Risk factors for clinically significant intra-ventricular hemorrhage in pregnancies complicated by preterm premature rupture of membranes.

Objectives: Preterm birth is a major cause of adverse perinatal outcomes, including intraventricular hemorrhage (IVH). IVH has been shown to contribute to lasting neurological disability, however the role of maternal characteristics and potentially modifiable risk factors that contribute to these outcomes have not been well defined. We sought to determine predictors of IVH in pregnancies complicated by early preterm premature rupture of membranes (PPROM). Study design: We performed a retrospective cohort study of all singleton pregnancies with early PPROM <32 weeks GA and delivery >22 weeks GA at University of Colorado Hospital (UCH) from 1/1/2007-12/31/2011. Clinically significant IVH (Grade III or IV) was the primary outcome of this study. To determine independent predictors of IVH we created a multivariate model including all univariate covariates with p-value of ≤ 0.10. Results: In our cohort (n=229), when adjusted for non-white race, younger maternal age and increased BMI were independent predictors of clinically significant IVH (OR=1.4 CI 1.04-1.79, p=0.03; OR 1.2 CI 1.04-1.33, p=0.01, respectively). Female gender was also found to be an independent predictor of poor 5 minute APGAR (OR=2.3 CI 1.06-5.28, p=0.04). Conclusions: In our cohort, infants born to younger mothers or mothers with higher BMI appear to be at increased risk for clinically significant IVH. Interestingly, on further analysis, we found that female newborns had a 2-fold greater risk of poor 5 minute APGAR of less than 7. Given these data, larger studies are warranted to examine modifiable and non-modifiable risk pregnancy that may be associated with IVH and subsequent adverse neurological outcomes in pregnancies complicated by early PPROM.

Patterns of placental pathology in preterm premature rupture of membranes.

Inflammation is associated with preterm premature rupture of membranes (PPROM) and adverse neonatal outcomes. Subchorionic thrombi, with or without inflammation, may also be a significant pathological finding in PPROM. Patterns of inflammation and thrombosis may give insight into mechanisms of adverse neonatal outcomes associated with PPROM. To characterize histologic findings of placentas from pregnancies complicated by PPROM at altitude, 44 placentas were evaluated for gross and histological indicators of inflammation and thrombosis. Student's t-test (or Mann-Whitney U-test), χ 2 analysis (or Fisher's exact test), mean square contingency and logistic regression were used when appropriate. The prevalence of histologic acute chorioamnionitis (HCA) was 59%. Fetal-derived inflammation (funisitis and chorionic plate vasculitis) was seen at lower frequency (30% and 45%, respectively) and not always in association with HCA. There was a trend for Hispanic women to have higher odds of funisitis (OR = 5.9; P = 0.05). Subchorionic thrombi were seen in 34% of all placentas. The odds of subchorionic thrombi without HCA was 6.3 times greater that the odds of subchorionic thrombi with HCA (P = 0.02). There was no difference in gestational age or rupture-to-delivery interval, with the presence or absence of inflammatory or thrombotic lesions. These findings suggest that PPROM is caused by or can result in fetal inflammation, placental malperfusion, or both, independent of gestational age or rupture-to-delivery interval; maternal ethnicity and altitude may contribute to these findings. Future studies focused on this constellation of PPROM placental findings, genetic polymorphisms and neonatal outcomes are needed.

Anti-inflammatory glucocorticoid action: inhibition of griPGHS, a new cyclooxygenase.

Our understanding of the anti-inflammatory actions of glucocorticoid hormones has significantly advanced in the past year with the discovery of a second cyclooxygenase gene which we call 'glucocorticoid-regulated inflammatory prostaglandin G/H synthase' (griPGHS). In mouse fibroblasts and human monocytes levels of griPGHS mRNA and protein rise dramatically in response to growth factors, cytokines, and oncogene activation. These inductions are markedly suppressed by the glucocorticoid hormone, dexamethasone. This stands in contrast to the behavior of the previously cloned cyclooxygenase (PGHS), which appears to be constitutively expressed. Thus far, data show that griPGHS is increased in many other systems where prostaglandin biosynthesis is regulated, including models of inflammation, tissue injury, and hormonal control of reproductive processes. Thus, griPGHS is likely to be a key mediator of many clinically relevant processes and as such represents an important target for both steroidal and nonsteroidal anti-inflammatory agents.

Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing.

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.

cDNA cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase.

The antiinflammatory glucocorticoids are potent inhibitors of cyclooxygenase, a key regulator of prostaglandin synthesis; yet, the mechanism(s) by which this occurs is not fully understood. We have cloned a 4.1-kilobase (kb) cDNA, distinct from the previously cloned cyclooxygenase (2.8 kb), that confers cyclooxygenase activity to transfected cells. The mRNA for this newly discovered cyclooxygenase is unique for its long 3' untranslated region containing many AUUUA repeats. Levels of the 4.1-kb cyclooxygenase mRNA are rapidly increased by serum or interleukin 1 beta in mouse fibroblasts and human monocytes, respectively, and decreased by glucocorticoids, whereas levels of the 2.8-kb cyclooxygenase mRNA do not change. Similar effects are seen in the presence of cycloheximide where the 4.1-kb, but not the 2.8-kb, mRNA is greatly superinduced. Thus, there are both constitutive (2.8 kb) and regulated (4.1 kb) cyclooxygenase species, the latter most likely being a major mediator of inflammation.

Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.

Several proinflammatory cytokines can increase prostaglandin E2 (PGE2) synthesis in a variety of cell types, constituting an important component of the inflammatory response. We demonstrate here that leukoregulin, a 50-kDa product of activated T lymphocytes, dramatically increases PGE2 synthesis in cultured human orbital fibroblasts. This up-regulation is mediated through an induction of prostaglandin-endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addition and are near maximal by 6 h, when they are 50-fold or higher above basal levels. The increase in PGHS-2 mRNA levels is partially blocked by cycloheximide, suggesting de novo synthesis of an intermediate protein may be required for a maximal leukoregulin response. Nuclear run-on studies indicate PGHS-2 gene transcription is up-regulated by leukoregulin 2-fold after 2 and 6 h. PGHS-2 protein, as assessed by Western blotting and two-dimensional protein gel analysis, is increased dramatically in orbital fibroblasts. This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE2 and cAMP levels following leukoregulin treatment is also blocked by indomethacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygenase inhibitor. The dramatic increase in cAMP levels causes marked alteration in orbital fibroblast morphology. PGHS-2 expression in dermal fibroblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in morphology. Both orbital and dermal fibroblasts express high levels of PGHS-1 mRNA and protein, the other abundant form of cyclooxygenase. In contrast to its effects on PGHS-2 expression, leukoregulin fails to alter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid production in human fibroblasts. The increased PGHS-2 expression elicited by leukoregulin in orbital fibroblasts may be a consequence of both transcriptional and post-transcriptional effects. These observations help clarify the pathogenic mechanism relevant to the intense inflammation associated with Graves' ophthalmopathy. Lymphocytes trafficked to orbital tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.

Relative activities of retrovirally expressed murine prostaglandin synthase-1 and -2 depend on source of arachidonic acid.

We have developed derivatives of mouse embryonic fibroblasts (10T1/2) and Chinese hamster ovary (AS52) cells that stably express high levels of murine prostaglandin synthase-1 or -2 (PGHS-1 or -2). The cDNAs were transferred using retroviral vectors and the resulting G418-resistant clones were analyzed for prostaglandin E2 (PGE2) production. Specific expression was confirmed by Western and Northern analyses. Enzyme activities, protein, and message levels peaked 1 (10T1/2) or 2 (AS52) days after seeding but decreased as cells became density arrested. Upon subculturing, enzyme activities returned to their initial high levels. With 10 microM exogenous arachidonic acid (AA) as the substrate, PGHS-1 activities were approximately 3- to 5-fold higher than PGHS-2 activities. Conversely, when exogenous AA was left out of the medium and only endogenous AA was available as substrate, enzyme activities were lower; but PGHS-2 activities were 5-fold (10T1/2) or 1.5-fold (AS52) higher than PGHS-1 activities. Following phorbol ester treatment to stimulate endogenous AA release, PGHS-2 activities increased over time and by 6 hours, were 4-fold (10T1/2) or 2-fold (AS52) higher than PGHS-1 activities. However, when calcium ionophore A23187 was used to stimulate endogenous AA release, maximum PGHS activities occurred within 30 min of treatment; PGHS-1 activities were equal to (10T1/2) or 2-fold higher (AS52) than PGHS-2 activities. Because these cell lines allow us to measure specific PGHS activity in intact cells, we were able to demonstrate that the relative activities of the two PGHS isozymes depend on the source of AA (exogenous versus endogenous) or biochemical stimulus used to mobilize endogenous AA (A23187 versus phorbol ester). These data suggest that PGHS-1 and PGHS-2 preferentially utilize different pools of AA and may be modulated through different stimulus-initiated pathways.