Search PubMed⌕ Search

Biomedical subjects

S Bernard

Publications and source records attributed to S Bernard.

At least 127 records · Page 7Linked to original sources
PubMed1990

Kinetics of the in vitro antibody response to transmissible gastroenteritis (TGE) virus from pig mesenteric lymph node cells, using the ELISASPOT and ELISA tests.

A method is described for in vitro studies of viral humoral immune responses in the pig. After oral immunization with transmissible gastroenteritis (TGE) coronavirus, antibody production from primed mesenteric lymph node cells was revealed by an in vitro boost with viral antigen. For the latter the leukocytes were co-cultured with UV-inactivated virus using a variety of different methods of antigenic stimulation. Enumeration of specific antibody-secreting cells (ASC) and titration of secreted anti-virus antibodies were performed with ELISASPOT (using 3-amino 9-ethyl carbazole as the peroxidase chromogen) and ELISA tests respectively, according to the Ig isotype. The results showed a close relationship between ASC numbers and secreted antibody titres. The best in vitro antibody synthesis was observed when the sensitized cells were maintained in contact with virus during the whole culture period. Antibody responses were defined by a kinetic profile characterized by a narrow peak, with a maximum occurring after 4 and 6 days of culture and with the IgA response appearing earlier than the IgG. This methodology, which analyses specific antibody responses at the cellular level, may permit studies on the mechanisms of Ig isotype regulation. Extended to leukocytes from other organs of the immune system, it may also constitute an in vitro model to study antibody responses expressed in different lymphoid tissues of the pig.

Animals↗
PubMed1990

Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1990

Lactogenic immunity to transmissible gastroenteritis (TGE) of swine induced by the attenuated Nouzilly strain of TGE virus: passive protection of piglets and detection of serum and milk antibody classes by ELISA.

Piglets of eight sows vaccinated by different routes with the attenuated TGE mutant coronavirus, Nouzilly (N) strain, and piglets from two field seropositive sows were challenged with a virulent TGE strain. On the day of challenge and 10 days after challenge, milk and serum samples from sows were analysed for their level of neutralizing antibodies, total immunoglobulin classes and TGE antibody classes by an ELISA. No direct relationship was seen between the level of protection of the litters and the titres of the different antibody classes on the day of challenge. However, an inverse correlation was seen 10 days after challenge between protection and the level of TGE antibodies.

Animals↗
PubMed1990

Contribution of molecular biology to the study of the porcine interferon system.

We have performed molecular studies on the pig interferon (IFN) system (i) to analyse the role played by endogenous IFN in neonatal viral enteritis such as transmissible gastroenteritis and possibly to obtain, via recombinant DNA technology, a new anti-infectious and immunomodulatory agent in this species, (ii) to characterize the structure and biological functions of the IFN-like antiviral activity produced by the porcine embryo at the time of implantation in the uterus. By probing porcine genomic libraries with human and porcine IFN-alpha probes to isolate related genes, we have shown that the porcine IFN-alpha multigene family included, like several other mammalian species, two subfamilies of related but distinct genes. Class I subfamily contains at least 11 loci, located on chromosome no. 1, among which nine have been cloned and two (potentially functional) sequenced. Class II subfamily, which is specifically expressed by the embryo of ruminants before implantation, contains at least seven loci among which six have been cloned. One of the sequenced class I loci: PoIFN-alpha 1 encodes a 189 amino acids (AA) preprotein. After removal of the sequence encoding the putative signal peptide (23 N-terminal AA) this gene was inserted into an Escherichia coli bicistronic expression vector allowing intracellular synthesis of mature porcine IFN-alpha 1 (methionyl IFN-alpha 1). Expression of the recombinant protein was optimized by insertion of a seven base pairs long random synthetic sequence in the intercistronic region, followed by cloning in E. coli and immunodetection of clones expressing high amounts of recombinant protein. The E. coli strain obtained produced high levels of a 18,000 Da protein exhibiting the same in vitro overall biological properties as leucocyte derived porcine IFN (LeuIFN). However, it had a stronger antiviral effect on porcine cells than LeuIFN. After immunoaffinity purification to a specific activity of 5-10 x 10(7) International Units (IU)/mg of protein, pharmacokinetic and pharmacological studies were realized to determine the in vivo half life of this rIFN-alpha in the pig. These experiments revealed no major toxic effects in newborn (given 5 x 10(6) IU/kg) or adult (1 X 10(6) IU/kg) pigs. A significant pyrogenic effect (+ 1.5 degrees C) was noted only in the adults.

Animals↗
PubMed1990

Interferon therapy of relapsed and refractory Hodgkin's disease: Cancer and Leukemia Group B Study 8652.

A phase II trial of alpha 2b-interferon in patients with relapsed or refractory Hodgkin's disease was conducted by the Cancer and Leukemia Group B. Nineteen patients were eligible for study. These patients had received at least two (median of four) previous chemotherapeutic programs and 79% had received prior radiation therapy. Three patients had undergone intensive chemotherapy and autologous bone marrow transplantation. The treatment regimen consisted of interferon-alpha 2b 10 X 10(6) IU/m2 subcutaneously three times per week. Only limited antineoplastic activity was seen in this heavily pretreated group of patients. There was one partial response and four patients had reduction in measurable disease not meeting the criteria for partial response. The drug was well tolerated. Toxicity was predominantly myelosuppression. Thrombocytopenia was particularly severe in patients with bone marrow involvement. The observed antineoplastic activity, albeit limited, in this heavily pretreated group of patients suggests a potential role for this agent in combination regimens in patients with earlier disease.

Adult↗
PubMed1989

Differences in glycosylation state of fibronectin from two rat colon carcinoma cell lines in relation to tumoral progressiveness.

We have investigated the biosynthesis and carbohydrate structure of fibronectin secreted by two rat colon carcinoma cell lines. The cell line Prob yields progressive tumors after s.c. injection in syngeneic BD IX rats while tumors developed by Regb cells disappear after 20 days. No difference was observed in the fibronectin biosynthesis from both cell lines; however, the glycosylation degree was higher in Regb than in Prob cells indicating probable differences in the posttransductional process. The analysis of the glycosylation nature shows that fibronectin doesn't bear O-linked carbohydrate chains. The fibronectin of progressive Prob cells is more sialylated than that of the regressive Regb ones. In addition, the tri- and tetraantennary glycans are more important in Prob, while the fucosylated triantennary glycans are three times higher in Regb cells. These differences in the glycosylation state of the fibronectins could explain their differential susceptibility to the proteases treatment. In fact, the low glycosylated fibronectin from the progressive Prob cells was more rapidly degraded by several proteases than that of regressive Regb cells. The identification of the specific sites of proteolytic cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the cell attachment domain as well as the collagen binding domain of Regb cell fibronectin are particularly protected against proteolytic degradation.

Adenocarcinoma↗
PubMed1989

Differences in the release of 5'-nucleotidase and alkaline phosphatase from plasma membrane of several cell types by PI-PLC.

1. We have compared the effect of phosphatidyl inositol specific phospholipase C (PI-PLC) on the attachment of both 5'-nucleotidase and alkaline phosphatase to the liver plasma membrane from different species. 2. Our results demonstrate differences in the susceptibilities of both enzymes to PI-PLC treatment in relation to their origin. 3. These results were confirmed by immunoblotting using polyclonal anti-5'-nucleotidase antibodies. 4. In addition, in a single animal, susceptibility of both enzymes to PI-PLC treatment is different from one tissue to another. 5. The different percentages of released enzymes could be explained either by a polymorphism in the anchoring of these proteins at the cell surface membrane, or by a different steric hindrance or environment at the cleavage site itself.

5'-Nucleotidase↗
PubMed1989

Natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis.

Our objective was to evaluate the level of passive protection against transmissible gastroenteritis (TGE) among 57 newborn piglets nursing from seven seropositive sows previously naturally infected with porcine respiratory coronavirus (PRCV). After challenge exposure we observed mortality rates of 44% for litters of seven PRCV-infected sows, 40% for litters of four sows orally immunized with the attenuated TGEV strain Nouzilly, and 91% for litters of seven seronegative susceptible sows. A blocking ELISA with two appropriate monoclonal antibodies distinguished serological responses of PRCV-infected sows from those of TGEV-immunized sows. The results suggest that natural infection of the sow with PRCV may induce a degree of protective lactogenic immunity against TGE.

Animals↗
PubMed1989

Immunochemical identification and biological characterization of cytotoxic necrotizing factor from Escherichia coli.

The aim of this study was to identify Escherichia coli cytotoxic necrotizing factor (CNF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and to investigate the possible dissociation of CNF from hemolysin (Hly), which is often produced by CNF-producing strains. CNF was purified from cell lysates of a CNF-producing strain by using ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and preparative nondenaturing PAGE. All eluates from successive longitudinal slices of a preparative polyacrylamide gel were tested for cytoxicity and analyzed by SDS-PAGE; CNF activity was quantitatively correlated with a protein of 115 kilodaltons (kDa). This procedure increased both cytotoxicity and lethal activity (about 300-fold). We then compared SDS-PAGE protein patterns of enriched lysates from eight field and mutant E. coli strains producing both CNF and Hly, Hly alone, or neither; the 115-kDa band was present solely in CNF-producing strains, irrespective of Hly production. A neutralizing antiserum was produced against unpurified CNF from strain BM2-1 and then extensively adsorbed with cells and extracts of a CNF-defective mutant from BM2-1. The adsorbed antiserum possessed antitoxin activity and neutralized both lethal and necrotic effects of cell lysates from all the CNF-producing strains tested. In an immunoblot of enriched extract from BM2-1, the adsorbed antiserum recognized, besides the 115-kDa protein, another protein of 59 kDa, which was present in the CNF-defective mutant from BM2-1 and was not associated with cytotoxicity. We can conclude from these findings that CNF is a protein of 115 kDa associated with both cytotoxicity and in vivo toxicity, distinct from Hly, and present in all presumed CNF-producing strains tested.

Animals↗
PubMed1989

Incidence and duration of chemotherapy-induced nausea and vomiting in the outpatient oncology population.

Nausea and vomiting are commonly recognized side effects of chemotherapy. However, the incidence and duration of these effects have not been systematically studied in a large outpatient oncology population. This survey was conducted over two consecutive 6-week periods in the adult oncology clinics of two university teaching hospitals. The objectives were: (1) to document the incidence and duration of chemotherapy-induced nausea and vomiting; (2) to identify variables that influence nausea and vomiting; and (3) to describe patterns of antiemetic prescribing and compliance. One hundred thirty-eight completed patient-maintained diaries were returned (70% response rate). Anticipatory nausea and vomiting were reported by 9.4% and 6.5% of patients, respectively. Fifty percent and 27% of patients reported nausea and vomiting, respectively, on the day chemotherapy was administered (day 1: acute nausea and vomiting phase). Percentages fell to 22% and 11% by day three and 14% and 2.5% on day 5. Of patients who reported nausea and vomiting during the five-day period, 52% and 33% experienced nausea and vomiting, respectively, during the delayed period only (days 2 through 5: delayed emesis phase). Emetogenicity of chemotherapy significantly influenced incidence and duration of those symptoms. Sixty-seven percent of patients reported taking antiemetics on one or more days during the survey period. Of patients who reported antiemetic use, 92% reported antiemetics on day 1, 51% on day 3, and 31% on day 5. At-home antiemetic use was related to the emetogenicity of chemotherapy received. Patients who receive moderate to strong emetogens as defined in this report should receive antiemetic therapy for a minimum of three days. Increasing the dose of antiemetic prescribed both in the clinic and at home may be of benefit.

Adult↗
PubMed1989

An improved pyrogallol red-molybdate method for determining total urinary protein.

We adapted the pyrogallol red-molybdate method for total urinary protein to the Cobas Bio centrifugal analyzer. The method is simple, rapid, sensitive, and inexpensive. Addition of 25 mg of sodium dodecyl sulfate per liter to the reagent modifies protein reactivities so that the chromogenicity of human gamma globulins is the same as that of albumin. Results by this method and a comparison method that included gel filtration and a modified biuret reaction correlated well (r = 0.951).

Adult↗
PubMed1988

Purification of bovine liver cytosolic 5'-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme.

Cytosolic 5'-nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP-Sepharose columns result in a 12,000-fold purification. The sequential elution of glycoproteins from the concanavalin-A-Sepharose column with methyl alpha-D-glucoside and methyl alpha-D-mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane-bound 5'-nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane-bound 5'-nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5'-nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes. The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [alpha,beta-CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [alpha,beta-CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5'-nucleotidase. All these observations indicate that the two isoenzymes are different.

5'-Nucleotidase↗
PubMed1988

Application of high-performance ion-exchange chromatography to the analysis of cytosolic glucocorticoid receptor.

The use of high-performance ion-exchange chromatography (HPIEC) on a Mono Q column was investigated for the analysis of glucocorticoid receptor. In the presence of 10 mM sodium molybdate, both liganded and unliganded glucocorticoid receptor were eluted as a single and sharp peak (0.32 M NaCl). In the absence of molybdate and after exposure to heat and salt, another peak of specifically bound radioactivity was eluted with 0.08 M NaCl. When HPIEC was performed in the absence of molybdate, two molecular forms of the liganded receptor were detected which eluted with 0.08 M NaCl (Stokes' radius Rs = 5.1 nm, s20,w = 4.6 S, calculated mol. wt Mr approximately 100,000) and 0.32 M NaCl (Rs = 7.3 nm, S20,w = 9.0 S, calculated Mr approximately 280,000). Analysis of both forms with mini-columns of DNA-Ultrogel, DEAE-Trisacryl and hydroxylapatite (HA-Ultrogel) confirmed the identity of the two peaks with transformed and non-transformed glucocorticoid-receptor complexes. These results suggest that HPIEC may provide a useful tool for the rapid resolution and quantification of receptor molecular forms.

Adrenalectomy↗
PubMed1988

Effects of 2'-deoxy-5-fluorouridine on regenerating liver following partial hepatectomy in the rat.

UNLABELLED: This study evaluated the effect of 2'-deoxy-5-fluorouridine (FUDR) on the regeneration of the liver following partial (68%) hepatectomy in the rat. Male Sprague-Dawley rats weighing between 190 and 240 g underwent partial hepatectomy under ether anesthesia. Twelve hours postoperatively rats received intraperitoneal injections of 2'-deoxy-5-fluorouridine or 0.9% NaCl solution as follows: Group I, 0.9% NaCl solution (n = 49); Group II, 89 mg 2'-deoxy-5-fluorouridine/kg of body weight (n = 25); and Group III, 178 mg 2'-deoxy-5-fluorouridine/kg of body weight (n = 24). Sham groups underwent celiotomy and liver palpation followed by 0.9% NaCl solution injections (n = 5) or low dose 2'-deoxy-5-fluorouridine (n = 5) and high dose 2'-deoxy-5-fluorouridine (n = 5). The regenerative ability of the liver was evaluated by weight and deoxyribonucleic acid synthesis in the liver remnant. RESULT: Both low and high dose 2'-deoxy-5-fluorouridine delayed the peak of deoxyribonucleic acid synthesis from 36 to 72 hr as compared to control animals which had maximal synthesis at 25 to 36 hr postoperatively (P less than 0.01). Weight of the liver remnants demonstrated a similar pattern. CONCLUSION: High doses of 2'-deoxy-5-fluorouridine administered intraperitoneally delay, but do not inhibit, liver regeneration following partial (68%) hepatectomy as reflected by DNA synthesis and weight of the remnant.

Analysis of Variance↗
PubMed1988

Testicular Ca++ ATPase activity in mice: effects of age and gonadotropin administration.

These studies describe a high affinity calcium (Ca++)-dependent ATPase in purified testicular plasma membranes, which exhibits increased activity from weaning age to adulthood. Administration of human chorionic gonadotropin (hCG; 5 IU) increased enzyme activity in 21-day old and pubertal (35 to 40-day old), but not in adult mice. In pubertal mice, these increases in testicular Ca++-ATPase activity were dose-related and evident 60 min after hCG administration. A second challenge dose of 5 IU hCG administered either 24, 48 hrs, or 5 days later, had no additional effect on Ca++ ATPase in purified testicular plasma membranes in these pubertal animals. The present findings indicate that testicular plasma membrane Ca++ATPase activity exhibits a developmental pattern concomitant with increased testicular steroidogenic activity during sexual maturation. Furthermore, enzyme activity is increased by gonadotropic stimulation and exhibits a refractoriness similar to that of androgen biosynthesis to repeated hCG stimulation.

Aging↗