Background and AimsEmerging evidence supports a strong bidirectional relationship between the gastrointestinal system and the central nervous system, referred to as the gut-brain axis. This axis has been proposed to be of particular importance in the context of neurodevelopmental disorders including autism spectrum disorder (ASD). While neurotransmitter dysregulation and synaptic dysfunction have been well documented in the brains of individuals with ASD, less is known about how these molecular changes manifest in the gutMethodsIn this study, we analyzed RNA signatures in biopsies from the ileum and colon of children with chronic GI symptoms, either with (cases) or without (controls) an autism diagnosis.ResultsGenes associated with serotonin signaling (AANAT, HTR1D, HTR3A, SLC6A4), dopamine synthesis and receptor function (DDC,DRD5), and glutamatergic pathways (GAD2, GRIK3, GLUD1, GRIN2C, SLC1A3, SLC38A1, GABRP, GABRB3, GRM4) were significantly altered in children with ASD, suggesting a broad disruption in neurotransmitter homeostasis in the gut. Additionally,several neuroactive compounds, such as NPY, GIP, NTS, AREG, GKN1, GHRL, LEP, PDYN, and EDN2, were also impacted,further implicating altered neuroimmune interactions and developmental pathways in gut tissues.ConclusionsThese findings highlight the potential role of the gut as a critical modulator of neurodevelopmental processes in ASD and suggest that gut-brain axis dysregulation may contribute to the ASD phenotype. This study provides new insights into the molecular mechanisms in the gastrointestinal tract in individuals with ASD and suggests novel therapeutic targets for restoring healthy gut-brain communication.

IntroductionRespiratory syncytial virus A (RSVA) and respiratory syncytial virus B (RSVB) may induce distinct airway host responses. We compared them with influenza A virus (IAV) and human metapneumovirus (hMPV) in airway organ tissue equivalents (OTEs) and developed the Relaxed Magnitude Altitude Score with Hub-Centric Network Analysis framework (RMAS-HCNA).MethodsOTEs were infected with RSVA-GFP5 or RSVB-GFP3 at low multiplicity of infection and profiled at 24 and 72 h using the NanoString nCounter Host Response Panel. Previous IAV and hMPV profiles were integrated. Analyses included source-matched differential expression, cross-virus ranking, network-based module selection, nested cross-validation, and Gene Ontology Biological Process enrichment.ResultsRSVA showed the strongest temporal expansion and developed a late response centered on intracellular transport, localization, and organelle organization. RSVB remained the most restricted response, shifting from early inflammatory and signaling-associated activity to a later interferon-stimulated profile. IAV maintained a broad antiviral response, whereas hMPV combined limited induction with extensive late downregulation of stress, catabolic, metabolic, and transport-related processes. A shared antiviral component was present across infections, while downregulated responses were largely virus dependent. Under five-fold outer and four-fold inner nested cross-validation, the selected genes achieved a mean outer-test Macro F1 of 0.859 and balanced accuracy of 0.888, compared with 0.604 and 0.616 for the all-gene comparator.DiscussionThe four viruses shared an antiviral foundation but followed distinct temporal and functional trajectories. The framework identified compact, biologically representative panels that generalized better than the complete measured gene set, although independent genome-wide and in vivo validation remains necessary.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a heterogeneous condition of uncertain etiology. We assessed bladder collagen characteristics in phenotypically characterized IC/BPS patient subgroups that may influence pathophysiology. Forty-four females (30 IC/BPS; 14 non-IC/BPS) were included. Patients were divided into groups based on Hunner lesions (HL; N=14 or non-HL; N=16) and anesthetic bladder capacity (BC) (BC≤500 cc; low BC; N=17 or BC>500 cc; non-low BC; N=13). Bladder biopsy tissue slides were stained with hematoxylin and eosin or picrosirius red and semi-quantitatively analyzed by a pathologist. CT-FIRE software was used to quantitatively assess lamina propria collagen. All patients with IC/BPS had lower collagen fiber density independent of subgroup (p<0.0001-0.0038) compared to controls. HL had more peri-muscular collagen accumulation (p=0.0061), more acute inflammation (p=0.0364), more severe chronic inflammation (p=0.0088), and narrower collagen fibers than non-HL and controls. Non-low-BC patients had lower collagen density (p=0.0075) and straighter collagen fibers (p=0.0127) than low BC. Low-BC patients had narrower collagen fibers than control (p=0.0096). IC/BPS, regardless of subgroup, is associated with a bladder lamina propria with diminished collagen density. HL, non-low-BC, and low-BC subgroups have unique collagen characteristics. These findings suggest a collagen fiber destruction and redistribution process, which differs by subgroup, and may contribute to pathophysiology of IC/BPS.

INTRODUCTION AND HYPOTHESIS:Hunner lesions (HL) are distinctive ulcerative bladder lesions characterized by a central scar and surrounding erythema. Traditionally, HL has been used as a clinical diagnostic tool for a subset of patients with interstitial cystitis/bladder pain syndrome (IC/BPS); however, emerging evidence supports a unique pathophysiology for IC/BPS with HL (HIC) that is distinct from IC/BPS without HL (NHIC). This review is aimed at synthesizing current literature on the etiology of HL/HIC and evaluate whether HIC and NHIC warrant classification as separate clinical conditions with distinct management strategies. METHODS:A comprehensive literature review using PubMed was conducted to identify literature surrounding HL etiology and pathophysiology. Case-control studies, cohort studies, experimental studies, case reports, and animal studies were included; editorial comments and literature reviews were excluded from this review. RESULTS:Emerging data suggest that a subset of patients might be predisposed to HIC, including alteration in major histocompatibility complex (MHC) molecules, HLA amino acid positions, and additional protein expression alterations detected by RNA sequencing. Hypoxia-inducible factor activation and chronic ischemia have also been shown to play a role in HL development. Both local and systemic inflammation are seen in HIC, with clonal B-cell expansion influencing the upregulation of inflammatory cytokines, chemokines, and lymphoplasmacytic cells. With additional validation these biomarkers have the potential to serve as a diagnostic tool. CONCLUSION:Hunner lesion with IC/BPS is a chronic condition likely caused by an upregulation of humoral immunity leading to an overexpression of inflammatory markers within the urothelium, suggesting an autoimmune-mediated pathology for this patient subgroup.

This study reports on the use of human liver tissue equivalents (hLTEs) fabricated using major cell types at ratios that recapitulate native liver structure, physiology, and function, to investigate transduction efficiency, cellular tropism, functional impact, and genotoxicity of 2 adeno-associated virus (AAV) serotypes, AAV5 and AAV3b, encoding eGFP under the strong cytomegalovirus (CMV) promoter to ensure ubiquitous transgene expression in all cells. Additionally, AAV5 encoding eGFP or a bioengineered FVIII transgene (lcoET3) under the phosphoglycerate kinase (PGK) promotor was used to identify unique pathways specific to lcoET3 and/or the effects of different promoters. Overall, AAV5 yielded higher eGFP expression, both AAVs transduced endothelial and stellate cells more efficiently than hepatocytes and Kupffer cells, and both altered liver function biomarkers. Differential gene expression analysis showed that multiple genes involved in hepatotoxicity/inflammation were significantly dysregulated, with each serotype, promoter, and transgene producing a distinct pattern of transcriptional alterations. Integration site analysis identified AAV integrations throughout the human genome, with some in the vicinity of multiple genomic loci associated with oncogenesis. Interestingly, numerous integrations were also found within the human mitochondrial genome. Therefore, hLTEs are a valuable platform for human-relevant safety assessment and to gain critical insights for developing safer and more effective liver-directed AAV gene therapy.

There is a critical need to understand pathophysiological mechanisms involved in injury from acute chlorine gas (Cl2) exposure. Limited information is available regarding the time course and mechanisms of injury after acute Cl2 exposure due to a lack of human clinical data and limited fidelity of pre-clinical animal models. We designed and integrated a Cl2 exposure platform to generate and deliver precise concentrations of Cl2 to a microfluidic human airway-on-a-chip microphysiological system. Chemical, biological, structural, and functional airway-on-a-chip responses to Cl2 exposure were characterized across multiple concentrations, exposure times, and post-exposure timepoints. Transcriptomics and metabolomics analyses delineated key molecular, cellular, and physiological pathways involved in the acute response to Cl2 exposure. This work represents a significant advancement towards high-throughput, human-relevant characterization of pulmonary toxicants and medical countermeasure development, addressing critical gaps in toxicology modeling while reducing reliance on animal studies.

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IntroductionEbola virus (EBOV) infection triggers intense host transcriptional responses that overlap extensively with those induced by other viral and bacterial pathogens. This overlap complicates the identification of EBOV-specific gene expression signatures and limits diagnostic specificity. Defining transcriptional markers that distinguish EBOV from other infections is essential for improving molecular diagnostics and advancing understanding of EBOV-specific host responses.MethodsWe developed a multi-step filtering framework using blood-derived RNA-Seq data from nonhuman primates and human cohorts organized into independent training and test sets. In the training cohort, differential expression analysis was performed using an edgeR-based GLMQL-MAS approach to identify EBOV-associated genes. Candidates were filtered against non-EBOV comparator datasets, including mpox virus, influenza, bacterial pneumonia, acute HIV-1 infection, and multiple SARS-CoV-2 variants, to remove broadly shared host-response genes. Genes included in the NanoString nCounter® Host Response Panel were additionally excluded. The resulting EBOV-specific signature was evaluated in independent EBOV and non-EBOV test cohorts using principal component analysis and logistic regression. Functional enrichment was assessed using KEGG pathways.ResultsInitial analysis identified numerous interferon-stimulated genes that were similarly upregulated across infections. After cross-infection filtering and NanoString exclusion, 281 EBOV-specific genes were identified. Optimization within the training cohort yielded a top-50 gene set that clearly separated EBOV from Non-EBOV samples. In the independent test cohort, classification performance improved substantially, with the F1 score increasing from 37.5% when all genes were used to 95.0% after applying the top-50 gene set. Enrichment analysis of the top-50 EBOV-specific genes revealed significant association with vascular, coagulation, secretory, and metabolic pathways. ADAMTS1 showed consistent upregulation in EBOV while remaining downregulated or inactive in comparator infections.DiscussionStructured cross-pathogen filtering enables identification of EBOV-specific transcriptional features beyond shared antiviral responses. The validated gene signature generalizes across independent cohorts and highlights biologically distinct pathways, which supports its potential utility for host-based diagnostic development.

AIMS:The objective of this study was to assess the duration and clinical correlates of positive response in interstitial cystitis/bladder pain syndrome patients undergoing therapeutic bladder hydrodistension (HOD) or HOD with electrofulguration (for those with Hunner lesions). METHODS:One hundred and twenty four women were enrolled in this prospective IRB-approved study. Participants answered a set of validated questionnaires at intake including chronic overlapping pain conditions (COPCs), brief pain index (BPI), and IC/BPS symptomatic questionnaires. These were repeated at 1, 2-, 3-, 6-, and 12-months posttreatment, together with a global response assessment (GRA). The primary outcome measure was the GRA score. A secondary analysis of patients who were strong responders to treatment was used to identify clinical correlates of positive response. RESULTS:At 1-month post-HOD, 62 patients (53.9%) were responders (i.e., reported persistent positive response to treatment). Further, 21/49 (42.9%) respondents continued to report a positive response at 12 months. Logistic regression analyses identified several correlates of strong response to therapy including absence of Huynner lesions (odds ratio (OR) = 3.629), lack of diagnosis of fibromyalgia (OR = 0.31), lower number of total sites of pain on BPI pain map (OR = 0.91), fewer total number of COPCs (OR = 0.36), and lower complex medical symptom inventory scale (CMSI; OR = 0.91). After false discovery rate correction, only the CMSI remained statistically significant. CONCLUSIONS:Therapeutic hydrodistension results in a clinically meaningful and sustained improvement in associated symptoms and pain for many patients. However, having higher scores on markers of a widespread pain/non-bladder centric phenotype was associated with a lower treatment success rate.

There is significant variation in interstitial cystitis/bladder pain syndrome (IC/BPS) biopsy processing and reporting. The objective of this study was to review pathology reports from a large IC/BPS patient cohort to identify differences in findings. We hypothesize that variation in IC/BPS bladder biopsy reporting might be most frequent when it comes to mast-cell counts. We performed a retrospective analysis of 461 diagnostic pathology reports collected from our IRB-approved prospective study of patients diagnosed with IC/BPS at the Urology Clinic at Wake Forest Baptist Hospital from October 2011 to July 2023 (IRB00018552). Data were assigned as continuous or categorical variables. Groups were compared using Student’s t test, Mann–Whitney, or Chi-squared tests. Staining strategy for mast-cell visualization differed between pathologists and included in order of frequency mast-cell tryptase (TPSAB1), CD117 (KIT), unspecified stain, a combination of stains, and toluidine blue. Mast-cell count was reported as a single number, range, or qualitatively. Pathologists used units of high-powered field (HPF), mm2, or did not specify. As expected, average mast-cell count per HPF was significantly lower than per mm2 across all stains (p < 0.0001). Average count with KIT was significantly lower than TPSAB1 (p < 0.0001). This trend remained significant when considering only KIT and TPSAB1 counts per HPF (p = 0.0007). Additionally, reports identified squamous metaplasia, acute inflammation, and/or chronic inflammation. There is a lack of standardization regarding histological analysis of bladder biopsies from patients with IC/BPS, leading to inconsistent data and confusion surrounding the significance of pathology report findings.

Pelvic venous disorders (PVDs) are a group of inter-related pathological diagnoses that can present with a range of symptoms including pelvic and urinary symptoms similar to those seen in interstitial cystitis/bladder pain syndrome (IC/BPS). The relationship between PVDs and IC/BPS in humans has yet to be evaluated. This study’s objectives are to determine the prevalence of PVD in patients with IC/BPS and to identify associated characteristics. Charts from our study on patients with IC/BPS undergoing hydrodistension (HOD) were screened for those with an MRI or CT scan of the abdomen/pelvis. Imaging findings suggestive of PVD were noted. Information regarding anesthetic bladder capacity (BC), Hunner lesion (HL) status, results for validated IC/BPS symptoms, and patient reports of comorbid non-urological associated syndromes (NUAS) known to co-occur with IC/BPS were collected. A total of 133 patients with IC/BPS had the appropriate studies for a diagnosis of PVD, of which 64 (48.1

Despite the well-known risks of tobacco use, tobacco exposure remains a major contributor to morbidity and mortality worldwide. Although cigarette use has declined, the popularity of novel tobacco products (NTPs), such as electronic cigarettes (ECs) and heated tobacco products (HTPs), has increased. Given the evolving landscape of the tobacco industry, robust in vitro models are needed to evaluate the potential for harm of novel products on the airways. We applied a 3D in vitro human airway organ tissue equivalent (OTE) model to evaluate its ability to characterize the acute effects of aerosol exposure from a combustible cigarette, an HTP, and two ECs using a VITROCELL VC1 Smoking Machine. Each product was tested using a nicotine-matched single exposure dose, providing a standardized benchmark relevant to real-world use. To deliver comparable amounts of nicotine (~35-38 μg), exposures ranged from 20 to 64 min depending on the product. Following exposure, OTEs were evaluated for cytotoxicity, oxidative stress, epithelial barrier function, ciliary function, inflammatory cytokine release, and inflammatory gene expression changes. Compared to cigarettes, NTP exposures resulted in reduced OTE cytotoxicity and inflammation. HTP exposure resulted in moderate cytotoxicity and oxidative stress, an increased inflammatory response, reduced epithelial barrier function, and temporary impairment of ciliary function. For the selected nicotine-matched dose, neither EC notably induced cytotoxicity nor inflammation or disrupted epithelial barrier or ciliary function. This work establishes a methodology for comparing NTPs using a physiologically relevant human in vitro model and supports further examination of NTPs using delivered nicotine as a benchmark.

Randomized clinical trials have resulted in few approved therapies for the treatment of interstitial cystitis/bladder pain syndrome and chronic prostatitis/chronic pelvic pain syndrome, collectively referred to as urologic chronic pelvic pain syndrome. Heterogenous patient populations, mismatches of treatments to patient phenotypes, non-specific outcomes and use of standard study designs not leveraging phenotypic heterogeneity might have contributed to the inability of previous trials to demonstrate existing efficacy. The Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network has identified important phenotypic characteristics associated with differential symptom severity and treatment responsiveness. Based on Multidisciplinary Approach to the Study of Chronic Pelvic Pain findings and external research, empirically informed strategies were generated for defining patient populations, specifying treatments and selecting primary outcomes for future randomized clinical trials in urologic chronic pelvic pain syndrome. Explicitly specifying the scope of eligibility criteria across heterogeneous patient subgroups defined by pain widespreadness, the presence of Hunner lesions, the presence of pain with bladder filling or relieved by voiding, the extent of chronic overlapping pain conditions, and pelvic floor tenderness is needed. Therapies should be selected based on the mechanism of action and relevance to the mechanism of pain and dominant symptomology that the patient experiences. Evidence suggests that pain and urinary symptoms should be evaluated separately. Promising trial designs for identifying effective therapies in this heterogeneous patient population include sequential multiple assignment randomized trials and adaptive designs. This Expert Recommendation from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain Research Network provides informed considerations and a greatly expanded foundation that can be used to refine the design of future therapeutic clinical trials in urologic chronic pelvic pain syndrome.