Involvement of the Inflammasome and Th17 Cells in Skin Lesions of Human Cutaneous Leishmaniasis Caused by Leishmania (Viannia) panamensis.

Localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) panamensis is an endemic disease in Panama. This condition causes ulcerated skin lesions characterized by a mixed Th1/Th2 immune response that is responsible for disease pathology. However, the maintenance of the in situ inflammatory process involves other elements, such as Th17 and inflammasome responses. Although these processes are associated with parasite elimination, their role in the increase in disease pathology cannot be discarded. Thus, the role in Leishmania infection is still unclear. In this sense, the present study aimed at characterizing the Th17 and inflammasome responses in the skin lesions of patients with LCL caused by L. (V.) panamensis to help elucidate the pathogenesis of this disease in Panama. Th17 and inflammasome responses were evaluated by immunohistochemistry (IHQ) in 46 skin biopsies from patients with LCL caused by L. (V.) panamensis. The Th17 immune response was assessed using CD3, CD4, RoRγt, IL-17, IL-6, IL-23, and TGF-ß1 antibodies, and the inflammasome response was assessed by IL-1ß, IL-18, and caspase-1 antibodies. The presence of the Th17 and inflammasome responses was evidenced by a positive reaction for all immunological markers in the skin lesions. An inverse correlation between the density of amastigotes and the density of RoRγt+, IL-17+, IL-1ß +, and caspase-1+ cells was observed, but no correlation between Th17 and the inflammasome response with evolutionary disease pathology was reported. These data showed the participation of Th17 cells and the inflammasome in the inflammatory response of the skin lesions of LCL caused by L. (V.) panamensis infection. These results suggest a role in the control of tissue parasitism of IL-17 and the activation of the NLRP3 inflammasome dependent on IL-1ß but cannot exclude their role in the development of disease pathology.

Differential Recruitment of Dendritic Cells Subsets to Lymph Nodes Correlates with a Protective or Permissive T-Cell Response during Leishmania (Viannia) Braziliensis or Leishmania (Leishmania) Amazonensis Infection.

Leishmania (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a large clinical and immunopathological spectrum in human disease; while La may be responsible for anergic disease, Lb infection leads to cellular hypersensitivity. To better understand the dichotomy in the immune response caused by these Leishmania species, we evaluated subsets of dendritic cells (DCs) and T lymphocyte in draining lymph nodes during the course of La and Lb infection in BALB/c mice. Our results demonstrated a high involvement of DCs in La infection, which was characterized by the greater accumulation of Langerhans cells (LCs); conversely, Lb infection led to an increase in dermal DCs (dDCs) throughout the infection. Considering the T lymphocyte response, an increase of effector, activated, and memory CD4(+) T-cells was observed in Lb infection. Interleukin- (IL-) 4- and IL-10-producing CD4(+)and CD8(+) T-cells were present in both La and Lb infection; however, interferon- (IFN-) γ-producing CD4(+)and CD8(+) T-cells were detected only in Lb infection. The results suggest that during Lb infection, the dDCs were the predominant subset of DCs that in turn was associated with the development of Th1 immune response; in contrast La infection was associated with a preferential accumulation of LCs and total blockage of the development of Th1 immune response.

Expression of Foxp3, TGF-ß and IL-10 in American cutaneous leishmaniasis lesions.

Regulatory T cells (Tregs) are a unique population of CD25+CD4+ T cells that regulate innate and adaptive immune responses and have the ability to control the excessive or misdirected effects of the immune system. This modulation involves different mechanisms, such as the suppression of T cell proliferation and cytokine production, the secretion of suppressive cytokines (IL-10 and TGF-ß) and the induction of effector T cell apoptosis in humans with infectious diseases such as Leishmania infections. The aim of this study was to evaluate the expression of Foxp3, IL-10 and TGF-ß through immunohistochemistry in 22 skin biopsies of patients with localized cutaneous leishmaniasis (LCL) caused by Leishmania (Viannia) spp. from an endemic area in pre-Amazonian area of Maranhão State, Brazil. The density of these markers was also analyzed according to the species of parasite and the progression of the disease. The cellular density was 234 cells/mm(2) for Foxp3+ cells, 357 cells/mm(2) for TGF-ß+ cells and 648 cells/mm(2) for IL-10+ cells in the studied skin lesions. The analysis of the cellular density of these immunological markers in relation to the species of Leishmania demonstrated that lesions caused by L. (V.) braziliensis had a lower density of Foxp3+ cells than lesions caused by L. (Viannia) spp. The expression of IL-10 was also lower in lesions caused by L. (V.) braziliensis. There were no significant differences in TGF-ß expression between the two groups. The evaluation of these markers according to the progression of the disease did not reveal any significant differences. These findings suggest that Treg Foxp3+ cells, IL-10, and TGF-ß play important roles in the immunopathogenesis of LCL and that these roles differ depending on the causal Leishmania species.

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model.

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4(+) and CD8(+) immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207(+), CD11c(+), CD4(+), CD8(+), iNOS(+) cellular densities. Cytokine (IFN-γ, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207(+) and CD11c(+) were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4(+) and CD8(+) were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS(+) was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-γ was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

Effects of salivary gland homogenate from wild-caught and laboratory-reared Lutzomyia longipalpis on the evolution and immunomodulation of Leishmania (Leishmania) amazonensis infection.

We investigated the effects of Lutzomyia longipalpis salivary glands homogenate of wild-caught and laboratory-reared vectors on the lesion evolution and immunomodulation of the infection caused by Leishmania (Leishmania) amazonensis. To compare the effect of both salivary glands homogenate (SGH), C57BL/6 mice were inoculated subcutaneously into the hind footpads or into the ear dermis with 10(6) promastigotes in the presence or not of SGH from wild-caught and laboratory-colonized sand flies. Comparing SGH groups, the lesion size was lower in mice co-inoculated with wild-caught SGH, as the parasitism and the infiltration of macrophages at the inoculation site. Wild-caught SGH also determined lower production of IL-4 and IL-10 but higher IL-12 levels compared with laboratory-reared SGH. Our findings address a probable bias by using SGH from laboratory-colonized sand flies instead of wild-caught vector SGH in studies concerning saliva effects. A possible mild influence of sand fly saliva in natural infections caused by Leishmania is also speculated, as infection is transmitted by wild and not by laboratory-reared vectors.

Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis.

The immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis were reviewed in the light of more recent features found in the clinical and immunopathological spectrum of American cutaneous leishmaniasis. It was shown a dichotomy in the interaction between these Leishmania species and human T-cell immune response; while L. (V.) braziliensis shows a clear tendency to lead infection from the localized cutaneous leishmaniasis (LCL), a moderate T-cell hypersensitivity form at the centre of the spectrum, toward to the mucocutaneous leishmaniasis (MCL) at the T-cell hypersensitivity pole and with a prominent Th1-type immune response, L. (L.) amazonensis shows an opposite tendency, leading infection to the anergic diffuse cutaneous leishmaniasis (ADCL) at the T-cell hyposensitivity pole and with a marked Th2-type immune response. Between the central LCL and the two polar MCL and ADCL, the infection can present an intermediary form known as borderline disseminated cutaneous leishmaniasis, characterized by an incomplete inhibition of T-cell hypersensitivity but with a evident supremacy of Th1 over Th2 immune response (Th1 > or = Th2). These are probably the main immunopathogenic competences of L. (V.) braziliensis and L. (L.) amazonensis regarding the immune response dichotomy that modulates human infection outcome by these Leishmania parasites.

Comparative studies of the anti-leishmanial activity of three Crotalus durissus ssp. venoms.

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus-Cdt, Crotalus durissus cascavella-Cdca, and Crotalus durissus collilineatus-Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70+/-1.72 microg/ml) in comparison with that of Cdca venom (IC50 value of 9.41+/-1.21 microg/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30+/-2.18 microg/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00+/-9.50 microg/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80+/-0.52 microg/ml and 19.95+/-4.21 microg/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80+/-2.21 microg/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.

Anti-leishmania activity of semi-purified fraction of Jacaranda puberula leaves.

The crude methanolic extract from leaves of Jacaranda puberula showed activity against Leishmania (Leishmania) amazonensis. The extract presented active against promastigote forms with an inhibitory concentration 50% (IC(50)) value of 88.0 mug/ml, but only moderated activity against amastigote forms; however in higher concentrations the extract showed cytotoxic effects. The bio-guided chromatographic fractionation the crude methanolic extract against amastigotes yielded a fraction with an IC(50) value of 14.0 mug/ml (without cytotoxic activity) in relation to the crude extract (IC(50) value, 359.0 microg/ml). These data indicate that J. puberula leaves contain active compounds, which should be further investigated for the development of new potential drugs against cutaneous leishmaniasis.

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs.

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of São Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical signs of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Araçatuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick-stained for optical microscopy. Direct immunofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific.

Interleukin-2-activated natural killer cells may have a direct role in the control of Leishmania (Leishmania) amazonensis promastigote and macrophage infection.

To study the role of Natural Killer (NK) cells in Leishmania infection, peritoneal macrophages from BALB/c mice were infected with Leishmania (Leishmania) amazonensis promastigotes and incubated with interleukin-2 (IL-2)-activated NK (A-NK) cells at different ratios of A-NK cells to infected macrophages (5:1, 1:1, 0.2:1). The A-NK cells were added either together with the parasites (0-h group) or 24 h later (24-h group). Morphological studies of the cultures revealed predominance of parasitic debris within macrophages that were in close contact with A-NK cells and the decrease in parasite recovery was directly proportional to the A-NK cell concentration used. Interferon-gamma (IFN-gamma) and IL-12 were detected in the supernatant at levels proportional to the A-NK cell concentration used. No significant difference was observed between the groups with respect to NO levels in the culture supernatant. When A-NK cells were added directly to the L. (L.) amazonensis promastigote cultures, the parasite recovery decreased proportional to the number of A-NK cells added. In vivo studies demonstrated smaller lesion sizes in animals inoculated with both parasites and A-NK cells compared with parasites alone. Histopathology of the skin lesions from animals receiving A-NK cells together with the parasites showed moderate parasitism and a nodular inflammatory infiltrate formed by mononuclear cells and a few vacuolized macrophages. In contrast, animals inoculated only with the parasites showed a highly parasitized dermis with infiltration of intensely vacuolized macrophages. These results demonstrate the role of A-NK cells in parasite lysis and in resistance of macrophages to L. (L.) amazonensis in the early phase of infection.

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice.

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 +/- 5.3 vs 5.3 +/- 1.5) at 7 days of infection (P<0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 +/- 24.9 vs 6.3 +/- 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 ± 5.3 vs 5.3 ± 1.5) at 7 days of infection (P < 0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 ± 24.9 vs 6.3 ± 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

Serum cytokine profile in the subclinical form of visceral leishmaniasis

The factors determining the development or not of visceral leishmaniasis (VL) have not been completely identified, but a Leishmania-specific cellular immune response seems to play a fundamental role in the final control of infection. Few studies are available regarding the production of cytokines in the subclinical form of VL, with only the production of IFN-g and TNF-a known. The aim of the present study was to identify immunological markers for the oligosymptomatic or subclinical form of VL. A prospective cohort study was conducted on 784 children aged 0 to 5 years from an endemic area in the State of Maranhão, Brazil, between January 1998 and December 2001. During 30 consecutive months of follow-up, 33 children developed the oligosymptomatic form of the disease and 12 the acute form. During the clinical manifestations, serum cytokine levels were determined in 27 oligosymptomatic children and in nine patients with the acute form using a quantitative sandwich enzyme immunoassay. In the subclinical form of VL, variable levels of IL-2 were detected in 52.3 percent of the children, IL-12 in 85.2 percent, IFN-g in 48.1 percent, IL-10 in 88.9 percent, and TNF-a in 100.0 percent, with the last two cytokines showing significantly lower levels than in the acute form. IL-4 was not detected in oligosymptomatic individuals. Multiple discriminant analysis used to determine the profile or combination of cytokines predominating in the subclinical form revealed both a Leishmania resistance (Th1) and susceptibility (Th2) profile. The detection of both Th1 and Th2 cytokine profiles explains the self-limited evolution accompanied by the discrete alterations observed for the subclinical form of VL.

Serum cytokine profile in the subclinical form of visceral leishmaniasis.

The factors determining the development or not of visceral leishmaniasis (VL) have not been completely identified, but a Leishmania-specific cellular immune response seems to play a fundamental role in the final control of infection. Few studies are available regarding the production of cytokines in the subclinical form of VL, with only the production of IFN-gamma and TNF-alpha known. The aim of the present study was to identify immunological markers for the oligosymptomatic or subclinical form of VL. A prospective cohort study was conducted on 784 children aged 0 to 5 years from an endemic area in the State of Maranhão, Brazil, between January 1998 and December 2001. During 30 consecutive months of follow-up, 33 children developed the oligosymptomatic form of the disease and 12 the acute form. During the clinical manifestations, serum cytokine levels were determined in 27 oligosymptomatic children and in nine patients with the acute form using a quantitative sandwich enzyme immunoassay. In the subclinical form of VL, variable levels of IL-2 were detected in 52.3% of the children, IL-12 in 85.2%, IFN-gamma in 48.1%, IL-10 in 88.9%, and TNF-alpha in 100.0%, with the last two cytokines showing significantly lower levels than in the acute form. IL-4 was not detected in oligosymptomatic individuals. Multiple discriminant analysis used to determine the profile or combination of cytokines predominating in the subclinical form revealed both a Leishmania resistance (Th1) and susceptibility (Th2) profile. The detection of both Th1 and Th2 cytokine profiles explains the self-limited evolution accompanied by the discrete alterations observed for the subclinical form of VL.

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection

Epidermal dendritic cells in murine lupus

O lúpus eritematoso sistêmico (LES) é uma síndrome auto-imune multissistêmica. O acometimento cutâneo é freqüente e constitui manifestaçao clínica importante. O polimorfismo das lesoes cutâneas presume o envolvimento de diversos fatores etiopatogênicos. As células de Langerhans epidérmicas (CLs) têm funçoes imunorregulatórias importantes e participam de um modo decisivo na fisiopatologia de várias doenças dermatológicas inflamatórias, existindo evidência de seu envolvimento da patogênese da lesao cutânea lúpica. O camundongo F1 (NZB x NZW) um dos mais completos modelos experimentais de LES, nao desenvolve lesoes de pele. A ausência de dados na literatura referentes as CLs no camundongo F1 (NZB x NZW) motivou-lhes a realizar o presente estudo, que demonstrou, de forma original, a presença de CLs na epiderme do modelo F1, tanto por técnica citoquímica quanto por microscopia eletrônica de transmissao. Utilizando a técnica citoquímica da ATPase, estudamos as CLs no camundongo F1 (NZB x NZW) antes e após irradiaçao com luz ultravioleta B (UVB). As CLs no modelo F1 apresentaram-se em maior número e com alteraçoes morfológicas (dendritos curtos e menos ramificados) quando comparadas com os animais dos grupos-controles (BALB/c e C57BL/6). A irradiaçao UVB induziu alteraçoes morfológicas e reduçao no número das CLs do camundongo F1 em extensao semelhante ao observado no camundongo BALC/c. Nossos resultados sugerem possível alteraçao funcional nas CLs do camundongo F1 (NZB x NZW) e fornecem evidências de que estas células nao apresentam maior sensibilidade à luz UVB em comparaçao às de outras raças de camundongos. Estudos adicionais sao necessários para se testar possível associaçao entre essas alteraçoes fenotípicas das CLs e a ausência de manifestaçoes cutâneas nos camundongos F1 (NZB x NZW)

Comparative and sequential histopathology of plasmodium chabaudi-infected balb/c mice

1. Rodent experimental models have been useful to study severe malaria but few serial and controlled studies have been conducted. In the presente investigation, we describe the histopathology of lethal and non-lethal rodent malaria induced by Plasmodium berghei and P. chabaudi. P. berghei malaria shows a uniformly lethal course, while P. chabaudi malaria produces a non-lethal acute infection with recovery and periodical recurdescences. Sequential histopathological changes were also characterized in P. chabaudi malaria to determine the evolution of the lesions. 2. P. berghei-infected mice have a more severe organ involvement and lower blood regenerative changes than P. chabaudi-infected mice. Two patterns of organ involvement were observed by cimparing the two infections. The first is related to nonspecific parasitized red blood cell clearance by liver and spleen. The second is related to specific changes due to a specific parasite strain interaction with the host, such as those found in the lungs. 3. Sequential changes in P. chabaudi-infected mice were characterized by perihepatocytic reticulin fiber deposition during the recovery from infection, which faded in subsequent stages. Other organs had a similar regressive evolution, except splenic lymphoid tissue which underwent histological restoration or even hypertrophy after depletion in the acute stage. No brain or heart lesions were observed in either model during the acute and subsequent stages. 4. P. chabaudi infection, whose histopathology is described here for the first time, should be useful as a non-lethal experimental model to study the evolution of histological alterations in malaria