Objective: Tumor necrosis factor receptor (TNFR)–associated periodic syndrome (TRAPS) is an autosomal‐dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. Unlike other autoinflammatory diseases in which anti‐TNF therapy is largely a successful treatment option, therapy with the anti‐TNF drug infliximab is often ineffective in patients with TRAPS. Moreover, in certain cases, infliximab actually triggers severe episodes of inflammation. The aim of this study was to elucidate the mechanisms underlying such a reaction.Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with TRAPS. Both caspase 3 activity and NF‐κB subunit activity were determined by enzyme‐linked immunosorbent assay. Cytokine secretion was assessed using a specific customized human multiplex bead immunoassay kit.Results: Unlike findings in controls, cells from a family of 9 patients, all of whom carried the T50M mutation in TNFRSF1A, failed to respond to infliximab through proapoptotic induction of caspase 3 activity. Instead, we observed enhanced antiapoptotic c‐Rel subunit activity, accompanied by a significant increase in secretion of the proinflammatory cytokines interleukin‐ 1β (IL‐1β), IL‐1 receptor, IL‐6, IL‐8, and IL‐12.Conclusion: Altered extracellular conformation of TNFRI, resulting from the T50M mutation in TNFRSF1A, results in failure of PBMCs to induce an apoptotic response to infliximab. We hypothesize that failure to shed infliximab‐bound TNF/TNFRI from the cell surface of cells from patients with the T50M mutation triggers c‐Rel activation, and that this leads to a marked increase in cytokine secretion and an increased proinflammatory response. In light of these findings, we strongly advise caution when prescribing infliximab as anti‐TNF therapy to patients with TRAPS.

There is no prognostic test to ascertain whether cervical intraepithelial neoplasias (CINs) regress or progress. The majority of CINs regress in young women, and treatments increase the risk of adverse pregnancy outcomes. We investigated the ability of a DNA methylation panel (the S5 classifier) to discriminate between outcomes among young women with untreated CIN grade 2 (CIN2). Baseline pyrosequencing methylation and human papillomavirus (HPV) genotyping assays were performed on cervical cells from 149 women with CIN2 in a 2-year cohort study of active surveillance. Twenty-five lesions progressed to CIN grade 3 or worse, 88 regressed to less than CIN grade 1, and 36 persisted as CIN1/2. When cytology, HPV16/18 and HPV16/18/31/33 genotyping, and the S5 classifier were compared to outcomes, the S5 classifier was the strongest biomarker associated with regression vs progression. The S5 classifier alone or in combination with HPV16/18/31/33 genotyping also showed significantly increased sensitivity vs cytology when comparing regression vs persistence/progression. With both the S5 classifier and cytology set at a specificity of 38.6% (95% confidence interval [CI], 28.4–49.6), the sensitivity of the S5 classifier was significantly higher (83.6%; 95% CI, 71.9–91.8) than of cytology (62.3%; 95% CI, 49.0–74.4; P = 0.005). The highest area under the curve was 0.735 (95% CI, 0.621–0.849) in comparing regression vs progression with a combination of the S5 classifier and cytology, whereas HPV genotyping did not provide additional information. The S5 classifier shows high potential as a prognostic biomarker to identify progressive CIN2.

Objective: Tumor necrosis factor receptor–associated periodic syndrome (TRAPS) is an autosomal‐dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. The cellular mechanisms by which mutations in this gene trigger inflammation are currently unclear. Because NF‐κB is the major intracellular signaling component inducing secretion of proinflammatory cytokines, we sought to determine whether differences in the clinical phenotype of patients with TRAPS may be attributable to variable effects of TNFRSF1A mutations on TNFRI expression, localization, or NF‐κB activity.Methods: Peripheral blood mononuclear cells were obtained from patients (following informed consent), and cellular nuclear and cytosolic fractions were generated by subcellular fractionation. Localization of IκBα and NF‐κB was determined by Western blotting of the resultant fractions. NF‐κB subunit activity was determined by enzyme‐linked immunosorbent assay analysis and confirmed by electrophoretic mobility shift assay. Subcellular localization of TNFRI was determined by immunofluorescence confocal microscopy or by immunoblotting following affinity isolation of plasma membrane by subcellular fractionation.Results: Cells from patients with the fully penetrant C73R mutation had marked activation of the proinflammatory p65 subunit of NF‐κB. In contrast, cells from patients with the low‐penetrant R92Q mutation displayed high levels of DNA binding by the p50 subunit, an interaction previously linked to repression of inflammation. Interestingly, although cells from patients with the C73R mutation have no TNFRI shedding defect, there was nonetheless an unusually high concentration of functional TNFRI at the plasma membrane.Conclusion: High levels of TNFRI at the cell surface in patients with the C73R mutation hypersensitizes cells to stimulation by TNF, leading to increased NF‐κB p65 subunit activation and an exaggerated proinflammatory response.

Background: Human papillomavirus (HPV)-based screening is rapidly replacing cytology as the cervical screening modality of choice. In addition to being more sensitive than cytology, it can be done on self-collected vaginal or urine samples. This study will compare the high-risk HPV positivity rates and sensitivity of self-collected vaginal samples using four different collection devices and a urine sample. Methods: A total of 620 women referred for colposcopy were invited to provide an initial stream urine sample collected with the Colli-Pee device and take two vaginal self-samples, using either a dry flocked swab (DF) and a wet dacron swab (WD), or a HerSwab (HS) and Qvintip (QT) device. HPV testing was performed by the BD Onclarity HPV Assay. Results: A total of 600 vaginal sample pairs were suitable for analysis, and 505 were accompanied by a urine sample. Similar positivity rates and sensitivities for CIN2+ and CIN3+ were seen for DF, WD, and urine, but lower values were seen for QT and HS. No clear user preferences were seen between devices, but women found urine easiest to collect, and were more confident they had taken the sample correctly. The lowest confidence in collection was reported for HS. Conclusions: Urine, a DF swab, and WD swab all performed well and were well received by the women, whereas the Qvintip and HerSwab devices were less satisfactory. Impact: This is the first study to compare five self-sampling methods in the same women taken at the same time. It supports wider use of urine or vaginal self-sampling for cervical screening.

Design of dual antagonists for the chemokine receptors CCR2 and CCR5 will be greatly facilitated by knowledge of the structural differences of their binding sites. Thus, we computationally predicted the binding site of the dual CCR2/CCR5 antagonist N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzohepten-8-yl] carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium (TAK-779), and a CCR2-specific antagonist N-(carbamoylmethyl)-3-trifluoromethyl benzamido-parachlorobenzyl 3-aminopyrrolidine (Teijin compound 1) in an ensemble of predicted structures of human CCR2 and CCR5. Based on our predictions of the protein-ligand interactions, we examined the activity of the antagonists for cells expressing thirteen mutants of CCR2 and five mutants of CCR5. The results show that residues Trp98^2.60 and Thr292^7.40 contribute significantly to the efficacy of both TAK-779 and Teijin compound 1, whereas His121^3.33 and Ile263^6.55 contribute significantly only to the antagonistic effect of Teijin compound 1 at CCR2. Mutation of residues Trp86^2.60 and Tyr108^3.32 adversely affected the efficacy of TAK-779 in antagonizing CCR5-mediated chemotaxis. Y49A^1.39 and E291A^7.39 mutants of CCR2 showed a complete loss of CCL2 binding and chemotaxis, despite robust cell surface expression, suggesting that these residues are critical in maintaining the correct receptor architecture. Modeling studies support the hypothesis that the residues Tyr49^1.39, Trp98^2.60, Tyr120^3.32, and Glu291^7.39 of CCR2 form a tight network of aromatic cluster and polar contacts between transmembrane helices 1, 2, 3, and 7.