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Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-11-09Bitstream added on 2014-06-13T19:05:02Z : No. of bitstreams: 1 dias_prp_dr_botfca.pdf: 1303201 bytes, checksum: ff13b531a76b1d9405f4151a52ea6f09 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Cucumber mosaic virus (CMV), uma espécie do gênero Cucumovirus, é um dos mais importantes vírus que infecta pimentão, causando prejuízos consideráveis na produção em todo o mundo. Quando da infecção precoce, em geral, ambas a qualidade e a quantidade de frutos produzidos são afetados. O vírus apresenta inúmeras estirpes capazes de infectar pimentão, diferindo na expressão dos sintomas. CMV pode infectar mais de 865 espécies de plantas, incluindo ervas daninhas, sendo transmitido por diversas espécies de afídeos de maneira não circulativa. Inseticidas são ineficazes para prevenir a disseminação da doença em virtude da forma de transmissão do vetor. No presente trabalho, verificou-se que o CMV foi o principal vírus identificado em campo. Vinte e três isolados de Capsicum spp. foram purificados biologicamente e caracterizados através de análises sorológica, biológica e molecular. Todos os 23 isolados da coleção foram classificados no subgrupo I do CMV, induzindo mosaico sistêmico, redução do desenvolvimento vegetativo e deformação foliar em Nicotiana glutinosa e Nicotiana tabacum 'Havana 425', diferindo apenas na intensidade de sintomas. Somente 8 isolados foram capazes de causar mosaico em Vigna unguiculata. Amplificação combinada com clivagem pela enzima Msp I foi eficiente para distinguir os subgrupos do CMV, resultando em banda de 500 pb somente para a amostra-controle do CMV II, dando origem a 3 fragmentos com 190, 150 e 120 pb, enquanto todos os outros isolados permaneceram com 488 pb e sem clivagem, correspondendo ao CMV-I. Não foi detectado RNA satélite em nenhum isolado do campo. A reação ao CMV de cultivares e híbridos comerciais de pimentão é desconhecida, mas tudo indica serem susceptíveis... . / Cucumber mosaic virus (CMV), a species of the genus Cucumovirus, is one of the most important virus that infect pepper, causing notable losses in pepper production worldwide. With early infection, in general, both quality and quantity of fruit produced will be affected. The virus exists as a number of strains capable of infecting pepper, differing in symptom expression. CMV can infect more than 865 plant species including many weed species and it is transmitted by many aphid species in a non-circulative manner, meaning that insecticides cannot prevent the spread of this disease. At this work, the CMV was the main virus identified in the field. Twenty-three CMV isolates from Capsicum spp. were biologically purified and characterized for serological, biological and molecular analysis. All 23 isolates from collection were found to belong to subgroup I. All isolates caused systemic mosaic, reduction of vegetative development and deformation in the leaf in N. glutinosa and N. tabacum 'Havana 425', differing in symptom intensity. Only 8 isolates were able to cause systemic mosaic in V. unguiculata. Amplification combined with Msp I cleavage was efficient to distinguish the CMV subgroups. This process resulted in a 500 pb for the CMV II control only, giving origin to three fragment with 190, 150 and 120pb, while all other isolates remained uncleaved with 488 pb, corresponding to the CMV-I isolates. It was not detect RNA satellite in a field isolates. Pepper comercial cultivars and hybrids reaction to CMV is unknowledge, but it seems to be susceptible. The identification of cultivated varieties or wild relatives of pepper that are better able to fend off attack by viral pathogens such as CMV is a critical first step towards developing resistant commercial varieties.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-27T10:31:50Z No. of bitstreams: 2 Dissertação - Rachel de Paula Santos Ribeiro - 2017.pdf: 12713551 bytes, checksum: c361c2531a99b684a3f4f3633f77b96c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-27T10:32:23Z (GMT) No. of bitstreams: 2 Dissertação - Rachel de Paula Santos Ribeiro - 2017.pdf: 12713551 bytes, checksum: c361c2531a99b684a3f4f3633f77b96c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-27T10:32:24Z (GMT). No. of bitstreams: 2 Dissertação - Rachel de Paula Santos Ribeiro - 2017.pdf: 12713551 bytes, checksum: c361c2531a99b684a3f4f3633f77b96c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-26 / Autoimmune diseases can be triggered by viruses, bacteria and parasites. However, the participation of these infectious agents in the etiology of vitiligo, it is a current research topic. In this study, the serum of 51 participants with vitiligo and 51 control subjects was analyzed for the presence of anti-Toxoplasma gondii (T.gondii) IgG, anti-herpes simplex (HSV) 1/2 IgG, anti-cytomegalovirus (CMV) IgG and anti-hepatitis C (HCV) IgG. / As doenças automimunes podem ser desencadeadas por vírus, bactérias ou parasitas. E o envolvimento destes agentes infecciosos na etiologia do vitiligo, é tema de intensa investigação atual. Neste estudo, o soro de 51 participantes com vitiligo e de 51 pessoas controle pareados por sexo e idade foi analisado para presença de imunoglobulinas IgG anti- Toxoplasma gondii (T.gondii), anti-herpes simples (HSV) 1/2, anti citomegalovírus (CMV) e anti-hepatite C (HCV).

L’infection congénitale à CMV est la première cause de retard psychomoteur et de surdité, toutes causes confondues. En France, la prévalence de l’infection congénitale à CMV en France est estimée entre 0,6 et 0,7%. Parmi les enfants présentant une infection symptomatique, 10-20% des nourrissons font une forme sévère et décèdent, et 50 à 60% développent des lésions cérébrales avec un handicap neurologique parfois lourd consécutif à une microcéphalie, des calcifications intracrâniennes, des dilatations ventriculaires ou des convulsions. Certains parmi ceux-ci développeront aussi des séquelles neurosensorielles consécutives à une atteinte visuelle, auditive ou vestibulaire. La première partie de ce travail permet de décrire l’histoire et la physiopathologie des lésions de l’oreille interne chez l’homme. Les lésions de l’oreille interne sont évaluées chez 6 fœtus ayant une infection congénitale à CMV, âgés de 21 à 35 semaines d’aménorrhée, et corrélées aux atteintes centrales et viscérales. Dans la cochlée, les lésions prédominent au niveau de la strie vasculaire alors que l’infection est moins fréquente au niveau des cellules de l’organe de Corti. Les lésions vestibulaires sont plus florides que les lésions cochléaires et prédominent au sein de l’épithélium non sensoriel, en particulier des cellules sombres du saccule et de l’utricule, et de l’épithélium des canaux semi-circulaires. Ces lésions du compartiment endolymphatique de l’oreille interne pourraient être responsables d’une altération de la sécrétion du potassium et ainsi d’une modification de l’homéostasie de la cochlée et du vestibule par atteinte de la strie vasculaire et des cellules sombres. Secondairement, cela pourrait être responsable d’une dégénérescence de l’organe de Corti et des organes sensoriels vestibulaires. La survenue de la surdité neurosensorielle et de l’atteinte vestibulaire dépend très certainement de la durée et de l’intensité de l’infection virale et de l’inflammation. La deuxième partie de ce travail analyse les atteintes du système nerveux central qui n'ont pas fait l'objet d'étude détaillée. Nous avons sélectionné 9 fœtus de 23 SA à 38 SA dont la gestation a été interrompue devant une suspicion d’infection congénitale par le CMV. Ils ont été étudiés sur le plan foetopathologique standard, neuropathologiques, immunohistochimiques et morphométriques. La réplication virale productive prédomine dans le cortex et dans la région périventriculaire. Le virus infecte préférentiellement les cellules GFAP-positive et nestine-positive, mais aussi à un moindre degré les neurones, les cellules endothéliales et la microglie. La réaction immune microgliale et cellulaire cytotoxique semble insuffisante pour contrer l’importance de la dissémination virale suggérant son immaturité. Les malformations corticales sont de mécanismes variés et il existe une atteinte préalablement méconnue des bulbes olfactifs. Une troisième partie est consacrée au développement d’un modèle murin d’infection congénitale par le CMV visant à mieux comprendre les mécanismes physiopathologiques d'atteintes auditives et vestibulaires et à définir les modalités thérapeutiques. Les souriceaux infectés par injection intra-amniotique de virus MCMV développent une surdité dont le profil évolutif s’apparente à la clinique humaine. Les lésions histologiques se caractérisent par des atteintes de la strie vasculaire, de l’organe de Corti et du ganglion spiral. Ces différents axes de recherche complémentaires permettent de comprendre les mécanismes centraux et périphériques participant aux handicaps neurosensoriels chez les enfants atteints d’infection congénitale par le CMV. Les études cliniques et physiopathologiques complémentaires qui en découlent permettront d'affiner les cascades lésionnelles mis en jeu et d'optimiser la prise en charge de ces enfants / Congenital CMV infection is the leading cause of mental retardation and neurosensory-hearing loss. In France, the prevalence of congenital CMV infection is estimated between 0.6 and 0.7%. Amongst the children presenting with a symptomatic infection,10-20% of newborns will have a severe manifestation and die and 50-60% will develop cerebral lesions with a neurological handicap due to microcephaly, intracranial calcifications, ventricular dilations or convulsions. Some amongst these will also develop neurosensory sequelae such as visual, auditory or vestibular lesions. The first part of this work describes the history and the physiopathology of the inner ear lesions in human. The lesions were evaluated in 6 fetuses presenting with a congenital CMV infection, aged 21 to 35 gestational weeks and correlated to central and visceral lesions. In the cochlea, the lesions predominated in the stria vascularis whereas the lesions were les frequent in the organ of Corti. The vestibular lesions were more florid than cochlear lesions and predominated in the non-sensory epithelium in particular the dark cells of the saccula and the utricule and the epithelium of the semi-circular canals. These lesions of the endolymphatic compartment may be responsible of the alteration of the potassium secretion in the inner ear and therefore a modification of the homeostasis of the cochlea and the vestibule by lesion of the stria vascularis and the dark cells. With time, this could be responsible of the degeneration of the organ of Corti and of the vestibular sensory organs. The onset of neurosensory hearing loss and vestibular disorder probably depend upon the duration and the intensity of viral infection and inflammation. The second part of this work analyses the central nervous system lesions that have seldom been detailed. We have selected 9 fetuses aged 23-28 gestational weeks whose pregnancy has been interrupted due to congenital CMV infection. Standard fetopathological examination was completed with neuropathological, immunohistochemical and morphometric assessment. Viral replication predominated in the cortex and in the periventricular zone. The virus infects preferentially GFAP positive and nestin positive cells, but also, to a lesser degree, neurons, endothelial cells and microglia. Immune microglial and cytotoxic cellular responses seem insufficient to counter the importance of the viral dissemination suggesting immaturity. Cortical malformations are due to several mechanisms and olfactory bulbs are frequently infected. The third part describes the development of a murine model for congenital CMV infection in order to better understand the physiopathological mechanisms of the auditory and vestibular lesions and to define the guidelines for management. Infected newborn mice developed neurosensory-hearing loss with characteristics similar to human deafness. Histological lesions were mainly observed in the stria vascularis, the organ of Corti and the spiral ganglia. These different complementary research axes led to a better understanding of the central and peripheral mechanisms participating in the neurosensory handicaps of children with congenital CMV infection. Further clinical and physipathogical studies will allow a more precise comprehension of the lesional cascades and a optimization of the medical management of these children

A infecção pelo Vírus da Imunodeficiência Humana (HIV) pode predispor à presença de fatores de risco cardiovascular e promover a ativação do sistema imunológico, contribuindo para a formação de lesões ateroscleróticas. A existência de coinfecções, como pelo Citomegalovírus (CMV) e a ativação imunológica inespecífica poderiam intensificar o processo de inflamação crônica e acelerar os danos vasculares decorrentes desta. O objetivo principal deste estudo foi avaliar se a recorrência da infecção pelo CMV, a magnitude da resposta imunológica específica ao CMV ou da ativação inespecífica de células T e monócitos associava-se ao aumento da espessura das camadas média e íntima das carótidas (cIMT) em crianças e adolescentes coinfectados pelo HIV e CMV. Consistiu-se de um estudo longitudinal, em que 40 crianças e adolescentes coinfectados pelo HIV e CMV foram acompanhados por 2 anos. Periodicamente avaliou-se a presença de recorrência do CMV, com o uso de detecção de DNA do CMV no soro por meio da técnica de Reação em Cadeia da Polimerase; a ativação imunológica perante este vírus com o ensaio do Quantiferon CMVR, a dosagem de anticorpos IgM e IgG contra o CMV. Também, nós medimos a ativação imunológica inespecífica de células T usando a dosagem do receptor I de TNF solúvel (sTNFRI) e a quantificação da presença HLADR+CD38+TCD8+; e a de monócitos por meio da dosagem de CD14 solúvel (sCD14). Para caracterizar adicionalmente as crianças estudadas, outros parâmetros foram registrados periodicamente: 1-Relativos à infecção pelo HIV: parâmetros clínicos e quantificação de linfócitos CD4+/CD8+ e de RNA-HIV; 2-Parâmetros antropométricos: Peso, estatura, circunferência abdominal e Índice de Massa Corporal (IMC); 3-Parâmetros Laboratoriais: lipoproteínas, glicemia, insulinemia, hemoglobina glicosilada e cálculo do índice HOMA IR. Devido à baixa incidência de recorrência da infeção pelo CMV (0,97/100 pessoas-mês) não foi possível analisar este fator no presente estudo. De maneira geral, na entrada do estudo aespessura da íntima/média das artérias carótidas da maioria (70%) dos adolescentes situava- se acima do Percentil 75 da distribuição de referência, sendo que durante o período de 2 anos não ocorreu incremento significativo da medida desse parâmetro arterial. Não foi identificada associação entre a magnitude da ativação da imunidade específica ao CMV e a evolução da cIMT ao longo de dois anos. Apesar de ter sido detectado que pequenos incrementos nos indicadores de ativação imunológica inespecífica (TNRFI, sCD14 e/ou HLADR+CD38+) associaram-se a discretas reduções da cIMT ao final de dois anos, a ativação imunológica de células T e monócitos não se associou ao incremento da espessura da íntima/média arterial durante esse período de tempo. Embora não tenhamos confirmado a nossa hipótese, dados obtidos nesse estudo podem se tornar referencia para planejamento de estudos mais amplos e com maior período de observação. / Human Immunodeficiency Virus (HIV) infection might predispose the presence of cardiovascular risk factors and promote the activation of the immune system contributing to the formation of atherosclerotic lesions. The presence of coinfections, such as by Cytomegalovirus, and the immunological unspecific activation may intensify the process of chronic inflammation and accelerate the vascular damage resulting from it. The main objective of this study was to evaluate whether the recurrence of CMV infection and the degree of the CMV-specific cellular immune response or the unspecific activation of T cells and monocytes were associated with the increase in the thickness of the carotid intima-media thickness (cIMT) in HIV/CMV-coinfected children and adolescents. A longitudinal study was carried out in which 40 HIV/CMV-coinfected children and adolescents were followed-up for 2 years. Periodically, it was determined the presence of CMV recurrence with the use of CMV-DNA detection in serum by Polymerase Chain Reaction; the specific immunological activation of this virus with the Quantiferon CMVR assay and the dosage of IgM and IgG antibodies against CMV. Also, we measured the nonspecific immunological activation of T cells using the soluble TNF receptor I (sTNFRI), and the presence of HLADR+CD38+TCD8+; and that of monocytes by soluble CD14 dosage (sCD14). To further characterize the studied children, other parameters were periodically evaluated: 1-HIV-related: clinical parameters and quantification of CD4+/CD8+ lymphocytes and HIV-RNA; 2-Anthropometric parameters: weight, height, abdominal circumference, and Body Mass Index (BMI); 3-Laboratory parameters: lipoproteins, fasting glucose, fasting insulinemia, glycosylated hemoglobin, and calculation of HOMA IR. Due to the low incidence of CMV recurrence (0.97/100 persons-month), it was not possible to analyze this factor in the present study. Overall, the cIMT of most (70%) adolescents were higher than the 75 percentile of the reference distribution at enrollment, and no significant increment occurred over a 2 year period. No association between the degree of CMV-specific immunity activation and the evolution of cIMT over 2 years was identified. Although it was found that small increments on the unspecific immunological activation markers (TNRFI,sCD14 and/or HLADR+CD38+) were associated with discrete reductions of the cIMT at the end of two years, the immunological activation did not associate with an increment of the carotid intima/media thickness over this time period. Even if we have not confirmed our hypothesis, the data obtained in this study can be used for planning bigger studies with a more prolonged observation period.

A infecção pelo Vírus da Imunodeficiência Humana (HIV) pode predispor à presença de fatores de risco cardiovascular e promover a ativação do sistema imunológico, contribuindo para a formação de lesões ateroscleróticas. A existência de coinfecções, como pelo Citomegalovírus (CMV) e a ativação imunológica inespecífica poderiam intensificar o processo de inflamação crônica e acelerar os danos vasculares decorrentes desta. O objetivo principal deste estudo foi avaliar se a recorrência da infecção pelo CMV, a magnitude da resposta imunológica específica ao CMV ou da ativação inespecífica de células T e monócitos associava-se ao aumento da espessura das camadas média e íntima das carótidas (cIMT) em crianças e adolescentes coinfectados pelo HIV e CMV. Consistiu-se de um estudo longitudinal, em que 40 crianças e adolescentes coinfectados pelo HIV e CMV foram acompanhados por 2 anos. Periodicamente avaliou-se a presença de recorrência do CMV, com o uso de detecção de DNA do CMV no soro por meio da técnica de Reação em Cadeia da Polimerase; a ativação imunológica perante este vírus com o ensaio do Quantiferon CMVR, a dosagem de anticorpos IgM e IgG contra o CMV. Também, nós medimos a ativação imunológica inespecífica de células T usando a dosagem do receptor I de TNF solúvel (sTNFRI) e a quantificação da presença HLADR+CD38+TCD8+; e a de monócitos por meio da dosagem de CD14 solúvel (sCD14). Para caracterizar adicionalmente as crianças estudadas, outros parâmetros foram registrados periodicamente: 1-Relativos à infecção pelo HIV: parâmetros clínicos e quantificação de linfócitos CD4+/CD8+ e de RNA-HIV; 2-Parâmetros antropométricos: Peso, estatura, circunferência abdominal e Índice de Massa Corporal (IMC); 3-Parâmetros Laboratoriais: lipoproteínas, glicemia, insulinemia, hemoglobina glicosilada e cálculo do índice HOMA IR. Devido à baixa incidência de recorrência da infeção pelo CMV (0,97/100 pessoas-mês) não foi possível analisar este fator no presente estudo. De maneira geral, na entrada do estudo aespessura da íntima/média das artérias carótidas da maioria (70%) dos adolescentes situava- se acima do Percentil 75 da distribuição de referência, sendo que durante o período de 2 anos não ocorreu incremento significativo da medida desse parâmetro arterial. Não foi identificada associação entre a magnitude da ativação da imunidade específica ao CMV e a evolução da cIMT ao longo de dois anos. Apesar de ter sido detectado que pequenos incrementos nos indicadores de ativação imunológica inespecífica (TNRFI, sCD14 e/ou HLADR+CD38+) associaram-se a discretas reduções da cIMT ao final de dois anos, a ativação imunológica de células T e monócitos não se associou ao incremento da espessura da íntima/média arterial durante esse período de tempo. Embora não tenhamos confirmado a nossa hipótese, dados obtidos nesse estudo podem se tornar referencia para planejamento de estudos mais amplos e com maior período de observação. / Human Immunodeficiency Virus (HIV) infection might predispose the presence of cardiovascular risk factors and promote the activation of the immune system contributing to the formation of atherosclerotic lesions. The presence of coinfections, such as by Cytomegalovirus, and the immunological unspecific activation may intensify the process of chronic inflammation and accelerate the vascular damage resulting from it. The main objective of this study was to evaluate whether the recurrence of CMV infection and the degree of the CMV-specific cellular immune response or the unspecific activation of T cells and monocytes were associated with the increase in the thickness of the carotid intima-media thickness (cIMT) in HIV/CMV-coinfected children and adolescents. A longitudinal study was carried out in which 40 HIV/CMV-coinfected children and adolescents were followed-up for 2 years. Periodically, it was determined the presence of CMV recurrence with the use of CMV-DNA detection in serum by Polymerase Chain Reaction; the specific immunological activation of this virus with the Quantiferon CMVR assay and the dosage of IgM and IgG antibodies against CMV. Also, we measured the nonspecific immunological activation of T cells using the soluble TNF receptor I (sTNFRI), and the presence of HLADR+CD38+TCD8+; and that of monocytes by soluble CD14 dosage (sCD14). To further characterize the studied children, other parameters were periodically evaluated: 1-HIV-related: clinical parameters and quantification of CD4+/CD8+ lymphocytes and HIV-RNA; 2-Anthropometric parameters: weight, height, abdominal circumference, and Body Mass Index (BMI); 3-Laboratory parameters: lipoproteins, fasting glucose, fasting insulinemia, glycosylated hemoglobin, and calculation of HOMA IR. Due to the low incidence of CMV recurrence (0.97/100 persons-month), it was not possible to analyze this factor in the present study. Overall, the cIMT of most (70%) adolescents were higher than the 75 percentile of the reference distribution at enrollment, and no significant increment occurred over a 2 year period. No association between the degree of CMV-specific immunity activation and the evolution of cIMT over 2 years was identified. Although it was found that small increments on the unspecific immunological activation markers (TNRFI,sCD14 and/or HLADR+CD38+) were associated with discrete reductions of the cIMT at the end of two years, the immunological activation did not associate with an increment of the carotid intima/media thickness over this time period. Even if we have not confirmed our hypothesis, the data obtained in this study can be used for planning bigger studies with a more prolonged observation period.

Cytomegalovirus (CMV)-specific T cells expand with CMV reactivation and are probably prerequisite for control and protection. Given the critical role STAT5A phosphorylation (pSTAT5A) in T cell proliferation, this study presents a simple and sensitive flow cytometric-based pSTAT5A assay to quickly identify CMV-specific T cell proliferation. We determined pSTAT5A in T cells treated with CMV-specific peptide mix (pp65 + IE1 peptides) from 20 healthy adult subjects and three immunodeficient patients with CARMIL-2 mutation. After stimulation, the percentage of pSTAT5A+ T cells in CMV-seropositive (CMV+) subjects significantly increased from 3.0% ± 1.9% (unstimulated) to 11.4% ± 5.9% (stimulated) for 24 h. After 7 days of stimulation, the percentage of expanded T cells amounted to 26% ± 17.2%. Conversely, the percentage of pSTAT5A+ T cells and T cell proliferation from CMV-seronegative (CMV−) subjects hardly changed (from 3.0% ± 1.3% to 3.7% ± 1.8% and from 4.3% ± 2.1% to 5.7% ± 1.7%, respectively). We analyzed the correlation between the percentage of pSTAT5A+ T cells versus (1) CMV-IgG concentrations versus (2) the percentage of expanded T cells and versus (3) the percentage of initial CMV-specific T cells. In immunodeficient patients with CARMIL-2 mutation, CMV-specific pSTAT5A and T cell proliferation were completely deficient. In conclusion, flow cytometric-based pSTAT5A assay represents an appropriate tool to quickly identify CMV-specific T cell proliferation and helps to understand dysfunctions in controlling other pathogens. Flow cytometric-based pSTAT5A assay may be a useful test in clinical practice and merits further validation in large studies.

Until recently, all in vitro drug susceptibility assays of cytomegalovirus (CMV) were performed in clinically irrelevant fibroblast cells. This study sought to test if drug susceptibility was affected by cell type. MRC-5 embryonic lung fibroblasts and ARPE-19 retinal pigmented epithelial cells were infected with BADrUL131-Y4 epithelial/fibroblast tropic virus under serial concentrations of ganciclovir (GCV) or maribavir (MBV). Virus was quantified using plaque reduction, GFP fluorescence, and yield reduction. Both drugs performed less efficiently in ARPE-19 cells. A cell type effect was observed for both plaque reduction and yield reduction assays with implications for the treatment of CMV retinitis as well as other manifestations of CMV Disease that involve non-fibroblast cell types.

Human cytomegalovirus infects approximately 50% of adults in the United States and in most cases is asymptomatic. However, in the case of immune compromised persons such as AIDS patients, transplant patients, and newborn babies, life threatening CMV disease can occur. The HCMV major immediate-early enhancer functions as a master regulatory switch, whose activation is essential for the expression of the major IE transactivating proteins, IE1 p72 and IE2 p86. While critical to the viral lifecycle, regulation of MIE enhancer activation is very complex and not yet fully understood. I characterized the role of cis-acting partners within the MIE enhancer that function to strengthen MIE gene expression and discovered a novel mode of post-IE enhancer regulation. These results add significantly to our understanding the inner workings of the HCMV MIE enhancer/promoter in lytically infected cells. The distal portion of the MIE enhancer is composed of two functionally redundant segments, which are necessary for MIE gene expression at low multiplicity of infection (MOI). Using an unbiased genetic approach I identified a previously unrecognized cis-acting TGGGCA/G repeat that is inextricably linked to GC-box repeats, which together form an enhancer-spanning network. This network of elements (TG network) is conserved in nonhuman primate CMV MIE enhancers. HCMV constructs lacking the entire enhancer TG network inadequately sustain MIE gene expression at low MOI at post-immediate early (IE) times of infection (≥8 h pi). An MIE enhancer-specified mode of post-IE regulation has not been described before and suggests a cis-regulatory code specialization that has evolved to sustain rather than to initiate MIE gene expression. I hypothesized that another cis-acting element(s) function together with the TG network to form a multi-network system that senses and integrates a variety of cellular environmental signals to modulate efficiency in the initiation and/or maintenance of MIE enhancer-dependent gene expression. Using recombinant viruses with mutations in either the cyclic AMP response element (CRE) network, NFkB network, TG network, or a combination of these networks, I show that the TG-C and TG-K partnerships are the most important for conferring the greatest level of MIE enhancer-dependent MIE gene expression, frequency and size of viral plaques in HFF cells, while the TG-K partnership is most important to DNT-2 cells. Additionally, I conclude that the C-K partnership functions through an alternate mechanism than that of the TG network. Together these results suggest that the strength of enhancement by cis-acting network pair interactions forms a multi-network system that modulates efficiency of MIE enhancer-dependent gene expression and which differs in relation to cell type during lytic infection.

L'immunothérapie cellulaire passive par injection de lymphopcytes T cytotoxiques offre des possibilités thérapeutiques nouvelles dans l'immunité anti-virale et anti-tumorale. Par rapport aux méthodes actuelles de stimulation utilisant des lignées présentatrices d'antigènes, nous essayons de développer une méthode alternative d'amplification des lymphocytes T CD8 à l'aide d'une combinaison de protéines recombinantes immobilisées sur billes.<br />L'interaction 4-1BB/4-1BBL (CD137/CD137L) constitue un des signaux de co-stimulation impliqués dans l'activation des lymphocytes T CD8 effecteurs. Le travail présenté ici décrit pour la première fois la production et la caractérisation d'une forme fonctionnelle de 4-1BBL recombinant soluble. De plus, nous montrons qu'il est possible d'amplifier in vitro des lymphocytes T mémoires anti-CMV et anti-EBV avec des complexes HLA-peptide associés à ce 4-1BBL ou à de l'anticorps anti-CD28. L'intérêt de la co-stimulation du 4-1BB est comparée à celle du CD28 dans les 2 contextes antigéniques étudiés.