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.

Abnormal expression of galactosyl(alpha 1-->3) galactose epitopes in squamous cells of the uterine cervix infected by human papillomavirus.

This study describes the presence of alpha-galactosyl epitopes on 12 cervical biopsy samples with features of human papillomavirus infection (HPV) and different stages of cervical intraepithelial neoplasia. An avidin-biotin-peroxidase assay with a monoclonal antibody recognizing gal(alpha 1-->3)gal residues was strongly positive in 5 of 12 cases. None of the controls stained (p = 0.02). Immunostaining was intense in the areas with the highest viral load (koilocytes and keratinocytes) and absent in malignant foci. Immunostaining was also absent in normal exo- and endocervical epithelium of 12 controls with no features of HPV infection. A faint background staining in cases and controls was evident, but similar. These initial findings suggest that alpha-galactosyl epitopes are expressed in cervical squamous cells infected with HPV, turning them vulnerable to lysis by natural anti-alpha-galactosyl antibodies.

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.

Successive generation of antigen-presenting, dendritic cell lines from murine epidermis.

Dendritic cells are specialized APCs that exhibit an extraordinary capacity to activate naive T cells. Langerhans cells (LC), as epithelial tissue-specific members of this family, play key roles in the induction of T cell-mediated immunity against environmental, infectious, and tumor-associated Ags in skin. A major limitation in studying the biology of dendritic cells or LC has been the absence of stable, long-term cell lines. To overcome this limitation, we have established a series of APC lines (XS series) from newborn BALB/c mouse epidermis. XS lines, which have grown for more than 12 mo in culture, exhibit high similarity to LC freshly procured from skin in terms of: a) tissue of derivation (epidermis), b) phenotype (lalow/CD45+/E-cadherin+/B7-1-), c) shape (elongated dendrites), and d) Ag-presenting profile (modest ability to activate naive, allogeneic T cells and remarkable ability to present a protein Ag to primed CD4+ T cells). The availability of XS lines as well as the methodologies used for their growth enhance our capability of studying the biology of LC at biochemical and molecular levels.

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.

Leprosy in women: characteristics and repercussions.

"Health is often measured in terms of low mortality; nevertheless, merely being alive is not a measure of the quality of life" H. Méndez Castellanos. Physiological, socioeconomic and cultural factors play important roles in the response of women to Mycobacterium leprae and in the impact of leprosy on their lives. They appear to develop stronger immunological responses to M. leprae than men, as suggested by lower incidence and less severe clinical forms of disease in most areas of the world, as well as stronger reactions of cell-mediated immunity after prophylactic vaccination. Genetic factors and physiological status including pregnancy, intercurrent infection and malnutrition might be among the factors which modulate this response. Women in leprosy-endemic areas of the world, with few exceptions, suffer from marked economic and social dependency and inferiority which can only be heightened by the social stigma associated with leprosy. Nevertheless, they bear an enormous responsibility for the health of their families, often as head of the household, and they often possess a unique capacity to influence community opinion. With the introduction of multidrug therapy, leprosy control throughout the world is no longer an unrealistic goal. Active vaccination may constitute the other factor necessary for eventual eradication of the disease. The incorporation of women at all levels into active roles in health care programs may constitute one of the decisive factors in the success or failure of leprosy control.

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.

Immunocytochemical characterization of immune cells in lesions of American cutaneous leishmaniasis using novel T cell markers.

Some recently defined lymphocyte immunophenotypes were determined in lesions of patients with American cutaneous leishmaniasis (ACL). New monoclonal antibodies have allowed the demonstration of cell surface antigens of T lymphocytes, such as CD45RA and CD45RO, which recognize different maturational stages of the same T CD4+ cell subgroup: 'virgin' (CD4+CD45RA+) and 'memory' (CD4+CD45RO+) T cells respectively. The CD4/CD8 cell ratios were higher in mucocutaneous leishmaniasis (MCL) than in localized cutaneous leishmaniasis (LCL) lesions. Diffuse cutaneous leishmaniasis (DCL) has the highest values of 'virgin' T cells; LCL and MCL patients have lower values, similar to each other. 'Memory' T cells were higher in MCL than in LCL or DCL. The ratio of 'memory'/'virgin' T cells was 7.9 for LCL, 9.6 for MCL and 2.5 for DCL. The highest value for IL-2 receptor positive cells (CD25) was observed in LCL, whereas single CD45RO-immunoreactive cells showed a peak value in DCL patients. HLA-DR+ cells were present in all three clinical forms of ACL. MCL patients showed a lack of epithelial Langerhans cell (CD1a+) in the nasal mucosa.

Leukocyte immunophenotypes in bronchoalveolar lavage fluid and peripheral blood of paracoccidioidomycosis, sarcoidosis and silicosis.

Leukocyte subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood of patients with paraccoccidioidomycosis, sarcoidosis and silicosis were characterized using monoclonal antibodies and an immunoperoxidase technique. In paraccocidioidomycosis, the number of T-helper/inducer CD4-positive lymphocytes was lower in peripheral blood than in BAL fluid. Additional analysis showed that the expression of HLA-DR was very similar in alveolar macrophages, lung and blood T-cells. In sarcoidosis and silicosis there were higher proportions of T-helper/inducer cells in peripheral blood than in BAL fluid. The alterations in the T-helper/inducer/T-suppressor/cytoxic CD4/CD8 ratio in sarcoidosis and silicosis were more appreciable in peripheral blood than in BAL fluid, contrasting with the results in paracoccidioidomycosis. The expression of HLA-DR by alveolar macrophages in sarcoidosis was the highest of all the disease studied. No statistically significant differences were observed between chronic multifocal and chronic unifocal paracoccidioidomycosis disease, stage II and stage III sarcoidosis, and chronic and accelerated silicosis. The three granulomatous diseases analyzed had a few alveolar macrophages expressing the CD4 molecule on their surface. These findings and the technique of analyzing both peripheral blood and BAL leukocyte subsets may help to understand the pathogenesis of interstitial lung diseases.

Linfocitos T vírgenes y T memoria en la leishmaniasis cutánea americana / Tlyphovytes virgins and TY memory in American cutaneous leishmaniasis

La caracterización in situ de subpoblaciones leucocitarias ha permitido evaluar la participación de los diferentes componentes celulares en la respuesta inmunológica a distintos agentes patógenos. En lesiones de leishmaniasis cutánea americana se ha podido demostrar que parte de la falla inmunológica de los pacientes con leishmaniasis difusa recae sobre la subpoblación de lifocitos cooperadores inductores CD4+, los cuales no son capaces de producir Interleucina-2. Nuevos anticuerpos monoclonales permiten subdividir a los linfocitos T CD4+ en linfocitos T vírgenes CD4+ CD45RA+ y linfocitos T memoria CD4+CD45RA. En el presente estudio, se demostró que el número de linfocitos T memoria es mayor en pacientes muco-cutáneos (LCM) que localizados (LCL) y difusos (LCD). La relación de linfocitos T memoria/T vírgenes fue de 7,9 para LCM y 2,5 para LCD. Esta relación es una nueva forma de evaluar la condición inmunológica de los individuos, y pudiera ser útil en la evaluación de esquemas terapéuticos