GK1 Improves the Immune Response Induced by Dendritic Cells of BALB/c Mice Infected with Leishmania mexicana Promastigotes.

PURPOSE: Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), and their capacity to activate the immune response has been widely used in immunotherapies against different diseases, predominantly cancer. However, they have not been so widely used in immunotherapies against infectious diseases. Leishmania mexicana is the causative agent of cutaneous leishmaniasis in Mexico, which can result in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). DCL is characterized by the incapability of the immune response to control the parasite, which thus disseminates to all teguments. Treatments against DCL have shown low efficacy, which is a reason why alternative therapies such as immunotherapies are promising. One adjuvant that has proven its effectiveness in immunotherapies against some cancers and infections is GK1, a component of the SPVac vaccine against porcine cysticercosis. GK1 has the capacity to elicit proinflammatory cytokines and chemokines from DCs and macrophages. METHODS: We pulsed bone marrow-derived dendritic cells (BMDCs) with GK1 and a lysate obtained from L. mexicana promastigotes and tested the efficacy of this combination against the infection of susceptible mice with L. mexicana. RESULTS: We found that BMDCs stimulated with GK1 and a lysate of L. mexicana promastigotes secreted IFN-γ and IL-12, and when they were adoptively transferred to BALB/c mice which were then infected with L. mexicana promastigotes, there was a reduction in the size of the lesion and in the parasite load. CONCLUSIONS: The adjuvant properties of GK1 along with parasite antigens may have a protective effect against the infection of BALB/c mice with L. mexicana.

Host and parasite responses in human diffuse cutaneous leishmaniasis caused by L. amazonensis.

Diffuse cutaneous leishmaniasis (DCL) is a rare form of leishmaniasis where parasites grow uncontrolled in diffuse lesions across the skin. Meta-transcriptomic analysis of biopsies from DCL patients infected with Leishmania amazonensis demonstrated an infiltration of atypical B cells producing a surprising preponderance of the IgG4 isotype. DCL lesions contained minimal CD8+ T cell transcripts and no evidence of persistent TH2 responses. Whereas localized disease exhibited activated (so-called M1) macrophage presence, transcripts in DCL suggested a regulatory macrophage (R-Mϕ) phenotype with higher levels of ABCB5, DCSTAMP, SPP1, SLAMF9, PPARG, MMPs, and TM4SF19. The high levels of parasite transcripts in DCL and the remarkable uniformity among patients afforded a unique opportunity to study parasite gene expression in this disease. Patterns of parasite gene expression in DCL more closely resembled in vitro parasite growth in resting macrophages, in the absence of T cells. In contrast, parasite gene expression in LCL revealed 336 parasite genes that were differently upregulated, relative to DCL and in vitro macrophage growth, and these transcripts may represent transcripts that are produced by the parasite in response to host immune pressure.

PD-L1 May Mediate T-Cell Exhaustion in a Case of Early Diffuse Leishmaniasis Caused by Leishmania (L.) amazonensis.

Introduction: Diffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the "anergic" pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry. Case presentation: A 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation. Conclusion: The dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient.

Importância da resolvinas na infecção por Leishmania Amazonensis / Importance of resolvins in Leishmania Amazonensis infection

A Leishmaniose Cutânea Difusa (LCD) é uma manifestação clínica. rara causada pela Leishmania amazonensis que é caracterizada por uma resposta celular. parasitária ineficiente e macrófagos intensamente parasitados nas lesões cutâneas.. Mediadores lipídicos e seus precursores desempenham um papel crucial durante a. infecção por Leishmania. Estudos prévios demonstram que pacientes com leishmaniose. tegumentar, exibem um distinto balanço de eicosanoides in situ e sistêmico.. Recentemente, demonstrou-se que mediadores lipídicos especializados na pró-resolução. desempenham um papel crítico na redução de processos inflamatórios patológicos. induzindo a restauração da homeostasia em diferentes modelos experimentais. Entre. esses mediadores, as resolvinas da série D exibem potente atividade anti-inflamatória e. imuno-regulatória que inclui a inibição da quimiotaxia leucocitária e bloqueio na. produção de citocinas pró-inflamatórias. No entanto, ainda é desconhecido se as. resolvinas desempenham um papel significativo no estabelecimento e persistência da. infecção por Leishmania. OBJETIVO: Nesse estudo, avaliamos os níveis circulantes. de Resolvina D1 (RvD1) em pacientes com leishmaniose tegumentar apresentando a. forma clínica cutânea localizada (LCL) ou difusa. RESULTADOS: Nossos resultados. demonstram que pacientes com LCD apresentam maiores níveis plasmáticos de RvD1. quando comparados a LCL ou controles endêmicos. Além disso, os níveis séricos de. RvD1 em pacientes com LCD se correlacionam positivamente com a Arginase I e TGF-. β, enquanto que inversamente com os níveis sistêmicos de TNF-α. Experimentos. adicionais in vitro utilizando macrófagos humanos revelaram que a RvD1 promove a. replicação intracelular da L. amazonensis por um mecanismo associado a indução da. enzima heme oxigenase-1. CONCLUSÃO: Os resultados sugerem que a via de. produção da RvD1 pode servir como uma potencial estratégia terapêutica para os. pacientes com LCD.

INTRODUCTION: Diffuse Cutaneous Leishmaniasis (DCL) is a rare clinical manifestation caused by Leishmania amazonensis that is characterized by an inefficient parasite-specific cellular responses and heavily parasitized macrophages in skin lesions. Lipid mediators and their precursors play a crucial role during Leishmania infection. Previous works have shown that patients with cutaneous leishmaniasis exhibit a distinct in situ and systemic balance of this eicosanoids. Recently, pro-resolution lipid mediators have been shown to play critical role in dampening pathological inflammatory processes to reestablish homeostasis in a diverse range of experimental settings. Among these mediators, resolvins from D series have been described to exhibit potent antiinflammatory and immune-regulatory activities that include inhibition of leukocyte chemotaxis and blockage on the production of proinflammatory cytokines. However, whether resolvins play significant roles in establishment and persistence of Leishmania infection is currently unknown. AIM: We addressed this question by assessing circulating levels of resolvin D1 (RvD1) in tegumentary leishmaniasis patients presenting localized cutaneous leishmaniasis (LCL) or diffuse disease. RESULTS: We found that DCL patients have higher plasma levels of RvD1 when compared with LCL patients or endemic controls. In addition, the levels of this mediator were positively correlated with arginase-I and TGF-β and were negatively correlated with TNF-α levels. Additional in vitro experiments using primary human macrophages revealed that resolvin D1 promotes the intracellular L. amazonensis replication for a mechanism dependent on induction of heme oxygenase-1 enzyme. CONCLUSION: These results indicate that targeting RvD1 could serve as potential strategy for DCL patients.

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis.

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.

Exposure of phosphatidylserine on Leishmania amazonensis isolates is associated with diffuse cutaneous leishmaniasis and parasite infectivity.

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of leishmaniasis, characterized by an inefficient parasite-specific cellular response and heavily parasitized macrophages. In Brazil, Leishmania (Leishmania) amazonensis is the main species involved in DCL cases. In the experimental model, recognition of phosphatidylserine (PS) molecules exposed on the surface of amastigotes forms of L. amazonensis inhibits the inflammatory response of infected macrophages as a strategy to evade the host immune surveillance. In this study, we examined whether PS exposure on L. amazonensis isolates from DCL patients operated as a parasite pathogenic factor and as a putative suppression mechanism of immune response during the infection. Peritoneal macrophages from F1 mice (BALB/c×C57BL/6) were infected with different L. amazonensis isolates from patients with localized cutaneous leishmaniasis (LCL) or DCL. DCL isolates showed higher PS exposure than their counterparts from LCL patients. In addition, PS exposure was positively correlated with clinical parameters of the human infection (number of lesions and time of disease) and with characteristics of the experimental infection (macrophage infection and anti-inflammatory cytokine induction). Furthermore, parasites isolated from DCL patients displayed an increased area in parasitophorous vacuoles (PV) when compared to those isolated from LCL patients. Thus, this study shows for the first time that a parasite factor (exposed PS) might be associated with parasite survival/persistence in macrophages and lesion exacerbation during the course of DCL, providing new insights regarding pathogenic mechanism in this rare chronic disease.

Glicoconjugados (GIPLs e LPGs) de Leishmania braziliensis: Modulação do Sistema Imune Inato e variaçõesna estrutura de carboidratos / Glycoconjugates (GIPLs and LPGS) of Leishmania braziliensis: Innate Immune System Modulation and variations the carbohydrate structure

Os glicoconjugados de Leishmania tem sido extensivamente estudados, mas ainda poucose sabe sobre o quanto polimorfismos intra e interespecíficos contribuem com o desenvolvimentodas diferentes imunopatologias das leishmanioses. Por este motivo, duas espécies de importânciaepidemiológica foram examinadas, L. braziliensis e L. infantum, agentes causadores dasleishmanioses cutânea e visceral, respectivamente. O LPG de L. braziliensis não possui cadeiaslaterais enquanto o LPG de L. infantum carrega em sua estrutura oligômeros de até três β-glicoses como cadeias laterais. Por outro lado a estrutura dos GIPLs destas espécies eradesconhecida e foi objeto de estudo deste trabalho.A análise estrutural dos GIPLs mostrou que L. infantum possui GIPLs pequenos e ricosem manose, sugerindo predominância de GIPLs do tipo I e híbridos enquanto L. braziliensisapresenta GIPLs grandes e ricos em galactose, sugestivo do tipo II. Para analisar o papel destasmoléculas na interação com o hospedeiro, macrófagos peritoneais murinos foram tratados comLPG ou GIPLs e a produção de nitrito, citocinas, bem como a ativação de MAPKs foramavaliados.De forma geral, macrófagos estimulados com LPG de L. braziliensis, demonstraram umaprodução maior de TNF-α, IL-1β, IL-6 e NO do que os estimulados com LPG de L. infantum,adicionalmente, células tratadas com LPG de ambas as espécies mostraram uma resposta proinflamatória mais proeminente. Além disto, os GIPLs mostraram a capacidade de inibir aprodução de IL-12 e NO em macrófagos estimulados com IFN-γ e LPS. Finalmente, osglicoconjugados destas duas espécies resultaram em uma cinética diferencial na ativação deMAPKs. O LPG de L. braziliensis mostrou uma ativação transiente enquanto o de L. infantumuma ativação gradual. Os GIPLs de ambas espécies falharam em ativar MAPKs

Glicoconjugados (GIPLs e LPGs) de Leishmania braziliensis: Modulação do Sistema Imune Inato e variaçõesna estrutura de carboidratos

Os glicoconjugados de Leishmania tem sido extensivamente estudados, mas ainda poucose sabe sobre o quanto polimorfismos intra e interespecíficos contribuem com o desenvolvimentodas diferentes imunopatologias das leishmanioses. Por este motivo, duas espécies de importânciaepidemiológica foram examinadas, L. braziliensis e L. infantum, agentes causadores dasleishmanioses cutânea e visceral, respectivamente. O LPG de L. braziliensis não possui cadeiaslaterais enquanto o LPG de L. infantum carrega em sua estrutura oligômeros de até três β-glicoses como cadeias laterais. Por outro lado a estrutura dos GIPLs destas espécies eradesconhecida e foi objeto de estudo deste trabalho.A análise estrutural dos GIPLs mostrou que L. infantum possui GIPLs pequenos e ricosem manose, sugerindo predominância de GIPLs do tipo I e híbridos enquanto L. braziliensisapresenta GIPLs grandes e ricos em galactose, sugestivo do tipo II. Para analisar o papel destasmoléculas na interação com o hospedeiro, macrófagos peritoneais murinos foram tratados comLPG ou GIPLs e a produção de nitrito, citocinas, bem como a ativação de MAPKs foramavaliados.De forma geral, macrófagos estimulados com LPG de L. braziliensis, demonstraram umaprodução maior de TNF-α, IL-1β, IL-6 e NO do que os estimulados com LPG de L. infantum,adicionalmente, células tratadas com LPG de ambas as espécies mostraram uma resposta proinflamatória mais proeminente. Além disto, os GIPLs mostraram a capacidade de inibir aprodução de IL-12 e NO em macrófagos estimulados com IFN-γ e LPS. Finalmente, osglicoconjugados destas duas espécies resultaram em uma cinética diferencial na ativação deMAPKs. O LPG de L. braziliensis mostrou uma ativação transiente enquanto o de L. infantumuma ativação gradual. Os GIPLs de ambas espécies falharam em ativar MAPKs

Human cutaneous leishmaniasis: interferon-dependent expression of double-stranded RNA-dependent protein kinase (PKR) via TLR2.

We investigated the type I interferon (IFN-1)/PKR axis in the outcome of the Leishmania (Leishmania) amazonensis infection, along with the underlying mechanisms that trigger and sustain this signaling pathway. Reporter assays of cell extracts from RAW-264.7 macrophages infected with L. (L.) amazonensis or HEK-293T cells cotransfected with TLR2 and PKR promoter constructions were employed. Primary macrophages of TLR2-knockout (KO) or IFNR-KO mice were infected, and the levels of PKR, IFN-1, and superoxide dismutase 1 (SOD1) transcript levels were investigated and compared. Immunohistochemical analysis of human biopsy lesions was evaluated for IFN-1 and PKR-positive cells. Leishmania infection increased the expression of PKR and IFN-ß on induction of PKR-promoter activity. The observed effects required the engagement of TLR2. TLR2-KO macrophages expressed low IFN-ß and PKR levels postinfection with a reduced parasite load. We also revealed the requirement of PKR signaling for Leishmania-induced IFN-1 expression, responsible for sustaining PKR expression and enhancing infection. Moreover, during infection, SOD1 transcripts increased and were also enhanced when IFN-1 was added to the cultures. Remarkably, SOD1 expression was abrogated in infected, dominant-negative PKR-expressing cells. Finally, lesions of patients with anergic diffuse cutaneous leishmaniasis exhibited higher levels of PKR/IFN-1-expressing cells compared to those with single cutaneous leishmaniasis. In summary, we demonstrated the mechanisms and relevance of the IFN-1/PKR axis in the Leishmania infection.

Leishmania mexicana lipophosphoglycan differentially regulates PKCalpha-induced oxidative burst in macrophages of BALB/c and C57BL/6 mice.

Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCalpha is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCalpha. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCalpha and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCalpha activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCalpha activity, whereas in the more resistant strain it augmented enzymatic activity 2.8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCalpha activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCalpha activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCalpha-induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.

Leishsmania (Leishmania) amazonensis infection: Muscular involvement in BALB/c and C3H.HeN mice.

Recent studies have provided some insights into Leishsmania (Leishmania) amazonensis muscular infection in dogs, although, muscular disease due to leishmaniasis has been poorly documented. The aim of our study was to evaluate involvement of Leishmania in muscular infection of two distinct mouse strains (BALB/c and C3H.He), with different genetic backgrounds. BALB/c mice, susceptible to Leishmania infection, showed, at the beginning of infection, a great number of infected macrophages among muscle fibers; however, in C3H.He resistant mice, muscle fibers were less damaged than in BALB/c mice, but some parasitized macrophages could be seen among them. A follow up of the infection showed an intense inflammatory infiltrate mainly composed of infected macrophages in BALB/c muscles and the presence of amastigotes within muscle fibers; while C3H.He mice exhibited a moderate inflammatory infiltrate among skeletal muscle fibers and an absence of amastigotes. Total destruction of muscles was observed in BALB/c mice in the late phase of infection (day 90) while C3H.He mice showed a process of muscle repair. We concluded that: (1) the muscles of BALB/c mice were more affected by leishmaniasis than those of C3/H.He mice; (2) Leishmania amastigotes are capable of infecting muscular fibers, as observed in BALB/c mice; (3) as inflammatory infiltrate is less intense in C3H.He mice these animals are capable of restoring muscular fibers.

Increase of NK cells and proinflammatory monocytes are associated with the clinical improvement of diffuse cutaneous leishmaniasis after immunochemotherapy with BCG/Leishmania antigens.

Diffuse cutaneous leishmaniasis (DCL) is characterized by disseminated lesions and the absence of a specific cellular immune response. Here, the immunochemotherapy outcome of a patient with DCL from Amazonian Brazil infected with Leishmania (Leishmania) amazonensis is presented. After several unsuccessful chemotherapy treatment regimens and many relapses, a monthly immunotherapy scheme of L. amazonensis PH8 plus L. (Viannia) braziliensis M2903 monovalent vaccines associated with Bacillus Calmette-Guerin (BCG) was established, one round of which also included an M2903 vaccine associated with intermittent antimonial treatment. Temporary healing of all lesions was achieved, although Leishmania skin tests were negative and interferon gamma was not detected in mononuclear cell cultures stimulated with Leishmania antigens. The frequencies of CD16 (+)CD56(+) NK cells (approximately 2x) and CD14 (+)CD16(+) proinflammatory monocytes (approximately 8x) increased in peripheral blood, and CD56 (+) lymphocytes were found infiltrating the lesions. An association between the increase of the frequency of innate immune system cells and the healing of lesions is shown, suggesting that this protocol of immunotherapy reduced the parasite load and activated NK cells and monocytes.

Diffuse cutaneous leishmaniasis responds to miltefosine but then relapses.

BACKGROUND: Diffuse cutaneous leishmaniasis (DCL), although rare, is profoundly incapacitating. At present there is no successful treatment for this progressive protozoan infection, which is associated with the absence of specific cell-mediated immunity (CMI) to Leishmania. This disease shares features with visceral leishmaniasis (VL), including specific CMI inactivity during active disease and a heavy parasitic burden, but VL responds well to treatment. Miltefosine is the first orally administered drug which has shown efficacy in the treatment of VL; it has not been adequately evaluated in the treatment of DCL. OBJECTIVES: To evaluate the efficacy of miltefosine in the treatment of DCL, using clinical, parasitological, histopathological and immunological criteria. METHODS: Sixteen patients with DCL were treated with miltefosine, 2.0-2.5 mg kg(-1) daily, for variable periods of time (75-218 days). Patients were hospitalized for the first month and evaluated every 2 weeks until the termination of treatment with routine laboratory chemistry, percentage clinical improvement, presence of parasites in skin smears, growth of parasites in culture medium and in hamsters, histopathological characteristics of the granulomas, adverse side-effects, and reactivity to leishmanin skin test antigen. Further cycles of treatment were given in some of these patients, particularly after suspension of treatment was followed by relapse. RESULTS: Patients showed dramatic clinical improvement and reduction in the parasite burden by day 15 after the initiation of treatment, which continued while treatment was maintained. By day 45, 15 patients showed 80-90% clinical improvement. Nevertheless, suspension of treatment was followed by the development of new lesions in all but one patient. Inoculation in hamsters was observed to be the most sensitive technique to detect persisting parasites. Adverse events were very mild. CONCLUSIONS: Miltefosine produced a dramatic clinical and parasitological response in patients with DCL and improvement continued during drug administration, but with a single exception all patients presented new lesions after suspension of treatment. There was no histological or skin test evidence to suggest the development of CMI during treatment, which may be an indispensable criterion for the evaluation of potentially effective drugs against DCL.

Low serum levels of dehydroepiandrosterone and cortisol in human diffuse cutaneous leishmaniasis by Leishmania mexicana.

Low levels of dehydroepiandrosterone (DHEA) and cortisol hormones produced by the suprarenal cortex have been associated with diseases involving chronic inflammation, low interferon (IFN)-gamma, and high interleukin (IL)-6. Diffuse cutaneous leishmaniasis (DL), a long-lasting intracellular parasitic infectious disease, can spread unknown levels of DHEA and cortisol. Serum concentrations of both were measured in 5 patients with DL, in 15 patients with localized lesions produced by Leishmania (LL), and in 20 healthy volunteers. Leishmania mexicana mexicana was identified as the causal agent in patients with DL and LL. Hormone levels were lower in DL compared with controls and LL. Furthermore, we detected a lower percentage of IFN-gamma-positive cells with higher levels of IL-6 and higher titers of anti-Leishmania antibodies in patients with DL, whereas patients with LL were similar to controls. These data suggest that patients with DL may be good candidates for DHEA and cortisol supplementation.

Monocyte cytokine and costimulatory molecule expression in patients infected with Leishmania mexicana.

Leishmania mexicana causes localized and diffuse cutaneous leishmaniasis. Patients with localized cutaneous leishmaniasis (LCL) develop a benign disease, whereas patients with diffuse cutaneous leishmaniasis (DCL) suffer from a progressive disease associated with anergy of the cellular response towards Leishmania antigens. We evaluated the production of the interleukins (IL) IL-12, IL-15, IL-18 and tumour necrosis factor-alpha (TNF-alpha) and the expression of the costimulatory molecules CD40, B7-1 and B7-2 in monocytes from LCL and DCL patients, stimulated in vitro with Leishmania mexicana lipophosphoglycan (LPG) for 18 h. LCL monocytes significantly increased TNF-alpha, IL-15 and IL-18 production, and this increase was associated with reduced amounts of IL-12. DCL monocytes produced no IL-15 or IL-18 and showed a decreasing tendency of TNF-alpha and IL-12 production as the severity of the disease increased. No difference was observed in the expression of CD40 and B7-1 between both groups of patients, yet B7-2 expression was significantly augmented in DCL patients. It remains to be established if this elevated B7-2 expression in DCL patients is cause or consequence of the Th2-type immune response that characterizes these patients. These data suggest that the diminished ability of the monocytes from DCL patients to produce cell-activating innate proinflammatory cytokines when stimulated with LPG is a possible cause for disease progression.

Experimental model of chronic osteomyelitis caused by Leishmania (L) amazonensis.

Experimental animal models have been used for the study of the physiopathogenesis of leishmaniasis, on some occasions with success, while in other situations such as bone alterations that accompany tegumentary leishmaniasis, especially in diffuse cutaneous form (DCL), the mechanisms are still unknown. In the present study, we determined these alterations in an animal model susceptible to Leishmania (L) amazonensis. Amastigotes of L. (L) amazonensis isolated from patients with diffuse cutaneous leishmaniasis (DCL) were inoculated into the hind paws of eight BALB/c mice, macroscopic and histopathological aspects were analyzed. After 90 and 120 days of evolution, histopathological analysis demonstrated a mononuclear cell infiltrate rich in plasma cells and intense parasitism of intra- and extra-medullary macrophages, with areas of bone necrosis and discrete involvement of cartilaginous tissue. The results show that the inflammatory process developed during L. (L) amazonensis infection might cause bone tissue destruction and secondarily affect the joints.

Differential effects of antigens from L. braziliensis isolates from disseminated and cutaneous leishmaniasis on in vitro cytokine production.

BACKGROUND: Disseminated leishmaniasis is an emerging infectious disease, mostly due to L. braziliensis, which has clinical and histopathological features distinct from cutaneous leishmaniasis. METHODS: In the current study we evaluated the in vitro production of the cytokines IFN-gamma, TNF-alpha, IL-5 and IL-10 by peripheral blood mononuclear cells (PBMC) from 15 disseminated leishmaniasis and 24 cutaneous leishmaniasis patients upon stimulation with L. braziliensis antigens genotyped as disseminated leishmaniasis or cutaneous leishmaniasis isolates. RESULTS: Regardless of the source of L. braziliensis antigens, PBMC from cutaneous leishmaniasis patients produced significantly higher IFN-gamma than PBMC from disseminated leishmaniasis patients. Levels of TNF-alpha by PBMC from cutaneous leishmaniasis patients were significantly higher than disseminated leishmaniasis patients only when stimulated by genotyped cutaneous leishmaniasis antigens. The levels of IL-5 and IL-10 production by PBMC were very low and similar in PBMCs from both disseminated leishmaniasis and cutaneous leishmaniasis patients. The immune response of each patient evaluated by the two L. braziliensis antigens was assessed in a paired analysis in which we showed that L. braziliensis genotyped as disseminated leishmaniasis isolate was more potent than L. braziliensis genotyped as cutaneous leishmaniasis isolate in triggering IFN-gamma and TNF-alpha production in both diseases and IL-5 only in cutaneous leishmaniasis patients. CONCLUSION: This study provides evidence that antigens prepared from genotypically distinct strains of L. braziliensis induce different degrees of immune response. It also indicates that both parasite and host play a role in the outcome of L. braziliensis infection.