CMV) e o outro com sepse por Psedomonas aeruginosa. Diante dos resultados obtidos, podemos observar que a infecção ativa pode ocorrer nos pacientes com LES, principalmente naqueles com a doença em atividade. Poucos estudos têm avaliado o impacto destas infecções no cuidado diário dos pacientes com LES. Acreditamos que este trabalho seja pioneiro e será de fundamental importância, contribuindo com este grupo de pacientes. Entretanto, futuros estudos deverão ser implementados com um número maior de pacientes e de coletas/paciente, principalmente naqueles com LES em atividade, que foram demonstrados com aqueles com fator de risco aumentado / Abstract: Systemic lupus erythematosus (SLE) is a connective tissue systemic pathology that presents itself in several ways, depending on the organ affected, the seriousness of the disease and patient¿s age, being influenced by racial factors, immunologic and environmental patterns. SLE patients have great predisposition to develop infections due to the immunosuppression induced by the disease itself and by the use of medicine in the treatment. Infections caused by herpesvirus, especially Human Cytomegalovirus (CMV) and Epstein-Barr (EBV), have been developed into several serious autoimmune diseases, including SLE. Herpesvirus 6 and 7 (HHV-6 and HHV-7) reactivation generally occurs in immunodepressed patients, but their roles are unclear. Herpesvirus infections have influence both on the beginning of the autoimmune process and on the aggravation of the disease progression. The patients that present high risks of developing herpesvirus related diseases can be identified using active infection detection techniques, such as the Nested polymerase chain reaction (Nested-PCR) and the CMV pp65 antigen detection (antigenemia). Depending on the case, the patients can receive treatment with antivirals. Face to the exposed, the objectives of this study were: to monitor the patients with SLE with regard to active infection by CMV using Nested-PCR and antigenemia techniques, and EBV, HHV-6, HHV-7 in serum, as well as to evaluate the clinic impact to these infections. There were included in this work blood samples of 71 patients that are being treated at the Department of Rheumatology, Faculty of Medical Sciences ¿ University of Campinas - UNICAMP, with a confirmed SLE diagnosis, given that 20/71 (28%) had active lupus (SLEDAI ? 8) and 51/71 (72%) of the patients didn¿t present lupic activity (SLEDAI < 8). Considering the blood samples researched, 10/71 (14%) were positive for the studied herpesvirus, and 90% of the patients with active infection presented lupus in activity (p ? 0,006). Active infection by CMV was observed in 4 patients (5,6%). HHV-7 was detected in 4 samples (5,6%). Two other patients presented double infection by CMV and HHV-7 (2,8%). Active infection by EBV and HHV-6 was not detected in any of the analyzed samples. Two patients have deceased, whose conditions developed into pulmonary sepsis (probable disease by CMV) and into Psedomonas aeruginosa sepsis, respectively. After analyzing the achieved results, we observe that active infection can appear in patients with SLE, especially in those with the disease in activity. Few studies have evaluated the impact of these infections on the daily care of patients with SLE. In this sense, we believe that this work is pioneer and that it will be of fundamental importance, contributing to this group of patients. However, future studies should be implemented, with a larger number of patients and samples, especially those with SLE in activity, which are the ones with increased risk factor as shown / Mestrado / Clinica Medica / Mestre em Clinica Medica