Expression of caveolin-1 in rat Leydig cells

Caveolin-1, the first member of caveolin family reported, is recognized as the structural component of caveola, a plasma membrane invagination or vesicles that are a subcompartment distinct from clathrin-coated pits. This protein is also known to be involved in cholesterol trafficking. The aim of this study was to determine the expression of caveolin-1 in adult rat Leydig cells. Testis sections incubated with an antibody to caveolin-1 showed, by immunohistochemistry, a moderate number of Leydig cells with different degrees of immunoreaction and a strong reaction in endothelial cells and in the lamina propia of seminiferous tubules.Caveolin- 1 was detected in the cell cytoplasm with a granular pattern and on the cell surface of Leydig cells cultured 24 h on uncoated, laminin-1 or type IV collagen coated coverslips. We also observed a milder reaction in 3 h cultures. Immunoreaction was also detected in Leydig cells with an antibody to tyrosine-phosphorylated caveolin-1. By double immunofluorescent technique, we observed co-localization of caveolin- I and 313-hydroxysteroid dehydrogenase. Western blot analysis revealed a band of about 22 kDa molecular weight that was recognized with both caveolin-1 and tyrosine-phosphocaveolin-1 antibodies. Caveolin-l is one of a few proteins with ademonstrated ability to bind cholesterol in vivo. In this context, the presence of caveolin- in Leydig cells may be related to cholesterol traffic--a rate limiting step in steroid biosynthesis.

Expression of caveolin-1 in rat Leydig cells

Caveolin-1, the first member of caveolin family reported, is recognized as the structural component of caveola, a plasma membrane invagination or vesicles that are a subcompartment distinct from clathrin-coated pits. This protein is also known to be involved in cholesterol trafficking. The aim of this study was to determine the expression of caveolin-1 in adult rat Leydig cells. Testis sections incubated with an antibody to caveolin-1 showed, by immunohistochemistry, a moderate number of Leydig cells with different degrees of immunoreaction and a strong reaction in endothelial cells and in the lamina propia of seminiferous tubules.Caveolin- 1 was detected in the cell cytoplasm with a granular pattern and on the cell surface of Leydig cells cultured 24 h on uncoated, laminin-1 or type IV collagen coated coverslips. We also observed a milder reaction in 3 h cultures. Immunoreaction was also detected in Leydig cells with an antibody to tyrosine-phosphorylated caveolin-1. By double immunofluorescent technique, we observed co-localization of caveolin- I and 313-hydroxysteroid dehydrogenase. Western blot analysis revealed a band of about 22 kDa molecular weight that was recognized with both caveolin-1 and tyrosine-phosphocaveolin-1 antibodies. Caveolin-l is one of a few proteins with ademonstrated ability to bind cholesterol in vivo. In this context, the presence of caveolin- in Leydig cells may be related to cholesterol traffic--a rate limiting step in steroid biosynthesis.(AU)

Immunodetection of cell adhesion molecules in rat Sertoli cell cultures.

PROBLEM: The presence of cell adhesion molecules (CAMs) in Sertoli cells has not been explored extensively. The expression of CAMs involved in cell-matrix and cell-to-cell interactions in Sertoli cell cultures was examined. METHOD OF STUDY: Immunohistochemical and Western blot techniques were applied to rat Sertoli cell cultures using specific antibodies to alpha 3, alpha 5, and alpha 6 integrin subunits; NCAM; and cadherins. RESULTS: Expression of alpha 3 and alpha 6 integrin subunits (mainly laminin receptors) and lack of expression of alpha 5 integrin subunit (fibronectin receptor) was observed in Sertoli cells by immunohistochemistry. These cells also expressed neural CAM (NCAM) and N-cadherin. By Western blot analysis, Sertoli cell extracts reacted with antibodies to alpha 3 integrin subunit revealed a band approximately 130 kDa, whereas no expression of alpha 5 integrin subunit was detected. Cell extracts incubated with antibodies to pan cadherin exhibited a band approximately 120 kDa, whereas bands of 180, 140, and 120 kDa were observed with antibodies to NCAM. CONCLUSION: New data about the expression of receptors for extracellular matrix proteins (alpha 3 and alpha 6 integrin subunits) as well as cell-to-cell adhesion molecules (NCAM and cadherins) are reported in rat Sertoli cell cultures.

Immunodetection of cell adhesion molecules and extracellular matrix proteins in rat Leydig cell cultures.

Cell adhesion molecules (CAMs) as well as extracellular matrix (ECM) proteins were identified in Leydig cell cultures using immunohistochemical and Western blot analysis. Leydig cells were isolated from 60-day-old rats and cultured for 4 days. For immunofluorescence and immunoperoxidase techniques, Leydig cells were incubated with antisera to ECM proteins (antibodies to laminin, type IV collagen and fibronectin); antisera to integrins (antibodies to beta 1, alpha 3, alpha 5 and alpha 6 integrin subunits) and antisera to cell-to-cell adhesion molecules (antibodies to N-CAM and N-cadherin). Results of the two immunohistochemical techniques were similar. Laminin and type IV collagen were detected in the perinuclear area of Leydig cell cytoplasm and cell processes as bright granular immunofluorescence or as a brown reaction product using the immunoperoxidase technique. Leydig cells expressed alpha 3 and alpha 6 integrin subunits (mainly laminin receptors), while no reaction was detected with antibodies to the alpha 5 integrin subunit (fibronectin receptor). Leydig cells also expressed cell-to-cell adhesion molecules such as N-CAM and N-cadherin. Using Western blot analysis, Leydig cell extracts incubated with antibodies to laminin revealed two bands of around 200 kDa, which is characteristic of laminin 1 light chains. A band with electrophoretic mobility similar to that of the alpha 2 (IV) collagen chain from EHS sarcoma and a band of around 230 kDa similar to fibronectin were also detected in Leydig cell extracts using specific antisera. Leydig cells incubated with antibodies to the alpha 3 integrin subunit revealed two bands below 120 kDa. Finally, Western blot results showed that Leydig cells expressed N-CAM as two faint bands of around 140 kDa and N-cadherin as a 120 kDa band. The present data suggest that Leydig cells in culture are able to synthesize ECM proteins and express ECM receptors (integrins), as well as cell-to-cell adhesion molecules such as N-CAM and N-cadherin.

[Anticytoplasm antibodies of polymorphonuclear neutrophils in Wegener's granulomatosis, other autoimmune diseases, and in patients undergoing hemodialysis]. / Anticuerpos anti-citoplasma de polimorfonuclear neutrófilo en granulomatosis de Wegener, en otras enfermedades autoinmunes y en pacientes hemodializados.

Systemic vasculitis are an heterogeneous group of diseases characterized by inflammatory infiltration and necrosis of blood vessel walls. Antineutrophil cytoplasmic antibodies (ANCA) with different immunofluorescent patterns (C or P) have been described as serological markers of some of these diseases and some types of glomerulonephritis. The presence of ANCA by immunofluorescence on normal fixed polymorphonuclear neutrophils was investigated in 182 patients. Results are depicted in Table 1. ANCA was present in 16/17 (94%) patients with Wegener Granulomatosis (W.G.) (ACR criteria) (p < 0.001). In 14 out of the 16 (82%), the pattern was ANCA-C (associated in 10 with ANCA-P) and only ANCA-P was observed in the remaining two. The presence of ANCA was associated with active disease: 15/16 samples of active patients and 3/9 of inactive patients were ANCA positive (p < 0.01). Among the other groups, ANCA-C was detected in only one patient with isolated subglottal stenosis. The specificity of ANCA-C for W.G. was 99%. ANCA-P was also detected in 3/49 (6%) patients with connective tissue disorders and in 3/63 (5%) patients in chronic hemodialysis with exclusive or predominant renal disease of unknown etiology. Three additional ANCA positive patients with known diagnosis (2 W.G. and 1 Systemic Lupus Erythematosus) were also in hemodialysis in the same unit. Thus, an ANCA related mechanism may be involved in the pathogenesis of approximately 10% of cases undergoing this procedure. None of 45 sera submitted for the detection of antinuclear antibodies were ANCA positive. Detection of ANCA (especially C pattern) may be of help in the diagnosis of W.G. and in monitoring clinical activity of the disease.

Anticuerpos anti-citoplasma de polimorfonuclear neutrófilo en granulomatosis de Wegener, en otras enfermedades autoinmunes y en pacientes hemodializados. / [Anticytoplasm antibodies of polymorphonuclear neutrophils in Wegeners granulomatosis, other autoimmune diseases, and in patients undergoing hemodialysis]

Systemic vasculitis are an heterogeneous group of diseases characterized by inflammatory infiltration and necrosis of blood vessel walls. Antineutrophil cytoplasmic antibodies (ANCA) with different immunofluorescent patterns (C or P) have been described as serological markers of some of these diseases and some types of glomerulonephritis. The presence of ANCA by immunofluorescence on normal fixed polymorphonuclear neutrophils was investigated in 182 patients. Results are depicted in Table 1. ANCA was present in 16/17 (94

) patients with Wegener Granulomatosis (W.G.) (ACR criteria) (p < 0.001). In 14 out of the 16 (82

), the pattern was ANCA-C (associated in 10 with ANCA-P) and only ANCA-P was observed in the remaining two. The presence of ANCA was associated with active disease: 15/16 samples of active patients and 3/9 of inactive patients were ANCA positive (p < 0.01). Among the other groups, ANCA-C was detected in only one patient with isolated subglottal stenosis. The specificity of ANCA-C for W.G. was 99

. ANCA-P was also detected in 3/49 (6

) patients with connective tissue disorders and in 3/63 (5

) patients in chronic hemodialysis with exclusive or predominant renal disease of unknown etiology. Three additional ANCA positive patients with known diagnosis (2 W.G. and 1 Systemic Lupus Erythematosus) were also in hemodialysis in the same unit. Thus, an ANCA related mechanism may be involved in the pathogenesis of approximately 10

of cases undergoing this procedure. None of 45 sera submitted for the detection of antinuclear antibodies were ANCA positive. Detection of ANCA (especially C pattern) may be of help in the diagnosis of W.G. and in monitoring clinical activity of the disease.

Cryostat sections with tissue amastigotes of Trypanosoma cruzi as a substrate for Chagas' disease serology.

Sera from 30 chronic chagasic patients together with 52 control samples (34 with other pathological conditions and 18 from normal individuals) were titrated by the indirect immunofluorescent technique (IFA) on Trypanosoma cruzi amastigotes. Acetone-fixed cryostat sections of skeletal muscle of Rockland mice 10 days post-infection with the RA isolate of T. cruzi were used as substrate. Results were compared with titres obtained by conventional IFA on epimastigotes. All 52 control sera had amastigote titres less than or equal to 2 double dilutions (dd) as compared with epimastigote values. Out of the 30 chagasic samples, differences were greater than or equal to 4 dd (less than or equal to 1 log) for 22, 3 dd for 5 and less than or equal to 2 dd for the remaining 3, when comparing amastigote and epimastigote titres. These results show that the use of amastigotes in cryostat sections of infected tissue for performing Chagas' serology in a simple, adequate and sensitive method.

Experimental myocarditis induced in Swiss mice by homologous heart immunization resembles chronic experimental Chagas' heart disease.

The Swiss mouse is considered a satisfactory model for experimental chronic chagasic myocarditis and there is some evidence of an immunopathologic mechanism in the development of this disease. To further support this conjecture, 45-day-old albino Swiss mice (40 animals) were immunized with homologous heart in complete Freund's adjuvant. As controls, 20 animals were likewise inoculated with allogeneic testis, as "non-related" antigen. Three mice from the former group died suddenly at 19-21 days postinoculation while the survivors were sacrificed at 60 days for serum samples, and histologic analysis of the heart and skeletal muscle. Electrocardiographic records were taken at Days 0, 30, and 60 postinoculation. Of myocardium-inoculated animals and testis-inoculated mice 33/37 (89%) and 1/20 (5%), respectively, exhibited myocarditis (P less than 0.001). Histologic lesions were highly reminiscent of those observed in chronic experimental Chagas' disease of Swiss mice. Antimuscle antibodies were seen, by indirect immunofluorescence employing cryostat sections, in 30/33 (91%) of the former group and in 3/20 (15%) of the latter (P less than 0.001), some of which recognized a surface antigen of primary cultured fetal rat myocardiocytes. Mice inoculated with myocardium also exhibited electrocardiographic abnormalities consisting in QRS interval widening. Results show that following an autoimmune experimental design the main features of chronic chagasic myocarditis may be reproduced in the Swiss mouse. This agrees with the likely role of an immunopathologic mechanism in heart damage due to Trypanosoma cruzi infection.

Infeccion experimental de un primate sudamericano, el Cebus sp, con la cepa XJ de virus Junin. / Experimental infection of a South American primate the Cebus sp, with XJ strain of Junin virus

La inoculacion por via im del Cebus sp con la cepa XJ de virus Junin ha demostrado que este primate sudamericano es sensible a la infeccion. Los animales infectados presentaron poliadenopatias generalizadas, disminucion de peso, viremia, virus en fauces y leuco-plaquetopenia. Todos desarrollaron una rapida respuesta inmune humoral y se recuperaron a partir de la 2a.semana pi. Sin embargo, uno de cuatro animales evidencio un cuadro neurologico tardio en presencia de anticuerpos circulantes. En su cerebro se pudieron observar depositos de gamma globulinas y complemento en estructuras capilares por inmunofluorescencia, asi como leves infiltrados linfocitarios meningeos focales. Este trabajo sugiere que el Cebus sp podria ser un modelo experimental util para dilucidar mecanismos inmunopatogenicos a nivel del sistema nervioso central inducidos por el virus Junin