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Biomedical subjects

D A Clark

Publications and source records attributed to D A Clark.

At least 109 records · Page 6Linked to original sources
PubMed1997

Maternal response to paternal trophoblast antigens.

PROBLEM: What is the function of the immunoglobulin (Ig) G antibody bound to trophoblast in normal pregnancy, and what is the antigen? METHOD: IgG was acid eluted from term human placental microvesicles and reacted with the antigen, R80K, left on the vesicles. The eluted antibody was used to detect the antigen on monocytes, lymphocytes, and lymphoblastoid cell lines. The eluted antibody is highly polymorphic, but monoclonal antibodies (mAbs) were made against conserved regions of the molecule. These also reacted with the murine equivalent of the human R80K and were used in inhibition studies of natural killer (NK) cell killing and the mouse abortion models, CBA x DBA2 F1 resorption in CBA females, the endotoxin-induced resorption model, and a sonic stress-induced murine resorption model. RESULTS: All 600 syncytiotrophoblast microvesicle preparations of human term placenta had IgG antibody bound, elutable at pH 3.0. The eluted antibody reacted with about 15% of unrelated human placentae. In horses mares make detectable antibody early in pregnancy, at about the time of implantation. The IgG antibody was bound to an 80-kDa protein (R80K) also detected on B lymphocytes and monocytes. In HLA homozygous lymphoblastoid B cell lines, which reacted with one or more eluted antibodies, had a pattern of cytotoxicity independent of HLA Class I; and as a single 80-kDa peptide chain, R80K did not resemble HLA molecules. Genetic studies in horses show that of the two paternal allotypes of R80K detectable by placental alloantibodies, only one, usually the grandpaternal one, is present in all the placentae of a sibship. Two of 26 eluted human antibodies had affinity for K562 and inhibited killing by human peripheral blood NK cells. One mAb, BA11, against a conserved site on R80K inhibited killing of K562, and also reacted with the murine R80K homologue. BA11 inhibited murine NK cell killing and virtually completely inhibited three NK cell-dependent mouse resorption models. CONCLUSION: R80K protein is a target molecule for NK cell activity expressed on all placentae. It has a polymorphic alloantigenic determinant completely covered with maternal antibody in all successful term pregnancies. In murine NK cell-dependent models of abortion, a mAb against a monomorphic determinant present in human and murine R80K prevents abortion very effectively. It seems that the R80K molecule must be covered with antibody to prevent NK attacks on trophoblast.

Animals↗
PubMed1997

Murine T cell determination of pregnancy outcome: I. Effects of strain, alphabeta T cell receptor, gammadelta T cell receptor, and gammadelta T cell subsets.

PROBLEM: T cells bearing alphabeta T cell receptor (TcR) and gammadelta TcR are present at the fetomaternal interface, and the latter, which express surface activation markers, can react with fetal trophoblast cell antigens. What is the role of these cells? METHOD: Using stress-abortion-prone DBA/2-mated CBA/J and abortion-resistant C57/B16 mice, alphabeta, gammadelta, and CD8+/- T cell subsets were measured in spleen and uterine decidua. The effect of immunization against abortion and administration of anti-TcR antibody in vivo was examined. Cytokine synthesis was measured by intracellular staining of Brefeldin A-treated cells. RESULTS: Abortion-prone matings showed an unexpected accumulation of gammadelta T cells beginning in the peri-implantation period and this was suppressed by immunization against abortion. The immunization deleted gammadelta T cells producing the abortogenic cytokines, TNF-alpha and gamma-interferon, and increased production of the anti-abortive cytokines, IL-10 and transforming growth factor-beta2 (TGF-beta2). Immunization also boosted the number of alphabeta T cells which were present in the decidua as early as 2 days after implantation. In vivo injection of GL4 (anti-delta) depleted gammadelta T cells producing Th1 cytokines in the peri-implantation period, and prevented abortions, whereas H57 (anti-beta) decreased the number of alphabeta T cells and led to 100% abortions. CD8+ T cells present in peri-implant decidua before onset of abortions were mostly alphabeta TcR+, although some were gammadelta+. Changes in gammadelta and alphabeta T cells in pregnancy were most dramatic in uterine tissue. CONCLUSION: Although decidual gammadelta T cells after formation of a distinct placenta and fetus produce anti-abortive TGF-beta2-like molecules and IL-10, prior events can lead to abortion. High local production of TNF-alpha and gamma-interferon develop during the peri-implantation phase because of an excessive increase in the Th1 cytokine+ subset of gammadelta cells; these cytokines may be contributed by other tissues in decidua, and the contribution of bioactive factors by gammadelta T cells may augment the cytokine pool. In contrast, alphabeta T cells (which may be inactivated by stress that causes abortions) may mediate the anti-abortive effect of alloimmunization. Alloimmunization involves a shift from a Th1 to a Th2 pattern in the gammadelta T cells in decidua.

Abortion, Spontaneous↗
PubMed1997

Prospective study of human herpesvirus 6, human herpesvirus 7, and cytomegalovirus infections in human immunodeficiency virus-positive patients.

Blood samples from human immunodeficiency virus (HIV)-positive patients were monitored for cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and HHV-7 by PCR. We detected CMV in 17% of the patients, HHV-6 in 6%, and HHV-7 in 3%. The viral loads of CMV were significantly higher than those of HHV-6 (P = 0.007) or HHV-7 (P = 0.01). Detection of CMV and HHV-6 was associated with low and high CD4 counts, respectively.

CD4 Lymphocyte Count↗
PubMed1997

Diagnosis of primary human herpesvirus 6 and 7 infections in febrile infants by polymerase chain reaction.

Primary human herpesvirus 6 (HHV-6) and 7 (HHV-7) infections were identified in febrile children by qualitative and quantitative polymerase chain reaction (PCR) assays. Diagnosis was based on the differential detection of viral DNA in peripheral blood mononuclear cells (PBMC), but not in saliva. Six of 41 febrile infants, but none of seven non-febrile controls, were identified with primary infections (three HHV-6, three HHV-7). These children had significantly higher viral loads in PBMC (HHV-6, median 24213 genomes/10(6) PBMC; HHV-7, median 6,040,000 genomes/10(6) PBMC) than DNA-aemic, saliva PCR positive children (HHV-6, median 1606 genomes/10(6) PBMC, p < 0.01; HHV-7, median 7089 genomes/ 10(6) PBMC, p < 0.05). Viral DNA was detected in serum by PCR in only 50% of primary infections. All three children with primary HHV-7 infection had febrile convulsions. Thus PCR, including quantitative assays, may identify primary HHV-6 and HHV-7 infections when an appropriate combination of clinical specimens is used.

DNA, Viral↗
PubMed1997

The effect of inhibitors of inducible nitric oxide synthase on chronic colitis in the rhesus monkey.

GI inflammation is associated with an increase in nitric oxide production and expression of the inducible isoform of nitric oxide synthase (iNOS). Using a spontaneous model of chronic colonic inflammation in rhesus monkeys, which shares morphological and clinical features with ulcerative colitis, we assessed the therapeutic benefit of administration of iNOS inhibitors. Sixteen colitic rhesus monkeys underwent an endoscopy procedure before commencement of the trial, and biopsies from three sites of the colon and plasma were collected. Monkeys were randomly assigned to three treatment groups and were administered by oral bolus 60 mg/kg/day L-N 6-(1-Iminoethyl) lysine, 60 mg/kg/day aminoguanidine or a placebo (0.9% NaCl) twice daily. Monkeys were sacrificed after 10 days, coIonic tissue from multiple sites was dissected and processed for histological and biochemical analysis. In rhesus colitis, diarrhea was characterized by a significant increase in fecal water content and daily fecal output. iNOS was localized immunohistochemically in plasma cells and neutrophils in the colonic mucosa and lamina propria, paralleled by enhanced iNOS gene expression determined by reverse-transcriptase polymerase chain reaction. Only L-N 6-(1-iminoethyl) lysine administration resulted in a significant reduction in systemic nitric oxide production, and neither of the iNOS inhibitors significantly reduced the histological inflammatory score nor ameliorated diarrheal symptoms. From these findings, we conclude that in this chronic, spontaneous model of colonic inflammation, administering iNOS inhibitors with this treatment regimen did not provide any major therapeutic benefit.

Administration, Oral↗
PubMed1997

Process characteristics of worry and obsessive intrusive thoughts.

One hundred and twenty-five university students were administered a battery of self-report measures to assess worry disposition and content, frequency of obsessive intrusive thoughts, and 10 conceptually derived appraisal dimensions of worry and obsessional intrusions. Process ratings indicated that the worrisome thoughts were considered more disturbing than the ego-dystonic intrusive thoughts. Worry was also distinguished by a focus on the possible consequences of negative events, whereas concern about the personal meaning of the thought was a unique process dimension for obsessive intrusive thoughts. Both content and process variables are important in differentiating worry and obsessive-like intrusive thoughts in a nonclinical population.

Adolescent↗
PubMed1996

Hypoglycemic activity of a series of alpha-alkylthio and alpha-alkoxy carboxylic acids related to ciglitazone.

The thiazolidinedione moiety of ciglitazone and its analogues can be replaced by an alpha-alkoxy or alpha-thioether carboxylic acid group. The influence of the nature of the R group, the length of the connector to the aromatic backbone of the molecule, and the stereochemistry have been studied. The most potent compounds have glucose-lowering activity at doses as low as 0.01 mg/kg.

3T3 Cells↗
PubMed1996

Inhibition of immunoprotective CD8+ T cells as a basis for stress-triggered substance P-mediated abortion in mice.

The embryo expresses paternal antigens foreign to the mother and therefore has been viewed as an allograft. The maternal immune system responds to paternal antigens on the "graft", and these responses are thought to protect pregnancy. However, pregnancy can be aborted by stress, which stimulates local production of TNF-alpha and inhibits TGF-beta 2-producing natural suppressor cell (NS) activity via a neurotransmitter substance P-dependent pathway. Immunization protects against stress-triggered abortion and CD8+ T cells appear to be required. The objective of the present study was to investigate the importance of CD8+ T cells in the prevention of stress-triggered abortion by immunization. Injection of anti-CD8 increased the abortion rate in nonimmunized mice and in immunized mice. Following anti-CD8 injection, stress failed to further increase the abortion rate; a similar high rate of abortion was seen in immunized and anti-CD8-injected mice. These data suggested that stress could act by neutralization and/or elimination of immunoprotective CD8+ T cell function. CD8+ T cells from pregnant mice have been reported to produce a 34-kDa suppressor factor, but we detected a 1.5- to 2-kDa suppressive factor in the HPLC fractions of supernatants obtained from nonstressed decidua, and this activity was abolished by stress and boosted by immunization with Balb/c cells.

Abortion, Spontaneous↗
PubMed1996

High levels of spermine in IVF medium as a negative predictor of subsequent success of embryo transfer.

PURPOSE: Our purpose was to test the prediction that spermine levels in IVF culture supernatants measure by rapid ELISA assay correlate with subsequent success or failure to establish a pregnancy. RESULTS: Forty-nine patients undergoing gonadotropin-stimulated ovulation after LHRH analogue treatment in the current study succeeded and 57 failed. With the exception of the first 4 hr of culture, where polyamine levels were slightly (but not significantly) higher in supernatants associated with subsequent success, higher levels of polyamines were predictive of failure. CONCLUSIONS: The result in this study differs from previous data obtained with women ovulated using clomiphene where low spermine levels (assayed in an immunosuppression assay in vitro) correlated with failure to establish pregnancy, and detectable levels correlated with success. Supplementation of IVF culture medium with spermine and/or spermidine appears unlikely to improve IVF success rates, where the success rate is already very good, and may possibly do harm. The possibility that different methods of ovulation affect subsequent polyamine production in vitro by fertilized oocytes merits further study.

Culture Media, Conditioned↗
PubMed1996

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase.

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.

Administration, Oral↗
PubMed1996

Fetal growth retardation in rats may result from apoptosis: role of peroxynitrite.

Administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) results in fetal growth retardation. This study was designed to further examine the influence of NO on fetal growth, specifically, the potential role of inducible NOS and to evaluate the possibility that apoptosis contributed to uteroplacental dysfunction. L-NAME administration caused a paradoxical increase in NO synthesis determined by direct detection of NO by electrochemistry, nitrite accumulation, and cGMP levels, indicating that a lack of NO was not the cause of the fetal growth retardation. Additionally, supplemental L-arginine or NO donors failed to reverse the effects of L-NAME on fetal and placental size. Administration of low dose endotoxin (30 micrograms/kg IP daily for 6 d) also caused significant reductions in fetal and placental size and increased NO synthesis comparable to that seen with L-NAME. Inducible NOS was constitutively expressed in the pregnant uterus (smooth muscle and epithelia) and placenta (sinusoids and macrophages) but was absent in the nonpregnant state as determined by RT-PCR and immunohistochemistry. Neither L-NAME nor endotoxin modified the expression of iNOS. In situ evidence for apoptosis (DNA fragmentation) was minimal to absent in control pregnant rats, but markedly evident in the placenta (decidua) and uterus of rats treated with L-NAME or endotoxin. Immunohistochemical evidence for nitrotyrosine, a marker for peroxynitrite formation, was absent in control rats but colocalized with apoptosis in the L-NAME and LPS groups. We conclude that L-NAME-induced fetal growth retardation is not due to a lack of NO, but as for endotoxin, results from a net reduction in cellular proliferation due to the induction of apoptosis, possibly in response to peroxynitrite formation.

Animals↗
PubMed1996

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats.

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1 alpha on lymphoid cells in the thymus, lymph node, spleen and heart allografts. ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W3/13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1 alpha on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecule which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1 alpha-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

Animals↗