Searching for Lower Female Genital Tract Soluble and Cellular Biomarkers: Defining Levels and Predictors in a Cohort of Healthy Caucasian Women

Background: High concentrations of pro-inflammatory cytokines have been previously observed in the genital fluids of women enrolled in microbicide trials and may explain observed increased HIV transmission in some of these trials. Although the longitudinal nature of these studies allows within-subject comparisons of post-product levels to baseline levels, the fact that the physiologic variations of these cytokines and other markers of immune activation are not fully defined in different populations, makes it difficult to assess changes that can be directly attributed to microbicide use as opposed to other biological and behavioural factors. Methods: Cervicovaginal lavage samples were collected from 30 healthy Caucasian and assayed for concentrations of ten cytokines/chemokines, total protein content and two antimicrobial proteins using a multiplex immunoassay and ELISA. Cellular markers were characterized by flow cytometry on mononuclear cells collected from the endocervix using flocked swabs. Bacterial quantification was performed using quantitative PCR. Results: Ectopy, menstrual cycle phase, prostate-specific antigen and presence of leucocytes in endocervical cells' supernatant were associated with the concentrations of cyto-/chemokines in cervicovaginal secretions. Approximately 3% of endocervical cells collected were monocytes of which a median of 52% (SD = 17) expressed both CD4 and CCR5 markers. Approximately 1% of the total cells were T-cells with a median of 61% (SD = 10) CD4 and CCR5 expression. Around 5% of the monocytes and 16% of the T-cells expressed the immune activation marker HLA-DR. Higher percentages of T-cells were associated with greater quantities of IL-1RA, GM-CSF and elafin. Conclusion: We demonstrate the presence of selected soluble and cellular immune activation markers and identify their predictors in the female genital tract of healthy women. Future clinical trials should consider ectopy, sexual activity, menstrual cycle phase and presence of bacterial species as possible confounders when evaluating the possible inflammatory effects of microbicide compounds

Mechanics of cutting manoeuvres by ostriches ('Struthio camelus')

We studied the strategies used by cursorial bipeds (ostriches) to maneuver during running. Eight ostriches were induced to run along a trackway and execute turns. Ground reaction forces and three-dimensional kinematics of the body and leg joints were simultaneously recorded, allowing calculation of joint angles and quasi-static net joint torques. Sidesteps, where the leg on the outside of the turn changes the movement direction, and crossovers using the inside leg, occurred with nearly equal frequency. Ostriches executed maneuvers using a simple control strategy that required minimal changes to leg kinematics or net torque production at individual joints. Although ostriches did use acceleration or braking forces to control body rotation, their morphology allowed for both crossovers and sidesteps to be accomplished with minimal net acceleratory/braking force production. Moreover, body roll and ab/adduction of the leg shifted the foot position away from the turn direction, reducing the acceleratory/braking forces required to prevent under-or over-rotation and aligning the leg with the ground reaction force

Correlates of the molecular vaginal microbiota composition of African women.

BACKGROUND: Sociodemographic, behavioral and clinical correlates of the vaginal microbiome (VMB) as characterized by molecular methods have not been adequately studied. VMB dominated by bacteria other than lactobacilli may cause inflammation, which may facilitate HIV acquisition and other adverse reproductive health outcomes. METHODS: We characterized the VMB of women in Kenya, Rwanda, South Africa and Tanzania (KRST) using a 16S rDNA phylogenetic microarray. Cytokines were quantified in cervicovaginal lavages. Potential sociodemographic, behavioral, and clinical correlates were also evaluated. RESULTS: Three hundred thirteen samples from 230 women were available for analysis. Five VMB clusters were identified: one cluster each dominated by Lactobacillus crispatus (KRST-I) and L. iners (KRST-II), and three clusters not dominated by a single species but containing multiple (facultative) anaerobes (KRST-III/IV/V). Women in clusters KRST-I and II had lower mean concentrations of interleukin (IL)-1α (p < 0.001) and Granulocyte Colony Stimulating Factor (G-CSF) (p = 0.01), but higher concentrations of interferon-γ-induced protein (IP-10) (p < 0.01) than women in clusters KRST-III/IV/V. A lower proportion of women in cluster KRST-I tested positive for bacterial sexually transmitted infections (STIs; ptrend = 0.07) and urinary tract infection (UTI; p = 0.06), and a higher proportion of women in clusters KRST-I and II had vaginal candidiasis (ptrend = 0.09), but these associations did not reach statistical significance. Women who reported unusual vaginal discharge were more likely to belong to clusters KRST-III/IV/V (p = 0.05). CONCLUSION: Vaginal dysbiosis in African women was significantly associated with vaginal inflammation; the associations with increased prevalence of STIs and UTI, and decreased prevalence of vaginal candidiasis, should be confirmed in larger studies

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins

Novel Cephalosporin conjugates display potent and selective inhibition of Imipenemase-type Metallo-β-Lactamases

Medicinal Chemistr

Pan-cancer functional analysis of somatic mutations in G protein-coupled receptors

G Protein-coupled receptors (GPCRs) are the most frequently exploited drug target family, moreover they are often found mutated in cancer. Here we used a dataset of mutations found in patient samples derived from the Genomic Data Commons and compared it to the natural human variance as exemplified by data from the 1000 genomes project. We explored cancer-related mutation patterns in all GPCR classes combined and individually. While the location of the mutations across the protein domains did not differ significantly in the two datasets, a mutation enrichment in cancer patients was observed among class-specific conserved motifs in GPCRs such as the Class A "DRY" motif. A Two-Entropy Analysis confirmed the correlation between residue conservation and cancer-related mutation frequency. We subsequently created a ranking of high scoring GPCRs, using a multi-objective approach (Pareto Front Ranking). Our approach was confirmed by re-discovery of established cancer targets such as the LPA and mGlu receptor families, but also discovered novel GPCRs which had not been linked to cancer before such as the P2Y Receptor 10 (P2RY10). Overall, this study presents a list of GPCRs that are amenable to experimental follow up to elucidate their role in cancer.Medicinal Chemistr

Non-Sexual Transmission of Trichomonas vaginalis in Adolescent Girls Attending School in Ndola, Zambia

OBJECTIVES: To identify risk factors for trichomoniasis among young women in Ndola, Zambia. METHOD: The study was a cross-sectional study among adolescent girls aged 13-16 years in Ndola, Zambia. Study participants were recruited from schools in selected administrative areas that represented the different socio-economic strata in town. Consenting participants were interviewed about their socio-demographic characteristics; sexual behaviour; and hygiene practices. Self-administered vaginal swabs were tested for Trichomonas vaginalis. HSV-2 antibodies were determined on serum to validate the self-reported sexual activity. RESULTS: A total of 460 girls participated in the study. The overall prevalence of trichomoniasis was 27.1%, 33.9% among girls who reported that they had ever had sex and 24.7% among virgins. In multivariate analysis the only statistically significant risk factor for trichomoniasis was inconsistent use of soap. For the virgins, none of the risk factors was significantly associated with trichomoniasis, but the association with use of soap (not always versus always) and type of toilet used (pit latrine/bush versus flush toilet) was of borderline significance. CONCLUSION: We found a high prevalence of trichomoniasis in girls in Ndola who reported that they had never had sex. We postulate that the high prevalence of trichomoniasis in virgins in Ndola is due to non-sexual transmission of trichomoniasis via shared bathing water and inconsistent use of soap

Acceptability of Carraguard Vaginal Microbicide Gel among HIV-Infected Women in Chiang Rai, Thailand

Background: Few studies of microbicide acceptability among HIV-infected women have been done. We assessed CarraguardH vaginal gel acceptability among participants in a randomized, controlled, crossover safety trial in HIV-infected women in Thailand. Methodology/Principal Findings: Participants used each of 3 treatments (Carraguard gel, methylcellulose placebo gel, and no product) for 7 days, were randomized to one of six treatment sequences, and were blinded to the type of gel they received in the two gel-use periods. After both gel-use periods, acceptability was assessed by face-to-face interview. Responses were compared to those of women participating in two previous Carraguard safety studies at the same study site. Sixty women enrolled with a median age of 34 years; 25 % were sexually active. Self-reported adherence (98%) and overall satisfaction rating of the gels (87% liked ‘‘somewhat’ ’ or ‘‘very much’’) were high, and most (77%) considered the volume of gel ‘‘just right.’ ’ For most characteristics, crossover trial participants evaluated the gels more favorably than women in the other two trials, but there were few differences in the desired characteristics of a hypothetical microbicide. Almost half (48%) of crossover trial participants noticed a difference between Carraguard and placebo gels; 33 % preferred Carraguard while 12 % preferred placebo (p=0.01). Conclusions/Significance: Daily Carraguard vaginal gel use was highly acceptable in this population of HIV-infecte

Correlates of self-reported offending in children with a first police contact from distinct socio-demographic and ethnic groups

<p>Abstract</p> <p>Background</p> <p>This study aims to identify risk factors for level of offending among childhood offenders from different socio-economic status (SES) neighborhoods and ethnic origins.</p> <p>Method</p> <p>Three groups of childhood first time police arrestees were studied using standardized instruments for individual and parental characteristics: native Dutch offenders from moderate to high SES neighborhoods, native Dutch offenders from low SES neighborhoods, and offenders of non-Western origin from low SES neighborhoods.</p> <p>Results</p> <p>All subgroups showed high rates of externalizing disorders (27.2% to 41.8%) and familial difficulties (25.7% to 50.5%). Few differences between neighborhoods were found in the prevalence and impact of risk factors. However, the impact of some family risk factors on offending seemed stronger in the low SES groups. Regarding ethnical differences, family risk factors were more prevalent among non-Western childhood offenders. However, the association of these factors with level of offending seemed lower in the non-Western low SES group, while the association of some individual risk factors were stronger in the non-Western low SES group. Turning to the independent correlation of risk factors within each of the groups, in the Dutch moderate to high SES group, 23.1% of the variance in level of offending was explained by ADHD and behavioral problems; in the Dutch low SES group, 29.0% of the variance was explained by behavioral problems and proactive aggression; and in the non-Western low SES group, 41.2% of the variance was explained by substance use, sensation seeking, behavioral peer problems, and parental mental health problems.</p> <p>Conclusions</p> <p>Thereby, the study indicates few neighborhood differences in the impact of individual and parental risk factors on offending, while individual and parental risk factors may differ between ethnic groups.</p

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies