Inflammatory Markers in Uterine Lavage Fluids of Pregnant, Non-Pregnant, and Intrauterine Device Implanted Mares on Days 10 and 15 Post Ovulation

Intrauterine devices (IUDs) are used in mares to suppress oestrous behaviour, but the underlying mechanism is yet to be elucidated. The presence of an embryo or an IUD prevents cyclooxygenase-2 (COX-2) and, subsequently, prostaglandin (PG) release and luteolysis. However, inflammation may also be involved. Endometrial inflammatory markers in uterine lavage fluid were measured on Day 10 (EXP 1, n = 25) and Day 15 (EXP 2, n = 27) after ovulation in inseminated mares, non-pregnant or pregnant, and in mares in which a small plastic sphere had been inserted into the uterus 4 (EXP 1) or 3 days (EXP 2) after ovulation. Uterine lavage fluid samples were analysed for nitric oxide (NO), prostaglandin E2 (PGE2) (only Exp. 1), prostaglandin F2α (PGF2α), inhibin A and cytokines, and blood samples for progesterone and oestradiol. On Day 10, the concentration of PGF2α was lower (p < 0.05) in the IUD group than in pregnant mares. The concentration of the modulatory cytokine IL-10 was significantly higher in the IUD group in comparison to non-pregnant mares, and inhibin A was significantly higher in IUD mares than in the pregnant counterparts on Day 15. The results suggest that the presence of IUD causes endometrial inflammation which is at a resolution stage on Day 15.

The Effect of Surgical Insertion and Proinflammatory Cytokines on Osteochondral Allograft Survival and Metabolism

Objective To investigate the responses of refrigerated osteochondral allograft cartilage (OCA) and fresh cartilage (FC), including cell survival and metabolism, to surgical impaction and proinflammatory cytokines. Design Osteochondral plugs (8 mm diameter) were harvested from prolonged-refrigerated (14-28 days) and fresh (≤24 hours postmortem) human femoral hemicondyles and subjected to a 0.2 N s pneumatic impaction impulse. Cartilage explants were removed from subchondral bone and randomized to 1 of 6 treatment groups: (1) Unimpacted control (UIC), (2) Impacted control (IC), (3) Impacted + interleukin (IL)-1β (0.1 ng/mL), (4) Impacted + IL-1β (0.1 ng/mL) + IL-6, (5) Impacted + IL-1β (10 ng/mL), and (6) Impacted + IL-1β (10 ng/mL) + IL-6. Samples were measured for cell viability, histology, and proteoglycan (PG) content at days 0, 2, 7, and 14 of culture. Results In UIC, cell viability was indistinguishable between OCA and FC and remained constant. Impaction alone decreased cell viability by 30% ( P < 0.01) in the OCA superficial layer and by 26% ( P < 0.01) in the entire tissue, but did not affect viability in FC. Cytokine addition did not further influence cell viability. Impaction alone did not affect PG synthesis. Addition of cytokines to impacted tissue decreased PG synthesis by ~3-fold in both tissue types in comparison with corresponding impacted controls ( P < 0.01). Throughout 2-week culture, PG release remained stable in all FC groups, but peaked at day 14 in OCA cartilage subjected to cytokines. Conclusions Mechanical impaction, mimicking surgical insertion, has a more profound effect on cell viability in OCA than in FC. Addition of proinflammatory cytokines further decreases OCA tissue metabolism and integrity.

Influence of monoolein on progesterone transdermal delivery

abstract This work aimed to investigate in vitro the influence of monoolein (MO) on progesterone (PG) transdermal delivery and skin retention. Information about the role of MO as an absorption enhancer for lipophilic molecules can help on innovative product development capable of delivering the hormone through the skin in a consistent manner, improving transdermal therapy of hormonal replacement. MO was dispersed in propylene glycol under heat at concentrations of 0% (control), 5% w/w, 10% w/w and 20% w/w. Then, 0.6% of PG (w/w) was added to each formulation. Permeation profile of the hormone was determined in vitro for 48 h using porcine skin in Franz diffusion cells. PG permeation doubled when 5% (w/w) of MO was present in formulation in comparison to both the control and higher MO concentrations (10% and 20% w/w). An equal trend was observed for PG retention in stratum corneum (SC) and reminiscent skin (E+D). PG release rates from the MO formulations, investigated using cellulose membranes, revealed that concentrations of MO higher than 5% (w/w) hindered PG release, which indeed negatively reflected on the hormone permeation through the skin. In conclusion, this work demonstrated the feasibility of MO addition (at 5% w/w) in formulations as a simple method to increase transdermal PG delivery for therapies of hormonal replacement. In contrast, higher MO concentrations (from 10% to 20% w/w) can control active release, and this approach could be extrapolated to other lipophilic, low-molecular-weight molecules.

Effect of the conceptus on uterine prostaglandin-F2α and prostaglandin-E2 release and synthesis during the periimplantation period in the pig

The present study was conducted to evaluate the effect of the conceptus on uterine prostaglandin-F2α (PGF2α) and prostaglandin-E2 (PGE2) release and the expression of prostaglandin synthase enzymes during the periimplantation period in the pig. A surgically generated model with conceptuses developing in only one of the uterine horns was created. The highest concentration of PGF2α and PGE2 was found in the gravid uterine horn, compared with the non-gravid horn and the intact horn of cyclic gilts. Endometrial concentration of both PGs in pregnant gilts was elevated regardless of the conceptus in the uterine horn, whereas only myometrial PGE2 concentration increased during pregnancy. Expression of prostaglandin-E2 synthase (mPGES-1) mRNA in the endometrium was upregulated during the oestrous cycle, while protein expression presented a similar pattern to that of PGE2 concentration in the uterine flushings. Prostaglandin-F2α synthase (PGFS) mRNA and protein expression in the endometrium did not differ between pregnancy and oestrous cycle but PGFS mRNA in the myometrium increased during pregnancy both in the gravid and the non-gravid uterine horns. We suggest a local effect of the conceptus on PG release pathways but also a more systemic effect within the whole uterus with regard to PG synthesis and accumulation in uterine tissues.

Leptin receptor expression and in vitro leptin actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct

In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2α and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2α, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2α release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2α production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system.

The effect of a diet supplemented with the n-6 polyunsaturated fatty acid linoleic acid on prostaglandin production in early- and late-pregnant ewes

Polyunsaturated fatty acids derived from the diet are incorporated into cell membranes where they act as precursors for prostaglandin (PG) synthesis. Linoleic acid (LA; 18:2 n-6) is a major constituent of plant oils and its consumption in Westernized populations is increasing. This study investigated the influence of LA on PG production by the uterus and placenta. Pregnant ewes were fed a control or an LA-enriched diet. Oxytocin (OT) was injected on day 45 (early) or day 133 (late) of gestation to measure the release of 13,14-dihydro-15-keto PGF2α (PGFM). Ewes were killed on day 46 or day 138 for collection of uterine intercaruncular endometrium and fetal allantochorion. Basal and stimulated PG release from explant cultures was assessed before and after in vitro treatment with OT, lipopolysaccharide (LPS), dexamethasone (DEX) or calcium ionophore (CaI). Expression of cyclooxygenase (COX)-1 and COX-2 was determined by Western blot in endometrium of late-gestation ewes. Circulating PGFM levels in vivo did not differ according to diet but there were highly significant differences in the release of PGs in vitro. Basal production of PGF2αand PGE2 by the endometrium and of PGE2 by the allantochorion were all higher in tissues from LA-supplemented ewes. Endometrial tissues produced more PG following OT and CaI treatment, whereas DEX inhibited production of both PGs at both stages of gestation. In allantochorion collected at day 46 LPS did not significantly alter PGE2 release and DEX increased output, whereas at day 138 LPS was stimulatory but DEX was inhibitory. These data show that a high-LA diet can significantly increase the ability of both endometrium and placental tissues to produce PGs in vitro. This effect of diet may only become apparent after a sustained period of PG release, so was not seen following the brief pulse caused by OT treatment in vivo. As COX protein levels were unaltered, the main influence was likely to be via conversion of LA to arachidonic acid, providing an increased supply of precursor. These results support previous studies which suggest that alterations in dietary polyunsaturated fatty acids may influence the time of labour.

Impact of combined NO and PG blockade on rapid vasodilation in a forearm mild-to-moderate exercise transition in humans

We tested the hypothesis that nitric oxide (NO) and prostaglandins (PGs) contribute to the rapid vasodilation that accompanies a transition from mild to moderate exercise. Nine healthy volunteers (2 women and 7 men) lay supine with forearm at heart level. Subjects were instrumented for continuous brachial artery infusion of saline (control condition) or combined infusion of NG-nitro-l-arginine methyl ester (l-NAME) and ketorolac (drug condition) to inhibit NO synthase and cyclooxygenase, respectively. A step increase from 5 min of steady-state mild (5.4 kg) rhythmic, dynamic forearm handgrip exercise (1 s of contraction followed by 2 s of relaxation) to moderate (10.9 kg) exercise for 30 s was performed. Steady-state forearm blood flow (FBF; Doppler ultrasound) and forearm vascular conductance (FVC) were attenuated in drug compared with saline (control) treatment: FBF = 196.8 ± 30.8 vs. 281.4 ± 34.3 ml/min and FVC = 179.3 ± 29.4 vs. 277.8 ± 34.8 ml·min−1·100 mmHg−1 (both P < 0.01). FBF and FVC increased from steady state after release of the initial contraction at the higher workload in saline and drug conditions: ΔFBF = 72.4 ± 8.7 and 52.9 ± 7.8 ml/min, respectively, and ΔFVC = 66.3 ± 7.3 and 44.1 ± 7.0 ml·min−1·100 mmHg−1, respectively (all P < 0.05). The percent ΔFBF and ΔFVC were not different during saline infusion or combined inhibition of NO and PGs: ΔFBF = 27.2 ± 3.1 and 28.1 ± 3.8%, respectively ( P = 0.78) and ΔFVC = 25.7 ± 3.2 and 26.0 ± 4.0%, respectively ( P = 0.94). The data suggest that NO and vasodilatory PGs are not obligatory for rapid vasodilation at the onset of a step increase from mild- to moderate-intensity forearm exercise. Additional vasodilatory mechanisms not dependent on NO and PG release contribute to the immediate and early increase in blood flow in an exercise-to-exercise transition.

Differential COX localization and PG release in Thy-1+ and Thy-1− human female reproductive tract fibroblasts

A key role exists for prostaglandins (PGs) in reproductive health, including fertility and parturition. However, the cellular sources and regulation of PG production by cyclooxygenase (COX) in the human female reproductive tract remain poorly understood. We recently reported that human female reproductive tract fibroblasts are divisible into distinct subsets based on their Thy-1 surface expression. Herein, we demonstrate that the expression, induction, and subcellular localization of COX-1 and COX-2 and the downstream PG biosynthesis are markedly different between these subsets. Specifically, Thy-1+ fibroblasts highly express COX-1, which is responsible for high-level PGE2 production, a feature usually attributed to the COX-2 isoenzyme. In contrast, COX-2, generally considered an inducible isoform, is constitutively expressed in the Thy-1− subset, which only minimally produces PGE2. The intracellular signaling pathways for COX regulation also differ between the subsets. Determination of differences in signal transduction, COX expression and localization, and PG production by human reproductive fibroblast subtypes supports the concept of fibroblast heterogeneity and the possibility that these subsets may play unique roles in tissue homeostasis and in inflammation.

Antibiotic-Induced Release of Lipoteichoic Acid and Peptidoglycan from Staphylococcus aureus: Quantitative Measurements and Biological Reactivities

ABSTRACT Antibiotics with different mechanisms of action may vary with respect to their effects on the release and immunostimulatory activities of cell wall fragments from gram-positive bacteria. Therefore, after Staphylococcus aureus was cultured for 4 h in the absence of antibiotics (control) and in the presence of β-lactam antibiotics (imipenem, flucloxacillin, or cefamandole) and protein synthesis-inhibiting antibiotics (erythromycin, clindamycin, or gentamicin), the lipoteichoic acid (LTA) and peptidoglycan (PG) levels in the bacterial supernatants were measured. β-Lactam antibiotics greatly enhanced the release of LTA and PG (4- to 9-fold and 60- to 85-fold, respectively), whereas protein synthesis inhibitors did not affect PG release and even inhibited the release of LTA compared to the amount of LTA released in control cultures. The capacity of β-lactam supernatants to stimulate the production of tumor necrosis factor alpha and interleukin-10 in human whole blood was significantly higher than that of protein synthesis inhibitor or control supernatants; the amounts of these cytokines released were directly proportional to the concentrations of PG and LTA in the supernatants. Enzymatic degradation of PG in the supernatants indicated that PG was mainly responsible for the observed biological reactivity.