WITHDRAWN: ALERRT's "BATH" Assessment Algorithm No Longer Meets the Needs of American Law Enforcement Personnel or the Public They Serve.

This article has been witdrawn at the request of the author(s). The Publisher apologizes for any inconvenience this may cause.

Toll-like receptors: Important immune checkpoints in the regression of cervical intra-epithelial neoplasia 2.

Toll-like receptors (TLRs) are innate immune defenders thought to be critical for the clearance of human papillomavirus (HPV) infections hence preventing the development of HPV-associated high-grade cervical intra-epithelial neoplasia (CIN2 or 3), a potential cervical cancer precursor. However, the role of TLRs in the regression of established cervical lesions, such as CIN2, is hindered by a lack of prospective design studies. Using SYBR green real-time PCR assays, we have examined the gene expression of TLR2, TLR3, TLR7, TLR8 and TLR9, in cytobrush collected endocervical cells of 63 women diagnosed with CIN2 at study entry (baseline) and followed over a 3-year period. Wilcoxon rank-sum test was used to examine the association between TLR expression levels, measured at baseline, and CIN2 outcome (regression vs. persistence/progression) over time. HPV genotyping was performed using Roche Linear Array Assay detecting 37 HPV types. Women with CIN2 regression showed significantly higher baseline levels of TLR2 (p = 0.006) and TLR7 (p = 0.007), as well as a non-significant trend for a higher TLR8 expression (p = 0.053) compared to women with CIN2 persistence/progression. Six women with CIN2 regression, who presented with an HR-HPV DNA-negative CIN2 lesion at study entry, had significantly higher baseline levels of TLR2 (p = 0.005), TLR7 (p = 0.013) and TLR8 (p = 0.012), compared to women with CIN2 persistence/progression, suggesting their role in clearance of HPV prior to clearance of the lesion. Our results confirm a key role of TLRs in regression of CIN2 and support the potential use of TLR-agonists for treatment of these lesions.

Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor.

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

Diversity of Cervicovaginal Cytokine Response to Incident Chlamydia trachomatis Infection Among a Prospective Cohort of Young Women.

PROBLEM: Animal, in vitro, and ex vivo studies have identified several cytokines involved in host immunity to genital Chlamydia trachomatis (CT) infection. However, in vivo cytokine responses are not well described. Our objectives were to document cervicovaginal cytokine levels and intrawoman cytokine changes during incident CT in a prospective cohort. METHODS: From our prospective cohort, 62 women had incident CT, comprising a CT-negative visit followed by a CT-positive visit. At these visits, cytokine protein levels (IL-6, IL-8, IL-1α, IL-1ß, MIP-1α, RANTES, IFN-γ) were measured using cervicovaginal lavages and the MILLIPLEX(™) /Luminex(®) multiplex assay. Quartiles were defined for each cytokine from all 124 visits. RESULTS: At the group level, RANTES was higher (P < 0.01) at the CT-positive visit than at baseline, but the other cytokines did not significantly differ. For intrawoman cytokine changes, women with a cytokine level that increased at least one quartile higher (going from baseline to the CT-positive visit) ranged between 26 and 53%. Women with a cytokine level staying in the same quartile ranged between 32 and 48%. Women with a cytokine level that decreased at least one quartile lower ranged between 15 and 37%. CONCLUSION: Intrawoman cervicovaginal cytokine changes during incident CT appear heterogeneous and may reflect differences in natural host immunity.

Development of a novel liquid bead array human papillomavirus genotyping assay (PGMY-LX) and comparison with linear array for continuity in longitudinal cohort studies.

Studies of the natural history of human papillomavirus (HPV) infection require reproducible, type-specific testing of the viral types that infect cervical tissue; Linear Array (LA) is one method that has been widely used. We sought to develop a cost-effective, high-throughput alternative using the same PGMY09/11 primer/probe system and offering sensitivity and specificity comparable to those with LA to ensure continuity in longitudinal studies. We report here on a Luminex-based approach, PGMY-LX, that offers type-specific detection of 33 oncogenic and nononcogenic types. Detection of HPV type-specific plasmid DNA was highly specific, with high signal-to-noise ratios for all types except nononcogenic type 40 and no cross-reactivity between types. Cohen's unweighted κ values for 378 clinical samples tested by both LA and PGMY-LX were ≥0.8 (range, 0.80 to 1.0) for 25 types, including oncogenic HPV types 16, 31, 33, 39, 45, 58, and 59 and possibly oncogenic types 53, 66, 73, and 82) and >0.7 (range, 0.74 to 0.79) for oncogenic types 18, 35, 51, and 56 and probable oncogenic type 68b, indicating substantial or better type-specific agreement between the two methods. The reproducibility by PGMY-LX of the types detected by LA varied from 94% when a single HPV type was present to 66% when multiple types were present. The interrun reproducibility for PGMY-LX varied from 98% for single-type infections to 85% for multiple-type infections. The high reproducibility of PGMY-LX and the type-specific agreement with LA allows PGMY-LX to be incorporated into longitudinal, cohort studies that have historically relied on LA.

Expression of nucleic acid-sensing Toll-like receptors predicts HPV16 clearance associated with an E6-directed cell-mediated response.

Toll-like receptors (TLRs), important in rapid clearance of incident human papillomavirus (HPV) infections, may also be important in shaping the adaptive response to persistent infections. We examined here the association between TLR expression and clearance of HPV16 infections following periods of persistence, using longitudinal TLR measurements and a time-to-clearance analysis, as well as the interaction between TLRs and adaptive, cell-mediated responses involved in clearance. TLR2, TLR3, TLR7, TLR8 and TLR9 mRNA expression were measured in cervical cytobrush samples by quantitative PCR. Responses to the HPV16 E6 and E7 oncoproteins were measured by an interferon-γ immunospot assay. Bivariable and multivariable Cox proportional hazard models were used to estimate the effect of TLRs on HPV16 clearance. Higher expression of TLR3 or TLR7 at an HPV16-positive visit was a significant (p ≤ 0.05) predictor of clearance by the following visit, in both unadjusted and adjusted (for smoking and oral contraceptive use) models. In women with, but not those without, a positive response to E6, higher expression of TLR3 (hazard ratio: 1.2 [95% confidence interval: 1.04-1.39], p = 0.012), TLR7 (1.39 [1.14-1.7], p = 0.001), TLR8 (1.37 [1.11-1.69], p = 0.003), or TLR9 (1.53 [1.13-2.08], p = 0.006) was significantly associated with clearance, revealing an important link between innate and adaptive immunity in the control of HPV infections following periods of persistence.

Computational and ¹³C investigations of the diazadienes and oxazadienes formed via the rearrangement of methylenecyclopropyl hydrazones and oximes.

Computational and further experimental investigations of the previously reported diazadienes, obtained via the rearrangement of methylenecyclopropyl hydrazone 1 are reported. Calculations at the CCSD(T)/cc-pVTZ//B3LYP/6-31G(d) level of theory indicate that the initially reported product 3 would, if formed, undergo rapid electrocyclic ring opening and, hence, would be unstable under the reaction conditions. Based on this computational prediction, further analysis of the (13)C NMR spectrum, previously attributed to 3, led to the revision of structure 3 to that of its N-tosylaminopyrrole constitutional isomer 11. Similarly, structure 8, formed in the rearrangement of oxime 6, was revised to that of N-hydroxypyrrole 12.

Cervical cytokines and clearance of incident human papillomavirus infection: Hawaii HPV cohort study.

Mechanisms for the control and resolution of human papillomavirus (HPV) infection of the cervix include the local production of cytokines, which control recruitment and function of cells integral to pathogen control. We established a cohort of women for long-term follow-up to examine the mucosal expression of antiviral (IFN-α2), Type-1 (IFN-γ, IL-12), regulatory (IL-10), and proinflammatory (IL-1α, IL-1ß, IL-6, IL-8, MIP-1α, and TNF) cytokines in association with the clearance of incident cervical HPV infection. Interviews and specimens for HPV DNA analysis and cytokine protein measurement were obtained at baseline and at 4-month intervals. A Cox proportional hazards model was used to study the relationship between clearance of 107 high-risk and 111 low-risk incident HPV infections and cytokine levels among 154 women. Positive changes from baseline levels of IL-10, IL-12, MIP-1α, and TNF were associated with significantly longer times to type-specific HPV clearance. Inverse trends in the hazard ratios associated with clearance of high-risk HPV infections were monotonic and significant for IL-12 (ptrend = 0.02) and TNF (ptrend = 0.02); the likelihood of high-risk HPV clearance was reduced by 65% and 67%, respectively, among women in the highest as compared with the lowest quartile of change from baseline. Our results suggest that in women with a nontransient cervical HPV infection, proinflammatory, Type-1, and regulatory cytokines are elevated, underscoring the long-term commitment of local immune mediators to viral eradication.

Measurement of mucosal biomarkers in a phase 1 trial of intravaginal 3% StarPharma LTD 7013 gel (VivaGel) to assess expanded safety.

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Interlaboratory reproducibility of female genital tract cytokine measurements by Luminex: implications for microbicide safety studies.

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-1ß, 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-1ß 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.

Association between toll-like receptor expression and human papillomavirus type 16 persistence.

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.

Higher levels of cervicovaginal inflammatory and regulatory cytokines and chemokines in healthy young women with immature cervical epithelium.

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-1ß, 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.

Increased levels of immune activation in the genital tract of healthy young women from sub-Saharan Africa.

OBJECTIVES: 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. DESIGN: A cross-sectional study nested in a phase 1 microbicide trial. METHODS: 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. RESULTS: 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]. CONCLUSION: 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.

Enantioselective alpha-trifluoromethylation of aldehydes via photoredox organocatalysis.

The first enantioselective, organocatalytic alpha-trifluoromethylation and alpha-perfluoroalkylation of aldehydes have been accomplished using a readily available iridium photocatalyst and a chiral imidazolidinone catalyst. A range of alpha-trifluoromethyl and alpha-perfluoroalkyl aldehydes were obtained from commercially available perfluoroalkyl halides with high efficiency and enantioselectivity. The resulting alpha-trifluoromethyl aldehydes were subsequently shown to be versatile precursors for the construction of a variety of enantioenriched trifluoromethylated building blocks.

Diminished IFN-gamma and IL-10 and elevated Foxp3 mRNA expression in the cervix are associated with CIN 2 or 3.

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

Synthesis of highly functionalized pyrrolidines via a selective iodide-mediated ring expansion of methylenecyclopropyl amides.

This manuscript describes a highly selective iodide-mediated, tandem Mannich/cyclization to afford trans-2,3-disubstituted pyrrolidines from methylenecyclopropyl amides in good to excellent yields and selectivities. The reaction scope has been drastically expanded to include a wide array of aromatic, heteroaromatic and alpha,beta-unsaturated imines, as well as a variety of methylenecyclopropyl amides. Additionally, mechanistic studies were carried out to ascertain the nature of the ring-opening/ring-closing mechanism using deuterated substrates. Results from these studies indicate that the primary mechanism is an S(N)2/S(N)2 ring opening/ring closing and that iodine- or iodide-mediated isomerization of the iodo enolate is likely occurring.

Validation of reference genes in cervical cell samples from human papillomavirus-infected and -uninfected women for quantitative reverse transcription-PCR assays.

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.

Determination of cytokine protein levels in cervical mucus samples from young women by a multiplex immunoassay method and assessment of correlates.

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.