Biomedical subjects

S G Coutinho

Publications and source records attributed to S G Coutinho.

At least 19 recordsLinked to original sources

PubMed2005

Flow cytometric analysis of cellular infiltrate from American tegumentary leishmaniasis lesions.

BACKGROUND: CD4+ and CD8+ T lymphocytes play different roles in the outcome of leishmaniasis. However, T-cell distribution in lesions shows significant variability in in situ immunocytochemical studies. OBJECTIVES: In this report flow cytometry was used to determine the predominant T-cell subsets in leishmaniasis lesions, and their relationship with Leishmania-responsive circulating T cells. PATIENTS AND METHODS: Mononuclear cells from lesions or peripheral blood (PBMC) of 34 cutaneous (CL), four mucosal (ML) and four disseminated leishmaniasis were phenotypically characterized by flow cytometry. Leishmania-responsive T cells were obtained after in vitro stimulation of PBMC with leishmanial antigens. RESULTS/CONCLUSIONS: Variable amounts of gammadelta lymphocytes were present in all lesions, with no association with duration of illness. The highest percentages of interleukin-2R- and interferon-gammaR-positive cells were observed in ML lesions and could render these T cells more susceptible to the effects of these cytokines. The distribution of intralesional T-lymphocyte subsets was quite variable (CD4+ > CD8+ = 18 cases, CD8+ > CD4+ = 12 cases and CD4+ congruent with CD8+ = 4 cases) without any association with clinical parameters, and could explain the controversy regarding proportions of these T-cell subsets in leishmaniasis lesions. Low percentages of Leishmania-reactive CD8+ T cells were observed in blood while an enrichment of CD8+ cells was shown in the inflammatory infiltrate, suggesting that local immunoregulatory factors could favour the recruitment and/or proliferation of local CD8+ lymphocytes. Increased percentages of CD8+ cells observed in older lesions are consistent with the hypothesis that they can mediate healing, although their involvement in tissue damage cannot be ruled out. It is possible that these mechanisms can influence the clinical outcome or even the response to therapy.

Adult↗

PubMed2002

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease.

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Animals↗

PubMed2001

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.

Adolescent↗

PubMed2001

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Adult↗

PubMed2000

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis.

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 +/- 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 +/- 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 +/- 3.8 and 15.3 +/- 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Adult↗

PubMed1999

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.

Adult↗

PubMed1998

Leishmania pifanoi amastigote antigen P-4: epitopes involved in T-cell responsiveness in human cutaneous leishmaniasis.

In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protein P-4 has been shown to induce significant protection against infection. Further, recent studies examining the response of peripheral blood mononuclear cells (PBMC) from Leishmania braziliensis-infected human patients have demonstrated that the P-4 protein selectively elicits a significant TH1-like response. Because a TH1-like response is associated with cure, epitope studies were conducted to further evaluate the human response to P-4. PBMC from confirmed cutaneous leishmaniasis patients infected with L. braziliensis in Rio de Janeiro, Brazil, an area where the disease is endemic, were examined for T-cell proliferation and/or cytokine production in response to whole-parasite homogenate, isolated P-4 protein, and/or P-4 peptides. Twenty of the 22 patients (91%) examined responded to the native P-4 protein by proliferation and/or gamma interferon (IFN-gamma) production. According to the proliferation data, PBMC from 14 patients (64%) were found to respond to the intact P-4 protein (stimulation index of >/=2.5). Fifty-seven percent of the P-4-responsive patients studied responded to at least one of the P-4 peptides; 11 individual peptides were found to elicit a proliferative response. Of 17 patients examined for cytokine production, no PBMC produced detectable interleukin-4 in response to P-4 protein or peptides. However, PBMC from 14 patients (82%) produced significant levels of IFN-gamma (>/=20 pg/ml) in response to native P-4 protein. Nineteen of the 23 peptides were found to elicit an IFN-gamma response from at least two patients. These data indicate that multiple epitopes spanning the entire P-4 molecule are responsible for the TH1-like immune response observed, indicating that the intact P-4 amastigote molecule, rather than selected peptides, may prove to be the most useful for leishmaniasis vaccine development.

Animals↗

PubMed1998

Immunologic patterns associated with cure in human American cutaneous leishmaniasis.

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

CD4-Positive T-Lymphocytes↗

PubMed1998

Cutaneous scars in American tegumentary leishmaniasis patients: a site of Leishmania (Viannia) braziliensis persistence and viability eleven years after antimonial therapy and clinical cure.

Two former patients treated for the cutaneous form of American tegumentary leishmaniasis were reviewed eight and 11 years, respectively, following clinical cure. We were able to isolate Leishmania parasites in a culture of material from the two scar biopsies, and in one of them the parasite was characterized as Leishmania (Viannia) braziliensis. In both cases, the histopathology revealed discreet hyperceratosis and a slight infiltrate of mononuclear cells surrounding and on the walls of the surface and deep dermal vessels. No amastigotes were seen on immunohistochemical or histopathologic examination. The Montenegro skin test result and the in vitro lymphoproliferative response to Leishmania antigen were positive, but no specific IgG and IgM antibodies were detected. Otorhinolaryngologic examination showed no macroscopic alteration in the mucosae. These findings are important for the evaluation and criteria of post-treatment cure.

Adult↗

PubMed1996

Leishmania amazonensis: the Asian rhesus macaques (Macaca mulatta) as an experimental model for study of cutaneous leishmaniasis.

As a means of assessing the usefulness of the Rhesus macaque (Macaca mulatta) as a nonhuman primate model for studying cutaneous leishmaniasis, monkeys were infected with Leishmania amazonensis. Variation in the level of susceptibility was found; however, animals inoculated with 10(8) promastigotes provided consistent results as indicated by an earlier onset and/or larger size of lesions. Three monkeys, which had recovered from skin lesions, were challenge-infected using the same parasite strain/dose; although these animals remained susceptible to homologous infection, lesion size was smaller and healed faster than in the initial infection. The immunologic features during infection were assessed. Levels of IgM and IgG antibodies to promastigote antigens rose during active infection and then declined; immunoblot analyses indicated that numerous leishmanial antigens (predominately >30 kDa) were recognized. Delayed type hypersensitivity (DTH) responses and proliferative responses (PBL) developed during active infection and/or rechallenge. Circulating peripheral T cell subpopulations varied throughout the course of infection. Initially (6-8 weeks p.i.), CD4+ T cells appear to predominate; subsequently (15-21 weeks p.i.), an increase in CD8+ T cells was observed. Pathologic analyses indicated that lesions contained amastigotes with a mononuclear infiltrate of macrophages, lymphocytes, and plasma cells, and formation of tuberculoid-type granulomas. As the progression and resolution of leishmanial infection in rhesus macaques are very similar to those observed in humans, this primate model could be employed for elucidating the mechanisms of protective immunity in cutaneous leishmaniasis.

Animals↗

PubMed1996

T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi amastigote antigens and Leishmania braziliensis promastigote antigens: immunologic patterns associated with cure.

Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-gamma and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. approximately or = 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. approximately or = 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-gamma and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis.

Animals↗

PubMed1994

Leishmania-reactive CD4+ and CD8+ T cells associated with cure of human cutaneous leishmaniasis.

Fourteen patients suffering from American cutaneous leishmaniasis were studied. Assays of the lymphocyte proliferative response induced in vitro by Leishmania braziliensis antigens were performed. After 5 days in culture, L. braziliensis-stimulated blast T cells were harvested for CD4+ and CD8+ phenotype analysis. When results before and at the end of therapy were compared, leishmaniasis patients showed an increase in the percentage of CD8+ blast T cells and a decline in the proportion of CD4+ blast T cells in cultures. The levels of gamma interferon in T-cell culture supernatants showed a tendency to increase when the patients were cured. These results show a pattern of higher proportions of Leishmania-reactive CD8+ T cells and lower proportions of Leishmania-reactive CD4+ T cells after cure.

Adult↗

PubMed1994

Does lipophosphoglycan enhance the T cell-stimulatory activity of lipophosphoglycan-associated proteins?

Peripheral blood mononuclear cells (PBMC) from 13 American tegumentary leishmaniasis (ATL) patients and two healthy controls were tested in in vitro proliferative response assays with crude lipophosphoglycan (LPG), purified LPG, LPG-associated proteins (LPGAP) and synthetic LPGAP from L. donovani. Cells from another group of six ATL patients were similarly tested with LPGAP obtained from L. major. L. major LPGAP was more stimulatory than L. donovani LPGAP. Crude LPG from L. donovani was significantly more stimulatory than all the other L. donovani antigens tested, including L. donovani LPGAP. The present results indicate that the association with the glycolipid (LPG) may enhance the antigenicity of LPGAP for human T lymphocytes.

Animals↗

PubMed1994

An experimental model of the production of metastases in murine cutaneous leishmaniasis.

An experimental investigation into the influence of artificially induced trauma in the production of leishmanial metastatic lesions and into the possible role played by Leishmania-reactive T cell populations in the metastatic process was carried out. Trauma was induced by incising a small cut into the shaved rump of Leishmania amazonensis-infected BALB/c mice. Ten days after the trauma, mice were killed to quantify the parasite load in the traumatic lesion or in the equivalent area in nontraumatized mice, by limiting dilution analysis. Results demonstrated that metastatic lesions occurred earlier in traumatized animals and that parasites could be detected sooner in traumatic lesions than in equivalent areas in nontraumatized mice. When lymph node cells from L. amazonensis antigen-immunized BALB/c mice were adoptively transferred intravenously to L. amazonensis-infected syngeneic mice, the parasite load in the metastatic lesions was greater in the animals that received La Ag-reactive T cells than in the controls. When CD4(+)- or CD8(+)-depleted T cell populations from La Ag-immunized mice were adoptively transferred to infected traumatized or nontraumatized animals, we observed that the metastatic lesions in CD4(+)-inoculated animals had a greater number of parasites than the lesions in mice from all other groups. Thus, a new and reliable mouse model for studying the mechanisms involved in leishmanial metastasis is described.

Animals↗

PubMed1992

Montenegro skin tests for American cutaneous leishmaniasis carried out on school children in Rio de Janeiro, Brazil: an indicator of transmission risk.

Montenegro skin tests were carried out in 1985 and 1987 on two groups of school children in the city of Rio de Janeiro. Group A consisted of 449 children residing in the Jacarepagua district, in areas where transmission of human and canine American cutaneous leishmaniasis (ACL) has been high; this group was considered to be the one at greatest risk of acquiring the infection through L. braziliensis. Group B consisted of 282 children from Bonsucesso, a suburb of Rio de Janeiro that is at a considerable distance from any area where ACL is endemic; this was a lowest risk group, and was thus used as a control. Analysis of the cutaneous test results showed that in Group A, 8.9% of the sample tested positive, whereas in Group B the result was only 2.1%. In group A there were no significant differences in the proportion of positives when analysed according to sex, age and the year when the tests were carried out. A study of the effects associated with place of residence together with other variables was carried out using log-linear regression analysis. It showed that effects arising from place of residence were maintained; that this was the only significant effect; and that it was independent of other variables. The testing of school children using the Montenegro intradermal test was shown to be a useful procedure in the characterization of localities in which there is a risk of ACL infection.

Adolescent↗

PubMed1992

Flow cytometry in the study of the interaction between murine macrophages and the protozoan parasite Leishmania amazonensis.

A flow cytometry method was adapted to study interaction between murine macrophages and Leishmania amazonensis. Using this method it was possible to detect internalization of parasites through an increase in macrophage granularity (side scatter), with the latter indicating the presence of parasites inside parasitophorus vacuoles. A quenching technique was used to confirm the feasibility of the method and to distinguish between internalized and externally attached parasites. Experiments using fixed-labeled and killed-unlabeled parasites gave similar results, demonstrating that granularity was an adequate parameter in the study of parasite-macrophage interaction, when compared with labeling methods. Experiments that measured internalization using only the increase in macrophage granularity as an indicator showed that living L. amazonensis was internalized to a greater extent than killed-unlabeled parasites. This finding suggests that the parasite has an active role in the process of internalization. The 2 methods in combination, flow cytometry and labeling, can be used to study murine peritoneal cell-L. amazonensis interactions and to sort phagocytosing and nonphagocytosing subpopulations of macrophages for further studies.

Animals↗

PubMed1991

Analysis of the human T cell responsiveness to purified antigens of Leishmania: lipophosphoglycan (LPG) and glycoprotein 63 (gp 63).

The responsiveness of peripheral blood lymphocytes from 15 cutaneous leishmaniasis patients and five healthy volunteers to purified surface antigens of Leishmania was studied. The following purified antigens were independently used as stimuli for lymphocyte proliferation in vitro: lipophosphoglycan of L. braziliensis, (Lb-LPG), lipophosphoglycan of L. mexicana (Lm-LPG), glycoprotein 63 of L. braziliensis (Lb-gp 63) and glycoprotein 63 of L. mexicana (Lm-gp 63). Among six patients tested with Lb-gp 63 and Lm-gp 63, two borderline responses and two positive responses were observed, respectively, all the others being negative. Lb-LPG and Lm-LPG induced positive responses in all patients tested (11 with Lb-LPG and five with Lm-LPG). A positive dose-response correlation was observed in the responses of the patients to both Lb-LPG and Lm-LPG. Significant differences (P less than 0.01) were found between the two groups (patients and healthy subjects) with regard to the responses to Lb-LPG. The proliferating cells after stimulation with Lb-LPG were shown to belong to the CD4+ T cell subset by flow cytometry. However, the following evidence suggests that the T cell responses were not induced by LPG itself but rather by protein contaminants in the LPG preparations: a highly purified Lb-LPG batch that had undergone additional purification induced positive responses in cells from only three out of six patients and a proteinase K-treated Lb-LPG batch did not stimulate any positive response among the same six patients.

Adult↗

PubMed1990

Trypanosoma cruzi: quantification in tissues of experimentally infected mice by limiting dilution analysis.

A limiting dilution assay (LDA) was developed for the quantification of Trypanosoma cruzi in the heart and blood of infected mice. Three groups of swiss mice were injected ip with "CL", "Colombiana," and "Y" strains. At 1-day intervals after infection, blood and the heart were removed. Serial blood dilutions in LIT medium were performed and distributed in four groups of 24 microplate wells. The growth of parasite was visually checked in an inverted microscope. It was found that curves of parasitemia obtained by parasite counting in a hemocytometer or estimated by LDA were similar. A similar method was used to quantify parasites in the heart of mice. The heart was cut, washed, dried, and its weight was determined. The heart pieces were disrupted by passage through a mesh stainless-steel screen into LIT. Serial dilutions of the heart homogenate were made in LIT and added to at least 24 replicate microplate wells. Parasites were detectable earlier in the heart of mouse infected with Y strain when compared to CL and Colombiana strains. Parasites were detected in the heart of mice of all strains by 6 days after infection. This LDA for quantification of T. cruzi permits a more precise evaluation of the number of living parasites in infected tissues.

Animals↗