Failure of E. coli bacteria to induce preterm delivery in the rat

Abstract Background We sought to develop a model of bacterially induced preterm delivery in rats to parallel similar models in mice. Methods Female Sprague-Dawley rats on day 17 of gestation (normal term = 21–22 days) were inoculated into the uterus with either 2 × 109 – 7 × 1010 killed E. coli organisms, 1 – 4 × 108 live E. coli or sterile solution. These inoculations were made either via trans-cervical catheter or by direct intrauterine injection at laparotomy. Animals were then observed for delivery for variable periods up to term. Necropsies were performed and fetal viability was assessed. Results No rats delivered prematurely after bacterial exposure (27 animals observed for at least 48 hours), and all animals followed to term (n = 3) delivered live pups. No dams exhibited signs of systemic illness. There was a statistically significant but small negative effect of killed E. coli on fetal viability (100% of 80 fetuses from 6 control pregnancies and 93% of 182 fetuses from 14 bacterially-treated pregnancies were alive at necropsy, p = 0.014). Live bacteria had a larger effect on fetal viability, with only 64% of 14 fetuses, 47% of 28 fetuses and 32% of 31 fetuses surviving after trans-cervical administration of 7 × 107, 2 × 108 and 4 × 108 E. coli, respectively. Conclusion Unlike mice, it has proven difficult to induce preterm labor in the rat using E. coli as a stimulating agent. The relevant literature is reviewed and hypotheses are offered to explain this phenomenon

Surfactant Protein (SP)-A Suppresses Preterm Delivery and Inflammation via TLR2

Toll like receptors (TLRs) are pattern-recognition molecules that initiate the innate immune response to pathogens. Pulmonary surfactant protein (SP)-A is an endogenously produced ligand for TLR2 and TLR4. SP-A has been proposed as a fetally produced signal for the onset of parturition in the mouse. We examined the effect of interactions between SP-A and the pathogenic TLR agonists lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C)) (ligands for TLR4, TLR2 and TLR3, respectively) on the expression of inflammatory mediators and preterm delivery. Three types of mouse macrophages (the cell line RAW 264.7, and fresh amniotic fluid and peritoneal macrophages, including macrophages from TLR4 and TLR2 knockout mice) were treated for up to 7 hours with pathogenic TLR agonists with or without SP-A. SP-A alone had no effect upon inflammatory mediators in mouse macrophages and did not independently induce preterm labor. SP-A significantly suppressed TLR ligand-induced expression of inflammatory mediators (interleukin (IL)-1β, tumor necrosis factor (TNF)-α and the chemokine CCL5) via a TLR2 dependent mechanism. In a mouse inflammation-induced preterm delivery model, intrauterine administration of SP-A significantly inhibited preterm delivery, suppressed the expression of proinflammatory mediators and enhanced the expression of the CXCL1 and anti-inflammatory mediator IL-10. We conclude that SP-A acts via TLR2 to suppress TLR ligand-induced preterm delivery and inflammatory responses.</p

Interleukin 1 receptor antagonist knockout mice show enhanced microglial activation and neuronal damage induced by intracerebroventricular infusion of human β-amyloid

BACKGROUND: Interleukin 1 (IL-1) is a key mediator of immune responses in health and disease. Although classically the function of IL-1 has been studied in the systemic immune system, research in the past decade has revealed analogous roles in the CNS where the cytokine can contribute to the neuroinflammation and neuropathology seen in a number of neurodegenerative diseases. In Alzheimer's disease (AD), for example, pre-clinical and clinical studies have implicated IL-1 in the progression of a pathologic, glia-mediated pro-inflammatory state in the CNS. The glia-driven neuroinflammation can lead to neuronal damage, which, in turn, stimulates further glia activation, potentially propagating a detrimental cycle that contributes to progression of pathology. A prediction of this neuroinflammation hypothesis is that increased IL-1 signaling in vivo would correlate with increased severity of AD-relevant neuroinflammation and neuronal damage. METHODS: To test the hypothesis that increased IL-1 signaling predisposes animals to beta-amyloid (Aβ)-induced damage, we used IL-1 receptor antagonist Knock-Out (IL1raKO) and wild-type (WT) littermate mice in a model that involves intracerebroventricular infusion of human oligomeric Aβ1–42. This model mimics many features of AD, including robust neuroinflammation, Aβ plaques, synaptic damage and neuronal loss in the hippocampus. IL1raKO and WT mice were infused with Aβ for 28 days, sacrificed at 42 days, and hippocampal endpoints analyzed. RESULTS: IL1raKO mice showed increased vulnerability to Aβ-induced neuropathology relative to their WT counterparts. Specifically, IL1raKO mice exhibited increased mortality, enhanced microglial activation and neuroinflammation, and more pronounced loss of synaptic markers. Interestingly, Aβ-induced astrocyte responses were not significantly different between WT and IL1raKO mice, suggesting that enhanced IL-1 signaling predominately affects microglia. CONCLUSION: Our data are consistent with the neuroinflammation hypothesis whereby increased IL-1 signaling in AD enhances glia activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae

Pretreatment with Pancaspase Inhibitor (Z-VAD-FMK) Delays but Does Not Prevent Intraperitoneal Heat-Killed Group B Streptococcus-Induced Preterm Delivery in a Pregnant Mouse Model

Caspases and apoptosis are thought to play a role in infection-associated preterm-delivery. We have shown that in vitro treatment with pancaspase inhibitor Z-VAD-FMK protects trophoblasts from microbial antigen-induced apoptosis. Objective. To examine whether in vivo administration of Z-VAD-FMK would prevent infection-induced preterm-delivery. Methods. We injected 14.5 day-pregnant-mice with heat-killed group B streptococcus (HK-GBS). Apoptosis within placentas and membranes was assessed by TUNEL staining. Calpain expression and caspase-3 activation were assessed by immunohistochemistry. Preterm-delivery was defined as expulsion of a fetus within 48 hours after injection. Results. Intrauterine (i.u.) or intraperitoneal (i.p.) HK-GBS injection led to preterm-delivery and induced apoptosis in placentas and membranes at 14 hours. The expression of calpain, a caspase-independent inducer of apoptosis, was increased in placenta. Treatment with the specific caspase inhibitor Z-VAD-FMK (i.p.) prior to HK-GBS (i.p.) delayed but did not prevent preterm-delivery. Conclusion. Caspase-dependent apoptosis appears to play a role in the timing but not the occurrence of GBS-induced preterm delivery in the mouse

Synergy Between Viral and Bacterial Toll-Like Receptors Leads to Amplification of Inflammatory Responses and Preterm Labor in the Mouse1

Toll-like receptors (TLRs) recognize molecular constituents of pathogens and activate host innate immune responses. TLR2 responds to Gram-positive organisms and components of their cell walls. TLR3 responds to double-stranded RNA (an intermediate in viral replication). A mouse macrophage cell line (RAW 264.7) and freshly obtained mouse peritoneal macrophages were treated in tissue culture for 5 or 10 h with either peptidoglycan (PGN; a TLR2 ligand, 1 μg/ml), polyinosinic:cytidylic acid (poly(I:C); a TLR3 ligand, 10 μg/ml), both PGN and poly(I:C), or neither. Total RNA was extracted, and RT-PCR was performed. A mouse model of preterm birth induced by intrauterine injection of TLR ligands was used to test in vivo effects. Compared to stimulation with either PGN or poly(I:C) alone, stimulation of macrophages with both ligands (whether simultaneously or sequentially) resulted in synergistic expression of inflammatory mediators, including inducible nitric oxide synthase, interleukin 1 beta, tumor necrosis factor alpha, and the chemokine CCL5 (RANTES). Using peritoneal macrophages obtained from mutant and control mice, this synergy was determined to be dependent upon TLR2 and the TLR-related intracellular adaptor proteins MYD88 and TICAM1 (TRIF). Simultaneous administration of both PGN and poly(I:C) to pregnant mice also produced dramatic synergy in the occurrence of preterm delivery. These results support a possible role for viral infection in preterm labor. Synergy in the mechanisms of parturition suggests the existence of a “two-hit” trigger mechanism that minimizes responses to stimuli of limited biological significance while providing an efficient amplification strategy for rapid activation of labor in response to multiple or more severe insults

Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates

Background: A mixture of phenol and guanidine isothiocyanate (“P/GI”, the principal components of TRIzol™ and similar products) is routinely used to isolate RNA, DNA, and proteins from a single specimen. In time-course experiments of cells grown in tissue culture, replicate wells are often harvested sequentially and compared, with the assumption that in-well lysis and complete aspiration of P/GI has no effect on continuing cultures in nearby wells. Methods: To test this assumption, we investigated morphology and function of RAW 264.7 cells (an immortalized mouse macrophage cell line) cultured in covered 96-well plates for 4, 8, or 24 h at varying distances from a single control well or a well into which P/GI had been deposited and immediately aspirated completely. Results: Time- and distance-dependent disruptions resulting from proximity to a single well containing trace residual P/GI were seen in cell morphology (blebbing, cytoplasmic disruption, and accumulation of intracellular vesicles), cell function (pH of culture medium), and expression of genes related to inflammation (Tnfα) and autophagy (Lc3b). There was no transcriptional change in the anti-apoptotic gene Mcl1, nor the pro-apoptotic gene Hrk, nor in P/GI-unexposed control cultures. LPS-stimulated cells incubated near P/GI had lower expression of the cytokine Il6. These effects were seen as early as 4 h of exposure and at a distance of up to 3 well units from the P/GI-exposed well. Conclusions: Exposure to trace residual quantities of P/GI in covered tissue culture plates leads to substantial disruption of cell morphology and function in as little as 4 h, possibly through induction of autophagy but not apoptosis. This phenomenon should be considered when planning time-course experiments in multi-well covered tissue culture plates.</p