Description of Bacterial RNA Transcripts Detected in Mycobacterium tuberculosis - Infected Cells from Peripheral Human Granulomas using Single Cell RNA Sequencing.

Mycobacterium tuberculosis (Mtb) remains a global human health threat and a significant cause of human morbidity and mortality. We document here the capture of Mtb transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Because of early literature suggesting the possible presence of polyadenylated transcripts in Mtb RNA, we included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples from patients presenting at the TB clinic, Port Moresby General Hospital, Papua New Guinea. We used 10X Genomics single-cell RNA sequencing transcriptomics pipeline, which initiates mRNA amplification with poly-T primers on ∼30-micron beads designed to capture, in this case, human mRNA associated with individual cells in the clinical samples. Utilizing the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences (rrs and rrl, respectively) and other bacterial gene transcripts in the cell culture and patient samples. We interpret Mtb reads associated with the host cell's unique molecular identifier (UMI) and transcriptome to indicate infection of that individual host cell. The Mtb transcripts detected showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least 1 sequence difference compared to the H37Rv reference genome. The data presented includes only bacterial sequences from patients with TB infections that were confirmed by the hospital pathology lab using acid-fast microscopy and/or GeneXpert analysis. The repeated, non-random nature of the sequence variations detected in Mtb rrs and rrl transcripts from multiple patients, suggests that, even though this appears to be a stochastic process, there is possibly some selective pressure that limits the types and locations of sequence variation allowed. The variation does not appear to be entirely artefactual, and it is hypothesized that it could represent an additional mechanism of adaptation to enhance bacterial fitness against host defenses or chemotherapy.

Serum miRNA improves the accuracy of a multivariate index assay for triage of an adnexal mass.

OBJECTIVE: To determine whether a multimodal assay combining serum microRNA with protein biomarkers and metadata improves triage assessment of an adnexal mass. METHODS: Serum samples from 468 training subjects (191 cancer cases and 277 benign adnexal mass controls or healthy controls) were analyzed for seven protein biomarkers and 180 miRNA. Circulating analyte data were combined with age and menopausal status (metadata) into a neural network model to classify samples as cases or controls. Forward regression with ten-fold cross-validation minimized the dimensionality of the model while maximizing linear separation between cases and controls. Model validation proceeded using both internal (44 cases and 56 controls) and external validation sets (51 cases and 59 controls). RESULTS: The total study population comprised 678 subjects, including 286 cases and 392 controls. Overall, 290 (43%) of the subjects were premenopausal. A panel of 10 miRNA delivered optimal performance when combined with protein and metadata features. The combined model improved the Receiver Operator Characteristic Area Under the Curve (ROC AUC) on the internal (AUC = 0.9; 95% CI 0.81-0.95) and external validation sets (AUC = 0.95; 95% CI 0.90-0.98) compared to miRNA alone or proteins plus metadata (without miRNA). On external validation, the combined model offered 92% sensitivity at 80% specificity overall, with 80% and 100% sensitivity for early and late-stage cancers, respectively, including 78% sensitivity for early-stage, serous ovarian cancers and 82% sensitivity for early-stage, non-serous cancers. CONCLUSIONS: A multimodal assay combining miRNA with protein biomarkers, age, and menopausal status improves surgical triage of an adnexal mass.

Association of Operative Approach With Postoperative Outcomes in Neonates Undergoing Surgical Repair of Esophageal Atresia and Tracheoesophageal Fistula.

INTRODUCTION: Minimally invasive surgery (MIS) is gaining traction as a first-line approach to repair congenital anomalies. This study aims to evaluate outcomes for neonates undergoing open versus MIS repairs for esophageal atresia/tracheoesophageal fistula (EA/TEF). METHODS: In this retrospective study, neonates undergoing EA/TEF repair from 2013 to 2020 were identified using the National Surgical Quality Improvement Program-Pediatric database. Proportions of operative approach (open vs. MIS) over time were analyzed. A propensity score-matched analysis using preoperative characteristics was performed and outcomes were compared including composite morbidity and reintervention rates (overall, major [thoracoscopy, thoracotomy], and minor [chest/feeding tube placement, endoscopy]) between operative approaches. Pearson's chi-square or Fisher's exact tests were used as appropriate. RESULTS: We identified 1738 neonates who underwent EA/TEF repair. MIS utilization increased over time. Pre-match, neonates undergoing open repair were more likely to be premature, lower weight, ventilator dependent, and have cardiac risk factors with higher severity. Post-match, the groups were similar and included 340 neonates per group. MIS repair was associated with longer median operative time (209 vs. 174 min, p < 0.001) and increased overall post-operative intervention rates (7.6% vs. 2.9%, p = 0.01). There were no differences in composite morbidity (24.4% vs. 25.0%, p = 0.86) outside of reintervention. CONCLUSION: MIS approach for neonates with EA/TEF appears to be associated with a higher rate of reinterventions. Further studies evaluating MIS approaches for the repair of EA/TEF are needed to better define short- and long-term outcomes. TYPE OF STUDY: Retrospective comparative study. LEVEL OF EVIDENCE: Level III.

Targeting NGF but not VEGFR1 or BDNF signaling reduces endometriosis-associated pain in mice.

INTRODUCTION: Endometriosis is a chronic inflammatory disease that affects âˆ¼10 % of women. A significant fraction of patients experience limited or no efficacy with current therapies. Tissue adjacent to endometriosis lesions often exhibits increased neurite and vascular density, suggesting that disease pathology involves neurotrophic activity and angiogenesis. OBJECTIVES: We aim to evaluate the potential for key tyrosine-kinase-receptor-coupled neurotrophic molecules to contribute to endometriosis-associated pain in mice. METHODS: Peritoneal fluid was collected from endometriosis patients undergoing surgery and the levels of NGF and VEGFR1 regulators (VEGFA, VEGFB, PLGF, and sVEGFR1) were quantified by ELISA. VEGFR1 regulator concentrations were used to calculate VEGFR1 occupancy. We used genetic depletion, neutralizing antibodies, and pharmacological approaches to specifically block neurotrophic ligands (NGF or BDNF) or receptors (VEGFR1, TRKs) in a murine model of endometriosis-associated pain. Endometriosis-associated pain was measured using von Frey filaments, quantification of spontaneous abdominal pain-related behavior, and thermal discomfort. Disease parameters were evaluated by lesion size and prevalence. To evaluate potential toxicity, we measured the effect of entrectinib dose and schedule on body weight, liver and kidney function, and bone structure (via micro-CT). RESULTS: We found that entrectinib (pan-Trk inhibitor) or anti-NGF treatments reduced evoked pain, spontaneous pain, and thermal discomfort. In contrast, even though calculated receptor occupancy revealed that VEGFR1 agonist levels are sufficient to support signaling, blocking VEGFR1 via antibody or tamoxifen-induced knockout did not reduce pain or lesion size in mice. Targeting BDNF-TrkB with an anti-BDNF antibody also proved ineffective. Notably, changing dosing schedule to once weekly eliminated entrectinib-induced bone-loss without decreasing efficacy against pain. CONCLUSIONS: This suggests NGF-TrkA signaling, but not BDNF-TrkB or VEGF-VEGFR1, mediates endometriosis-associated pain. Moreover, entrectinib blocks endometriosis-associated pain and reduces lesion sizes. Our results also indicated that entrectinib-like molecules are promising candidates for endometriosis treatment.

Extracorporeal life support use in patients with bronchopulmonary dysplasia: A single center case series.

PURPOSE: Preterm pediatric patients with bronchopulmonary dysplasia (BPD) represent a subgroup previously deemed high risk candidates for ECLS (extracorporeal life support) due to suspected high mortality or increased post ECLS morbidity. The aim of this study was to determine outcomes for patients with an established history of BPD who subsequently required ECLS. METHODS: A single center retrospective review was performed between 01/2010-06/2022 for patients less than 2 years of age, born prematurely (<32 weeks) with a subsequent diagnosis of BPD, and who required ECLS for respiratory failure. Demographic and clinical data, including ECLS data, were collected. Speech, language, feeding/swallowing, cognitive, hearing, vision, or motor function deficits were obtained with a median follow up of 42 months following discharge. RESULTS: Nineteen patients met criteria. The median birth weight and gestational age was 0.86 kg (IQR 0.73, 1.0) and 26 weeks (IQR 25, 27), respectively. The median chronological age at cannulation was 12.1 months. The most common etiologies for respiratory failure requiring ECLS were viral (68.4%) and bacterial (21.1%) pneumonia. Survival to decannulation was 78.9% (15/19) and survival to hospital discharge was 63.2% (12/19). Amongst survivors to discharge, 42% (5/12) required new or additional home oxygen and 50% (6/12) were noted to have neurodevelopmental/behavioral concerns on follow up at 1 year with 25% (3/12) with concerns beyond a year. CONCLUSION: Patients with underlying BPD who require ECLS have comparable mortality and long-term neurodevelopmental outcomes to non-BPD patients with respiratory failure. This information can be useful when considering ECLS candidacy and providing family counseling.

Single Cell Analysis of Peripheral TB-Associated Granulomatous Lymphadenitis.

We successfully employed a single cell RNA sequencing (scRNA-seq) approach to describe the cells and the communication networks characterizing granulomatous lymph nodes of TB patients. When mapping cells from individual patient samples, clustered based on their transcriptome similarities, we uniformly identify several cell types that known to characterize human and non-human primate granulomas. Whether high or low Mtb burden, we find the T cell cluster to be one of the most abundant. Many cells expressing T cell markers are clearly quantifiable within this CD3 expressing cluster. Other cell clusters that are uniformly detected, but that vary dramatically in abundance amongst the individual patient samples, are the B cell, plasma cell and macrophage/dendrocyte and NK cell clusters. When we combine all our scRNA-seq data from our current 23 patients (in order to add power to cell cluster identification in patient samples with fewer cells), we distinguish T, macrophage, dendrocyte and plasma cell subclusters, each with distinct signaling activities. The sizes of these subclusters also varies dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations and macrophage/dendrocyte populations were negatively correlated with NK cell populations. In addition, we also discovered that the scRNA-seq pipeline, designed for quantification of human cell mRNA, also detects Mtb RNA transcripts and associates them with their host cell's transcriptome, thus identifying individual infected cells. We hypothesize that the number of detected bacterial transcript reads provides a measure of Mtb burden, as does the number of Mtb-infected cells. The number of infected cells also varies dramatically in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomas, including many interactions between stromal or endothelial cells and the other component cells, such as Collagen, FN1 and Laminin,. In addition, other more selective communications pathways, including MIF, MHC-1, MHC-2, APP, CD 22, CD45, and others, are identified as originating or being received by individual immune cell components.

Variables Affecting CA15.3 Tumor Antigen Expression and Antibodies against It in Female National Health and Nutritional Survey Participants.

BACKGROUND: Cancers of ductal origin often express glycoprotein mucin 1 (MUC1), also known as CA15.3, with higher levels leading to poor prognosis. Conversely, anti-MUC1 antibodies develop in some patients, leading to better prognosis. We sought to identify epidemiologic factors associated with CA15.3 antigen or antibody levels. METHODS: Levels of CA15.3 antigen and anti-CA15.3 IgG antibodies were measured in archived sera from 2,302 mostly healthy women from the National Health and Nutritional Survey; and epidemiologic predictors of their levels were examined using multivariate and correlational analyses. RESULTS: Among racial groups, Black women had the highest levels of CA15.3 antigen and lowest levels of antibodies. Increasing body mass index and current smoking were associated with low anti-CA15.3 antibody levels. Low CA15.3 antigen levels were seen in oral contraceptive users and high levels in women who were pregnant or lactating at the time of blood collection, with the latter group also having high antibody levels. Past reproductive events associated with high antigen levels included the following: later age at menarche, having given birth, and history of endometriosis. Lower antigen levels were seen with increasing duration of OC use. Anti-CA15.3 antibody levels decreased with an increasing estimated number of ovulatory years. CONCLUSIONS: Key determinants of CA.15.3 antigen or antibody levels include the following: race, body mass index, smoking, later menarche, childbirth, number of ovulatory cycles, and endometriosis. IMPACT: This study supports the premise that known epidemiologic factors affecting risk for or survival after MUC1-expressing cancers may, at least partially, operate through their association with CA15.3 antigen or antibody levels.

Clinical and immunophenotype correlating with response to immunotherapy in paediatric patients with primary liver carcinoma. A case series.

BACKGROUND: Paediatric hepatocellular carcinomas (HCC) traditionally arise in the context of a normal structural and functional liver and carry a dismal prognosis. While chemotherapy is the frontline standard, there is emerging interest in the study of immunotherapies for paediatric patients with relapsed/refractory disease. There is limited data to support whether immunotherapies will be of utility in this patient population. METHODS: Six paediatric patients (median age:16 years, range: 12-17 at the time of treatment) with advanced hepatocellular neosplams, either conventional hepatocellular or fibrolamellar carcinoma, were treated with immunotherapy. Patients were consented to institutional genomic profiling and biobanking protocols. Baseline samples and serial tissue samples, when available, were evaluated for somatic mutation rate, actionable gene mutations, and pan-immune bulk RNA expression profiling. Results were correlated with clinical course. FINDINGS: Three patients responded to checkpoint inhibition: one achieved a complete, durable response and the other two, prolonged stable disease. Three additional patients progressed. Diagnostic tissue from the complete responder demonstrated a higher relative mutational burden and robust immune infiltrate. Pre-treatment samples from the three responders demonstrated decreased expression of genes associated with T-cell dysfunction. INTERPRETATION: A subset of patients with primary paediatric hepatocellular tumours will respond to immunotherapy. Immunotherapies are currently under prospective study for relapsed/refractory liver tumours in paediatric patients. Results from this report support the prospective collection of serial serum and tissue samples which may further identify genomic and immunophenotypic patterns predictive of response. FUNDING: This work was supported by Philanthropic funds (Pan Mass Challenge, Team Angus and Team Perspective).

Effect of positive social comparative feedback on the resting state connectivity of dopaminergic neural pathways: A preliminary investigation.

Positive social comparative feedback is hypothesized to generate a dopamine response in the brain, similar to reward, by enhancing expectancies to support motor skill learning. However, no studies have utilized neuroimaging to examine this hypothesized dopaminergic mechanism. Therefore, the aim of this preliminary study was to investigate the effect of positive social comparative feedback on dopaminergic neural pathways measured by resting state connectivity. Thirty individuals practiced an implicit, motor sequence learning task and were assigned to groups that differed in feedback type. One group received feedback about their actual response time to complete the task (RT ONLY), while the other group received feedback about their response time with positive social comparison (RT + POS). Magnetic resonance imaging was acquired at the beginning and end of repetitive motor practice with feedback to measure practice-dependent changes in resting state brain connectivity. While both groups showed improvements in task performance and increases in performance expectancies, ventral tegmental area and the left nucleus accumbens (mesolimbic dopamine pathway) resting state connectivity increased in the RT + POS group but not in the RT ONLY group. Instead, the RT ONLY group showed increased connectivity between ventral tegmental area and primary motor cortex. Positive social comparative feedback during practice of a motor sequence task may induce a dopaminergic response in the brain along the mesolimbic pathway. However, given that absence of effects on expectancies and motor learning, more robust and individualized approaches may be needed to provide beneficial psychological and behavioral effects.

Chitosan Coating with Rosemary Extract Increases Shelf Life and Reduces Water Losses from Beef.

This study aimed to evaluate the influence of films based on chitosan and rosemary extract on the physicochemical, microbiological, and oxidative characteristics of beef. Refrigerated steaks of Longissimus dorsi were distributed in a factorial arrangement (4 × 4) into four treatments consisting of four edible films (control; chitosan; chitosan + 4% rosemary extract; and chitosan + 8% rosemary extract) and four days of aging (0, 2, 4, and 8 days). Incorporating 4% or 8% rosemary extract into the chitosan film improved the characteristics of the films in terms of moisture absorption and elasticity. The edible coatings with chitosan and rosemary extract and the different days of aging increased the tenderness and decreased the lipid oxidation of beef. In addition, the chitosan films containing rosemary extract increased the water-holding capacity and decreased the cooking losses of beef. The films containing 4% and 8% rosemary extract decreased the development of mesophilic and psychrotrophic bacteria and Staphylococcus ssp. in beef. We recommend incorporating 4% rosemary extract into chitosan-based coatings to preserve the quality of refrigerated beef.

Use of Sodium Thiosulfate as an Otoprotectant in Patients With Cancer Treated With Platinum Compounds: A Review of the Literature.

PURPOSE: Hearing loss occurs in 50%-70% of children treated with cisplatin. Scientific efforts have led to the recent approval of a pediatric formula of intravenous sodium thiosulfate (STS) for otoprotection by the US Food and Drug Administration, the European Medicines Agency, and the Medicines and Health Regulatory Authority in the United Kingdom. To inform stakeholders regarding the clinical utility of STS, the current review summarizes available literature on the efficacy, pharmacokinetics (PK), and safety of systemic STS to minimize cisplatin-induced hearing loss (CIHL). DESIGN: A comprehensive narrative review is presented. RESULTS: Thirty-one articles were summarized. Overall, systemic STS effectively reduces CIHL in the preclinical and controlled clinical study settings, in both adults and children with cancer. The extent of CIHL reduction depends on the timing and dosing of STS in relation to cisplatin. Both preclinical and clinical data suggest that systemic STS may affect plasma platinum levels, but studies are inconclusive. Delayed systemic administration of STS, at 6 hours after the cisplatin infusion, does not affect cisplatin-induced inhibition of tumor growth or cellular cytotoxicity in the preclinical setting, nor affect cisplatin efficacy and survival in children with localized disease in the clinical setting. CONCLUSION: Systemic administration of STS effectively reduces the development and degree of CIHL in both the preclinical and clinical settings. More studies are needed on the PK of STS and cisplatin drug combinations, the efficacy and safety of STS in patients with disseminated disease, and the ability of STS to prevent further deterioration of pre-established hearing loss.

Off-label prescribing of immune checkpoint inhibitor therapy at a single pediatric cancer center.

BACKGROUND: Immune checkpoint inhibitors (ICI) have improved outcomes in a variety of adult cancers and are prescribed with increasing frequency across oncology. However, patterns of off-label use of ICI in pediatrics remain unclear. METHODS: This is a single-institution, retrospective cohort study evaluating off-label ICI use in pediatric and young adult patients with cancer treated at our institution from 2014 to 2022. Response was based on clinician assessment derived from clinical records. Immune-related adverse events (iRAEs) were classified according to CTCAE v5.0. RESULTS: We identified 50 unique patients treated with off-label ICI (28 with solid tumors, 20 with central nervous system (CNS) tumors, 2 with hematologic malignancies). At time of ICI initiation, only five patients (10%) had localized disease, and all but one patient was treated in the relapsed/refractory setting. All patients were treated with the FDA-approved weight-based dosing recommendations. Overall, there was disease control in 21 patients (42%), with best response including one complete response (melanoma), two partial responses (high-grade glioma, CNS nongerminomatous germ cell tumor), and 18 patients with stable disease. Forty-four patients (88%) eventually experienced disease progression. Among 22 patients (44%) experiencing iRAEs, 10 (20%) had a grade ≥3 irAE, 12 (24%) required corticosteroids, and 14 (28%) required ICI discontinuation. irAE occurrence was associated with significantly improved progression-free survival (HR 0.35; 95% CI: 0.18 to 0.68; p = 0.002) and overall survival (HR 0.33; 95% CI: 0.17 to 0.66; p = 0.002). CONCLUSIONS: At our institution, ICI was most commonly prescribed in the relapsed/refractory setting to patients with metastatic disease. The treatment was generally well-tolerated in the pediatric population. The overall response rate was low, and the majority of patients eventually experienced disease progression. A few patients, however, had durable treatment responses. Further studies are needed to identify which pediatric patients are most likely to benefit from ICI.

Examining chromatin heterogeneity through PacBio long-read sequencing of M.EcoGII methylated genomes: an m6A detection efficiency and calling bias correcting pipeline.

Recent studies have combined DNA methyltransferase footprinting of genomic DNA in nuclei with long-read sequencing, resulting in detailed chromatin maps for multi-kilobase stretches of genomic DNA from one cell. Theoretically, nucleosome footprints and nucleosome-depleted regions can be identified using M.EcoGII, which methylates adenines in any sequence context, providing a high-resolution map of accessible regions in each DNA molecule. Here, we report PacBio long-read sequence data for budding yeast nuclei treated with M.EcoGII and a bioinformatic pipeline which corrects for three key challenges undermining this promising method. First, detection of m6A in individual DNA molecules by the PacBio software is inefficient, resulting in false footprints predicted by random gaps of seemingly unmethylated adenines. Second, there is a strong bias against m6A base calling as AT content increases. Third, occasional methylation occurs within nucleosomes, breaking up their footprints. After correcting for these issues, our pipeline calculates a correlation coefficient-based score indicating the extent of chromatin heterogeneity within the cell population for every gene. Although the population average is consistent with that derived using other techniques, we observe a wide range of heterogeneity in nucleosome positions at the single-molecule level, probably reflecting cellular chromatin dynamics.

Subgroups of pelvic pain are differentially associated with endometriosis and inflammatory comorbidities: a latent class analysis.

ABSTRACT: Chronic pelvic pain is heterogeneous with potentially clinically informative subgroups. We aimed to identify subgroups of pelvic pain based on symptom patterns and investigate their associations with inflammatory and chronic pain-related comorbidities. Latent class analysis (LCA) identified subgroups of participants (n = 1255) from the Adolescence to Adulthood (A2A) cohort. Six participant characteristics were included in the LCA: severity, frequency, and impact on daily activities of both menstruation-associated (cyclic) and non-menstruation-associated (acyclic) pelvic pain. Three-step LCA quantified associations between LC subgroups, demographic and clinical variables, and 18 comorbidities (10 with prevalence ≥10%). Five subgroups were identified: none or minimal (23%), moderate cyclic only (28%), severe cyclic only (20%), moderate or severe acyclic plus moderate cyclic (9%), and severe acyclic plus severe cyclic (21%). Endometriosis prevalence within these 5 LCA-pelvic pain-defined subgroups ranged in size from 4% in "none or minimal pelvic pain" to 24%, 72%, 70%, and 94%, respectively, in the 4 pain subgroups, with statistically significant odds of membership only for the latter 3 subgroups. Migraines were associated with significant odds of membership in all 4 pelvic pain subgroups relative to those with no pelvic pain (adjusted odds ratios = 2.92-7.78), whereas back, joint, or leg pain each had significantly greater odds of membership in the latter 3 subgroups. Asthma or allergies had three times the odds of membership in the most severe pain group. Subgroups with elevated levels of cyclic or acyclic pain are associated with greater frequency of chronic overlapping pain conditions, suggesting an important role for central inflammatory and immunological mechanisms.

Plasma microRNA expression in adolescents and young adults with endometriosis: the importance of hormone use.

Introduction: Prior studies have investigated the diagnostic potential of microRNA (miRNA) expression profiles for endometriosis. However, the vast majority of previous studies have only included adult women. Therefore, we sought to investigate differential expression of miRNAs among adolescents and young adults with endometriosis. Methods: The Women's Health Study: from Adolescence to Adulthood (A2A) is an ongoing WERF EPHect compliant longitudinal cohort. Our analysis included 64 patients with surgically-confirmed endometriosis (96% rASRM stage I/II) and 118 females never diagnosed with endometriosis frequency matched on age (median = 21 years) and hormone use at blood draw. MicroRNA measurement was separated into discovery (10 cases and 10 controls) and internal replication (54 cases and 108 controls) phases. The levels of 754 plasma miRNAs were assayed in the discovery phase using PCR with rigorous internal control measures, with the relative expression of miRNA among cases vs. controls calculated using the 2-ΔΔCt method. miRNAs that were significant in univariate analyses stratified by hormone use were included in the internal replication phase. The internal replication phase was split 2:1 into a training and testing set and utilized FirePlex miRNA assay to assess 63 miRNAs in neural network analyses. The testing set of the validation phase was utilized to calculate the area under the curve (AUC) of the best fit models from the training set including hormone use as a covariate. Results: In the discovery phase, 49 miRNAs were differentially expressed between endometriosis cases and controls. The associations of the 49 miRNAs differed by hormone use at the time of blood draw. Neural network analysis in the testing set of the internal replication phase determined a final model comprising 5 miRNAs (miR-542-3p, let-7b-3p, miR-548i, miR-769-5p, miR-30c-1-3p), yielding AUC = 0.77 (95% CI: 0.67-0.87, p < 0.001). Sensitivity in the testing dataset improved (83.3% vs. 72.2%) while the specificity decreased (58.3% vs. 72.2%) compared to the training set. Conclusion: The results suggest that miR-542-3p, let-7b-3p, miR-548i, miR-769-5p, miR-30c-1-3p may be dysregulated among adolescent and young adults with endometriosis. Hormone use was a significant modifier of miRNA dysregulation and should be considered rigorously in miRNA diagnostic studies.

Pelvic pain symptoms and endometriosis characteristics in relation to oxidative stress among adolescents and adults with and without surgically-confirmed endometriosis.

Background: While the majority of reproductive-aged females will experience pelvic pain during their lives, biological mechanisms underlying pelvic pain are not well understood. We investigated associations between pelvic pain symptoms and oxidative stress among people with and without surgically-confirmed endometriosis. Methods: Using an enzyme-linked immunosorbent assay, we measured 8-Hydroxy-2'-deoxyguanosine (8-OHdG) in urine samples and corrected for creatinine levels in 434 surgically-confirmed endometriosis participants compared to 605 participants never diagnosed with endometriosis. At enrollment, participants reported details of their pelvic pain symptoms. Linear regression was used to compute geometric mean (GM) creatinine-corrected 8-OHdG levels with 95% confidence intervals (CI) among all participants and those with and without endometriosis separately, adjusting for potential confounders. Interactions by surgically-confirmed endometriosis status were tested by Wald statistics. Results: No trends in 8-OHdG were observed among those with or without endometriosis for severity or frequency of dysmenorrhea, acyclic pelvic pain, dyspareunia or pain with bowel movements. Among endometriosis participants, lower 8-OHdG levels were observed for participants with any white, blue/black, or brown lesions (GM=76.7 versus 82.9 ng/mg; p=0.10), which was primarily driven by lower levels of 8-OHdG for any blue/black lesions (GM=72.8 versus 81.6 ng/mg; p=0.05). Conclusion: While no associations were observed between 8-OHdG and pelvic pain symptoms, future research is needed to assess how other pathways of oxidative damage, e.g. through proteins or lipids, may affect endometriosis-associated symptoms. Additionally, further research is needed to understand differences in oxidative stress among endometriosis lesion sub-phenotypes.

Plasma proteins and persistent postsurgical pelvic pain among adolescents and young adults with endometriosis.

BACKGROUND: Noninvasive biomarkers that predict surgical treatment response would inform personalized treatments and provide insight into potential biologic pathways underlying endometriosis-associated pain and symptom progression. OBJECTIVE: To use plasma proteins in relation to the persistence of pelvic pain following laparoscopic surgery in predominantly adolescents and young adults with endometriosis using a multiplex aptamer-based proteomics biomarker discovery platform. STUDY DESIGN: We conducted a prospective analysis including 142 participants with laparoscopically-confirmed endometriosis from the Women's Health Study: From Adolescence to Adulthood observational longitudinal cohort with study enrollment from 2012-2018. Biologic samples and patient data were collected with modified World Endometriosis Research Foundation Endometriosis Phenome and Biobanking Harmonization Project tools. In blood collected before laparoscopic ablation or excision of endometriosis, we simultaneously measured 1305 plasma protein levels, including markers for immunity, angiogenesis, and inflammation, using SomaScan. Worsening or persistent postsurgical pelvic pain was defined as having newly developed, persistent (ie, stable), or worsening severity, frequency, or persistent life interference of dysmenorrhea or acyclic pelvic pain at 1-year postsurgery compared with presurgery. We calculated odds ratios and 95% confidence intervals using logistic regression adjusted for age, body mass index, fasting status, and hormone use at blood draw. We applied Ingenuity Pathway Analysis and STRING analysis to identify pathophysiologic pathways and protein interactions. RESULTS: The median age at blood draw was 17 years (interquartile range, 15-19 years), and most participants were White (90%). All had superficial peritoneal lesions only and were treated by excision or ablation. One-year postsurgery, pelvic pain worsened or persisted for 76 (54%) of these participants with endometriosis, whereas pelvic pain improved for 66 (46%). We identified 83 proteins associated with worsening or persistent pelvic pain 1-year postsurgery (nominal P<.05). Compared with those with improved pelvic pain 1-year postsurgery, those with worsening or persistent pelvic pain had higher plasma levels of CD63 antigen (odds ratio, 2.98 [95% confidence interval, 1.44-6.19]) and CD47 (odds ratio, 2.68 [95% confidence interval, 1.28-5.61]), but lower levels of Sonic Hedgehog protein (odds ratio, 0.55 [95% confidence interval, 0.36-0.84]) in presurgical blood. Pathways related to cell migration were up-regulated, and pathways related to angiogenesis were down-regulated in those with worsening or persistent postsurgical pelvic pain compared with those with improved pain. When we examined the change in protein levels from presurgery to postsurgery and its subsequent risk of worsening or persistent postsurgical pain at 1-year follow-up, we observed increasing levels of Sonic Hedgehog protein from presurgery to postsurgery was associated with a 4-fold increase in the risk of postsurgical pain (odds ratio [quartile 4 vs 1], 3.86 [1.04-14.33]). CONCLUSION: Using an aptamer-based proteomics platform, we identified plasma proteins and pathways associated with worsening or persistent pelvic pain postsurgical treatment of endometriosis among adolescents and young adults that may aid in risk stratification of individuals with endometriosis.

Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo.

BACKGROUND: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. METHODS: We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. RESULTS: NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. CONCLUSIONS: These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages.

Predicting response to and relapse after treatment of trichotillomania with the Comprehensive Behavioral model (ComB).

Prior studies of behavior therapy for trichotillomania (TTM) have shown that response is variable, and relapse after treatment discontinuation is common. Little information is available concerning prognostic factors capable of predicting individual differences in response or maintenance of improvement. The present study is a secondary analysis of a randomized controlled trial (N = 36) of the Comprehensive Behavioral (ComB) model of treatment for TTM (Carlson et al., 2021). We investigated age, disorder history, pre-treatment symptom severity, longest prior period of abstinence from pulling, and Emotion and Intention hair pulling styles as predictors of initial response. We studied age, disorder history, pre-treatment symptom severity, longest prior period of abstinence from pulling, and post-treatment symptom severity or hair-pulling abstinence as predictors of relapse following treatment. Older age significantly predicted lower TTM severity following treatment. Lower pre-treatment severity significantly predicted lower severity of TTM at the 3-month follow-up.