Vicente de Paulo Coelho Peixoto de Toledo

Evaluation of the stability and immunogenicity of autoclaved and nonautoclaved preparations of a vaccine against American tegumentary leishmaniasis

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.

Vaccine for prophylaxis and immunotherapy, Brazil

0 ver the last 10 years, 153,283 cases of American cutaneous leishmaniasis (ACL) were reported in Brazil. ACL incidence has been estimated to be around 20,000 new cases per year over the last five years,’ characterizing this disease as highly endemic in many parts of the country. Prevention of ACL is based largely on avoiding contact with the vector, a method not always feasible because of the way the disease is transmitted. Contrary to what has been observed in visceral leishmaniasis, the complex epidemiology combined with the problems associated with drug treatment (prolonged treatment time and numerous side effects, in addition to drug resistance) make prophylaxis against ACL a serious health problem in countries affected by the disease. Due to the peridomiciliary habits of the only vector of American visceral leishmaniasis known to date (Lutzomyin hgipalpis) and the fact that the disease is relatively easy to detect in the main reservoir, the domestic dog, effective prophylactic measures such as patient treatment, insecticide spraying, elimination of the reservoirs, and epidemiological surveillance are usually successful in American visceral leisl~maniasis.2,‘? Unfortunately, this is not the case in ACL. Due to the sylvatic nature of both the vectors (many sandfly species have been identified as possible vectors) and reservoirs (most of them, still not identified),4 effective prophylactic measures are rarely effective in this form of leishmaniasis.5 Since most of the infections are acquired inside the forest, measures such as insecticide spraying and elimination of the reservoirs are virtually unfeasible. In addition, the possibility of development of insecticide resistance’ in some sandfly species has also to be taken into consideration, not to mention the severe risks of environmental contamination associated with such procedures. ACL is, thus, an

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.

Leishmania (Leishmania) chagasi is not vertically transmitted in dogs

The most frequent and most important mode of human or canine visceral leishmaniasis (CVL) transmission is through the bite of infected sand flies. This study investigates Leishmania (Leishmania) chagasi vertical transmission in offspring of naturally infected dogs. Thus 63 puppies from 18 female dogs with CVL were used. Parasite presence was evaluated through parasitologic and histopathologic examination of lymphatic organs, as well as polymerase chain reaction (PCR) on samples from adults (milk, uterus, placenta, spleen, liver and bone marrow) and offspring (spleen, liver, lymph nodes and bone marrow). PCR sensitivity and specificity were calculated using a microscope as the gold standard on samples of bone marrow, spleen and liver. Specificity was 100% for all organs and sensitivity was 100% for bone marrow, 71.4% for spleen and 66.6% for liver. Bone marrow smears (n = 63), histopathology and imprint of spleen (n = 25), liver (n = 25) and lymph nodes (n = 25) were performed to evaluate congenital transmission in the 63 offspring. PCR was done on 92 samples collected from 56 of the offspring. No test performed on the offspring was positive. It was not possible to confirm vertical transmission of CVL (95% confidence interval for the observed prevalence), despite positive PCR in the placenta of seropositive adults.

A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

CD4-CD8-αβ and γδ T cells display inflammatory and regulatory potentials during human tuberculosis

T-cells play an important role controlling immunity against pathogens and therefore influence the outcome of human diseases. Although most T-lymphocytes co-express either CD4 or CD8, a smaller T-cell subset found the in the human peripheral blood that expresses the αβ or γδ T-cell-receptor (TCR) lacks the CD4 and CD8 co-receptors. These double negative (DN) T-cells have been shown to display important immunological functions in human diseases. To better understand the role of DN T-cells in human Mycobacterium tuberculosis, we have characterized their frequency, activation and cytokine profile in a well-defined group of tuberculosis patients, categorized as severe and non-severe based on their clinical status. Our data showed that whereas high frequency of αβ DN T-cells observed in M. tuberculosis-infected patients are associated with disease severity, decreased proportion of γδ DN T-cells are found in patients with severe tuberculosis. Together with activation of CD4+ and CD8+ T-cells, higher frequencies of both αβ and γδ DN T-cells from tuberculosis patients also express the chronic activation marker HLA-DR. However, the expression of CD69, an early activation marker, is selectively observed in DN T-cells. Interestingly, while αβ and γδ DN T-cells from patients with non-severe tuberculosis display a pro-inflammatory cytokine profile, characterized by enhanced IFN-γ, the γδ DN T-cells from patients with severe disease express a modulatory profile exemplified by enhanced interleukin-10 production. Overall, our findings suggest that αβ and γδ DN T-cell present disparate immunoregulatory potentials and seems to contribute to the development/maintenance of distinct clinical aspects of TB, as part of the complex immunological network triggered by the TB infection.

Peripheral blood mononuclear cells immunophenotyping in pulmonary tuberculosis patients before and after treatment.

Tuberculosis (TB) is a lung disease caused by Mycobacterium tuberculosis. The interaction between the bacillus and the host may lead to a protective cellular immune response. In the present study, we propose the “in vitro” evaluation of this cellular immune response in patients with tuberculosis before and after chemotherapic treatment. Eleven patients with TB and 9 asymptomatic subjects with tuberculin skin test negative (TST–) (purified protein derivative (PPD) ≤10 mm) were evaluated. The peripheral lymphocytes of the subjects were analyzed utilizing the following surface markers: CD3+, CD4+, CD8+, CD19+, CD25+, CD56+, CD14+, CD16+ and HLA‐DR+. At the end of the treatment, symptomatic patients presented a significant predominance (P<0.05) of CD4+ lymphocytes, a significant decrease (P<0.05) in activated CD8+ T cells and a significant increase (P<0.05) in the marker CD19+. A predominance of mature NK cells and a significant decrease (P<0.05) in NKT cells were observed. Also observed was a trend toward decrease in immunoregulatory T cells and a predominance of pro‐inflammatory macrophages. TST– subjects presented a predominance of CD4+ over CD8+ and predominance of CD19+ and of mature NK cells in comparison to the group of patients, both before and after treatment. Thus, several cell types, such as CD4+ and CD8+ T cells, NK cells and their subpopulations, NKT cells and pro‐inflammatory macrophages could act in a synergic way to control the growth and multiplication of M. tuberculosis.

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil.

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.

Pulmonary tuberculosis: evaluation of interferon-gamma levels as an immunological healing marker based on the response to the Bacillus Calmette-Guerin

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis whose interaction with the host may lead to a cell-mediated protective immune response. The presence of interferon gamma (IFN-gamma) is related to this response. With the purpose of understanding the immunological mechanisms involved in this protection, the lymphoproliferative response, IFN-gamma and other cytokines like interleukin (IL-5, IL-10), and tumor necrosis factor alpha (TNF-alpha) were evaluated before and after the use of anti-TB drugs on 30 patients with active TB disease, 24 healthy household contacts of active TB patients, with positive purified protein derivative (PPD) skin tests (induration > 10 mm), and 34 asymptomatic individuals with negative PPD skin test results (induration < 5 mm). The positive lymphoproliferative response among peripheral blood mononuclear cells of patients showed high levels of IFN-gamma, TNF-alpha, and IL-10. No significant levels of IL-5 were detected. After treatment with rifampicina, isoniazida, and pirazinamida, only the levels of IFN-gamma increased significantly (p < 0.01). These results highlight the need for further evaluation of IFN-gamma production as a healing prognostic of patients treated.

Natural killer cell subpopulations in putative resistant individuals and patients with active Mycobacterium tuberculosis infection.

Herein, we intended to perform flow‐cytometric analyses of peripheral blood NK‐cell subsets in patients with active tuberculosis (TB) and those putative resistant subjects displaying positive tuberculin skin test (TST+) and compared with TST− healthy controls. Our findings demonstrated distinct phenotypic features in TST+ as compared with TB. While lower values of NK‐cells with increased frequency of CD3−CD16+ CD56− and CD3−CD16−CD56+ subsets besides lower frequency of CD3−CD16+ CD56+ NK‐cells was observed in TST+, unaltered levels of NK‐cells with increased levels of CD3−CD16+ CD56− NK‐cells with lower frequency of CD3−CD16+ CD56+ NK‐cells was found in TB. Additional analysis highlighted a shift towards increased levels of CD3−CD16−/+CD56bright NK‐cells as the hallmark of TST+, whereas unaltered frequency was observed in TB. Increased levels of CD3+CD56+ cells were observed in both TST+ and TB. Further focusing on the monocyte/NK‐cell network, we have reported that enhanced frequency of CD14+ CD16+ monocytes particularly observed in TST+. Outstanding were the distinct correlation profiles observed between CD3−CD16−CD56+ NK‐cells and CD3+ CD56+ cells CD14+ CD16+ monocytes for TST+ and TB. These data suggested that high levels of CD3−CD16−CD56+ NK‐cells aside CD14+ CD16+ monocytes as well as non‐concurrent increment of CD3+ CD56+ cells, may be involved in protective mechanisms in putative tuberculosis‐resistant individuals. On the other hand, the basal levels of macrophage‐like monocytes despite its positive correlation with increased levels of CD3+ CD56+ cells may count for the lack of the protective immunity in patients with active tuberculosis. Further studies focusing on the cytokine profiling of peripheral blood innate immunity cells before and after chemotherapic treatment are currently under evaluation.