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Biomedical subjects

P Sanchez

Publications and source records attributed to P Sanchez.

At least 109 records · Page 6Linked to original sources
PubMed1985

Stimulation of ribonucleic acid and deoxyribonucleic acid synthesis in spermatogenic cells by their coculture with Sertoli cells.

Germ cells were isolated from 30-day-old rat testes and purified by a Percoll gradient. Germ cells were cultured for 24 h to eliminate contaminant cells by utilizing the property of many nongerm cells of attaching to plastic surfaces. Germ cells were subsequently seeded on top of 7-day-old Sertoli cell monolayers. RNA and DNA synthetic activities were estimated by the rate of incorporation of [3H]uridine and [3H] thymidine into trichloroacetic acid-precipitable material. Germ cells increased RNA synthesis from 54,053 +/- 22,824 to 168,019 +/- 57,137 dpm/2 h X 10(6) cells (mean +/- SD) between 4 and 24 h of coculture, respectively (P less than 0.01), while they decreased this activity from 32,150 +/- 6,800 to 6,014 +/- 5,243 dpm when they were cultured alone for the same periods. Coculture of germ cells with either a cell derived from the adult bovine aortic endothelial cells (ABAE), or rat fibroblasts in primary culture prevented the fall in RNA synthesis but did not stimulate it. Addition of high concentrations of lactate to the culture medium did not have any affect in germ cells cultured alone but produced a slight stimulation in germ cells cocultured with ABAE cells, which was much smaller than the effect observed during coculture with Sertoli cells. Germ cells in coculture with Sertoli cells also increased their DNA synthesis from 2,186 +/- 765 at 4 h to 9,679 +/- 4,057 dpm [3H]thymidine/2 h X 10(6) cells at 24 h (P less than 0.05). When Sertoli cells were treated with FSH (1 microgram/ml), the synthesis of both RNA and DNA of germ cells cocultured with these cells was significantly higher than for germ cells cocultured with nontreated Sertoli cells. The system of coculture of Sertoli cells and germ cells consisted of the reassociation of the two previously isolated cells in a coculture, followed by their separation which permitted the effect of the coculture on the individual cell type to be assessed. By using this technique is was possible to obtain evidence suggesting that a chemical messenger other than lactate might be involved in the stimulatory effect of Sertoli cells on germ cells and that FSH increased this putative chemical messenger.

Animals↗
PubMed1985

Relationship between hCG receptors disappearance and steroid accumulation during long term hCG stimulation in porcine Leydig cell cultures.

LH-hCG receptors and steroidogenesis can be maintained for more than a week in porcine Leydig cell culture (chemically defined medium). Free receptors and steroid accumulation were measured following long time exposure to hCG. As expected, the percent of receptor disappearance increases with the hCG concentration in the incubation medium. However, the steroidogenesis was also proportional to hCG concentration for 24 hours as well as 48 hours. A 50% receptor disappearance was obtained for 0.5 ng hCG/ml which led to a steroid accumulation of 50% of the maximal accumulation obtained with 100 ng/ml. A very significative positive correlation (rho) was observed between receptor occupancy (internalized and/or irreversibly occupied receptors) and steroid accumulation (T and DHAS) during a 24 or 48 hour period (rho = 0.97 and 0.98 for T, and rho = 0.96 and 0.94 for DHAS). Following 24 or 48 hour exposure to increasing hCG concentration, the cells were acutely stimulated by 25 ng hCG/ml for 4 hours. The steroidogenic response obtained to this acute stimulation was inversely proportional to the concentration of hCG during the previous period. A negative correlation was found between receptor occupancy and acute steroidogenic response (rho : -0.96 and -0.98 for T). These results indicate that during long term stimulation as well as during acute restimulation the absolute number of occupied receptors conditioned the steroidogenic response. These results do not fit into the concept of spare receptors and suggest that in these immature cells all receptors are potentially active in the regulation of steroidogenesis.

Animals↗
PubMed1985

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. I. Protective cross-immunity between transplantable tumor lines.

Dimethylbenz[a]anthracene (DMBA)-induced transplantable fibrosarcomas in B2 homozygous chickens (SC line) grow progressively in normal chickens, but are rejected by chickens immunized previously with irradiated tumor cells and Corynebacterium parvum. Tumor-immune chickens resist challenge by the immunizing tumor lines as well as by some, but not all, fibrosarcoma lines. The pattern of cross-reactivity between four DMBA-induced transplantable tumor lines was examined in detail. Ability to reject a tumor challenge correlated very well (p less than 0.001) with the presence of delayed-type hypersensitivity (DTH) to that tumor. Immunization with one of two of the DMBA-induced lines tested also caused rejection of transplantable tumors developed from methylcholanthrene-induced and benzo(a)pyrene-induced primary fibrosarcomas. Although immunization with tumor caused DTH to chicken embryo fibroblasts (CEF), immunization with CEF failed to cause protective immunity or DTH to tumors. Presence of protective immunity, where tested, also correlated with the ability of spleen cells from immune donors to inhibit tumor growth in Winn tests. Humoral immunity exhibited even greater cross-reactivity than did cellular immunity. Distinct patterns of cross-reactivity were nevertheless observed with respect to the serum antibodies as detected in ELISA. Two of these patterns were also observed in several sera from primary tumor-bearing chickens, both including reactivity with CEF. Such reactivity was absent from normal chicken sera.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence.

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

Selective and polyclonal induction of high levels of lambda light chain-bearing immunoglobulins in BALB/c and SJL mice.

Polyclonal B cell activation is V gene independent and consequently results in the unselected expression of both V lambda and V kappa genes. In an attempt to selectively and polyclonally trigger lambda light chain-bearing B cells, we immunized mice with low doses of lipopolysaccharide (LPS) coupled to antibodies specific for the constant region of lambda 1 chains. Analyses of the sera of treated mice revealed that LPS anti-lambda 1 selectively triggers lambda-but not kappa-bearing B cells to high rate Ig secretion. Surprisingly, immunized SJL mice (rlo lambda 1 phenotype) mounted a lambda + response comparable to that detected in mice with the r + lambda 1 phenotype. Because we could definitively establish that the majority of the induced antibodies belong the lambda 1 subtype, our results have strong implications both for the study of the repertoire of lambda 1-bearing B cells and for our understanding of the functional mechanism(s) by which the r lambda 1 locus acts.

Animals↗
PubMed1983

The selection and maintenance of the V region determinant repertoire is germ-line encoded and T cell-independent.

The number of lipopolysaccharide-sensitive precursor cells synthesizing immunoglobulin (Ig) which reacts with the monoclonal anti-M460 antibody F6(51) has been determined in the spleen and in the bone marrow of different strains of mice. These precursor frequencies fall into two quantitatively different groups. The first group includes mice with the same Igh haplotype as BALB/c animals (Igha). In this group, spleen cells contained between 1:10(4) to 1:5 X 10(4) B cell precursors secreting Ig which bound F6(51). The second level of precursor was obtained with animals with allotypic haplotypes other than Igha. These values were too low to allow accurate frequency determinations. The frequency of these cells in mice of the latter group, however, increased dramatically when these animals were hyperimmunized with the monoclonal anti-M460 antibody. Similar results were obtained when the frequencies were determined using the Ig- fraction of bone marrow cells. Surprisingly, the numbers of lipopolysaccharide-sensitive B cell precursors secreting F6(51)-binding Ig in spleen cells of nude mice was found to be similar to the one in splenocytes of normal mice, and even in this case, the frequencies reflected the genetic background of the animals tested. Taken together these data support the notion that the establishment and the maintenance of the M460 idiotypic repertoire is germ-line encoded and independent of regulatory T cells.

Animals↗
PubMed1983

Allotypic restriction of the expression of MOPC460 idiotope after immunization with either anti-2,4-dinitrophenyl (DNP) or anti-idiotypic antibodies.

The MOPC460 idiotype is expressed in mice with the IghCa allotypic haplotype after anti-2,4-dinitrophenyl (DNP) immunization. We have previously shown that two monoclonal syngeneic anti-idiotypic antibodies (IDM 92-13 and IDM 41-27) define two distinct idiotopes (the 460.92 and the 460.41) on the M460 idiotype. The current study demonstrates that only one idiotope (460.92) is recurrently expressed after antigen immunization in IghCa positive mice and also that, immunization against the monoclonal anti-idiotypic molecules induces the synthesis of 460.92 idiotope positive anti-DNP antibodies. However, the detection of such molecules is only possible when animals with the IghCa allotypic haplotype are immunized with the IDM 92-13 molecules. Immunization of mice with either of the two monoclonal anti-idiotypic antibodies never results in the synthesis 460.41 positive molecules. Therefore, whatever protocol of immunization used, the expression of 460.92 was allotypic restricted.

2,4-Dinitrophenol↗
PubMed1983

Incomplete expression of the MOPC 460 idiotype in the sera of BALB/c mice immunized either with DNP antigen or with anti-idiotypic antibodies.

The MOPC 460 idiotype, as defined by polyclonal probes, has been described as a recurrent marker among the anti-DNP antibodies synthetized by IghCa mice. In this paper, we demonstrate, using syngeneic monoclonal anti-idiotypic probes, that only a part of the idiotopes of this idiotype are indeed recurrently expressed in BALB/c mice (IghCa) after immunization with DNP antigen. We will also show that the immunization of BALB/c mice with monoclonal anti-idiotypic antibody specific for the recurrent determinant results firstly in the synthesis of anti-DNP antibodies and secondly in the expression of the same recurrent M460 idiotope present on a part of induced anti-DNP molecules. Contrary to this, the immunization with the monoclonal anti-idiotypic antibody specific for the private idiotope never resulted in the synthesis of anti-DNP antibodies. These results clearly suggest that, after DNP or anti-idiotypic immunization, the M460 idiotype is not expressed in its entirety.

Animals↗
PubMed1983

Androgen production in primary culture of immature porcine Leydig cells.

The present paper examines the steroidogenic responsiveness of immature porcine Leydig cells in primary culture. Both testosterone (T) and dehydroepiandrosterone sulfate (DHAS) secretion were measured under basal conditions and after stimulation with human chorionic gonadotropin (hCG) (25 ng/ml). In medium supplemented with insulin, transferrin, epidermal growth factor (3H) and 0.1% calf serum, cells survived 3-5 days in culture. The production of steroids (under hCG stimulation) is poor on day 0-1 of the culture. On day 2-4 basal T and DHAS levels are 1.9 and 17.0 ng/10(6) cells/24 h. The addition of hCG stimulated T and DHAS production 19- and 6-fold respectively and the average productions were 37 and 109 ng/10(6) cells/24 h. Increasing the serum to 0.5% did not change the viability of the cultures, but increased hCG stimulated T and DHAS production (183 and 188 ng/10(6) cells/24 h). The addition of alpha-tocopherol (vitamin E) to 0.1% calf serum led to a 4-fold increase in stimulated T production (142 ng/10(6) cells/24 h) and maintained full cell viability for more than 5 days. Measurement of 3 beta-ol steroid dehydrogenase activity indicates that the amount of enzyme is 4 times higher at day 2 than at day 0 and 1 (with or without hCG), suggesting a spontaneous maturation of the cells in culture. This might explain the increased T production with time in culture. In cumulative experiments (24 h) the cells do not seem to be desensitized to hCG stimulation following prolonged exposure to 25 ng hCG since the daily steroid production is increasing with time in culture. However, kinetic studies show that steroidogenesis is not linear over a 24 h period. In cumulative experiments the steroid production stops between 12 and 16 h following hCG exposure (5 and 100 ng/ml) and resumes following a medium change. These results suggest that some inhibitory compounds are accumulated in the medium and are controlling the Leydig cell function. Moreover high doses of hCG (100 ng/ml) result in a lower production of steroids and an earlier plateau in the case of DHAS. These results demonstrate that porcine Leydig cells can live and differentiate in hormone- and vitamin-supplemented medium and that auto-feedback mechanisms inhibiting steroid accumulation take place under in vitro conditions.

3-Hydroxysteroid Dehydrogenases↗
PubMed1982

Idiotype regulation: evidence for the involvement of Igh-C-restricted T cells in the M-460 idiotype suppressive pathway.

Allotypic restriction of idiotype suppression has been shown in several idiotypic systems. It has also been demonstrated that naive BALB/c mice contain naturally occurring suppressor T cells capable of inhibiting the M-460 idiotype expressed on BALB/c B cells, and that T cells from allotype-congenic mice have no effect on this expression. Since we have previously shown that upon polyclonal activation B cells of all mice tested are capable of secreting M-460-positive anti-2,4,6-trinitrophenyl (TNP) antibodies, we postulated that idiotype suppressor cells may recognize idiotypes in conjugation with products of the Igh loci. In the present study we tested this hypothesis. The data clearly showed that regardless of their allotypic haplotype all strains of mice do possess suppressor cells capable of recognizing the M-460 idiotype. However, mixing experiments indicated that the functional potential of these suppressor cells can only be expressed when T and B lymphocytes come from allotype-matched animals. Efficient suppression in those cultures containing allotype-matched but incompatible B and T cells at the I-J region of the H-2 locus could be detected. Finally, using allotypic recombinant strains of mice, we found evidence that the restrictive elements in idiotypic suppression are the product of genes mapping in the Igh-C locus, or closely linked to it.

Animals↗
PubMed1982

Esophageal manometry: a benefit and cost analysis.

Esophageal manometric study has gained tremendous popularity over the past decade. However, the contribution of this diagnostic technology has not been critically evaluated. The purpose of this report is, therefore, to determine how frequently esophageal manometry alters the clinical diagnosis and treatment and to assess the cost of new information. The patients reviewed in this report consisted of 363 consecutive referrals. Each completed a questionnaire, had an esophagogram, and underwent an esophageal manometric study for the evaluation of dysphagia, heartburn, and/or chest pain of unexplained etiology. To determine the clinical contribution of manometry, diagnoses before and after the study were compared. On the basis of symptoms and radiologic data, specific clinical entities were diagnosed in 36 patients. Manometric study did not confirm the diagnosis of achalasia in four of the 27 patients referred with this diagnosis and resulted in 19 additional specific diagnoses. Manometry changed the course of treatment in 14 cases, eight additional patients with achalasia received treatment, and four false-positive patients were spared inappropriate treatment. Moreover, two patients with simultaneous esophageal motor disorder and chest pain were spared further investigation. It is concluded that esophageal manometry altered the clinical diagnosis in 6% and changed the course of treatment in 4% of the population studied. Esophageal manometry is beneficial in patients with chest pain, dysphagia, and those in whom diagnosis of achalasia is suspected, but is of little benefit in patients with chronic heartburn. Assuming the cost per study to be +250, the cost of the study was +3945 per alteration of diagnosis and +6482 per alteration of treatment.

Cost-Benefit Analysis↗
PubMed1982

Specificity and idiotypic analysis of monoclonal antibodies directed against the MOPC460 idiotype.

Eight syngeneic anti-idiotypic hybridomas (IDMs) have been obtained against the BALB/c myeloma protein MOPC460 which displays anti-TNP activity. The study of their anti-idiotype specificity allowed us to distinguish them into two groups which define the presence of at least two idiotypic determinants or idiotopes in the MOPC460 idiotype. The biochemical analysis of the monoclonal antibodies is consistent with this dichotomy. This analysis, in fact, showed a striking correlation between anti-idiotypic specificity and biochemical characteristics of the monoclonal antibodies. Consequently, the idiotypic specificities of three of these hybridomas were studied. In accordance with what is expected, our results clearly indicate a strong idiotypic similarity for hybridomas belonging to the same group and a lack of idiotypic cross-reactivity.

Animals↗
PubMed1981

Recognition of MOPC-460 variable region determinants by polyclonally distributed triggering receptors on B lymphocytes.

Purified M460 protein-induced untreated and anti-Thy 1.2 and complement-treated murine spleen cells to proliferate and differentiate in vitro. Fc receptor complexing was not responsible for the phenomenon, since M460 Fab fragments retained the B cell triggering activity. Activation was inhibitable by the addition of DNP-glycine to cultures and could also be detected by using spleen cells from various strains of mice, including the C3H/HeJ, which is genetically nonresponding to LPS. Hybrid molecules were constructed between the M460 proteins and the products of 2 myelomas that do not activate B cells, namely, X25 and X24. The only hybrid molecules that retained triggering activity were (L460-Hx25)2. Interestingly, the same molecule was also the only one recognized by an anti-M460 monoclonal antibody (F6(51)) that has been previously shown to induce B cells to proliferate and differentiate. The data suggest the existence on B lymphocytes of a network of polyclonally distributed mitogen receptors that share determinants cross-reacting with immunoglobulin idiotypes.

Animals↗
PubMed1977

[Desensitization by human chorionic gonadotropin (hCG): demonstration of a negative control of gonadotropin receptors by hCG at the level of testicular Leydig cells].

A single injection of hCG (500 IU) to intact and hypophysectomized prepubertal rat increases plasma testosterone levels and decreases hCG receptors in the testicular Leydig cells for 4 to 5 days. In intact animals testosterone exhibit a rapid increase within hours and a delayed paradoxical increase between 60 and 96 hours. In hypophysectomized animals the first peak is not present. In both intact and hypophysectomized rats hCG receptors are almost undetectable between 10 and 96 hours following hCG injection. Since receptor occupancy cannot account for this phenomenon it is concluded that hCG is exerting a negative control on its own receptors.

Animals↗