Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi.

Canine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density. Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8(+) T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8(+) lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)-II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8(+) T cells re-emphasizes the role of the T cell-mediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4(+) and CD8(+) T cells) and lower MHC-II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.

Flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis.

Leishmaniasis, caused by infection with the protozoan parasite Leishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigen Leishmania homolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time with Leishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-gamma) production by several different sources, listed in order of contribution: CD4(+) T lymphocytes, CD4(-), CD8(-) lymphocytes, and CD8(+) T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4(+) T lymphocytes (IFN-gamma(+) and IL-10(-)/IL-4(-)), Tc1 CD8(+) T cells (IFN-gamma(+), and IL-10(-)/IL-4(-)), and a high frequency of TNF-alpha-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment.

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine

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%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) 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.

Histopathology of human American cutaneous leishmaniasis before and after treatment.

Chemical therapy for the treatment of leishmaniasis is still inadequate, and a number of drugs and therapeutic programs are being tested. Besides treatment, the ultimate goal is an effective cure, and histopathological analyses of the lesion cicatrices constitute an important measure of treatment success, or otherwise, in this respect. In this paper, we describe histopathological patterns in cases of American cutaneous leishmaniasis in 32 patients from the municipality of Caratinga, Minas Gerais, Brazil, before and after treatment with the following therapeutic methods: 1) leishvacin + glucantime; 2) leishvacin + BCG associated with glucantime; 3) glucantime; 4) leishvacin + BCG. Lesion fragments were collected from all patients by biopsy prior to, and approximately 30 days after, each treatment which resulted in a clinical diagnosis of cure. Following the analysis of slides, the preparations were described from a histopathological point of view and grouped taking into account the prevalence or significance of the characteristic elements. This process resulted in the following classification: 1. exudative reaction (ER); 2. exudative giant cell reaction (EGCR); 3. exudative productive reaction (EPR); 4. exudative productive giant cell reaction (EPGCR); 5. exudative productive necrotic reaction (EPNR); 6. necrotic exudative reaction (NER); 7. productive exudative reaction (PER), 8. productive giant cell reaction (PGCR); 9. productive exudative giant cell reaction (PEGCR); 10. productive exudative giant cell granulomatous reaction (PEGCGR); 11. productive reaction (PR) and 12. productive cicatricial (cure) reaction (PCR). After this analysis, it was noted that clinical cure did not always coincide with histopathological cure.

Histopathology of human American cutaneous leishmaniasis before and after treatment

Chemical therapy for the treatment of leishmaniasis is still inadequate, and a number of drugs and therapeutic programs are being tested. Besides treatment, the ultimate goal is an effective cure, and histopathological analyses of the lesion cicatrices constitute an important measure of treatment success, or otherwise, in this respect. In this paper, we describe histopathological patterns in cases of American cutaneous leishmaniasis in 32 patients from the municipality of Caratinga, Minas Gerais, Brazil, before and after treatment with the following therapeutic methods: 1) leishvacin + glucantime; 2) leishvacin + BCG associated with glucantime; 3) glucantime; 4) leishvacin + BCG. Lesion fragments were collected from all patients by biopsy prior to, and approximately 30 days after, each treatment which resulted in a clinical diagnosis of cure. Following the analysis of slides, the preparations were described from a histopathological point of view and grouped taking into account the prevalence or significance of the characteristic elements. This process resulted in the following classification: 1. exudative reaction (ER); 2. exudative giant cell reaction (EGCR); 3. exudative productive reaction (EPR); 4. exudative productive giant cell reaction (EPGCR); 5. exudative productive necrotic reaction (EPNR); 6. necrotic exudative reaction (NER); 7. productive exudative reaction (PER), 8. productive giant cell reaction (PGCR); 9. productive exudative giant cell reaction (PEGCR); 10. productive exudative giant cell granulomatous reaction (PEGCGR); 11. productive reaction (PR) and 12. productive cicatricial (cure) reaction (PCR). After this analysis, it was noted that clinical cure did not always coincide with histopathological cure.

A quimioterapia para a leishmaniose não é satisfatória e existem hoje, várias drogas e esquemas terapêuticos em teste. Além do tratamento ideal, busca-se um critério de cura efetivo, onde a análise da histopatologia da cicatriz poderá ser de grande valia. Este trabalho propõe caracterizar o padrão histopatológico de casos humanos de leishmaniose tegumentar americana, em 32 pacientes do município de Caratinga-MG, antes e após o tratamento com os seguintes métodos terapêuticos: 1) leishvacin + glucantime; 2) leishvacin + BCG associado ao glucantime; 3) glucantime; 4) leishvacin + BCG. Foram colhidos fragmentos das lesões de todos os pacientes, através de biópsias, antes e após o tratamento, com diagnóstico de cura. Após análise das lâminas, as preparações foram descritas, do ponto de vista histopatológico, e agrupadas levando em conta a prevalência e a significância do elemento característico. Tal processo resultou na classificação: 1. reação exsudativa; 2. reação exsudativa giganto-celular; 3. reação exsudativa produtiva; 4. reação exsudativa produtiva giganto-celular; 5. reação exsudativa produtiva necrótica; 6. reação necrótica exsudativa; 7. reação produtiva exsudativa; 8. reação produtiva giganto-celular; 9. reação produtiva exsudativa giganto-celular; 10. reação produtiva exsudativa giganto-celular granulomatosa; 11. reação produtiva e 12. reação produtiva cicatricial (cura histopatológica). Observamos após tal análise, que nem sempre a cura clínica coincide com a cura histopatológica.

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

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b, c/CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activation of CD18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

The kinetics of Montenegro skin tests in dogs inoculated with an antileishmaniasis vaccine: A histopathologic study

A intradermorreaçäo de Montenegro (IRM) é um método de diagnóstico muito utilizado para a Leishmaniose Tegumentar Americana (LTA). Entretanto, säo relativamente poucos os trabalhos a respeito das alteraçöes texturais provocadas pelo antígeno, sobretudo em cäes. Estudou-se a histopatologia da cinética do teste cutâneo de Montenegro em cäes vacinados experimentalmente com vacina antileishmaniose tegumentar americana (vacina anti-LTA) 6, 12, 24, 48 e 72 horas, 7 e 14 dias após a administraçäo do antígeno. O quadro histopatológico geral segue o padräo observado nas inflamaçöes inespecíficas. Nas primeiras 24 horas, observou-se, geralmente, o exsudato menos intenso e predominantemente composto de granulócitos neutrófilos; após 48 e 72 horas, mais acentuado e constituído principalmente de células mononucleares. Aos 7 e 14 dias a reaçäo tendia a ser menos intensa

Immunocytochemical identification of Leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies

O método da peroxidase-antiperoxidase foi utilizado para estudar as propriedades imunocitoquímicas de Leishmanias e de amastigotas do Trypanosoma cruzi, in situ, após os tecidos terem sido submetidos a diferentes tipos de fixaçäo. Anti-soros foram obtidos de coelhos cronicamente infectados com três cepas de T. cruzi ou imunizados com L. mexicana amazonensis e L. braziliensis guyanensis e aplicados nos cortes histológicos de 5*m de espessura. Os antígenos de T. cruzi foram coroados muito bem pelos três soros anti-T. cruzi e pelos dois soros anti-Leishmania com diluiçöes entre 1:1.000 e 1:2. Diferentemente, os antígenos de Leishmania foram revelados pelos soros anti-Leishmania somente em baixas diluiçöes, ou seja, entre 1:60 e 1:160 enquanto que os soros anti-T. cruzi, mesmo nestas diluiçöes baixas quando usados para revelar Leishmania. Embora näo haja explicaçäo clara para esta reaçäo imunocitoquímicacruzada "reversa-monodirecional" entre Leishmania e amastigotas de T. cruzi os resultados do presente trabalho mostram que anticorpos policlonais contra diferentes espécies de Leishmania, quando usados para detecçäo imunocitoquímica de Leishmania e T. cruzi in situ, reagem mais fortemente com amastigotas de T. cruzi do que com espécies homólogas

Avaliacao de antigenos de Cysticercus cellulosae no imunodiagnostico da cisticercose humana pela hemaglutinacao indireta. / Evaluation of Cysticercus cellulosae antigens in the immunodiagnosis of human cysticercosis by indirect hemagglutination

Os Autores avaliaram os antigenos de Cysticercus cellulosae para o imunodiagnostico da cisticercose humana pela reacao de hemaglutinacao indireta. Observaram que os antigenos de escolex (ES) e ES pico II sao mais especificos que os antigenos de C.cellulosae total (CC), liquido vesicular e de membrana (ME). Usando soros de pacientes com cisticercose comprovada parasitologicamente, obtiveram uma sensibilidade de 0,80 para o ES, 0,84 para o ES II e especificidade 1,0, quando os soros foram usados na diluicao 1:8 para o antigeno ES e 1:16 para o antigeno ES II

American cutaneous leishmaniasis: disappearance of amastigotes from lesions during antimonial therapy.

Durante o tratamento com antimonial de Nmetil glucamina (Glucantime) dez pacientes com forma cutanea de leishmaniose tegumentar americana foram observados, para se determinar o tempo de desaparecimento de amastigotas das lesoes. Antes do tratamento todos os pacientes apresentavam teste de Montenegro positivo e amastigotas em esfregacos realizados por aposicao de material biopsiado das lesoes. O isolamento de parasitos da lesao em meio de NNN foi positivo em sete pacientes e em oito os cortes histologicos mostraram presenca de amastigotas. Durante as 10 primeiras doses de glucantime todos os pacientes tiveram culturas negativas. Amastigotas foram detectadas em esfregacos por aposicao ate a quarta dose e nos cortes histologicos ate a sexta dose de Glucantime. Os aspectos histopatologicos antes do tratamento sao brevemente descritos. Em apenas 2 pacientes observou-se durante o tratamento modificacoes histopatologicos dignas de nota: com o aparecimento de fibrose extensiva e hiperplasia pseudo carcinomatosa estes casos como os demais evoluiram para a cicatrizacao e cura das lesoes. Em vista dos resultados obtidos, os Autores concluem que a cura clinica das lesoes, pelo menos nas formas de leishmanioses cutaneas no Vale do Rio Doce, e ainda o melhor criterio para interromper o tratamento com antimoniato de N-metil glucamina, no esquema de tratamento utilizado no presente trabalho