Antigens from Leishmania amastigotes inducing clinical remission of psoriatic arthritis.

A first generation vaccine (AS100-1) was manufactured with protein from four cultured Leishmania species, which proved to be effective in the treatment of psoriasis. A single blind trial on 3,132 psoriasis patients revealed 508 (16.2%) subjects with psoriatic arthritis (PsA) that received AS100-1 antigens. The study group was distributed according to percent psoriasis area and severity index (PASI) reduction from PASI 10 to PASI 100. All groups decreased in arthritis score (AS), tender joints counts and nail changes after treatment; the highest decreased in the PASI 100 group. Relapses of psoriasis and PsA had PASI and AS lower than initial values before treatment. Clinical remissions were at lower doses and less time, after the second course of treatment. Peripheral blood mononuclear cells (PBMC) lymphocyte subsets (LS) varied with PASI range (1-10, 11-20 and 21-72). Pre-treatment, absolute values of gated LS: CD4+, CD8+HLA-, CD8+HLA+, CD8+CD3-, CD8+CD3+ decreased in PBMC as PASI increased, suggesting migration from the blood to the skin. In contrary to the previous finding, the following LS: CD8+CD4-, CD3+CD8-, HLA+CD8-, CD19, CD8+CD4+ and membrane surface immunoglobulin IgA+, IgD+, IgM+, IgE+, and IgG+ increased in PBMC as PASI increased suggesting activation and proliferation by unknown antigens creating a homeostatic cycle between skin/joints and peripheral blood. After nine doses of AS100-1, the following LS: CD8+CD3+, CD8+HLA+, CD3+CD8-, CD4+CD8-, CD8+HLA-, HLA+CD8-, CD8+CD3-, CD19+, CD8+CD4-, CD8+CD4+, IgA+, IgD+, IgM+, IgE+, and IgG+ decreased significantly as compared with values before treatment. The LS decreased stops the vicious cycle between skin/joints and blood explaining clinical remission of lesions.

Antigens from Leishmania amastigotes induced clinical remission of psoriasis.

While injecting volunteers in Venezuela with a vaccine for prevention of leishmaniasis, we observed 100% clinical remission of a psoriatic lesion in one subject. Subsequently, the vaccine (AS100) was evaluated in psoriatic patients with an open label, single center study. The study was conducted in 2,770 subjects and included plaque (79%), guttate (10%), plaque and guttate (10%), palm/plantar, erythrodermia, inverse, plaque and arthritis and nail psoriasis. Baseline PASI compared with post-treatment values were: PASI 100, 23%; PASI 75, 45%; PASI 50, 13%; PASI 10, 9% and

Overview of some biomedical research projects in tropical medicine conducted at the Instituto Venezolano de Investigaciones Cientificas.

The Instituto Venezolano de Investigaciones Cientificas (IVIC) is a government-funded multidisciplinary academic institution dedicated to research, development and technology in many areas of knowledge. Biomedical projects and publications comprise about 40% of the total at IVIC. In this article, we present an overview of some selected research and development projects conducted at IVIC which we believe contain new and important aspects related to malaria, ancylostomiasis, dengue fever, leishmaniasis and tuberculosis. Other projects considered of interest in the general area of tropical medicine are briefly described. This article was prepared as a small contribution to honor and commemorate the centenary of the Instituto Oswaldo Cruz.

Epidemiologic characterization of American cutaneous leishmaniasis in an endemic region of eastern Venezuela.

The status of American cutaneous leishmaniasis was investigated from 1985 to 1991 to provide an epidemiologic characterization of the disease in Bergantin, a rural community in the northeastern part of Anzoátegui State, Venezuela. The study revealed the presence of the infection during the period analyzed, with an average incidence of 50.2 cases per 10,000 inhabitants and this number has increased 1.5 times during the last two years. Three villages where clinical cases had been recorded were selected for a comparison of their prevalence data. These villages comprise the human population in the high and low altitude limits of Bergantin. Immunologic assessment of the inhabitants used two different antigen preparations to examine responses to parasites associated with the cutaneous and visceral forms of the disease. The leishmanin skin test (LST) was used in a sample of 276 individuals (46.3% of the inhabitants) and resulted in an overall positivity of 16.7%. The percentage of LST positivity varied with age and sex, yet analysis of this response and the prevalence for each village reflected the specific characteristics of these localities. La Montaña, situated at 800 meters above sea level, had the highest prevalence (800 cases per 10,000 inhabitants) and the most positive LST response (21.2%) in comparison with the two other villages situated at a lower altitude (300 meters above sea level).

Comparison of chagasic and non-chagasic myocardiopathies by ELISA and immunoblotting with antigens of Trypanosoma cruzi and Trypanosoma rangeli.

Trypanosoma cruzi associated myocardiopathy, or Chagas disease, continues to be a serious problem in Venezuela, for which there is neither a vaccine nor a cure. In order to learn more about the humoral immune response to trypanosomal antigens, and to try to identify dominant antigens, we used ELISA and immunoblotting to study the reactivity of sera from patients with chagasic and non-chagasic myocardiopathies, against surface and secreted proteins from T. cruzi and T. rangeli. Both species are found in the same insect vector, but only T. cruzi is thought to be pathogenic in vertebrates. The ELISA results fell into three patterns: (1) high reactivity values with both T. cruzi and T. rangeli surface and secreted proteins; (2) high values to T. cruzi but low values with T. rangeli; and (3) high values to T. rangeli and low values with T. cruzi. This finding that some chagasic sera react more strongly against T. rangeli than against T. cruzi is intriguing, and warrants further investigation. When chagasic sera were tested on Western blots of total extracts of T. cruzi and T. rangeli, the pattern of reactive bands was similar against both parasites, but no two sera showed an identical pattern. Furthermore, there was no correlation between a particular immunoblotting pattern and either the antibody titer, or the severity of the disease. Several T. cruzi and T. rangeli antigens were recognized by sera from healthy controls as well as from patients with other tropical diseases endemic in Venezuela. Overall, our results suggest that the humoral immune response to trypanosomal antigens is complex, and no single antigen may be the determining factor in the pathogenesis of chagasic myocardiopathy.

A comparison of the molecular biology of trypanosomes and leishmaniae, and its impact on the development of methods for the diagnosis and vaccination of leishmaniasis and Chagas disease.

Our main interest have focused on Chagas disease and Leishmaniasis, working in the areas of: 1--The molecular biology of Trypanosomes and Leishmaniae, and 2--The immunology of Chagas disease, cutaneous leishmaniasis and visceral leishmaniasis. In this article we summarize the work realized in the last 20 years in the Immunobiology Laboratory at the IVIC with special emphasis in the development of a vaccine against leishmaniasis that is being currently used in a field trial in human beings of the endemic area of Guatire, Miranda State, Venezuela.

Variability of Trypanosoma cruzi epimastigote surface antigens with changes in the temperature of the cultures.

Trypanosoma cruzi epimastigotes changed their pattern of surface proteins when the temperature of growth rose from 30 degrees C to 34 degrees C. Challenge of mice with blood-form trypomastigotes produced high parasitemias when animals were immunized with surface proteins from epimastigotes cultured at 30 degrees C and with Nonidet P-40-extracted epimastigotes pellets cultured at 34 degrees C. However, low parasitemias were recorded after immunization with surface proteins from epimastigotes cultured at 34 degrees C or Nonidet P-40-extracted epimastigotes pellets at 30 degrees C. The lowest parasitemia, together with the longest survival time and absence of immunosuppression, was observed after immunization of mice with the product extracted with non-ionic detergent from epimastigotes grown at 30 degrees C and treated with tosyl-L-lysine-chloromethyl ketone.

Splenocyte membrane changes and immunosuppression during infection and reinfection with Trypanosoma cruzi.

Antisera to epi- and trypomastigote forms of Trypanosoma cruzi were used to detect trypanosome antigens on the surface of lymphocytes from infected mice. Only the anti-trypomastigote serum could recognize antigens expressed transiently on the splenocyte membranes from infected animals. The number or structural configuration of Concanavalin A receptors was similarly affected and a clear correlation was seen between these two types of membrane changes and the immunosuppression to mitogens and SRBC presented by the infected mice. Reinfected animals did not show evidences of trypanosome proliferation in blood or tissues nor trypomastigote antigens on splenocytes, but presented a less intense, transient immunosuppression as measured by responsiveness to mitogens and SRBC, suggesting that the primed immune system can eliminate the new parasite inoculum before the host is immunosuppressed and also that the liberation of strong immunosuppressor trypomastigote antigens induce the new state of suppression.

Differential growth requirements of several Leishmania spp. in chemically defined culture media.

17 strains of Leishmania from 4 species: brasiliensis, mexicana, donovani and garnhami have been continually cultured at 26 degrees C, in the absence of proteins, in a medium containing salts, glucose, D-ribose, 2-deoxyribose, hemin, tricine, HEPES, 34 amino acids and intermediates of amino acid metabolism, 23 vitamins, 6 nucleotides and tetrahydrofolic acid. A wide variation in growth requirements was observed among leishmaniae which permitted the preparation of different minimum culture media for each Leishmania spp. Virulence of parasites was maintained after 30 passages in these chemically defined media. The requirements for differentiation to amastigotes also varied among the species as a function of the temperature of incubation and the protein content of the culture medium. Bovine serum albumin tryptic peptides substituted fetal bovine serum as growth factors at 30-34 degrees C.

Trypanosoma cruzi: growth requirements at different temperature in fetal bovine serum or peptide supplemented media.

An enriched synthetic medium with low molecular weight peptides allows Trypanosoma cruzi epimastigotes to grow at 26-37 C. Using this medium, the growth requirements of T. cruzi were compared at different temperatures. When supplemented with fetal bovine serum or serum peptides, nine amino acids were absolutely required from the first passage, while additional amino acids and amino acid precursors were needed to support growth during a second passage. Five amino acids (beta-alanine, glutamine, cysteine, ornithine, and threonine) were also required absolutely at temperatures ranging between 30 and 37 C. Nine vitamins were needed at all temperatures, while ascorbic acid and ergocalciferol were not necessary at any temperature. The remaining amino acids and vitamins showed a variable role as growth factors depending on the temperature increase. In peptide supplemented media, requirements for amino acids and their precursors, as well as vitamins and nucleotides, increased markedly when compared with the protein supplemented medium. A peptide composed of one glutamic acid, two alanines, and one lysine can substitute for serum for trypanosomal growth at all temperatures. Several minimum media have been prepared in which epimastigote forms of T. cruzi can grow at 26-37 C for more than 10 passages.

Protein and nucleotide contamination of bovine liver catalase used in culture medium explains growth of Trypanosoma cruzi.

Commercially available bovine liver catalase has been used to supplement chemically defined medium for growth of Trypanosoma cruzi. The protein extract was found to be contaminated with 25 to 30 protein bands as well as DNA and RNA polymers.

Immunization of hamsters with TLCK-killed parasites induces protection against Leishmania infection.

Hamsters immunized with N-p-tosyl-L-lysine-chloromethyl ketone TLCK-treated L. brasiliensis brasiliensis (LB) from culture, infected with LB amastigotes presented: a gradual increase in T and B cell responsiveness to mitogens by lymph node lymphocytes, and an increased response to concanavalin A with no changes for dextran sulphate and pokeweed mitogen in splenocytes. Absence of parasites in lymph nodes after 6 weeks post-infection and a nodule 4 times smaller than that of infected control animals. The nodule was undetectable after 70 days of infection. Hamsters preimmunized with TLCK-treated L. donovani (LD) from culture did not show suppression of the blastogenic response to mitogens of spleen and lymph node cells after infection with LD amastigotes and survived for more than one year, whereas infected, unimmunized animals died five months after infection. Animals preimmunized with culture parasites (LB or LD) treated with phenyl-methyl-sulphonyl-fluoride (PMSF) and infected with LB or LD amastigotes did not show any protective effect.

Differential effect of culture epimastigotes and blood-form Trypamastigotes on normal mouse splenocyte responsiveness to mitogens

Tripomastigotas de sangue da cepa Y de T. cruzi mostraram uma forte inibiçäo da resposta de transformaçäo blástica a mitógenos de células T e B, nas estirpes C3H/He, C57BL/6 e BALB/cJ de camundongos, enquanto epimastigotas de cultura da cepa Y mantidos em meio que permite o crescimento dos parasitas a 26-, 30-, 34- e 37-C mostraram um forte efeito estimulante, que foi inclusive maior que o efeito dos mitógenos isolados. Os efeitos de inibiçäo e de estimulaçäo foram dependentes da dose. O efeito estimulante dos epimastigotas também foi dependente da temperatura, encontrando-se maiores índices de estimulaçäo à medida que a temperatura da cultura dos parasitas foi aumentada. Parasitas vivos, metabolicamente ativos, parecem ser necessários para a obtençäo de uma maior estimulaçäo dos linfócitos, o que sugere um papel potencial dos metabólitos segregados como ativadores policlonais dos linfócitos dos camundongos

Differential effect of culture epimastigotes and blood-form trypomastigotes on normal mouse splenocyte responsiveness to mitogens.

Blood form trypomastigotes of the Y strain of T. cruzi, produced a strong inhibition of the blastogenic response to T and B cell mitogens, of the C3H/He, C57BL/6 and BALB/cJ strains of mice, while culture epimastigotes of the Y strain kept in a medium that allows parasite growth at 26 degrees, 30 degrees, 34 degrees and 37 degrees C produced a strong stimulatory effect that was even higher than the effect of the mitogens alone. Both the inhibitory or the stimulatory effects were dose-dependent. The stimulatory effect of epimastigotes was also temperature-dependent producing increased stimulation indexes as the temperature of parasite cultures was raised. Metabolically active, living parasites seemed to be necessary for an improved lymphocyte stimulation suggesting a potential role of secreted metabolites as polyclonal activators of mouse lymphocytes.

Serum protein from Leishmania brasiliensis infected hamster that suppress lymphocyte response of normal hamster lymphocytes.

Immunosuppression in Leishmania brasiliensis (LB) or L. donovani (LD) infected hamsters is correlated with the appearance of two serum protein bands found at 21, 60, 68 and 76 days post LB-infection and with eight bands at 21 days post-LD-infection probably of host origin. A protein band from LB-infected hamster serum isolated by electrofocusing, suppressed the blastogenic response of normal lymphocytes to T and B cell mitogens.

Suppression of humoral immunity and lymphocyte responsiveness during experimental Trypanosoma cruzi infections.

Ratones C3H/He y C57B1/6 inoculados con 500 tripomastigotes de la cepa Y de T.cruzi muestran durante la fase aguda de la enfermedad, parasitemia, aumento del bazo y ganglios linfaticos asi como parasitos intracelulares en el corazon. Analisis de las celulas presentes en ganglios linfaticos y bazo presenta aumento de celulas IgM e IgG. Cuando la esplenomegalia es mayor, alrededor del dia 17 postinfeccion, los linfocitos esplenicos mostraron un descenso marcado en la respuesta a mitogenos de celulas B y T, antigenos de T. cruzi y celulas formadoras de placas contra globulos rojos de carnero. Celulas de bazo o celulas esplenicas adherentes a plastico, obtenidas de ratones durante la fase aguda de la infeccion suprimen la respuesta a mitogenos de linfocitos normales. Durante la fase cronica, desaparece la parasitemia, los parasitos intracelulares en el corazon y disminuye la esplenomegalia. Estos cambios preceden a la recuperacion de la respuesta a mitogenos y antigenos de T.cruzi en linfocitos esplenicos de C57B1/6.Esta recuperacion es solo parcial en C3H/He, los cuales son mas sensibles a la infeccion. Tambien se encuentra recuperacion de la respuesta humoral durante la fase cronica