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Biomedical subjects

C L Librach

Publications and source records attributed to C L Librach.

14 recordsLinked to original sources
PubMed2000

HLA-G has a concentration-dependent effect on the generation of an allo-CTL response.

Human leucocyte antigen (HLA) -G is expressed on trophoblast cells during pregnancy, suggesting a role in protection of the semiallogeneic fetus. Published data suggest that HLA-G protects a cell against natural killer cell lysis. It has been hypothesized that HLA-G may also protect the fetus by preventing allo-cytotoxic T lymphocyte (CTL) responses. To test this hypothesis, we assayed the effects of various concentrations of purified HLA-G on CTL response in a mixed lymphocyte culture (MLC) system. We found that concentrations > or =0.1 microg/ml of HLA-G suppressed the allo-CTL response by 30-100% over the control, but, paradoxically, concentrations of 0.01-0.05 microg/ml of HLA-G augmented the allo-CTL response by 25-50% over the control. Concentrations < or = 0.001 microg/ml HLA-G had no effect. Addition of HLA-G to preprimed allo-CTL effector cells did not affect their killing ability. Allo-CTL suppressive doses of HLA-G induced a T helper type 2 (Th2) cytokine response, whereas allo-CTL-enhancing doses of HLA-G induced a Th1-type cytokine response. HLA-G purified from first-trimester placenta does not affect allo-proliferative responses nor does it alter the percentage of CD4+ or CD8+ T cells in MLCs. These findings support a potential role for HLA-G-mediated suppression of allo-CTL formation in normal pregnancies. In addition, the effects observed at lower concentrations of HLA-G may have interesting implications for the condition of pre-eclampsia in which concentrations of this HLA class I molecule are reduced.

Antibodies, Monoclonal↗
PubMed1996

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Antibodies, Monoclonal↗
PubMed1996

Embryonic human leukocyte antigen-G expression: possible implications for human preimplantation development.

OBJECTIVE: To investigate further the association between human leukocyte antigen G (HLA-G) expression in human embryos and other factors known to influence IVF pregnancy outcome. SETTING: A university-based tertiary referral center (The Toronto Hospital). INTERVENTIONS: Nontransferred embryos at the two- to four-cell stage were obtained from patients undergoing IVF and were cultured in Ham's F-10 medium supplemented with 10% human sera or cocultured with ovarian cancer cells in the same medium. Embryos that reached blastocyst stage (n = 148) were analyzed by reverse transcriptase-polymerase chain reaction for HLA-G and beta 2 microglobulin (beta 2m) expression. Statistical analysis was performed to identify possible factors associated with variability of expression. RESULTS: Approximately 40% of studied blastocysts had detectable expression of both HLA-G and beta 2m messenger RNA. In 46% of blastocysts, beta 2m alone was observed. Interestingly, sibling embryos from patients that became pregnant were significantly more likely to express HLA-G than embryos from patients that did not conceive as a result of their IVF cycles. No association was found between HLA-G expression and culture conditions, patients age, or infertility diagnosis. CONCLUSION: The population of embryos obtained through IVF is heterogeneous in expression of HLA-G and beta 2m, which may reflect overall health of the embryos. Blastocysts showing positive HLA-G expression may have increased viability and implantation potential, although the underlying mechanisms remain to be elucidated.

Base Sequence↗
PubMed1995

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Amino Acid Sequence↗
PubMed1994

Interleukin-1 beta regulates human cytotrophoblast metalloproteinase activity and invasion in vitro.

During early human pregnancy, fetal cytotrophoblasts rapidly invade the uterus. This process has many similarities to tumor invasion, except that the extent and the timing of cytotrophoblast invasion are carefully regulated. Therefore, this system is particularly useful for studying mechanisms that regulate invasive processes. Previously, we showed that production and activation of the 92-kDa type IV collagenase (matrix metalloproteinase(MMP)-9) is necessary for cytotrophoblast invasion in vitro. In other systems, interleukin (IL)-1 beta is an important regulator of matrix-degrading metalloproteinases. Therefore, we investigated trophoblast production of IL-1 beta and its receptors, as well as the effects of this cytokine on cytotrophoblast metalloproteinase activity and invasion. The results showed that release of IL-1 beta parallels the invasive potential of the cytotrophoblasts; the highest levels are produced by first trimester cells and the lowest levels by term cells. Immunoprecipitation showed that cytotrophoblasts express the 80-kDa type I IL-1 receptor, suggesting that autocrine effects are possible. IL-1 beta stimulated trophoblast MMP-9 secretion (by a mechanism that required nascent mRNA and protein synthesis) as well as metalloproteinase activity and invasion of Matrigel. Increasing (by lipopolysaccharide treatment) or decreasing (by glucocorticoid treatment) IL-1 beta production had parallel effects on MMP-9 secretion, metalloproteinase activity, and invasion. Because IL-1 beta and corticosteroids are present in high concentrations at the maternal-fetal interface, normal trophoblast invasion may be regulated, in part, by their opposing actions. In contrast, stimulation of cytotrophoblast IL-1 beta secretion by lipopolysaccharide may play a role in the sequela of infected fetal membranes.

Collagenases↗
PubMed1992

Localization and regulation of the activin-A dimer in human placental cells.

Subunits of activin and inhibin and their mRNAs are present in human placental and decidual cells. However, evidence for the presence of intact activin dimers in the human placenta and their regulation has been lacking. Using a monoclonal antibody raised against the human activin-A dimer, we examined the cellular localization of immunoreactive activin-A dimer in human placentas of different gestational ages (8-41 weeks). In addition, we determined the effects of culture and various potential regulators on the cellular accumulation of immunoreactive activin-A dimer in trophoblast cells from human first trimester placentas. Activin-A dimer was found in both cyto- and syncytiotrophoblast cells of all gestational ages studied. Immunoreactive activin-A also was detected in placental Hofbauer cells in first and second trimester placentas as well as in cells of the placental membranes. Exposure of these cells to cAMP, GnRH, activin, inhibin, transforming growth factor-beta, dexamethasone, and interleukin-1 did not significantly change the intensity of immunostaining for activin-A dimer. These results together with previous data suggest that placental cells are a source of activin-A and that activin-A may be a paracrine and/or endocrine regulator of feto-maternal interactions during pregnancy.

Activins↗
PubMed1991

92-kD type IV collagenase mediates invasion of human cytotrophoblasts.

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.

Antibody Specificity↗
PubMed1988

Weights of fetuses with autosomal trisomies at termination of pregnancy: an investigation of the etiologic factors of low serum alpha-fetoprotein values.

In order to determine whether the low values of maternal serum alpha-fetoprotein observed with autosomal trisomies are associated with smaller fetal weights, 50 fetuses with Down syndrome (trisomy 21), 10 with trisomy 18, and 65 normal control fetuses, all aborted in the second trimester of pregnancy, were compared. The mean multiple of the median maternal serum alpha-fetoprotein was found to be 0.79 +/- 0.61 for fetuses with Down syndrome and 0.50 +/- 0.26 for those with trisomy 18, both results being significantly lower than results from the control fetuses (0.97 +/- 0.86). No significant difference in the weight distribution between fetuses with Down syndrome and control fetuses, corrected for gestational age, was found. By contrast, fetuses with trisomy 18 had a significantly lower weight distribution compared with that of the control fetuses (p less than 0.001). A linear relationship was found in normal fetuses between maternal serum alpha-fetoprotein values and fetal weight at a given gestational age. Fetal weight does not seem to account for the lower maternal serum alpha-fetoprotein levels seen in fetuses with Down syndrome but may partially account for the lower levels seen in fetuses with trisomy 18.

Abortion, Induced↗
PubMed1986

The influence of prolonged pregnancy on infant development at one and two years of age: a prospective controlled study.

Isolated reports of developmental disturbances following prolonged pregnancy led us to compare, prospectively, at 1 and 2 years of age, infants born after normal term gestations with those born after prolonged pregnancies (exceeding 294 days). The infants were subgrouped according to their physical condition at birth, that is, normal or dysmature (mild or advanced dysmaturity). Infant assessments included: (1) height and weight, (2) hospitalizations, and (3) mental development by the Griffiths Mental Development Scales. Follow-up testing was obtained on 130 term control infants and 89 infants of prolonged pregnancies at 1 year of age and 111 term control infants and 76 infants of prolonged pregnancies at 2 years of age. At 1 and 2 years the general intelligence quotient, physical milestones, and intercurrent illnesses for normal infants and those of prolonged pregnancies were not significantly different.

Body Height↗
PubMed1986

Microsurgical reversal of sterilization: a six-year study.

A prospective study of 113 personal consecutive microsurgical reversals of female sterilization during the 6-year period from 1979 to 1984 was carried out to determine factors affecting the pregnancy rate. The sterilizations were performed by laparoscopic unipolar coagulation in 54% of the patients, by the Pomeroy technique in 28%, by fimbriectomy in 8%, by the Irving operation in 5%, and by clips or rings in 4%. In the group with no minimum follow-up period, 50% had intrauterine pregnancies and 5% had ectopic gestations. Eighty-nine patients had at least 12 months of follow-up after reversal surgery. This group is studied in detail. Factors affecting the pregnancy rate were length of tube, type of sterilization performed, anastomotic site, and availability of both tubes for reconstruction. Age, parity, and interval from sterilization to reversal surgery did not affect the pregnancy rate. Fifty percent of the intrauterine pregnancies were conceived within 6 months of reversal surgery.

Abortion, Spontaneous↗
PubMed1984

Genetic amniocentesis in seventy twin pregnancies.

Genetic amniocenteses were performed in 70 twin pregnancies over an 11-year period. Both sacs were successfully sampled in 49 of 62 patients (79%). The success rate was decreased (68%) with two placentas (anterior and posterior) and was improved with gestational age greater than or equal to 17 weeks (88%) and with ultrasound visualization of the septum (86%). Of three spontaneous abortions, two were attributed to amniocentesis (chorioamnionitis). When twin pregnancy is diagnosed in a patient with an indication for genetic amniocentesis, a careful reevaluation and discussion of risk factors with the couple are recommended.

Adult↗
PubMed1983

Spontaneous reverse haemolytic plaque formation. II. The role of T cells and monocytes in regulating immunoglobulin secretion by human peripheral blood B cells.

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94%; this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10-1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1, this significantly reduced or abolished spontaneous IgG plaques, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for monocytes, OKM1 and FMC17, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors.

Animals↗
PubMed1982

Rapid replicate reverse haemolytic plaque assay directly in microwells used for tissue culture.

The reverse haemolytic plaque assay was adapted as a micromethod using microwells of flat-bottomed microtitre trays. To microwells containing the lymphocytes under test were added protein A-coupled ox erythrocytes, developing antisera directed against any class of immunoglobulin, and guinea pig complement absorbed with protein A-coupled erythrocytes. Plaques were scored by counting with a stereoscan microscope. The method is applicable both to spontaneous and mitogen-induced plaque formation and, as a further development of the technique to differentiated B cells in pokeweed mitogen-driven cultures, plaques were enumerated directly in the same wells of the microtitre tray as were employed for culture of the B cells. The method allows for several hundred plaque assays at one sitting.

Antibody-Producing Cells↗