Memoria para optar al título profesional de Bioquímico / El virus HTLV-I (Virus Linfotrópico humano Tipo I) es el agente etiológico de dos patologías principales, la Mielopatía asociada al HTLV-I/Paraparesia Espástica Tropical (HAM/TSP), una axonopatía neurodegenerativa, donde existe falla en el transporte axonal y la Leucemia de células T del adulto (ATL). No se ha detectado infección por HTLV-I en neuronas, desconociéndose el mecanismo por medio del cual se produce la neurodegeneración en HAM/TSP. Puesto que el reservorio principal del virus son los linfocitos T CD4+, algunos productos virales secretados por estos linfocitos infectados infiltrados en el sistema nervioso central, podrían afectar vías de transducción de señales relacionadas con la reorganización de citoesqueletos y transporte axonal, produciendo el daño axonal de distal a proximal observado en pacientes HAM/TSP. Los productos secretados por células MT2 (linfocitos infectados con HTLV-I) provocan retracción en células de neuroblastoma SH-SY5Y diferenciadas y disminuyen la velocidad de crecimiento neurítico en células PC12 durante su diferenciación a tipo neuronal. Entre las proteínas virales secretadas, se encuentra Tax, la disminución de la velocidad de crecimiento neurítico en células PC12, podría estar mediada por esta proteína viral a través de la vía transduccional que involucra a semaforinas y sus receptores Plexinas. Como un ejemplo, en el caso de Sema3A-Plexin A, un aumento de la actividad de Cdk5 causa hiperfosforilación de Tau y CRMP-2. El fallo en el transporte axoplásmico, podría explicarse por una desregulación de la fosforilación de las subunidades de los motores moleculares kinesina KIF5 y dineína citoplasmática Dync en este escenario. En este trabajo, se propuso estudiar el efecto de los productos secretados desde linfocitos infectados que incluyen Tax sobre el proceso de diferenciación neuronal. Para esto, se utilizó la línea celular de feocromocitoma de rata, células PC12 diferenciables a tipo neuronal por acción de NGF. La Hipótesis planteada es la siguiente: “La exposición a productos secretados de linfocitos infectados por HTLV-I provoca un aumento en el estado de fosforilación de proteínas motoras en modelo neuronal de células PC12” El primer objetivo desarrollado, fue determinar si Tax es uno de los productos virales secretados desde medio de cultivo de células MT-2 responsable del efecto de retardo de crecimiento neurítico de PC12. La adición al cultivo celular de anticuerpos monoclonales anti-Tax mostró que se bloqueó este efecto de retardo de crecimiento, lo que apunta a que este efecto se debe a la acción de Tax presente en el medio de secreción de las células MT-2. El segundo objetivo, contempló estandarizar protocolos de inmunodetección selectivos para determinar las subunidades asociadas al control de funcionalidad de kinesina KIF5 y dineína citoplasmática en células PC12. Los análisis por Western blot mostraron que las cantidades de las subunidades de kinesina KIF5 (cadena pesada y liviana) y dineína citoplasmática (cadena intermedia) presentes en las células PC12, no varían durante el período de diferenciación estudiado. Esto significa, que Tax no estaría alterando su expresión proteica. El tercer objetivo planteado fue comparar el grado de fosforilación de las subunidades de proteínas motoras en células expuestas a productos secretados desde células MT-2 con la condición control con medio de cultivo de la línea celular K562. Al no disponer de anticuerpos contra las formas fosforiladas de estas proteínas motoras, se procedió a separarlas mediante la técnica de electroforesis de geles en dos dimensiones. Este objetivo requirió inicialmente estandarizar esta técnica y luego se emplearon las muestras de lisados de PC12 provenientes del tercer día de diferenciación que habían sido sometidas al medio condicionado MT-2 y al de K562. Estos análisis no mostraron diferencias significativas en las migraciones electroforéticas, que podrían dar cuenta de cambios del estado de fosforilación de estas subunidades motoras. No obstante, estos resultados no se puede descartar que existan diferencias en el grado de fosforilación de las subunidades de estos motores moleculares, puesto que las diferencias en el grado de fosforilación pueden ser muy menores para ser observadas como cambios en la migración electroforética. Se debiera usar un método inmunológico más sensible empleando anticuerpos que reconozcan fosforilaciones determinadas estas proteínas / Effect of secretable proteins from HTLV-I infected lymphocytes on phosphorylation of motor proteins in PC12 cells as a model of neuronal culture HTLV-I virus (Human T-cell lymphotropic virus type I) is the etiologic agent of two main pathologies, the Myelopathy associated to HTLV-I/Tropical Spastic Paraparesis (HAM/TSP), a neurodegenerative axonopathy with impairment of axonal transport, and the Leukemia of adult T-cells (ATL). No infection in neurons has been detected, being unknown the mechanisms of the neurodegeneration in HAM/TSP. Lymphocytes T-CD4+ represent the main reservoir of the virus. Therefore, some secreted products from these infected lymphocytes infiltrated into the central nervous system could produce alteration of signal transduction pathways related with cytoskeletons and axonal transport, thus accounting for the distal to proximal axonal damage observed in HAM/TSP patients. Secreted products from MT-2 cells (HTLV-I infected lymphopcytes cell line) produce retraction in differentiated neuroblastoma SH-SY5Y cells and reduced the rate of neurite growing during PC12 cell differentiation to neuronal type. Among the viral secretable proteins is Tax, hence, the reduction of PC12 neurite growing could be mediated by this viral protein through transduction signalings that involves semaphorins and their receptor Plexins. As an example, in the case of Sema3A-Plexin A, an increase in Cdk5 activity causes Tau and CRMP-2 hyperphoshorylations. The failure of axonal transport could be explained by deregulation of phosphorylation of molecular motor subunits kinesin KIF5 and cytoplasmatic dynein Dync. In this work it was proposed to study the effect of secreted products, including Tax, from infected lymphocytes on the neuronal differentiation process. We employed the rat pheochromocytoma cell line, PC12 cells during NGF-differentiation to neuronal type. The Hypothesis stated expresses that: “Exposure to secreted products from HTLV-I infected lymphocytes causes an increase in the phosphorylation state of motor proteins in a neuronal model of PC12 cells” The first goal developed was to determine if Tax was one of the viral secreted products to the culture medium of MT-2 cell line responsible of retardation of PC12 neurite growing. Addition to the cell culture monoclonal antibodies anti-Tax blocked the effect on retardation rate of neurite growing, this pointed to the action of Tax present in the secretion medium of MT-2 cells. The second goal consisted in the standardization of selective protocols for immunodetection of subunits associated with the functional control of kinesin KIF5 and cytoplasmic dynein present in PC12 cells. Western blots analyses showed that levels of kinesin KiF5 (heavy and light chains) and cytoplasmic dynein (intermediate chain) present in PC12 cells did not change during the followed period of differentiation. This result suggests that their protein expression is not altered by Tax. The third goal included the comparison of phosphorylation levels of the subunits of the motor proteins exposed to MT-2 cell secreted products with the control condition with K562 cell line medium. Due to non-availability of the antibodies against phosphorylated forms of motor proteins, we separated them by two-dimensional gel electrophoresis. This goal initially required the standardization of the technique, and then PC12 lysates obtained at third day of differentiation in the presence of either MT-2 or K562 cell culture medium were used. These analyses did not show significant differences in the electrophoresis migrations of KiF5 and dynein that could account for changes in the phosphorylation levels of these subunits of motor proteins. In spite of our results, it cannot be discarded the existence of differences in the phosphorylation degree of the molecular motor subunits because the differences in the phosphorylation degree could be minors and undistinguishable by electrophoretic migration changes. Thereby, it should be used more sensitive immunological methods employing antibodies capable of distinguish motor proteins phosphorylations

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Human T-lymfotropt virus (HTLV) är ett C-typ onkovirus som tillhör familjen Retroviridae som huvudsakligen angriper T-lymfocyter och orsakar leukemi och andra autoimmuna sjukdomar. I den nuvarande kliniska diagnostiken används Enzyme-linked immunosorbent assay och ibland vid screeningsmetoden uppträder ospecifika reaktioner. På senare tid har flera metoder baserade på real-tids PCR utvecklats för att bestämma halten av virala genom i patienter. Syftet med denna studie var att sätta upp och optimera en realtids-PCR för detektion av humant T-lymfotropt virus typ-1 och 2. Inför optimering av realtids-PCR extraherades DNA från MT-4 och MO-T cellinjer.  Under optimering av realtids-PCR användes SYBR Green och smältpunktsanalys där flera komponenter bl.a. templat-, primer-, magnesium- koncentrationer samt annealing/elongeringstemperaturen modifierades. Efter avslutad optimering utfördes probetitrering för att specifikt skilja ut HTLV-1 och HTLV-2. Amplifieringsprodukten verifierades med Sangersekvensering. För att hitta den lämpligaste metoden för extrahering av DNA från helblod testades olika extraheringsmetoder för DNA-preparering. Efter färdig optimering såg PCR-programmet ut enligt följande: 95ºC i 3 min följt av 45 cykler med 95ºC i 3s och 60ºC i 10s. De optimala koncentrationerna av de olika reagenserna var 0,5µM HTV-F5, 0,7µM HTV-R4, 0,2µM HTLV-P1 och 0,1µM HTLV-P2.  Den optimala extraktionsmetoden från helblod visades vara med MagNA Pure Compact med 500µL helblod. Den färdig optimerade PCR-metoden är en semi-kvanitativ metod som fungerar väl för detektion av HTLV 1 och 2, men vidareutveckling av metoden krävs innan den kan tas i bruk för klinisk diagnostik.

O HTLV-1 (Human T-Lymphotropic Virus) foi o primeiro retrovírus descoberto. Sua patogenia é relacionada à infecção das células T CD4+ e T CD8+ e sua disseminação depende da expansão clonal destas células. A imortalização celular e a resposta imune inflamatória direta contra o vírus levam os pacientes a desenvolverem a leucemia/ linfoma de células T do adulto (ATL) e paraparesia espástica tropical/mielopatia (TSP/HAM) respectivamente. Ainda que o vírus seja conhecido desde 1980, não existem trabalhos na literatura que evidenciem possíveis manifestações bucais associadas. Alguns estudos clínico-epidemiológicos, realizados em regiões altamente endêmicas para o vírus, apontam a possibilidade de associação entre o HTLV e a síndrome de Sjögren (SS). Este estudo objetivou conhecer melhor uma população HTLV+ identificando possíveis alterações estomatológicas. Foram avaliados 139 pacientes do Instituto de Infectologia Emilio Ribas, sendo que 112 (80,5%) eram HTLV-1+, 26 eram (18,7%) HTLV-2 + e 1 paciente era soropositivo para ambos os tipos virais. Entre os pacientes HTLV-1+, 88 (64,7%) eram assintomáticos e 48 (35,3%) apresentavam TSP/HAM. As alterações bucais mais freqüentes foram: xerostomia (26,5%), candidíase (25,4%), língua fissurada (22,1%) e língua depapilada (12,4%). Modelos de regressão logística multivariada confirmaram a TSP/HAM como um fator de risco independente para xerostomia (p=0,02), apresentando, pacientes TSP/HAM+, 3 vezes mais chances de desenvolver xerostomia quando comparados com pacientes sem TSP/HAM (OR=2,69; 95%IC=1,17-6,17). / HTLV-1 (human T-lymphotropic virus), the first retrovirus discovered, is associated with adult T cell leukemia/lymphoma (ATL) and tropical spastic paraparesis / HTLV associated myelopathy (TSP / HAM). Clinical studies and case reports in endemic areas showed the development of oral ALT and Sjögren`s syndrome in this patients. The aim of this study was to investigate the oral cavity of HTLV seropositive patients in São Paulo city. The present study was approved by the Institute of Infectious Diseases Emílio Ribas ethics committee. All patients answered a questionnaire designed for the study. Demographic and clinical data were recorded and then analyzed using Epi info (3.3.4 version) and SPSS Statistical Package for Social Sciences (v16.0). One hundred and thirty nine oral examinations were performed, 112 (80,5%) were HTLV-1 +, 26 were (18,7%) HTLV-2 + and one patient presented both types of HTLV. Sixty four (56,7%) were asymptomatic HTLV-1 seropositive patients, fourty nine (43,3%) patients were positive for HTLV-1 and TSP/HAM. HIV and HCV co-infection and comorbities were observed in 110 (79,1%) cases. Xerostomia (26,5%), candidosis (25,4%), oral fissured tongue (22,1%) and papillary atrophy of the tongue (12,4%) were the most prevalent oral manifestations found in these patients. Models of multivariate logistic regression confirmed the TSP / HAM as an independent risk factor for xerostomia (p = 0.02). Patients with TSP / HAM + were 3 times more likely to develop xerostomia when compared with patients without TSP / HAM (OR = 2.69, 95% CI = 1.17-6.17).

O HTLV-1 (Human T-Lymphotropic Virus) foi o primeiro retrovírus descoberto. Sua patogenia é relacionada à infecção das células T CD4+ e T CD8+ e sua disseminação depende da expansão clonal destas células. A imortalização celular e a resposta imune inflamatória direta contra o vírus levam os pacientes a desenvolverem a leucemia/ linfoma de células T do adulto (ATL) e paraparesia espástica tropical/mielopatia (TSP/HAM) respectivamente. Ainda que o vírus seja conhecido desde 1980, não existem trabalhos na literatura que evidenciem possíveis manifestações bucais associadas. Alguns estudos clínico-epidemiológicos, realizados em regiões altamente endêmicas para o vírus, apontam a possibilidade de associação entre o HTLV e a síndrome de Sjögren (SS). Este estudo objetivou conhecer melhor uma população HTLV+ identificando possíveis alterações estomatológicas. Foram avaliados 139 pacientes do Instituto de Infectologia Emilio Ribas, sendo que 112 (80,5%) eram HTLV-1+, 26 eram (18,7%) HTLV-2 + e 1 paciente era soropositivo para ambos os tipos virais. Entre os pacientes HTLV-1+, 88 (64,7%) eram assintomáticos e 48 (35,3%) apresentavam TSP/HAM. As alterações bucais mais freqüentes foram: xerostomia (26,5%), candidíase (25,4%), língua fissurada (22,1%) e língua depapilada (12,4%). Modelos de regressão logística multivariada confirmaram a TSP/HAM como um fator de risco independente para xerostomia (p=0,02), apresentando, pacientes TSP/HAM+, 3 vezes mais chances de desenvolver xerostomia quando comparados com pacientes sem TSP/HAM (OR=2,69; 95%IC=1,17-6,17). / HTLV-1 (human T-lymphotropic virus), the first retrovirus discovered, is associated with adult T cell leukemia/lymphoma (ATL) and tropical spastic paraparesis / HTLV associated myelopathy (TSP / HAM). Clinical studies and case reports in endemic areas showed the development of oral ALT and Sjögren`s syndrome in this patients. The aim of this study was to investigate the oral cavity of HTLV seropositive patients in São Paulo city. The present study was approved by the Institute of Infectious Diseases Emílio Ribas ethics committee. All patients answered a questionnaire designed for the study. Demographic and clinical data were recorded and then analyzed using Epi info (3.3.4 version) and SPSS Statistical Package for Social Sciences (v16.0). One hundred and thirty nine oral examinations were performed, 112 (80,5%) were HTLV-1 +, 26 were (18,7%) HTLV-2 + and one patient presented both types of HTLV. Sixty four (56,7%) were asymptomatic HTLV-1 seropositive patients, fourty nine (43,3%) patients were positive for HTLV-1 and TSP/HAM. HIV and HCV co-infection and comorbities were observed in 110 (79,1%) cases. Xerostomia (26,5%), candidosis (25,4%), oral fissured tongue (22,1%) and papillary atrophy of the tongue (12,4%) were the most prevalent oral manifestations found in these patients. Models of multivariate logistic regression confirmed the TSP / HAM as an independent risk factor for xerostomia (p = 0.02). Patients with TSP / HAM + were 3 times more likely to develop xerostomia when compared with patients without TSP / HAM (OR = 2.69, 95% CI = 1.17-6.17).

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 188-225).

Made available in DSpace on 2013-08-07T18:41:35Z (GMT). No. of bitstreams: 1 000392212-Texto+Completo-0.pdf: 1435232 bytes, checksum: c5e320809f8271743be3719e2d01aeda (MD5) Previous issue date: 2007 / Introduction and Objectives: HTLV-I/II viruses have a special tropism for infecting T cells and inducing spontaneous lymphocyte proliferation. Leukemia and inflammatory states such as neurological manifestations are associated with HTLV-I/II infections and treatment is usually based on antiinflammatory drugs including glucocorticoids. Although steroid resistance has been reported, it is unknown whether this condition is related to the viral infection itself or treatment. Here, we investigated whether spontaneous cell proliferation is associated to T-cell sensitivity to glucocorticoids (dexamethasone – DEX). Materials ad Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from assinthomatic drug-free HTLV-I (n=18) and HTLVII (n=10) infected patients, as well as from healthy subjects (Control, n=11) and cultivated with and without phytohemmaglutinin (PHA 1%).Cells were also cultivated with PHA 1% and several concentrations of DEX (10-9 a 10-4 M). Results and Discussion: Patients with HTLV-I/II showed similar unstimulated and stimulated T-cell proliferation as well as comparable sensitivity to dexamethasone in vitro. There were no group differences in the frequency of glucocorticoid responders versus non-responders. However, T cells of patients with spontaneous proliferation were unresponsive to mitogenic stimulation and remarkably more resistant to dexamethasone than cells of patients with normal proliferation. These data suggest that the poor clinical response to steroids may be associated to spontaneous cell proliferation during HTLV infection. / Introdução e Objetivos: As infecções por HTLV-I/II se caracterizam pelo tropismo viral por infectar células T humanas e promover um estado de proliferação espontânea deste tipo celular. Leucemia e doenças inflamatórias graves, com destaque para as doenças neurológicas, estão fortemente associadas às infecções por HTLV-I/II e o tratamento usual destas patologias envolve a administração de antiinflamatórios da classe dos glicocorticóides. Embora haja relatos de resistência farmacológica à terapia com glicocorticóides, não se sabe ao certo se essa condição se deve à infecção viral ou ao hospedeiro. Neste estudo, avaliou-se a relação entre proliferação celular espontânea e sensibilidade de linfócitos T aos efeitos dos glicocorticóides (dexametasona - DEX). Materiais e Métodos: Células mononucleares de sangue periférico foram isoladas de pacientes assintomáticos e livres de tratamento, com infecção por HTLV-I (n=18) e HTLV-II (n=10), e foram cultivadas na presença e na ausência do mitógeno fitohemaglutinina (PHA 1%).Células foram também cultivadas com PHA 1% e concentrações variadas de DEX (10-9 a 10-4 M). Para fins comparativos, as mesmas avaliações foram realizadas em sujeitos saudáveis (Controle, n=11). Resultados e Discussão: Os pacientes com HTLV-I/II apresentaram taxas similares de proliferação estimulada (PHA 1%) e não-estimulada (PHA 0%), bem como sensibilidade comparável à DEX. Não houve diferença na freqüência de indivíduos resistentes versus sensíveis à DEX entre os grupos HTLV-I e HTLV-II. Entretanto, linfócitos T de pacientes com proliferação espontânea não responderam ao estímulo mitogênico por PHA e foram mais resistentes à modulação por DEX do que as células de pacientes com proliferação normal. Os resultados aqui apresentados sugerem que a baixa resposta clínica à terapia com glicocorticóides pode estar associada a um estado de proliferação celular espontânea decorrente da infecção por HTLV.