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Biomedical subjects

E Dupont

Publications and source records attributed to E Dupont.

At least 109 records · Page 6Linked to original sources
PubMed1994

Immunocytochemical localization and biological activity of 3 beta-hydroxysteroid dehydrogenase in the central nervous system of the frog.

The enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) catalyzes biosynthesis of progesterone (P) and all precursors of glucocorticoids, mineralocorticoids, androgens, and estrogens. Despite the broad interest raised by neurosteroids, the cellular localization of 3 beta-HSD has never been investigated in the brain. We took advantage of the availability of an antiserum raised against human placental 3 beta-HSD to determine the distribution of 3 beta-HSD-immunoreactive structures in the brain of the frog Rana ridibunda by the indirect immunofluorescence technique. Three populations of 3 beta-HSD-immunoreactive cell bodies were observed in the hypothalamus, namely, in the rostral region of the preoptic nucleus, the dorsal infundibular nucleus, and the dorsal part of the ventral infundibular nucleus. A dense network of 3 beta-HSD-immunoreactive nerve fibers was visualized in the dorsal area of the diencephalon, that is, in the lateral neuropil, the corpus geniculatus lateralis, and the nucleus posterolateralis thalami. Reversed-phase HPLC analysis of frog hypothalamic extracts combined with RIA detection showed the presence of substantial amounts of immunoreactive steroids coeluting with P and 17-hydroxyprogesterone (17OH-P). The synthesis of delta 4-3-keto-steroids in the frog hypothalamus was investigated using the pulse-chase technique with 3H-pregnenolone (3H-delta 5P) as a precursor. The formation of five tritiated metabolites of 3H-delta 5P was observed, one of which coeluted with 17OH-P. Conversion of 3H-delta 5P into this radioactive metabolite was significantly reduced by trilostane, a specific inhibitor of 3 beta-HSD. Immunodetection of newly synthesized steroids in HPLC fractions of hypothalamic extracts, using 17OH-P antibodies, revealed the existence of an immunoreactive steroid that exhibited the same retention time as synthetic 17OH-P. The present study provides the first immunocytochemical mapping of 3 beta-HSD, a key enzyme of the steroid biosynthetic pathway, in the CNS of a vertebrate. The data also demonstrate for the first time biosynthesis of neurosteroids in the brain of a nonmammalian vertebrate.

17-alpha-Hydroxyprogesterone↗
PubMed1993

Gap junctional intercellular communication in mouse lung epithelial cell lines: effects of cell transformation and tumor promoters.

Gap junctional intercellular communication (GJIC) is reduced by neoplastic transformation and treatment with tumor promoters in many types of cells but few data exist for the lung. GJIC was therefore evaluated in non-transformed (C10) and transformed (E9, 82-132, and PCC4) mouse lung epithelial cell lines and in C10 cells treated with tumor promoters. GJIC was assessed by fluorescent dye microinjection (dye-coupling). Dye-coupling levels were highest in C10 cells (85-90% communicating cells) followed by 82-132 cells (40-50%), E9 cells (15-20%), and PCC4 cells (3-10%). Indirect immunofluorescent staining with anti-gap junction protein (connexin) antibodies revealed that C10 cells expressed gap junctions comprised of connexin43, but not connexin32 or connexin26. The tumor promoters, butylated hydroxytoluene (BHT), 12-O-tetradecanoylphorbol-13-acetate (TPA), and p,p'-dichlorodiphenyltrichloroethane (DDT), inhibited dye-coupling in C10 cells but phenobarbital (PB) did not. BHT promotes mouse lung tumor formation, PB does not, while the effects of TPA and DDT on lung tumor development have not been reported. These data indicate that cell transformation and certain tumor promoters reduce GJIC in mouse lung epithelial cells and demonstrate correlations between the in vitro inhibition of GJIC and lung tumor promotion.

Animals↗
PubMed1993

[Torticollis treated with botulinum toxin].

The effect of botulinum toxin A treatment was studied in patients with idiopathic torticollis. Twenty patients with idiopathic torticollis received electromyographically guided intramuscular botulinum toxin A into the hyperactive neck muscles. In all, 48 treatments were given. The injections were repeated with intervals of 11 to 35 weeks (mean 18.7 weeks). The grade of improvement was estimated subjectively using a visual analogue scale. An overall improvement of 55% compared with the status before treatment was found. The best result obtained in each individual patient varied from 20% to 84% (mean 66%). The effect of the treatment began to wear off after eight to 22 weeks (mean 11.5 weeks). The side effects consisted of short term dysphagia in two patients. The administration of botulinum toxin in idiopathic torticollis is a safe and beneficial treatment with few side effects. The EMG guidance of injections proved to be helpful as it restricted the injections into muscles with electromyographic hyperactivity, thereby economizing the amount of toxin given. It is doubtful if double-blind studies of botulinum toxin can be undertaken due to the marked muscular wasting and weakness caused by the injections.

Adult↗
PubMed1993

Restoration of gap-junctional intercellular communication in a communication-deficient rat liver cell mutant by transfection with connexin 43 cDNA.

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.

Animals↗
PubMed1993

Ontogeny of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) in rat testis as studied by immunocytochemistry.

The enzymatic complex 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) is a step essential in the biosynthesis of all biologically active steroids, including androgens. In order to obtain information about the expression of 3 beta-HSD during testis development, we have localized this enzyme by light microscope immunocytochemistry during fetal and postnatal periods of development in the rat. In fetal testis, the enzyme was first detected in a few Leydig cells on the 17th day of gestation. The number of labeled cells and intensity of labeling increased with fetal development. From days 19 to 21 of gestation, strongly immunoreactive Leydig cells were arranged in clusters between seminiferous tubules. During the first days following birth, the number and size of positive cells rapidly decreased so that on postnatal days 5 and 10 only small, poorly stained cells could be seen. Fifteen days after birth, positive Leydig cells appeared more numerous and localized in peri- and intertubular spaces. At the onset of puberty, the intensity of labeling markedly increased. After puberty, and during adulthood, both strongly and weakly stained Leydig cells were mainly localized in intertubular spaces. Since the fluctuations in 3 beta-HSD content revealed in the present study by immunocytochemistry appear similar to those already observed for androgen secretion, such data suggest that regulation of 3 beta-HSD by trophic hormones might play an important role in regulating testicular androgen production during fetal as well as postnatal development.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Tumor necrosis factor alpha and interleukin 6 plasma levels in infected cirrhotic patients.

BACKGROUND: Patients with liver cirrhosis disclose both increased production and decreased metabolism of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). The present study analyzes the characteristic pattern of these cytokines during sepsis in cirrhotics. METHODS: TNF-alpha and IL-6 plasma levels, measured during 15 days from the onset of cirrhotic decompensation or of the septic event, were compared between 14 infected patients with liver cirrhosis, 18 uninfected decompensated cirrhotic patients, and 35 septicemic patients devoid of liver disease. Cytokines were measured using immunoassays. RESULTS: In infected cirrhotics, initial levels of both TNF-alpha and IL-6 were significantly higher than in noninfected cirrhotic patients (P < 0.0001) or in septicemic patients devoid of cirrhosis (P < 0.001). Initial IL-6 plasma levels (threshold, 200 pg/mL) showed 89% specificity and 100% sensitivity in discriminating cirrhotic decompensation due to infection from that caused by other factors. TNF-alpha and IL-6 plasma levels remained significantly higher for many days in infected cirrhotic patients compared with the other two groups. CONCLUSIONS: Both the profoundly increased initial levels of TNF-alpha and IL-6 and their persistence over days after sepsis onset seem characteristic of the cirrhotic patients. The exact relationship between prolonged exposure to TNF-alpha and poor prognosis in these patients is unknown, but it might represent a unique opportunity for the use of anti-TNF-alpha antibodies during sepsis.

Aged↗
PubMed1993

Ontogenesis of 3 beta-hydroxysteroid dehydrogenase delta 5-delta 4 isomerase in the rat ovary as studied by immunocytochemistry and in situ hybridization.

The enzyme complex 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5-4-ene isomerase (3 beta-HSD) is involved in the biosynthesis of all classes of active steroids, namely glucocorticoids, mineralocorticoids, and sex steroids. To obtain more information about the age-specific expression and localization of 3 beta-HSD during development in the rat ovary, two complementary cytochemical techniques were used, immunocytochemical localization with antibodies against purified human placental 3 beta-HSD, and 3 beta-HSD mRNA localization achieved by in situ hybridization with a [35S]-labeled cDNA encoding 3 beta-HSD. In the fetal ovary, no significant immunolabeling or hybridization signal could be observed. The first expression of the enzyme was observed 6 days after birth, immunolabeling as well as autoradiographic reaction being found in a few interstitial gland cells. At the 10th day of postnatal life, the theca interna cells of growing follicles appeared to be labeled with both techniques. The granulosa cells of growing follicles also exhibited hybridization signal, whereas no immunostaining could be detected in these cells at any of the time intervals studied. At puberty, 3 beta-HSD was localized in theca interna and granulosa cells of the growing and mature follicles as well as in the interstitial gland and corpus luteum cells. Similar results were obtained in 40-day-old and adult animals. The present data indicate a progressive expression of 3 beta-HSD in different morphological structures during ovarian development in the rat.

Aging↗
PubMed1993

HLA-B locus DNA typing: detection of B*7801 and seven additional alleles by BW6-specific exon 2 amplification.

A molecular approach to type a new HLA-B5 antigen, HLA-BSNA, characterized by its unusual association with the public determinant BW6, referred to as B*7801, has been designed. Antigens disclosing serological identity with SNA, BX1 and Te76 were also investigated. Based upon HLA-B exon 2 group-specific PCR, the following procedure was established: 5' and 3' primers were designed by targetting the codons 11-12 (AM) and 81-83 (LRG), respectively, in exon 2 (alpha 1 domain). The 5' primer discriminates with HLA-A, -C genes and pseudogenes, while the 3' primer detects the sequence encoding the BW6 epitope (NLRG) and discriminates it from the BW4 epitope. The combination of this pair of primers specifically amplifies 26 HLA-B alleles. Oligotyping for B*7801 was performed using a combination of two non-radioactively labeled SSO probes identifying positions T45 and D74 in exon 2. To resolve ambiguous hybridization patterns, an additional set of probes was used. The specificity of this BW6-group-specific amplification procedure was investigated on 150 genomic DNA samples. Among them, we obtained 94 amplified DNA products which were tested with eight SSOs. Beside B*7801, the following B alleles could be defined: B*0801, B*35, B*5401, B*5501-2, B*5601-2, B*1501-7 (including B62, B75 and B72) and B*4601. SNA, BX1 and Te76 DNA samples gave similar hybridization pattern providing a clue to the identity of these antigens. This "one PCR and two probes" procedure represents a simple oligotyping strategy which can also be applied to type many other HLA-B specificities.

Alleles↗
PubMed1993

Immortalized hypothalamic luteinizing hormone-releasing hormone neurons express a connexin 26-like protein and display functional gap junction coupling assayed by fluorescence recovery after photobleaching.

Expression of gap junction proteins was studied in the LHRH neuronal cell line, GT1-7, as a first step in defining the signalling mechanisms responsible for the pulsatile secretion of LHRH. GT1-7 cells were found to express a connexin 26-like protein that comigrated with mouse liver connexin 26 and that reacted with connexin 26-specific antibodies on Western blots. Immunofluorescent staining revealed punctate staining in a fraction of the cells, often present at points of apparent contact with neighboring cell bodies or processes. Fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein loaded GT1-7 cells showed dye coupling among 20-30% of cells that made contact with other cells, suggesting the presence of functional gap junctions in this cell line.

Alkaline Phosphatase↗
PubMed1993

Severe hemolysis due to a donor anti-D antibody after heart-lung transplantation. Association with lung and blood chimerism.

Hemolysis due to donor-derived red cell antibodies is a potential complication after minor ABO-mismatched solid organ transplantation. It has also been described in Rh-positive recipients receiving organs from previously isoimmunized Rh-negative donors. We report on a group O, Rh(D)-positive patient who received a heart-lung transplant from a group O, Rh(D)-negative donor presenting an anti-D antibody. Severe hemolytic anemia developed in the early postoperative period and lasted for 3 mo. Anti-D antibodies that were of donor origin were found in bronchoalveolar lavage fluid and serum. In addition, using the polymerase chain reaction for human leukocyte antigen class II genotyping, we were able to demonstrate the presence of a mixed population of donor and recipient cells in the lungs and the peripheral blood of the recipient. The chimeric state in the blood persisted until at least the fifty-seventh postoperative day. This first case report of hemolysis due to Rh(D) antibody after heart-lung transplantation emphasizes the need for routine red cell antibody screening of donors and for close monitoring of signs of red cell destruction in the recipient in cases of pretransplant donor isoimmunization.

Adult↗
PubMed1993

Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality.

PURPOSE: Cytokines have been associated with the development of sepsis and diffuse tissue injury following septic or endotoxic challenges in humans. Furthermore, relative organ-system dysfunction, not specific organ dysfunction, appears to predict outcome from critical illness. We hypothesized that persistence of inflammatory cytokines within the circulation, reflecting a generalized systemic inflammatory response, is associated with multiple-system organ failure (MSOF) and death from critical illness. In addition, since hepatic function is central to host-defense homeostasis, we further reasoned that critically ill patients with hepatic cirrhosis would have an increased incidence of MSOF and death following sepsis associated with a persistence of cytokines in the blood. PATIENTS AND METHODS: We measured serum levels of tumor necrosis factor (TNF), interleukin (IL) 1, IL-2, IL-6, and interferon gamma (IFG) serially for the first 48 h following the onset of hypotension (systolic blood pressure < 90 mm Hg) thought likely to be due to sepsis in all patients presenting to one ICU. These data were correlated with initial severity of shock and retrospective determination of septic or nonseptic origin, preexistent hepatic cirrhosis, subsequent development of MSOF, and outcome. RESULTS: Fifty-three specific episodes of shock in 52 patients were recorded (35 septic and 18 nonseptic episodes). Mortality was higher in septic patients (41 vs 17 percent, p < 0.01), as was the development of MSOF (29 vs 6 percent, p < 0.001), incidence of cirrhosis (21 vs 0 percent, p < 0.01), and TNF levels over the study interval (p < 0.01). Nonseptic patients also had an initial elevation in TNF over 48-h levels (p < 0.05) that were higher than serum levels reported for normal subjects (chi 2, p < 0.05). There was no relation between peak TNF level and outcome. Sixty-seven percent of the cirrhotic patients had development of MSOF and died, while only 30 percent of the noncirrhotic patients had development of MSOF or died (p < 0.05). The TNF and IL-6 levels in patients who had MSOF or who died were both elevated and did not decrease over time independent of presence or absence of sepsis (p < 0.01). Similarly, IL-6 levels after 12 h were higher in cirrhotic patients than in noncirrhotic septic patients (p < 0.05). No elevation in IL-1, IL-2, or IFG was seen in any patient subpopulation. CONCLUSIONS: TNF and IL-6 serum levels are higher in septic than in nonseptic shock, but the persistence of TNF and IL-6 in the serum rather than peak levels of cytokines predicts a poor outcome in patients with shock.

Cytokines↗
PubMed1993

Characterization of gap junctional communication-deficient mutants of a rat liver epithelial cell line.

To understand the mechanism(s) regulating gap junctional communication, we isolated gap junctional intercellular communication-deficient (GJIC-) mutant clones of a rat liver epithelial cell line, WB F-344, which is hypoxanthine-guanine phosphoribosyl transferase deficient (HGPRT-). The cells were exposed to a mutagenesis regimen and cocultured with the wild type HGPRT+ cells. Four GJIC- and one positive clones were characterized in the present study. Northern analysis of RNA isolated from both mutant and parental cells showed a single RNA species of about 3.0 kb which hybridized to connexin43 (Cx43) cDNA. Western blot analysis confirmed the expression of this junctional protein in all these clones. However, in the GJIC- clones the slowest migrating band corresponding to a hyperphosphorylated form, P2, of Cx43 protein (approximately 46 kDa) was absent suggesting that loss of this phosphorylated form of Cx43 may be involved in the failure of the mutants to establish cell-cell communication. Immunofluorescence analysis of the mutants did not reveal any differences in the distribution and localization of Cx43 between GJIC+ and GJIC- clones suggesting that the loss of phosphorylation did not affect the membrane association of this protein. Taken together, these data suggest that one mechanism for the loss of communication in these GJIC- mutants may be the consequence of a change in the intrinsic phosphorylation state of Cx43 protein.

Animals↗
PubMed1993

[Prevention of risks related to blood transfusions].

During the past decade, the major fears of transfusion medicine were blood-transmitted viral diseases (hepatitis, AIDS). By applying strict rules at each step of blood donation, it can now be considered that this risk is reduced to a minimal level. Furthermore, new technologies (leukocyte removal filters, cytapheresis devices) and strict immuno-hematological procedures (detection of preformed antibodies, use of blood with well-defined phenotype) will reduce morbidity due to immunological incompatibility. These techniques should provide a safe transfusion service in the near future.

Blood Group Antigens↗
PubMed1992

Detrimental role of donor-recipient HLA-DQ5 and -DQ6 disparities on cadaver kidney graft survival.

Donor-recipient incompatibility (D + R -) for HLA-DQ1, but not for -DQ2 or -DQ3, is associated with an adverse effect on cadaver kidney graft survival. Until now, however, DQ1 recipients of DQ1-negative kidneys (D - R +) have not been differentiated from DQ1-identical donor-recipient pairs (D + R +) and splits of DQ1, DQ5 and DQ6, have not been studied in that respect. From our data (480 transplantations performed from January 1980 to December 1990), three donor-recipient DQ combinations (D + R +, D - R +, D + R -) were formed for each of four DQ specificities (DQ2, DQ3, DQ5, DQ6). As DR-DQ linkage disequilibrium is well conserved in caucasoid individuals, DQ specificities were inferred from the associated DR specificities. Graft survival rate (%) was significantly lower for the DQ5 D + R - and the DQ6 D - R + combinations when compared with the other corresponding DQ combinations, whereas no significant difference was observed between the DQ2 and DQ3 combinations. In conclusion, if DQ1 plays a prominent role in kidney graft survival, the effects of its splits appear dissociated: DQ5 could be a marker of high antigenicity and DQ6 a marker of high responsiveness.

Cadaver↗
PubMed1992

Immunoglobulin A and interleukin 6 form a positive secretory feedback loop: a study of normal subjects and alcoholic cirrhotics.

Patients with alcoholic liver cirrhosis (ALC) have high serum levels and spontaneous in vitro production of immunoglobulin (Ig) A. Deposits of IgA are also found in liver sinusoids. Increased interleukin 6 (IL-6) production is another feature of this disease. This study shows a linear correlation between increased lipopolysaccharide (LPS)-induced IL-6 production and increased spontaneous IgA and IgG secretion by peripheral blood mononuclear cells (PBMCs). PBMCs and purified monocytes isolated from healthy control subjects and patients with ALC contain elevated IL-6 messenger RNA levels and produce IL-6 in response to stimulation with soluble polymeric IgA (p-IgA) or attached monomeric IgA (m-IgA) but not with soluble m-IgA. The addition of monospecific antibody to human IL-6 inhibits spontaneous IgA production by PBMC. This inhibition is more pronounced in patients with ALC. These data provide evidence that IgA, possibly by attachment to cells possessing Fc alpha receptors and secreting IL-6, is involved in the production of this major mediator and the amplification of Ig secretion. Circulating IgA and IgA deposits could therefore initiate a process of autoamplification implicated in the development of hypergammaglobulinemia in ALC.

Cells, Cultured↗
PubMed1992

Molecular characterization of a recombinant HLA-DR1/DR2 haplotype.

Serologic analysis of two families identified an HLA-DR haplotype in which DR1 and DR2 cosegregated. DNA-RFLP analysis of these families with an HLA-DRB probe revealed a pattern of hybridization suggestive of a recombination between DR1 and DR15. Following amplification, cloning, and nucleotide sequencing of HLA-DRB-gene second-exon DNA sequences, three DRB amplification products associated with the novel haplotype were identified: these corresponded to DRB1*0101, DR2 pseudogene, and DRB5*0101. Clones representing the DRB1*1501 and DR1 pseudogenes were not identified: oligonucleotide typing with DRB1*1501-specific probes confirmed the absence of this gene within the DR1/DR2 haplotype. We postulate that the DR1/DR2 haplotype represents a recombinant between those of DR1-Dw1 and DR15-Dw2, and that the crossing-over may have been between the DRB1*0101 gene and the DR2 pseudogene. This is further supported by DNA-RFLP analysis with HLA-DQB and DQA CDNA probes, which revealed conserved linkage genes between the DQB1*0501, DQA1*0101, and DRB1*0101 genes.

Base Sequence↗