Staphylococcus aureus Infection of Human Gestational Membranes Induces Bacterial Biofilm Formation and Host Production of Cytokines.

Staphylococcus aureus, a metabolically flexible gram-positive pathogen, causes infections in a variety of tissues. Recent evidence implicates S. aureus as an emerging cause of chorioamnionitis and premature rupture of membranes, which are associated with preterm birth and neonatal disease. We demonstrate here that S. aureus infects and forms biofilms on the choriodecidual surface of explanted human gestational membranes. Concomitantly, S. aureus elicits the production of proinflammatory cytokines, which could ultimately perturb maternal-fetal tolerance during pregnancy. Therefore, targeting the immunological response to S. aureus infection during pregnancy could attenuate disease among infected individuals, especially in the context of antibiotic resistance.

Mono-ethylhexyl phthalate stimulates prostaglandin secretion in human placental macrophages and THP-1 cells.

BACKGROUND: Diethylhexyl phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure, which is widespread in the US, increases preterm birth risk; however, the mechanisms driving this relationship are unclear. Because cyclooxygenase-2 (COX-2) dependent prostaglandin synthesis is implicated in preterm birth, we evaluated effects of mono-2-ethylhexyl phthalate (MEHP), the active metabolite of DEHP, on prostaglandin E2 (PGE2) synthesis and COX expression in human placental macrophages (PM). In addition, responses in PM were compared to those in a human macrophage-like cell line, THP-1. METHODS: PM and THP-1 cells were treated for 2, 4, 8, or 24 h with MEHP concentrations ranging from 10 to 180 micromolar. PGE2 concentrations were assessed in culture medium using ELISA, and COX expression was determined by western blot. RESULTS: Treatment of PM and THP-1 cells with 180 micromolar MEHP for 24 h significantly increased PGE2 release. Co-treatment of PMs or THP-1 cells with 180 micromolar MEHP and the non-selective COX inhibitor indomethacin reduced MEHP-stimulated PGE2 production. Similarly, co-treatment of PM and THP-1 cells with the COX-2 selective inhibitor NS-398 resulted in a significant decrease in PGE2, suggesting that MEHP-stimulated PGE2 is dependent specifically on increased COX-2 expression. Western blot analysis revealed a significant increase in COX-2 expression in PM and THP-1 cells treated with 180 micromolar MEHP, and no changes in COX-1 expression, supporting the role of COX-2 in MEHP-stimulated PGE2 synthesis. CONCLUSIONS: The findings from this study are the first to demonstrate phthalate-stimulated PGE2 synthesis in PM and warrant future studies into COX-2-dependent prostaglandin synthesis as a mechanism of toxicant-associated preterm birth.

Mono-2-ethylhexyl phthalate induces oxidative stress responses in human placental cells in vitro.

Di-2-ethylhexyl phthalate (DEHP) is an environmental contaminant commonly used as a plasticizer in polyvinyl chloride products. Exposure to DEHP has been linked to adverse pregnancy outcomes in humans including preterm birth, low birth-weight, and pregnancy loss. Although oxidative stress is linked to the pathology of adverse pregnancy outcomes, effects of DEHP metabolites, including the active metabolite, mono-2-ethylhexyl phthalate (MEHP), on oxidative stress responses in placental cells have not been previously evaluated. The objective of the current study is to identify MEHP-stimulated oxidative stress responses in human placental cells. We treated a human placental cell line, HTR-8/SVneo, with MEHP and then measured reactive oxygen species (ROS) generation using the dichlorofluorescein assay, oxidized thymine with mass-spectrometry, redox-sensitive gene expression with qRT-PCR, and apoptosis using a luminescence assay for caspase 3/7 activity. Treatment of HTR-8 cells with 180µM MEHP increased ROS generation, oxidative DNA damage, and caspase 3/7 activity, and resulted in differential expression of redox-sensitive genes. Notably, 90 and 180µM MEHP significantly induced mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2), an enzyme important for synthesis of prostaglandins implicated in initiation of labor. The results from the present study are the first to demonstrate that MEHP stimulates oxidative stress responses in placental cells. Furthermore, the MEHP concentrations used were within an order of magnitude of the highest concentrations measured previously in human umbilical cord or maternal serum. The findings from the current study warrant future mechanistic studies of oxidative stress, apoptosis, and prostaglandins as molecular mediators of DEHP/MEHP-associated adverse pregnancy outcomes.

Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species.

INTRODUCTION: The dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA). METHODS: DCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). MEHP was incubated with carboxy-H2DCFDA in cell-free solutions of Hank's buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H2DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells). RESULTS: MEHP did not increase fluorescence in solutions of carboxy-H2DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180µM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS containing 25µM TBBPA exhibited concentration-dependent increased fluorescence with 5-100µM carboxy-H2DCFDA (p<0.05), but not 1µM carboxy-H2DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5-25µM) (p<0.05), as we had observed in cell-free buffer. DISCUSSION: MEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H2DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H2DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.

Development of a rat pilocarpine model of seizure/status epilepticus that mimics chemical warfare nerve agent exposure.

We developed a rat pilocarpine seizure/status epilepticus (SE) model, which closely resembles 1.6-2.0 x LD50 soman exposure, to analyse the molecular mechanism of neuronal damage and to screen effective neuroprotectants against cholinergic agonist and chemical warfare nerve agent (CWNA) exposure. Rats implanted with radiotelemetry probes capable of recording electroencephalogram (EEG), electrocardiogram (ECG), temperature, and physical activity were treated with lithium chloride (5 mEq/kg, im), followed 24 h later by (ip) doses of pilocarpine hydrochloride. Based on radiotelemetry analysis, a dose of 240 mg/kg (ip) pilocarpine generated seizure/SE analogous to 1.6-2.0 x LD50 of soman. The model was refined by reducing the peripheral convulsions without affecting the central nervous system (CNS) by administering methylscopolamine bromide (1 mg/kg, ip), an anti-cholinergic that does not cross the blood-brain barrier. However, when methylscopolamine bromide was administered, a higher dose of pilocarpine (320 mg/kg, ip) was required to generate the equivalent seizure/SE. Histopathology data indicated that pilocarpine induces significant damage to the hippocampal region of the brain, with similar neuropathology to that of 1.6-2.0 x LD50 soman exposure. There was a reduction in body temperature after the administration of pilocarpine, as observed in organophosphate (OP) nerve agents exposure. The heart-rate of pilocarpine-treated animals increased compared to the normal range. The pilocarpine seizure/SE model was also reproducible in the absence of lithium chloride. These results support that pilocarpine seizure/SE model is useful in studying the molecular mechanisms of neuropathology and screening neuroprotectants following cholinergic agonist and CWNA exposure.

Histone acetylase inhibitor trichostatin A induces acetylcholinesterase expression and protects against organophosphate exposure.

The biological effects of organophosphorous (OP) chemical warfare nerve agents (CWNAs) are exerted by inhibition of acetylcholinesterase (AChE), which prevents the hydrolysis of the neurotransmitter acetylcholine, leading to hypercholinergy, seizures/status epilepticus, respiratory/cardiovascular failure, and potentially death. Current investigations show that bioscavenger therapy using purified fetal bovine AChE in rodents and non-human primates and the more recently tested human butyrylcholinesterase, is a promising treatment for protection against multiple LD(50) CWNA exposures. Potential impediments, due to the complex structure of the enzyme, purification effort, resources, and cost have necessitated alternative approaches. Therefore, we investigated the effects of transcriptional inducers to enhance the expression of AChE to achieve sufficient protection against OP poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses the chromatin, thereby increasing the binding of transcription factors and mRNA synthesis, was evaluated for induction of AChE expression in various neuronal cell lines. Dose-response curves showed that a concentration of 333 nM TSA was optimal in inducing AChE expression. In Neuro-2A cells, TSA at 333 nM increased the extracellular AChE activity approximately 3-4 fold and intracellular enzyme activity 10-fold. Correlating with the AChE induction, TSA pre-treatment significantly protected the cells against exposure to the organophosphate diisopropylfluorophosphate, a surrogate for the chemical warfare agents soman and sarin. These studies indicate that transcriptional inducers such as TSA up-regulate AChE, which then can bioscavenge any organophosphates present, thereby protecting the cells from OP-induced cytotoxicity. In conclusion, transcriptional inducers are prospective new methods to protect against CWNA exposure.