Histopathological and immunohistochemical aspects of American cutaneous leishmaniasis before and after different treatments* / Aspectos histopatológicos e imuno-histoquímicos da leishmaniose tegumentar americana antes e depois de diferentes tratamentos

The histopathology and immune responses of the healing process of leishmaniasis are still poorly studied. This study aimed to examine the histopathological and immunological aspects of lesions of patients with cutaneous leishmaniasis before and after different therapeutic methods. We studied 23 individuals grouped according to the treatments: Glucantime, Glucantime + Leishvacin and Glucantime + Leishvacin associated with Bacillus Calmette-Guerin. For analysis of the histopathological changes present in the dermis and epidermis, histological sections were stained with hematoxylin and eosin. The samples were immunostained before and after treatment to analyze the expression of interferon (IFN)-γ, interleukin (IL) 12, IL-10 and IL-4. Before treatment the presence of intense infiltrates of mononuclear cells was noticed and after treatment, even with a diagnosis of clinical cure, the subjects still showed a moderate inflammatory process. In the immunohistochemical analyses, we noticed a difference between the cytokines, with increased expression of cytokines IFN-γ and IL-12 compared to IL 10 and IL-4, both before and after treatment and, comparatively, the difference in this expression was more intense before treatment. However, the cytokine expression analyzed by treatment group showed no statistically significant difference. We conclude that a clinical cure does not always coincide with the histopathological ...

A histopatologia e as respostas imunológicas do processo de cura da leishmaniose são ainda pouco estudadas. Este estudo teve como objetivo avaliar os aspectos histopatológicos e imunológicos das lesões de pacientes com leishmaniose tegumentar, antes e após diferentes métodos terapêuticos. Foram estudados 23 indivíduos agrupados de acordo com os tratamentos: Glucantime, Glucantime + Leishvacin e Glucantime + Leishvacin associado com Bacilo Calmette-Guerin. Para a análise das alterações histopatológicas presentes na derme e epiderme, cortes histológicos foram corados com hematoxilina e eosina. Para avaliar a expressão de interferon (IFN)-γ, interleucina (IL) 12, IL-10 e IL-4 foi utilizada a técnica de imuno-histoquímica antes e após o tratamento. Antes do tratamento houve um intenso infiltrado de células mononucleares, após o tratamento, mesmo com um diagnóstico de cura clínica, apresentou-se ainda um moderado processo inflamatório. Na análise imuno-histoquímica, notamos uma diferença entre as citocinas, com expressão aumentada de citocinas IFN-γ e IL-12 em comparação com IL-10 e IL-4 tanto antes quanto depois do tratamento, e comparativamente, a diferença nesta expressão mostrou-se mais intensa antes do tratamento. No entanto, a expressão das citocinas analisadas por grupo de tratamento não mostraram diferenças ...

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06 percent and 85.9 percent, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

Immunotherapy, immunochemotherapy and chemotherapy for American cutaneous leishmaniasis treatment

O tratamento de primeira escolha para leishmaniose tegumentar americana é o antimonial pentavalente. Embora este tratamento seja na maioria das vezes efetivo e indicado, devem ser consideradas as desvantagens tais como efeitos colaterais, longa duracão do tratamento e contra-indicacão para cardiopatas, nefropatas, idosos, grávidas e outras condicões. Com o advento da vacina antileishmaniose tegumentar americana para fins profiláticos e terapêuticos, associando-a ou não a outros fármacos, muitas pesquisas têm sido desenvolvidas, sendo a vacina a principal entre os atuais recursos no tratamento e prevencão da leishmaniose tegumentar americana. Em conclusão, a associacão do antimônio com a vacina (imunoquimioterapia) apresentou o mesmo índice de cura em relacão ao tratamento padrão (100%), e ainda reduziu o volume do sal em 17,9% e o tempo de cura significativamente, de 87 para 62 dias; conseqüentemente, reduzindo os efeitos colaterais.

Aspectos da ecologia e do comportamento de flebotomíneos em área endêmica de leishmaniose visceral, Minas Gerais / Aspects of the ecology and behaviour of phlebotomines in endemic area for visceral leishmaniasis in State of Minas Gerais

O comportamento e hábitos alimentares de algumas espécies da flebotomíneos têm sido útil na compreensão da epidemiologia das leishmanioses. No município de Porteirinha (MG), foram realizadas capturas mensais sistematizadas utilizando-se 28 armadilhas luminosas tipo CDC, durante o período de janeiro a dezembro de 2002. Foram capturadas 14 espécies de flebotomíneos, totalizando 1.408 exemplares. De acordo com o ambiente, os resultados obtidos mostraram que o peridomicílio apresentou a maior (53,3 por cento) porcentagem dos espécimens encontrados na região, embora parte (46,7 por cento) da fauna também tenha sido encontrada no intradomicílio. O repasto sanguíneo de 38 fêmeas de Lutzomyia longipalpis, provenientes do campo, foi identificado através da reação de precipitina. Os resultados indicam que Lutzomyia longipalpis foi a espécie predominante (65,1 por cento), mostrando-se oportunista, podendo sugar uma ampla variedade de vertebrados.

Evaluation of enzyme-linked immunosorbent assay using crude Leishmania and recombinant antigens as a diagnostic marker for canine visceral leishmaniasis

The performances of ELISA assays with different antigen preparations, such as Leishmania amazonensis or L. chagasi lysates and the recombinant antigens rK-39 and rK-26, were compared using sera or eluates from dried blood collected on filter paper to detect anti-Leishmania antibodies in dogs from a visceral leishmaniasis-endemic area in Brazil. Of 115 IFAT-reactive dogs at 1:40 titre, 106 (92.2 percent) were positive in parasitological exams (skin and/or spleen). These animals were compared to healthy animals (n = 25), negative for IFAT at a titre of 1:40 and parasitological exams. The sensitivities of crude and recombinant antigens were similar and remarkably high for both sera and eluates (97-100 percent). Specificity was higher than 96 percent for sera and eluates for different antigens, except for L. chagasi antigen using eluates (88 percent). Concordance values among the tests were higher either for sera or eluates (J = 0.95-1.00). High concordances were observed between sera and eluates tested with different antigens (kappa = 0.93-0.97). Crude and recombinant antigens identified different clinical phases of canine leishmaniasis. These results show that eluates could be used in canine surveys to identify L. chagasi infection. Recombinant antigens added little when compared to crude antigen in identifying positive dogs. Cross-reactivity with other diseases whose distribution often overlaps VL-endemic areas is a limitation of crude antigen use however.

Identification and purification of immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine (Leishvacin®)

Immunogenic proteins from nonliving promastigote polyvalent Leishmania vaccine against American tegumentary leishmaniasis (Leishvacin®), produced by Biobrás (Biochemistry of Brazil ), Montes Claros, State of Minas Gerais, Brazil, were identified and purified by polyacrylamide electrophoresis gel and electroelution. C57BL/10 mice were vaccinated with proteins with estimated molecular weights of 42, 46, 63, 66, 73, 87, 97, and 160kDa in three doses of 30æg of each protein at 15-day intervals combined with 250æg of Corynebacterium parvum followed by a challenge infection with 10(5) infective promastigotes from Leishmania (Leishmania) amazonensis. The ability of these proteins to induce immune response and protection was analyzed. No statistical difference was observed in the level of IFN-g induced by proteins in vaccinated groups in comparison with control groups. Six months after challenge infection, protection levels of 28.57; 42.86; 57.14; 42.86; 42.86, 57.14; 42.86 and 57.14 percent were demonstrated for each purified protein

Anticorpos antipromastigotas vivas de Leishmania (Viannia) braziliensis, detectados pela citometria de fluxo, para identificaçäo da infecção ativa na leishmaniose tegumentar americana / Anti-live Leishmania (Viannia) braziliensis promastigote antibodies, detected by flow cytometry, to identify active infection in american cutaneous leishmaniasis

Neste estudo, descrevemos etapas iniciais de padronizaçäo de uma nova metodologia para detecçäo de anticorpos antipromastigotas vivas de Leishmania (Viannia) braziliensis, pela citometria de fluxo e a análise de sua aplicabilidade para estudos clínicos. Foram avaliados 39 indivíduos com sorologia convencional (RIFI) positiva para leishmaniose, classificados quanto à ausência/presença de lesäo (L- e L+). Os resultados foram expressos sob a forma de percentual de parasitas fluorescentes positivos (PPFP). A análise dos dados, na diluiçäo 1:1.024, permitiu distinguir 95 por cento dos pacientes L+ como um grupo de alta reatividade (PPFP>50 por cento) e 72 por cento dos indivíduos L- como um grupo de baixa reatividade (PPFP<=50 por cento). A análise dos títulos da reaçäo de imunofluorescência indireta näo demonstrou nenhuma relaçäo com a ausência/presença de lesäo. Em conjunto, nossos dados sugerem a aplicabilidade da citometria de fluxo na identificaçäo dos casos de infecçäo ativa, o que näo tem sido possível através das reaçöes sorológicas convencionais

Vaccination of C57BL/10 mice against cutaneous leishmaniasis using killed promastigotes of different strains and species of Leishmania

Antigenic extracts from five Leishmania stocks were used to vaccinate C57BL/10 mice. The Leishvacin® and PH8 monovalent vaccine yielded the highest IFN-gamma levels in the supernatants of spleen cell culture from vaccinated animals. Each single strain immunized group showed evidence of protective immunity six months after the challenge with promastigotes of Leishmania (Leishmania) amazonensis. No differences were detected between the vaccinated groups. It can be concluded that vaccines composed of single Leishmania stocks can provide protection to C57BL/10 mice against L. (L.) amazonensis infection

Atypical Mucocutaneous Leishmaniasis Caused by Leishmania braziliensis in an Acquired Immunodeficiency Syndrome Patient: T-cell Responses and Remission of Lesions Associated with Antigen Immunotherapy

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5 percent) most of them with CD8+ phenotype (33 percent). Very low CD4+ cells (2.2 percent) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells

Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis

In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guérin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacin. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 mu/g of each protein at 15 days interval. One hundred mu/g of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 105 infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86 percent of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57.14 percent of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43 percent protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7.60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14 percent of protection was found after the first challenge and 28.57 percent after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis

Leishmaniose visceral canina estudo parasitológico e histopatológico em olhos de cäes: parte I / Canine visceral leishmaniosis parasitological and histopathological study in dog's eyes: part I

Vários autores têm relatado o envolvimento ocular externo em cäes naturalmente infectados com Leihmania infantum e Leishmania donovani. Entretanto, ainda näo foram realizados estudos dos olhos de cäes no nosso meio. Objetivando investigar a possibilidade de parasitismo ocular, quatro cäes controle e 12 cäes com leishmaniose visceral foram examinados, todos provinientesda regiäo metropolitana de Belo Horizonte, MG, Brasil.O diagnóstico foi realizado através de sorologia convencional (RIFI e ELISA) e exame parasilógico de aspirado de medula óssea. Para o isolamento do parasito foi realizada a inoculaçäo de aproximadamente 100 ml de humor aquoso e/ou corpo vítreo em meio de cultura de NNN/LIT, que foi mantido a ...

Leishmaniose visceral canina estudo imunológico em olhos de cäes: parte II / Canine visceral leishmaniasis immunological study in dog's eyes

O quadro clínico da leishmaniose visceral canina se assemelha muito ao quadro clínico da leishmaniose visceral humana. Embora estas alteraçöes sejam amplamente abordadas em praticamente todos os sistemas no cäo, o estudo das alteraçöes imunológicas näo têm sido profundamente estudados. Aproximadamente 20ml de humor aquoso e corpo vítreo foram utilizados para exames imunológicos através do ensaio imunoenzimático (ELISA) para detecçäo de anticorpos anti leishmania (IgM e IgG) e 10ml de humor aquoso foram utilizados para dosagem de proteína pela micro técnica de Lowry. Detectou se anticorpos anti leishmania (IgM e IgG) em níveis que variaram de 1:80 a 1:2560 no humor aquoso e/ou corpo vítreo dos animais infectados e menores que 1:5 nos animais controle...

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

A resposta imune induzida por uma proteina recombinante de Leishmania (Leishmania) amazonensis de 33 kD (Larp33) foi avaliada em linfocitos de individuos vacinados com a Leishvacin e em camundongos atraves de vacinacao. Larp33 foi expressa em Escherichia coli apos clonagem de um fragmento genomico de L. (L.) amazonensis de 2,2 kb no vetor pDS56-6His. Larp33 foi reconhecida por anticorpos IgG presentes no soro de individuos vacinados com Leishvacin e induziu proliferacao em linfocitos desses individuos em niveis comparaveis ao antigeno total de Leishmania...

Histopathology and immunocytochemical study of type 3 e and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

Os objetivos deste trabalho visaram uma analise comparativa das alteracoes histopatologicas e da expressao de CR3 e CR4 no figado e baco de caes natural e experimentalmente infectados com L. chagasi. As lesoes histopatologicas fundamentais observadas principalmente nos caes naturalmente infectados foram: os granulomas epitelioides hepaticos, a hiperplasia e a hipertrofia das celulas de Kupffer, dos foliculos de Malpighi e das celulas monucleadas da polpa vermelha do baco. Os cortes de figado e baco corados pela tecnica de imunocitoquimica mostraram a presenca dos antigenos CD11b,cCD18 nos animais controles e infectados, sem diferencas qualitativas e quantitativas no figado...

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishamnia

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of y-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50 por cento of the immunized animals were protected for six months.