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.

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.

Double immunogold staining method for the simultaneous ultrastructural localization of regulatory peptides.

Recent studies have suggested that the morphological characteristics of secretory granules contained within endocrine cells and nerves may be determined largely by their chemical composition. The use of the immunogold staining (IGS) method, which is based on the adsorption of colloidal gold to immunoglobulins, has been used in our laboratory to demonstrate a wide range of intracellular antigens at both the light and electron microscope levels. In this study we have applied a modification of the IGS method for the simultaneous detection of two separate antigens in a single tissue section, using a variety of region-specific antisera to different peptides. Peptide antisera were raised in rabbits or in guinea pigs and these were applied simultaneously or sequentially to grid-mounted ultrathin tissue sections. Antigenic sites were visualized at the electron microscope level using antisera raised in goats, adsorbed to gold particles of 12, 20, or 40 nm. Using this technique we have attempted to investigate the coexistence of multiple antigens in single tissue sections, in particular in single granules; the topographic distribution of molecular forms within one single granule or granule population; the heterogeneity of peptidergic neurons and also the heterogeneity of peptide content in morphologically similar granules. The double immunogold staining procedures described here have proved to be extremely effective for the simultaneous ultrastructural localization of two antigens (peptide-peptide; peptide-propeptide) on a single tissue section. The further development of this technique may provide useful information on neuroendocrine cell dynamics in normal and diseased states.

Electron immunocytochemical localization of enkephalin-like material in catecholamine-containing cells of the carotid body, the adrenal medulla, and in pheochromocytomas of man and other mammals.

Enkephalin-like immunoreactivity has been localized to electron-dense secretory granules of cat and piglet carotid bodies and adrenal medullae, horse adrenal medulla, and also to human adrenal medulla and pheochromocytomas using a gold-labeled antibody technique performed at the electron microscopic level. The same granules were also demonstrated to exhibit dopamine-beta-hydroxylase-like immunoreactivity, which suggests a granular colocalization of amines and peptides in catecholamine-storing cells.

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.

Density of epidermal Langerhans cells in psoriasis patients treated with an aromatic retinoid (RO 10-9359). An immunoperoxidase study using anti-T6 and anti-Ia monoclonal antibodies.

Immunocytochemical techniques using antibodies to the specific T6 and Ia (Major Histocompatibility Complex, class II, human HLA-Dr) antigens were used to determine the densities of epidermal Langerhans cells (LC) in psoriasis patients treated with the aromatic retinoid RO 10-9359. Fourteen patients were treated with the aromatic retinoid and were skin biopsied before, during and after therapy. Two psoriasis patients receiving PUVA (systemic 8-methoxypsoralen + UVA irradiation) were included in the study. The results showed an increase in LC numbers during aromatic retinoid administration, which coincided with an improvement in the clinical severity of the lesions. At the end of retinoid administration the LC numbers were similar to those found in the initial psoriatic plaques. The density of Ia+ LC, in comparison with T6+ LC in the epidermis of psoriatic plaques were significantly different. Dendritic and non-dendritic Ia+ cells were also observed in the dermis of the plaques. Unlike aromatic retinoid treated patients, PUVA treated patients showed a decrease of both T6+ and Ia+ epidermal LC by the middle of therapy, a total absence of immunoreaction by the end of therapy, and a return to normal skin values a few weeks after treatment. This immunocytochemical study helps in distinguishing between dendritic and other possible Ia-expressing cells from the infiltrate that may penetrate the epithelium. These results do not conclusively demonstrate the role of LC in the pathogenesis of psoriasis. Other factors, such as the interrelationship with other immune response cell types and alterations in the lymphokine cascade may be important.

Mononuclear cell subpopulations and infiltrating lymphocytes in erythema dyschromicum perstans and vitiligo.

Erythema dyschromicum perstans (EDP) and vitiligo are two cutaneous pigmentary dermatoses of unknown etiology. In the present study, the leukocyte infiltrates in the affected skin of EDP and vitiligo patients were studied using the avidin-biotin (ABC) immunoperoxidase technique and monoclonal antibodies which recognise the following mononuclear cell subgroups: T-suppressor/cytotoxic (CD8-Leu-2), T-helper (CD4 = OKT4), T-suppressor + macrophages (Leu-15), Pan T (CD3 = Leu-4), macrophages (Leu-M3) and Langerhans cells (CD1 = Leu-6), and other cellular markers such as Ia antigens and the Interleukin-2 receptor (CD25 = TAC). The immunocytochemical analysis showed a selective accumulation of CD3+, CD8+, Leu-15-, T-cytotoxic cells in the epidermis of both EDP and early lesions of vitiligo. In addition, an increase in the number of epidermal Langerhans cells (CD1+) was observed in some cases of EDP and vitiligo. The CD4/CD8 ratios in affected and uninvolved skin for both disorders were not significantly different, although values lower than unity were only observed in the infiltrates of affected skin. Ia antigen positivity was observed in the dendritic cells of the dermis and epidermis, as well as in most of the lymphoid cells within the infiltrates for both diseases. Macrophages (Leu-M3) in EDP dermal infiltrates were generally found adjacent to extracellular melanin pigment. Lymphocytes expressing TAC (CD25) surface antigens were also present in the dermal infiltrates. These morphological observations suggest a possible immune cell participation in the dyschromia of such cutaneous disorders.

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.

Leukocyte subsets in the granulomatous response produced after inoculation with Mycobacterium leprae-BCG in lepromatous patients.

Leukocyte subsets present in the granulomatous response produced after the inoculation of a mixture of Mycobacterium leprae and BCG in lepromatous leprosy patients were characterized in situ using monoclonal antibodies and an immunoperoxidase technique. The granuloma produced after M. leprae-BCG inoculation showed a distribution pattern similar to tuberculoid granulomas. T lymphocytes bearing the CD8 phenotype (T cytotoxic/suppressor) were sequestered to the periphery of the epithelioid tubercles and T helper-inducer CD4+ lymphocytes were distributed throughout the infiltrate. Langerhans cells CD1+ were increased in the epidermis, and in dermis they were localized mainly in the mantle surrounding the granuloma. Most of the dermal infiltrate produced after the inoculation or M. leprae-BCG expresses the HLA-DR antigen. Similarly, most keratinocytes were also positive to this MHC antigen. The granulomatous response to BCG was similar to the inoculation of a mixture of M. leprae-BCG, however acid-fast bacilla were still present. The inoculation of M. leprae produced a macrophage granuloma with no clearing of the bacilla which resembles the lepromatous leprosy granuloma.

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.

Cryptosporidiosis in Venezuelan children with acute diarrhea.

Thirteen of 120 Venezuelan children with acute diarrhea were found to be excreting Cryptosporidium oocysts in their stools. This confirms that Cryptosporidium can infect immunocompetent children, and the relatively high frequency found suggests that this protozoan may be an important cause of diarrhea in Venezuela.

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.

Immunogold staining procedure for the localisation of regulatory peptides.

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.

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.

Immunocytochemical detection of Entamoeba histolytica.

Human anti-Entamoeba histolytica immunoglobulin was used to detect Entamoeba histolytica in 74 positive samples from several different sources, using an indirect immunoperoxidase method. In 73 samples, the protozoan was easily identified. Trophozoites and cysts of all cultured Entamoeba strains examined were strongly stained, and as few as 3 trophozoites per microscope slide could be detected. In addition, 51 negative control samples were also tested and non-specific reactions were not observed. These preliminary results show that this method is both sensitive and specific, and can easily detect trophozoites and cysts of different E. histolytica strains.

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.

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.

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.

[Immunohistochemical localization of S-100 protein in granular cell myoblastoma]. / Localización inmunohistoquímica de la proteína S-100 en el mioblastoma de células granulares.

Three cases of granular cell myoblastoma have been studied in order to determine the presence and distribution of the S-100 specific protein in the neoplastic cells, using immunocytochemical staining techniques, through the modified avidin-biotin method. Positive immunostaining was observed in the three cases studied. The comparative study of various control cases histogenetically originating from neuroectoderm (melanoma) and specifically from Schwann cells, as also the presence of strongly positive staining in Schwann cells of peripheral nerve fibres situated inside and outside the tumor, support the concept of the neurogenic origin of this interesting tumor.