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Ovine herpesvirus 2 (OvHV-2) is a gamma herpesvirus and is the causative agent of lymphoproliferative disease – sheep-associated malignant catarrhal fever in susceptible ruminants, including cattle. Sheep become persistently infected but do not show apparent clinical infection. MCF is characterized by marked T cell hyperplasia and proliferation of unrestricted cytotoxic large granular lymphocytes (LGLs) which leads to necrosis of infiltrated tissues and generally causes death of the host. Little is known about the underlying molecular basis of MCF pathogenesis or what controls the differences in clinical outcome of infection in two closely-related host species. MicroRNAs (miRNAs) constitute a large family of small, ~22nt, noncoding RNA molecules that regulate gene expression by targeting messenger RNAs post-transcriptianally in eukaryotes and viruses. Herpesvirus encoded miRNAs have been shown to play a role in regulating viral and cellular processes including cell cycle and may have a role in pathogenesis. OvHV-2 has also been found to encode for at least 46 OvHV-2 miRNAs in an immortalized bovine LGL cell line. 23 of these miRNAs have also been validated by northern blot analysis and RT qPCR. It was hypothesised that these OvHV-2 miRNAs may regulate viral and cellular genes expression and may play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 miRNAs have functional targets within viral and host cell genes. Bio-informatic analysis has predicted several targets for these OvHV2 miRNAs in the 5’ and 3’ UTRs of several virus genes. Luciferase inhibition assay confirmed that out of 13 selected predicted targets, three (two targets ORF73 and one within ORF50) were positive and functional. A fourth predicted target was also found functional (ORF20), but its functionality could not be confirmed by knocking out the target site. A newly developed technique Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was also used to identify miRNAs bound targets within cattle and sheep genome. High throughput sequencing and analysis of the hybrid data revealed many target genes. Four of those targeted genes, were validated by luciferase inhibition assays and three were found to be targeted by OvHV-2 miRNAs. This study gives the first evidence of viral miRNAs bound to their targets in cattle and sheep cells, by a highly sensitive technique-CLASH and provides a tool for studying differences in pathogenesis of two closely-related host species.

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Risk of skin cancer and lymphoproliferative diseases in patients after kidney transplantation Author: Kristína Sidorová Tutor: doc. PharmDr. Josef Malý, Ph.D. Consultant: Mgr. Barbora Vaňková Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradec Králové, Charles University Introduction and aims: Organ transplantations (Tx) are associated with the lifelong use of immunosuppressive therapy (IS), which carries with it, among other risks, an increased incidence of malignancies. The most common malignancies after Tx are skin tumors, post-transplant lymphoproliferative diseases (PTLD) are also more common in transplant patients. The aim of the study was to determine the incidence of skin tumors and PTLD in patients with kidney Tx within one transplant center and to analyze the risk factors associated with these diseases. Methods: Retrospective longitudinal study was conduted in the Teaching Hospital Hradec Králové. The study included patients from the age of 18 who had overcome kidney Tx until 24 April 2019, were registered in a transplant clinic in Hradec Králové and had a medical record in the hospital information system. Data collection from medical records took place from 15 April 2019 to 31 December 2019. Collected data included demographic characteristics, family history,...

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Base teórica: doenças linfoproliferativas crônicas de linhagem B (DLPC-B) são neoplasias clonais que afetam linfócitos B maduros. A tirosina quinase de Bruton (do inglês Bruton’s tyrosine kinase, BTK) é uma proteína essencial para o desenvolvimento, diferenciação e sinalização nos linfócitos B. Ki-67 é uma proteína nuclear associada à proliferação celular. A avaliação de proteínas envolvidas nas vias de sinalização oncogênicas pode levar ao aprimoramento do diagnóstico, tratamento e definição de prognóstico das DLPC-B. Objetivo: avaliar a expressão de BTK e Ki-67 em linfócitos de portadores de DLPC-B. Métodos: para avaliação de BTK foi realizado um estudo transversal; foi avaliada a expressão de BTK em amostras de pacientes saudáveis e de pacientes com diagnóstico de DLPC-B. Para avaliação de Ki-67 foi realizado um estudo transversal. As amostras foram marcadas com CD45 FITC e CD19 APC para identificação dos linfócitos B. Após a lise das hemácias, foi realizada marcação citoplasmática de BTK PE e/ou Ki-67 PerCP-Cy5.5. O percentual de expressão e a intensidade de fluorescência média (IFM) dos marcadores avaliados foram determinados nos linfócitos B. A análise estatística foi realizada com testes de correlação de Pearson e Spearman entre BTK ou Ki-67 e as demais variáveis clínicas e laboratoriais, e ANOVA seguido por teste post hoc de Bonferroni para comparações entre grupos. Foi considerado resultado significante quando P < 0,05. Resultados: não foram observadas diferenças na expressão de BTK e não houve associação entre a expressão de BTK e as variáveis clínicas avaliadas. A expressão de Ki-67 foi maior nos grupos linfoma do manto, linfoma de Burkitt e linfoma difuso de grandes células B em relação aos demais; após análise multivariada, a IFM de Ki-67 foi associada à IFM de CD38. Conclusão: no presente trabalho, a expressão de BTK em DLPC-B foi similar a de linfócitos B normais e a expressão de Ki-67 foi maior nas DLPC-B com curso clínico mais agressivo. / Background: mature B-cell neoplasms (MBCN) are clonal neoplasms that affect mature B-cell lymphocytes. Bruton’s tyrosine kinase (BTK) is an essential protein for the development, differentiation and signaling in B-cell lymphocytes. Ki-67 is a nuclear protein associated to cellular proliferation. Evaluation of proteins involved in oncogenic signaling pathways can lead to improvement in the diagnosis, treatment and prognosis definition in MBCN. Objective: to evaluate the expression of BTK and Ki-67 in lymphocytes of MBCN patients using flow cytometry. Methods: a cross-sectional study was conducted for BTK assessment; BTK expression was assessed on healthy patients samples and MBCN samples. For evaluation of Ki-67 a cross-sectional study was conducted. Samples were stained with CD45 FITC and CD19 APC for identification of B-cell lymphocytes. After lysis of red blood cells, cytoplasmic staining of BTK PE and/or Ki-67 PerCP-Cy5.5 was performed. Percentage of expression and mean fluorescence intensity (MFI) of the markers were determined in B-cell lymphocytes. Statistical analysis was performed with Pearson and Spearman correlation tests between BTK and Ki-67 and the other clinical and laboratory variables, and ANOVA followed by post-hoc Bonferroni test for comparisons between groups. Results were considered significant when P < 0.05. Results: no differences in BTK expression were identified and there was no association between BTK expression and clinical variables evaluated. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma and diffuse large B-cell lymphoma cases; after multivariate analysis, MFI of Ki-67 was associated with MFI of CD38. Conclusions: in this study, BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes and Ki-67 expression was higher in MBCN with more aggressive clinical courses.

Le virus Epstein-Barr (VEB) est fortement associé au développement de syndromes lymphoprolifératifs (SLP) en greffe pédiatrique. Ce virus a la capacité d’immortaliser les lymphocytes B et de provoquer leur prolifération incontrôlée chez l’hôte immunodéprimé. Plusieurs études démontrent que le cycle lytique du virus jouerait un rôle primordial dans la genèse des SLP en produisant des particules virales pouvant infecter les cellules B adjacentes. Chez un individu immunodéprimé, ces cellules B nouvellement infectées peuvent donner naissance à une expansion lymphocytaire. Le projet présenté dans ce mémoire fait partie d’un programme de recherche visant à élucider le rôle de l’infection productive par le VEB dans le développement des SLP. L’objectif précis de ce projet est de développer un anticorps monoclonal chimère contre la glycoprotéine gp350 du VEB dans le but de neutraliser le virus et d’ainsi prévenir son entrée dans les cellules B. Notre laboratoire a construit une version chimère de l’anticorps monoclonal murin 72A1, lequel se lie à la gp350 et bloque l’infection. Les premiers essais ont révélé la présence de chaînes non fonctionnelles (aberrantes) dans l’hybridome produisant l’anticorps 72A1. La construction de la chaîne légère authentique est maintenant complète alors que celle de la chaîne lourde est toujours en cours. Le processus de caractérisation de l’anticorps chimère inclura des essais de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Dans cette optique, une lignée cellulaire exprimant de façon stable la gp350 a été établie. Notre anticorps chimère anti-gp350 pourrait éventuellement être utilisé comme thérapie préventive chez les greffés présentant un risque élevé de SLP en empêchant l’infection des cellules B adjacentes. / The Epstein-Barr virus (EBV) is associated with B-cell post-transplant lymphoproliferative disease (PTLD). EBV has the unique property of immortalizing B lymphocytes, thereby causing their uncontrolled proliferation in an immunocompromised host. Certain evidence suggests that EBV productive infection may play a primary role in the genesis of PTLD by generating virus particles which can infect bystander B cells. In an immunocompromised individual, these infected B cells may then give rise to expanding B-cell clones. The project presented in this thesis is part of a research program seeking to elucidate the role of EBV productive infection in the genesis of PTLD. The specific aim of this work was to design a chimeric monoclonal antibody against the EBV envelope glycoprotein gp350 in order to neutralize the virus, thereby preventing entry into B cells. Our laboratory constructed a chimeric version of the murine monoclonal antibody, 72A1, which binds to gp350 and blocks infection. The initial cloning attempts revealed the presence of nonfunctional (aberrant) transcripts in the hybridoma line producing the 72A1 antibody. The chimeric version of the authentic light chain is now completed while the chimeric heavy chain construction is ongoing. As part of the characterisation process for the chimeric antibody, a cell line stably expressing surface gp350 was generated. This gp350-expressing cell line will be used for antibody dependent cellular cytotoxicity assays (ADCC). This anti-gp350 chimeric antibody could be useful as a preventive therapy in transplant patients at high risk for PTLD by blocking the infection of bystander B cells.

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.