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ABSTRACT: The aim of this study was to examine the effect of the 3% StarPharma LTD 7013 gel (VivaGel) on mucosal immune markers hypothesized to be associated with HIV-1 acquisition.
Phase 1, placebo-controlled, randomized, double-blind clinical trial was performed in 54 young women in the United States and Kenya. Participants used carbopol gel with and without (placebo) StarPharma LTD 7013 twice daily over 14 days. Cervical specimens were collected for cytokines, chemokines, T cells, and dendritic cells at days 0, 7, 14, and 21. A negative binomial regression model was used to assess differences between study arms.
Several mucosal immune parameters were increased in the VivaGel arm compared with placebo. For cytokines D7, IL-6 (P = 0.05); D 14, interferon gamma (P = 0.03), IL-2 (P = 0.04), IL-5 (P = 0.003), and IL-10 (P = 0.001) were increased. On D7, CD8+/CD69+ T cells tended to be increased (P < 0.08); limiting analysis to visits without blood or bacterial vaginosis, these findings were stronger as follows: at D7, CD8+/CD69+ T cells were increased in the VivaGel arm (P < 0.005), as were CD4+/CD69+ cells (P = 0.001) and CD4+/CCR5+ T cells (P = 0.01). The changes described for D7 and 14 were no longer seen at D21.
Markers associated with inflammation and epithelial damage were reversibly elevated in the VivaGel arm compared with the placebo arm after 7-14 days of twice daily product use.
JAIDS Journal of Acquired Immune Deficiency Syndromes 11/2011; 59(2):134-40. · 4.43 Impact Factor
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ABSTRACT: The interlaboratory reproducibility of cytokine measurements from cervicovaginal samples by Luminex has not been reported. Using cervicovaginal lavage specimens collected on three study days from 12 women participating in a Phase I microbicide study, we measured a panel of eight cytokines in three independent laboratories. Four (IFN-γ, IL-10, IL-17, and TNF) were below the limit of detection in the majority (85%) of samples in either two or all three laboratories, an observation that may guide analyte selection for future studies. Good interlaboratory agreement (intraclass correlation coefficient, r>0.7) in absolute levels was observed for IL-1β, IL-6, and IL-8, while poor agreement was seen for IFN-α2 (r=0.47). When considering within-subject change from baseline (pre-product, at study-day 0) to either post-product visit (study-days 7 and 14), IL-1β and IL-6 exhibited good interlaboratory agreement (r>0.7), while IFN-α2 and IL-8 did not. Future studies addressing the clinical utility of specific biomarkers of inflammation for microbicide trials should consider reproducibility in the context of defining biologically meaningful thresholds of change for candidate biomarkers, ensuring that such change can be reliably distinguished from background variability.
Cytokine 07/2011; 56(2):430-4. · 3.02 Impact Factor
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ABSTRACT: The mechanisms involved in mucosal immune control of cervical human papillomavirus (HPV) infection remain ill defined. Because toll-like receptors (TLRs) are key players in innate immune responses, we investigated the association between TLR expression and viral persistence or clearance in young women with incident infections with oncogenic HPV types 16 or 51. Messenger RNA expression of TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8 and TLR9 was measured by quantitative reverse transcription-PCR using human endocervical specimens, collected before and after viral acquisition, in a cohort well characterized for HPV infections. Wilcoxon rank sum test was used to compare the change seen from preinfection to incident infection between women who subsequently cleared infection with those who did not. HPV 16 infections that cleared were significantly (p < 0.05) associated with an increase in expression of the four viral nucleic acid-sensing TLRs (TLR3, TLR7, TLR8 and TLR9) as well as TLR2 upon viral acquisition. Similar associations were not observed for HPV 51. In women who subsequently cleared their HPV 16 infection, changes in TLR1, TLR3, TLR7 and TLR8 expression levels between preincident and incident visits were significantly correlated with parallel changes in the levels of interferon-α2, measured by immunoassay in cervical lavage specimens. This study suggests that dampened TLR expression in the cervical mucosa is a type-specific mechanism by which HPV 16 interferes with innate immune responses, contributing to viral persistence, and that TLR upregulation and resultant cytokine induction is important in subsequent viral clearance.
International Journal of Cancer 02/2011; 128(4):879-86. · 5.44 Impact Factor
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ABSTRACT: Young women aged 15-24 years have the highest rates of sexually transmitted infections (STIs). The vulnerability of adolescents is often attributed to risky sexual behaviors, whereas biological factors affecting mucosal immunity are poorly understood. The objective of this cross-sectional study was to examine associations between the type of cervical epithelium and protein levels of 11 cervicovaginal cytokines and chemokines in non-pregnant healthy young women. Cervical epithelial types were viewed on colpophotography and measured quantitatively using computerized planimetry. We selected 16 women with immature epithelium (predominantly columnar and early/mid squamous metaplasia), and 16 women with mature epithelium (predominantly squamous epithelium). Cytokine levels were measured in cervicovaginal lavage samples by MILLIPLEX™ MAP Human Cytokine/Chemokine multiplex immunoassay. Bivariate Box-Cox regression models compared cytokine levels between immature and mature groups. Multivariate Box-Cox models adjusted separately for age, years since menarche, days since last menses, years of sexual activity, number of lifetime sexual partners, HPV infection, hormonal contraceptive use, smoking, bacterial vaginosis by Nugent's criteria, and polymorphonuclear cells on wet prep. The mean age was 19.2 years. Women with immature epithelium demonstrated significantly higher levels of IL-1α, IL-1β, IL-6, IL-8, MIP-1α, RANTES, TNFα, IL-10, IL-12 and IFNγ (each p<0.01), compared to women with mature epithelium. Results remained highly significant in the multivariate models. Cytokine profiles in the healthy state may foreshadow differential responses to pathogens. Cervical epithelial type should be measured in clinical studies involving cervicovaginal immune markers.
Journal of Reproductive Immunology 11/2010; 88(1):66-71. · 2.97 Impact Factor
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ABSTRACT: To determine whether healthy, young women in sub-Saharan Africa have a more activated immune milieu in the genital tract (i.e. activated CD4 T cells) than a similar population in the United States.
A cross-sectional study nested in a phase 1 microbicide trial.
Cervical cytobrushes were collected from 18 to 24-year-old women in San Francisco, California, USA (n = 18) and Kisumu, Kenya (n = 36) at enrollment into a phase 1 microbicide trial. All participants tested negative for HIV, herpes simplex virus 2, gonorrhea, chlamydia, and trichomonas, and had abstained from sex for at least 7 days prior to enrollment. Cryopreserved T-cell populations were assayed by flow cytometry in a central laboratory. Secretory leukocyte protease inhibitor levels were assayed in cervicovaginal lavage samples. The Wilcoxon rank-sum test was used to compare immune parameters between sites.
The total number of endocervical CD4(+) T cells was slightly higher in participants from San Francisco, but participants from Kisumu had a substantially higher number and proportion of CD4(+) T cells expressing the early activation marker CD69, with and without the HIV coreceptor C-C chemokine receptor type 5, and a greater proportion of activated CD8(+) T cells. Median (interquartile range) genital levels of secretory leukocyte protease inhibitor were lower in participants from Kisumu compared with those from San Francisco [190 (96-519) vs. 474 (206 817) pg/ml, P < 0.03].
Activated mucosal T cells were increased in the genital tract of young, sexually transmitted infection/HIV-free Kenyan women, independent of common genital coinfections, and secretory leukocyte protease inhibitor levels were reduced. The cause of these mucosal immune differences is not known, but could partly explain the high HIV incidence in young women from sub-Saharan Africa.
AIDS (London, England) 08/2010; 24(13):2069-74. · 4.91 Impact Factor
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ABSTRACT: Cervical mucosal expression of cytokines involved in mediating cellular immunity is believed to influence the persistence of human papillomavirus (HPV) infection, a necessary prerequisite for the development of cervical intraepithelial neoplasia (CIN). Additionally, regulatory T (Treg) cells are increasingly understood to be important modulators of cellular immunity. Using quantitative RT-PCR, we measured, in cross-sectional design, the cervical mRNA expression of IFN-gamma, IL-10, and IL-12, as well as the Treg transcription factor Forkhead box P3 (Foxp3), in a cohort of young women representing CIN 1, 2, and 3, as well as benign histology. Higher levels of IFN-gamma and IL-10 were significantly (p <or= 0.05) associated with decreased odds of having high-grade cervical disease (CIN 2 or 3) in multivariate logistic regression models. In contrast, higher levels of mucosal Foxp3 expression were associated with increased odds of having CIN 2 or 3 (p = 0.004). In a multivariate model including cervical infection with HPV16 and/or another high-risk HPV type, Foxp3 remained higher in the CIN 2/3 group, but the difference was notably less significant (p = 0.05). These findings support a model in which diminished cellular immunity in the cervical mucosa and mucosal enrichment of Treg cells both contribute to the development of high-grade lesions.
International Journal of Cancer 10/2008; 124(6):1379-83. · 5.44 Impact Factor
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ABSTRACT: Reference genes for quantitative reverse transcription-PCR (qRT-PCR) studies must be validated for the cell type studied and should be stable between the groups that represent the independent variable in an experimental design. We sought to identify the reference genes in cervical cell specimens showing the most stable expression between human papillomavirus (HPV)-infected and -uninfected women without high-grade cervical intraepithelial neoplasia. Using endocervical cells collected by cytology brush and Sybr green-based qRT-PCR, eight candidate genes were screened for amplification efficiency, specificity, and overall stability (by use of geNorm software). The five most stable genes were then further evaluated both for overall stability (geNorm) and intergroup stability (by use of NormFinder software) in specimens from HPV-negative and HPV-positive women. The combination of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and RPLP0 was the most stable overall, with a geNorm stability measure of 0.603. The intergroup analysis showed GAPDH to be the most stable single gene and RPLP0 to be second most stable and also showed that these genes represent the most stable two-gene combination, with a NormFinder stability value of 0.130. The fact that these two distinct approaches identified the same pair of genes provides added confidence that, when the focus is on HPV infection, a normalization factor derived from these two genes is likely to be appropriate.
Clinical and vaccine immunology: CVI 09/2008; 15(9):1369-73. · 2.37 Impact Factor
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ABSTRACT: Cytokines in cervical mucus are likely to play important roles in controlling pathogens. The cervical mucosal environment is complex, however, with many endogenous and exogenous factors that may affect cytokine levels. We used a multiplex, suspension-array-based immunoassay method to measure 10 proinflammatory (interleukin-1beta [IL-1beta], IL-6, and IL-8) and immunoregulatory (gamma interferon [IFN-gamma], IL-2, IL-4, IL-5, IL-10, IL-12, and IL-13) cytokines in cervical mucus specimens collected via ophthalmic sponge from 72 healthy, nonpregnant women and correlate their levels with biologic and behavioral covariates in a cross-sectional design. Proinflammatory and immunoregulatory cytokines were readily detected, although proinflammatory cytokines were present at markedly higher levels than were immunoregulatory cytokines. Among the covariates examined, the most striking finding was the significant (P < or = 0.05) association between depressed levels of the cytokines IFN-gamma, IL-1beta, IL-6, and IL-10 and cigarette smoking. Also, nonsignificant trends toward lower cytokine levels were found in the settings of incident and persistent human papillomavirus infection. The ready detection of proinflammatory cytokines may be reflective of the female genital tract as an anatomic site that is constantly exposed to immunogenic stimulation. Cigarette smoking appears to downregulate cytokine responses in the cervical mucosa, which may help explain the implicated role of tobacco use as a cofactor for cervical cancer development.
Clinical and vaccine immunology: CVI 01/2008; 15(1):49-54. · 2.37 Impact Factor
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Journal of the American Chemical Society 03/2007; 129(6):1482-3. · 9.91 Impact Factor
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Chemical Reviews 02/2007; 107(1):174-238. · 40.20 Impact Factor
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ABSTRACT: A novel and efficient route to exo-beta,gamma-unsaturated lactams from substituted and non-substituted secondary methylenecyclopropyl amides is reported. Subsequent modification of the resulting exo-beta,gamma-unsaturated lactams provides access to several pharmaceutically relevant scaffolds. [reaction: see text].
Organic Letters 12/2006; 8(24):5521-4. · 5.86 Impact Factor
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ABSTRACT: Measurements of mucosal immune parameters in the uterine cervix are potentially influenced by numerous factors, including infections, endogenous and exogenous hormones, semen, and nicotine and its metabolites in cervical mucus. The objective of this study was to examine correlates of immunoregulatory cytokine mRNA expression in cervical cytology samples in a cross-sectional design. Samples, collected at study entry by cervical cytology brush from 368 women aged 13-21 enrolled in a longitudinal study of the natural history of human papillomavirus (HPV) infection, were tested by quantitative RT-PCR for expression of IFN-gamma, IL-4, IL-10, and IL-12. In a multivariate analysis, elevated levels of IFN-gamma, IL-10, and IL-12 were significantly (p < 0.05) associated with several variables, including current C. trachomatis infection, recent intercourse, and current oral contraceptive pill use. Suppressed IL-4 and IL-10 levels were associated with cigarette smoking within the last 24 h. Time since last menstrual period did not affect any of the cytokines; in a substudy of weekly cytokine variability, however, IL-10 showed a non-significant trend toward higher levels around the time of menstruation.
Journal of Clinical Immunology 05/2006; 26(3):222-32. · 3.08 Impact Factor
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ABSTRACT: [reaction: see text] A concise and enantioselective synthesis of (-)-(alpha)-kainic acid in 13 steps with an overall yield of 15% is reported. The pyrrolidine kainoid precursor with the required C2/C3 trans stereochemistry was prepared with excellent diastereoselectivity (>20:1) via a MgI(2)-mediated ring expansion of a tertiary methylenecyclopropyl amide. A selective hydroboration was then employed to set the remaining stereochemistry at the C4 position en route to (-)-(alpha)-kainic acid.
Organic Letters 08/2005; 7(14):3045-7. · 5.86 Impact Factor
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ABSTRACT: [reaction: see text] A highly diastereoselective MgI(2)-mediated ring expansion of methylenecyclopropane amides to functionalized pyrrolidines has been developed using chiral aromatic sulfinimines. The 2,3,4-trisubstituted pyrrolidines were isolated in generally good to excellent yields and in excellent diastereoselectivities for aromatic and heterocyclic sulfinimines.
Organic Letters 10/2004; 6(19):3309-12. · 5.86 Impact Factor
