Detection of the 43,000-molecular-weight glycoprotein in sera of patients with paracoccidioidomycosis

The 43,000-molecular-weight (43K) soluble glycoprotein was detected in sera of patients with paracoccidioidomycosis by the immunoblot technique by using as the probe rabbit monospecific antisera to this fraction. The 43K antigen was present before treatment in sera of patients with the acute (juvenile) form; it started to disappear from circulation after 10 months of chemotherapy, and it was undetectable afer 2 years of treatment. In the chronic cases, the 43K antigen was detected in patients without treatment, and it was absent in the healed cases. The detection of the 43K protein specific to Paracoccidioides brasiliensis may be important for its diagnostic value as well as for modulation of the host immune response.

Rabbit antibody responses to experimental infestation with Dermatobia hominis.

The three larval stages of Dermatobia hominis (Linnaeus) have been evaluated for their immunogenicity by ELISA and immunodiffusion (ID) using sera from experimentally infested rabbits. During a primary infestation, first instar D. hominis were found to cause most reaction and allowed the earliest diagnosis by ELISA. An inhibition of the antibody response against second and third instars was observed. The inhibition disappeared after departure of the larvae from the host. In experimentally immunized hosts the antibody response, following challenge, was highest against second and third instar antigens. Antibody remained elevated during the infestation but fell immediately after the larvae had left the host.

Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

Utilidade dos métodos imuno-histoquímicos para o diagnóstico anátomo-patológico.

In order to determine the value of immunohistochemical staining methods for the morphologic diagnosis, we studied 949 histologic specimens sent for consultation to the Immunohistochemistry Laboratory of Department of Pathology of the Medical School of Botucatu in the period 1984-1989. All case were submitted to the immunoperoxidase staining with the methods PAP or ABC. Immunohistochemical stains confirmed the original morphologic diagnosis in 468 cases (49.3%); made the definitive diagnosis from a list of differential diagnostic possibilities in 244 cases (25.7%); provided contributory information in 74 cases (7.8%); were non-contributory in 114 cases (12%) and rendered an unsuspected diagnosis in 49 cases (5.2%). In some cases with non-contributory information the differences in methods of fixation might have led to suboptimal preservation of tissue antigens. The immunohistochemical staining may provide important and sometimes essential informations for definitive diagnosis. This technique was particularly useful for differential diagnosis between carcinoma, lymphoma and melanoma.

Pulmonary paracoccidioidomycosis in immunized mice

Paracoccidioidomycosis was induced in immunized (IM) and non-immunized (NI) mice. The histopathology, the number of fungi in the lungs, the cellular (footpad test - FPT and macrophage inhibition factor assay - MIF) and humoral (immunodiffusion test) immune response were investigated serially postinfection. In the IM mice, at days 1 and 3, there was intense and predominant macrophagic-lymphocytic alveolitis with loose granulomatous reaction; at day 30, inflammation was mild. In the NI group, up to day 3, the lesions were focal; later there was formation of extensive epithelioid granuloma. The number of fungi in IM mice were always smaller than those of NI group. Immunization alone induced positive FPT and MIF indices with low titer of antibody. After infection, there was a significant decrease of the FPT indices in the IM group, which we interpreted as desensitization due to trapping of sensitized lymphocytes in the lungs. In conclusion, (1) The lesional pattern of pulmonary paracoccidioidomycosis in IM mice was similar to that of a hypersensitivity pneumonitis. This reaction was probably effective in reducing the extension of the infection and decrease the number of fungi. (2) In this model, pulmonary resistance against P. brasiliensis seems to be related to local and systemic delayed-type hypersensitivity reaction. © 1992 Kluwer Academic Publishers.

Peripheral giant cell granuloma. An immunohistochemical and ultrastructural study.

OBJECTIVE: To study the nature of multinucleated and mononuclear cells from peripheral giant cell granuloma (PGCG). MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded sections of 40 cases of PGCG were immunohistochemically stained for vimentin, alpha I-antichymotrypsin, CD68, S-100 protein, lysozyme, leucocyte common antigen (LCA), factor VIII-related antigen and muscle cell actin. Six cases of PGCG were also studied by transmission electron microscopy. RESULTS: Vimentin, alpha I-antichymotrypsin and CD68 were expressed in both the mononuclear and multinucleated giant cells. Dendritic mononuclear cells, positive for S-100 protein, were noted in 67.5% of the lesions, whereas lysozyme and leucocyte common antigen were detected in occasional mononuclear cells. Ultrastructural examination showed mononuclear cells with signs of phagocytosis and sometimes interdigitations with similar cells. Others presented non-specific characteristics and the third type exhibited cytoplasmic processes and occasional Birbeck granules. Some multinucleated giant cells showed oval nuclei, abundant mitochondria and granular endoplasmic reticulum whereas others presented with irregular nuclei and a great number of cytoplasmic vacuoles. CONCLUSIONS: Immunohistochemical and ultrastructural results suggest that PGCGs of the jaws are composed mainly of cells of the mononuclear phagocyte system and that Langerhans cells are present in two thirds of the lesions.

Alterações imunitárias na cirrose hepática alcoólica+.

The alcoholic liver cirrhosis usually causes overall immunological changes which might be attributed to either the hepatic disease itself, to ethanol action and/or to malnourishment of the patient. These immune abnormalities comprise both cellular and humoral immunity, consisting of increased immunoglobulin levels, depressed late-skin response to antigens, lowered proliferative response of lymphocytes to mitogens, lower plasma levels of complement proteins (C3 and C4) and by either lower (IL2 and gamma IF) or increased (IL1, TNF, IL6 and IL8) cytokine levels. Parallel to the systemic immune suppression found in most patients, there is also a concomitant local, genetically based, immune stimulation at the liver level which leads to hepatic self-aggression. The systemic immune-suppression could be responsible for periodical infections or neoplasia found in these patients. The possible factors for the immune exhaustion are: a) lower hepatic clearance of toxins and/or bacteria; b) lower hepatic synthesis of complement components; c) cytokines (IL2 and gamma IF) deficiencies, and d) deficiencies of nutrients related to the antioxidant and/or immune defense mechanisms. The immune stimulation of the liver self aggression is characterized by the preferential migration of cytotoxic T cell and neutrophils to the liver, following stimulatory factors such as Mallory bodies, acetaldehyde and/or antibodies. Moreover, the local increase of cytokines (IL1, TNF, IL6 and IL8) levels would be liable for the local phagocyte chemotaxy (IL8) or part of liver injury (TNF) eased by the lower antioxidant defense of the cirrhotic liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical study of a Paracoccidioides brasiliensis polysaccharide-like antigen

The polysaccharide antigen from P. brasiliensis has been largely employed in serologic tests, as well as in skin tests, to evaluate cellular immunity. SDS-PAGE analysis of this antigen has revealed a variability in the number of bands exhibited by isolates SN, 265, 339, 113 and 18 (7 to 16 bands). The antigens obtained from isolates 2, PTL, 192 and Adel showed two or three bands. Glycoprotein analysis demonstrated a broad region between 50 and 90 kDa. Major bands of 48 and 30 kDa were present in almost all antigens. Optimal complement fixing dilution appears to be unaffected by the number of bands presented by different antigens. The immunoblot analysis revealed that the 90 and 30 kDa bands were mainly recognized by sera from paracoccidioidomycosis patients. Bands of high molecular weight were also recognized by most of the sera studied. Sera from histoplasmosis recognized the 94 kDa band. In conclusion, although the isolates exhibit quantitative variability in the number of fractions, it is possible to use only one or two samples given the greatest frequency of reactivity is seen in the 30 and 90 kDa fractions.

Antigen distribution in mucocutaneous biopsies of human paracoccidiomycosis

The authors studied the distribution of Paracoccidioides brasiliensis antigen(s) in human skin and oral mucosa. In biopsies obtained from untreated patients showing the chronic form of the disease, the authors demonstrated the P. brasiliensis antigen using two polyclonal immune sera raised in rabbits, one against the exoantigens of P. brasiliensis and the other against a 43-kDa glycoprotein. Langerhans' cells were detected through double immunolabeling using an anti-S100 protein monoclonal antibody. Double labeling immunohistochemistry showed that both of the immune sera labeled the yeast cells in the center of the granuloma and those transmigrating through the epithelial layer equally well. Granulomas exhibited the P. brasiliensis antigen permeating cells, mainly at the periphery of the granulomatous inflammation. The P. brasiliensis antigen(s) accumulated in the macrophages but not in the Langerhans' cells. P. brasiliensis antigens, detected by antiserum against parasite exoantigens, were also deposited between basal keratinocytes, but not in the granular cells, in 47% of the biopsies. P. brasiliensis antigens, as assessed by immunoelectron microscopic techniques, are present in the cytoplasm of the yeast cells in the host tissues. Antigens are transported to the cell membrane and later excreted through the cell wall. Antigenic deposits are also seen at the fungus-host interface.

Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Use of lipopolysaccharides to characterize Bradyrhizobium spp.

Three methods of extraction of lipopolysaccharide (LPS) were compared-the conventional hot phenol-water method with 40% phenol, a modified form of this method using 10% phenol, and the hot saline method. Good recovery of LPS was achieved by each of the three methods, with the LPS found in the aqueous phase with the two phenol-based procedures. The application of SDS-PAGE to the LPS extracts, followed by silver staining, showed similar banding with all three methods of extraction. When the hot saline extraction LPS fraction from eight strains of Bradyrhizobium spp. and eight strains of Bradyrhizobium japonicum was compared with SDS-PAGE, characteristic profiles were achieved. Serological analysis of eight strains of Bradyrhizobium spp., using antisera prepared against whole cells in agglutination reactions, showed extensive sharing of antigens. When antisera was prepared using outer membrane LPS, extracted by the hot saline method, the amount of cross-reaction was reduced greatly. The results indicated that LPS provide an efficient means of obtaining monospecific antisera to be used for serological identification of strains of Bradyrhizobium spp. and that the hot saline extraction method is recommended for a fast, simple and efficient way to obtain LPS and characterize this bacterium.

Both psoriasis and benign migratory glossitis are associated with HLA-Cw6

This study was undertaken to investigate human leucocyte antigen (HLA) associations with benign migratory glossitis and psoriasis in Brazilian patients and particularly to determine whether benign migratory glossitis is also associated with HLA-Cw6, the classical association observed in psoriasis. The results showed a highly significant association of Cw6 with both psoriasis and benign migratory glossitis, with this antigen being present in 59.% of the patients with psoriasis, in 43.8% of the patients with benign migratory glossitis, and in only 12.6% of the controls. Other significant positive associations, although at a lower significance level, were with B13, both in psoriasis and in benign migratory glossitis, and with B17, only in psoriasis. To our knowledge, this is the first report on the association of Cw6 with benign migratory glossitis. We believe that this finding reinforces the concept of a pathogenetic relationship between benign migratory glossitis and psoriasis.

Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis

The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al. [1] (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM). Fava Netto antigen (AgF) was tested in parallel as a control antigen. The study was conducted with mice and guinea pigs infected with P. brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups. The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively. The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals. The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals. Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR). Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens. The in vitro cell immune response showed variable results for the various experimental models, i.e. significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients. Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.

Specific components found in circulating immune complexes (CIC) in paracoccidioidomycosis

Circulating immune complexes (CIC) from 15 paracoccidioidomycosis (PCM) patient sera and from 20 healthy control sera were analysed. After CIC precipitation, solubilization and acid treatment, only a little reactivity to P. brasiliensis antigens was found in the free antibodies from PCM-CIC. This result has suggested that there were antibodies with a high affinity bound to fungus components. Dissociated CIC were fractionated in a column of Sephacryl S300 and the fractions that probably contained antigens were pooled and applied to an affinity column, prepared with mouse anti-gp43 monoclonal antibody. Using ECL-Western blotting assay two polypeptide with apparent mass of 43 and 62 kDa were found.

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment 1), we analyzed two antigen lots of two human isolates (Bt1 and Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh and Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment 2), we compared the antigenic profile of three antigen hatches from three human isolates (Bt1, Bt2 and Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment 1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment 2, the reference system for P brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.