published_or_final_version / Microbiology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 158-171).

Human herpesvirus 6 (HHV-6) infections occur in > 95% of humans. Primary infection, which occurs in early childhood as an asymptomatic illness or manifested clinically as roseola infantum, leads to a state of subclinical viral persistence and latency. Reactivation of latent HHV-6 is common after liver transplantation, possibly induced and facilitated by allograft rejection and immunosuppressive therapy. Since the vast majority of humans harbor the virus in a latent state, HHV-6 infections after liver transplantation are believed to be mostly due to endogenous reactivation or superinfection (reactivation in the transplanted organ). In a minority of cases, however, primary HHV-6 infection may occur when an HHV-6 negative individual receives a liver allograft from an HHV-6 positive donor. The vast majority of documented HHV-6 infections after liver transplantation are asymptomatic. In a minority of cases, HHV-6 has been implicated as a cause of febrile illness with rash and myelosuppression, hepatitis, pneumonitis, and encephalitis after liver transplantation. In addition, HHV-6 has been associated with a variety of indirect effects such as allograft rejection, and increased predisposition and severity of other infections including cytomegalovirus (CMV), hepatitis C virus, and opportunistic fungi. Because of the uncommon nature of the clinical illnesses directly attributed to HHV-6, there is currently no recommended HHV-6-specific approach to prevention. However, ganciclovir and valganciclovir, which are primarily intended for the prevention of CMV disease, are also active against HHV-6 and may prevent its reactivation after transplantation. The treatment of established HHV-6 disease is usually with intravenous ganciclovir, cidofovir, or foscarnet, complemented by reduction in the degree of immunosuppression. This article reviews the current advances in the pathogenesis, clinical diagnosis, and therapeutic modalities against HHV6 in the setting of liver transplantation.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

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

Human herpesvirus 6A (HHV-6A) has yet to be definitively linked to a specific disease. This is due in part to the ubiquitous nature of the virus. Humanized Rag2-/-γc-/- (Rag-hu) mice were tested to determine if these were a suitable animal model to study the virus. Both cell-free and cell-associated virus was used for infection and both were found to be efficient at infecting the mice. Viral DNA was found in the plasma and cellular blood fractions, bone marrow, lymph node, and thymus, indicating successful infection and propagation of the virus in vivo. The CD3+CD4- population was depleted, while the CD3-CD4+ was increased in infected animals. The CD3-CD4+CD8- and CD3+CD4+CD8- populations were depleted and the CD3+CD4+CD8+ population increased when analysis was gated upon CD4+ cells. The CD3-CD4+CD8+ population expanded and the CD3-CD4+CD8- population was reduced when analysis was gated on the CD3- population. Additional flow cytometry analysis revealed increases in CD4+CD8+ double positive cells in the peripheral blood of cell-free infected mice, which could indicate improper T cell selection and a premature departure of these cells from the thymus, possibly contributing to autoimmunity. Previous research has shown that HIV and HHV-6A may have a synergistic effect on one another and that HHV-6A may act as a cofactor in the progression to AIDS. After determining the Rag-hu mouse model was suitable for studying HHV-6A infection, a coinfection of HHV-6A and HIV-1 was performed. Coinfected mice had fewer thymocytes when compared with the HIV-1 only, mock-infected, and to a lesser extent HHV-6A only groups which could indicate increased cell death in the coinfected group as well as possible disruptions in migration of cells, either causing cells to be sequestered in the bone marrow and unable to migrate to the thymus, or causing premature egress of the cells in the thymus due in part to premature upregulation of CCR7, both of which would explain the smaller cellular populations found in the coinfected mouse thymi. Additional studies were performed to determine if a preferential targeting existed between HHV-6A and HIV-1 as these viruses are found simultaneously coinfecting the same cell. Preferential targeting was not observed by cell-associated migration assay, but increased migration of HHV-6A-infected cells was observed in a CCL21 dependent manner. These studies have provided useful information about HHV-6A and its relevance to HIV/AIDS as well as a possible mechanism of the involvement of HHV-6A in multiple sclerosis (MS) and other autoimmune diseases.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T20:07:57Z (GMT). No. of bitstreams: 1 Thomasini_RonaldoLuis_M.pdf: 1552471 bytes, checksum: 69e020e777011a1dab1c773d2466694a (MD5) Previous issue date: 2007 / Resumo: Neste estudo, 29 pacientes adultos, transplantados de fígado foram monitorados (até 180 dias pós-transplante) para infecções ativas por HCMV, HHV-6 e HHV-7 usando Nested-PCR (N-PCR). O protocolo de imunossupressão foi baseado na combinação de esteróides e ciclosporina e profilaxia com ganciclovir não foi usada. Aciclovir foi usado como profilaxia para o Herpes simples. Um grupo controle foi estudado, N-PCR em DNA extraído de PBL e soro de 53 indivíduos sadios foram realizados. Destes indivíduos, 8 amostras de saliva foram coletadas para isolamento do HHV-7 e produção de controle positivo. DNA do HHV-7 foi detectado em 87,5% de saliva, em 28,3% de PBL e 0% de soro. Isolamento do vírus mostrou ser 100% correlato com N-PCR. Soro foi considerado a amostra de escolha para detectar infecção ativa por HHV-7. DNA do HHV-6, HHV-7 e HCMV foram, frequentemente, detectados em pacientes após transplante hepático (65,5%, 51,7% e 48,2%, respectivamente). A maioria dos pacientes com infecção ativa por mais que um vírus é infectado de forma seqüencial e não concorrente. Infecção ativa por HHV-7 ocorreu em muitos casos antes da infecção ativa por HCMV e/ou HHV-6 indicando que ele poderia ser um fator para reativação daqueles vírus. Nested-PCR para HCMV teve valor preditivo positivo de 50% e valor preditivo negativo de 100% para infecção sintomática. Neste estudo, o HCMV foi relacionado disfunção do enxerto, HHV-6 foi associado com pneumonite, encefalite, disfunção e rejeição do enxerto e predisposição para infecção oportunista. Em pacientes livres de HCMV e/ou HHV-6, nenhuma manifestação clínica nem achados laboratoriais significativos foram relacionados ao HHV-7. O tratamento antiviral com ganciclovir foi considerado satisfatório para o HCMV, mas para o HHV-6 e HHV-7, os dados não foram conclusivos. O aciclovir poderia ter demonstrado uma limitada atividade contra o HHV-7 / Abstract: In this study, 29 adult liver transplant patients were monitored (until day 180th posttransplantation) for HCMV, HHV-6 e HHV-7 active infections using Nested-PCR (N-PCR). Immunosuppression protocol was based on combinations of steroids and cyclosporine and no ganciclovir prophylaxis was used. Aciclovir was employed as Herpes simplex prophylaxis. A control group was studied; N-PCR in DNA extracted from PBL and serum of 53 healthy individuals was carried out. From these individuals, 8 samples of saliva were collected to design a positive control to N-PCR. HHV-7 DNA was detected in 87.5% of saliva, in 28.3% of PBL and 0% of serum. Virus isolation showed to be 100% correlated with N-PCR. Serum was considered to be the sample of choice to detect HHV-7 active infection. HHV-6, HHV-7 and HCMV DNA were frequently detected in patients after liver transplant (65.5%, 51.7% and 48.2%, respectively). The results show that few patients remain negative to active infection with betaherpesviruses after liver transplantation. Most of the patients with active infection with more than one virus were infected sequentially and not concurrently. HHV-7 active infection occurred in most of cases prior HCMV and HHV-6 active infections indicating that it could be a factor to reactivation of these viruses. HCMV Nested-PCR presented positive predictive value of 50% and negative predictive value of 100%. In this study, HCMV was related with graft dysfunction, HHV-6 was associated with pneumonitis, encephalitis, liver dysfunction, graft rejection and predisposition to opportunist infections. In HCMV and/or HHV-6 free patients, no clinical manifestation nor significant laboratory findings was related to HHV-7. Ganciclovir Antiviral treatment was considered satisfactory to HCMV symptomatic infections but to HHV-6 and HHV-7, no conclusive data was found. Aciclovir could be a limited activity against HHV-7 / Mestrado / Farmacologia / Mestre em Farmacologia

Orientador: Sandra Cecília Botelho Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T10:22:52Z (GMT). No. of bitstreams: 1 Thomasini_RonaldoLuis_D.pdf: 2991233 bytes, checksum: 570abfb75508069faffb192f2c6c5dcd (MD5) Previous issue date: 2011 / Resumo: Periodontite humana crônica é um processo inflamatório caracterizado por denso acúmulo de células imunes no tecido periodontal. A periodontite pode levar a perda do dente no paciente e a patogênese desta doença não é completamente conhecida. Este estudo testou a hipótese de que as células do infiltrado inflamatório podem abrigar betaherpesviruses e estes vírus estão ligados á subpopulação específicas de linfócitos. Fragmentos de tecido periodontal foram obtidas de pacientes afetados por periodontite e de indivíduos saudáveis. Imuno-histoquímica foi realizada para a contagem de células CD19+, céulas CD3+ e células CD4+ e CD8+. Reação em cadeia da polimerase e imuno-histoquímica foram realizados para detectar citomegalovírus, herpesvirus humanos 6 e 7 nas amostras. Como esperado, os tecidos coletados de indivíduos saudáveis não apresentaram nível significativo de infiltrado inflamatório e, portanto, foram excluídos dos procedimentos de imunofenotipagem. Os resultados mostraram que células CD19+ foram discretamente predomiantes sobre as células CD3+ no tecido periodontal afetado, mas estatisticamente não significativo. A subpopulação CD4+ de linfócitos estava significativamente em maior número que a subpopulação CD8+ de linfócitos (P=0,004), nas amostras. Citomegalovírus e herpesvírus humano 7 foram encontrados em locais afetados, mas não no tecido coletado de indivíduos saudáveis (P=0,04 e P=0,04, respectivamente). Herpesvirus humano 6 foi raramente detectado. Foi encontrado correlação entre citomegalovírus com menor relação de CD19+/CD3+ (P=0,003) e herpesvirus humano 7 com menor relação CD19+/CD3+ (P=0,003) e maior relação de CD4+/CD8+ ( P=0,002). Imuno-histoquímica foi negativa para citomegalovírus, herpesvirus humano 6 e herpesvirus humano 7 em todas as amostras. Este estudo mostra que citomegalovírus e herpesvírus humano 7 podem estar presentes em regiões afetadas pela periodontite, mas são incomuns em regiões saudáveis. Além disso, este estudo sugere que citomegalovírus pode ser relacionado ao infiltrado inflamatório, com predomínio de células CD3+ e, herpesvirus humano 7 pode estar relacionado ao infiltrado inflamatório com predomínio de células CD4+. Os dados sugerem que citomegalovírus e herpesvírus humano 7 podem estar presentes no infiltrado inflamatório, em estado de latência. No entanto, outros métodos deveriam ser realizados para confirmar esta hipótese / Abstract: Human chronic periodontitis is an inflammatory process characterized by dense accumulation of immune cells in the periodontal tissue. The periodontitis can lead to loss of teeth in the patient and the pathogenesis of this disease is not completely known. This study tested the hypothesis that cells within inflammatory infiltrate can harbor betaherpesviruses and these viruses are linked to specific lymphocyte subpopulation. Biopsies of periodontal tissue were taken from periodontitis affected and from healthy subjects. Immunohistochemistry was performed to count CD19+ cells, CD3+ cells, CD4+ and CD8+ cell subsets. Polymerase chain reaction and immunohistochemistry were performed to detected cytomegalovirus, human herpesvirus 6 and 7 in the samples. As expected, tissues collected from healthy subjects presented no significant level of inflammatory infiltration and therefore were excluded from immunostaining procedures. The results showed that CD19+ cells had discrete predominance over CD3+ cells in the periodontitis affected tissue but not statistically significant. CD4+ lymphocyte subset were significantly higher then CD8+ lymphocyte subset (P=0.004) in the samples. Cytomegalovirus and human herpesvirus 7 were found in affected sites but not in tissue collected from healthy subjects (P=0.04 and P=0.04, respectively). Human herpesvirus 6 was rarely detected. We found a correlation between cytomegalovirus with lower CD19+/CD3+ ratios (P=0.003) and human herpesvirus 7 with lower CD19+/CD3+ ratio (P=0.003) and higher CD4+/CD8+ ratios (P=0.002). Imunohistochemistry was negative for cytomegalovirus, human herpesvirus 6 and human herpesvirus 7 in the total of samples. This study shows that cytomegalovirus and human herpesvirus 7 can be present in periodontitis affected sites but are uncommon in healthy sites. Moreover, this study suggests that cytomegalovirus can be related to inflammatory infiltrate with predominance of CD3+ cells and, human herpesvirus 7 can be related to inflammatory infiltrate with predominance of CD4+. The data suggest that cytomegalovirus and human herpesvirus 7 could be present in the inflammatory infiltrate in latent state. However, different methods should be performed to confirm this hypothesis / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

<p dir="ltr">Viruses have long been investigated for their possible associations with a multitude of neurodegenerative diseases. Recently, herpesviruses, such as human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV), have been of growing interest as potential triggers or contributors in the pathogenesis of Alzheimer’s disease (AD) and other causes of dementia. The role that herpesviruses play in these disease processes remains unclear, with evidence for the infectious hypothesis of AD often ambiguous and strongly debated. Obtaining definitive evidence for the role of herpesviruses in AD and other dementia-related diseases remains challenging due to many factors, including the complexities of multifactorial diseases, the unique lifecycle of herpesviruses, and the lack of translational <i>in vivo</i> models of herpesvirus infection and AD. We investigated the potential association of herpesviruses with these diseases through the application of bioinformatic analyses and molecular techniques to human whole genome sequences (WGS) and the utilization of a nonhuman primate model, the common marmoset <i>(Callithrix jacchus).</i></p><p dir="ltr">We observed a higher prevalence of HHV-6 partial integration in synucleinopathies associated with dementia compared to other cohorts. We characterized Callitrichine herpesvirus 3 (CalHV-3) in the common marmoset as a translational model of gammaherpesvirus infection, highlighting similarities to human EBV infection. We described beta-amyloid (Ab) pathology in the brains of herpesvirus infected and uninfected marmosets. In marmosets infected with CalHV-3, we noted lower plasma Ab42:40 ratios and GFAP levels and a positive correlation between viral load and Ab42, Ab40, and total tau. These findings suggest that herpesviruses may play a role in dementia-related neurodegenerative diseases through multiple different mechanisms, including the integration of viral genomes and the alteration or augmentation of disease biomarkers.</p>

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