Inducible nitric oxide synthase and cytokine pattern in lesions of patients with American cutaneous leishmaniasis.

American tegumentary leishmaniasis has three forms: localized (LCL), found in resistant individuals; diffuse (DCL), found in susceptible individuals; and intermediate cutaneous leishmaniasis (ICL), found in individuals with exacerbated immunity. We evaluated cytokines and inducible nitric oxide synthase (iNOS) in lesions of LCL, ICL and DCL using immunohistochemistry. LCL granulomas showed a preponderance of interferon (IFN)-gamma and interleukin (IL)-12 expression, whereas ICL granulomas had more IL-4-, IL-10- and mainly transforming growth factor (TGF)-beta1-expressing cells. Higher densities of iNOS+ cells were observed in ICL and LCL than in DCL. iNOS was also expressed in keratinocytes of LCL and ICL lesions, and in epidermal dendritic cells of ICL lesions. In LCL and ICL, most keratinocytes expressed IL-12 and a portion expressed IFN-gamma. IL-12+ and IFN-gamma+ dendritic cells were absent or sparse in LCL and ICL epidermis. Our results show the importance of iNOS, IL-12 and INF-gamma in LCL and ICL lesions, emphasizing the existence of a mixed cytokine pattern in ICL different from the Th1 and Th2 responses established in LCL and DCL lesions.

Acute immobilization stress induces clinical and neuroimmunological alterations in experimental murine cutaneous leishmaniasis.

BACKGROUND: The skin is an important component of the neuroendocrine-immune axis. Several studies have shown that stress exacerbates skin disorders, affecting the function of sebaceous glands, keratinocytes, epidermal Langerhans cells and other cells, having an impact on the pathogenesis of many immunologically associated skin diseases. In American cutaneous leishmaniasis, we have shown the importance of the epidermis as a regulatory site, with the key participation of Langerhans cells. OBJECTIVES: To analyse the effect of acute immobilization stress on Langerhans cells, substance P (SP), calcitonin gene-related peptide (CGRP) and the natural course of infection in a murine model of cutaneous leishmaniasis. METHODS: BALB/c mice, susceptible to Leishmania infection, were placed under acute stress by immobilization (confinement) for 2 or 8 h before inoculation with L. mexicana (MHOM/BZ/82/BEL21). An avidin-biotin immunoperoxidase technique was used for cell and neuropeptide identification. RESULTS: The stressed animals became more susceptible to the parasite infection, which was manifested by acceleration and exacerbation of the lesions. In addition, the stressed animals showed morphological alterations (spherical bodies and shortened dendrites) and decreased numbers of epidermal Langerhans cells, when compared with control L. mexicana-infected mice. Mice stressed for 8 h showed greater and antidromic immunoreactivity to CGRP and SP at the time of infection. Moreover, the single inoculation of parasites caused a decrease of CGRP innervation. CONCLUSIONS: Acute immobilization stress induces an immunosuppressive state that further favours Leishmania invasion in susceptible animals.

Nitric oxide and cellular immunity in experimental cutaneous leishmaniasis.

We examined the local and systemic production of nitric oxide (NO) and the pattern of cytokine during the course of Leishmania mexicana infection in susceptible BALB/c and resistant C57BL/6 mice. NO derivatives were measured in serum, and the expression of inducible nitric oxide synthase (iNOS), interferon (IFN-gamma), interleukin (IL-4) and epidermal Langerhans cells (LC) was measured in the lesions by immunohistology. Circulating NO concentrations, iNOS+ cell density, IFN-gamma+ Th1 cells and CD205+ Langerhans cells were higher in early lesions of resistant C57BL/6 mice. In contrast, susceptible BALB/c mice developed chronic and progressive lesions with a predominance of IL-4+ Th2 cells. In both susceptible and resistant mice, lesion size and lymph node volume followed a similar course. The early local and systemic production of NO in resistant mice may be related with the premature production of IFN-gamma observed, contributing to the resolution of the lesion.

Microwave irradiation for rapid epidermis-dermis separation and improved epidermal cell immunodetection.

We explored the effects of microwave irradiation on epidermal-dermal separation and subsequent immunostaining of epidermal cells. Epidermal sheets were obtained after incubation in 0.02 M EDTA in PBS and microwave irradiation with 4 pulses of 420 watts for 5 sec, with a total incubation period of 4 min. The control epidermal sheets were immunostained for Langerhans cells and dendritic epidermal T cells using a conventional immunoperoxidase method. The experimental immunodetection of these cells was assisted by incubating the primary antibodies for 10 min at 70 watts. We showed a simple and rapid method for separation of the epidermal-dermal junction and immunostaining of epidermal cells with optimal morphological preservation.

Intermediate or chronic cutaneous leishmaniasis: leukocyte immunophenotypes and cytokine characterisation of the lesion.

The American cutaneous forms of leishmaniasis include immune-responder individuals with localised cutaneous leishmaniasis (LCL) and non-responder individuals with diffuse cutaneous leishmaniasis (DCL). Patients with intermediate or chronic cutaneous leishmaniasis (ICL) have increased morbidity due to the length of their illness, atypical forms and areas of compromise. In the present study, we evaluated the expression of the leukocyte antigens (CD4, CD8, CLA: cutaneous lymphocyte antigen, CD69, CD83 and CD1a) and cytokines (IFN-gamma, IL-4, IL-10 and TGF-beta 1) in the lesions of patients with ICL (n = 18) using an immunocytochemical procedure. ICL results were compared with the information for LCL (n = 19) and DCL (n = 4). The numbers of CD4+ and CD8+ T cells in ICL were similar to those of LCL lesions, but significantly different (P < or = 0.05) from DCL lesions. LCL lesions have about half the numbers of early activated CD69+ cells as ICL, but most are CLA+ skin homing memory T cells, whereas ICL lesions have the highest number of CD69+ T cells, but about one-third of these cells expressed CLA. This suggests that the granuloma of ICL patients contains many activated T cells that are unprimed to cutaneous-launched antigens, thus contributing to an aberrant immune response. In contrast, DCL granulomas presented the lowest numbers of activated CD69+ and CLA+ cells, associated with the characteristic tolerogenic state of these patients. The immunolocalisation of cytokines showed a mixed cytokine pattern in ICL lesions with many positive cells for IL-10, TGF-beta 1, IL-4 and IFN-gamma, with a preponderance of the first two, and different from the prevalent Th1 and Th2 responses associated with LCL and DCL lesions, respectively. CD1a+ Langerhans cells were decreased (P < or = 0.05) in both ICL (271 +/- 15 cells/mm2) and DCL (245 +/- 19 cells/mm2) as compared to LCL (527 +/- 54 cells/mm2) epidermis. The percentage of IL-10+ epidermal Langerhans cells in ICL (33.69), from the total CD1a+ population, was higher than in LCL (17.45). In addition, fewer CD83+ primed Langerhans cells were present in ICL epidermis. The diminished participation of epidermal Langerhans cells, causing a defective signalling by the epidermis, in ICL lesions may account for the tissue-damaging state observed in these patients.

[Immunopathology of American tegumentary leishmaniasis]. / Inmunopatología de la leishmaniasis tegumentaria Americana.

The complex cell mediated immune response in parasitic diseases can be evaluated in different body compartments. In the present work we describe the results of studies of peripheral blood lymphocytes and the cutaneous lesions in the three clinical forms of tegumentary leishmaniasis. These clinical forms, that constitute a clinical, histological and immunological spectrum, are: localized cutaneous leishmaniasis (LCL), diffuse cutaneous (DCL9, and the intermediate forms that include mucous and verrocous leishmaniasis (MCL). The overall results suggest that the cytokine pattern of lymphocytes in the blood and lesions of LCL, the self-limiting form of the disease, is T-helper type 1. This leads to the cure of the lesions in these patients, either spontaneously or after appropriate therapy. The disseminated disease in DCL patients is resistant to chemotherapy, and is characterized by a Th2 cytokine pattern, with a an absence of IL-2 AND ifn-gamma production when the lymphocytes are specifically stimulated by leishmanial antigen. This is probably why these patients are unable to control the infection, and allows the cutaneous dissemination of the parasite. The intermediate MCL form is characterized by destructive lesions of the oral and nasopahryngeal mucosas, with a tendency to the chronicity of the infection. The cytokine pattern of MCL patients is a mixture of Type 1 and Type 2 responses. This may be responsible for the progression of the disease in these patients, as it is possible that when both types of cytokines are produced, the Type 2 responses can predominate over the Type 1 and the disease is maintained in a chronic, although activated, state. The examination of the cutaneous lesions demonstrated that the epidermis in the DCL lesions is deficient in ICAM-1 accessory signals and MHC-II expression by keratinocytes, and presents a variable number of Langerhans cells. In MCL lesions, the expression of ICAM-1 and MHC-II is elevated, and Langerhans cells are absent in the damaged epithelium. The epidermis of LCL lesions show ICAM-1 in patches and MHC uniformly expressed by keratinocytes. DCL lesions are characterized by low CD4/CD8 and memory/naive T cell ratios, low numbers of T gamma delta cells, and an apparent defect in the expression of LFA-1 directional receptors. The cytokine patterns are Th1 and Th2, with the latter predominating. MCL granulomas manifest high CD4/CD8 and memory/naive Tcel ratios, low numbers of T gamma delta, a high coefficients of cellular adhesion, with a mixed Th1/Th2 cytokine pattern. LCL granulomas are characterized by a normal CD4/CD8 ratio, a high memory/naive cell ratio, numerous groups of T gamma delta, a high expression of directional receptors, and Th1/Th0 cytokine patterns. We discuss the results in the context of the immunological effects, both in immunotherapy and immunoprophylasis, of the combined vaccine of BCG plus promastigotes of Leishmania. In immunotherapy we demonstrate that the combined vaccine simulates a Th1 response in LCL patients. As an immunoprophylactic vaccine in healthy individuals from an endemic area of leishmaniasis, it stimulates a Th1 response (positivity in the Montenegro cutaneous reactions, proliferative responses in vitro and production of IFN-gamma), with a low specific antibody response. This demonstrates that the combined vaccine is potentially useful both in the treatment of LCL patients, as well as being potentially protective applied as immunoprophylaxis in the control of leishmaniasis.

Immunocytochemical and histopathologic characterization of lesions from patients with localized cutaneous leishmaniasis caused by Leishmania panamensis.

Localized cutaneous leishmaniasis (LCL) in Colombia is caused primarily by Leishmania panamensis, a different species from those reported in Brazil, French Guiana, and Venezuela. Because different parasites may elicit disparate immune responses, the present study was undertaken to establish the leukocyte participation in the immune response against L. panamensis. Epidermal and dermal immune complexes were studied using an avidinbiotin immunoperoxidase technique and specific monoclonal antibodies. In LCL, the epidermis showed keratinocytes expressing intercellular adhesion molecule-1, a universal expression of human leukocyte antigen-DR, and a hyperplasia of CD1a+ Langerhans cells. The dermal granuloma observed had a mean +/- SEM value for the CD4/CD8 ratio of 0.80 +/- 0.06. The expression of the activation molecules CD25 (interleukin-2 receptor) and CD18 (lymphocyte function-associated antigen-1 beta), 10.5% and 38.1% respectively, suggests that many cells are primed and proliferating. Most T cells in the granuloma expressed alpha beta T cell receptor (TCR) (40.3%) whereas only a few (6.7%) expressed gamma delta TCR. The results show that Colombian LCL patients possessed the appropiate activation and accessory signals from immunocompetent cells to trigger the effector phase of the immune response and eventually eliminate the parasite.

T cell-mediated terminal maturation of dendritic cells: loss of adhesive and phagocytotic capacities.

Dendritic cells (DC) are a specific subset of APC characterized by the potent ability to activate immunologically naive T cells. We have observed previously that the murine epidermis-derived DC line XS52 undergoes a set of profound changes upon Ag-specific interaction with T cells, including IL-1 beta secretion acquired expression of CD86, and lost expression of CD115 (CSF-1 receptor) and proliferative responsiveness to CSF-1. These changes, which appear to reflect a critical transition during Ag presentation, have been termed T cell-mediated "terminal maturation" of DC, Here we report that XS52 cells also lose their adhesive and phagocytotic capacities during this event. XS52 cells, ordinarily adhere to petri dishes and phagocytose latex heads, as has been reported for DC freshly procured from spleen and skin. Importantly, XS52 cells lose both capacities after 3 to 24 h of incubation with HDK-1 T cells (keyhole limpet hemocyanin-specific TH1 clone) or with 5S8 T cells (dinitrobenzene sulfonate specific Th0 clone) in the presence of Ag. By contrast, incubation with T cells alone or with Ag alone has minimal effects, indicating that this regulation required both T cells and Ag. With respect to mechanisms, several lines of evidence suggest this IFN-gamma, which is secreted by T cells, serves as the primary mediator in down-regulating both capacities. Our observations illustrate a unique mechanism by which responding T cells upon Ag-specific activation by DC, suppress the machinery of Ag uptake through the elaboration of IFN-gamma.

Hydrogen peroxide mediates UV-induced impairment of antigen presentation in a murine epidermal-derived dendritic cell line.

Ultraviolet-B (290-320 nm) radiation is known to impair the antigen-presenting cell (APC) function of Langerhans cells (LC), skin-specific members of the dendritic cell (DC) family. We sought to address mechanisms of this effect, focusing on the role played by hydrogen peroxide. For this purpose, we used a newly established murine DC line, XS52, which resembles epidermal LC in several respects. The APC capacity of XS52 cells, using two different CD4+ T cell clones as responders, was inhibited significantly (> 50%) by exposure to UV radiation (unfiltered FS20 sunlamps) at relatively small fluences (50-100 J/m2). Ultraviolet radiation also inhibited growth factor-dependent proliferation of XS52 cells. On the other hand, cell surface phenotype was relatively well preserved after irradiation; expression levels of B7-1 and B7-2 were reduced slightly, while other molecules (e.g. Ia, CD54, CD11a and CD18) were not affected. With respect to the role played by hydrogen peroxide, pretreatment with purified catalase (900 U/ mL) prevented UV-induced inhibition of APC function. Short-term exposure to 3 mM H2O2 or t-butyl H2O2 mimicked UV radiation by inhibiting APC function. Finally, intrinsic catalase activity was substantially lower in XS52 cells compared with Pam 212 keratinocytes. These results indicate that the generation of hydrogen peroxide alone is sufficient to produce some, but not all, of the deleterious effects of UV radiation on DC derived from the skin.

Comparative ultrastructure and immunolabeling of MHC-II antigens of alveolar macrophages obtained from patients with paracoccidioidomycosis and other lung diseases.

Samples of alveolar macrophages (AM) obtained by bronchoalveolar lavage from patients with either paracoccidioidomycosis, silicosis, sarcoidosis, or allergic alveolitis were investigated by electron microscopy and immunocytochemistry to compare cellular ultrastructure and expression of MHC-II antigens in the AM cell surface. All samples of AM obtained from patients with these pathologies showed heterogeneous structural features. Although, this morphological diversity is also present in AM of healthy donors, our observations seem to indicate that in the diseases studied this morphofunctional diversity is associated with additional ultrastructural characteristics inherent to each disease. In paracoccidioidomycosis the proportion of vacuolated macrophages is significantly lower than in other diseases; this might indicate that in paracoccidioidomycosis the proportion of activated AM is smaller. We observed significant differences in the expression of MHC-II antigens. Silicosis, sarcoidosis, and allergic alveolitis do not differ significantly in the quantity of immunolabeled AM or in the distribution of the label. The percentage of AM from paracoccidioidomycosis that exhibit the MHC-II molecule is very low with poor immunolabeling. In this disease the low expression of the MHC-II molecule could be related to a decrease of the antigen presenting function by AM.

Inadequate epidermal homing leads to tissue damage in human cutaneous leishmaniasis.

Leishmaniasis is a model disease for the study of immunoregulatory mechanisms associated with host resistance and susceptibility. In this article, Felix Tapia and colleagues propose that defects in the signaling properties of the epidermis can result in the generation of either a chronic granulomatous response, which is unable to eliminate the parasite, or a proinflammatory mucocutaneous response and tissue damage.

Enumeration of selected leukocytes in the small intestine of BALB/c mice infected with Cryptosporidium parvum.

Neonatal, suckling BALB/c mice inoculated with Cryptosporidium parvum produce an infection characterized by continuous shedding of oocysts that spontaneously clears by the time the animals are three weeks of age. Neonatal mice were used to characterize the leukocyte subgroups present in Peyer's patches from the ileum and jejunum of Cryptosporidium-infected and healthy mice. After infection, ileal Peyer's patches showed a predominant CD8+ response, with abundant monocytes-macrophages (MOMA-2+) and nonlymphoid dendritic cells (NLDC-145+ cells). In contrast, jejunal Peyer's patches showed more T lymphocytes than ileal patches, with a predominance of CD4+ cells and many dendritic NLDC-145+ cells and MOMA-2+ cells. The present results showed that ileal and jejunal Peyer's patches are functionally different in response to Cryptosporidium parasites. These findings suggest a preferential involvement of jejunal Peyer's patches in T cell-dependent immunity against the parasite, whereas ileal patches may be associated with B cell expansion and maturation.

Adhesion molecules in lesions of American cutaneous leishmaniasis.

Accessory signals, which include adhesion molecules, MHC-II molecules and cytokines, are necessary to foster the interaction between memory T cells and epidermal cells, that is required to promote cutaneous inflammatory responses. American cutaneous leishmaniasis (ACL) is characterized by a spectrum of immunological manifestations, and is a prototype disease for the study of regulatory mechanisms involved in immune protection against protozoal infection. In the present study, we show that diffuse cutaneous leishmaniasis (DCL) epidermis contains keratinocytes that do not express ICAM-1 and HLA-DR molecules. Langerhans cells (LC) are within normal values or somewhat lower, and a very few cells expressing the HB15 molecule--a new described member of the Ig superfamily--are found in such lesions. Mucocutaneous leishmaniasis (MCL) epithelium shows an increased expression of ICAM-1 and HLA-DR molecules, few HB15+ cells, and an absence of epithelial LC. Localized cutaneous leishmaniasis (LCL) epidermis displays ICAM-1+ keratinocytes organized in patches, a uniform expression of HLA-DR, hyperplasia of LC, and numerous HB15+ cells. In all forms of the disease, infiltrating T cells express more LFA-1 beta than LFA-1 alpha, but LFA-1 beta+ T cells are more abundant in LCL granulomas. In contrast, there are more LFA-1 alpha+ T cells in DCL and MCL than in LCL granulomas. LCL lesions also show the highest numbers of HB15+ cells within the granuloma. These results indicate the importance of adhesion molecules in ACL lesions, and open new possibilities for therapeutic schemes oriented towards the control of cell migration.

The clinical and immunological spectrum of American cutaneous leishmaniasis.

American cutaneous leishmaniasis is characterized by a spectrum of clinical manifestations. These include localized, often self-healing single lesions, intermediate forms which frequently produce mucosal lesions and often show exaggerated delayed-type hypersensitivity (DTH), and the rare diffuse cutaneous leishmaniasis in which no reaction of protective cell-mediated immunity or DTH can be demonstrated. Clinical, pathological and immunological studies have begun to unravel some of the mechanisms associated with different disease manifestations, dependent on complex interactions between the host immune response, measured in terms of indices including lymphocyte subsets and lymphokines in vitro and within active lesions, and different species of Leishmania.

Determination of the cytokine profile in American cutaneous leishmaniasis using the polymerase chain reaction.

The lymphokine profiles were determined in the skin lesions of the three distinct clinical forms of American cutaneous leishmaniasis (ACL), using a reverse transcriptase polymerase chain reaction (RT-PCR) and primers for various lymphokines. The message for interferon-gamma (IFN-gamma), tumour necrosis factor-beta (TNF-beta), and IL-8 was expressed in the three clinical forms of ACL. IL-1 beta mRNA was expressed in most localized (LCL) and mucocutaneous (MCL) leishmaniasis, but in only few of the diffuse cutaneous leishmaniasis (DCL). IL-2 mRNA was detected in about half of the lesions, with more prominent values for MCL. IL-4 mRNA was present in most lesions from the three clinical forms, but markedly increased in DCL. IL-5 and IL-10 mRNAs were expressed in all MCL and in half of the DCL lesions and weakly expressed in LCL lesions. IL-10 mRNA was more abundant in MCL lesions. In contrast, IL-6 and TNF-alpha mRNAs were expressed in a large number of LCL. In MCL, IL-6 mRNA was expressed in most cases and TNF-alpha mRNA in all the cases. In DCL, IL-6 mRNA was absent and TNF-alpha mRNA was weakly expressed. These results suggest that most T cells present in the MCL and DCL lesions secrete a mixture of type 1 and type 2 cytokine patterns, but in DCL granulomas type 2 cytokines predominate. In LCL the cytokine patterns show a mixture of type 1 and type 0 with a preponderance of IFN-gamma over IL-4, and low levels of IL-5 and IL-10. The lack of IL-6 and TNF-alpha mRNAs, and the low expression of IL-1 beta in DCL lesions suggest a defect in the antigen-processing cells that may account for the state of unresponsiveness in these patients.

Gonadotrophin-releasing hormone immunoreactivity in the brain of the tropical freshwater fish, Pygocentrus notatus (Teleostei-Characidae).

The distribution of GnRH in the brain of the teleost Pygocentrus notatus was demonstrated with the avidin-biotin peroxidase immunocytochemical method using highly specific antibody against synthetic mammalian GnRH. Optimal immunoreaction was obtained using: 1) Bouin's fluid for fixation; 2) repeated incubation with primary antiserum; 3) the use of a detergent in the dilution buffer; 4) the high sensitivity of the avidin-biotin immunoperoxidase method with the cobalt intensification of 3-3' diaminobenzidine tetrahydrochloride; and 5) the use of primary antibody with high specificity. GnRH-immunoreactive (GnRH-ir) in cells and/or axons was observed in all main brain regions. In the forebrain, GnRH-ir was located in a network extending from the caudal part of the olfactory bulb to the telencephalon. GnRH-ir fibres were also observed in the optic tectum, cerebellum and hypothalamus. Two groups of neuronal cell bodies were identified. One group was located in the antero-ventral telencephalon corresponding to the nucleus olfactoretinalis. The second group was found in the rostrodorsal hypothalamus. No GnRH-ir material was detected in the pituitary gland, thus confirming the results of previous studies on brain GnRH-ir distribution obtained by radioimmunoanalysis in this species. These results demonstrate a high degree of similarity between the GnRH systems of P. notatus and other teleost species.

The cutaneous lesion in American leishmaniasis: leukocyte subsets, cellular interaction and cytokine production.

Interactions between immunocompetent cells require the participation of T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). These interactions are mediated by interlinking cytokines, which are important in determining the type of immune response. In the present study, we have shown that in American cutaneous leishmaniasis (ACL) lesions, most infiltrating T cells expressed the alpha beta TCR including those selectively migrating to the epidermis. In contrast, gamma delta T cells were abundant in localized (LCL) and scarce in muco-cutaneous (MCL) and diffuse (DCL) cutaneous leishmaniasis, suggesting a role in effective granulomas. There were differences in the expression of LFA-1 alpha and beta subunits, with most cells expressing LFA-1 beta. The ratio LFA-1 beta/LFA-1 alpha was higher in LCL (11.8:1) than in MCL (3.3:1) and DCL (2.4:1). Similar results were observed in Leishmania mexicana-infected C57BL/6 mice. DCL lesions showed a higher proportion of LFA-1 alpha+ cells than MCL and LCL lesions. A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of the cytokine profiles showed that most T cells present in the MCL and DCL lesions secrete a mixture of Type 1 and Type 2 cytokine patterns, but in DCL granulomas predominate the Type 2 cytokines. In LCL the cytokine patterns show a preponderance of INF gamma over IL-4, and low levels of IL-5 and IL-10, suggesting a Type 1 cytokine profile.

The cutaneous lesion in American leishmaniasis: leukocyte subsets, cellular interaction and cytokine production

Interactions between immunocompetent cells require the participation of T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). These interactions are mediated by interlinking cytokines, which are important in determining the type of immune response. In the present study, we have shown that in American cutaneous leishmaniasis (ACL) lesions, most infiltrating T cells expressed the alpha beta TCR including those selectively migrating to the epidermis. In contrast, gamma delta T cells were abundant in localized (LCL) and scarce in muco-cutaneous (MCL) and diffuse (DCL) cutaneous leishmaniasis, suggesting a role in effective granulomas. There were differences in the expression of LFA-1 alpha and beta subunits, with most cells expressing LFA-1 beta. The ratio LFA-1 beta/LFA-1 alpha was higher in LCL (11.8:1) than in MCL (3.3:1) and DCL (2.4:1). Similar results were observed in Leishmania mexicana-infected C57BL/6 mice. DCL lesions showed a higher proportion of LFA-1 alpha+ cells than MCL and LCL lesions. A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of the cytokine profiles showed that most T cells present in the MCL and DCL lesions secrete a mixture of Type 1 and Type 2 cytokine patterns, but in DCL granulomas predominate the Type 2 cytokines. In LCL the cytokine patterns show a preponderance of INF gamma over IL-4, and low levels of IL-5 and IL-10, suggesting a Type 1 cytokine profile

Epidermal compromise in American cutaneous leishmaniasis.

In American cutaneous leishmaniasis (ACL), Leishmania parasites enter the epidermis of the host via the bite of infected sandflies. Immune responses against the parasite vary from "effective" in localized (LCL) to a state of "selective anergy" in diffuse (DCL) cutaneous leishmaniasis, whereas the intermediate muco-cutaneous form (MCL) is characterized by an exacerbated cell-mediated immunity. We have shown that in LCL epidermis, Langerhans cells (LC) are increased, HLA-DR is universally expressed and intercellular adhesion molecule-1 (ICAM-1) immunoreactivity is distributed in patches. In addition, mRNA for IL-1 beta, IL-8, TNF alpha, TNF beta, and INF gamma may be detected in epidermal sheets by reverse transcriptase followed by polymerase chain reaction (RT-PCR). In contrast, DCL epidermis shows fewer LC than LCL epidermis, and expression of ICAM-1, HLA-DR, and IL-1 beta mRNA cannot be detected. MCL lesions show a mucosal epithelium lacking LC, but ICAM-1 is universally expressed. The clinical manifestations of ACL can be reproduced experimentally in different strains of inbred mice. In healthy mice, we have shown a positive correlation between LC and dendritic epidermal T cells (DETC) numbers. This correlation was not, however, observed in L. mexicana-infected mice, suggesting that infection alters the balance between the two cell types. In addition, agents that modulate LC and DETC cell densities change the development of experimental leishmaniasis. These results suggest that the epidermis is essential in determining the type of immune response that is developed against the Leishmania parasites.

American Leishmania spp. and Trypanosoma cruzi: galactosyl alpha(1-3) galactose epitope localization by colloidal gold immunocytochemistry and lectin cytochemistry.

Patients with Chagas' disease or different clinical forms of leishmaniasis (cutaneous or visceral) have elevated galactosyl alpha (1-3)galactose antibodies. Using colloidal gold immunocytochemistry--monoclonal antibody gal-13 (specific for lipid-linked galactosyl alpha (1-3)galactose residues) and anti-nidogen antibodies and lectin cytochemistry (Bandeiraea simplicifolia IB4), both techniques specific for demonstrating galactosyl alpha (1-3)galactose residues--we have found terminal disaccharide residues on the Trypanosoma cruzi external surface of Vero cell-derived trypomastigotes but not in intact epimastigotes (although disrupted epimastigotes strongly stained), in the lips of the flagellar pocket, and on the parasitic side exactly opposite to the flagellar pocket in amastigote and promastigote forms of American Leishmania. These results resemble those obtained using anti-laminin antibodies in both trypanosomatids. In addition, results obtained with anti-nidogen antibodies seem to recognize in Trypanosoma cruzi and American Leishmania culture forms another different unknown terminal disaccharide. These results confirm the presence of terminal galactosyl alpha (1-3)galactose residues in both trypanosomatids, and that rabbit anti-laminin antibodies are indeed also recognizing galactosyl alpha (1-3)galactose residues as demonstrated for human circulating antibody. The presence of abundant galactosyl alpha (1-3)galactose residues on Trypanosomatid family members suggests a specific unknown role in parasite physiology for this terminal disaccharide.