Occult cytomegalovirus in vivarium-housed mice may influence transplant allograft acceptance.

We have recently shown that latent murine cytomegalovirus (MCMV) can influence murine transplant allograft acceptance. During these studies we became aware that vivarium-housed control mice can acquire occult MCMV infection. The purpose of this investigation was to confirm occult MCMV transmission and determine the timing, vehicle, and possible consequences of transmission. Mice arriving from a commercial vendor were negative for MCMV both by commercial serologic testing and by our nested PCR. Mice housed in our vivarium became positive for MCMV DNA 30-60 days after arrival, but remained negative for MCMV by commercial serologic testing. To confirm MCMV we sequenced PCR products for several genes and showed >99% homology to MCMV. Further sequence analyses show that the occult MCMV is similar to a laboratory strain of MCMV, but the vehicle of transmission remains unclear. Control tissues from historical experiments with unexplained graft losses were evaluated for occult MCMV, and mice with unexplained allograft losses showed significantly higher incidence of occult MCMV than did allograft acceptors. Deliberate infection with very low titer MCMV confirmed that viral transmission can occur without measurable virus specific antibody or T-cell responses. These data suggest that vivarium-housed mice can develop occult MCMV that is missed by currently available commercial serologic testing, and that these infections may influence transplant allograft acceptance.

Allogeneic stimulation causes transcriptional reactivation of latent murine cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well-described complication of transplantation that may be caused by allogeneic stimulation, immunosuppression, or both. These studies were performed to determine if allogeneic stimulation alone is sufficient to reactivate latent CMV. BALB/c mice latently infected with Smith strain murine CMV (MCMV) received allograft (n = 8), allograft plus cortisol (n = 5), or isograft (n = 4) skin. All allograft recipients rejected their grafts within 9 to 12 days of transplantation. Three weeks after grafting, recipients were evaluated for MCMV reactivation, and all allograft recipients (8/8) showed MCMV reactivation, while no isografts had reactivation (0/4). Surprisingly, cortisol therapy blocked MCMV reactivation (0/5). These data suggested that allogeneic stimulation alone can trigger systemic reactivation of latent CMV. Although immunosuppression is thought to contribute to reactivation, certain agents that impair NF-kappaB activation may actually reduce reactivation.

Disruption of murine cardiac allograft acceptance by latent cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well-described complication of solid organ transplantation. These studies were performed to (1) determine if cardiac allograft transplantation of latently infected recipients results in reactivation of CMV and (2) determine what impact CMV might have on development of graft acceptance/tolerance. BALB/c cardiac allografts were transplanted into C57BL/6 mice with/without latent murine CMV (MCMV). Recipients were treated with gallium nitrate induction and monitored for graft survival, viral immunity and donor reactive DTH responses. Latently infected allograft recipients had approximately 80% graft loss by 100 days after transplant, compared with approximately 8% graft loss in naïve recipients. PCR evaluation demonstrated that MCMV was transmitted to cardiac grafts in all latently infected recipients, and 4/8 allografts had active viral transcription compared to 0/6 isografts. Latently infected allograft recipients showed intragraft IFN-alpha expression consistent with MCMV reactivation, but MCMV did not appear to negatively influence regulatory gene expression. Infected allograft recipients had disruption of splenocyte DTH regulation, but recipient splenocytes remained unresponsive to donor antigen even after allograft losses. These data suggest that transplantation in an environment of latent CMV infection may reactivate virus, and that intragraft responses disrupt development of allograft acceptance.

Blockade NF-kappaB activation prohibits TNF-alpha-induced cyclooxygenase-2 gene expression in ED27 trophoblast-like cells.

Among the many functions of trophoblast cells is the production of prostaglandins (PGs) for governing several fetoplacental vascular functions during gestation and the triggering of events leading to parturition. Recent evidence suggests that pro-inflammatory cytokines such as tumour necrosis factors (TNF-alpha) induce PG formation via cyclooxygenase-2 (COX-2), a highly inducible enzyme whose gene is regulated at least in part by inducible transcription factor NF-kappaB. To examine the mechanism by which COX-2-driven PG biosynthesis occurs in trophoblast cells, we utilized the immortalized trophoblast-like cell line ED(27). These cells exhibit many of the properties of villous or extravillous trophoblasts and produce large amounts of PGs in response to TNF-alpha. We demonstrated that challenge of ED(27)cells with TNF-alpha caused binding of the NF-kappaB complex to its kappaB site followed by increased accumulation of COX-2 transcripts. In addition, the inhibitor of NF-kappaB, IkappaB-alpha, became phosphorylated and was rapidly degraded in cytokine-treated cells; this process was abolished by co-incubation with the proteasome inhibitor, MG-132. Finally, when cells were pre-incubated with MG-132 and then challenged with TNF-alpha, PG formation was attenuated in a concentration-dependent manner. These data indicate that, in ED(27)trophoblast-like cells isolated from the first-trimester placenta, TNF-alpha treatment leads to activation of NF-kappaB and subsequent transcription of the COX-2 gene.

Interleukin-1 alpha, epidermal growth factor, and transforming growth factor-beta exhibit differential kinetics on endothelin-1 synthesis in amnion cells.

OBJECTIVE: To investigate the effects of three cytokines, interleukin-1 alpha (IL-1 alpha), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta), on the regulation of endothelin-1 (ET-1) mRNA and protein production in human amnion cells. METHODS: Human amnion cells were harvested from uncomplicated pregnancies undergoing elective cesarean delivery at term and grown in primary monolayer culture. Cells were treated with IL-1 alpha, EGF, and TGF-beta for dose-response and time course experiments. Northern analysis was used to determine ET-1 mRNA expression, and enzyme-linked immunosorbent assay was used for ET-1 peptide determination. RESULTS: Interleukin-1 alpha, EGF, and TGF-beta induced the expression of ET-1 mRNA and protein in a dose- and time-dependent fashion. The kinetics of ET-1 mRNA production did not differ markedly with respect to the inducing cytokine, but the kinetics of ET-1 protein production was quite different. Interleukin-1 alpha and EGF stimulated a rapid increase in ET-1 that peaked by 24 hours, and the levels declined to just above the detection limit by 72 hours. In contrast, TGF-beta-stimulated cells showed modest ET-1 production at early times (4-24 hours) and then gradually increased and peaked at 72 hours. CONCLUSIONS: Cytokines modulate the expression of ET-1 mRNA and its cognate protein in human amnion cells. The differential kinetics of ET-1 peptide expression in amnion cells suggests that ET metabolism as well as synthesis contribute to the net expression of endothelin in amnion.

Coordinate expression of inducible nitric oxide synthase and cyclooxygenase-2 genes in uterine tissues of endotoxin-treated pregnant mice.

OBJECTIVES: Our purpose was to investigate the relationship between expression of cyclooxygenase-2 and inducible nitric oxide synthase genes after labor induction with bacterial lipopolysaccharide in a murine model of preterm parturition. STUDY DESIGN: Pregnant C57B1/6 mice were given Escherichia coli lipopolysaccharide (20 micrograms per mouse) by intraperitoneal injection on day 16 of gestation, and the animals were followed up for signs of labor. Control mice received an equivalent volume of 0.9% saline solution. The latency from lipopolysaccharide injections until appearance of the first pup was recorded. Two separate groups of mice were given either aminoguanidine or indomethacin (5 mg/kg intragastric) 24 hours before induction of preterm labor. In a separate set of experiments mice were treated with lipopolysaccharide as described and were killed at intervals from 0.5 to 72 hours and intrauterine tissues (uterus, placenta, and fetal membranes) were removed and snap frozen in liquid nitrogen. Total protein and ribonucleic acid were extracted for Western and Northern blot analysis of cyclooxygenase-2 and inducible nitric oxide synthase protein and messenger ribonucleic acid, respectively. RESULTS: Northern blots from uterine, placental, and fetal membrane tissues of lipopolysaccharide- and saline solution-treated mice revealed that cyclooxygenase-2 and inducible nitric oxide synthase messenger ribonucleic acid transcripts were rapidly (within 0.5 to 2 hours) up-regulated after lipopolysaccharide administration but were unchanged in mice injected with saline solution. Immunoblot analysis with isoform-specific antibodies revealed that both enzymes were expressed in uterus, placenta, and fetal membranes in a coordinated fashion with peak expression seen at 6 to 8 hours. Although the steady-state accumulation of messenger ribonucleic acid transcripts encoding cyclooxygenase-2 and inducible nitric oxide synthase peaked at 6 hours and declined to baseline by 16 hours after injection with lipopolysaccharide, expression of cyclooxygenase-2 and inducible nitric oxide synthase was sustained through the period when premature delivery was observed. Nitric oxide-dependent cyclooxygenase-2 and inducible nitric oxide synthase expression was demonstrated by the elimination of accumulation of both messenger ribonucleic acid transcripts in mice pretreated with aminoguanidine before injection with lipopolysaccharide. CONCLUSIONS: These data indicate that nitric oxide synthesis may be a prerequisite for subsequent stimulation of cyclooxygenase-2 and inducible nitric oxide synthase gene expression. Taken together, the data suggest that cyclooxygenase-2 and inducible nitric oxide synthase are expressed in a coordinated manner in the uterus of endotoxin-challenged pregnant mice and that their enzymatic products may contribute to the signaling of uterine activity or cervical changes culminating in expulsion of the fetus.

Interleukin-1 receptor antagonist blocks interleukin-1-induced expression of cyclooxygenase-2 in endometrium.

OBJECTIVE: Our purpose was to test the hypothesis that the interleukin-1 receptor antagonist can inhibit interleukin-1-induced prostaglandin production and de novo expression of the inducible cyclooxygenase-2 isoform in a human endometrial epithelial cell line. STUDY DESIGN: A continuous line of human endometrial epithelial cells was established from a hysterectomy specimen from a nonmalignant uterus. Cells were maintained as a monolayer culture in medium 199 supplemented with 10% fetal bovine serum and 50 micrograms/ml gentamicin. Cultures were treated with cytokines (interleukin-1 alpha or interleukin-1 beta, interleukin-1 receptor antagonist, or tumor necrosis factor-alpha), and media were collected for analysis of prostaglandin E2 and prostaglandin F2 alpha) by radioimmunoassay, whereas cells were harvested for ribonucleic acid and protein extractions and subsequent Northern blot or Western blot analyses, respectively. RESULTS: When endometrial cells were incubated with interleukin-1 alpha or interleukin-1 beta, each cytokine was shown to stimulate the production of prostaglandin E2 and prostaglandin F2 alpha in a time- and dose-dependent fashion, with interleukin-1 alpha being far more potent than interleukin-1 beta. Interleukin-1 receptor antagonist inhibited interleukin-1 alpha- and interleukin-1 beta-induced prostaglandin formation, with 50% inhibitory concentration values of 30 ng/ml for prostaglandin E2 and 90 ng/ml for prostaglandin F2 alpha. When Northern blots of interleukin-1 alpha-treated cells were probed with a complementary deoxyribonucleic acid fragment specific for either cyclooxygenase-1 or cyclooxygenase-2, rapid de novo induction of cyclooxygenase-2 messenger ribonucleic acid was observed; however, cyclooxygenase-1 expression was constant regardless of interleukin-1 alpha concentration or incubation time. Coincubation of cells with interleukin-1 alpha (10 ng/ml) and cycloheximide caused superinduction of cyclooxygenase-2 messenger ribonucleic acid but had no effect on the expression of cyclooxygenase-1 messenger ribonucleic acid. Actinomycin D completely abolished interleukin-1 alpha-induced cyclooxygenase-2 messenger ribonucleic acid expression, suggesting that the cytokine caused transcriptional activation of the cyclooxygenase-2 gene. Experiments were conducted to examine whether interleukin-1 receptor antagonist could suppress interleukin-1-induced cyclooxygenase-2 expression. Cells were preincubated for 30 minutes with interleukin-1 receptor antagonist and then challenged with interleukin-1 alpha. Northern and Western analyses revealed that interleukin-1 receptor antagonist blocked interleukin-1 alpha-induced expression of cyclooxygenase-2 messenger ribonucleic acid transcripts and the subsequent appearance of cyclooxygenase-2 protein. Interleukin-1 receptor antagonist had no effect on the constitutive expression of cyclooxygenase-1 messenger ribonucleic acid and protein. Interleukin-1 receptor antagonist failed to alter prostaglandin E2 formation in response to tumor necrosis factor-alpha, indicating that the antagonist is specific for interleukin-1 family cytokines. Finally, interleukin-1 receptor antagonist acted as a partial agonist in some experiments in that relatively high concentrations (> 100 ng/ml) caused a modest increase in prostaglandin E2 and F2 alpha production. CONCLUSIONS: These data indicate that interleukin-1 receptor antagonist is a potent inhibitor of interleukin-1-induced arachidonic acid metabolism and could possibly serve as an endogenous or exogenous modulator of interleukin-1 action in the endometrial epithelium.

Tumour necrosis factor-alpha induces cyclo-oxygenase-2 gene expression in first trimester trophoblasts: suppression by glucocorticoids and NSAIDs.

Tumour necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine which stimulates the synthesis and release of prostaglandins (PGs) in several in vitro and in vivo models of preterm labour. While TNF-alpha simulated PG production has been described in decidual, amnion and myometrial cells, to date no studies have focused on the role of TNF-alpha in the stimulation of arachidonic acid metabolism in placental trophoblast cells. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in PG biosynthesis and is expressed de novo during cellular activation by cytokines. To test whether TNF-alpha alters expression of COX-2, trophoblasts from first trimester chorionic vili were cultured as a continuous cell line and treated with TNF-alpha alone or with TNF-alpha and dexamethasone (Dex). Total RNA and protein were extracted from the trophoblasts and subjected to Northern and immunoblot analysis, respectively. Northern blots were hybridized with a 32P-labelled probe encoding the COX-2 cDNA and immunoblots were incubated with anti-COX-2 antibodies. There was a time- and dose-dependent increase in COX-2 mRNA and protein expression in cells stimulated with TNF-alpha. The effect of TNF-alpha on COX-2 mRNA and protein expression was inhibited by dexamethasone (Dex). To examine the production of PGE2 and PGF(2 alpha), specific RIAs were performed on culture media from similarly stimulated cells. PG accumulation after TNF-alpha stimulation occurred in a time- and dose-dependent fashion with a similar inhibition of PG accumulation after Dex exposure. To be certain that TNF-alpha stimulated PGE2 production was, indeed, a result of COX-2 induction, RIAs were carried out with the COX-2-selective inhibitor NS-398. Cells stimulated with the NS-398 after TNF-alpha exposure demonstrated suppression of TNF-alpha-stimulated PGE2 formation. The results suggest that TNF-alpha elicits part of its pathophysiologic effects in preterm labour via alterations in COX-2 gene expression within the placental microenvironment.

Genistein suppresses EGF-induced prostaglandin biosynthesis by a mechanism independent of EGF receptor tyrosine kinase inhibition.

This study demonstrated that genistein, a selective tyrosine kinase inhibitor, blocked PGE2 production in human A431 and WISH cells and murine 3T3 cells in response to epidermal growth factor and platelet-derived growth factor. Blockade of growth factor-induced PGE2 production was dose-dependent (IC50 approximately equal to 7-8 microM). Genistein also abolished PGE2 formation in response to calcium ionophores, A23187 and ionomycin, and the phorbol ester, phorbol myristate acetate. Moreover, genistein-treated A431 and WISH cells incorporated significantly less [3H]arachidonic acid into membrane phospholipids than control cells. Finally, genistein decreased the specific activity of prostaglandin H2 synthase prepared from A431 cells, WISH cells, and ram seminal vesicle. The IC50 of genistein for inhibition of prostaglandin H2 synthase specific activity extracted from A431 and WISH cells approximated that half-maximal inhibitory concentration in the whole cell assay. These data indicate that genistein may interfere with arachidonic acid metabolism at several key points by a mechanism(s) that is independent of its inhibitory action on receptor tyrosine protein kinases. Taken together, these results also suggest that caution should be exercised when drawing conclusions about the putative role of tyrosine kinases in signal transduction events using genistein as a pharmacological blocker.

Insulin and glucose modulate glucose transporter messenger ribonucleic acid expression and glucose uptake in trophoblasts isolated from first-trimester chorionic villi.

OBJECTIVE: Our purpose was to determine the effects of insulin and glucose on glucose transport and expression of GLUT1 glucose transporter messenger ribonucleic acid in first-trimester human trophoblast-like cells. STUDY DESIGN: First-trimester human trophoblast-like cells were maintained as a continuous cell line. For 2[3H]deoxy-D-glucose uptake and messenger ribonucleic acid studies the cells were incubated in the presence or absence of insulin (10(-7) to 10(-11) mol/L) or D-glucose (0 to 50 mmol/L) for 0 to 24 hours. Glucose transport was measured by incubating cells with 0.1 mmol/L 2[3H]deoxy-D-glucose for 5 minutes. Specific uptake was determined by incubating companion cultures with 10 mumol/L cytochalasin B. The cells were then solubilized with sodium hydroxide and the radioactivity counted. Data were expressed as nanomoles of 2[3H]deoxy-D-glucose transported per milligram of protein per 5 minutes and analyzed by one-way analysis of variance with post hoc testing by the method of Tukey. GLUT1 messenger ribonucleic acid was measured by Northern blotting of total ribonucleic acid samples hybridized to a phosphorus 32-labeled complementary deoxyribonucleic encoding the rat GLUT1 glucose transporter. As a control for loading efficiency, blots were stripped and rehybridized to a 40-mer phosphorus 32-labeled beta-actin oligonucleotide probe. RESULTS: Insulin treatment resulted in a dose-dependent increase in the transport of 2[3H]deoxy-D-glucose at 24 hours (p < 0.001 at 10(-7) mol/L). This change was first detected at 12 hours of incubation. These data closely paralleled the insulin-induced increase in GLUT1 messenger ribonucleic acid seen in Northern blots. In contrast to insulin, increasing concentrations of D-glucose did not change the transport of 2[3H]deoxy-D-glucose. However, when cells were incubated in low concentrations of D-glucose (0 or 1 mmol/L), an enhancement in the uptake of 2[3H]deoxy-D-glucose (p < 0.001) was observed. Kinetic studies indicated that D-glucose augmentation of 2[3H]eoxy-D-glucose uptake was significant at 9 hours (p < 0.05). The effects of D-glucose on GLUT1 messenger ribonucleic acid expression paralleled the uptake of 2[3H]deoxy-D-glucose, although the modulation of GLUT1 messenger ribonucleic acid levels by glucose was much less pronounced than in insulin-treated cells. CONCLUSION: Although it has been assumed that the placenta has a limited role in influencing glucose transport to the fetus, our in vitro data demonstrate that both insulin and glucose can modulate glucose transport at the cellular level of the placental trophoblast. Thus maternal insulin and glycemic status may influence the expression of GLUT1, the major trophoblast glucose transporter protein, therefore directly affecting first-trimester placental glucose transport. These in vitro data may help explain the association between maternal glucose abnormalities and impaired fetal development during the first trimester when placental GLUT1 messenger ribonucleic acid expression is at its peak.

Interleukin-1 beta induces cyclooxygenase-2 in cultured human decidual cells.

PROBLEM: The purpose of this study was to examine the hypothesis that interleukin-1 beta (IL-1 beta)-elicited increases in decidual prostaglandin E2 and F2 alpha (PGE2 and PGF2 alpha) biosynthesis are due to the de novo expression of the inducible isoform of cyclooxygenase (i.e., COX-2). METHOD: Primary human decidual cell cultures were established from term placentas delivered by cesarean section. After 8 days in vitro, when the cultures secreted immunoreactive prolactin, the cells were incubated for 24 h in serum-free medium, and then challenged with IL-1 beta from 1 to 48 h. PGE2 and PGF2 alpha content in the media were measured by specific radioimmunoassays. RESULTS: IL-1 beta stimulated a time-dependent enhancement in PGE2 and PGF2 alpha production, with PGF2 alpha synthesis predominating over PGE2. IL-1 beta also induced a dose-dependent increase in the output of both arachidonic acid metabolites. When Northern blots of IL-1 beta-treated and control cells were probed with cDNAs encoding either COX-1 or COX-2 isoforms or an oligonucleotide probe encoding a portion of the human beta-actin, we detected a time- and dose-dependent increase in the steady-state levels of COX-2, but not COX-1 or beta-actin mRNA transcripts. Moreover, the expression of COX-2 mRNA in IL-1 beta-stimulated cells was superinduced by preincubation with cycloheximide, but completely abolished by actinomycin D. CONCLUSIONS: Taken together, the data suggest that COX-2 mRNA expression is largely responsible for the robust increase in PG formation seen in IL-1 beta-treated decidual cells.

Interleukin-1 beta regulates the inducible cyclooxygenase in amnion-derived WISH cells.

The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in amnion-derived WISH cell cultures when stimulated by interleukin-1 beta (IL-1 beta). Confluent monolayer cultures of WISH cells were incubated with human recombinant IL-1 beta (0.001-10 ng/ml) for 0-24 hours, while control cells received medium alone. PGE2 production was measured by specific radioimmunoassay. IL-1 beta enhanced the production of PGE2 in a dose- and time-dependent manner with enhanced production detectable by 2 h following exposure. Immunoblot analysis using isoform-specific antibodies showed that the inducible cyclooxygenase enzyme, i.e., COX-2, was expressed by 2 h in IL-1 treated cells, while the constitutive COX-1 remained unaltered in its expression. Northern blot analysis demonstrated that COX-2 mRNA expression was not detected in untreated cells, but became evident after a 30-min exposure to IL-1 beta (10 ng/ml). COX-1 mRNA was detected under basal conditions and did not increase significantly following IL-1 beta treatment. The close parallel between the kinetics of COX-2 mRNA and protein expression and PGE2 accumulation in the medium, as well as the constitutive, unregulated nature of the COX-1 isoform, indicates that cytokine-driven PGE2 formation in WISH cells may be mediated by de novo expression of the novel COX-2 enzyme.

Insulinlike growth factors. Their regulation of glucose and amino acid transport in placental trophoblasts isolated from first-trimester chorionic villi.

The transport of glucose and amino acids from the maternal to fetal circulation through the placenta is critical to the delivery of fuel for normal fetal growth and development. Little information indicates that transplacental glucose or amino acid transport is influenced by hormones or polypeptide growth factors. We developed a continuous cell line of cytotrophoblastlike cells derived from first-trimester human chorionic villi as a model system to study the regulation of glucose and amino acid transport by insulinlike growth factors (IGFs). Using immunocytochemical and biochemical criteria, the cells were shown to manifest a trophoblastlike phenotype. The cells were maintained in serum-supplemented medium until confluent, at which time they were shifted to serum-free medium for one day. Experiments were initiated by transferring the cells to glucose-free assay buffer and incubating them with IGF-I, IGF-II or insulin. Glucose uptake was measured by the transport of 2-deoxy-D-[1,2-3H]glucose (2[3H]DG) in the presence or absence of cytochalasin B, which has been shown to competitively inhibit glucose uptake. IGF-I, IGF-II and insulin each enhanced 2[3H]DG transport in a dose-dependent fashion. Amino acid transport was measured by incubation of the cells with IGF-I for 60 minutes, followed by a 5-minute challenge with alpha-[methyl-3H]aminoisobutyric acid. IGF-I caused a dose-dependent increase in uptake of the amino acid analog. Radioreceptor assays using [125I]insulinlike growth factor I ([125I]IGF-I) demonstrated that the trophoblast-derived cells contained high-affinity, saturable receptors for IGF-I that also bound IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of functional insulin-like growth factor-I receptors by human amnion cells.

OBJECTIVES: The purpose of the study was to investigate whether amnion cells contain functional insulin-like growth factor-I receptors. STUDY DESIGN: To test whether human amnion cells contain insulin-like growth factor-I receptors, radioligand binding studies, affinity cross-linking studies, and Northern blot analysis were conducted in primary amnion cells and in an immortal amnion cell line (WISH). To test whether the insulin-like growth factor-I receptors on amnion cells are functional, cytochalasin B-inhibitable 2-deoxyglucose uptake was measured after stimulating the cells with insulin-like growth factor-I. RESULTS: Radioligand binding studies demonstrated that primary amnion cells and WISH cells contained a single class of high-affinity receptors with an apparent dissociation constant of 0.18 +/- 0.04 nmol/L and a receptor concentration of 79 +/- 26.2 fmol/mg protein and dissociation constant of 0.44 +/- 0.03 nmol/L and a receptor concentration of 33.3 +/- 6.45 fmol per 106 cells, respectively. Affinity cross-linking studies revealed two major insulin-like growth factor-I binding sites, 135 and 270 kd. Both primary amnion cells and WISH cells exhibited cytochalasin B-inhibitable tritiated 2-deoxyglucose uptake in response to insulin-like growth factor-I treatment. Finally, treatment of WISH cells caused tyrosine phosphorylation of three proteins (molecular weight, 116, 95.4, and 83.5 kd) was observed by Western blotting with antiphosphotyrosine antibodies. CONCLUSION: These results provide the first evidence that human amnion epithelial cells contain functional high-affinity insulin-like growth factor-I receptors that mediate glucose transport.

Transforming growth factor-beta potentiates epidermal growth factor-induced prostaglandin E2 production in amnion cells.

Primary cultures of human amnion cells and the amnion-derived cell line WISH were used to evaluate the hypothesis that transforming growth factor-beta (TGF-beta) can modulate epidermal growth factor (EGF)- induced prostaglandin E2 (PGE2) production. Cells were preincubated for 1 hr with TGF-beta (0.0001-10 ng/ml) and then incubated in the presence or absence of EGF (10 ng/ml) for 4 hrs. TGF-beta alone did not stimulate PGE2 synthesis at any dose examined. However, when primary cultures of amnion cells or WISH cells were preincubated with TGF-beta and then challenged with EGF, there was a potentiation of PGE2 production that was much greater than the additive values of TGF-beta or EGF alone. These data suggest that EGF-induced PGE2 production by amnion cells can be modulated by low concentrations of TGF-beta.

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.

Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides.

The enzyme exonuclease I from Escherichia coli hydrolyzes successive nucleotides from the 3'-termini of single-stranded deoxyribonucleotide homopolymers. When the reaction is stopped after partial hydrolysis, only intact starting material and small oligomers can be isolated. The distribution of oligomeric products varies with the base composition of the polymer but the largest oligomer that can be isolated from the reaction of exonuclease I with homopolymers of deoxyadenylate, deoxythymidylate, or deoxycytidylate is a decamer. These results suggest a model in which exonuclease I possesses at least two nucleotide binding sites. When both sites are filled, with 11-mers and longer polymers, the enzyme does not dissociate from the polymer during hydrolysis. When, with smaller oligomers, only a single site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation. The kinetics of the reactions of exonuclease I with purified polydeoxyriboadenylates of defined size distributions have been investigated. The maximum rates of hydrolysis are nearly independent of polymer size while the apparent Michaelis constants are inversely proportional to the polymer size. A simple steady state model yields a kinetic equation that is consistent with our results. Competition experiments indicate that the rate at which exonuclease I associates with the 3'-terminus of a polydeoxyribonucleotide is independent of the polymer's chain length.

Asteroidal source of meteorites.

The evolution of asteroidal orbits initially near the Kirkwood Gap at the 1:2 commensurability with Jupiter's period provides a mechanism for the production of meteorites from the asteroid belt without excessive velocity change. The resulting yield ( approximately 10(9) grams per year) and the orbital elements of Earth-crossing objects are in agreement with observational data on meteorites.