Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Impaired Th1 Response Is Associated With Therapeutic Failure in Patients With Cutaneous Leishmaniasis Caused by Leishmania braziliensis.

BACKGROUND: Leishmania skin test (LST) evaluates the delayed type hypersensitivity to Leishmania antigens (LA) and has been used for diagnosis of cutaneous leishmaniasis (CL). In CL patients LST is usually positive but a small percentage have negative LST. The aim of this study was to determine the clinical and immunologic features and response to antimony therapy in LST-negative CL patients. METHODS: We compare the clinical presentation, response to therapy, and immune response of CL patients with negative vs positive LST. RESULTS: The clinical presentation was similar in both groups but LST-negative patients had a lower cure rate. In the lesions, LST-negative patients displayed less inflammation and necrosis, and higher frequency of CD8+ T cells. Mononuclear cells from LST-negative patients had a poor T helper 1 cell (Th1) response but levels of interleukin-1ß (IL-1ß), IL-6, IL-17, granzyme B, and metalloproteinase-9 (MMP-9) were similar to the LST-positive group upon stimulation with LA. Leishmania internalization and killing by macrophages were similar in both groups. Cure of disease was associated with restoration of Th1 response. CONCLUSIONS: In LST-negative patients, impaired Th1 response is associated with therapeutic failure. Increased frequency of CD8+ T cells and high production of inflammatory cytokines, granzyme B, and MMP-9 contributes to immunopathology.

Granzyme B Produced by Natural Killer Cells Enhances Inflammatory Response and Contributes to the Immunopathology of Cutaneous Leishmaniasis.

BACKGROUND: Skin lesions from patients infected with Leishmania braziliensis has been associated with inflammation induced by cytotoxic CD8+ T cells. In addition, CD8+ T cell-mediated cytotoxicity has not been linked to parasite killing. Meanwhile, the cytotoxic role played by natural killer (NK) cells in cutaneous leishmaniasis (CL) remains poorly understood. METHODS: In this study, we observed higher frequencies of NK cells in the peripheral blood of CL patients compared with healthy subjects, and that NK cells expressed more interferon-γ, tumor necrosis factor (TNF), granzyme B, and perforin than CD8+ T cells. RESULTS: We also found that most of the cytotoxic activity in CL lesions was triggered by NK cells, and that the high levels of granzyme B produced in CL lesions was associated with larger lesion size. Furthermore, an in vitro blockade of granzyme B was observed to decrease TNF production. CONCCLUSIONS: Our data, taken together, suggest an important role by NK cells in inducing inflammation in CL, thereby contributing to disease immunopathology.

Early Cutaneous Leishmaniasis Patients Infected With Leishmania braziliensis Express Increased Inflammatory Responses After Antimony Therapy.

Background: Early cutaneous leishmaniasis (ECL) is characterized by a nonulcerated papular lesion and illness duration less than 30 days. Approximately 4 weeks later, the cutaneous leishmaniasis (CL) ulcers appear. We were surprised to find that failure after antimony therapy (Sb5) is higher in ECL than CL. We hypothesize that the inflammatory response in ECL patients may increase during Sb5 therapy, which leads to treatment failure. Methods: A cohort of 44 ECL patients infected by Leishmania braziliensis was established to evaluate the response to Sb5 and to compare immunologic responses in ECL patients with CL and healthy subjects. Results: A hierarchical clustering based on cytokine levels showed a weak positive correlation between proinflammatory cytokine levels and those patients that failed Sb5 treatment. Although Sb5 therapy decreased interferon-γ and tumor necrosis factor levels in CL patients, we were surprised to find that an increase in these cytokines was observed in ECL patients. Moreover, interleukin (IL)-10 was less able to down-modulate immune responses in ECL. Conclusions: The enhanced production of proinflammatory cytokines, due in part to the decreased ability of IL-10 to down-modulate immune response during therapy in ECL, promotes the development and persistence of leishmania ulcer despite antimony therapy.

Cytotoxic activity in cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a chronic disease caused by species of the protozoan Leishmania and characterised by the presence of ulcerated skin lesions. Both parasite and host factors affect the clinical presentation of the disease. The development of skin ulcers in CL is associated with an inflammatory response mediated by cells that control parasite growth but also contribute to pathogenesis. CD8+ T cells contribute to deleterious inflammatory responses in patients with CL through cytotoxic mechanisms. In addition, natural killer cells also limit Leishmania infections by production of interferon-γ and cytotoxicity. In this review, we focus on studies of cytotoxicity in CL and its contribution to the pathogenesis of this disease.

Intermediate monocytes contribute to pathologic immune response in Leishmania braziliensis infections.

Ulcer development in patients with cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is associated with high levels of tumor necrosis factor (TNF). We found that early after infection, before ulcer development, the frequency of CD16(+) (both intermediate [CD14(+)CD16(+)] and nonclassical [CD14(dim)CD16(+)]) monocytes was increased in the peripheral blood of patients with L. braziliensis, compared with uninfected controls. These results suggest that CD16(+) monocytes might promote disease. Also, we found that intermediate monocytes expressed CCR2 and that increased levels of CCL2 protein were present in lesions from patients, suggesting that intermediate monocytes are more likely than nonclassical monocytes to migrate to the lesion site. Finally, we found that the intermediate monocytes produced TNF. Our results show that intermediate monocytes are increased in frequency soon after infection; express CCR2, which would promote their migration into the lesions; and, owing to their production of TNF, can enhance the inflammatory response.

In Situ versus Systemic Immune Response in the Pathogenesis of Cutaneous Leishmaniasis.

The role of the immune response in the pathogenesis of cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis is predominantly carried out via blood cells. Here, we evaluate whether cytokine production by peripheral blood mononuclear cells (PBMCs) reflects what has been documented at the lesion site. The participants included 22 CL patients diagnosed with a positive PCR. PBMCs were stimulated for 72 h with a soluble leishmania antigen (SLA). Biopsies obtained from the edge of the ulcers were incubated for the same period. Cytokines in supernatants were assessed via ELISA. TNF, IL-1ß, IL-6, IL-17, and granzyme B (GzmB) were higher in the supernatants of biopsies than in PBMCs, but IFN-γ was higher in the supernatants of PBMCs than in biopsies. There was a positive correlation between IFN-γ and TNF in PBMCs, and an inverse correlation between TNF and IL-10 in the cells from the lesion site. A strong correlation between IL-1ß, IL-17, and GzmB was observed in the biopsies, and a positive correlation was detected between these cytokines and the lesion size. Our results indicate that the immune response in L. braziliensis lesions is different from that observed in peripheral blood, and our data suggest that in addition to IL-1ß and GzmB, IL-17 participates in the pathology of CL.

Assessment of Immune and Clinical Response in Patients with Mucosal Leishmaniasis Treated with Pentavalent Antimony and Pentoxifylline.

Mucosal leishmaniasis (ML) is a severe form of tegumentary leishmaniasis associated with a persistent inflammatory response. High levels of TNF, IFN-γ, CXCL9 and CXCL10 are found in ML patients, and the association of pentoxifylline with antimony is more effective in decreasing the healing time in ML patients when compared to antimony alone. The present study aimed to investigate the existence of a correlation between cytokine and chemokine production and ML severity and evaluate the potential value of cytokine and chemokine production as marker of therapeutic response in ML patients. This prospective study included 86 subjects in an area of endemic Leishmania braziliensis transmission. Patients diagnosed with ML were classified into clinical stages ranging from I to V according to disease severity. TNF, IFN-γ, CXCL9 and CXCL10 levels were quantified in the supernatant of the mononuclear cell cultures by ELISA before and after treatment with antimony alone or antimony plus pentoxifylline. The median TNF level in the group with mild disease (Stages I-II) was 1064 pg/mL (142-3738 pg/mL), while, in the group with moderate or severe disease (Stages III-V), it was 1941 pg/mL (529-5294 pg/mL) (p = 0.008). A direct correlation was observed between ML clinical severity and levels of TNF production (r = 0.44, p = 0.007). Patients who were treated with antimony and pentoxifylline healed significantly faster than those treated with antimony alone (52 vs. 77 days, hazard ratio = 0.60; 95% confidence interval = 0.38-0.95, p = 0.013). Therapeutic failure was higher in the group that received antimony alone (25% vs. 7%; p = 0.041). There was a significant decrease in CXCL9 after therapy of ML in both groups (p = 0.013; p = 0.043). TNF levels are associated with the severity of mucosal diseases, and pentoxifylline associated with antimony should be the recommended therapy for ML in countries where liposomal amphotericin B is not available.

High Anti-Leishmania IgG Antibody Levels Are Associated With Severity of Mucosal Leishmaniasis.

Background: Mucosal leishmaniasis (ML), the most inflammatory form of tegumentary leishmaniasis, is predominantly caused by Leishmania braziliensis. The disease is characterized by the development of lesions, mainly in the nasal mucosa. An exacerbated inflammatory response has been associated with the presence of destructive and disfiguring lesions, with stages of severity ranging from small nodulations to the complete destruction of the nasal pyramid architecture. As Leishmania is an intracellular parasite, most immunological studies have emphasized the cell-mediated immune response, while relatively few studies aimed to investigate the role antibodies in protection against, or the pathology of ML. Methods: Patients with a confirmed diagnosis of ML were classified according to clinical staging criteria. Serum levels of Leishmania-specific IgG, IgG1 and IgG2 antibodies were determined by ELISA before and after treatment with antimony or antimony plus pentoxifylline. Results: Patients in stages IV and V produced higher concentrations of IgG and IgG1 antibodies when compared to those in stage I and II. Significant reductions were seen in the concentrations of IgG and IgG2 antibodies in most patients who responded well to treatment. Conclusions: Our data demonstrate an association between IgG antibody titers and the severity of mucosal disease. The observed reduction in antibody production after successful treatment in most patients preliminarily indicates that these tests can be used to aid in the assessment of therapeutic response.

Anti-Leishmania IgG is a marker of disseminated leishmaniasis caused by Leishmania braziliensis.

BACKGROUND: In this study, we determined the accuracy of anti-Leishmania IgG and IgG subclasses to distinguish clinical forms of American tegumentary leishmaniasis (ATL) and and determined the relationship between antibodies levels with cytokine production and severity of ATL. METHODS: Participants were 40 patients with cutaneous leishmaniasis (CL), 20 patients with mucosal leishmaniasis (ML), 20 patients with disseminated leishmaniasis (DL), and 20 individuals with subclinical Leishmania braziliensis infection (SC). Diagnosis was performed by DNA of L. braziliensis or IFN-γ production in SC. IgG and subclasses of IgG to soluble Leishmania antigen and cytokine levels in supernatants of mononuclear cells were detected by ELISA. RESULTS: IgG was detected in 95%, 95%, and 100% of patients with CL, ML, and DL, respectively. Higher levels of anti-Leishmania IgG and IgG2 were seen in DL compared to CL, ML, and SC. ROC analysis confirmed the ability of IgG to distinguish DL from the other clinical forms. A direct correlation was observed between IgG titers and levels of IFN-γ and CXCL10 in CL and DL, and IgG2 antibodies were correlated with the number of lesions in DL. CONCLUSIONS: High anti-Leishmania IgG and IgG2 levels are characteristic of DL, and while IgG was correlated with pro-inflammatory cytokines, IgG2 was direct correlated with the number of lesions.

Evaluation of the Ability of Miltefosine Associated with Topical GM-CSF in Modulating the Immune Response of Patients with Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) due to L. braziliensis is associated with an exaggerated inflammatory response and tissue damage. Miltefosine is more effective than pentavalent antimony (Sbv) in the treatment of CL, and here, we evaluate the ability of Sbv, miltefosine, and GM-CSF administered intravenously, orally, or topically, respectively, to modify the immune response. Patients were treated with miltefosine plus GM-CSF, miltefosine plus placebo, or Sbv. Mononuclear cells were stimulated with soluble Leishmania antigen (SLA) on day 0 and day 15 of therapy, and cytokine levels were determined in supernatants by ELISA. The lymphocyte proliferation and oxidative burst were evaluated by flow cytometry, and the degree of infection and Leishmania killing by optical microscopy. Proliferation of CD4+ T cells were enhanced in patients using miltefosine and in CD8+ T cells when GM-CSF was associated. Enhancement in the oxidative burst occurred in the miltefosine plus GM-CSF group on day 15 of therapy. Moreover, the number of L. braziliensis in infected monocytes on day 15 as well as the percentage of infected cells was lower after 48- and 72-hour culture in cells from patients treated with miltefosine plus GM-CSF. In addition to the ability of miltefosine to kill Leishmania, the changes in the immune response caused by miltefosine and GM-CSF may increase the cure rate of CL patients using these drugs.

A Potential Role for Mononuclear Phagocytes in Cutaneous Ulcer Development in Human Immunodeficiency Virus-Leishmania braziliensis Coinfection.

Skin ulcer development in cutaneous leishmaniasis due to Leishmania braziliensis infection is associated with a mononuclear cell infiltrate and high levels of tumor necrosis factor (TNF). Herein, we show that despite the absence of Leishmania-driven TNF, a cutaneous leishmaniasis patient with acquired immunodeficiency syndrome developed a skin ulcer. The presence of mononuclear phagocytes and high levels of TNF, chemokine (C-C motif) ligand 2 (CCL2), and metalloproteinase-9 in tissue are identified as potential contributors to immunopathology observed in L. braziliensis-infected patients.

Matrix metalloproteinase 9 production by monocytes is enhanced by TNF and participates in the pathology of human cutaneous Leishmaniasis.

INTRODUCTION: Cutaneous leishmaniasis (CL) due to L.braziliensis infection is characterized by a strong inflammatory response with high levels of TNF and ulcer development. Less attention has been given to the role of mononuclear phagocytes to this process. Monocytes constitute a heterogeneous population subdivided into classical, intermediate and non-classical, and are known to migrate to inflammatory sites and secrete inflammatory mediators. TNF participates in the induction of matrix metalloproteinases (MMPs). MMP-9 is an enzyme that degrades basal membrane and its activity is controlled by the tissue inhibitor of metalloproteinase. METHODS: Mononuclear cells were obtained from ex-vivo labeling sub-populations of monocytes and MMP-9, and the frequency was determined by flow cytometry. Culture was performed during 72 hours, stimulating the cells with SLA, levels of MMP-9 and TIMP-1 in the supernatants were determined by ELISA. RESULTS: We observed that cells from CL lesions secrete high amounts of MMP-9 when compared to healthy subjects. Although MMP-9 was produced by monocytes, non-classical ones were the main source of this enzyme. We also observed that TNF produced in high level during CL contributes to MMP-9 production. CONCLUSIONS: These observations emphasize the role of monocytes, TNF and MMP-9 in the pathogenesis of L. braziliensis infection.

Cytotoxic activity in cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a chronic disease caused by species of the protozoan Leishmania and characterised by the presence of ulcerated skin lesions. Both parasite and host factors affect the clinical presentation of the disease. The development of skin ulcers in CL is associated with an inflammatory response mediated by cells that control parasite growth but also contribute to pathogenesis. CD8+ T cells contribute to deleterious inflammatory responses in patients with CL through cytotoxic mechanisms. In addition, natural killer cells also limit Leishmania infections by production of interferon-γ and cytotoxicity. In this review, we focus on studies of cytotoxicity in CL and its contribution to the pathogenesis of this disease.