Peptidoglycan induces necrosis and regulates cytokine production in murine trophoblast stem cells.

PROBLEM: Intrauterine bacterial infection during pregnancy may lead to adverse outcome. The objective of this study was to assess whether peptidoglycan (PGN) derived from Gram-positive bacteria induces trophoblast stem (TS) cell death or alters TS cell cytokine production. METHOD OF STUDY: Toll-like receptor (TLR) transcript expression was assessed by RT-PCR. Protein expression was determined by confocal microscopy or flow cytometry. 7-Aminoactinomycin D (7-AAD) staining was used to assess TS cell death. Morphological features of cell death were evaluated by transmission electron microscopy. The presence of cleaved caspase-3 and high mobility group box 1 (HMGB1) protein was examined by Western blot. Cytokine levels in cell supernatants were determined using a mouse cytokine 23-plex panel. RESULTS: Toll-like receptor 2 and TLR4 protein was expressed from the 1-cell stage through the blastocyst stage of murine embryo development. Murine TS cells expressed TLR2 and TLR6 but not TLR1 or TLR4 RNA. Only TLR2 protein was detected at the plasma membrane of TS cells. PGN induced TS cell death by a caspase-3-independent mechanism. The cell death pathway induced by PGN was morphologically consistent with necrosis. Finally, PGN induced HMGB1 release and increased MIP-1ß secretion while inhibiting the constitutive release of RANTES. CONCLUSION: Peptidoglycan-induced TS cell necrosis and the subsequent release of HMGB1 and MIP-1ß may regulate an infection-induced inflammatory response at the maternal-fetal interface and thus may play a role in the pathogenesis of infection-associated pregnancy complications.

Tissue-specific deletion and discontinuous loss of heterozygosity are signatures for the mutagenic effects of ionizing radiation in solid tissues.

The mouse Aprt locus on chromosome 8 was used as the selectable target for the study of spontaneous and ionizing radiation-induced mutations in kidney epithelia and ear fibroblasts. Fifty-two Aprt heterozygous mice were exposed to 7.5 Gy of (137)Cs-gamma radiation on their right sides, and Aprt-deficient clones were isolated from enzymatically digested tissues at times ranging from 1 day to 14 months after irradiation. A statistically significant increase in the mutant frequencies for the irradiated tissues was observed when compared with the spontaneous mutant frequencies for the nonirradiated tissues. A molecular analysis of spontaneous mutations observed for the nonirradiated tissues revealed tissue-specific differences; apparent chromosome loss was common in kidney mutants but infrequent in the ear mutants, whereas apparent deletions were common in the ear mutants but not detected in the kidney mutants. For the irradiated kidneys, apparent deletions were observed commonly demonstrating that these events are markers for ionizing radiation mutagenesis in this tissue. All of the loss of heterozygosity (LOH) tracts observed in the spontaneous mutants were continuous, but discontinuous LOH patterns were observed in 6--8% of ionizing radiation-induced ear and kidney cell mutants. Work with kidney-derived cell lines showed that discontinuous LOH is a novel signature for delayed ionizing radiation mutagenesis. Considered together, these results suggest that ionizing radiation-induced mutations in vivo can result from both direct and delayed mutagenic effects.