Comparison of LPS-stimulated release of cytokines in punch versus transwell tissue culture systems of human gestational membranes

Abstract Background Cytokine signaling within the amnionic, chorionic and decidual extraplacental gestational membranes plays an important role in membrane rupture and the timing of birth. The predominant in vitro explant culture system for evaluating cytokine induction in human gestational membranes has been the free-floating biopsy punch culture. Punch systems have been used to investigate the impact of various toxicants, pharmaceuticals and genetic variation on expression of pro-inflammatory cytokines. More recently, a dual compartment transwell culture system has been developed that more closely mimics the intrauterine compartment. The current study compares these two systems with respect to release of pro- and anti-inflammatory cytokines in response to lipopolysaccharide (LPS), a model stimulant. Methods Tissue samples were exposed to 100 ng/ml LPS for 12 h and cytokines were measured by ELISA. Data are expressed as increase relative to non-treated controls. Results Levels of interleukin-6 increased in punch culture medium samples to a significantly greater extent (34.2 fold) compared with medium from transwell cultures in the amnion (6.6 fold) or choriodecidual (7.1 fold) compartments. Interleukin-8 also showed a significantly greater induction in punch (4.8 fold) than transwell amnion (1.6 fold) or choriodecidual (1.7 fold) samples. The anti-inflammatory interleukin-10 showed a significant difference between punch (36.5 fold) and transwell amnion (15.4 fold) samples, but no difference was observed between punch and transwell choriodecidual (28.5 fold) samples. Neither interleukin-1beta nor tumor necrosis factor-alpha (TNF-alpha) showed a significant difference between the punch and transwell samples. Conclusions These results indicate that the pattern of LPS-stimulated cytokine release from gestational membranes in vitro depends on the culture system used, confounding comparisons of studies that use different gestational membrane culture systems to study inflammatory responses.http://deepblue.lib.umich.edu/bitstream/2027.42/78253/1/1477-7827-8-121.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78253/2/1477-7827-8-121.pdfPeer Reviewe

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Increased contraction frequency in rat uterine strips treated In Vitro with o,p′-DDT

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Tissue-specific cytokine release from human extra-placental membranes stimulated by lipopolysaccharide in a two-compartment tissue culture system

<p>Abstract</p> <p>Background</p> <p>The extra-placental gestational membranes secrete cytokines in response to bacteria and other infectious agents, with potentially adverse consequences for pregnancy. The present study used lipopolysaccharide (LPS) as a prototype endotoxin to investigate the pattern of stimulated cytokine release from the amniotic and choriodecidual sides of full-thickness human gestational membranes in a two-compartment tissue culture system.</p> <p>Methods</p> <p>Gestational membranes were collected from healthy non-laboring caesarean deliveries at term. Full-thickness membranes from each placenta were cut into pieces, mounted on Transwell frames, and placed in culture wells to create a two-compartment culture with the gestational membranes serving as the barrier between compartments. The LPS (100 ng/ml) was added to the amniotic, choriodecidual or both chambers of the culture, and cytokines were assayed in the medium of the amniotic and choriodecidual chambers after 8 h of LPS exposure. Cytokine concentrations were analyzed by two-way analysis of variance for effects of treatment and side specificity of cytokine release from the membranes.</p> <p>Results</p> <p>LPS exposure on the choriodecidual side of the membranes significantly increased TNF-alpha, IL-6, IL-10 and IL-8 in the choriodecidual compartment, whereas TNF-alpha was the only cytokine observed to increase in the amniotic compartment. When LPS treatment was to the amniotic side of the membranes, there were significant increases in TNF-alpha and IL-6 in the amniotic compartment as well as increased concentrations of TNF-alpha, IL-6 and IL-8 in the choriodecidual compartment; however, there were no statistically significant differences for IL-10 in either compartment. No statistically significant differences were observed for IL-1beta, TGF-beta or IL-4 concentrations in response to LPS, regardless of the exposure modality.</p> <p>Conclusion</p> <p>The amnion and choriodecidua exhibited distinct patterns of response to LPS with evidence of inflammatory signaling across the layers of the gestational membranes. These results suggest a complicated network of signaling within the gestational membranes, in which cytokine- and tissue-specific responses to inflammatory stimulation may have important implications for maintaining pregnancy in the challenge of microbial invasion of the uterine compartment.</p

Sustained inhibition of rat myometrial gap junctions and contractions by lindane

BACKGROUND: Gap junctions increase in size and abundance coincident with parturition, forming an intercellular communication network that permits the uterus to develop the forceful, coordinated contractions necessary for delivery of the fetus. Lindane, a pesticide used in the human and veterinary treatment of scabies and lice as well as in agricultural applications, inhibits uterine contractions in vitro, inhibits myometrial gap junctions, and has been associated with prolonged gestation length in rats. The aim of the present study was to investigate whether brief exposures to lindane would elicit sustained inhibition of rat uterine contractile activity and myometrial gap junction intercellular communication. METHODS: To examine effects on uterine contraction, longitudinal uterine strips isolated from late gestation (day 20) rats were exposed to lindane in muscle baths and monitored for changes in spontaneous phasic contractions during and after exposure to lindane. Lucifer yellow dye transfer between myometrial cells in culture was used to monitor gap junction intercellular communication. RESULTS: During a 1-h exposure, 10 micro M and 100 micro M lindane decreased peak force and frequency of uterine contraction but 1 micro M lindane did not. After removal of the exposure buffer, contraction force remained significantly depressed in uterine strips exposed to 100 micro M lindane, returning to less than 50% basal levels 5 h after cessation of lindane exposure. In cultured myometrial myocytes, significant sustained inhibition of Lucifer yellow dye transfer was observed 24 h after lindane exposures as brief as 10 min and as low as 0.1 micro M lindane. CONCLUSION: Brief in vitro exposures to lindane have long-term effects on myometrial functions that are necessary for parturition, inhibiting spontaneous phasic contractions in late gestation rat uterus and gap junction intercellular communication in myometrial cell cultures

Characterization of o,p'-DDT-stimulated contraction frequency in rat uterus in vitro

Exposure to organochlorine pesticides, including DDT, has previously been associated with premature birth. Using an improved protocol to characterize dose and time dependent responses, the present report extends a preliminary finding by this laboratory that o,p'-DDT directly stimulates uterine contractility. Contraction frequency was determined in longitudinal uterine strips from pregnant rats under isometric force conditions. Following equilibration, the uterine strips were monitored for a 1-hr baseline period, then treated with o,p'-DDT or ethanol (solvent control) for 3 hr, followed by 3 hr without test substance. During exposure to 100 [mu] o,p'-DDT, the frequency of contraction significantly increased by 66% relative to matched controls. After removal of o,p'-DDT from the medium, the frequency of contraction continued to increase in uterine strips exposed to 50 and 100 [mu] o,p'-DDT. A dose effect was clearly observed during the post-treatment period, with 50 and 100 [mu] o,p'-DDT significantly increasing contraction frequency by 39 and 104% relative to controls. No significant differences in contraction frequency were observed with 10 [mu] o,p'-DDT during any test period. These data show that o,p'-DDT directly stimulated isometric contractions in rat uterine strips.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29112/1/0000150.pd

Refined localization of human connexin32 gene locus, GJB1, to Xq13.1

Connexins are the peptide subunits of gap junctions that interconnect cells to allow the direct, intercellular transfer of small molecules. Recently, the human connexin32 gene (locus designation GJB1) has been regionally mapped by three other laboratories to Xp11-q13, Xcen-q22, and Xp11-q22. The smallest region of overlap from these studies is Xcen-q13. By using a series of somatic cell hybrid mapping panels and a rat connexin32 cDNA probe, we have localized the human GJB1 locus to a much smaller region in proximal Xq13.1, in interval 8, as described by Lafreniere et al. (8).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30018/1/0000386.pd

Urinary Phthalate Metabolites in Relation to Preterm Birth in Mexico City

Background: Rates of preterm birth have been rising over the past several decades. Factors contributing to this trend remain largely unclear, and exposure to environmental contaminants may play a role. Objective: We investigated the relationship between phthalate exposure and preterm birth. Methods: Within a large Mexican birth cohort study, we compared third-trimester urinary phthalate metabolite concentrations in 30 women who delivered preterm (< 37 weeks of gestation) with those of 30 controls (≥ 37 weeks of gestation). Results: Concentrations of most of the metabolites were similar to those reported among U.S. females, although in the present study mono-n-butyl phthalate (MBP) concentrations were higher and monobenzyl phthalate (MBzP) concentrations lower. In a crude comparison before correcting for urinary dilution, geometric mean urinary concentrations were higher for the phthalate metabolites MBP, MBzP, mono(3-carboxylpropyl) phthalate, and four metabolites of di(2-ethyl-hexyl) phthalate among women who subsequently delivered preterm. These differences remained, but were somewhat lessened, after correction by specific gravity or creatinine. In multivariate logistic regression analysis adjusted for potential confounders, elevated odds of having phthalate metabolite concentrations above the median level were found. Conclusions: We found that phthalate exposure is prevalent among this group of pregnant women in Mexico and that some phthalates may be associated with preterm birth