Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation.

Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte-endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution.

Physiologic uterine inflammation and labor onset: integration of endocrine and mechanical signals.

The mechanisms underlying the preparation of the uterus for labor are not fully understood. Our previous studies have shown that during pregnancy, the uterine muscle (myometrium) undergoes dramatic phenotypic modulation culminating in term labor. The current review will discuss the cellular mechanisms involved in the regulation of myometrial activity and its modulation by endocrine signals and by mechanical stimulation of the uterus by the growing fetus. In particular, the contribution of uterine inflammation to the onset of labor will be described. We provide evidence that increased production of cytokines/chemokines in pregnant myometrium is associated with uterine occupancy and regulated by progesterone, suggesting the integration of mechanical and endocrine signals. Myometrial cells can actively participate in the inflammatory process in the uterus through the release of multiple proinflammatory cytokines and chemokines, providing a strong signal for activation of immune cells, their subsequent infiltration into pregnant uterus, and the initiation of labor.

Design and synthesis of a long-wavelength latent fluorogenic substrate for salicylate hydroxylase: a useful fluorimetric indicator for analyte determination by dehydrogenase-coupled biosensors.

Salicylate hydroxylase (SHL) catalyzes the production of catechol (plus CO(2) and H(2)O) from salicylate, NADH, and O(2). Coimmobilization of SHL with a NAD(P)(+)-dependent dehydrogenase in front of a Clark-type oxygen electrode has been investigated in the development of a general type of dehydrogenase-based biosensors that can detect various biological analytes; however, currently, no fluorophores are available for these applications. We synthesized the first new long-wavelength latent fluorogenic substrate SHLF (3) for SHL. In the presence of NADH and under aerobic conditions, SHL catalyzes the decarboxylative hydroxylation of SHLF followed by a quinone-methide-type rearrangement reaction concomitant with the ejection of a fluorescence coumarin 2, which is spontaneous and irreversible at physiological temperatures in aqueous media. The fluorescence signal generated by this process is specific and, in the near red spectral region with an emission maximum at 595 nm, is suppressed by salicylic acid. The fluorescence response of SHLF is insensitive to various biological reactive oxygen species (ROS) and reductants. Furthermore, SHLF is a sensitive fluorimetric indicator for analyte determination in the SHL-coupled dehydrogenase assay in which NAD(+) is converted to NADH. This novel fluorescence assay detected 3-hydroxybutyrate and cholesterol in the nanomolar range and is more sensitive than the current SHL-dehydrogenase amperometric sensors, making it applicable to the construction of a fiber-optic fluorescence biosensor for clinical diagnostic uses.

RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation.

The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.