Effector memory CD4(+) T cells differentially express activation associated molecules depending on the duration of American cutaneous leishmaniasis lesions.

A high number of Leishmania-responder T cells is found in cutaneous leishmaniasis lesions, suggesting that important immunological events occur at the site of infection. Although activated, cytotoxic and regulatory T cells infiltrating into lesions may influence disease pathogenesis, the role of the T cell differentiation pattern of lymphocytes in lesions is unknown. Our aim was to investigate whether the phase of lesion development (early or late) is influenced by the functional status of cells present in inflammatory infiltrate. Activation, cytotoxity and T cell differentiation molecules were evaluated in lesion mononuclear cells by flow cytometry. The frequency of T cells was correlated with the lesion area (r = 0·68; P = 0·020). CD4(+) CD25(+) T cells predominated over CD4(+) CD69(+) T cells in early lesions (less than 30 days), whereas late lesions (more than 60 days) exhibited more CD4(+) CD69(+) T cells than CD4(+) CD25(+) T cells. The duration of illness was correlated positively with CD4(+) CD69(+) (r = 0·68; P = 0·005) and negatively with CD4(+) CD25(+) T cells (r = -0·45; P = 0·046). Most CD8(+) T cells expressed cytotoxic-associated molecules (CD244(+) ), and the percentages were correlated with the lesion area (r = 0·52; P = 0·04). Both CD4(+) and CD8(+) effector memory T cells (TEM -CD45RO(+) CCR7(-) ) predominated in CL lesions and were significantly higher than central memory (TCM -CD45RO(+) CCR7(+) ) or naive T cells (CD45RO(-) CCR7(+) ). An enrichment of TEM cells and contraction of naive T cells were observed in lesions in comparison to blood (P = 0·006) for both CD4(+) and CD8(+) T cells. Lesion chronicity is associated with a shift in activation phenotype. The enrichment of TEM and activated cytotoxic cells can contribute to immune-mediated tissue damage.

Cytotoxic cell involvement in human cutaneous leishmaniasis: assessments in active disease, under therapy and after clinical cure.

Cutaneous leishmaniasis (CL) is an important public health issue worldwide. The control of Leishmania infection depends on cellular immune mechanisms, and the inflammatory response may contribute to pathogenesis. A beneficial role of CD8(+) T lymphocytes has been proposed; nevertheless, other studies suggest a cytotoxic role of CD8(+) T lymphocytes involved in tissue damage, showing controversial role of these cells. The goal of the current study was to understand the immunopathology of CL and determine the profile of cytotoxic cells--such as CD4(+) T, natural killer and natural killer T cells--that might be involved in triggering immunological mechanisms, and may lead to cure or disease progression. The frequencies of cytotoxic cell populations in peripheral blood, obtained from patients with active disease, during treatment and after clinical healing, were assessed by flow cytometry. Cytotoxicity could not be related to a deleterious role in Leishmania braziliensis infection, as patients with active CL showed similar percentages of degranulation to healthy individuals (HI). Cured patients exhibited a lower percentage of degranulating cells, which may be due to a downregulation of the immune response. The understanding of the immunopathological mechanisms involved in CL and the commitment of cytotoxic cells enables improvements in therapeutic strategies.

Antigen-triggered interferon-γ and interleukin-10 pattern in cured mucosal leishmaniasis patients is shaped during the active phase of disease.

An exacerbated type 1 response to leishmanial antigens is the basis of tissue destruction observed in mucosal leishmaniasis (ML). After therapy, a persistent production of high levels of inflammatory cytokines can confer a poor prognosis. Herein we investigated whether the clinical conditions defined during the active phase of ML affect the magnitude of long-term anti-Leishmania immune response. Twenty clinically cured ML cases were studied. Peripheral blood mononuclear cells (PBMC) were cultured with L. braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), concanavalin-A (Con-A) or medium alone, and the lymphocyte proliferative response and cytokine secretion were quantified. Medical records were reviewed for Montenegro skin test (MST) during diagnosis, duration of ML disease or time elapsed after clinical cure. The duration of disease was correlated positively with MST (r = 0·61). Lb-Ag induced interferon (IFN)-γ was correlated positively with duration of illness (r = 0·69) as well as the frequency of secreting cells [enzyme-linked immunospot (ELISPOT)] assay. No association was observed for Tg-Ag or Con-A. Disease duration was correlated negatively with interleukin (IL)-10 production (r = -0·76). Moreover, a negative correlation between length of time after clinical cure and TNF levels (r = -0·94) or the IFN-γ : IL-10 ratio (r = -0·89) were also seen. We suggest that the magnitude of the IFN-γ inflammatory response triggered by ML can be driven by the time of leishmanial antigens exposition during the active phase of the disease. This pattern could persist even long-term after cure. However, despite IFN-γ levels, the decrease of the TNF and IFN-γ : IL-10 ratio reflects the control of proinflammatory responses achieved by cure of ML, possibly preventing disease relapses.

Decrease in anti-Leishmania IgG3 and IgG1 after cutaneous leishmaniasis lesion healing is correlated with the time of clinical cure.

For better efficiency in the establishment of American tegumentary leishmaniasis clinical cure, the World Health Organization suggests that the clinical criteria are supported by serologic data. The present study aims to investigate the dynamics of IgG subclass production in clinical evolution post-treatment of cutaneous leishmaniasis (CL). Paired sera from 23 subjects with CL resulting from Leishmania braziliensis infection were studied during the active lesion phase (aCL) and after clinical cure post-therapy (hCL), which included an alternative protocol with a low dose of antimony. Anti-Leishmania IgG and its subclasses were measured using ELISA, and the immunoglobulin levels were correlated with patients' clinical data. All of the subjects were clinically healed and did not present relapse during follow-up. Serum levels of anti-Leishmania IgG (r = -0·79; P < 0·0001), IgG1 (r = -0·64, P < 0·001) and IgG3 (r = -0·42, P < 0·045) in hCL were negatively correlated with the duration of clinical cure. After 24 months of clinical cure, 73% of samples were negative for IgG1 and 78% were negative for IgG3. In conclusion, the detection of serum anti-Leishmania IgG1 and IgG3 is an improved laboratory strategy to aid in the decision of interruption of the ambulatory follow-up of CL patients.

Therapeutic failure in American cutaneous leishmaniasis is associated with gelatinase activity and cytokine expression.

Cutaneous lesions caused by Leishmania braziliensis infection occasionally heal spontaneously, but with antimonials therapy heal rapidly in approximately 3 weeks. However, about 15% of the cases require several courses of therapy. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are gelatinases that have been implicated in other chronic cutaneous diseases and skin re-epithelialization. These enzymes are controlled by their natural inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] and by some cytokines. Uncontrolled gelatinase activity may result in intense tissue degradation and, consequently, poorly healing wounds. The present study correlates gelatinase activity to therapeutic failure of cutaneous leishmaniasis (CL) lesions. Our results demonstrate an association between gelatinase activity and increased numbers of cells making interferon (IFN)-γ, interleukin (IL)-10 and transforming growth factor (TGF)-ß in lesions from poor responders. Conversely, high levels of MMP-2 mRNA and enhanced MMP-2 : TIMP-2 ratios were associated with a satisfactory response to antimonials treatment. Additionally, high gelatinolytic activity was found in the wound beds, necrotic areas in the dermis and within some granulomatous infiltrates. These results indicate the importance of gelatinase activity in the skin lesions caused by CL. Thus, we hypothesize that the immune response profile may be responsible for the gelatinase activity pattern and may ultimately influence the persistence or cure of CL lesions.

Psoriasis affects individuals of African descent and white Brazilians similarly.

INTRODUCTION: Cultural, socio-demographic and environmental factors such as tropical climate and exposure to sun could have an impact on the incidence or clinical course of psoriasis. Here we describe the main clinical aspects of psoriasis in Brazilian patients and also investigate whether any particular feature can distinguish the disease occurring in Brazil from that occurring in other countries. MATERIAL AND METHODS: We recorded the clinical features of 151 psoriasis patients seen in a Brazilian public dermatological care unit between 2006 and 2008. RESULTS: Males and females were similarly affected. The reported races were as follows: whites, 47 cases (41.6%), interracial individuals (mixed race), 42 cases (37.2%) and blacks, 24 cases (21.2%). Chronic plaque-type psoriasis was the most prevalent clinical form (110 cases, 72.8%) followed by palm and sole involvement (21 cases, 13.9%). CONCLUSIONS: We demonstrated that psoriasis in these Brazilian subjects was similar to that observed in subjects from other countries, but interracial and black populations were affected as much as whites. Considering the high rate of interracial populations among Brazilians we cannot exclude the possibility that Afro-descendants may have inherited Caucasian genes associated with psoriasis. Poor socio-economic conditions of Afro-descendants can limit their possibilities of receiving adequate treatments, impairing their health-related quality of life.

The skin homing receptor cutaneous leucocyte-associated antigen (CLA) is up-regulated by Leishmania antigens in T lymphocytes during active cutaneous leishmaniasis.

The cutaneous leucocyte-associated antigen receptor (CLA) can direct Leishmania-specific T lymphocytes towards inflamed skin lesions. Homing receptors [CLA, lymphocyte-associated antigen 1 (LFA-1) or CD62L] were analysed in lymphocytes from blood and cutaneous leishmaniasis (CL) lesions. CL patients with active lesions (A-CL) presented lower levels of T lymphocytes expressing the CLA(+) phenotype (T CD4(+) = 10.4% +/- 7.5% and T CD8(+) = 5.8% +/- 3.4%) than did healthy subjects (HS) (T CD4(+) = 19.3% +/- 13.1% and T CD8(+) = 21.6% +/- 8.8%), notably in T CD8(+) (P < 0.001). In clinically cured patients these percentages returned to levels observed in HS. Leishmanial antigens up-regulated CLA in T cells (CLA(+) in T CD4(+) = 33.3% +/- 14.1%; CLA(+) in T CD8(+) = 22.4% +/- 9.4%) from A-CL but not from HS. An enrichment of CLA(+) cells was observed in lesions (CLA(+) in T CD4(+) = 45.9% +/- 22.5%; CLA(+) in T CD8(+) = 46.4% +/- 16.1%) in comparison with blood (CLA(+) in T CD4(+) = 10.4% +/- 7.5%; CLA(+) in T CD8(+) = 5.8% +/- 3.4%). Conversely, LFA-1 was highly expressed in CD8(+) T cells and augmented in CD4(+) T from peripheral blood of A-CL patients. In contrast, CD62L was not affected. These results suggest that Leishmania antigens can modulate molecules responsible for migration to skin lesions, potentially influencing the cell composition of inflammatory infiltrate of leishmaniasis or even the severity of the disease.

Characterization of human T lymphocyte-mediated immune responses induced by a vaccine against American tegumentary leishmaniasis.

Forty-three Brazilians were immunized against American tegumentary leishmaniasis using a vaccine made of whole antigens from killed promastigotes of five American dermotropic Leishmania strains. None of the immunized subjects had a positive reaction in the Montenegro skin test (leishmanin) before vaccination, and 74% developed positive reactions in the skin test after vaccination. The proliferative responses of peripheral blood mononuclear cells (PBMC) induced by antigens from dermotropic Leishmania species were significantly higher after vaccination than before vaccination. However, with antigens from L. chagasi (a causative agent of American visceral leishmaniasis), there was no significant difference between the proliferative responses obtained before and after vaccination. Interferon-gamma was detected in the supernatants of L. braziliensis antigen-stimulated PBMC cultures after vaccination (but not before vaccination). One year after vaccination, PBMC were obtained from eight of the immunized individuals and stimulated with L. braziliensis antigens in proliferative response assays. In all cases, the majority of the responding cells were CD8+ T cells, in contrast to the results of a group of patients with active lesions of tegumentary leishmaniasis, whose L. braziliensis-reactive cells were mainly of the CD4+ T cell phenotype.

Clinical analysis and parasite genetic diversity in human immunodeficiency virus/Chagas' disease coinfections in Brazil.

To evaluate the possible role of parasitemia on Chagas' disease reactivation in Chagas' disease/human immunodeficiency virus (HIV) coinfection cases and the impact of HIV coinfection on Trypanosoma cruzi genetic diversity, 71 patients with Chagas' disease (34 HIV+ and 37 HIV-) were surveyed. Moreover, 92 T. cruzi stocks from 47 chronic chagasic patients (29 HIV+ and 18 HIV-) were isolated and analyzed by multilocus enzyme electrophoresis and a random amplified polymorphic DNA procedure. High parasitemia appeared to play a major role in cases of Chagas' disease reactivation. In HIV+ patients, the genetic diversity and population structure (clonality) of T. cruzi was similar to that previously observed in HIV- patients, which indicates that immunodepression does not modify drastically genotype repartition of the parasite. There was no apparent association between given T. cruzi genotypes and specific clinical forms of Chagas' disease/HIV associations.

Cellular and humoral immune responses of a patient with American cutaneous leishmaniasis and AIDS.

The lymphocyte responsiveness to leishmanial antigens and its influence on the course of cutaneous leishmaniasis was studied in a patient with AIDS-associated American cutaneous leishmaniasis caused by Leishmania braziliensis. The patient had cutaneous disseminated erythematous papules or nodules and mucosal lesions as well as moniliasis and weight loss. The patient had a poor delayed-type hypersensitivity to leishmanial antigens, showing 3 mm of induration. The cellular immune responses were studied in vitro by lymphocyte proliferative assays induced by leishmanial antigens and concanavalin A. The T cell phenotypes were analysed by flow cytometry. The peripheral blood mononuclear cells before proliferation showed an inversion of the CD4/CD8 ratio (0.28:1). The lymphoproliferative responses to antigen and mitogen were very low (indices < 2.5). The blast-like cell phenotypes after antigen stimulation in culture were: CD3+ 44.8%, CD4+ 7.53% and CD8+ 17.45%. In AIDS patients the decrease in the pool of CD4+ cells, and consequent diminution of the CD4/CD8 ratio, produced by HIV infection provokes a generalized immune depression. The patient's disseminated clinical picture was probably related to the inability of his T cell-mediated immune responses to control the spread of Leishmania infection.

Long-term follow-up of co-infected HIV and Trypanosoma cruzi Brazilian patients.

Three cases of Trypanosoma cruzi-HIV co-infected haemophiliacs are described. Parasitological (xenodiagnosis, haemoculture, PCR) and immunological (CD4+ and CD8+ T cell counts, in vitro lymphoproliferative responses) studies were performed. Hybridization of isolated parasites with a specific probe confirmed the T. cruzi aetiology. We observed that despite the high parasitaemia, no clinical or parasitological evidence of T. cruzi reactivation was detected. CD4+ T cells decreased with time in two patients and the lymphocyte proliferative response to T. cruzi was very low in all patients. These data suggest that T. cruzi infection may have a long silent course in immunosuppressed HIV patients. Therefore, this parasitic infection should be investigated in any AIDS patient coming from areas endemic for Chagas' disease.

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.

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.

Can interferon-gamma and interleukin-10 balance be associated with severity of human Leishmania (Viannia) braziliensis infection?

Suitable levels of interferon (IFN)-gamma and interleukin (IL)-10 seem to favour the outcome of cutaneous leishmaniasis (CL), while high IFN-gamma and low IL-10 production are associated with severity of mucosal leishmaniasis (ML). Considering that cytokine balance is important for the maintenance of protective responses in leishmaniasis, our aim was to investigate leishmanial antigens-induced IFN-gamma and IL-10 levels maintained in healed individuals who had different clinical outcomes of Leishmania infection. Thirty-three individuals who recovered from L. braziliensis infection were studied: cured CL (CCL), cured ML (CML), spontaneous healing of CL (SH) or asymptomatic individuals (ASY). Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) in culture supernatants of L. braziliensis-stimulated peripheral blood mononuclear cells (PBMC). IFN-gamma levels were higher in CML (7593 +/- 5994 pg/ml) in comparison to SH (3163 +/- 1526 pg/ml), ASY (1313 +/- 1048 pg/ml) or CCL (1897 +/- 2087 pg/ml). Moreover, cured ML cases maintained significantly lower production of IL-10 (127 +/- 57.8 pg/ml) in comparison to SH (1373 +/- 244 pg/ml), ASY (734 +/- 233 pg/ml) or CCL (542 +/- 375 pg/ml). Thus, a high IFN-gamma/IL-10 ratio observed in CML can indicate unfavourable cytokine balance. On the other hand, no significant difference in the IFN-gamma/IL-10 ratio was observed when CCL individuals were compared to SH or ASY subjects. In conclusion, even after clinical healing, ML patients maintained a high IFN-gamma/IL-10 secretion profile in response to leishmanial antigens. This finding can explain a delayed down-modulation of exacerbated inflammatory responses, which can be related in turn to the necessity of prolonged therapy in ML management. Conversely, lower IFN-gamma/IL-10 balance observed in CCL, SH and ASY individuals can represent a better-modulated immune response associated with a favourable prognosis.

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.

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.