Anandamide binds to cannabinoid receptors and plays several central and peripheral functions. The aim of this work was to study the possible role for this endocannabinoid in controlling sperm-oviduct interaction in mammals. We observed that bull sperm and bovine oviductal epithelial cells express cannabinoid receptors, CB1 and CB2, and fatty acid amide hydrolase, the enzyme that controls intracellular anandamide levels. A quantitative assay to determine whether anandamide was involved in bovine sperm-oviduct interaction was developed. R(C)-methanandamide, a non-hydrolysable anandamide analog, inhibited sperm binding to and induced sperm release from oviductal epithelia. Selective CB1 antagonists (SR141716A or AM251) completely blocked R(C)-methanandamide effects. However, SR144528, a selective CB2 antagonist, did not exert any effect, indicating that only CB1 was involved in R(C)-methanandamide effect. This effect was not caused by inhibition of the sperm progressive motility or by induction of the acrosome reaction. Overall, our findings indicate for the first time that the endocannabinoid system is present in bovine sperm and oviductal epithelium and that anandamide modulates the sperm-oviduct interaction, by inhibition of sperm binding and induction of sperm release from oviductal epithelial cells, probably by activating CB1 receptors.

Genital tract bacterial infections could induce abortion and are some of the most common complications of pregnancy; however, the mechanisms remain unclear. We investigated the role of prostaglandins (PGs) in the mechanism of bacterial lipopolysaccharide (LPS)-induced pregnancy loss in a mouse model, and we hypothesized that PGs might play a central role in this action. LPS increased PG production in the uterus and decidua from early pregnant mice and stimulated cyclooxygenase (COX)-II mRNA and protein expression in the decidua but not in the uterus. We also observed that COX inhibitors prevented embryonic resorption (ER). To study the possible interaction between nitric oxide (NO) and PGs, we administered aminoguanidine, an inducible NO synthase inhibitor. NO inhibited basal PGE and PGF 2␣ production in the decidua but activated their uterine synthesis and COX-II mRNA expression under septic conditions. A NO donor (S-nitroso-N-acetylpenicillamine) produced 100% ER and increased PG levels in the uterus and decidua. LPS-stimulated protein nitration was higher in the uterus than in the decidua. Quercetin, a peroxynitrite scavenger, did not reverse LPS-induced ER. Our results suggest that in a model of septic abortion characterized by increased PG levels, NO might nitrate and thus inhibit COX catalytic activity. ER prevention by COX inhibitors adds a possible clinical application to early pregnancy complications due to infections. embryonic resorption ͉ uteri ͉ decidua ͉ peroxynitrite ͉ sepsis M aternal infections could cause abortion in humans (1, 2), but their mechanism is not clear. Spontaneous and cytokineboosted abortion rates have been linked to exposure to lipopolysaccharide (LPS) in the environment (3). Bacteria could enter the uterus with ejaculate or by intestinal absorption (4). Previously, we developed a mouse model to study LPS-induced pregnancy loss (5). LPS (1 g/g i.p.) injected on day 7 of pregnancy produced 100% embryonic resorption (ER) at 24 h, with fetal expulsion at 48 h. Nitric oxide (NO) produced by inducible NO synthase (iNOS) plays a key role in ER (5). LPS produced systemic effects but did not affect the mothers' survival or future pregnancies.Prostaglandin (PG) biosynthesis is catalyzed by cyclooxygenase (COX) I and II, the later being inducible by proinflammatory agents such as cytokines and LPS (6-8). It is well known that PGs mediate septicemic signs and symptoms of Gram-negative bacterial infections and stimulate contractility of the myometrium (9, 10). Thus, PGs are considered to be effective abortifacients and are important mediators of LPS-induced ER and preterm labor. Silver et al. (11) showed that deciduae from LPS-treated mice produce inflammatory eicosanoids as PGE 2 , PGF 2␣ , and thromboxane B 2 and that indomethacin (Indo), a nonselective COX inhibitor, prevents abortion.A significant body of experimental evidence suggests a relationship between NO and PGs (12, 13), particularly in pathophysiologic events associated with gestation. In our laboratory we found that epidermal...

The tumor microenvironment is acidic as a consequence of upregulated glycolysis and poor perfusion and this acidity, in turn, promotes invasion and metastasis. We have recently demonstrated that chronic consumption of sodium bicarbonate increased tumor pH and reduced spontaneous and experimental metastases. This occurred without affecting systemic pH, which was compensated. Additionally, these prior data did not rule out the possibility that bicarbonate was working though effects on carbonic anhydrase, and not as a buffer per se. Here, we present evidence that chronic ingestion of a non-volatile buffer, 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA) with a pKa of 6.9 also reduced metastasis in an experimental PC3M prostate cancer mouse model. Animals (n = 30) were injected with luciferase expressing PC3M prostate cancer cells either subcutaneously (s.c., n = 10) or intravenously (i.v., n = 20). Four days prior to inoculations, half of the animals for each experiment were provided drinking water containing 200 mM IEPA buffer. Animals were imaged weekly to follow metastasis, and these data showed that animals treated with IEPA had significantly fewer experimental lung metastasis compared to control groups (P < 0.04). Consistent with prior work, the pH of treated tumors was elevated compared to controls. IEPA is observable by in vivo magnetic resonance spectroscopy and this was used to measure the presence of IEPA in the bladder, confirming that it was orally available. The results of this study indicate that metastasis can be reduced by non-volatile buffers as well as bicarbonate and thus the effect appears to be due to pH buffering per se.

Accurate preclinical predictions of the clinical efficacy of experimental cancer drugs are highly desired but often haphazard. Such predictions might be improved by incorporating elements of the tumor microenvironment in preclinical models by providing a more physiological setting. In generating improved xenograft models, it is generally accepted that the use of primary tumors from patients are preferable to clonal tumor cell lines. Here we describe an interdisciplinary platform to study drug response in multiple myeloma (MM), an incurable cancer of the bone marrow. This platform uses microfluidic technology to minimize the number of cells per experiment, while incorporating 3D extracellular matrix and mesenchymal cells derived from the tumor microenvironment. We used sequential imaging and a novel digital imaging analysis algorithm to quantify changes in cell viability. Computational models were used convert experimental data into dose-exposure-response "surfaces" which offered predictive utility. Using this platform, we predicted chemosensitivity to bortezomib and melphalan, two clinical MM treatments, in 3 MM cell lines and 7 patient-derived primary MM cell populations. We also demonstrated how this system could be used to investigate environment-mediated drug resistance and drug combinations that target it. This interdisciplinary preclinical assay is capable of generating quantitative data that can be used in computational models of clinical response, demonstrating its utility as a tool to contribute to personalized oncology. Major Findings By designing an experimental platform with the specific intent of generating experimental parameters for a computational clinical model of personalized therapy in multiple myeloma, while taking in consideration the limitations of working with patient primary cells, and the need to incorporate elements of the tumor microenvironment, we have generated patient-individualized estimations of initial response and time to relapse to chemotherapeutic agents.