Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

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

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

Thesis (doctor of medicine)--Rijksuniversiteit te Groningen, 1979.

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.

The human body is exposed to a multitude of prevalent viruses, requiring ongoing surveillance and protection by the immune system. Maintenance of human anti-viral adaptive immunity in diverse tissue sites is determined by a multitude of factors and critical for long-term protection against repeat exposure to viral infection. Yet, studies of anti-viral immunity have primarily been limited to animal studies and studies of peripheral blood in humans. Studies in mice have demonstrated that memory T cells in tissues provide superior protection against viral infection compared to circulating T cells, particularly tissue-resident memory T cells (TRM), which remain in tissues long-term without re-entering circulation. However, much remains to be understood about how anti-viral immune responses are maintained in human tissues and how adaptive immune cells in various tissues sites function upon re-exposure to viral antigens. We have established a human tissue resource through a collaboration with LiveOnNY, a local organ procurement organization, to obtain blood and multiple lymphoid and mucosal sites from donors of all ages. Using this tissue resource, we employed comprehensive cellular and molecular analysis to investigate tissue immunity to three prevalent but distinct viruses—influenza A, CMV, and SARS-CoV-2. We compared CD8+ T cells recognizing ubiquitous and longstanding viruses influenza A and CMV across multiple tissue sites of 58 organ donors ages 1-78 years in order to elucidate how covariates of virus, tissue, age, and sex impact the anti-viral immune response. Using flow cytometry, T cell receptor repertoire sequencing, functional assays, and single-cell transcriptional profiling, we showed that virus specificity and tissue localization are the primary drivers of anti-viral T cell immune responses in the human body, with age and sex further influencing T cell subset differentiation. Specifically, virus specificity correlated with virus-specific T cell distribution, memory subset differentiation, and clonal repertoire, while tissue localization determined overall subset distribution and functional responses. We further investigated the tissue-localized immune response to emergent SARS-CoV-2. By examining multiple tissues of organ donors who had recovered from natural infection by SARS-CoV-2, we showed that adaptive memory immune responses persisted months after infection, with memory T and B cells preferentially localized in the lung and lung-associated lymph node. Persisting memory cell populations included tissue-resident T and B cells, particularly in the lung, as well as germinal center B cells in the lung-associated lymph node along with follicular helper T cells, indicating ongoing generation of humoral immunity. Together, these findings highlight the importance of tissue-localized anti-viral immunity and help to define characteristics of site-specific protective immunity that may be leveraged for the development of more effective treatment and prevention strategies.

The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection. The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 17, 2009). Includes bibliographical references.

The overall aim of this thesis was to determine how natural killer (NK) cells regulate virus infections in vivo. Anti-viral mechanisms by which NK cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of adult C57BL/6 mice were first studied, revealing different mechanisms of control in different organs. Three days post-infection, MCMV titers in the spleens of perforin-deficient (perforin 0/0) mice were higher than in wild type controls, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers but not in spleen titers. Depletion of IFN-γ in adult C57BL/6 mice by injections with mAbs to IFN-γ resulted in an increase in viral titers in the liver but not in the spleen. Analyses using IFN-γ-receptor-deficient (IFN-γR0/0) mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that even though the donor spleen cells could respond to IFN-γ, the depletion of NK cells in a recipient environment where the host cells could not respond to IFN-γ caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-γ has the ability to induce a variety of cells to produce nitric oxide (NO), and administrating the nitric oxide synthase (NOS) inhibitor Nω-monomethyl-L-arginine (L-NMA) into MCMV-infected adult C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. These data indicate that in adult C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, whereas in the liver the production of IFN-γ by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-1r (Cmv-1-resistant) locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver. The ability of adoptively transferred cells to protect suckling mice from MCMV was another model used to study the mechanisms utilized by NK cells in the regulation of MCMV. Adoptive transfers of 129, C57BL/6 and perforin 0/0 spleen cells or lymphokine-activated killer (LAK) cells into 4 - 6 day old MCMV-infected C57BL/6 suckling mice significantly lowered the splenic MCMV titers in these mice compared to the infected controls. Adoptive transfers of C57BL/6 spleen cells into MCMV-infected 129 suckling mice also decreased the amount of MCMV in the 129 suckling mice, but C57BL/6 spleen cells could not regulate MCMV synthesis when adoptively transferred into 129/IFN-γR0/0 suckling mice. These results suggest that, in the suckling mouse model, the regulation of MCMV by the adoptively transferred NK cells is via an IFN-γ-dependent, perforin-independent, Cmv-1-independent mechanism. The Cmv-1 gene locus resides within the NK gene complex, in close proximity to the Ly49 NK cell receptor family. Analyses were carried out to determine if any of the 4 known Ly49 NK cell receptors (Ly49A, C, D and G2) played a role in the control of MCMV synthesis by NK cells. Studies comparing the expression of the different Ly49 NK cell subsets in the spleen and the peritoneal cavity revealed that there were differences in the distribution of the Ly49 receptors on NK1.1+ cells. Three days post-MCMV infection, the percentage of NK1.1+- Ly49+ NK cells in the spleen and the peritoneal cavity were different than in naive controls. Within the splenic NK1.1+ population, increases in NK1.1+ -Ly49A+ and NK1.1+-Ly49G2+ cells but decreases in NK1.1+-Ly49C+ and NK1.1+-Ly49D+ cells were observed. These changes in the spleen were accompanied by a concomitant decrease in NK1.1+ - Ly49A+ cells and increases in NK1.1+-Ly49C+, NK1.1+-Ly49D+ and NK1.1+-Ly49G2+ cells within the NK1.1+ population in the peritoneal cavity. These data suggest that 3 days post-MCMV infection, there may be movement of NK cells between the different organs. The role of Ly49 NK cell receptors in the regulation of MCMV was tested using adult C57BL/6 mice depleted of single or multiple Ly49 NK cell subsets. These in vivo depletions did not affect the ability of the residual NK cells to regulate MCMV synthesis. LAK cells sorted into the different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice lowered the splenic MCMV titers in these mice. Together, these results indicate that even though there is a redistribution of the Ly49 NK cell subsets during MCMV infection, the presence or absence of anyone of the 4 tested Ly49 NK cell receptors does not affect the regulation of MCMV by NK cells. However, there remain a possibility that one of the undefined Ly49 receptors or an untested NK cell receptor may be important in the control ofMCMV. Most of the cloned NK cell receptors have been shown to bind to MHC class I molecules, and MHC class I antigens have been implicated as modulators of target cell sensitivity to NK cell-mediated lysis. The regulation of virus infections and the fate of NK cells and their natural targets was examined in β2-microglobulin-deficient mice [β2m (-/-)], which have defective MHC class I expression. Infections with either the NK cell-sensitive MCMV or the NK cell-resistant lymphocytic choriomeningitis virus (LCMV) significantly augmented NK cell activity in either C57BL/6 or β2m (-/-) mice. Depletion of NK cells in vivo with antiserum to asialo GM1 markedly enhanced the synthesis of MCMV but had no effect on the synthesis of LCMV in either strain of mouse. Adoptively transferred β2m (-/-) spleen cells lowered splenic MCMV titers in C57BL/6 suckling mice, not unlike adoptively transferred C57BL/6 spleen cells. Analysis of naturally NK cell-sensitive thymocyte targets from these virus-infected β2m (-/-) mice revealed no cell surface expression of class I MHC detectable by conformation-dependent or -independent antibodies, but the virus infections enhanced class I expression on thymocytes from C57BL/6 mice. The sensitivity of C57BL/6 thymocytes to NK cell-mediated lysis was markedly reduced after in vivo poly inosinic:cytidylic (poly I:C) treatment or viral infection; in contrast, the sensitivity of the β2m (-/-) thymocytes was significantly less affected by poly I:C or viral infection. These data indicate that the normal expression of MHC class I antigens on NK cells or their targets is not required for the anti-viral functions of NK cells against an NK-sensitive virus (MCMV) nor do they protect an NK-resistant virus (LCMV) from the anti-viral activity of NK cells. Together, the data presented in this thesis help to further our understanding of the mechanisms utilized by NK cells in the control ofMCMV in both adult and suckling mice, and also help clarify the roles played by Ly49 NK cell receptors and MHC class I molecules in the regulation of MCMV.

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