Orientador: Sergio Tadeu Martins Marba / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T12:12:01Z (GMT). No. of bitstreams: 1 Kallas_SheiladeLima_M.pdf: 1394129 bytes, checksum: 6a73ee8e82058acb673cea32eee2b643 (MD5) Previous issue date: 2008 / Resumo: Os objetivos do estudo foram determinar a prevalência da infecção congênita por citomegalovírus em recém-nascidos internados na unidade de internação neonatal do CAISM-UNICAMP, avaliar a eficácia da Nested-PCR para citomegalovírus em sangue estocado em papel filtro e descrever variáveis epidemiológicas da população estudada. Foi realizado um estudo epidemiológico transversal, onde foram selecionados recém-nascidos internados na unidade de internação neonatal durante o período de 01 de Abril de 2005 a 30 de Junho de 2006. Foram coletadas amostras de urina dos recém-nascidos e calculada a prevalência da infecção congênita por CMV pelo método diagnóstico da Nested PCR. Para validação do teste diagnóstico, foram coletados, concomitantemente com a urina, amostras de sangue dos recém-nascidos, estocados em papel filtro, e realizada a Nested-PCR. Os resultados da Nested-PCR em papel filtro foram comparados com os da urina e foram calculados sensibilidade, especificidade, valores preditivos positivo e negativo. Dados epidemiológicos da população foram colhidos pela revisão de prontuários. Foram obtidos os seguintes resultados: sensibilidade 75% , especificidade 99,4%, valores preditivos positivo 60,0% e negativo 99,7%. A prevalência da infecção congênita por CMV foi de 1,2%. A média de idade materna foi de 25,5 anos. A maior parte das mães, 40,9%, foram procedentes de Campinas; 86,3% delas realizaram pré-natal, 96,2% das que realizaram o pré-natal não realizaram sorologia para CMV, 65,3% dos partos foram por cirurgia cesariana, sendo que em 46% das cesáreas a indicação foi sofrimento fetal agudo. Sobre os recém-nascidos, 74,3% deles eram pré-termos, 57% pesavam menos que 2 kg, sendo a média de idade gestacional de 34,2 semanas. Concluimos que a prevalência da infecção congênita foi baixa e que o método diagnóstico Nested-PCR em sangue estocado em papel filtro teve alta especificidade e moderada sensibilidade / Abstract: The aims of this study were: to determine the prevalence of congenital infection by cytomegalovirus in newborn patients of the intensive neonatal care unit of the CAISM-UNICAMP Hospital; to evaluate the efficacy of Nested PCR diagnostic test in blood stored on filter paper and to describe epidemiological characteristics of this population. A cross section study was made including newborns admitted in the intensive care unit from April first, 2005 to June 30, 2006. Urine samples were collected from newborns and the prevalence was determined by Nested-PCR. To evaluate the efficacy of Nested PCR in blood stored on filter paper, the Nested-PCR in urine samples were compared to those in blood samples. Sensibility, specificity, positive and negative predictive values were calculated. Epidemiological characteristics of the population were described. The following results were observed: sensibility: 75%; specificity: 99, 4%; positive predictive values: 60, 0% and negative predictive values: 99,7%. The prevalence of congenital cytomegalovirus infection, calculated by Nested-PCR in urine samples was 1,2% in this period. The average of mother age was 25,5 years old.The most of mothers (40,9%) were from Campinas; 86,3% of them had prenatal care; 96,2% of the mothers who had prenatal care didn¿t had CMV serology; 65,3% of the delivery were by caesarean, and the reason of caesarean was acute fetal suffering in 46%. The newborn characters were: 74,3% were premature babies, 57% had weight less than 2 kilograms and the average of birth age was 34,2 weeks. We concluded that the prevalence of the congenital infection by CMV was low and that Nested PCR diagnostic test in blood stored on filter paper is highly specific and moderately sensible / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Os objetivos do estudo consistiram em verificar o impacto da infecção congênita por CMV ao nascer e avaliar potenciais preditores prognósticos de anormalidades tardias em uma coorte de criança s com esta infecção. Métodos: Por meio de uma triagem neonatal, 66 de 11.957 crianças foram identificadas como portadoras de infecção congênita por CMV. Todas as crianças infectadas sintomáticas e assintomáticas foram avaliadas ao nascimento por meio de exame físico detalhado, avaliação auditiva, fundoscopia ocular, neurossonografia transfontanelar (NTF) realizada por neurorradiologista infantil e quando alterada, foram submetidas à ressonância magnética de encéfalo (RMc). Análise de regressão logística foi realizada para verificar a associação de potenciais fatores de risco para ocorrência de surdez neurossensorial relacionada ao CMV e/ou presença de achados anormais em NTF e/ou RMc. A análise da Curva ROC foi utilizada para avaliar a associação da carga do DNA do CMV expresso em log10, com a presença de sintomas ao nascer, achados anormais em NTF e/ou RMc e ocorrência de surdez neurosensorial. Resultados: Em 66 crianças infectadas, os sinais clínicos sugestivos de infecção congênita ao nascer foram observados em 8/66 (12,12%; IC95%: 5,74-23,03), sendo considerados sintomáticos. As 58 crianças restantes foram considerados assintomáticos. A surdez neurossensorial foi observada em 8/66 (12,12%; IC95%: 5,74-23,03%) das crianças. Destas, 4/58 (6,9%; IC95%: 2,23- 17,54%) e 4/8 (50%; IC95%: 17,44-82,55%) crianças eram assintomáticas e sintomáticas, respectivamente. Achados de NTF e/ou RMc sugestivas de infecção congênita foram observadas em 7 de 8 crianças sintomáticas (87,5%; IC95%: 46,67-99,34%). Dentre as 58 assintomáticas, 53 (92,4%) completaram todas as avaliações e destas 29/52 apresentaram alterações sugestivas de infecção congênita em NTF e/ou RMc (55,7%; IC95%: 41,41 - 69,27%). O achado mais comum foi a vasculopatia lenticuloestriada acompanhado ou não de cistos subpendimários (13/29: 43.3%). Outros achados anormais foram os cistos subpendimários como achado isolado (11/29: 37.9%), calcificação única ou periventricular com ou sem áreas de gliose (4/29: 13,8%) e ventriculomegalia (4/29: 13,8%). Análise de regressão logística mostrou que apenas a presença de sintomas clínicos ao nascer foi preditivo para a ocorrência de surdez neurossensorial. Achados de neuroimagens (NTF e/ou RMc) foram observados em 3 de 4 crianças assintomáticas (75%) enquanto 26 (54,2%) de 48 crianças sem surdez tinham avaliações normais de NTF e/ou RMc (p=0,42; RR:2,38: IC95%: 0,26-21,39). Nenhum dos outros fatores de risco foram independentemente associados com surdez. A presença de plaquetopenia e/ou níveis altos de gama glutamil transferase (?GT) foi associado com a presença de achados anormais em NTF e/ou RMc uma análise univariada. O poder discriminatório pela determinação do ponto de corte do valor da carga do DNA do CMV foi avaliada pela área abaixo da curva ROC (AUC) e não houve associação entre a carga viral e a ocorrência de surdez e/ou achados anormais de NTF e/ou RMc. Conclusões: Embora a triagem neonatal da infecção congênita por CMV permita identificar a maioria das crianças infectadas que são clinicamente assintomáticas ao nascer, uma proporção significante destas crianças poderá ser beneficiada por uma avaliação do sistema nervoso central através de NTF, uma vez que, achados anormais são muito frequentes. Embora não tenha sido possível determinar fatores independentemente preditivos de ocorrência de surdez neurossensorial, os achados de NTF podem ser potenciais fatores preditivos de anormalidades tardias em crianças assintomáticas. / The objectives of this study were to verify the impact of congenital cytomegalovirus (CMV) infection at birth and to evaluate potential prognostic predictors of late abnormalities in a cohort of children with this infection. Methods: By means of a CMV neonatal screening, 66 of 11.957 infants were identified as congenitally infected. Infants with and without clinical abnormalities detectable at birth underwent physical examination, cranial ultrasound performed by a paediatric radiologist and/or cranial magnetic resonance imaging, ocular fundoscopy, and hearing evaluation using evoked otoacoustic emissions and auditory brainstem response. Logistic regression analysis was carried out to verify the association between the risk factors for occurrence of hearing loss related to CMV and/ or abnormal cranial ultrasound findings. ROC curve was plotted using the log10 value of CMV DNA load to evaluate the association between viral load and clinical symptoms at birth, abnormal cranial ultrasound findings and hearing loss. Results: Of all 66 infected children, the clinical signs suggestive of congenital infection at birth were observed in 8/66 (12.12%; IC95%: 5.74- 23.03) symptomatic infants. Sensorineural hearing loss was observed in 8/66 (12,12%; IC95%: 5.74-23.03%) children. Of these, 4/58 (6,9%; IC95%: 2.23-17.54%) and 4/8 (50%; IC95%: 17,44-82.55%) children were asymptomatic and symptomatic, respectively. Cranial ultrasound findings suggestive of congenital infection were observed in 7 of the 8 symptomatic children (87.5%; IC95%: 46.67-99.34%). Among the 58 asymptomatic infants, 53 underwent complete evaluation and 29/52 had abnormal cranial ultrasound results (55,7%; IC95%: 41.41 - 69,27%). The most prevalent findings was lenticulostriate vasculopathy with subependymal pseudocysts present in 13 of the /29 (43.3%) infants with cranial ultrasound. Other abnormal findings were isolated subependymal pseudocysts (11/29: 37.9%); single or periventricular calcifications and/or gliosis (4/29: 13.8%); and ventriculomegaly (4/29: 13,8%). Logistic regression analysis showed that only the presence of clinical findings predicted the occurrence of hearing loss. Cranial ultrasound findings were observed in 3 of asymptomatic infants (75.0%) while 26 (54.2%) of 48 infants with no hearing loss had abnormal imaging features (p=0,42; RR:2,38: IC95%: 0,26-21,39). None of the other factors risk were independely associated with development of hearing loss. The presence of thrombocytopenia and/or high level of gamma-glutamyltranspeptidase (?GT) was associated with cranial ultrasound findings on univariated analysis. No discrimination power was achieved using the area under the ROC curve to verify the association between CMV DNA load in the urine of the infected children and the developing of hearing loss, presence of cranial ultrasound findings and clinical signs at birth. Conclusions: Although a neonatal screening of cCMV will identify the majority of infected infants who are clinically asymptomatic, a significant proportion of them could benefit from a central nervous system image evaluation, since abnormal findings are frequent. Althoug it was not possible to determine risk factors that are independently associated to development of sensorineural hearing loss, cranial ultrasound findings could be a potential prognostic markers of adverse outcomes of congenital CMV in asymptomatic infants.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T05:17:03Z (GMT). No. of bitstreams: 1 Silva_AnaCarolinaGuardiada_M.pdf: 824536 bytes, checksum: dc87f957a639ab7206e1a727070c8b0e (MD5) Previous issue date: 2008 / Resumo: O Citomegalovírus (CMV) e o Herpesvírus humano 6 são vírus universais pertencentes à subfamília dos betaherpesvírus. Esses vírus permanecem latentes, podendo ser reativados por um período de imunossupressão, como acontece em pacientes submetidos a transplantes de fígado. O CMV é um importante patógeno oportunista, que influencia negativamente esses pacientes. O HHV-6 é um vírus linfotrópico, alem de infectar outras células como monócitos e células endoteliais, usando o receptor celular CD-46. A reativação do HHV-6 tem sido associada com a do CMV e rejeição do enxerto. Nos transplantados de fígado a reativação do HHV-6 tem aparecido junto com a infecção do CMV. O CMV tem sido associado como importante causa de mortalidade e morbidade nos transplantados de órgãos sólidos. Esses vírus podem causar disfunção no enxerto, supressão da medula e pré-disposição para a doença por CMV. Este estudo detectou o DNA do CMV e HHV-6 em 41 transplantados de fígado usando a Nested- PCR. Este método foi escolhido por ser mais sensível e possibilitar a genotipagem. Também analisamos a co-infecção e o impacto clínico desses vírus nos transplantados hepáticos. 145 biópsias foram analisadas (41 - biópsias de doador e 104 - biópsias pós-transplante). 23 (15.8%) das 145 foram positivas para o CMV e 53 (36.5%) positivas para o HHV-6. 19 (13%) tiveram a co-infecção na mesma amostra. 21 pacientes tiveram rejeição ao enxerto e desses 16 tiveram infecção viral. A presença desses vírus observado, nas biópsias hepáticas dos doadores e no pós- transplante, sugere que as infecções no pré-transplante são importante via de transmissão desses vírus aos receptores, causando episódios de rejeição. / Abstract: Cytomegalovirus (CMV), Human Herpesvirus-6 (HHV-6), belong to the ß-herpesvirus subfamily. These viruses can be reactivated from latency during immunosuppression. period especially after liver transplantation, CMV has been the most important opportunistc infection that negatively influences the outcome of patients. HHV-6 is a lymphotropic virus, but it may also infect other cells, such as monocytes and epithelial cells, using the CD46-molecule as a cellular receptor. HHV-6 reactivations are often seen associated with CMV infection and allograft rejection. In liver transplant patients, HHV-6 reativations are frequently found together with CMV infection. CMV has been implicated as an important causes of morbidity and mortality among solid organ transplant patients. Both have been related to graft dysfunction, bone morrow suppression, and predisposition to CMV disease. In this study, CMV and HHV-6 DNA were detected in 41 liver transplant patients, using nested polymerase chain reaction (PCR). This method was chosen because increase the sensibility and with the products we can be classified into CMV genotypes. We also evaluate the co-infection and the clinical impact between those virus in liver transplant patients. 145 biopsies were tested, (41 - liver donor biopsies and 104 - liver post- transplant), Twenty three (15,8%) of 145 liver biopsies were CMV- PCR positive and fifty three (36,5%) of 145 were positive HHV-6- PCR. Nineteen (13%) of 145 biopsies were both CMV and HHV-6 positive. 21 patients had allograft rejection and 16 had infection for this virus. With the presence of the viruses observed in the samples of the donor and post-transplant, suggests that pre-transplant HHV-6 and CMV infection may be a risk factor post-transplant. They had associated with allograft refection. / Mestrado / Mestre em Farmacologia

Orientadores: Raquel Silveira Bello Stucchi, Sandra Cecília Botelho Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T21:51:59Z (GMT). No. of bitstreams: 1 Milan_Arlete_D.pdf: 3275520 bytes, checksum: 25bbae8b034967286ca090efbc210090 (MD5) Previous issue date: 2013 / Resumo: O transplante de fígado tornou-se a terapia mais eficaz para o tratamento dos pacientes com doença hepática terminal, sendo que o sucesso pode ser limitado por complicações infecciosas no primeiro ano pós-transplante. O citomegalovírus é o tipo de infecção viral mais comum e está relacionado com a morbidade e mortalidade. A infecção bacteriana é uma séria complicação em pacientes transplantados de fígado. O Herpesvírus humano 8 tem uma distribuição global heterogênea, poucos estudos existem em pacientes submetidos à transplante de fígado. Nosso objetivo foi correlacionar a infecção do citomegalovírus e do Herpesvírus Humano 8 com a ocorrência de infecções bacterianas nos pacientes submetidos à transplante de fígado. Foram incluídos pacientes monitorados por seis meses para a detecção da infecção do citomegalovírus e do Herpesvírus Humano 8. As amostras foram realizadas no momento da cirurgia e semanalmente até o segundo mês, quinzenalmente no terceiro mês e mensalmente do quarto ao sexto mês. A infecção por citomegalovírus foi definida por antigenemia positiva (> três células) ou dois testes positivos de Nested-Reação em Cadeia de Polimerase com intervalo de 30 dias associada a sintomas clínicos. A metodologia para o diagnóstico de infecção bacteriana foi por meio de cultura de urina e de sangue através de testes bioquímicos e automatizado BacT / ALERT® 3 D - VITEK® (bioMérieux, França). A investigação para o Hespesvírus Humano 8 foi através da Nested-Reação em Cadeia de Polimerase da região Open Reading Frame-26 e confirmado pela sorologia da imunoglobulina G - Enzyme Linked Immunosorbet Assay the Advanced Biotechnologies Incorporated (Maryland, EUA). Teste do qui-quadrado foi utilizado para as variáveis dicotômicas com diferenças significativas quando p < 0,05. Cinquenta pacientes foram acompanhados no período de fevereiro de 2008 a janeiro de 2010. Vinte e um (42%) pacientes tiveram infecção bacteriana. Dezesseis pacientes (32%) apresentaram infecção por citomegalovírus . Dos 16 pacientes, 13 (81%) mostraram infecção bacteriana concomitante. Trinta e quatro pacientes (68%) não tiveram infecção por citomegalovírus e deles, 8 (24%) tiveram infecções bacterianas. Não encontramos em nossa casuística positividade para o Herpesvírus Humano 8. As infecções bacterianas pós-transplante de fígado foram associadas a infecção ativa pelo citomegalovírus / Abstract: Liver transplantation has become the most effective therapy for the treatment of patients with end-stage liver disease but its success can be limited by infectious complications during the first year post-transplant. Cytomegalovirus is the most common viral infection and it is associated with morbidity and mortality. Bacterial infection is a serious complication in liver transplant patients. Human Herpesvirus 8 has a global distribution heterogeneous; there are few studies in patients undergoing liver transplantation. Our objective was to correlate infection of cytomegalovirus and with the occurrence of bacterial infections in patients undergoing liver transplantation. This study included patients monitored for six months for detection of cytomegalovirus and of Human Herpesvirus 8 infection. The sample collections were performed at the time of surgery, weekly until the second month, twice a month in the third month, and monthly from the fourth to the sixth month. Cytomegalovirus infection was defined by positive antigenemia (> three cells) or two positive Nested-Polymerase Chain Reaction tests associated with clinical symptoms. The methodology for the diagnosis of bacterial infection was through biochemical tests and the automated BacT / ALERT® 3 D - VITEK® (bioMérieux, França) for identification and antibiogram using samples of urine and blood cultures. Research for Human Herpesvirus 8 was by Nested-Polymerase Chain Reaction region Open Reading Frame-26 and confirmed by serology IgG - Enzyme Linked Immunosorbet Assay the Advanced Biotechnologies Incorporated (Maryland, EUA). Chi-square test was used for dicotomic variables with significant differences when p < 0,05. Fifty patients were followed up from february 2008 to january 2010. Twenty-one (42%) patients had bacterial infection. Sixteen patients (32%) had citomegalovírus infection. Of the 16 patients, 13 (81%) showed concomitant bacterial infection. Thirty-four patients (68%) had no citomegalovírus infection, and of them, 8 (24%) had bacterial infections. Not found in our sample positive for Human Herpesvirus 8. Bacterial infections after liver transplantation were associated with cytomegalovirus active infection / Doutorado / Clinica Medica / Doutora em Clínica Médica

L'infection congénitale par le cytomégalovirus humain (HCMV) est la première cause de séquelles acquises du système nerveux central (CNS). Elle est responsable de surdités neurosensorielles, de paralysies cérébrales ou d'anomalies neuro-développementales graves (0,1% des naissances) telles que des microcéphalies ou des anomalies de gyration. Pour étudier les effets de l'infection par le HCMV sur le développement cérébral, nous utilisons des cellules souches neurales (NSC) humaines dérivées de cellules souches embryonnaires (ES), ainsi que des coupes histologiques de cerveaux fœtaux infectés. Notre travail a porté sur l'analyse des conséquences de l'infection sur un facteur de transcription essentiel lors du développement cérébral, le Peroxisome Proliferator-Activated Receptor gamma (PPARg). Nous avons démontré que l'infection par le HCMV diminuait la neuronogénèse, en association avec une augmentation des niveaux d'expression et d'activité de PPARg. En accord avec ces résultats, nous avons montré que le niveau d'expression de l'acide 9-hydroxyoctadecadienoique (9-HODE), un agoniste connu de PPARg était augmenté dans les NSC infectées. En outre, l'ajout de 9-HODE dans les NSC reproduit l'effet de l'infection sur PPARg conduisant à une augmentation du nombre de cellules positives pour l'antigène viral IE parmi les NSC infectées. De plus, nous avons démontré que : (1) l'activation pharmacologique ou l'expression ectopique de PPARg suffisent pour perturber la neuronogénèse de NSC non infectées ; (2) le traitement de NSC non infectées par le 9-HODE diminue la différenciation des NSC ; (3) le traitement de NSC infectées par du T0070907, un inhibiteur de PPARg restaure un taux normal de différenciation. Le rôle crucial de PPARg dans les pathologies fœtales liées à l'infection a été souligné par la mise en évidence de sa translocation nucléaire au sein des zones germinatives de cerveaux fœtaux infectés congénitalement par le HCMV (N=20), mais pas dans les cas contrôles. Nous avons également identifié un des gènes cibles de PPARg dans le cerveau infecté: LIS1, le gène de la lissencéphalie classique, dont l'expression est également augmentée dans les NSC infectées, de façon dépendante de l'activité de PPARg. Nous avons mis en évidence que l'expression de LIS1 était augmentée de façon massive dans les cerveaux fœtaux infectés congénitalement par le HCMV (N=6) par rapport aux cas contrôles (N=3). Ceci pourrait jouer un rôle central dans la physiopathologie, car il est connu que toute perturbation de l'expression de LIS1 conduit à des anomalies importantes de la migration neurale et au développement d'un phénotype dit "lissencephaly-like". L'ensemble de nos données révèle le rôle clé de PPARg dans la neuronogénèse et la pathophysiologie de l'infection congénitale par le HCMV. Elles ouvrent la voie à une meilleure compréhension des mécanismes régissant les phénotypes pathologiques, notamment concernant le rôle de LIS1 dans les anomalies de la migration neurale. / Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1 % of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses. We investigated the outcome of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARg, a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARg levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARg agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARg activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARg was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV infected NSCs with the PPARg inhibitor T0070907 restored a normal rate of differentiation. The role of PPARg in the disease phenotype was strongly supported by the immunodetection of nuclear PPARg in brain germinative zones of congenitally infected fetuses (N=20), but not in control samples. We also identified LIS1 as one of the target genes for PPAR??in the infected brain. Levels of LIS1, the gene of classical lissencephaly, were strongly increased in infected NSC, presumably resulting from increased PPAR? activity. The relevance of this finding was further supported by our demonstration of a massive increase in the immunodetection in LIS1 fetal brains congenitally infected with HCMV (N = 6), relative to control cases (N = 3). Indeed, it is well known that overexpression of LIS1 is responsible for significant abnormalities of neural migration and development of a lissencephaly-like phenotype. Altogether, our findings reveal a key role for PPARg in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARg gene targets in the infected brain.

Sterols play many important roles in physiology, including maintaining cell membrane integrity, and producing vitamin D and steroid hormones. Recent studies implicate sterol metabolism in the host innate immune response. Previous work, based on transcriptional profiling studies of mouse cytomegalovirus (MCMV) infection of primary bone-marrow-derived macrophages (BMDM, MΦ), uncovered a previously uncharacterized role of interferon in regulating the cholesterol pathway. Notably, Toll-like receptor (TLR) induced interferon modulates the suppression of SREBP2 (Sterol Regulatory Element-Binding Protein 2) activation, the master transcription factor for sterol biosynthesis. This finding resulted in the downregulation of the sterol biosynthesis pathway. However, how interferon is molecularly linked to sterol metabolism, and what part of the pathway mediates the antiviral effect remains unknown. The central hypothesis of the thesis is that the antiviral effect of interferon is in part mediated by secondary sterol metabolites and the dependency of viral replication on the host mevalonate branch of the sterol biosynthesis pathway. To test this hypothesis, my studies have examined the components of the host sterol pathway and their respective roles in influencing viral replication. Paradigmatically, I used MCMV and BMDM to explore the host- metabolic-virus interactions. Specifically, my findings address the question of how MCMV replication depends on the sterol biosynthesis pathway, and how the pathway is modulated by interferon as an antiviral response. In Chapter 2, the importance of the sterol biosynthesis pathway for viral replication was investigated using a combination of gene silencing and pharmacological inhibitors. These studies demonstrated that resistance to viral infection through suppressing the cholesterol pathway is not due to a requirement of the virus for cholesterol itself, but instead involves the mevalonate-isoprenoid arm of the pathway. This branch of the pathway chemically links lipids to specific host proteins (protein prenylation). These results suggest a new role for the mevalonate arm during viral infection. In Chapter 3, I examined what part of the sterol pathway mediates the antiviral effects. Oxysterols are natural modulators of sterol biosynthesis, and are produced by the oxidation of cholesterol by the enzyme cholesterol hydroxylase. Oxysterol suppression of SREBP2 activation leads to transcriptional repression of the sterol biosynthesis pathway. Additionally, oxysterols also modulate cholesterol homeostasis through cholesterol efflux. My studies led to identifing cholesterol-25-hydroxylase (Ch25h) as an interferon-stimulated gene (ISG). CH25H oxidizes cholesterol to produce a soluble oxysterol metabolite, 25-hydroxycholesterol (25-HC). Treatment of cells with 25-HC resulted in antiviral effects against MCMV and MHV-68. 25-HC was found to have no effects on MCMV entry into the host cell, but rather mediated inhibition of viral gene transcription. In addition, 25-HC-specific antiviral effect partially involved the suppression of the isoprenoid pathway, rather than cholesterol efflux. This work uncovered a physiological role for 25-HC as a sterol-lipid effector of an innate immune pathway. The antiviral activity of 25-HC in a lipid replete condition was found to occur at a concentration higher than the concentration required to inhibit SREBP2 activation. This implies that the antiviral effects of 25-HC is independent of SREBP2 in sterol replete conditions. Conversely, the antiviral action of 25-HC was signifi enhanced in cells under sterol-depleted conditions, suggesting that the antiviral effect of 25- HC is likely mediated through multiple processes involving SREBP2 dependent and independent mechanisms. These sterol dependent and independent mechanisms are examined in Chapter 4, using pathway expression profiling and pharmacological synergy studies. These studies showed that 25-HC suppression of the isoprenoid synthetic pathway is crucial in controlling infection, but also highlighted that other 25-HC dependent antiviral mechanisms are likely to exist. The inhibition of the mevalonate-isoprenoid arm by statins and 25-HC clearly demonstrated that MCMV replication dependents on protein prenylation. Chapter 5 investigation showed that either chemical inhibition of geranylgeranylation of host proteins or limiting mevalonate production led to restriction of MCMV replication. Importantly, through a series of systematic loss of function siRNA screenings demonstrated that specific host RabGTPases mediating vesicular transport pathways play vital roles in the replication and the assembly of the virus. This finding provides new mechanistic insights in to the dependency of cytomegalovirus replication on the host cell trafficking pathways and lays the groundwork for further definition of this important aspect of host-viral interactions. In summary, the overall findings of this research support the original hypothesis, by highlighting the importance of the host mevalonate-isoprenoid pathway, and provide further definition of the mechanisms and components linking sterol metabolism with interferon mediated antiviral effect.

Human cytomegalovirus (HCMV) infection is the major cause of illness and death in immunocompromised patients. HCMV is the most common cause of congenital viral infection in humans. A polymerase chain reaction (PCR) method was developed for the rapid detection of CMV in urine. Several parameters of the PCR procedure were optimized to reduce time and improve sensitivity. By eliminating the extraction of DNA from clinical specimens, reducing the number of amplification cycles, utilization of the "hot start" PCR procedure and direct detection of PCR product by ethidium bromide fluorescence staining, a procedure was developed which could be performed in less than 3 hours. Comparison studies using cell culture and direct detection of CMV by PCR on urine specimens were performed. Sensitivity was further examined to determine if inhibitors of the PCR reaction were present in urine.

L’infection à cytomégalovirus (CMV) cause des pathologies graves en absence d’immunité. Les lymphocytes T (LT) γδ participent à la réponse anti-CMV puisqu’ils s’amplifient dans le sang de patients transplantés rénaux concomitamment à une diminution de la charge virale. D’autre part, l’amplification T γδ est associée à un risque moindre de cancers cutanés chez ces patients. Nous avons choisi d’utiliser le modèle murin de l’infection à CMV afin d’étudier la capacité des LTγδ à protéger les souris contre l’infection et le cancer.Nous avons montré qu’en absence de LTαβ dans des souris TCRα-/- (αβ-γδ+), différentes sous populations de LTγδ s’amplifient dans les organes cibles du CMV. Le contrôle de la charge virale observé in situ suite à leur amplification protège les souris TCRα-/- des dommages hépatiques/pulmonaires et de la mort, alors que les souris CD3ε-/- (αβ-γδ-) succombent à l’infection. Enfin, l’effet protecteur des LTγδ est également observé en absence de NK, de LTαβ et de LB, montrant l’importance que peuvent avoir ces cellules dans un contexte d’immunodéficience touchant les autres acteurs immunitaires.Nous avons montré la capacité du CMV à inhiber la croissance de tumeurs coliques (MC38) et de mélanomes (B16F10) implantés en sous-cutané dans des souris immunodéficientes, révélant un rôle anti-tumoral du CMV indépendant de l’immunité et des LTγδ. La permissivité au CMV de ces lignées tumorales suggère un effet direct du virus, par apoptose (B16F10) ou par un mécanisme encore indéterminé (MC38). Enfin, une inhibition comparable est observée pour une lignée carcinomateuse humaine, présupposant un effet indirect du virus sur le microenvironnement tumoral. / Cytomegalovirus causes serious pathologies in immune-compromised hosts. γδ T cells increase in the peripheral blood of renal transplant recipients concomitantly to a decrease of CMV viral antigenemia, indicating that they participate to the immune response against CMV. Moreover, γδ T cell amplification is associated with a reduced risk of skin cancer in these patients. We chose to use the mouse model of CMV infection to study the capacity of γδ T cells to protect mice against CMVinfection and cancer.We showed that in the absence of αβ T cells in TCRα-/- mice (αβ-γδ+), different γδ T cell subsets are increased in CMV target organs. A concomitant decrease of viral load was observed in TCRα-/- mice which survived CMV infection, in contrast to CD3ε-/- mice which died and displayed damage to the lungs and liver. γδ T cell antiviral protective effect was also observed in the absence of NK, αβ T and B cells, showing the crucial role that these cells could play in immunodeficient contexts where other immune players are compromised.We showed the ability of CMV to inhibit the growth of subcutaneous colonic tumors (MC38) and melanomas (B16F10) in immunodeficient mice, thus revealing an anti-tumor role of CMV independently of immunity and γδ T cells. CMV was able to infect these tumor cell lines in accordance with a direct anti-tumor effect of the virus, through apoptosis (B16F10) or by means of a still unresolved mechanism. Finally, CMV also inhibits the growth of human colonic tumors, leading to the hypothesis that a viral-mediated indirect anti-tumor effect could also operate.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T11:25:10Z (GMT). No. of bitstreams: 1 Tavares_CarlaAparecida_M.pdf: 2590689 bytes, checksum: f1ca87aa51c2db9aa3fe97a2330a029c (MD5) Previous issue date: 2006 / Resumo: A reativação da infecção pelo Citomegalovírus humano (HCMV) e pelo Poliomavírus (BKV) no uroendotélio, vem sendo relacionada a complicações como a Cistite Hemorrágica (CH) em receptores de transplante de células progenitoras hematopoéticas (TCPH), o que representa um fator de risco para estes pacientes. Este estudo prospectivo de 41 receptores de TCPH teve como objetivos, detectar a infecção ativa pelo HCMV no sangue e pelo BKV em amostras de sangue e urina após TCPH, usando as técnicas de antigenemia (AGM), citologia urinária e Reação em Cadeia da Polimerase tipo Nested (¿Nested-PCR") para se verificar a participação do HCMV e BKV como possíveis fatores de risco para CH e o impacto clínico destas viroses nestes pacientes. O monitoramento dos receptores de TCPH foi baseado em coletas de sangue para realização de AGM e "Nested-PCR" para detecção do HCMV e Citologia urinária para verificar células Decoy como um marcador de replicação viral, e também "Nested-PCR" de urina e sangue para diagnóstico de replicação do poliomavírus de todos os receptores (independente de Citologia urinária positiva ou negativa). Nos receptores estudados, a freqüência de AGM positiva para HCMV foi de 63,4%, com "Nested-PCR" positivo de 78%. A doença pelo HCMV ocorreu em 8/41 (21,1%) dos receptores, dos quais 1/8 (12,5%) veio a óbito. Dos 41 receptores 14 (34,1 %), tiveram BKV detectado na urina e 13 (31,7%) no sangue, todos os receptores que apresentaram infecção ativa pelo BKV tiveram também pelo HCMV, sendo que 16 (39%) evoluíram com CH. Estes resultados sugerem que o BKV juntamente com o HCMV em receptores de TCPH pode estar envolvidos na patogênese da CH, sendo assim, medidas preventivas baseadas na inibição da replicação viral de ambos os vírus poderão minimizar o impacto clínico da cistite hemorrágica nesse grupo de transplantados / Abstract: The reactivation of inrection by Human Cytomegalovirus (HCMV) and by Polyomavirus (BKV) in the uroendothelium, has been related with haemorrhagic cystitis (HC) in haematopoietic stem cell transplantation (HSCT) receptors, which represents a factor of risk for those patients. This prospective study of 41 HSCT receptors has the purpose of detect the active HCMV infection in the blood and BKV in blood and urine samples after HSCT, using antigenemia assay (AGM), urine cytology and Nested polymerase chain reaction ("Nested-PCR"), to verify the chance of participation of HCMV and BKV as factor of risk for HC and the clinic impact of these virus on those patients. The HSCT receptor' s monitoring was based in blood collection for AGM and "Nested-PCR" to detect HCMV and urine cytology to verify decoy celIs as a marker of BK virus replication, and "Nested-PCR" on urine and blood in alI receptors (independently of positive or negative cytology) as welL In the HSCT receptors, the frequency of positive AGM for HCMV was 63,4%, with a positive "Nested-PCR" of 78%. The HCMV disease occurred in 8/41 (21,1%) of the receptors, and 1/8 (12,5%), dead. Among the 41 receptors, 14 (34,1%) had BKV detected in the urine and 13 (31,7%) in the blood. All the receptors that showed active infection by BKV, had active infection by HCMV, as well, and 16 (39%) developed HC. These results suggest that BKV and HCMV, together, in HSCT receptors might be involved in HC pathogenesis. Being thus , prevents measurements based on viral replication inhibition for both vírus could minimize the clinic impact of hemorrhagic cystitis on those transplanted grou / Mestrado / Mestre em Farmacologia

Orientador: Sandra Cecilia Botelho Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T10:34:08Z (GMT). No. of bitstreams: 1 Costa_ClaudiaRaquelCantarelli.pdf: 4518268 bytes, checksum: fb54bd9384d947b72d5d29ed906ae70f (MD5) Previous issue date: 2009 / Resumo: O cytomegalovirus humano (HCMV) e o herpesvirus humano 6 (HHV-6) são ß-herpesvirus com homologia superior a 67% e alta soroprevalência na população adulta. A infecção primaria por estes herpesvirus ocorre comumente na infância e é normalmente subclinica, ou pode causar mononucleose (HCMV) ou exantema súbito (HHV-6) sendo resolvidos na maioria dos casos sem complicações. Após a infecção primária os vírus permanecem no hospedeiro por toda vida podendo ser reativado de seu estado de latência em indivíduos adultos imunocomprometidos como os receptores de células tronco hematopoiéticas (TCTH). A reativação ou reinfecção por estes vírus causam serias complicações em pacientes submetidos ao transplante de células tronco hematopoiéticas como pneumonia intersticial, febre, gastroenterite, mielossupressão, encefalite e doença do enxerto contra o hospedeiro (GVHD). A reativação do HHV-6 após o transplante é associada com o desenvolvimento de infecções oportunistas, doença causada pelo citomegalovírus humano e possíveis episódios de rejeição aguda. Com efetivos tratamentos antivirais disponíveis, um monitoramento adequado destes vírus distinguindo entre latência e reativação é critico para estes pacientes. Monitoramos 30 pacientes submetidos à TCTH quanto a infecção ativa por HCMV e HHV-6 pelas técnicas de nested-PCR em soro e células, PCR- em tempo real em soro e células e transcrição reversa acoplada a nestedPCR (RT-nPCR). 29 pacientes (96,66%) apresentaram infecção ativa por HCMV sendo 21 pacientes (70%) pela nested-PCR em células, 17 pacientes(56,66%) pela neste-PCR em soro ,23 pacientes(76,67%) pela PCR em tempo real em células,19 pacientes (63,33%) pela PCR em tempo real em soro e 15 pacientes (53,3%) pela RT-nPCR. 25pacientes (83,33%) apresentaram infecção ativa por HHV-6, sendo 14 pacientes (46,7%) pela nested-PCR em células, 2 pacientes(6,6%) pela PCR em tempo real em células,23 pacientes (76,67%) %) pela PCR em tempo real em soro e 9 pacientes (30%) pela RT-nPCR. Todos os pacientes que apresentaram infecção ativa por HCMV apresentaram também presença do HHV-6, e 25 pacientes (83,33%) apresentaram co-infecção HCMV/HHV-6, sendo a infecção por HHV-6 precoce em relação ao HCMV. O presente estudo encontrou também associação entre infecção ativa por HCMV e doença do enxerto contra o hospedeiro. / Abstract: Human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) are ß-herpesvirinae extremely closely related with a homology > 67% with a high seroprevalence in the adult population. Primary infection commonly appears in early childhood and is usually subclinical, or may cause mononucleosis (HCMV) or febrile illness, including exanthema subitum (HHV-6), solving, in the majority of cases, without complications. After primary infection, the viruses persist in the infected individual through life and can be reactivated from their state of latency in immunocompromised hosts. Reactivation or reinfection causes severe clinical diseases in patients who underwent hematopoietic stem cell transplantation, like interstitial pneumonia, fever, gastroenteritis, myelossupression, encephalitis and graft-versus-host-disease (GVHD). A potential increase in virulence of HHV-6 in the course of a simultaneous CMV reactivation, leading to a great risk of CMV-associated disease. In this present study, 30 patients who received HSCT were monitoring for active HCMV and HHV-6 infection by Nested PCR in serum and peripheral blood leukocytes (PBL) samples, real time PCR in serum and PBL and RT-nPCR. In 29 patients (96,66%) active HCMV infection was detected: 21 patients (70%) by PBL nested-PCR, 17 patients (56,66%) by serum neste-PCR, 23 patients(76,67%) by PBL real-time-PCR,19 patients (63,33%) by serum real-time-PCR and 15 patients (53,3%) by RT-nPCR. In 25 patients (83,33%) active HHV-6 infection was detected : 14 patients (46,7%) by PBL nested-PCR, 2 patients(6,6%) by PBL real time -PCR,23 patients (76,67%) by serum real time-PCR and 9 patients (30%) by RT-nPCR. In all patients who had active HCMV infection, HHV-6 DNA was detected. 25 patients (83,33) had HCMV/HHV-6 co-infection, and the active HHV-6 infection was detected earlier in the majority of the cases. Our results showed a correlation between GVHD and active HCMV infection and detection of active HCMV infection by serum nested-PCR and PBL and serum real time-PCR. / Universidade Estadual de Campi / Ciencias Basicas / Doutor em Clínica Médica