A pele e as mucosas constituem as primeiras barreiras na defesa contra infecções e os macrófagos são componentes essenciais do sistema imune inato, importante neste aspecto. O envolvimento destas células pode ser verificado em grande percentual das imunodeficiências primárias. Desta forma, a avaliação da função fagocitária é de extrema relevância para o reconhecimento dos distúrbios imunológicos que acometem a pele. O objetivo do presente estudo foi avaliar a metodologia laboratorial para a detecção de defeitos funcionais dos fagócitos. Para isto foram estabelecidos os seguintes testes laboratoriais: Nitro Blue Tetrazolium (NBT), Dihidrorodamina (DHR), quimiotaxia, fagocitose e a aderência de S. aureus e C. albicans por citometria de fluxo (CF), além de morte intracelular de S. aureus e C. albicans (CF). Para verificar a integridade do sistema complemento realizou-se ensaios hemolíticos para as vias clássica e alternativa (CH50 e AP50). A metodologia proposta foi aplicada em indivíduos normais para a padronização dos testes. O burst oxidativo avaliado pelo teste da dihidrorodamina (DHR) foi aplicado em 101 indivíduos saudáveis e em paralelo, 50 indivíduos sadios para o teste do NBT. Os mesmos testes foram realizados em pacientes com Candidíase mucocutânea crônica (CMC) (n=9 ), Candidíase persistente (n=5), Suspeita de distúrbios de fagócitos (SDF) (n=14), Doença Granulomatosa Crônica (DGC)(n= 7) e portadores de DGC (n=5). A quimiotaxia foi padronizada em 34 controles para neutrófilos estimulados com Lipopolissacarídeo de E.Coli (LPS) e 5 com fungo Candida albicans. A técnica de fagocitose e aderência de patógenos foi padronizada com os mesmos estímulos (n=7 para fungos/n=5 para bactéria). Após a padronização, o ensaio foi aplicado em pacientes com candidíase persistente (n=5 para bactéria e n=5 para fungo) e em pacientes com CMC (n= 3 para bactéria e n=4 para fungo). Os ensaios de fagocitose e morte intracelular (capacidade bactericida e fungicida) foram padronizados em 18 indivíduos sadios para bactérias e os ensaios de morte intracelular para S. aureus foi aplicado em pacientes com CMC (n=5), com CP (n=6), com SDF (n =9) e com DGC (n=2), para os ensaios de fagocitose com morte intracelular para fungos foram utilizados 22 indivíduos saudáveis e após a padronização do ensaio foram aplicados em pacientes com CMC (n=8), pacientes com CP ( n= 7), pacientes com DGC (n=2) e indivíduos com SDF (n= 13) O ensaio de DHR foi padronizado e estabelecido em 80% de intensidade de fluorescência para células estimuladas com PMA e 15% de intensidade de fluorescência para células sem estímulo. Nos resultados do DHR encontrou-se diferença significativa no grupo de DGC (n=7)(P= 0,0001), no grupo de portadores (n=5)(P=0,0005) e no grupo de SDF (n=14)(P= 0,0053). O ensaio do DHR foi repetido após 24 horas da coleta (n=7), não se verificando alteração da resposta. A quimiotaxia mostrou diferença significativa entre C (n=4) vs SDF (n=3)(P=0,0001) e pacientes com CMC apresentaram redução da capacidade quimiotática para bactérias (n=3)e fungos (n= 4) com soro autólogo (P= 0,0246 e P=0,0109, respectivamente). Na fagocitose e aderência de bactérias inativadas ,os grupos de CMC, CP E SDF não mostraram diferenças significativas com bactérias não opsonizadas ou opsonizadas com soro AB e apresentaram menor índice de fagocitose (C x CMC)(P=0,0357) quando foram opsonizadas com soro autólogo. Na fagocitose e aderência de fungos inativados, controles e grupos de pacientes apresentaram resposta semelhante com fagocitose preservada. Os ensaios de morte intracelular para bactérias não opsonizadas houve menor expressão de fagocitose no grupo de C x SDF (P=0,0044). Na capacidade bactericida verificou-se diferença significativa entre os grupos CxCMC (P=0,0403). A opsonização das bactérias com soro AB foi significativamente diferente entre os grupos CxCP (P=0,0129) e CxSDF (P=0,0048) e com capacidade bactericida diferente entre grupos CxCP (P=0,0258) e CxSDF (P=0,0205). Na avaliação da fagocitose de bactérias opsonizadas com soro autólogo foi verificada diferença significativa entre os grupos CxCP (P=0,0013) e CxSDF (P=0,0048). Não houve diferença na capacidade bactericida dos grupos de pacientes com o controle. Os ensaios de fagocitose e morte intracelular para fungos sem opsonização não mostrou diferença estatisticamente significativa. A morte intracelular mostrou-se diferente para o grupo CxCMC (P=0,0155) e quando opsonizado com soro AB houve diferença CxCP (P=0,0369). A fagocitose com opsonização por soro autólogo significativa no grupo CxSDF (P=0,0001) e um paciente de CMC com sua fagocitose comprometida quando comparado com o controle do dia. A morte intracelular foi diferente nos grupos CxCMC (P=0,0018) e CxCP (p=0,0203). Não houve diferença estatisticamente significativa à avaliação do complemento. O ensaio do DHR mostrou ser sensível e preciso para o diagnóstico de DGC e portadores de DGC, porém pode detectar outras alterações de fagócitos. O ensaio de aderência e fagocitose mostraram-se variáveis dificultando a padronização de valores de normalidade e exclusão de defeitos. Ensaios de fagocitose com morte intracelular mostraram-se como a melhor forma de detectar distúrbios de fagócitos além do diagnóstico de DGC. A aplicação de controles do dia mostrou-se necessária e importante para a detecção de defeitos funcionais. O presente trabalho mostrou que a avaliação de distúrbios de fagócitos por morte intracelular por citometria de fluxo pode ser aplicado em outras situações clínicas com comprometimento imunológico / Skin and mucosa are part of the first barriers in the defense against infections, and the macrophages are essential components of the innate immune system, important when related to this aspect. The involvement of these cells can be seen in a large percentage of the primary immunodeficiencies. Therefore, the assessment of the phagocitary function is extremely important for the recognition of immunological disorders which affect the skin. The present study focus on the evaluation of the laboratorial methodology for the detection of functional defects of phagocytes. For this the following laboratorial tests were established: Nitro Blue Tetrazolium (NBT), chemotaxis, phagocytosis and adherence of S. aureus and C. albicans through flow cytometry (FC), besides the intracellular death of S. aureus and C. albicans (FC). To assess the integrity of the complement system hemolytic assays were performed for the classic and alternative pathways (CH50 and AP50). The proposed methodology was applied to normal individuals for the standardization of the assays. The oxidative burst evaluated through the dihydrorodamine essay (DHR) was applied to 101 healthy individuals and in parallel, 50 healthy individuals for the NBT assay. The same assays were performed on patients with Chronic mucocutaneous candidiasis (CMC)(n=9), persistent candidiasis (n=5), Phagocytes disorders suspicious (PDS) (n=14), Chronicle granulomatous disease (CGD)(n=7) and CGD carriers (n=5). Chemotaxis was standardized using 34 controls for neutrophils stimulated by lipopolisacharydes from e. coli (LPS) and 5 by C. albicans. Phagocytosis and adherence of pathogens were standardized using the same stimuli (n=7 for fungi and n=5 for bacteria). Following the standardization, the assay was applied to patients with persistent candidiasis (n=5 for fungi and n=5 for bacteria) and on patients with CMC (n=4 for fungi and n=3 for bacteria). Phagocytosis and intracellular death assays (bactericidal and fungicidal capacity) were standardized using 18 healthy individuals for bacteria and the intracellular death assays for S. aureus were applied on patients suffering from CMC (n=5), from PC (n=6), from PDS (n=9) and from CGD (n=2), for the phagocytosis with fungi intracellular death assays 22 healthy individuals were used, and following the standardization the assay was applied to patients suffering from CMC (n=8), from PC (n=7), from CGD (n=2) and PDS individuals (n=13). The DHR assay was standardized and established according to fluorescence intensity 80% for cells stimulated by PMA and fluorescence intensity 15% for cells without stimuli. In the DHR results a significant difference in the CGD group (n=7)(P= 0,0001), in the carriers group (n=5)(P=0,0005) and in the PDS group (n=14)(P= 0,0053) was found. The DHR assay was performed once again 24 hours after the sample collection (n=7) and no changes in the response were seen. Chemotaxis showed a significant difference between C (n=4) vs PDS (n=3)(P=0,0001) and patients suffering from CMC showed decreased ability in the chemotaxis of bacteria (n=3) and fungi (n=4) with autologous serum (P= 0,0246 e P=0,0109, respectively). In the phagocytosis and adherence of inactivated bacteria, the CMC, PC and PDS groups showed no significant differences with non-opsonizated bacteria or opsonizated with AB serum and presented a lower phagocytosis level (C x CMC)(P=0,0357) when they were opsonizated by autologous serum. In the phagocytosis and adherence of inactivated fungi, controls and patient groups presented a similar response with preserved phagocytosis. In the intracellular death assays for non-opsonizated bacteria there was a lower phagocytosis expression in the C x SDF group (P=0,0044). In the bactericidal ability a significant difference between the groups C x CMC was seen (P=0,0403). The opsonization of bacteria with AB serum showed a significant difference among the groups C x CP (P=0,0129) and C x SDF (P=0,0048) and with different bactericidal ability among the groups C x CP (P=0,0258) and C x SDF (P=0,0205). In the evaluation of the phagocytosis of bacteria opsonizated by autologous serum a significant difference among the groups C x CP (P=0,0013) and C x SDF (P=0,0048) was seen. There was no difference between the bactericidal ability of the patients group and control group. The phagocytosis and intracellular assays for fungi without opsonization presented no significant statistical difference. Intracellular death was different for the C x CMC group (P=0,0155) and when opsonizated by AB serum difference was shown C x CP (P=0,0369). The phagocytosis with opsonization by autologous serum presented significant difference in the C x SDF group (P=0,0001) and in a CMC patient with compromised phagocytosis when compared with the daily control. Intracellular death was different in the C x CMC (P=0,0018) and C x CP (p=0,0203) groups. There was no significant statistical difference according to the complement evaluation. The DHR assay was seen as very sensitive and precise for the diagnosis of CGD, however it can detect other phagocyte alterations. The phagocytosis and adherence assay varied a lot making the standardization of normal values and defects exclusion very difficult. Phagocytosis with intracellular death assays showed the best performance to detect phagocytes disorders besides CGD diagnosis. The use of daily controls was seen as very necessary and important to detect functional disorders. This study demonstrated that phagocytes disorder evaluation through intracellular death using flow cytometry can be applied to other clinical situations which are immunologically compromised

Resumo. Foi realizado um estudo epidemiológico para avaliação dos fatores determinantes para a transmissão da LTA. O estudo foi realizado na área urbana, periurbana e rural da cidade de Itambaracá, incluindo a Vila Rural e as localidades de Porto Almeida e São Joaquim do Pontal, as duas últimas próximas à área impactada pelas hidrelétricas de Canoas I e II, no rio Paranapanema. Capturou-se de fevereiro de 2004 a junho 2006, 3.187 flebotomíneos. As espécies predominantes foram Nyssomyia neivai (34,36%), Pintomyia pessoai (32,57%), Migonemya migonei (11,61%), Nyssomyia whitmani (8,82%) e Pintomyia fischeri (2,73%) todas elas com capacidade de transmissão da doença. Para Ny neivai, houve predominância de machos e para as outras espécies vetoras, as fêmeas prevaleceram, com diferença estatística significante (p<0,001). As espécies mais abundantes, segundo o índice de abundância das espécies padronizado, foram Ny neivai, Pi. pessoai, Ny whitmani, Br. brumpti, Mg migonei e Pi fischeri. As maiores freqüências e diversidade das espécies foram encontradas na localidade de Porto Almeida (PA), seguida por São Joaquim do Pontal (SJP). Ny neivai apresentou médias sazonais mais elevadas e na distribuição mensal, picos em fevereiro, maio e outubro. A maior média horária desta espécie foi registrada das 19:00 às 20:00 h (1,2) mas mostrou-se ativa das 23:00 às 10:00 h da manhã. A prevalência de Acs da classe IgG para LTA em RIFI, na população humana foi de 6,73%, nos caninos de 1,75% e nos equídeos de 16,0 %. Em EIE/ELISA, a prevalência para os caninos foi de 16,49%. Na PCR, todos os cremes leucocitários correspondentes à soros reagentes a Acs da classe IgG (humanos), e Ac totais (eqüinos e caninos) foram negativos para Leishmania sp. No inquérito sócio econômico, verificou-se que a população entrevistada em sua maioria pertence à etnia branca e à faixa etária de 30-50 anos, recebem em torno de dois salários mínimos, possuem ocupações voltadas à agricultura e pecuária, nível de escolaridade fundamental (1º grau), habita casas de alvenaria, possuem animais domésticos com seus abrigos próximos às casas. Realizam atividades de trabalho e lazer em áreas de risco em proximidades às matas, áreas com animais silvestres e domésticos e com presença de matéria orgânica, que são fatores atrativos aos flebotomíneos. A população entrevistada, de uma forma geral, desconhece os aspectos da transmissão da LTA. Observou-se em todas as localidades amostradas a presença de flebotomíneos, com predominância de cinco espécies com capacidade vetorial de agentes da LTA, destacando-se Ny. Neivai , incluindo os peridomicílios. Houve transmissão ativa da doença, ocorrendo dois casos notificados e autóctones, em área de baixa freqüência de espécimes. A soroprevalência na população humana (6,73%) foi considerável, embora seja uma área de baixa ocorrência da doença. Nas localidades onde havia um foco mais recente da doença, em SJP, os cães e eqüinos apresentaram sorologia positiva. A alta prevalência de reagentes na sorologia observada em eqüídeos sugere que estes animais poderiam ser usados como sentinela para a detecção precoce da circulação do agente da LTA nas áreas estudadas. / Abstract. An epidemiological study was carried out in the urban area of Itambaracá and rural areas of the same municipality, including Vila Rural, Porto Almeida and São Joaquim do Pontal, to investigate the phlebotomine fauna and behavioral aspects of its species and also some determining factors of the transmission of American cutaneous leishmaniasis (ACL). The last two localities mentioned are situated close to the areas impacted by the Canoas I and II hydroelectric plants on the Paranapanema river. A total of 3,187 phlebotomines were captured between February 2004 and June 2006. Nyssomyia neivai predominated (34.36%), followed by Pintomyia pessoai (32.6%), Migonemyia migonei (11.6%), Nyssomyia whitmani (8.8%) and Pintomyia fischeri (2.7%), all of them implicated in the transmission of the disease. Regarding Ny. neivai, males predominated over females which may indicate the proximity of its breeding places to the capture sites. As for all the other vector species, the females were predominant, with significant statistical differences (p < 0,001). Ny neivai, Pi. pessoai, Ny. whitmani, Br. brumpti, Mg. migonei and Pi. fischeri presented the highest values for the species standardized abundance index. The greatest frequencies and diversity of the species were found at Porto Almeida (PA), a locality situated close to a well preserved forest fragment and secondly at São Joaquim do Pontal (SJP), with degraded forest. Of all the species, Ny. neivai presented the highest monthly averages with the main peak occurring in successive months of April. Its greatest hourly average was registered at 19:00-20:00 hours, but the species presented activity from 23:00 p.m. to 10:00 a.m. The prevalence of Ac IgG class, serum-reagents for Leishmania obtained with RIFI from the human, canine and equine populations was 6.73% (20/297), 1.75% (5/285) and 16% (8/50), respectively. A prevalence of 16.4% was observed by the EIE/ELISA test only for the canine population. The buffy-coat of all the reagent human and animal blood samples was tested by the polymerase chain reaction (PCR) to investigate the presence of Leishmania, with negative results. In the socioeconomic survey, it was found that the majority of the people interviewed belonged to the white ethnic and 30-50 year-old groups, earning two minimum salaries, exercising occupations associated with agriculture and livestock breeding, of fundamental school level, inhabiting brick houses and keeping pets and domestic animals in and around them. They exercise their work and leisure activities in risk areas close to forest fragments, thus in areas containing both wild and domestic animals with the presence of organic matter, which attract the sandflies. The population interviewed was, in general, ignorant of the facts involved in ACL transmission. Sandflies were observed at all the sites sampled, with the predominance of five vectors of the ACL agents, with Ny. neivai, the main vector in all three areas, also being captured in the peridomiciles. Active transmission of the disease, with two cases registered, was observed at sites with very low frequency of this sandfly. The serum prevalence in the human population (6.73%) was considerable, despite this being an area of few registered ACL cases. In the localities in which there was a more recent focus of the disease, in SJP, the dogs and equines presented positive serology. The high prevalence of equine serum reagents suggests that these animals could be used as sentinels for the early detection of the presence of the ACL agent in the area studied.

Introdução. As leishmanioses são doenças infecciosas, causadas por protozoários do gênero Leishmania, transmitidos por insetos da subfamília Phlebotominae (Diptera, Psychodidae). No Estado de São Paulo a incidência da leishmaniose tegumentar americana (LTA) e da leishmaniose visceral americana (LVA) está aumentando tanto em regiões rurais como urbana e periurbanas. O Parque Estadual da Cantareira (PEC) é uma Unidade de Conservação e abrange parte da Serra da Cantareira considerada a maior floresta urbana nativa do mundo e abrange parte dos municípios de São Paulo, Caieiras, Mairiporã e Guarulhos. Objetivos. Investigar a incidência de leishmaniose nos municípios que incluem áreas do Parque Estadual da Cantareira e identificar a fauna de flebotomíneos (Diptera, Psychodidae, Phlebotominae) e seu comportamento no Parque Estadual da Cantareira (PEC) e Parque Estadual Alberto Löfgren (PEAL) e área do entorno, inferir sobre as espécies importunas ao homem e que atuam na transmissão de leishmaniose na região. Metodologia. Estimou-se a incidência das leishmanioses na população humana por meio de dados obtidos no Centro de Vigilância Epidemiológica do Estado de São Paulo (CVE/SP) e Instituto Brasileiro de Geografia e Estatística (IBGE). Para o estudo dos insetos as 6 capturas foram feitas com armadilhas automáticas luminosas (CDC) e de Shannon branca e preta entre janeiro a dezembro de 2009 em ambiente de mata e peridomicilio nos dois parques e também no seu entorno, município de Mairiporã, para se obter informações sobre abundância, riqueza, diversidade, equitabilidade ,similaridade, sazonalidade das espécies e infecção natural por flagelados, também foi investigado ritmo horário noturno, comparação de atratividade da armadilha Shannon branca e preta e antropofilia das espécies. Resultados. Não houve casos de LVA notificados para a região dentro do período estudado. O coeficiente de incidência médio acumulado/100.000 hab/ano (CI) para LTA no período de 2000 a 2009 para os municípios da região estudada foi Mairiporã (2,08), Caieiras (0,03), Guarulhos (0,008) e São Paulo (0,03). A fauna flebotomínea compreendeu 12 espécies: Brumptomyia cardosoi, Brumptomyia carvalheiroi, Expapillata firmatoi, Migonemyia migonei, Pintomyia bianchigalatiae, Pintomyia fischeri, Pintomyia monticola, Psathyromyia lanei, Psathyromyia pascalei, Psathyromyia pestanai, Psychodopygus ayrozai e Psychodopygus lloydi. Foram obtidos os seguintes valores para índice de abundância de espécie padronizado: Pi. fischeri (0,813), Mg. migonei (0,571), Br. cardosoi (0,396), Pa. pascalei (0,341) e Ps. ayrozai (0,308). A similaridade das espécies de mata e peridomicilio foi de 0,59. Os ecótopos de mata e peridomicílio com os maiores índices de riqueza e diversidade foram respectivamente, Trilha das Figueiras e em um canil de um morador do PEC. Nas armadilhas Shannon branca e preta capturou-se uma média de 217,46 insetos/armadilha e 33,45 insetos/hora; a razão de fêmea/macho de Pi. 7 fischeri foi (preta=15,08:1,0; branca = 11,43:1,0), Mg. migonei (preta = 0,57:1,0; branca = 1,65:1,0). Fêmeas de Pi. fischeri foram significativamente mais atraídas para Shannon preta (2 = 8,3; p < 0,005) e Mg. migonei para branca (2 = 59,8; p < 0,001). Picos horários de atividade para fêmeas de Pi. fischeri e Mg. migonei foram diferentes em relação às estações do ano: Pi. fischeri foi mais atraída pelas armadilhas Shannon preta e branca na primeira metade da noite durante o verão, outono e inverno e na primavera ocorreu o oposto. As análises de correlação de Pearson mostraram uma correlação positiva e estatisticamente significante em relação as média das temperaturas mensais de 7 e 15 dias anteriores ao dia de captura. Nenhuma das 60 fêmeas dissecadas apresentou flagelados. Conclusões. O município de Mairiporã em comparação com os municípios abrangidos pelo PEC tem um CI muito mais elevado. Para Brumptomyia carvalheiroi é o primeiro registro para a região da Serra da Cantareira. As altas freqüências de Pi. fischeri e Mg.migonei nas armadilhas de Shannon em peridomicílio do PEC permitem inferir sobre a antropofilia destas espécies, causando incômodo aos moradores e que podem estar implicadas na transmissão do agente da leishmaniose tegumentar na região / Introduction. Leishmaniases are infectious diseases caused by protozoa of the genus Leishmania, transmitted by insects of the subfamily Phlebotominae (Diptera, Psychodidae). In the São Paulo State the incidence of American visceral (AVL) and cutaneous leishmaniases (ACL) is increasing in rural, urban and periurban areas. The Parque Estadual da Cantareira (PEC) is a conservation area and covers part of the Serra da Cantareira, considered the largest native urban forest in the world and covers parts of the municipalities of São Paulo, Caieiras, Mairiporã and Guarulhos. Objectives. To investigate the incidence of leishmaniasis in the municipalities that include areas of the Serra da Cantareira and to identify the phlebotomine fauna and its behavior in PEC and the Parque Estadual Alberto Löfgren (PEAL) and the surrounding area, identifying species annoying to humans and which can transmit Leishmania spp. in the region. Methodology. We estimated the incidence of leishmaniasis in the human population using data obtained from the Epidemiological Surveillance Center of São Paulo (CVE/SP) and the Brazilian Institute of Geography and Statistics (IBGE). The insects were captured with automatic light traps from January to December 2009 in the 9 forest and peridomiciliary environments of PEC and its surroundings (in the municipality of Mairiporã) and also in forest of the PEAL, to obtain information on abundance, richness, diversity, evenness, and seasonal variations. In a peridomicile of the PEC captures with black and white Shannon traps were undertaken to: investigate the nocturnal activity per hourly period; the attractiveness of these colors; the natural infection of the sandflies by flagellates, and infer the anthropophily of the species. Results. No case of AVL had been notified in the area. The accumulated average incidence rate /100,000 in hab. (AAIR) for ACL during the period 2000 to 2009 for the municipalities studied was Mairiporã (2.08), Caieiras (0.03), Guarulhos (0.008) and São Paulo (0.03). The phlebotomine fauna comprised 12 species: Brumptomyia cardosoi, Brumptomyia carvalheiroi, Expapillata firmatoi, Migonemyia migonei, Pintomyia bianchigalatiae, Pintomyia fischeri Pintomyia monticola, Psathyromyia lanei, Psathyromyia pascalei, Psathyromyia pestanai, Psychodopygus ayrozai and Psychodopygus lloydi. Pi. fischeri was the most abundant species, the standardized index of abundance of species (SIAS) = 0.813, followed by Mg. migonei (SIAS = 0.571), Br. cardosoi (0.396), Pa. pascalei (0.341) and Ps. ayrozai (0.308). The similarity of species in the forested and peridomiciliary environments was 0.59. The ecotypes of forest and peridomicile with the highest levels of richness and diversity were respectively, the Figueiras trail and a kennel near a residents house. In the black and white Shannon traps averages of 217.46 insects/trap and 33.45 insects/hour were captured, the female/male rate of Pi. fischeri being, in black, 15.08:1.0 and, in white, 11.4:1.0, of Mg. migonei , 10 in black ,0.6:1.0 and, in white, 1.7:1.0. Comparing the attractiveness of the black and white Shannon traps for females, those of Pi. fischeri were significantly more attracted to the black (2 = 8.3, p < 0.005) and Mg. migonei to the white (2 = 59.8, p <0.001). Females of Pi. fischeri were more attracted to black and white Shannon traps during the first half of the night in summer, autumn and winter and in the spring it was the opposite. The Pearsons correlation test showed a statistically significant positive correlation of the average monthly temperatures during the 7 and 15 days immediately preceding the day of capture. None of the 60 females dissected presented flagellates. Conclusions. The municipality of Mairiporã presented a much higher AAIR of ACL than the municipalities covered by PEC. This is the first record of Br. carvalheiroi for the region of Serra da Cantareira. The high frequencies of Pi. fischeri and Mg.migonei in Shannon traps in the peridomicile are suggestive of their anthropophily, and account for the bother they cause residents and the danger of their possible implication in the transmission of ACL in the region

A pele é exposta a inúmeros agentes, dentre os quais destaca-se a radiação ultravioleta (RUV), que pode energizar os cromóforos celulares da pele e estes reagindo com o oxigênio molecular podem resultar na geração das espécies reativas de oxigênio (EROs). Nestas circunstâncias, mesmo possuindo um sistema de defesa antioxidante, a concentração de radicais livres pode aumentar, rompendo o equilíbrio pró oxidante/antioxidante, levando ao estresse oxidativo na pele. Neste contexto, o uso tópico de formulações adicionadas de substâncias naturais tem sido um eficiente modo para enriquecer o sistema protetor cutâneo endógeno, reduzindo os danos oxidativos causados pela RUV na pele. Sendo assim, o presente trabalho teve como objetivo estudar o potencial fotoquimioprotetor do extrato e da fração de média polaridade de Inga edulis e desenvolver formulações estáveis física e quimicamente a fim de selecionar a que prover melhor liberação, penetração e retenção dos componentes do extrato e/ou da fração na epiderme viável. A partir do perfil cromatográfico foi possível observar que o extrato possui compostos comuns à fração e alguns deles foram identificados, sendo o composto vicenina-2 o escolhido para ser monitorado nos estudos de penetração e retenção cutânea. Dentre as formulações estudadas a formulação a base de Hostacerin SAF® adicionada da fração foi a que proveu a maior penetração/retenção do composto vicenina-2 e de compostos antioxidantes na epiderme viável e também foi considerada estável por todos os parâmetros avaliados no estudo de estabilidade preliminar. A fração de Inga edulis, veiculada por esta formulação, mostrou alta atividade antioxidante frente aos métodos estudados, apresentou baixa citotoxidade em cultura celular de fibroblastos, sendo a quantidade citotóxica estabelecida muito acima da quantidade de vicenina-2 retida na epiderme viável. A fração também apresentou fotoestabilidade frente a altas doses de radiação UVA e UVB quanto à citotoxidade, capacidade de reduzir o radical sintético DPPH? e de sequestrar o ânion superóxido in vitro. Assim, a formulação gel-creme com Hostacerin SAF® adicionada de 1% da fração de média polaridade do Inga edulis mostrou ser muito promissora e deve ser melhor estudada, visando futura aplicação como formulação fotoquimiopreventiva. / The skin is exposed to numerous agents, among which stands out the ultraviolet radiation (UVR) that can energize the chromophores of cell skin and these react with molecular oxygen resulting in the generation of reactive oxygen species (ROS). In these circumstances, the endogenous antioxidant defense system would be impaired leading to a pro oxidant state named oxidative stress. In this context, the topical formulations added with natural substances has been an efficient way to aid endogenous skin protective system, minimizing the oxidative damage caused by UV rays. Thus, the aim of the present work was to study the potential photochemopreventive activity of the extract and medium polarity fraction of Inga edulis and develop formulations physically and chemically stable in order to select the penetration and retention of the components of the extract and / or fraction oin the viable epidermis. The chromatographic profiles showed that the some compounds of the extract are also present in the fraction and some of them were identified. The compound vicenin-2 was chosen to be monitored in studies of cutaneous penetration and retention. Among the formulations studied, the formulation Hostacerin SAF® added to the fraction provided the highest penetration / retention of the compound vicenin-2 and antioxidant compounds in viable epidermis and also was considered stable at all parameters evaluated in the study of preliminary stability. The fraction of Inga edulis, conveyed by this formulation, showed strong antioxidant activity in the methods studied and showed low cytotoxicity in cell culture of fibroblasts. The cytotoxicity concentration was greater than the amount of vicenin-2 retained in viable epidermis. The fraction also showed photostability when it was exposed to high doses of UVA and UVB. The irradiated fraction showed no change in cytotoxicity and antioxidant activity. Thus, the formulation Hostacerin SAF® added with 1% of medium polarity fraction of Inga edulis proved very promising and should be further studied for a future application in photochemoprevention.

Die Hämoxygenase 1 (HO-1) ist ein Stressprotein mit antientzündlichen, immunsupprimierenden und zytoprotektiven Eigenschaften, welche in vielen Tiermodellen nachgewiesen wurden. Die zugrunde liegenden Mechanismen sind wenig bekannt. Diese Arbeit demonstriert erstmalig, dass die physiologische Induktion von HO-1 wichtig für die Limitierung von T-Zell-abhängigen Hautentzündungen ist. So führt der HO-1-Inhibitor, Zinn-Protoporphyrin IX (Sn-PPIX), zu einer verstärkten Hautentzündung im Mausmodell. Die pharmakologische Induktion von HO-1 durch Kobalt-Protoporphyrin IX, Co-PPIX, hemmt dagegen die Entzündung in DNFB- bzw. TMA-induzierten murinen Kontaktallergiemodellen sowohl bei Verabreichung von Co-PPIX während der Sensibilisierung als auch vor der Auslösung. Bemerkenswerterweise hemmt eine Co-PPIX-Behandlung die Antigen-induzierte T-Zellproliferation ex vivo in Milzzellen von behandelten Mäusen und in vitro in humanen mononukleären Zellen des peripheren Blutes. Da eine HO-1-Induktion durch Co-PPIX nur in Monozyten und in aus Monozyten abgeleiteten myloischen Dendritischen Zellen (MDDC), nicht aber in T-Zellen, beobachtet wurde, fokussierten alle weiteren Untersuchungen auf Antigen-präsentierende Zellen. HO-1-Induktion durch Co-PPIX reduziert die Expression von MHC-Klasse II und akzessorischen Molekülen und steigert die Phagozytose und den oxidativen Burst von Monozyten. Die immunphänotypische Differenzierung und Maturierung von MDDC wird gehemmt. Funktionsteste zeigen eine Reduktion der Expression und Sekretion von proinflammatorischen und immunstimulatorischen Zytokinen, während die Sekretion des antientzündlichen Zytokins IL-10 gesteigert ist. Die Fähigkeit der MDDC zur Antigenpräsentation gegenüber T-Helferzellen ist für Allo- und Recallantigene stark herabgesetzt. Mittels adenoviraler HO-1-Transduktion von MDDC konnte die Spezifität der Effekte bestätigt werden. Diese Daten zeigen, dass eine verstärkte HO-1-Aktivität die Dendritischen Zellen zu einem unreifen und immunkompromittierten Phänotyp verändert und weisen darauf hin, dass die HO-1-Induktion einen wichtigen Ansatz für die Hemmung der zellulären Immunität und für die Behandlung von T-Zell-abhängigen Hautentzündungen darstellt. / Heme oxygenase 1 (HO-1) is an antiinflammatory stress protein. Its immunosuppressive and cytoprotective activities have been demonstrated in several animal models. The underlying mechanisms, however, are poorly understood. This study demonstrates for the first time that the physiological induction of HO-1 is important for the limitation and resolution of T cell-dependent skin inflammation. So, the HO-1 inhibitor, tin protoporphyrin IX (Sn-PPIX), augments cutaneous inflammation in mouse model. Moreover, pharmacologic HO-1 induction by the potent HO-1 inducer, cobaltic protoporphyrin IX (Co-PPIX), inhibits inflammation when applied around sensitization or before challenge in murine DNFB- and TMA-induced contact hypersensitivity models. Remarkably, Co-PPIX treatment inhibits antigen-driven T cell proliferation both ex vivo in murine splenocytes and in vitro in human peripheral blood mononuclear cells. Since induction of HO-1 mRNA and protein was found in monocytes and monocyte-derived myeloid dendritic cells (MDDC) but not T cells, further investigations focused on antigen-presenting cells. HO-1 induction by Co-PPIX depresses monocytic MHC class II and accessory molecule expression whereas phagocytosis and respiratory burst activities are augmented. Moreover, HO-1 induction inhibits the immunophenotypic differentiation and maturation of MDDC. Functional analysis revealed a decreased proinflammatory cytokine production whereas secretion of the antiinflammatory cytokine IL-10 is increased. Remarkably, the antigen-presenting capacity of MDDC for T-helper cells is diminished both for allo- and for recall-antigens. Adenoviral HO-1 transduction of MDDC confirmed that the effects are mediated by HO-1. These data indicate that an enhanced HO-1 activity switches myeloid DCs to an immature and functionally compromised phenotype and suggest that HO-1 induction represents an important approach for depressing T cell immunity and for the treatment of T cell-dependent skin inflammation.

Ziel dieser Arbeit war die Identifizierung von Tumor-assozierten T-Zellepitopen (TATE) in kutanen T-Zelllymphomen (CTCL) und die Charakterisierung der Tumour-spezifischen zytotoxischen Immunabwehr. Zwei Tumor-spezifische T-Zellklone wurden aus den Tumor-infiltrierenden Lymphozyten (TIL) eines CTCL-Patienten etabliert. Die potentiellen natürlichen T-Zellepitope und Mimotope (synthetische Epitope ohne natürliche Korrelate aber mit meist hohen T-Zellaktivitäten) für diese Klone wurden mit einer kombinatorischen Peptidbibliothek identifiziert. Die Qualität und Quantität der T-Zellaktivitäten waren bei den jeweiligen Peptiden unterschiedlich. Die funktionellen Aviditäten varierten dabei um 3 Größenordnungen. Bei den einzelnen Peptiden korrelierte die Zytolyse und Zytokinsekretion nicht immer. Mit einigen Mimotopen wurden CTCL-Patienten für therapeutische Zwecke vakziniert. Die Frequenzen der Mimotop-spezifischen T-Zellen erhöhten sich während der ersten Vakzinationszyklen und tumorizide Aktivitäten konnten nachgewiesen werden. Diese ersten klinischen Anwendungen der Mimotope zeigen die Möglichkeit solcher Mimotope die sonst unzureichende Immunabwehr zu modulieren. Die Identifizierung neuer TATE ermöglichte die weitere Untersuchung der Tumor-spezifischen T-Zellen in der Peripherie und im Tumor des Patienten. Diese Analysen zeigten, dass diese Zellen im peripheren Blut aktiv aber im Tumor inaktiv waren. Die TILs waren vom effektor-memory Phänotyp expremierten aber nur schwach oder z. T. keine der Moleküle mit Effektorfunktion. Das immunsuppressive Zytokin TGF-beta könnte eine wichtige Rolle bei dieser unzureichenden Immunabwehr bei CTCL spielen. / The major goals of this work was the identification of tumour-associated T cell epitopes (TATE) in cutaneous T cell lymphoma (CTCL) and the characterisation of the tumour-specific cytolytic immune response. Two tumour-specific cytolytic T cell clones were established from the tumour-infiltrating lymphocytes (TIL) of one CTCL-patient. The potential natural T cell epitopes and mimotopes (epitopes without natural correlates but with more T cell stimulating capacity) for these T cell clones were identified using a combinatorial peptide library. The quantity and quality of the T cell response was different. The functional avidity of the peptides differed more than 3 orders of magnitude. The cytolysis and cytokine release did not correlate for each peptide. Some of the mimotopes were injected into CTCL-patients for therapeutic purpose. The frequency of the mimotope-specific T cell increased during the first vaccination cycles and a tumouricidal capacity could be observed. This first clinical application of the mimotopes showed the capacity of the mimotopes for the modulation of weak anti-tumour immune response. The identification of the new TATE allowed further characterisation of the tumour-specific T cells in the periphery and in the tumour of the patient. High frequency of the tumour-specific T cells could be detected in the tumour but they failed to show effector functions in comparison to the tumour-specific T cells in the peripheral blood. The tumour-specific T cells had the effector memory phenotype but expressed none or less amount of the cytolytic effector molecules. The reason for the suboptimal anti-tumour response in CTCL could be the immunesuppressive cytokine TGF-beta.

In den letzen Jahren gab es durch epidemiologische und molekularbiologische Studien vermehrt Hinweise, dass kutane humane Papillomviren (HPV) ursächlich an der Entstehung nicht-melanozytärer Hauttumore (engl. NMSC) beteiligt sind. Ziel der vorliegenden Arbeit war die Identifizierung molekularer Mechanismen der viralen Proteine E6 und E7 kutaner HPV-Typen. Die E6 oder E7 Gene der verschiedenen HPV-Typen 1, 4, 5, 8, 20, 38 und RTRX7 wurden untersucht. Natürliche Wirtszellen dieser Viren, humane primäre Keratinozyten (HPK) der Haut, wurden mit rekombinanten, für E6 oder E7 kodierenden Retroviren infiziert. Die Analysen erfolgten in Monolayer-Kultur (undifferenzierte Keratinozyten) oder in organotypischen Hautmodellen (Induktion der Keratinozytendifferenzierung). Die Expression von E6 oder E7 führte in Monolayer-HPK zu einer Verlängerung der Lebensspanne und zu einer deutlich erhöhten Verdoppelungsrate. Eine Telomeraseaktivierung, die charakteristisch für immortale Zellen ist, wurde nur in HPV 8 E6 positiven HPK nachgewiesen. In organotypischen Hautmodellen induzierte das E7 Protein von HPV 1, 4 und 38 starke Veränderungen in der Differenzierung sowie eine Zunahme der Proliferation. Weiterhin wurde eine Aufhebung der normalen Zellzykluskontrolle in suprabasalen HPV 5 E7 oder HPV 8 E7 beobachtet. Hinweise auf ein starkes invasives Potential von E7-infizierten HPK wurden für HPV 8 E7 bestätigt und für HPV 4 E7, HPV 38 E7 und RTRX7 E7 erweitert. Molekulare Mechanismen der viralen Gene E6 und E7 kutaner HPV unterscheiden sich von mukosalen Typen. Das Mehrstufenmodell der Karzinogenese beinhaltet eine Reihe fundamentaler Zelltransformationen, die für eine Tumorgenese nötig sind. In dieser Arbeit beschriebene Mechanismen der Modulation der Zelldifferenzierung und Zellproliferation durch die kutanen HPV-Typen 4, 5, 8 und 38 können unter Umständen zur Induktion und Progression früher Stadien von Plattenepithelkarzinomen (SCC) beitragen. / In the last years epidemiologic and molecular biological studies accumulated increasing evidence that cutaneous human papillomaviruses are etiologically involved in the formation of non-melanoma skin cancer (NMSC). The presented work aims to identify the underlying molecular mechanisms of the viral proteins E6 and E7 of cutaneous HPV types. The E6 and E7 genes of the different HPV types 1, 4, 5, 8, 20, and RTRX7, which are in vivo associated with cutaneous benign or malignant lesions, were studied. Natural host cells of these viruses, human primary keratinocyts (HPK) of the skin, were infected with recombinant E6 and E7 encoding retroviruses. The following analyses were performed in monolayer culture (non-differentiated keratinocytes) or in organotypic skin culture (induction of keratinocyte differentiation). The expression of E6 and E7 elongated the life span of monolayer HPK and significantly increased the doubling rate. An activation of the telomerase, characteristic for immortalized cells, was only detected in HPV 8 E6 positive cells. In organotypic skin cultures E7 of HPV 1, 4 and 38 induced drastic changes in differentiation and proliferation. Additionally an impairment of the normal cell cycle control in suprabasale HPV 5 E7 and 8 E7 cultures was seen. Hints for a strong invasive potential of E7 infected HPK were proven for HPV 8 E7 and expanded to HPV 4 E7, HPV 38 E7 and RTRX E7. The viral E6 and E7 genes of cutaneous and mucosal HPV types exhibit different molecular mechanisms. The multistep model of carcinogenesis includes a series of fundamental cell transformations necessary for tumorigenesis. Mechanisms for the modulation of cell differentiation and proliferation by cutaneous HPV types 4, 5, 8 and 38 described in this work could potentially contribute to the induction and progression of early stages of squamous cell carcinoma.

Zusammenfassung Die molekularbiologischen Untersuchungen der variablen Genabschnitte der Immunglobulingene von Lymphomzellen zweier Patienten mit kutanen B-Zell-Lymphomen wurden in der vorliegenden Arbeit mittels Einzel-Zell-PCR-Technik durchgeführt. Die erfolgte Analyse galt bevorzugt dem Aspekt der intraklonalen Diversität. Die Sequenzanalysen der Ig-Gene für die leichte Kette aus den insgesamt 100 untersuchten Tumorzellen des Patienten WS ergaben, daß zwischen den Zellen aus drei voneinander entfernt lokalisierten Gewebeproben intraklonale Diversität vorlag. Aufgrund der Mutationsanalysen und der Zuordnung der Subklone zu den Entnahmeorten ist eine evtl. antigengetriebene Entwicklung des Tumorklons zu vermuten. Bei Patient LB konnte trotz des großen Aufwandes, der Untersuchung von insgesamt 126 Zellen aus acht räumlich getrennten Biopsien, keine intraklonale Diversität für die schwere Kette der Immunglobulingene aufgezeigt werden. Die Sequenzunterschiede der leichten Kette beruhen auf einer einzigen stummen Mutation im Bereich der CDR 1, die keinen funktionellen Einfluß auf die Aminosäuresequenz hat und deshalb geringe prognostische Bedeutung besitzt. In beiden Fällen lagen hypermutierte Immunglobulingene vor. Die Tumorzellen des Patienten WS sind aus molekularbiologischer Sicht Keimzentrumszellen, die des Patienten LB eher Nachkeimzentrumszellen zuzuordnen. Die gewonnenen Erkenntnisse zeigen, daß bei der Untersuchung und Erforschung der Pathogenese von kutanen B-Zell-Lymphomen eine gewonnene Gewebeprobe nicht repräsentativ für den gesamten Tumor sein muß. Veröffentliche Ergebnisse, bei denen Monoklonalität anhand einer Biopsie postuliert wurde, müssen daher initial überdacht werden (74;76;77;84). Es ist folglich in Betracht zu ziehen, daß bei einer exakten molekularbiologischen Zuordnung der Tumorzellen zu ihrem Entwicklungsstadium und ihrer Herkunft räumliche als auch zeitliche Faktoren zu beachten sind. Eine chronologische Untersuchung könnte aufzeigen, ob andauernde Mutationen in den Tumorzellen stattfinden. Die Mikromanipulation und die Einzel-Zell-PCR-Technik sind elegante Methoden, um intraklonale Sequenzunterschiede aufzuzeigen und einen Beitrag für die molekularbiologische Erforschung der Pathogenese von kutanen B-Zell-Lymphomen zu leisten. Eine Kontrolle von malignoproliferativen Krankheiten, ob es unter den heutigen Therapiemöglichkeiten gelingt, den malignen Tumorklon vollständig zu beseitigen oder Aussagen über Progredienz und Prognose zu treffen, wäre ein interessanter Einsatzbereich dieser Methode. / abstract The molecularbiological investigation of the immunoglobulin variable region genes of two patients suffering on primary cutaneous B-cell-lymphoma was carried out using single-cell- PCR technic. The main focus lay on the aspect on the aspect of detecting intraclonal diversity. The sequence nucleotid-analysises of the immunoglubulin light chain genes obtained from a total of 100 investigated tumor B-cells in patient WS revealed intraclonal diversity among cells isolated from three spatially separated tissue samples. Considering the mutational pattern and the subclones in dependence on the tumorcells, taken from different topographical tumorsides, an antigen-driven clonal expansion could be supposed. Despite extensive efforts and the analysis of altogether 126 tumor B-cells out of eight spatially different localised biopsies, no intraclonal diversity could be observed for the immunoglobulin heavy chain rearrangement of second patient LB. Sequence differences of the Ig light chain based on only a single silent mutation within the CDR1 region. This mutation has no functional affect of the amino acid sequence and therefore little prognostical significance. In both cases the immunoglobulin genes were somatically hypermutated in compromise to the germ-line gene. From the molecularbiological point of view, the tumor cells of patient WS descended from germinal center cells, whereas the tumor B-cells of patient LB rather can be characterized as post-germinal center cells. The results clearly demonstrate, that the investigation of a single tissue sample of primary cutaneous B-cell lymphoma using micromanipulation and single cell-PCR technic may not be representative for the whole tumor. Publications concluding monoclonality with no intraclonal diversity from the analysis of a single biopsie therefore have to be interpreted with caution (74;76;77;84). Accordingly, an appropiate molecularbiological characterization of tumor B-cells regarding their developmental stage and origin has to consider spatial and time dependant aspects. Only the investigation of sequential biopsies may reliably detect ongoing mutations in tumor B-cells. The combination of micromanipulation and single cell PCR represents an elegant method to observe intraclonal diversity. The technic will give contribution to molecularbiological investigation of the pathogenesis of primary cutaneous B-cell lymphomas. It could be of interest to apply this method to the evaluation of current therapies with respect to its capability to eradicating the malignant cell clone completely, and to draw conclusions on disease progression and prognosis.

Em áreas endêmicas para leishmaniose visceral, a AIDS aumenta o risco de aparecimento desta doença de cem a mil vezes. Conhecem-se muitos estudos sobre leishmaniose visceral (LV) em HIV/AIDS no Mediterrâneo, mas, pouco sobre leishmaniose tegumentar (LT) em HIV/AIDS. No Brasil não se constatou, até o momento, um aumento de casos de co-infecção HIV-Leishmania, mas, isto pode ser conseqüente ao subdiagnóstico por várias causas incluindo o não conhecimento da apresentação clínica nesses casos. Pela prevalência maior da LT em relação à LV no Brasil, é de se supor que ocorram com freqüência casos de co-infecção com a forma tegumentar de leishmaniose no nosso meio. Como no Brasil não dispomos de estudos de descrição sistemática da apresentação clínica nos casos de co-infecção, um parâmetro importante visando o diagnóstico, realizou-se um estudo transversal e descritivo, durante um período de dois anos a partir de fevereiro de 2002, em dois centros de tratamento de doenças infecciosas em São Paulo. Partimos de uma população alvo de 113 pacientes com sorologia positiva para HIV, procedentes de área endêmica para leishmanioses, 69 com algum sintoma sugestivo de leishmaniose visceral ou tegumentar e 44 assintomáticos. Em dez pacientes foi diagnosticada leishmaniose por encontro de parasitos ou antígeno de Leishmania em tecidos, maioria (8/10) por busca ativa, sendo cinco casos de LV e cinco de LT. Baseados nesses casos, observamos que, o contato com área endêmica de leishmaniose mesmo por um período curto como duas semanas deva ser valorizado, principalmente se foi recente. Houve dificuldade no encontro de casos porque a leishmaniose não é considerado como diagnóstico diferencial de infecções intercorrentes nos casos de HIV/AIDS no nosso meio, houve presença concomitante de outras doenças oportunistas mais prevalentes e manifestações clínicas atípicas ou semelhantes a infecções oportunistas. xvi Os casos de LV apresentaram manifestações clássicas, enquanto que os casos de LT apresentaram lesões diversas, desde úlcera única até lesões múltiplas e polimórficas (placas infiltrativas, pápulas, nódulos, máculas), com tendência à disseminação atingindo mucosa e pele, incluindo área genital (em quatro dos cinco pacientes com LT). Em dois pacientes observaram-se aparecimento de lesões (num caso) e piora (noutro caso), após início da terapia antirretroviral altamente efetiva (HAART), com características sugestivas de síndrome inflamatória de recuperação imune. Quanto à classificação de HIV segundo CDC, oito pacientes eram C3 e dois B3 e todos apresentavam contagem de linfócitos T CD4+ menor que 200 células/?l. A sorologia para leishmaniose foi positiva em todos os casos de LV mas, positiva em três dos cinco casos de LT. O teste de Montenegro foi negativo nos dois casos de LV onde o teste foi realizado e positivo em todos os três pacientes de LT submetidos ao teste. No tratamento da leishmaniose, freqüentemente os pacientes apresentaram efeitos adversos aos medicamentos e obervou-se mortalidade alta (40%) e freqüência alta de recidiva nos pacientes sobreviventes (100% dos LT e 66% dos LV) / In endemic areas of visceral leishmaniasis (VL), AIDS increases the risk for VL in 100 to 1000 times. There are many studies on VL in HIV/AIDS patients from the Mediterranean basin but data on tegumentary leishmaniasis (TL) are scarce. In Brazil, no increase in the number of cases of HIV/Leishmania co-infection was reported so far, but it may be due to subdiagnosis owing to various reasons including ignorance on clinical features of the co-infected patients.. Since the prevalence of TL is greater than VL in Brazil, we presume that cutaneous or mucosal leishmaniasis would be frequent in cases of coinfection in our country. Since there are no studies on systematic description of clinical manifestations of HIV/Leishmania co-infection in Brazil, an important basis for diagnosis, study was done for a period of two years from February 2002 aiming description of clinical manifestations in a transversal study in two infectious disease reference centers in Sao Paulo city, Brazil. The target population was constituted by 113 HIV positive patients that reported some kind of contact with endemic area for leishmaniasis, 69 being with some symptoms suggestive of visceral or tegumentary leishmaniasis, and 44 asymptomatic. In ten patients diagnosis of leishmaniasis was established by the detection of amatigotes or Leishmania antigens in tissue samples, five presented VL and five TL. Eight of 10 were found by active search. Based in these cases, we noted that contact with endemic area, even for short time like two weeks has to be considered in the diagnosis, mainly if it occurred recently. The search for these cases were difficult because leishmaniasis is not considered within differential diagnosis ofopportunistic infections in HIV positive patients among physicians, and in addition more prevalent opportunistic infections were concomitantly present and the clinical features were atypical for leishmaniasis or similar to other opportunistic infections. Clinical manifestation of VL was classic, while that of TL was quite variable, ranging from single to multiple ulcers, with polimorphic xviii presentations (papules, nodules, infiltrative plaques, macules) that tended to disseminate to the other areas of skin and mucosa, including genital area (in four from five TL patients). In two patients appearance of lesions (in one case) and worsening of tegumentary manifestations (in another case) after introduction of highly active anti-retroviral therapy (HAART) were observed that were suggestive of the so called immune reconstitution inflammatory syndrome. Concerning CDC classification of HIV, eight patients were C3 and two B3, and all presented CD4+ T cell count lower than 200/?l. All the VL patients had anti-Leishmania antibody test positive, however in TL three of five cases of was positive. Leishmanin skin (Montenegro) test was negative in two cases of VL submitted to this test, and positive in all three TL patients evalued. Frequent toxicity and adverse events with treatments were observed, as well as high mortality (40%), and high frequency of relapses among survivors (100% of TL and 66% of VL cases)

Os antimaláricos, cloroquina e hidroxicloroquina, têm sido usados há décadas com bons resultados terapêuticos para o tratamento do lupus eritematoso e são considerados medicações seguras, muito embora, haja preocupação em relação à retinotoxicidade, notadamente com a cloroquina. O objetivo deste trabalho foi avaliar a ocorrência dos efeitos adversos desencadeados pelo tratamento com 250mg/d de difosfato de cloroquina em doentes com lupus eritematoso, dando ênfase à retinotoxicidade. Foram estudados 350 doentes e reavaliados seus respectivos prontuários, que datavam de 1980 a 2003. Os doentes foram acompanhados no ambulatório de colagenoses da Divisão de Dermatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. A ocorrência dos efeitos adversos foi de 35,7%, sendo que 17,4% decorreram de alterações oculares. Em 12% dos doentes ocorreu pigmentação retiniana sugestiva de retinopatia antimalárica, em 3,1% depósitos corneanos e, em 2,3%, sintomas visuais agudos. Em 10% dos doentes houve alterações gastrointestinais: epigastralgia (6%), náuseas e vômitos (3,7%) e diarréia (0,3%). Alterações dermatológicas ocorreram em 3,4% dos doentes: rash cutâneo no início do tratamento (2%), exacerbação de quadro de psoríase pré-existente (0,3%) e pigmentação cutânea (1,1%). Ocorreram ainda cefaléia (2,9%), alterações neuromusculares (1,7%) com quadro gripal símile no início do tratamento (1,1%), neuropatia sensitiva (0,3%) e miopatia compatível com miastenia (0,3%) e, sintomas neuropsiquiátricos (0,3%). A droga foi suspensa devido aos efeitos adversos em 22,9% dos doentes, principalmente, em decorrência de alterações oculares, gastrointestinais e dermatológicas. A reavaliação oftalmológica de 12% dos doentes com pigmentação retiniana, confirmou a retinopatia antimalárica em apenas 2,6%, o que demonstrou uma tendência à valorização de alterações retinianas inespecíficas, discretas e unilaterais, com indicação desnecessária da suspensão da droga em 9,4% dos doentes. Não ocorreram casos de retinopatia antimalárica avançada com lesão do tipo bull-eye. Não houve associação estatisticamente significativa entre a ocorrência de efeitos adversos e alterações retinianas com dose diária de difosfato de cloroquina por quilo de peso e com o tipo clínico do lupus eritematoso. As alterações retinianas foram estatisticamente significativas nos doentes acima de cinqüenta anos quando comparado ao grupo abaixo dos cinqüenta anos, possivelmente pela dificuldade em diferenciar as alterações iniciais da retinopatia antimlárica daquelas decorrentes da degeneração macular senil. O controle oftalmológico foi realizado em intervalo médio de 10,5 meses, demonstrando que o controle anual foi eficaz para o acompanhamento dos doentes. Nove doentes foram expostas durante o primeiro trimestre gestacional, não ocorrendo casos de mal formação fetal / Antimalarial agents, chloroquine and hydroxichloroquine, have been used for decades leading to good therapeutic outcomes at treatment approach for lupus erythematosus and are considered safe medication; however, the main concern is retina toxicity, especially with chloroquine. The purpose of the present study was to conduct analysis of the occurrence of adverse effects, triggered by use of 250 mg/d of chloroquine diphosphate at treatment for lupus erythematosus, especially retina toxicity. We analyzed 350 patients and reviewed their medical charts, from 1980 to 2003. The patients were followed up by the outpatient unit of collagenosis, Division of Dermatology, Hospital das Clinicas, Medical School, University of São Paulo. The occurrence of adverse effects was 35.7%, and eye affections were detected in 17.4% of patients. Impairment of retina pigmentation suggestive of antimalarial retinopathy occurred in 12%, cornea deposits in 3,1%, and acute visual symptoms in 2.3%. Gastrointestinal affections were detected in 10% of patients: epigastralgia (6%), nausea and vomiting (3.7%) and diarrhea (0.3%). Dermatological affections occurred in 3.4% of patients: skin rash in the beginning of treatment (2%), exacerbation of preexisting psoriasis (0.3%) and skin pigmentation (1.1%). We also detected headache (2.9%), neuromuscular disorders (1.7%) with flu-like episode at the beginning of treatment (1,1%), sensitive neuropathy (0,3%) and myopathy compatible with myasthenia (0.3%) and neuropsychiatric symptoms (0.3%). Discontinuation of drugs owing to side effects occurred in 22.9% of the patients, being that the main affections were eye, gastrointestinal and dermatological occurrences. Ophthalmologic reevaluation of retina pigmentation affections occurred in 12% of the patients, but we confirmed antimalarial retinopathy only in 2.6%, detecting a tendency to value nonspecific, discreet and unilateral affections, which generated unnecessary recommendations for discontinuation of drug in 9.4% of the patients. There were no cases of advanced retinopathy with bull-eye type lesion. There was no statistically significant association between occurrence of adverse effects and retina affections with daily dose per kg of chloroquine diphosphate and the differents types of lupus erythematosus. In patients over the age of 50, there was statistically significant increase in number of retina affections when compared to the group aged below 50 years, possibly owing to difficulty to differentiate between initial affections in antimalarial retinopathy from those resultant from senile macular degeneration. Ophthalmologic control was conducted on average after 10.5 months, showing that annual follow-up was effective to keep track of patients. Nine of the patients were exposed during the first gestational trimester and there were no cases of fetal malformations