Treatment of rat nephrotoxic nephritis. Use of 5-fluorouracil or methotrexate-5-fluorouracil association

To evaluate the effect of 5-fluorouracil (F) and methotrexate-5-fluorouracil association (MTX-F) on nephrotoxic nephritis, seven groups of 10 rats were inoculated with anti-rat glomerular basement membrane serum (AGBMS); five groups were treated with different doses of F, beginning on the 2nd or the 6th day, one group with MTX-F beginning on the 2nd day and one group (control) with distilled water. Twenty-four hour proteinuria was determined weekly until the 71st day. The kidneys were examined histologically and by immunofluorescence. The group treated with F (1.3 mg/100 g body weight) developed a severe glomerulonephritis similar to the control group; (b) the groups treated with F (2.0 mg/100 g body weight) or with MTX-F showed progressively lower proteinuria, less severe histological changes and less intense fluorescence due to autologous antibodies. The best results were observed in the MTX-F group and in the F group treated from the 6th day. These groups presented at the 71st day proteinuria of 84 and 91 mg as compared to 312 mg in the control group, and minimal histological lesions as compared to glomerulosclerosis and tubular atrophy in the control group. We concluded that either F or MTX-F produced significant improvement of nephrotoxic nephritis due to inhibition of autologous antibody production.

Serology of paracoccidioidomycosis. II. Correlation between class-specific antibodies and clinical forms of the disease

Untreated and previously treated patients with paracoccidioidomycosis were studied for: (i) serum levels of total IgG, IgM and IgA immunoglobulins, by radial immunodiffusion and Paracoccidioides brasiliensis (Pb) antibodies, by indirect immunofluorescence; (ii) correlation between their levels with the clinical forms of the disease; (iii) correlation between the serum titres obtained by tube precipitin with those of anti-Pb IgG, IgM and IgA. In the untreated group, serum IgG levels were significantly increased in patients with the more systemic forms of the disease, especially the acute progressive form. Serum IgA levels were significantly increased in all patients with no statistical difference between clinical forms. Serum IgM levels were normal in all patients. Anti-Pb IgG, IgA and IgM were detected in 97·5%, 32·5% and 45·0% of all cases, respectively. There was a sharp tendency towards higher levels of anti-Pb IgG among those with the acute progressive form (83·4%) in relation to the chronic, more localized forms, mixed form (68·0%) and isolated organic form (55·5%). In the untreated and previously treated group sera, there was positive correlation between the level of anti-Pb IgG and positivity for the tube precipitin test, suggesting that the precipitin-type antibodies are of the IgG class. Broadly, the present data demonstrate a polyclonal activation of the humoral immune system in paracoccidioidomycosis, with a positive relationship between serological results and severity of the disease. © 1984.

Serology of paracoccidioidomycosis - I. Evaluation of the indirect immunofluorescent test

The purposes of the present work were: i) to study the positivity indices and compare titers obtained with the indirect immunofluorescence (II), tube precipitation (TP), complement fixation (CF) and double immunodiffusion on agar gel (ID) tests in the sera of 196 patients with paracoccidioidomycosis before treatment, and ii) to compare the initial titers of II with those obtained 1 year or more after treatment. II was the most sensitive serologic reaction (85.2%), and the positivity indices for CF, ID and TP were 67.7%, 66.0% and 50.0%, respectively. The sera tended to show parallel mean titers in II, CF and TP tests. One year after treatment there was a fall in titers of II in 66.2% of patients. The data, taken as a whole, demonstrate the usefulness of the indirect immunofluorescent test and the importance of using 2 or more serologic tests for the diagnosis and monitoring of patients with paracoccidioidomycosis. © 1985 Martinus Nijhoff/Dr W. Junk Publishers.

Emprego da imunofluorescência direta no estudo das alterações de mucosa de palato, compatíveis com candidíase atrófica crônica, em indivíduos portadores de próteses dentárias totais muco-suportadas.

Histopathology by hematoxilin-eosin (HE) and periodic acid Schiff (PAS), concomitant direct immunofluorescence (DI) against total human immunoglobulins and against Candida albicans, was effectuated in 25 persons dentures wearers. In 5 persons without chronic athrofic candidiasis (CAC) clinical signals in the palate the HE showed wise inflammatory elements in the connective tissue and the PAS marked the continuous basal layer, the intra-cellular grains of granular layer and the uniform parakeratin on epithelial surface. In 20 others, with palatal signals of CAC, in the HE was frequent the features encountered in Candida infected and PAS revealed, beside descontinuous lamina basal and epithelial surface covered by tide and discontinuous parakeratin, the presence of round bodies few largers that presents in the granular layer, casually isolated in the medial portion of ret pegs and connective papillae. In the first 5 persons the DI against total human immunoglobulins not showed signals of the humoral immunologic phenomena, the same was valid to others 20 patients with CAC clinical aspects. However the DI with conjugate against C. albicans in the 20 cases with CAC signals revealed suitable aspects of the structures assumed by Candida in tissues. Cultures of samples obtained of the persons with CAC signals was positive in 100% to Candida, 70% presumptively albicans, against 80% of positiviness to generus Candida, 67% presumptively albicans, in the persons without CAC signals.

Infection and apparent invasion of Vero cells by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis probably uses many different mechanisms to establish itself in the host and cause disease. In this work, we assess an in vitro model system which uses cultured mammalian cells to investigate the virulence factors of P. brasiliensis. We were able to demonstrate an invasion process of the yeast form of this fungus in Vero cell cultures. We deduced that the overall invasive process involved three steps: adhesion, followed by invasion of individual epithelial cells and spread to adjacent cells.

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.

IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: Comparison of counterimmunoelectrophoresis and complement fixation

IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started. Antibodies against P. brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation. Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses). The highest efficiency percentages were found with IgG and IgA- ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%). Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA. IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01). Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels. This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

Characterization of a monoclonal antibody recognizing mast cells

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

Influence of canine brain decomposition on laboratory diagnosis of rabies.

Canine brains infected with rabies virus were submitted to decomposition by being left at room temperature of 25 to 29 degrees C for up to 168 h. At 24 h intervals, brain fragments were analyzed by immunofluorescence (IF) and by the mouse intracerebral inoculation (MI) test to confirm the diagnosis of rabies and to measure the putrefaction effect on the accuracy of the diagnosis. Forty eight h after the beginning of the experiment, the MI test showed signs of impairment with four negative results, while after 72 h, 100% of the results were negative to the MI test and only one result was negative to the IF test, indicating that the threshold period for accurate diagnosis is 24 to 48 h before putrefaction. The authors recommend the shipment of suspected cases of rabies to the laboratory for confirmation, but the use of putrid materials for diagnosis is meaningless because of false-negative results.

The detection of Toxoplasma gondii by comparing cytology, histopathology, bioassay in mice, and the polymerase chain reaction (PCR)

The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the São Manuel abattoir, São Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%). © 2001 Elsevier Science B.V.

Morphological aspects of Culex quinquefasciatus salivary glands

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm. © 2003 Elsevier Ltd. All rights reserved.

Soroprevalência de Toxoplasma gondii em rebanhos caprinos no Estado de São Paulo

Four hundred forty-two serum samples were collected from dairy goats in seven regions of São Paulo State. These were tested for Toxoplasma gondii antibodies using the indirect immunofluorescent antibody test. Sixty-four (14,5%) serologically positive animals were found from all these goat farms studied.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.