Inflammation of the female reproductive tract increases susceptibility to HIV-1 and other viral infections and, thus, it becomes a serious liability for vaginal products. Excessive release of proinflammatory cytokines may alter the mucosal balance between tissue destruction and repair and be linked to enhanced penetration and replication of viral pathogens upon chemical insult. The present study evaluates four surface-active microbicide candidates, nonoxynol-9 (N-9), benzalkonium chloride (BZK), sodium dodecyl sulfate, and sodium monolaurate for their activity against human sperm and HIV, and their capacity to induce an inflammatory response on human vaginal epithelial cells and by the rabbit vaginal mucosa. Spermicidal and virucidal evaluations ranked N-9 as the most potent compound but were unable to predict the impact of the compounds on vaginal cell viability. Interleukin (IL)-1 release in vitro reflected their cytotoxicity profiles more accurately. Furthermore, IL-1 concentrations in vaginal washings correlated with cumulative mucosal irritation scores after single and multiple applications (P < 0.01), showing BZK as the most damaging agent for the vaginal mucosa. BZK induced rapid cell death, IL-1 release, and IL-6 secretion. The other compounds required either more prolonged or repeated contact with the vaginal epithelium to induce a significant inflammatory reaction. Increased IL-8 levels after multiple applications in vivo identified compounds with the highest cumulative mucosal toxicity (P < 0.01). In conclusion, IL-1, IL-6, and IL-8 in the vaginal secretions are sensitive indicators of compound-induced mucosal toxicity. The described evaluation system is a valuable tool in identifying novel vaginal contraceptive microbicides, selecting out candidates that may enhance, rather than decrease, HIV transmission.

A study was undertaken to estimate the activities of the key enzymes of glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle in purified rat spermatocytes and spermatids, which have been shown to die in glucose-containing medium and require lactate/pyruvate for maintaining normal ATP concentrations. The aim was to elucidate the changes in the glycolytic and oxidative potential of germ cells undergoing meiosis. Pachytene spermatocytes and round spermatids from adult rat testis were purified to ϳ90% purity by trypsin digestion followed by a combination of centrifugal elutriation and Percoll density gradient centrifugation. After the purity and viability of these cells had been established, their contents of hexokinase, phosphofructokinase, lactate dehydrogenase (LDH) and LDH-X of glycolysis, glucose 6-phosphate dehydrogenase of the pentose phosphate pathway and citrate synthase, aconitase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of the TCA cycle were estimated. These enzymes were also estimated in epididymal spermatozoa for comparison with the testicular germ cells. The results indicate greater activity of glycolytic and pentose phosphate pathway enzymes in spermatocytes than in spermatids, which exhibited greater activity of TCA cycle enzymes than the former. The difference in activity was statistically significant for most of the enzymes studied. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes and significantly lower activity of pentose phosphate pathway and TCA cycle enzymes than did the testicular germ cells. We conclude that the unusual dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilise lactate, pyruvate, or both, while retaining a better ability to utilise glucose. Spermatozoa with the greatest glycolytic potential and the lowest TCA cycle activity appear to be 'programmed' to utilise exclusively glucose/fructose for energy.

In somatic cells, RHOA mediates actin dynamics through a GNA13-mediated signaling cascade involving RHO kinase (ROCK), LIM kinase (LIMK), and cofilin. RHOA can be negatively regulated by protein kinase A (PRKA), and it interacts with members of the A-kinase anchoring (AKAP) family via intermediary proteins. In spermatozoa, actin polymerization precedes the acrosome reaction, which is necessary for normal fertility. The present study was undertaken to determine whether the GNA13-mediated RHOA signaling pathway may be involved in acrosome reaction in bovine caudal sperm, and whether AKAPs may be involved in its targeting and regulation. GNA13, RHOA, ROCK2, LIMK2, and cofilin were all detected by Western blot in bovine caudal sperm. Overlay, immunoprecipitation, and subsequent mass spectrometry analysis identified several RHOA-interacting proteins, including proacrosin, angiotensin-converting enzyme, tubulin, aldolase C, and AKAP4. Using overlay and pulldown techniques, we demonstrate that phosphorylation of AKAP3 increases its interaction with the RHOA-interacting proteins PRKAR2 (the type II regulatory subunit of PRKA, formerly RII) and ropporin (ROPN1, a PRKAR2-like protein, or R2D2). Varying calcium concentrations in pulldown assays did not significantly alter binding to R2D2 proteins. These data suggest that the actin-regulating GNA13-mediated RHOA-ROCK-LIMK-cofilin pathway is present in bovine spermatozoa, that RHOA interacts with proteins involved in capacitation and the acrosome reaction, and that RHOA signaling in sperm may be targeted by AKAPs. Finally, AKAP3 binding to PRKAR2 and ROPN1 is regulated by phosphorylation in vitro.

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.

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SummaryUlcerative colitis (UC) and Crohn's disease (CD) are considered to be immunologically mediated disorders that share certain features with murine models of colitis. Whether any of these models are physiologically relevant to the human condition remains controversial. The hypothesis is that increased amounts of antibodies neutralizing transforming growth factor (TGF)-b, interleukin (IL)-2 or IL-10 create a relative immunodeficient state in inflammatory bowel disease (IBD) that predisposes to disease. To evaluate this, serum samples from patients with UC or CD and from normal healthy individuals were studied by enzyme-linked immunosorbent assays. Antibodies recognizing TGF-b were most prevalent in UC (P < 0·01); anti-IL-10 antibodies were elevated in CD (P < 0·05), while anti-IL-2 antibodies were the same for all three groups. Importantly, the percentage of IBD patients with at least one of the antibody levels greater than any control value was 30% for UC and 33% for CD. To verify the presence of these antibodies, immobilized TGF-b was exposed to UC sera and the attached proteins identified by Western blot assay. The proteins proved to be exclusively immunoglobulin (Ig) G. To evaluate the neutralizing activity of these antibodies, cytokine-specific IgG from subjects in each group of patients was incubated with TGF-b, IL-2 or IL-10 before addition to a bioassay with changes in viability determined by a colorimetric analysis. Antibodies from most individuals in all three groups neutralized the action of each cytokine. This study shows that about one-third of IBD patients may have a relative deficiency of TGF-b, IL-2 or IL-10 due to an increase in neutralizing antibodies in their sera.

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