Cytomegalovirus (CMV) is a common viral pathogen that influences the outcome of liver transplantation. In addition to the direct effects of CMV syndrome and tissue-invasive diseases, CMV is associated with an increased predisposition to acute and chronic allograft rejection, accelerated hepatitis C recurrence, and other opportunistic infections, as well as, reduced overall patient and allograft survival. Risk factors for CMV disease are often interrelated, and include CMV D+/R-serostatus, acute rejection, female gender, age, use of high-close mycophenolate mofetil and prednisone, and the overall state of immunity. In addition to the role of CMV-specific CD4+ and CD8+ T lymphocytes, there are data to suggest that functionality of the innate immune system contributes to CMV disease pathogenesis. In one study, liver transplant recipients with a specific polymorphism in innate immune molecules known as Toll-like receptors were more likely to develop higher levels of CMV replication and clinical disease. Because of the direct and indirect adverse effects of CMV disease, its prevention, whether through antiviral prophylaxis or preemptive therapy, is an essential component in improving the outcome of liver transplantation. In the majority of transplant centers, antiviral prophylaxis is the preferred strategy over preemptive therapy for the prevention of CMV disease in CMV-seronegative recipients of liver allografts from CMV-seropositive donors (D+/R-). However, the major drawback of antiviral prophylaxis is the occurrence of delayed-onset primary CMV disease. In several prospective and retrospective studies, the incidence of delayed-onset primary CMV disease-ranged from 16% to 47% of CMV D+/R- liver transplant recipients. Current data suggests that delayed-onset CMV disease is associated with increased mortality after liver transplantation. Therefore, optimized strategies for prevention and novel drugs with unique modes of action are needed. Currently, a randomized controlled clinical trial is being performed comparing the efficacy and safety of maribavir, a novel benzimidazole riboside, and oral ganciclovir as prophylaxis against primary CMV disease in liver transplant recipients. The treatment of CMV disease consists mainly of intravenous (IV) ganciclovir, and if feasible, a reduction in the degree of immunosuppression. A recent controlled clinical trial demonstrated that valganciclovir is as effective and safe as IV ganciclovir for the treatment of CMV disease in solid organ (including liver) transplant recipients. In this article, the author reviews the current state and the future perspectives of prevention and treatment of CMV disease after,liver transplantation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20072号 / 医博第4165号 / 新制||医||1018(附属図書館) / 33188 / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 小川 誠司, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Cytomegalovirus (CMV) is a member of the Herpesviridae family and is known to infect people of all ages. In most cases, CMV infection is asymptomatic, and the virus is cleared from the host without showing any significant symptoms. However, 1 out of 200 babies are born with congenital CMV infection, which affects multiple organs, including the brain, liver, spleen, lung, and inner ear. One long-term health problem in 1 out 5 babies born with congenital CMV infection is hearing loss. The progression of CMV-associated hearing loss in the first two years of life may lead to developmental delays in language, learning, and communication. Currently, for serological testing of CMV in patients older than 12 months, real-time polymerase chain reaction (rtPCR) is used. Although the rtPCR method quantitatively detects the CMV, this method is expensive and needs highly skilled technicians to perform the assay. Therefore, there is a need for a cost-effective, simple, and rapid diagnostic tool that can help detect CMV in newborn babies and prevent CMV-mediated pathology in pediatric as well as in future adult conditions. In recent scientific studies, gold nanoparticles (AuNP) of 100 nm, 40 nm, and 15 nm sizes have shown promising results in detecting various viruses. In our study, we developed an AuNP-based colorimetric assay for detecting CMV in pediatric-derived urine samples. For comparison purposes, the pediatric urine samples were screened through quantitative PCR (qPCR) for positive and negative CMV determination. In our assay, we explored the ability of different-sized nanoparticles to detect CMV in pediatric urine samples. Using purified CMV virions as a positive control, we have shown that AuNP can effectively detect the presence of CMV in pediatric urine samples. Therefore, this colorimetric assay may be a basis for a useful diagnostic tool for detecting CMV in newborn babies. It is a rapid and non-invasive high-throughput assay for screening for the presence of CMV at the point of care and may help provide better therapeutic outcomes for pediatric patients.

In patients after allogeneic haematopoietic stem cell transplantation (HSCT) is a human cytomegalovirus (CMV) one of the most important viral pathogens. Its detailed characteristic could provide information about the impact of each CMV genotype on overall survival of the patient, and some serious complications, such as graft versus host disease (GvHD). This thesis deals with retrospective genetic analysis of samples from 1877 patients transplanted at the Clinic of Pediatric Hematology and Oncology, University Hospital Motol and the Institute of Hematology and Blood Transfusion since 2002. DNA from biological samples (especially whole blood) was isolated kit Qiagen DNA Blood Mini or Qiagen DNA Mini and samples were prospectively detected presence of CMV DNA. Samples were subsequently stored at -20 řC. Genotyping was performed using real-time PCR technologies to the genes of 2 structural proteins glycoprotein B, glycoprotein H and using sequence specific primers and probes. In 1343 samples (71.6%) from 390 patients there was only one strain of CMV; in 256 (13.6%) samples from 113 patients have detected mixed infection caused by two or more strains of CMV. The most common genotype demonstrated in "single" infection was in pediatric and adult patients gB1/gH2 detected in 118 (28.4%) patients. Most...

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

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Le cytomégalovirus (CMV), un β-herpes virus, est considéré comme un modèle d'immuno-évasion virale. Il s'agit d'un agent pathogène opportuniste fréquent chez les patients immunodéprimés et une cause majeure de malformations congénitales lors de l'acquisition in utero. Le CMV code pour des protéines (i) qui empêchent la présentation de l'antigène aux lymphocytes T αβ notamment par l'inhibition de l'expression des molécules HLA-I et (ii) qui suppriment les fonctions des cellules NK en imitant ou en régulant à la baisse les ligands des récepteurs NK (NKR). Ces mécanismes d'évasion ne devraient pas affecter les lymphocytes T γδ dont la reconnaissance antigénique est indépendante du HLA-I, et d’ailleurs leur réponse au CMV a été largement rapportée dans de nombreux contextes physiopathologiques. Notre objectif était de comprendre comment les mécanismes d’immuno-évasion du CMV affectent la réponse des lymphocytes T γδ. Nous avons utilisé des adénovirus recombinants exprimant chacun des quatre gènes du CMV impliqués dans l’inhibition de l’expression du HLA-I, et un mutant du HCMV déficient pour ces 4 gènes (CMV-∆US). Nous avons observé une induction de l'expression de HLA-I par l'adénovirus control, et une inhibition par US2, US3 et US11. Lors de l'utilisation de CMV-∆US, les cellules infectées exprimaient beaucoup plus de HLA-I que les cellules infectées par CMV-WT. De façon intéressante et à l’opposé des lymphocytes T αβ, les lymphocytes T γδ produisent plus d'IFNy en présence de fibroblastes infectés par le CMV-WT, qu’avec des fibroblastes infectés par CMV-∆US. Ces résultats indiquent que les molécules HLA-I régulent les lymphocytes T γδ grâce à des mécanismes qui sont en cours d'investigation dans notre équipe. Les processus d'échappement immunitaire développés par le CMV pourraient ainsi favoriser la réponse des lymphocytes T γδ par rapport à celle des lymphocytes T αβ et expliquer le rôle important des cellules T γδ dans le contrôle du virus chez les individus immunodéprimés. D'autre part, les acides nucléiques et les protéines du HCMV ont été trouvés dans les tissus tumoraux, mais la relation précise entre le HCMV et le cancer reste un sujet de débat. La plupart du temps, HCMV est décrit comme un virus oncomodulateur avec un rôle pro-tumoral. Notre objectif était d'utiliser le modèle de la souris pour tester in vivo l'impact de CMV de souris (MCMV) sur la croissance des cellules tumorales. Nous avons observé que MCMV pourrait inhiber la croissance de tumeurs sous-cutanées de côlon MC38 chez les souris immunodéficientes. Encore plus surprenant lorsque l'on considère la spécificité d’espèce des CMV, l'infection par le MCMV inhibe de la même façon la croissance des cellules cancéreuses du côlon humain HT29, qui n’est pas affectée par le HCMV. In vitro, les protéines MCMV précoces (IE-1) sont détectées dans des cellules cancéreuses humaines et murines après l'infection. Cependant, peu de cellules cancéreuses sont retrouvées positives pour le MCMV dans les tumeurs HT29 prélevées sur des souris infectées par le MCMV. De manière surprenante, le MCMV inhibe la prolifération des cellules cancéreuses de côlon humain contrairement au HCMV. De plus, la transcription de l'interféron β humain est induite après une infection par le MCMV. Cette induction n'a pas été observée après l'infection par le HCMV. En conclusion, nos données suggèrent un potentiel effet anti-tumoral de MCMV sur les cellules cancéreuses du côlon humain (HT29), qui pourrait être au moins partiellement médiée par l'interféron β. Ces résultats ouvrent la voie à l'utilisation potentielle du MCMV en tant que traitement du cancer du côlon humain. / Cytomegalovirus (CMV), a Beta Herpes virus, is considered as a paradigm for viral evasion.It is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. CMV encodes molecules to prevent antigen presentation to αβ T cells through inhibition of MHC Class I expression and to suppress NK cell functions by mimicking or down-regulating ligands of NK receptors (NKR). These evasion mechanisms are not expected to affect γδ T cells and, as a matter of fact, their response to CMV has been widely reported in many different physiopathological contexts as well as in CMV-seropositive healthy donors (Dechanet et al, 1999)( Scheper, 2013). Our aim was to understand how CMV induce γδ T cell response. We used recombinant adenoviruses expressing each of the four US genes, and a mutant HCMV deleted for these 4 genes (CMV-DUS). We observed an induction of HLA-I expression by the control adenovirus, and an inhibition by US2, US3 and US11. When using CMV-DUS, infected cells expressed much more native HLA-I than CMV-WT infected cells. Interestingly and in sharp contrast to αβ T cells, γδ T cell were activated to produce IFNg when cultured with fibroblasts infected with CMV-WT, but not when fibroblasts were infected with CMV-DUS. These results indicate that HLA-I molecules regulate γδ T cells through mechanisms that are under investigation in our team. The immune escape processes developed by CMV could thus promote γδ over αβ T cell response and explain the important response of γδ T cells to the virus in immunosuppressed individuals.

Neutralisierende Antikörper sind entscheidend in der Eindämmung der Virusinfektion, indem sie den Eintritt in die Wirtszelle hemmen bzw. die Aktivierung der Komplementkaskade initiieren. Distinkte wirtseigene Oberflächenrezeptoren für die Fc-Domäne von IgG (FcyR) sind für die Kommunikation von humoraler und zellulärer Immunantwort verantwortlich. Auch Mitglieder der Herpesviren kodieren für Fc-bindende Proteine, die Kandidaten für immunevasive Funktionen darstellen könnten. Der Nachweis der HCMV-kodierten Fc-bindenden Proteine gp34 und gp68 als Bestandteil der Virushülle lies auf eine immunevasive Funktion hinsichtlich neutralisierendem IgG und Komplement-vermittelter Virolyse schließen, wie für den HSV-1-kodierten FcyR gE beschrieben. Weder für gp34 noch für gp68 konnte in vitro ein hemmender Effekt auf Neutralisation und Virolyse beobachtet werden. In unserem Labor wurde jedoch gezeigt, dass gp34 und gp68 selektiv die IgG-abhängige Aktivierung zellulärer FcyR inhibieren. Die glykosylierungsunabhängige Ligandenbindung von gp34 und gp68 wies auf unterschiedliche Interaktionsmechanismen zwischen den zellulären und den viralen FcyR hin. Mithilfe eines mutierten Fc-Fragments konnte für gp68 eindeutig eine mit HSV-1 gE überlappende Bindestelle an IgG identifiziert werden. Die für die Ligandenbindung erforderlichen Aminosäuren 71-292 von gp68 binden Fc in einer 2:1 Stöchiometrie, wobei die N-, nicht aber die O-Glykosylierung des vFcyRs essentiell sind. Darüber hinaus formt gp34 auf infizierten Zellen und auf der Virushülle kovalente Homooligomere. gp34-Cysteinpunktmutanten auf Basis der für die Bindung notwendigen Aminosäuren 24-140 lassen vermuten, dass die Oligomerisierung Voraussetzung für die Fc-Bindung ist. Im Gegensatz zu gp68 scheint der Mechanismus der Fc-Bindung von gp34 einzigartig unter den bekannten Fcy-Rezeptoren zu sein. Diese Ergebnisse lassen vermuten, dass trotz redundanter Expression der HCMV-FcyR der Bindungs- und Wirkungsmechanismus selektiv ist. / Neutralizing IgGs play a key role in diminishing virus infectivity by inhibiting the entry into host cells. Additionally, IgG-bound particles may be inactivated by virolysis through the activation of complement. Surface receptors specific for the Fc domain of IgG represent host proteins, connecting humoral and cellular immune responses. Also members of the herpes virus family code for proteins with Fc binding properties, implying functions that could intervene with antibody-dependent effector mechanisms. The presence of gp34 and gp68 on the virion membrane raised the question whether they are able to inhibit neutralising IgG and complement-mediated virolysis. The HSV-1-encoded FcyR gE was described to affect neutralisation and virolysis. Despite extensive analysis, there were no implications found that gp34 or gp68 interfere with neutralising IgG or virolysis in vitro. However, our lab could demonstrate that gp34 and gp68 selectively inhibit the IgG-dependent activation of the different host FcyRs. In contrast to the cFcyRs Fc recognition by gp34 and gp68 occurs independently of N-linked glycosylation of IgG, which points to a different binding mechanism among host and viral FcyRs. By taking advantage of a mutated Fc fragment, overlapping binding regions of the HSV-1 gE and gp68 were identified. For gp68 the amino acids 71-292 including the N-glycans are strictly required for Fc binding in a 2:1 stoichiometry. Interestingly, gp34 forms covalently linked homo-oligomers in infected cells and on the virion. Based on the minimal binding domain comprising the amino acids 24-140 of gp34, targeted cysteine exchange mutants revealed that oligomer formation by gp34 is absolutely required for Fc binding. In contrast to gp68, the Fc binding characteristics of gp34 appears to be unique among the known FcyRs. These findings allow us to postulate that even if the HCMV-encoded FcyRs are redundantly expressed the mechanistic details and binding properties are selective.