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Biomedical subjects

L Lustig

Publications and source records attributed to L Lustig.

At least 19 recordsLinked to original sources
PubMed2006

Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis.

BACKGROUND: Studies on experimental autoimmune orchitis (EAO) have helped to elucidate immunological mechanisms involved in testicular damage. We previously demonstrated that EAO is characterized by lymphomononuclear cell infiltrates and apoptosis of spermatocytes and spermatids expressing Fas and TNFR1. The aim of this work was to characterize the pathways involved in germ cell apoptosis in EAO and to determine the involvement of the Bcl-2 protein family in this process. METHODS AND RESULTS: EAO was induced in rats by immunization with testicular homogenate (TH) and adjuvants, whereas control (C) rats were injected with saline solution and adjuvants. Testis of EAO rats showed procaspase 8 cleavage products (western blot) with high caspase 8 activity. Cytochrome c content increased in the cytosol and decreased in the mitochondrial fraction of testis from EAO rats compared with C, concomitant with increased caspase 9 activity. Bax was mainly expressed in spermatocytes and spermatids and Bcl-2 in basal germ cells (immunohistochemistry). Baxbeta isoform content increased in EAO rat testis compared with C, whereas content of Baxalpha remained unchanged (western blot). However, Baxalpha content decreased in the cytosol and increased in the mitochondrial and endoplasmic reticulum (ER)-enriched fractions of testis from EAO rats compared with C (western blot). Bcl-2 content also increased in the testes of EAO rats. CONCLUSIONS: Our results demonstrated that extrinsic, mitochondrial and possibly ER pathways are inducers of germ cell apoptosis in EAO and that Bax and Bcl-2 proteins modulate this process.

Animals↗
PubMed2005

Involvement of CD44 in leukocyte recruitment to the rat testis in experimental autoimmune orchitis.

Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of the seminiferous tubules with germ cells that undergo apoptosis and sloughing. The aim of this study was to determine the role of CD44 in testicular leukocyte recruitment in EAO. The biological functions of CD44 have been attributed to the generation of a functionally active hyaluronan-binding phenotype. Orchitis was induced in Sprague-Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund's adjuvant using Bordetella pertussis as co-adjuvant. Control rats (C) injected with saline and adjuvants and normal (N) untreated rats were also studied. CD44 expression was analyzed by flow cytometry in peripheral blood mononuclear cells (PBMC) and lymph node cells isolated from rats at different times after the first immunization. We observed an increase in the mean fluorescence intensity of both samples in the C and experimental (E) groups only after the immunization period. A significant decrease in percentage of CD44+PBMC and in mean fluorescence intensity was observed in rats with orchitis compared with the C group. By in vitro hyaluronic acid-binding assay we demonstrated that the percentage of PBMC adhesion was higher in the E group compared with the C and N groups. By immunohistochemistry, we observed a significant increase in the number of CD44+cells in the testicular interstitium of rats with severe orchitis compared with the N and C groups. These results suggested that the CD44 molecule is involved in the homing of lymphomonocytes into the testes of rats with autoimmune orchitis.

Acute Disease↗
PubMed2001

Correlation between inhibin secretion and damage of seminiferous tubules in a model of experimental autoimmune orchitis.

The aim of the present study was to evaluate inhibin secretion in rats with autoimmune orchitis. As we have previously described, experimental autoimmune orchitis (EAO) induced in rats by active immunization with testis homogenate and adjuvants is characterized by an interstitial mononuclear cell infiltrate and sloughing of the germinal epithelium. At 120 days after the first immunization 60% of the rats exhibited a severe orchitis with large areas of aspermatogenic seminiferous tubules in which only spermatogonia and Sertoli cells with cytoplasmic vacuolization remained attached to the tubular wall. None of the untreated (N) or control (C) rats revealed pathological alterations. Sixty percent decrease in testis weight was observed in rats with EAO compared with N or C groups. A 3-fold increase in serum FSH levels was observed in rats with EAO compared with N or C groups (19.8+/-3.7 vs 5.6+/-0.3 and 5.9+/-0.1 ng/ml respectively). A significant decrease in inhibin B levels was observed in rats with EAO when compared with N or C groups (40+/-4.6 vs 207+/-38.8 and 221.4+/-28.6 pg/ml respectively). An inverse correlation between inhibin B and FSH serum levels and a direct correlation between inhibin B and testis weight were found. Strong expression of the inhibin alpha-subunit in Sertoli cells of untreated and control rats was observed; this subunit was undetectable or poorly detectable in rats with orchitis. Positive staining for the inhibin alpha-subunit was also observed in Leydig cells of all groups studied. In conclusion, using a model of autoimmune orchitis our results show that circulating inhibin B levels and inhibin alpha-subunit expression in Sertoli cell cytoplasm closely correlate with the degree of damage of the germinal epithelium.

Analysis of Variance↗
PubMed2000

Endometrial dating and determination of the window of implantation in healthy fertile women.

OBJECTIVE: To reassess endometrial morphological criteria of normality identifying the best morphological and molecular "implantation window" indicators in normal women. DESIGN: Prospective clinical study. SETTING: Assisted reproductive unit. PATIENT(S): Fourteen healthy volunteers. INTERVENTION(S): Blood sampling for LH, E(2), and progesterone (P4) determinations. Daily vaginal ultrasounds. Two endometrial biopsies per volunteer, 7 days apart, during luteal phase. MAIN OUTCOME MEASURE(S): Endometrial dating, pinopodes formation, immunohistochemical determination of integrins (alphavbeta3, alpha4beta1), leukemia inhibitory factor (LIF), interleukin-1 receptor type I (IL-1R tI), mouse ascites Golgi (MAG), the transmembrane mucin (MUC-1), and P4 receptor expression. RESULT(S): In 26 of 28 biopsies observers agreed; in two biopsies there was a discrepancy (difference of 72 hours). With use of LH peak, 24 of 26 samples were in phase, and 2 were 3 days behind. Pinopodes appeared on days 20-21 and persisted through day 28 in small groups or larger areas. beta3 Integrin was highly expressed in luminal and glandular epithelium from day 22 through 28; 48 hours thereafter pinopodes appeared. alpha4 Subunit exhibited luminal epithelium reaction positivity on days 22-23 and glands on days 18-23. LIF and IL-1R tI showed weak, erratic expression. MAG antibodies showed luminal epithelium expression up to day 22 and glands up to day 25. MUC-1 showed positivity during the whole luteal phase. P4 receptors were positive through day 20 and at the end of the luteal phase. CONCLUSION(S): The three most cited markers that frame the window of implantation do not correlate in our material. Pinopodes are present from day 20 on; beta3 and alpha4 integrin subunits indicate a window opening on days 22-23.

Adult↗
PubMed2000

Autoantibodies to cryptic epitopes elicited by infection with lactate dehydrogenase-elevating virus.

Lactate dehydrogenase-elevating virus (LDV) produces a permanent infection in mice with a B-lymphocyte polyclonal activation leading to hypergammaglobulinaemia. Since LDV specifically suppressed antibodies to native epitopes in CBA/Ht, but not BALB/c, mice immunized against a protein antigen, we explored the relationship between such a change in antibody specificity and the expression of autoantibodies under the influence of LDV. Again in CBA/Ht, but not BALB/c, mice we observed another effect of LDV: the sera from infected CBA/Ht mice were found by enzyme-linked immunosorbant assay to contain antibodies to various mouse tissue extracts. Immunoblots revealed a large spectrum of autoantigens that differed markedly between animals. Western-blot competition experiments showed that the protein autoantigens had to be denatured to react with most of the autoantibodies. Despite the presence of these autoantibodies directed to cryptic epitopes, no specific tissue lesions could be ascribed to the autoimmune response elicited by LDV infection, since both mouse strains showed mild inflammatory reactions in liver and kidney.

Animals↗
PubMed2000

Passive immunization with anti-laminin immunoglobulin G modifies the integrity of the seminiferous epithelium and induces arrest of spermatogenesis in the guinea pig.

In the testis, the base of the Sertoli cells is in contact with the basement membrane matrix, in which the laminins constitute the major noncollagenous components. We have previously demonstrated that antibodies against a preparation enriched in basement membranes of seminiferous tubules (STBM) or a noncollagenous fraction of STBM passively transferred induced modifications to the basement membranes and focal sloughing of the seminiferous epithelium in the rat. In the present report, we tested the effect of passive immunization with anti-laminin IgG on the limiting membrane of the seminiferous tubules, spermatogenesis, and maintenance of the blood-testis barrier in the adult guinea pig. Rabbit antibodies to laminin 1 (IgG fraction) were injected in adult male guinea pigs (GP). Nonimmunized GP and GP immunized with normal rabbit serum IgG were used as controls. Measurements of variations in the diameter and lumen of the tubules and in the size of individual components of the tubular limiting membrane showed that the highest percentage of tubules with reduced lumen occurred 30 days after passive immunization with anti-laminin, when the limiting membrane was thickest and lesions to the seminiferous epithelium were most severe. The lesions included thickening of the limiting membrane, infolding in the basal lamina, deposits of immune complexes coincident with sloughing of pachytene spermatocytes and spermatids, and vacuolization of the Sertoli cells. Mononuclear cell infiltration of the tubules was rare. Permeability tracer studies revealed that Sertoli cell tight junctions remained impermeable. Fifty and 80 days after treatment, the basement membrane of the tubules and the progression of the spermatogenesis were normal. Passive immunization with anti-laminin IgG provided a valuable experimental model for the in vivo study of the influence of the basement membrane on the issue of spermatogenesis and the integrity of the seminiferous epithelium.

Animals↗
PubMed2000

Effect of testicular macrophage conditioned media from rats with autoimmune orchitis on Leydig cell function.

PROBLEM: The aim of this study was to investigate the influence of immune-activated testicular macrophages obtained from rats with autoimmune orchitis (EAO) on Leydig cell steroidogenesis. METHOD OF STUDY: EAO was induced in rats by active immunization with testis homogenate and adjuvants. Testicular and peritoneal macrophages from rats with EAO were isolated and cultured for 24 hr. Testosterone (T) production by purified Leydig cells incubated in vitro with macrophage-conditioned media (CM) from rats with EAO or control rats was measured. RESULTS: An increase in T production by Leydig cells incubated with CM from testicular, but not peritoneal, macrophages of rats with EAO was observed. This increase was dose-dependent up to a concentration of 30% CM; proportions higher than 35% exhibited an inhibitory effect. CONCLUSIONS: Immune-activated testicular macrophages obtained from rats with EAO induced both stimulatory and inhibiting steroidogenic effects on Leydig cells in vitro and not the exclusively inhibitory action that has widely been attributed to activated macrophages. This dual effect probably depends on the ability of these cells to synthesize different molecules that may exert opposite effects.

Animals↗
PubMed1999

Serotonin in golden hamster testes: testicular levels, immunolocalization and role during sexual development and photoperiodic regression-recrudescence transition.

Serotonin (5-HT) is found in the gonads and accessory reproductive organs of several species. The golden (Syrian) hamster is a seasonal breeder. Exposure of male adult hamsters to short days for 14 weeks results in a severe gonadal regression, while after a photoinhibition period of 22 weeks a spontaneous testicular recrudescence occurs. The aim of this study was to investigate the presence of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the gonads of golden hamsters, its immunolocation and its physiological role in the testis. The influence of age and photoperiod was also analyzed. Hamsters of 23, 36, 46, 60 and 90 days of age were kept in long photoperiod (LP: 14:10 h light/dark), and adult animals were exposed either to LP or to short photoperiod (SP: 6:18 h light/dark) for 14 and 22 weeks. Testicular parenchyma and capsule levels of 5-HT and 5-HIAA increased significantly at ages of 36 and 60-90 days, but decreased markedly during the exposure of adult hamsters to SP for 14 and 22 weeks. Mast cells were found exclusively in the testicular capsule. The testicular number of mast cells increased concomitantly with age, but decreased in adult hamsters exposed to SP. Mast and Leydig cells presented 5-HT-positive immunoreactivity. During sexual maturation as well as during the transfer of adult hamsters from LP to SP, the 5-HIAA/5-HT ratio showed the highest values in active adult animals, indicating that the increase in testicular 5-HT levels in adulthood is accompanied by an augment in 5-HT turnover. In vitro basal and hCG-stimulated testosterone production was significantly inhibited in presence of physiological concentrations of 5-HT. In conclusion, the present studies demonstrate the existence of 5-HT in mast cells and Leydig cells of hamster testes, as well as describe an inhibitory action of this neurotransmitter on gonadal testosterone production. Furthermore, the age-dependent and photoperiodic-related changes detected in testicular 5-HT levels suggest that this neurotransmitter might act as an important local modulator of the action of gonadotropins on steroidogenesis during sexual development and during the photoperiodic regression-recrudescence transition in the golden hamster.

Aging↗
PubMed1998

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.

Animals↗
PubMed1997

Increased testosterone production in vitro by Leydig cells from rats with severe autoimmune orchitis.

We have previously observed (M. O. Suescun et al., 1994, Journal of Andrology, 15, 442-448) that rats with autoimmune orchitis (EAO) exhibit increased testosterone production in vitro by isolated testes. The aim of the present study was to determine whether the increase in testosterone production correlated with an enhanced number of Leydig cells and/or enhanced steroidogenic capacity per Leydig cell. For this purpose, EAO was induced in adult Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. At 80 days after the primary immunization, 60% of rats presented with severe testicular damage characterized by sloughing of the seminiferous epithelium, seminiferous tubule atrophy and interstitial mononuclear cell infiltration. At 160 days after the first immunization, testicular lesions were more severe. A morphometric study, by light microscopy, showed an increase in the number of Leydig cells in rats with EAO (45% increase at 80 days and 50% at 160 days). By electronmicroscopy, testicular sections of rats with EAO revealed the presence of numerous Leydig cells closely associated with macrophages. Most Leydig cells exhibited ultrastructural features of active steroid secreting cells. The steroidogenic capacity of Percoll-purified Leydig cells from tests of rats with EAO, killed at 80 and 160 days, was evaluated. Leydig cells from rats with EAO exhibited an enhanced steroidogenic response to hCG in vitro at 80 days (38%) and an increase in basal (77%) and post-hCG testosterone production (115%) at 160 days compared to controls. However, these cells were less sensitive to hCG. In conclusion, the results indicate that the enhancement of in-vitro testosterone production observed in rats with EAO is accounted for both by the increased number of Leydig cells and by the increased testosterone production of each Leydig cell.

Animals↗
PubMed1996

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.

Animals↗
PubMed1994

Alterations in cardiac muscarinic acetylcholine receptors in mice with autoimmune myocarditis and association with circulating muscarinic receptor-related autoantibodies.

Hearts from mice hyperimmunized with cardiac tissue were studied to evaluate the expression and biological activity of muscarinic cholinergic receptors and immunoglobulin G deposits along the immunization period. Mice were sacrificed at 10 day intervals from the first injection up to day 100. Simultaneously, the activity of autoantibodies against muscarinic receptors on normal hearts was also examined in sera. Hearts with autoimmune myocarditis showed a muscarinic receptor-related dysfunction, with an impaired response to exogenous muscarinic agonists and a significant reduction in muscarinic binding sites, both effects being maximum at 40-50 days post-immunization. In addition, serum or immunoglobulin G from mice with myocarditis were able to interact with muscarinic acetylcholine receptors displaying a partial agonist effect. Autoimmune sera and immunoglobulin G reduced heart contractility while inhibited 3H-QNB binding to cardiac acetylcholine receptors in a concentration dependent manner showing the highest effects at days 40-50 and decreased progressively thereafter. The development of muscarinic receptor-related cardiac dysfunction may be associated with the presence of circulating antibodies having muscarinic receptor activity. These studies are of relevance to clinical conditions such as Chagas' disease, where immunological processes involving the cholinergic system are considered to cause cardiomyopathy.

Animals↗
PubMed1994

Participation of cytoskeleton in the effect of antilaminin IgG on cardiac cholinoceptors.

1. We have previously demonstrated a molecular relationship between laminin and cardiac cholinoceptors. 2. We have now explored the participation of cytoskeletal proteins in the interaction between an antilaminin IgG with cardiac cholinoceptors. 3. Antilaminin IgG, whilst it specifically reacts with laminin molecules was able to induce cardiac cholinoceptor activation; acting like an agonist, decreasing cyclic AMP concentrations, reducing heart contractility and increasing phosphoinositide turnover. 4. Antilaminin IgG also interfered with the binding of a radiolabelled muscarinic antagonist, [3H]-quinuclidinyl benzilate. Colchicine and cytochalasin B, drugs that are able to prevent microfilament and microtubule polimerization, impaired the binding of antilaminin IgG to muscarinic cholinoceptors. 5. Cytochalasin B but not colchicine modified the muscarinic cholinoceptor effects mediated by regulatory G proteins (cyclic AMP and contractility) induced by antilaminin IgG. 6. It was demonstrated, by immunofluorescence, that none of these disrupting drugs altered the specific recognition of the antibody by its antigen. 7. These data indirectly suggest the participation of the cytoskeleton in the laminin and cholinergic receptor association.

Animals↗
PubMed1993

Phenotypic characterization of lymphocytic cell infiltrates into the testes of rats undergoing autoimmune orchitis.

Experimental autoimmune orchitis (EAO) was induced in adult Wistar rats by active immunization with a testicular homogenate (TH) and adjuvants. Fifty per cent of the immunized rats developed EAO. Testicular damage became evident at 50 days after the primary immunization and increased in severity at 80 days. Phenotypic characterization of T-cell subsets (CD4+ and CD8+) and Ia+ cells was performed on cryostat sections of testis obtained from normal rats, rats immunized with adjuvant (control group) and rats immunized with TH and adjuvants (experimental group) at 50 and 80 days. Labelled cells were only detected in the interstitial area; no labelled cells were observed inside the seminiferous tubules with any of the monoclonal antibodies used (W3/25, OX-8, OX-6). A significant increase in the numbers of CD4+ and CD8+, as well as of Ia+ cells, were observed in the testis of rats with severe EAO at 80 days after the first immunization. Rats of the same experimental group without testicular damage showed no major differences compared to rats from the control group, with the exception of a lower number of CD8+ cells. Variations in the lymphocyte subsets in lymph nodes draining the site of immunization showed the opposite pattern to that observed in the testis. In conclusion, these data suggest the traffic of specifically sensitized lymphocytes from lymph nodes to the testis and an active role of CD4+, CD8+ and Ia+ cells in the pathogenesis of EAO in the rat.

Animals↗
PubMed1993

[Integrins: a family of cell adhesion receptors].

Integrins are a family of cell adhesion molecules (CAM's) that mediate the communication between the intracellular and the extracellular compartments. The growing interest in CAM's is due to the essential role they play in cell-cell and cell-matrix recognition processes. These receptors are formed by a non-covalently associated glycoprotein complex of two distinct polypeptide chains, called, alpha and beta. The association of different subunits results in the formation of, at least, 16 different integrins that provide cells with a great versatility in their adhesion properties. An integrin molecule comprises a cytoplasmic domain that interacts with the cytoskeleton, a transmembranous domain and an extracellular domain that binds to one or more ligands. beta 1, beta 2 and beta 3, are the best characterized integrin subfamilies; they are expressed, in different amounts, in epithelial and endothelial cells, leukocytes, fibroblasts and platelets. b1 integrins are essentially involved in cell-extracellular matrix interactions and beta 2 subfamily in leukocyte-leukocyte and leukocyte-endothelial cell communications. The integrin subfamily beta 3 mediates the adhesion of platelets with fibrinogen and other ligands. During embryonic development, integrins in association with other CAM's, play an essential role in cell migration and morphogenesis. Moreover, in processes like inflammation, wound healing and thrombosis, integrins and other CAM's mediate the interactions among the injured tissue and circulating cells. In two genetic diseases like the leukocyte adhesion deficiency and the Glanzmann's thrombasthenia an impairment in leukocyte-endothelial cell interactions and platelet aggregation is detected, due to deficiencies or abnormalities in beta 2 or beta 3 integrin subfamilies, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1992

The California Maternal Serum alpha-Fetoprotein Screening Program: the role of ultrasonography in the detection of spina bifida.

Between January 1988 and June 1990, 161 cases of open spina bifida were identified by the California Maternal Serum alpha-Fetoprotein Screening Program. Eight percent of these cases were not diagnosed by an initial ultrasonographic evaluation. Three defects were not recognized until birth. Ultrasonography is inadequate to identify all cases of open spina bifida.

California↗