Co-infecção pelo vírus da hepatite G em pacientes infectados pelo Leihsmania chagasi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Estudo clínico-laboratorial e histopatológico de cães naturalmente infectados por Leishmania chagasi com diferentes graus de manifestação física

Canine Visceral Leishmania (CVL) is an important zoonotic disease that has a world wide distribution and has a large impact on public health on the American Continent, especially in Brazil, where the nature of endemic diseases in humans affects a large part of the nation. The influence of the prevalence of CVL in the increased rate of human cases in endemic areas and in the unleashing of epidemic outbreaks shows the need for a more profound understanding, that would generate significant advances in the current measures used to control the reservoirs of sickness that are practiced by the Programa Nacional de Vigilância e Controle da Leishmaniose Visceral. The present work describes and compares the clinical-laboratorial and histopathological findings of twenty-three dogs that were naturally infected by Leishmania chagasi, from endemic areas in metropolitan Natal, Rio Grande do Norte, Brazil. These animals, that were selected and given physical and serological exams (IFI and ELISA rK-39), were classified according to the degree of clinical severity and had blood samples drawn (whole blood and serum) for a complete hemogram and a coagulogram to be done as well as biochemical tests for kidney and liver function. The confirmation of infection by L. chagasi was done after the euthanasia of the animals, through the direct demonstration of the parasite in the impression of the spleen and liver crowned with GIEMSA and through a cultivation by means of NNN/Schneider. According to the clinical evaluation, the animals were classified as asymptomatic (7), oligosymptomatic (7) and polysymptomatic (9). Among the animals that were chosen to be autopsied, there were 2 asymptomatic, 3 oligosymptomatic and 3 polysymptomatic, for the purpose of studying their histopathology, having collected fragments of the spleen, liver, kidneys and skin and were fixed in 10% tamponed formol. The comparison between the average parameters of the clinical-laboratory tested animals in the groups was done through the Student t test (a<0.05). The main clinical signals observed were lymphadenomegaly, alopecy, dermatitis, exfoliation, cutaneous ulcers, onicogriphosis and emaciation. The main clinical-laboratorial alterations established, mainly in the polysymptomatic group, were anemia, hyperproteinemia, hyperglobulinemia, alterations in the albumin/globulin ratio and increased ALT activity. Renal alterations were not verified (urea and creatinine levels were normal). Thrombocytopenia was observed in three clinical groups. However, the other indicators of coagulation function (TAP and TTPA) did not have abnormal variations. There were inflammatory infiltrations and leishmania amastigotes in the skin of polysymptomatic dogs, however, they were not found in the skin of asymptomatic animals. Hypertrophy and hyperplasia of the phagocyte mononuclear system, leishmania amastigote parasites were found in the macrophages, extramedullary hematopoiesis and degenerative alterations were detected in the spleen and liver of 8 of the animals submitted to histopathological exams. In accord with these results, it was demonstrated that the expected alterations in the hematological and biochemical parameters in function of their viscerotropic nature of CVL are mainly observed in the more advanced stages of the disease. The absence of inflammatory infiltration and parasite load in the skin suggest that infected animals without symptoms may have an importance irrelevant to the infectiousness of the vector

Flebotomíneos (Diptera: Psychodidae) em um sistema agroflorestal da Região Metropolitana de Natal, Rio Grande do Norte, Brasil

Sandflies stand out as important vectors of leishmaniasis. The females need to ingest blood meals, enabling them to transmit protozoa of the genus Leishmania, which may give rise to visceral leishmaniasis (VL) or American tegumentary leishmaniasis (ATL), in addition to transmitting other parasites. Leishmaniasis are important infirmities, distributed worldwide, whose infection results from the interaction of reservoir animals, the vector insect, parasitic protozoa and the healthy host. In the state of Rio Grande do Norte (RN), Brazil, these insects are important transmitters of VL, which usually presents in the most serious form. It occurs mainly in metropolitan areas, with the dog as its main reservoir and Lutzomyia longipalpis as the vector. ATL is most present in the highland areas of the state. In addition to hematophagia, engaged in by the females, both sexes need to ingest carbohydrates, which are essential to the sand flies energy requirements and may interfere in the development of Leishmania. The aim of this study was to determine the occurrence and abundance of sand flies in different environments on the farm belonging to the Empresa de Pesquisas Agropecuárias do RN (Institute of Agricultural Research of RN), in the municipality of Parnamirim, in order to relate this occurrence with climatological and biological references and eating habits. Three consecutive monthly collections were carried out with CDC traps in a fragment of the Atlantic Forest, in a residence, on a goat breeding farm and on cashew, dwarf and giant coconut, mango, banana, eucalyptus, acacia and bean plantations. A total of 1241 sandflies from eight species (Lutzomyia evandroi, Lutzomyia longipalpis, Lutzomyia shannoni, Lutzomyia sordellii Lutzomyia walkeri, Lutzomyia wellcomei, Lutzomyia whitmani, and Lutzomyia intermedia) were collected, most in the forest environment. L. longipalpis, the main VL transmitter, was confirmed as a species adapted to anthropic environments, whereas others such as L. wellcomei, the vector of ATL, occurred predominantly in forests. Carbohydrate characterization of the sand flies and plants of the region demonstrated that a number of exotic plants such as hay and eucalyptus may play some role in the adaptation of these species to modified environments. Breeding in laboratory showed a mean biological cycle of 53.5 days from egg to adulthood for L. shannoni and the possibility of diapause behavior in L. wellcomei. This study serves as a source of information that may contribute to the epidemiological vigilance of tegumentary and visceral leishmaniasis in the state, given that it analyzes the bioecology of transmitting species, as well as their potential to adapt to new environments

Comportamento adaptativo de Lutzomyia longipalpis (Diptera: Psychodidae) às áreas urbanas da cidade de Teresina-PI.

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae) is currently appointed as the main vector of visceral leishmaniasis in the Americas. The growth of cities in areas originally endemics to American Visceral Leishmaniasis (AVL) resulted in the spread of the disease at the same time that observed the adaptation of this species to the urban environment.Changes in behavior of L.longipalpis that enabled the adapt to increasing losings of biodiversity, as well as the frequent exposure of the vector to insecticides evident in urban areas, could justify the increasing population of the species and consequently the spread of disease for these environments .Thus, we selected sixty houses spread among three areas with increasing stages of occupation of an area endemic for AVL in Teresina-PI. We evaluated the correlation between the density of L.longipalpis captured and different aspects, such as population density of animals, vegetation cover and socio-economic aspects in each house. In addition to the correlations, the feeding preference of the vector between the predominant plant species in the neighborhoods, as well as the presence of metabolic mechanisms of resistance among the captured insects were tested. The results showed that over the growing occupations, represented by three areas, L.longipalpis demonstrate its adaptive nature through an apparent opportunistic behavior in relation to sources of carbohydrates and blood. On the evolutionary point of view, this behavior may have favored its vector competence in urban areas among the limited presence of food sources, as well as in various environments encountered.

Antígenos da forma amastigota de Leishmania chagasi identificados por dupla varredura da biblioteca de cDNA

Control of human visceral leishmaniasis in endemic regions is hampered in part by the lack of knowledge with respect of the role reservoirs and vector. In addition, there is not yet an understanding of how non-symptomatic subclinical infection might influence the maintenance of infection in a particular locality. Of worrisome is the limited accessibility to medical care in places with emerging drug resistance. There is still no available protective vaccine either for humans or other reservoirs. Leishmania species are protozoa that express multiple antigens which are recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the causative agent of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T and T-dependent B cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second step screen for their ability to cause proliferation and IFN-γ responses of T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The clones encoded part of the coding sequence of glutamine synthetase, transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these protein Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against Leishmania

Efeito do sildenafil na lesão renal por isquemia-reperfusão em ratos

A lesão renal causada pelo processo isquemia-reperfusão ocorre em várias intervenções cirúrgicas, como transplantes de rins, cirurgias vasculares renais e na nefrectomia parcial. Devido ao processo isquêmico, ocorre disfunção do órgão e morte celular. Com o objetivo de avaliar o efeito do sildenafil, em ratos, administrado previamente à isquemia renal e reperfusão, em avaliações cintilográficas e histopatológicas, foram utilizados vinte e quatro ratos Wistar, aleatoriamente distribuídos em dois grupos. Os animais receberam 0,1 ml IV 99mTecnécio-Etilenodicisteína, realizando-se a cintilografia renal inicial e, em seguida, foram submetidos à laparotomia, provocando-se isquemia no rim esquerdo, com oclusão da artéria renal, durante 1 hora, com posterior reperfusão. Os animais do grupo sildenafil receberam previamente 1mg/kg de sildenafil via oral, 60 minutos antes da isquemia. O grupo controle recebeu somente solução salina. Após a isquemia e reperfusão, metade dos animais de cada grupo foi avaliada com 24 horas e a outra metade, com sete dias, com nova cintilografia renal. Após eutanásia, com superdose de anestésico, os rins foram retirados e submetidos a exame histopatológico. Empregou-se avaliação estatística com o teste t de Student e com teste não-paramétrico de Mann-Whitney. Foi observado no rim esquerdo do grupo controle um déficit funcional nas imagens cintilográficas, após sete dias, comparativamente ao respectivo estudo cintilográfico inicial (p<0,05). Nos rins esquerdos dos animais do grupo controle (24 horas pós-isquemia), ocorreu um maior grau de necrose celular quando comparados ao grupo tratado com o sildenafil (p<0,05). A cintilografia e a histopatologia demonstraram que o sildenafil exerceu ação protetora dos rins, após episódio de isquemia-reperfusão renal normotérmica. Deve ressaltar-se o x caráter interdisciplinar desta tese, que contou com a participação ativa de profissionais das áreas de cirurgia, medicina nuclear, urologia, patologia e estatística

Associação entre fatores nutricionais e a resposta frente à infecção por Leishmania chagasi

Leishmania chagasi infection presents a wide spectrum of clinical outcomes, ranging from asymptomatic self resolving infection to disease, visceral leishmaniasis (VL). The exact mechanisms that lead the evolution of infection to disease are not understood. It is believed that malnutrition is a risk factor associated with VL development, although there are few human studies in the area. We aimed to assess the nutritional factors associated with the response to L. chagasi infection in Rio Grande do Norte. The study was conducted from December 2006 to January 2008. 149 children were assessed: 20 active VL cases, 33 children with VL history, 40 DTH+ asymptomatic children and 56 DTH-. Nutritional status was assessed using z scores for Weight/Age, Weight/Height, Height/Age, Body Mass Index (BMI), and mid-upper arm circumference/height (MUAC/height). Vitamin A status was determined by serum retinol concentrations and the modified-relative-dose-esponse test (MRDR). Breastfeeding time and birth weight were also evaluated. VL children presented compromised nutritional status when compared to the other groups using BMI and MUAC/age, with means -1,53 ± 1,10 and -1,48 ± 1,28 z scores, respectively (ANOVA, p < 0,05). VL children also showed lower vitamin A levels: 43% presented serum retinol < 20 µg/dL and 15% MRDR > 0,060. Birth weight was inverserly associated with the risk to belong the VL group (β = -0,00; OR = 0,84; 95% CI 0,73 - 0,99; p = 0,047), whereas more breastfeeding time was directly associated with the risk to belong to the DTH+ group (β = 0,02; OD = 1,16; 95% CI 1,01 - 1,33; p = 0,036). The nutritional variables evaluated were associated with the response to the L. chagasi infection, with malnutrition and compromised vitamin A status as markers of children who present with VL. Higher birth weight was associated with protection to disease, and higher breastfeeding time was associated with increased likelihood of an asymptomatic infection. The results show that modifiable nutritional aspects in the study population are associated with the response to the L. chagasi infection

Fatores genéticos e étnicos relacionados à evolução da infecção por Leishmania infantum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Determinantes envolvidos na resposta imune celular humana à infecção por Leishmania infantum chagasi

Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-γ (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-γ produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients

Aspectos clínicos e imunológicos da infecção por Leishmania Infantum Chagasi em cães do Rio Grande do Norte / Paula Vivianne Souza de Queiroz. Natal/RN

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Aspectos ambientais e sociais envolvidos na transmissão da L. chagasi no município de Parnamirim/RN

Visceral leishmaniasis hás adapted in the past 20 years to periurban and urban areas, and in Natal, Rio Grande do Norte, it became endemic. Thid study aimed to evaluate the environmental and social aspectsof Leishmania chagasi infection and its epidemiologic transmission chain in an urban, periurban and rural area of Parnamirim-RN. A study with three sections was conducted: Section 1: Sectional study of the human and canine infection by L. chagasi and its environmental and social determinants. Section 2: Observational longitudinal cohort to evaluate the dynamics of the canine infection. Section 3: Longitudinal study to evaluate the behavior of Lu. Longipalpis vector and the seasonal factors related to its dynamics. To include in the study the hauses were randomly selected and georreferenciated. Montenegro skin test was done in the human population and blood samples were collected for anti-Leishmania antibody detection. The canine population was examinated for L. chagasi infection by RIFI, ELISA and ELISA for rK39. An entomologic surveillance was monthly done with CDC light trapsin 10 houses of each locality. Quantitative and qualitative analyses was done using STATISC 6.0. Probality and prediction maps were done using ArcGis 9.0 model. In the human population L. chagasi infection was associated with the area of the hause, age, sex, population densyti, vegetation, kind of the floor of thr hause, water and resides destiny. In the canine population L. chagasi infection was associated with the breed, size, time of living in the hause, presence of dogs in the neighborhood, presence of horses and donkeys in the neighborhood, vegetation, kind of the floor and walls of the hause. The human infection was associated with canine infction only when analyzed taking into account the locality. In the prospective study, serum conversion and antibody lost observed in 30,8% and 22% of the animals examined, respectively. The human infection rate by L. chagasi was 24,6%, by the presence of anti-Leishmania antibody and 38,6% by the Montenegro skin test. The canine infection rate 32,5% by the presence of anti-Leishmania antibody. The vector Lu longipalpis showed an atypical behavior. These results indicate that environmental and social factors are important variables associated with L. chagasi infection in humans and canines, with punctual association of thr last two. Control measures of the infection on the studied points are necessary, in the aim to reduce the endemic focus of the disease in the study area. This research was carried out in a multidisciplinary involving the categories of: doctor, biologist, veterinarian, statistical, pharmaceutical and biochemical

IL-10 na patogênese da infecção por Leishmania infantum

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Câncer do colo uterino: rastreamento, marcador biológico e polimorfismo genético

Existem fortes evidências de que os programas de rastreamento baseados em citologia resultaram em diminuição significativa da incidência e mortalidade por câncer do colo do útero, no entanto, um excesso substancial de tratamento de lesões intraepiteliais de baixo grau que dificilmente progrediriam para carcinoma cervical resulta da baixa especificidade do tradicional rastreio citológico. A detecção precoce das lesões através do rastreamento citológico e a avaliação do grau histológico em espécimes cervicais são fundamentais, entretanto não permitem identificar quais pacientes terão maior probabilidade de progressão para lesões de alto grau e carcinoma invasivo. A busca de potenciais marcadores de prognóstico; objetivando o entendimento da progressão das lesões intraepiteliais é de suma importância. Acredita-se que fatores imunoregulatórios, imunogenéticos e proteínas do ciclo celular estejam intimamente envolvidos no processo de carcinogênese. Considerando o exposto, a proposta do projeto foi inicialmente avaliar a eficácia da citologia oncológica no rastreamento do câncer cervical, foi investigado ainda o polimorfismo do gene do fator de transcrição FOXP3 e a expressão da proteína do ciclo celular P63 (P63) associados respectivamente a diagnóstico e prognóstico das lesões cervicais. Em um primeiro momento foi realizado estudo transversal que envolveu 3194 mulheres. As participantes foram submetidas à citologia e biópsia de colo dirigida por colposcopia e os resultados foram comparados para verificar-se a acurácia do teste de Papanicolaou na detecção de lesões intraepiteliais e câncer cervical. Posteriormente, realizou-se estudo comparativo do tipo observacional estratificado em três grupos: Grupo 1: 16 casos com diagnóstico histopatológico de metaplasia/cervicite, considerados normais, Grupo 2: 11 casos com lesão de baixo grau (LSIL) e Grupo 3: 15 casos com lesão de alto grau (HSIL) ou carcinoma epidermoide de colo. Um total de 42 participantes respondeu a um questionário epidemiológico padronizado sobre as características demográficas, hábitos pregressos, história reprodutiva e de comportamento sexual. Após exame colposcópico, foram coletados fragmentos de espécimes cervicais para a pesquisa da expressão proteica da P63 por imunohistoquímica. Amostras de sangue periférico foram coletadas para extração do DNA e detecção do polimorfismo do gene FOXP3. No primeiro estudo em que se avaliou a acurácia do teste de Papanicolaou, encontrou-se sensibilidade de 0,83, valor preditivo positivo (VPP) de 0,77 e especificidade de 0,23 no rastreamento das lesões cervicais e câncer de colo. viii Melhores resultados foram observados quando se avaliou a acurácia diagnóstica para lesões de alto grau e carcinoma com VPP de 0,99 e especificidade de 0,84.No estudo subsequente onde se comparou a expressão da proteína P63 observou-se maior número de núcleos marcados no grupo com lesões intraepiteliais de alto grau e câncer quando comparado ao grupo com biópsias negativas (p=0,0004). No último estudo pesquisou-se a associação do polimorfismo do gene FOXP3 com lesões intraepiteliais cervicais sendo evidenciada maior prevalência do genótipo heterozigoto, CT, no grupo com lesões de colo na histopatologia (p=0,027). Mulheres com lesões intraepiteliais de baixo ou alto grau e câncer de colo de útero apresentam maior expressão da proteína P63 e maior prevalência de genótipo heterozigoto do gene FOXP3 em comparação com as sem lesões cervicais. A associação da pesquisa da expressão da proteína e do polimorfismo do gene pode tornar os exames utilizados atualmente para a avaliação diagnóstica e prognóstica das lesões de colo uterino mais efetivos em detectar quais as mulheres com maior risco para progressão para câncer

Identificação dos princípios ativos presentes no extrato etanólico de cereus jamacaru e avaliação em ratos dos possíveis efeitos tóxicos e/ou comportamentais da exposição prolongada

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Ação da β-1,3 glucana no sistema imune de camundongos com sepse por Candida albicans

The frequency of disseminated candidiasis caused by yeast has enhancing in intensive care unit. Despite the availability of new antifungal drugs, C. albicans sepsis mortality causes can be as high as 30-40%. So, it has been needed to looking for a new therapeutic medicament that helps in treatment and prevention of this infection. Previous data that demonstrated that particulated β-glucan stimulates the immune system and experiments of this work were conducted to investigating if β-glucan extracted from Saccharomices cerevisiae, could modified the evolution of mouse model C. albicans systemic infection. Balb/c mice with sepsis and β-1,3 glucan treated or not were analyzed the influence of β-1,3 glucan in survival of the animals, in the fungal burdens in kidney, in the production of urea and TNF even in the histopathology of kidney. The experiments shown that the infected animals a nd glucan treated had great survival (p<0,05), less unit form colony in kidney and normal levels of urea. In the kidney histopathology of not glucan treated animals it has seen more lesions when compared with treated animals. So we conclude that β-1,3 glucan could stimulate the immune system against disseminated C. albicans