Se reporta dos casos de pancreatitis secundaria a la infección por citomegalovirus confi rmado por reacción en cadena de la polimerasa en tiempo real (PCR-RT) en pacientes portadores del virus de la inmunodefi - ciencia humana (VIH). Se descartaron otras causas mediante exámenes auxiliares. Ambos pacientes fueron tratados con ganciclovir y se obtuvo una mejoría tanto clínica como en los exámenes auxiliares. Esta patología no debe pasar desapercibida en pacientes VIH positivos a pesar de no presentar la característica clínica de pancreatitis aguda / We report two cases of pancreatitis secondary to cytomegalovirus infections which were tested by reverse transcription polymerase chain reaction (RT-PCR) in patients with human immunodefi ciency virus (HIV). Other causes were ruled out by laboratory fi ndings. Both patients were treated with ganciclovir and improved clinically and as indicated by laboratory fi ndings. This condition should not be ignored in HIV-positive patients in spite of the absence of the clinical characteristics of acute pancreatitis.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality amongst immuno-compromised individuals and is the leading cause of congenital diseases amongst newborn infants. Mouse CMV (MCMV) infection of inbred mice has been extensively used as a model for HCMV pathogenesis and host-virus interaction. Macrophages are a key target cell type in the pathogenesis of human and mouse CMV infections. Macrophages are semi-permissive to CMV infection, however, the nature of this restrictive mechanism of infection is open for investigation. In this thesis, I hypothesized that macrophage permissivity is determined by the dynamic interplay of the innate response during the immediate-early (IE) period of infection. To test this hypothesis, I first developed and validated a flow cytometry based assay. In MCMV infected macrophages, I found heterogeneous expression from the major IE promoter (MIEP) leading to the development of a refractory subpopulation for IE expression. I further developed a computational modelling approach to help elucidate the dynamics of infection during this period. Modelling work revealed that the occurrence of refractory subpopulation could be caused by either 1) pre-existence of heterogeneous permissivity of macrophages prior to infection or 2) through an emergent process. Experimental testing of the models shows that the heterogeneous IE expression of homogeneously infected macrophages is caused by an emergent process. MCMV infection using type I interferon receptor and signal transducers and activator of transcription 1 (Stat1) knockout macrophages reveals that the emergence of refractory subpopulation is predominantly mediated by type I interferon through Stat1. Comparative molecular analysis between progressively infected and refractory subpopulations reveals that MCMV MIEP activation in the refractory subpopulation is stochastically inhibited by high expression of type I interferon induced antiviral components.

Introduction : l’infection par le cytomégalovirus (CMV) humain est la plus fréquente des infections après greffe d'organe. Des effets indirects à long terme sont fortement suspectés mais restent encore largement incompris. Notre travail de thèse s’est intéressé à mieux comprendre les conséquences de l’infection du donneur par le CMV sur la réponse immunitaire du receveur et sur le devenir de son greffon. Résultat : nous avons initialement rapporté que l'infection du donneur (D+) par le CMV est un facteur de risque indépendant de perte de fonction du greffon rénal particulièrement si le receveur est également séropositif avant la greffe (D+R+ comparé aux D+R-). Le risque est fortement majoré en cas de mésappariement complet en HLA de classe I entre le receveur et son donneur. Puis nous avons analysé le rôle du greffon infecté dans le développement de la réponse lymphocytaire anti-CMV. Nous avons rapporté pour la première fois que la superinfection CMV entraine une augmentation du nombre de LT CD8 répondeurs spécifiques du CMV à distance de la transplantation, à condition que le donneur et le receveur partagent des identités HLA-I. De plus nous avons montré chez le sujet D+R- que l'expansion des lymphocytes T CD8 anti CMV restreints par le HLA-A2 nécessite l'expression de ce HLA par le donneur. Ces résultats ensemble indiquent le rôle des cellules du donneur dans l’inflation des LT CD8 anti-CMV à distance de la greffe. Dans un troisième travail, nous avons montré qu’un polymorphisme du gène de Programmed Cell Death 1 (PD-1.3) influe sur la survie des greffons rénaux et pulmonaires D+, les patients porteurs de l’allèle variant A ayant un meilleur pronostic que les patients homozygotes GG. Nos données indiquent aussi que les patients homozygotes AA ont un plus grand nombre de lymphocytes anti-CMV producteurs d'IFN-ɣ, suggérant que ce polymorphisme pourrait être associé à une dysfonction de la réponse immunitaire spécifique anti-CMV. Conclusion : ensemble ces données suggèrent pour la première fois que la qualité de la réponse lymphocytaire cytotoxique anti-CMV pourrait être importante pour contrôler la réplication virale dans le greffon et les lésions induites par cette dernière. Ainsi nous proposons deux mécanismes à l’origine du développement des lésions liées à l'infection à CMV dans le rein: défaut de reconnaissance des cellules allogéniques infectées en cas de mésappariement complet en HLA de classe I et une dysfonction LT CD8 anti-CMV. / Background: cytomegalovirus (CMV) is the leading cause of viral infection after solid organ transplantation. Despite a large body of literature, the effects of chronic cytomegalovirus (CMV) infection on graft outcome remain controversial.Results: we first reported that donor CMV infection (D+) was an independent risk factor of kidney graft loss, especially in pretransplant infected recipients (R+). In addition, we observed that full HLA-I mismatching was an important determinant of this risk. In a second study, we focused on effect of donor CMV infection on anti-CMV specific immune response. We reported that CMV superinfection greatly increased the number of anti-CMV IFN-ɣ-producing T cells, provided that donor and recipient shared at least one HLA-I identity. Then in D+R- HLA-A2-expressing recipients, we compared the number of anti-CMVpp65 CD8+T cells restricted by HLA-A2 depending on whether the donor expressed or not HLA-A2. Patients who received non-HLA-A2 kidneys developed very few anti-CMVpp65 T-cells restricted by HLA-A2 as compared to those who received an HLA-A2-expressing kidney. This result indicated that presentation of CMV peptides by donor cells was crucial to stimulate the expansion of pp65-specific memory CD8 T cells. Finally, we established that a SNP in the Programmed Cell Death 1 gene (PD-1.3) influenced D+ kidney and lung transplants survival, while it was also associated with the level of anti-CMV specific T-cell response. Conclusion: taken together, these data suggest that anti-CMV specific immune response is pivotal to control infection within the graft and prevent subsequent organ damages. We propose two mechanisms to explain effect of donor CMV infection on graft outcome: (1) inability of anti-CMV CD8 T cells to recognize donor-infected cells in case of full HLA-I mismatching, (2) dysfunction of anti-CMV CD8 T cells after transplantation in some patients, highlighted by our genetic study.

Reproduction de : Thèse d'exercice : Médecine : Nancy 1 : 2003. / Thèse : 03NAN1119. Titre provenant de l'écran-titre.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 85-92. Also available online.

Thesis (M. Phil.)--University of Hong Kong, 1997. / Includes bibliographical references (leaves 154-177).

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

miRNAs are a species of small-regulatory RNA that post-transcriptionally regulate gene expression via the RNA induced silencing complex (RISC). They are encoded ubiquitously among animals and plants, and have recently been shown to be encoded by the majority of herpesviruses. It seems likely that herpesvirus encoded miRNAs have evolved as a tool for the manipulation of host-cellular and viral-gene expression during infection. Human cytomegalovirus (HCMV) is a clinically important herpesvirus that represents a significant cause of morbidity and mortality in the immune-compromised. HCMV encodes as many as 25 miRNAs during infection, but the function of the majority of these is not known. Identifying the targets of HCMV miRNAs will not only establish a basis for understanding the role of miRNAs within the context of HCMV infection, but also provide a means for discovering novel host-virus interactions. Using RISC immunoprecipitation and siRNA screening, host-cellular targets of viral miRNAs that play important roles in the biology of HCMV were identified. ATP6VOC, a key component of the vacuolar-ATPase, was shown to be a target of miR-US25-1 and subsequent siRNA knockdown of ATP6VOC resulted in the almost complete inhibition of infectious virion production. Despite this, ATP6VOC knock-down did not inhibit viral entry, DNA synthesis, or gene expression, highlighting a possible role for ATP6VOC in the assembly and egress of HCMV. A critical step in HCMV assembly and egress is the formation of the juxta-nuclear virion assembly compartment (VAC). The HCMV VAC is derived from host-cellular endocytic and secretory vacuoles, and is crucial for the efficient nuclear egress of nucleocapsids, cyotplasmic tegumentation, final envelopment, and the egress of mature virions. Using siRNA knock-down, immunofluorescence-microscopy, and western-blot analysis, a crucial role for ATP6VOC and v-ATPase function in the formation of the VAC was demonstrated. siRNA knock-down of ATP6VOC resulted in a failure in the reorganisation of trans-golgi and early-endosomal compartments during infection, resulting in a failure in VAC formation. These findings demonstrate a crucial role for ATP6VOC during infection, and in so doing identify a novel host factor that is required for HCMV assembly.

O citomegalovírus (CMV), está entre os principais agentes infecciosos que acometem pacientes transplantados renais. A infecção por CMV está relacionada ao status sorológico do doador e receptor, bem como o tipo e intensidade da imunossupressão utilizada. A infecção, e em especial a doença citomegálica, determinam aumento da morbi-mortalidade após o transplante. O espectro da doença varia desde formas assintomáticas até a doença sistêmica grave com comprometimento de vários órgãos. A doença por CMV é diagnosticada através da evidência laboratorial de infeção, associada a quadro clínico compatível. A técnica da antigenemia identifica a presença do antígeno viral p65 em leucócitos do sangue periférico através de reação de imunoperoxidase utilizando-se anticorpos monoclonais. O objetivo principal deste trabalho foi o de determinar a incidência de infecção por CMV em uma coorte de pacientes transplantados renais usando a antigenemia como ferramenta diagnóstica. Secundariamente buscou-se avaliar o impacto desta infecção nas sobrevidas dos pacientes e dos enxertos em 6 anos de acompanhamento. No período de inclusão no estudo, janeiro de 1994 a fevereiro de 1995, foram realizados 74 transplantes renais na Santa Casa de Porto Alegre–RS. As amostras de sangue para a detecção da antigenemia foram obtidas semanalmente durante a internação hospitalar e posteriormente, sempre que houvesse suspeita clínica de infecção por citomegalovírus. Das 229 amostras analisadas, 51 (22,3%) foram positivas, em 24 pacientes, dos quais 41,6% (10/24) evoluíram de forma assintomática, 33,3% (8/24) apresentaram sintomas leves, e 25% (6/24) desenvolveram sintomas compatíveis com doença citomegálica. Desta forma, coorte estudada, a incidência de infecção e doença por CMV foram de 33,3% e 8,4%, respectivamente. Não houve associação entre as doses de imunossupressores, o uso de anticorpos monoclonais e número de episódios de rejeição com o desenvolvimento de infecção e doença por CMV. Nos transplantes realizados com doadores vivos, a incidência de infecção por CMV nos receptores de rins de doadores com sorologia positiva foi 61,9%, e nos receptores de doadores com sorologia negativa foi 14,3% (p=0,005). Os transplantes com receptores com sorologia negativa transplantados com rins de doadores soropositivos apresentaram incidência significativamente maior de infecção (75%) e doença (75%) por CMV do que os receptores com sorologia positiva transplantados com órgãos de doadores com soronegativos, 13,3% e 0%, respectivamente (p<0,05). A sensibilidade da antigenemia em detectar os pacientes que desenvolveram doença citomegálica foi de 100% e a especificidade foi 72,7%, com valor preditivo positivo de 25% e valor preditivo negativo de 100%. No grupo de pacientes que apresentou doença por CMV, ao término do seguimento, ocorreu um número significativamente mais elevado de perdas do enxerto (85%) do que no grupo de pacientes em que a infecção foi assintomática (29%), acarretando impacto negativo nas curvas de sobrevida de enxertos e pacientes (LogRank; p<0,05). A antigenemia mostrou ser uma ferramenta diagnóstica importante no manejo dos pacientes transplantados renais, possibilitando o diagnóstico precoce da infecção e auxiliando na identificação dos pacientes infectados que estão sob maior risco de desenvolvimento da doença. / Cytomegalovirus (CMV) is a major infectious agent in renal allograft recipients. CMV infection is related to the serologic status of the donor and the recipient, as well as to the type and dosage of immunossupression that the recipient is submitted to. Infection and specially disease due to CMV have determined a rise in morbidity and mortality after transplantation. The spectrum of the disease ranges from completely assymptomatic all the way up to a severe systemic disease with multiple organ compromise. CMV disease is diagnosed through laboratory evidence of infection associated with a compatible clinical presentation. Antigenemia technique identifies the presence of the p65 viral antigen in peripheral blood leukocytes through a peroxidase reaction, using monoclonal antibodies. The objective of this work was to determine the incidence of infection caused by CMV in a cohort of renal transplanted patients, using the antigenemia as a diagnostic tool. Furthermore, the outcome of graft and patients in a 6 year follow-up period was evaluated. During the period of inclusion in the study (January 1994 to February 1995), 74 renal transplants were performed at Santa Casa de Misericórdia, Porto Alegre, RS. Blood samples for detection of antigenemia were obtained weekly during the patient’s in-hospital period, and whenever there was a clinical suspicion of CMV infection afterwards. Of the 229 analyzed samples, 51 (22.3%) were positive in 24 patients, of which 41.6% (10/24) presented no symptoms, 33.3% (8/24) had mild symptoms, and 25% (6/24) developed symptoms compatible with CMV disease. In this cohort, the incidence of infection and CMV disease was 33.3% and 8.4%, respectively. There was no association between the dosage of immunosuppressive agents, the use of monoclonal antibodies and the number of rejection episodes with the development of infection or disease caused by CMV. In living related transplants, the incidence of CMV infection in receptors of serum positive donors was 61.9%, and in the recipients of serum negative donors it was 14.3% (p=0.005). Serum negative recipients transplanted with organs of serum positive donors presented a significantly greater incidence of infection (75%) and disease (75%) than the serum negative recipients transplanted with organs of serum negative donors, 13,3 % and 0% respectively (p<0.05). The sensibility of antigenemia to detect the patients that developed CMV disease was 100%, and the specificity was 72.7%, with a positive predictive value of 25% and a negative predictive value of 100%. At the end of the follow-up, in the group of patients who presented CMV disease the incidence of graft loss was significantly higher than the one observed in the group of patients who remained assymptomatic (85% versus 29% respectively), having a negative impact in the survival curve for both grafts and patients (LogRank; p<0.05). Antigenemia proved to be an important tool in the assessment of renal transplant patients, permitting the early diagnosis of disease, and the identification of infected patients that are at risk for the development of disease.

O citomegalovírus (CMV), está entre os principais agentes infecciosos que acometem pacientes transplantados renais. A infecção por CMV está relacionada ao status sorológico do doador e receptor, bem como o tipo e intensidade da imunossupressão utilizada. A infecção, e em especial a doença citomegálica, determinam aumento da morbi-mortalidade após o transplante. O espectro da doença varia desde formas assintomáticas até a doença sistêmica grave com comprometimento de vários órgãos. A doença por CMV é diagnosticada através da evidência laboratorial de infeção, associada a quadro clínico compatível. A técnica da antigenemia identifica a presença do antígeno viral p65 em leucócitos do sangue periférico através de reação de imunoperoxidase utilizando-se anticorpos monoclonais. O objetivo principal deste trabalho foi o de determinar a incidência de infecção por CMV em uma coorte de pacientes transplantados renais usando a antigenemia como ferramenta diagnóstica. Secundariamente buscou-se avaliar o impacto desta infecção nas sobrevidas dos pacientes e dos enxertos em 6 anos de acompanhamento. No período de inclusão no estudo, janeiro de 1994 a fevereiro de 1995, foram realizados 74 transplantes renais na Santa Casa de Porto Alegre–RS. As amostras de sangue para a detecção da antigenemia foram obtidas semanalmente durante a internação hospitalar e posteriormente, sempre que houvesse suspeita clínica de infecção por citomegalovírus. Das 229 amostras analisadas, 51 (22,3%) foram positivas, em 24 pacientes, dos quais 41,6% (10/24) evoluíram de forma assintomática, 33,3% (8/24) apresentaram sintomas leves, e 25% (6/24) desenvolveram sintomas compatíveis com doença citomegálica. Desta forma, coorte estudada, a incidência de infecção e doença por CMV foram de 33,3% e 8,4%, respectivamente. Não houve associação entre as doses de imunossupressores, o uso de anticorpos monoclonais e número de episódios de rejeição com o desenvolvimento de infecção e doença por CMV. Nos transplantes realizados com doadores vivos, a incidência de infecção por CMV nos receptores de rins de doadores com sorologia positiva foi 61,9%, e nos receptores de doadores com sorologia negativa foi 14,3% (p=0,005). Os transplantes com receptores com sorologia negativa transplantados com rins de doadores soropositivos apresentaram incidência significativamente maior de infecção (75%) e doença (75%) por CMV do que os receptores com sorologia positiva transplantados com órgãos de doadores com soronegativos, 13,3% e 0%, respectivamente (p<0,05). A sensibilidade da antigenemia em detectar os pacientes que desenvolveram doença citomegálica foi de 100% e a especificidade foi 72,7%, com valor preditivo positivo de 25% e valor preditivo negativo de 100%. No grupo de pacientes que apresentou doença por CMV, ao término do seguimento, ocorreu um número significativamente mais elevado de perdas do enxerto (85%) do que no grupo de pacientes em que a infecção foi assintomática (29%), acarretando impacto negativo nas curvas de sobrevida de enxertos e pacientes (LogRank; p<0,05). A antigenemia mostrou ser uma ferramenta diagnóstica importante no manejo dos pacientes transplantados renais, possibilitando o diagnóstico precoce da infecção e auxiliando na identificação dos pacientes infectados que estão sob maior risco de desenvolvimento da doença. / Cytomegalovirus (CMV) is a major infectious agent in renal allograft recipients. CMV infection is related to the serologic status of the donor and the recipient, as well as to the type and dosage of immunossupression that the recipient is submitted to. Infection and specially disease due to CMV have determined a rise in morbidity and mortality after transplantation. The spectrum of the disease ranges from completely assymptomatic all the way up to a severe systemic disease with multiple organ compromise. CMV disease is diagnosed through laboratory evidence of infection associated with a compatible clinical presentation. Antigenemia technique identifies the presence of the p65 viral antigen in peripheral blood leukocytes through a peroxidase reaction, using monoclonal antibodies. The objective of this work was to determine the incidence of infection caused by CMV in a cohort of renal transplanted patients, using the antigenemia as a diagnostic tool. Furthermore, the outcome of graft and patients in a 6 year follow-up period was evaluated. During the period of inclusion in the study (January 1994 to February 1995), 74 renal transplants were performed at Santa Casa de Misericórdia, Porto Alegre, RS. Blood samples for detection of antigenemia were obtained weekly during the patient’s in-hospital period, and whenever there was a clinical suspicion of CMV infection afterwards. Of the 229 analyzed samples, 51 (22.3%) were positive in 24 patients, of which 41.6% (10/24) presented no symptoms, 33.3% (8/24) had mild symptoms, and 25% (6/24) developed symptoms compatible with CMV disease. In this cohort, the incidence of infection and CMV disease was 33.3% and 8.4%, respectively. There was no association between the dosage of immunosuppressive agents, the use of monoclonal antibodies and the number of rejection episodes with the development of infection or disease caused by CMV. In living related transplants, the incidence of CMV infection in receptors of serum positive donors was 61.9%, and in the recipients of serum negative donors it was 14.3% (p=0.005). Serum negative recipients transplanted with organs of serum positive donors presented a significantly greater incidence of infection (75%) and disease (75%) than the serum negative recipients transplanted with organs of serum negative donors, 13,3 % and 0% respectively (p<0.05). The sensibility of antigenemia to detect the patients that developed CMV disease was 100%, and the specificity was 72.7%, with a positive predictive value of 25% and a negative predictive value of 100%. At the end of the follow-up, in the group of patients who presented CMV disease the incidence of graft loss was significantly higher than the one observed in the group of patients who remained assymptomatic (85% versus 29% respectively), having a negative impact in the survival curve for both grafts and patients (LogRank; p<0.05). Antigenemia proved to be an important tool in the assessment of renal transplant patients, permitting the early diagnosis of disease, and the identification of infected patients that are at risk for the development of disease.