Hélio A. Lessa

Up-regulation of Th1-type responses in mucosal leishmaniasis patients.

ABSTRACT The cytokine profile produced by peripheral blood mononuclear cells (PBMC) in response to leishmania antigens and the ability of interleukin-10 (IL-10) and transforming growth factor β (TGF-β) to modulate the immune response were evaluated in 21 mucosal leishmaniasis patients. Patients with mucosal disease exhibited increased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion and decreased IL-10 secretion compared to patients with classical cutaneous leishmaniasis. CD4+ Th1 cells were the main source of IFN-γ and TNF-α production in mucosal leishmaniasis patients. Evaluation of cytokine gene expression in PBMC of these patients showed that there was strong up-regulation of IFN-γ transcripts upon stimulation with leishmania antigen, in contrast to the baseline levels of IL-10 mRNA. IL-10 suppressed IFN-γ production by 48% in cell cultures from cutaneous leishmaniasis patients and by 86% in cell cultures from healthy subjects stimulated with purified protein derivative, whereas in similar conditions IL-10 suppressed IFN-γ production by 19% in cell cultures from mucosal leishmaniasis patients stimulated with leishmania antigen. TGF-β suppressed IFN-γ levels to a greater extent in healthy subjects than in mucosal leishmaniasis and cutaneous leishmaniasis patients. These data indicate that a poorly modulated T-cell response in mucosal leishmaniasis patients leads to production of high levels of proinflammatory cytokines, such as IFN-γ and TNF-α, as well as a decreased ability of IL-10 and TGF-β to modulate this response. These abnormalities may be the basis for the pathological findings observed in this disease.

Cytokine profile and pathology in human leishmaniasis

The clinical spectrum of leishmaniasis and control of the infection are influenced by the parasite-host relationship. The role of cellular immune responses of the Th1 type in the protection against disease in experimental and human leishmaniasis is well established. In humans, production of IFN-gamma is associated with the control of infection in children infected by Leishmania chagasi. In visceral leishmaniasis, an impairment in IFN-gamma production and high IL-4 and IL-10 levels (Th2 cytokines) are observed in antigen-stimulated peripheral blood mononuclear cells (PBMC). Moreover, IL-12 restores IFN-gamma production and enhances the cytotoxic response. IL-10 is the cytokine involved in down-regulation of IFN-gamma production, since anti-IL-10 monoclonal antibody (mAb) restores in vitro IFN-gamma production and lymphoproliferative responses, and IL-10 abrogates the effect of IL-12. In cutaneous and mucosal leishmaniasis, high levels of IFN-gamma are found in L. amazonensis-stimulated PBMC. However, low or absent IFN-gamma levels were observed in antigen-stimulated PBMC from 50% of subjects with less than 60 days of disease (24 +/- 26 pg/ml). This response was restored by IL-12 (308 +/- 342 pg/ml) and anti-IL-10 mAb (380 +/- 245 pg/ml) (P < 0.05). Later during the disease, high levels of IFN-gamma and TNF-alpha are produced both in cutaneous and mucosal leishmaniasis. After treatment there is a decrease in TNF-alpha levels (366 +/- 224 pg/ml before treatment vs 142 +/- 107 pg/ml after treatment, P = 0.02). Although production of IFN-gamma and TNF-alpha might be involved in the control of parasite multiplication in the early phases of Leishmania infection, these cytokines might also be involved in the tissue damage seen in tegumentary leishmaniasis.

Decreased In Situ Expression of Interleukin-10 Receptor Is Correlated with the Exacerbated Inflammatory and Cytotoxic Responses Observed in Mucosal Leishmaniasis

ABSTRACT Human infection with Leishmania braziliensis can lead to cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). We hypothesize that the intense tissue destruction observed in ML is a consequence of an uncontrolled exacerbated inflammatory immune response, with cytotoxic activity. For the first time, this work identifies the cellular sources of inflammatory and antiinflammatory cytokines, the expression of effector molecules, and the expression of interleukin-10 (IL-10) receptor in ML and CL lesions by using confocal microscopy. ML lesions displayed a higher number of gamma interferon (IFN-γ)-producing cells than did CL lesions. In both ML and CL, CD4+ cells represented the majority of IFN-γ-producing cells, followed by CD8+ cells and CD4− CD8− cells. The numbers of tumor necrosis factor alpha-positive cells, as well as those of IL-10-producing cells, were similar in ML and CL lesions. The effector molecule granzyme A showed greater expression in ML than in CL lesions, while inducible nitric oxide synthase did not. Finally, the expression of IL-10 receptor was lower in ML than in CL lesions. Thus, our data identified distinct cytokine and cell population profiles for CL versus ML patients and provide a possible mechanism for the development of ML disease through the demonstration that low expression of IL-10 receptor is present in conjunction with a cytotoxic and inflammatory profile in ML.

Oral Pentoxifylline Combined with Pentavalent Antimony: A Randomized Trial for Mucosal Leishmaniasis

BACKGROUND Mucosal leishmaniasis is associated with intense tissue damage and high tumor necrosis factor-alpha production. Therapeutic failure occurs in up to 42% of cases; patients who experience treatment failure will require >1 pentavalent antimony (Sb(v)) course or alternative drugs to achieve a cure. We previously showed that an inhibitor of tumor necrosis factor-alpha (pentoxifylline) combined with Sb(v) cured 90% patients refractory to monotherapy with Sb(v). METHODS A double-blind, placebo-controlled trial involving 23 patients with mucosal leishmaniasis evaluated the efficacy of pentoxifylline when administered in association with Sb(v), compared with Sb(v) treatment alone. Eleven patients were randomized to receive Sb(v) plus oral pentoxifylline for 30 days, and 12 patients received Sb(v) plus oral placebo. The criterion for cure was a complete healing of lesions. RESULTS All patients in the pentoxifylline group experienced a cure with 1 course of Sb(v), whereas 5 (41.6%) of 12 patients in the placebo group required a second course of Sb(v) (P=.037). The healing time +/- standard deviation in the pentoxifylline group was 83+/-36 days, compared with 145+/-99 days in the placebo group (P=.049). No relapses were documented in either group at the 2-year follow-up visit. CONCLUSIONS The addition of pentoxifylline to Sb(v) in mucosal leishmaniasis reduces the healing time significantly and prevents the need for further courses of Sb(v).

Multiclonal Leishmania braziliensis Population Structure and Its Clinical Implication in a Region of Endemicity for American Tegumentary Leishmaniasis

ABSTRACT In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CPs geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.

Mucosal leishmaniasis: epidemiological and clinical aspects

UNLABELLED Leishmaniasis has been documented in several countries, with an estimated prevalence of 12 million people and an incidence at around 400,000 new cases per year. Leishmaniasis in the New World is one the major endemic diseases in Brazil and Latin America. OBJECTIVE The aim of this study was to add to the current knowledge on mucosal leishmaniasis, bringing the experience of the Immunology and Otolaryngology Departments in the Professor Edgar Santos University Hospital of the Federal University of Bahia. CONCLUSION Cutaneous leishmaniasis is the most common form of New World Leishmaniasis; mucosal legions may occur simultaneously or after years of disease. Mucosal leishmaniasis is caused mainly by L. braziliensis braziliensis; although the nasal mucosa is the most affected area, lesions may be found on the lips, mouth, pharynx and larynx. In addition to parasite-related factors, the host immune response may be involved in the pathogenicity of lesions in mucosal leishmaniasis.

Leishmaniose mucosa: aspectos clínicos e epidemiológicos

Leishmaniasis has been documented in several countries, with an estimated prevalence of 12 million people and an incidence at around 400,000 new cases per year. Leishmaniasis in the New World is one the major endemic diseases in Brazil and Latin America. OBJECTIVE: The aim of this study was to add to the current knowdlegde on mucosal leishmaniasis, bringing the experience of the Imunology and Otolaryngology Departments in the Professor Edgar Santos University Hospital of the Federal University of Bahia. CONCLUSION: Cutaneous leishmaniasis is the most common form of New World Leishmaniasis; mucosal legions may occur simultaneously or after years of disease. Mucosal leishmaniasis is caused mainly by L. braziliensis braziliensis; although the nasal mucosa is the most affected area, lesions may be found on the lips, mouth, pharynx and larynx. In addition to parasite-related factors, the host immune response may be involved in the pathogenicity of lesions in mucosal leishmaniasis.

Mucosal leishmaniasis patients display an activated inflammatory T-cell phenotype associated with a nonbalanced monocyte population.

Leishmania braziliensis is a parasite that can induce at least two clinical forms of leishmaniasis in humans: cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). In humans, the specific mechanisms that determine which form will develop following infection are not well established. In this study, peripheral blood mononuclear cells from 17 CL and 9 ml patients were compared both ex vivo and after culture with soluble leishmania antigen (SLA). Patients with ML presented a higher frequency of activated T cells as measured by ex vivo frequencies of CD4+CD69+, CD4+CD28–, CD4+CD62L– and CD8+CD69+ than those with CL. Moreover, after stimulation with SLA, patients with ML presented a higher frequency of TNF‐α‐producing CD4+ and CD14+ cells than CL individuals. While CL patients displayed a positive correlation between the frequency of IL‐10 and TNF‐α‐producing monocytes, the ML patients did not. This lack of a positive correlation between IL‐10‐producing and TNF‐α‐producing monocytes in ML patients could lead to a less controlled inflammatory response in vivo. These results corroborate with a model of an exacerbated, unregulated, immune response in ML patients and point to key immunomodulatory leucocyte populations and cytokine networks that may be involved in the development of immunopathology in ML patients.

Geographic clustering of leishmaniasis in northeastern Brazil.

Different forms of this disease are spreading rapidly in distinct geographic clusters in this region.

A proposed new clinical staging system for patients with mucosal leishmaniasis

Mucosal leishmaniasis (ML) occurs mainly in areas where Leishmania braziliensis is transmitted. It affects predominantly the nasal mucosa and, in more severe forms, can lead to significant tissue destruction. There is no standard method for grading the severity of disease. We categorised 50 patients with ML according to a proposed new clinical staging system. Their age ranged from 10 to 86 y (mean ± SD: 36 ± 16 y) and 43 (86%) patients were male. The different degrees of evolution of mucosal disease, from the initial stage to the more severe long-term cases, enabled ML to be graded into five stages. Stage I is characterised by nodular lesions of the mucosa without ulceration. Stage II is represented by superficial mucosal ulcerations with concomitant fine granular lesions. Stage III is characterised by deep mucosal ulcerations with granular tissue formation. In stage IV there are irreversible lesions leading to perforation of the cartilaginous nasal septum with necrosis. In stage V the nasal pyramid is compromised with alterations of facial features as a consequence of severe tissue destruction. These stages may be useful in characterising the severity of the lesion and optimising therapeutic outcome.