Journal of Leukocyte Biology

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

The cell-intrinsic complement system has emerged as an important orchestrator of a variety of cell-physiological processes, with complement components interacting with intracellular effector systems to regulate cellular responses to pathogens or noxious stimuli. For instance, intracellular C5 signaling through a mitochondrial C5a receptor (C5aR1) controls IL-1{beta} production in human monocytes and macrophages. Here, we investigated whether cell-intrinsic C3 similarly regulates inflammatory responses in macrophages. In LPS-stimulated C3 knockout THP-1-derived macrophages, interferon (IFN)-{beta} production was increased, accompanied by elevated expression of interferon-stimulated genes and enhanced secretion of IFN-induced cytokines and chemokines. C3-deficient cells showed increased phosphorylation of IRF3 at Ser396 and a stabilization of the interaction between IRF3 and TBK1, along with enhanced IRF3 dimerization and nuclear translocation. TBK1 phosphorylation was unaffected, indicating that C3 limits IRF3-TBK1 complex formation rather than upstream TBK1 activation. Small-molecule inhibitors of complement factors B and D restored full-length C3 abundance in LPS-stimulated primary human macrophages, consistent with inhibition of the C3 convertase. It also reduced LPS-induced IFN-{beta} production in primary human macrophages and THP-1 cells, suggesting that full-length, uncleaved C3 suppresses IFN-{beta} production. Collectively, these findings identify cell-intrinsic C3 as a suppressor of IFN-{beta} production in human macrophages, highlighting the importance of the cell-intrinsic complement system in fine-tuning inflammatory responses to pathogens.

Background: Transplant arterial injury is an underlying feature of acute organ transplant rejection and is a main cause of late heart transplant failure. The role of the gut microbiota, and especially specific microbial components of this community, in controlling immune responses that cause this aspect of rejection is poorly understood. Methods: We utilized a murine aortic interposition model of transplant arterial injury to investigate the role of the gut commensal bacteria, Akkermansia muciniphila, in controlling immune responses in transplant arteries. Results: Early life treatment of female mice with broad spectrum antibiotics, which delayed colonization of the intestinal tract with bacteria until after weaning, led to the development of dysbiosis in adults that was characterized by the absence of A. muciniphila. This was related to an elevation in systemic levels of CCL2 and a reduction in the immunomodulatory short-chain fatty acid, propionate. When transplant arterial injury was examined, there was more arterial injury indicative of acute rejection and increased intimal thickening reflective of transplant arteriosclerosis in grafts from dysbiotic mice compared to controls. Dysbiosis also increased macrophage accumulation early after transplantation in dysbiotic mice. Notably, restoring A. muciniphila in the gut microbiota of dysbiotic mice through voluntary oral administration in infants ameliorated macrophage-mediated transplant arterial injury. Conclusions: A. muciniphila is an immunomodulatory component of the gut microbiota that protects against vascular injury and pathology in organ transplantation.

Introduction: C3 glomerulopathy (C3G) is driven by fluid-phase alternative complement pathway dysregulation, with emerging evidence linking glomerular neutrophil infiltration to disease severity. Neutrophil extracellular traps (NETs) are implicated in other forms of glomerulonephritis. However, their participation in the pathogenesis of C3G remains undefined. Methods: Kidney biopsies from 33 patients with C3G (15 with dense deposit disease [DDD] and 18 with C3 glomerulonephritis [C3GN]) were compared with 15 anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) biopsies as a neutrophil-rich disease control in this retrospective cross-sectional study. Glomerular neutrophils and NETs were identified using immunofluorescence, staining for myeloperoxidase, citrullinated histone H3, peptidyl arginine deiminase-4, and DNA. Supervised machine learning was used to quantify glomerular NET formation, and the data were correlated with kidney function at time of biopsy using linear regression. Results: Intraglomerular NETs were abundant and detected in the majority of glomeruli in C3G biopsies. Compared with AAV, C3G showed a significantly higher fraction of neutrophils forming NETs, despite similar neutrophil counts per glomerulus. NET abundance was similar in DDD and C3GN. In exploratory analyses, a greater proportion of glomeruli containing NETs was associated with lower kidney function (estimated glomerular filtration rate) at biopsy, and this association remained significant after adjustment for age, C3G subtype, and interstitial fibrosis. Conclusions: These observations demonstrate that intraglomerular NETs are a common and prominent observation in C3G and are associated with reduced kidney function at biopsy. These findings raise the possibility that NET deposition in glomeruli is a previously unrecognized driver of glomerular injury in C3G.

Despite the success of chimeric antigen receptor (CAR) T cell therapy for treatment of B cell acute lymphoblastic leukemia (B-ALL), its translation to acute myeloid leukemia (AML) has been hindered by limited efficacy and significant toxicity. Interferon-gamma (IFN{gamma}) blockade with emapalumab has recently emerged as a promising strategy to mitigate CAR T cell-related toxicities in B cell malignancies, based on evidence that IFN{gamma} is largely dispensable for optimal CAR T cell activity in B-ALL. Whether IFN{gamma} signaling is similarly non-essential in the AML context remains unclear. Here, we demonstrate that disruption of the IFN{gamma} axis impedes anti-AML CAR T cell function and prevents upregulation of target antigen CD123, the apoptotic mediator Fas, and the adhesion molecule ICAM-1 on AML cells. Conversely, exogenous IFN{gamma} enhances CAR T cell cytotoxicity and increases CAR T cell avidity for AML targets. These findings identify IFN{gamma} as a critical mediator of CAR T cell efficacy against AML by promoting increased target antigen expression, enhanced cytotoxicity, and stable CAR T/tumor interactions. Our results suggest that therapeutic IFN{gamma} blockade, including with emapalumab, may compromise CAR T cell responses in AML and should be approached with caution in this disease context.

Recurrent urinary tract infection represents a substantial unmet public health in women. Local administration of estradiol has been shown to reduce recurrence, however in vitro models of the female urinary tract remain limited and the mechanisms underlying the effects of estradiol are incompletely understood. Here, we describe a novel iPSC organoid differentiation protocol and its application to establish a multilayered transwell barrier culture model. Estradiol treatment resulted in reduced expression of innate antimicrobial peptides and cytokines, together with increased expression of demannosylation pathways. Treatment of transwell cultures with a combination of female sex hormones reduced endogenous CXCL8 signaling, independently of a 24-hour uropathogenic Escherichia coli (UPEC) challenge. To our knowledge, this is the first iPSC organoid-derived model of the urinary tract, which provides a platform for investigating interactions between the urothelium, urobiome and hormonal environment.

Liver metastases (LM) remain a major cause of death from different cancer types, in particular malignancies of the gastrointestinal tract. Liver metastases predict a poor response to immunotherapy due, among others, to the immunotolerant microenvironment (ME) of the liver and loss of local and systemic cytotoxic T cells. Thus, strategies that can reprogram the immune ME of the liver and restore cytotoxic T cell reactivity are being sought. We previously reported that estrogen signaling blockade impedes the growth of LM by reducing MDSC accumulation and monocyte/macrophage polarization. The aim of this study was to elucidate the underlying mechanism(s) and assess whether estrogen signaling in the myeloid lineage was driving the immunotolerant ME of LM. To this end, we generated mice with conditional myeloid cell-specific deletions of estrogen receptors (ER) or ER{beta} and analyzed in these mice the effect of ER loss on the liver immune ME and the outgrowth of LM. In mice with ER, but not with ER{beta} deletion, we observed a marked reduction in the growth of murine colon carcinoma MC-38 liver metastases as compared to their respective controls. Flow cytometry and immunohistochemistry revealed a decrease in macrophages that were polarized to the pro-tumorigenic M2-like phenotype and a concomitant increase in activated CD8+ T and NK cells relative to controls. Bulk RNAseq analysis performed on hepatic immune cells infiltrating the liver revealed changes in the expression of key cytokines/chemokines mediating immune cell recruitment, activation and polarization, including Ccl5 (upregulated) and Csf1 (downregulated). Taken together, the data suggest that ER signaling in myeloid-derived cells programs the immune landscape and contributes to an immunosuppressive and metastases-growth permissive ME in the liver.

Antigen presenting cells (APC) can bind HIV-1 and subsequently trans infect CD4+ T cells. In comparison to direct (cis) infection of CD4+ T cells by free virus, APC-mediated HIV-1 trans infection is significantly more efficient and requires lower virus titers. As such, B cell-mediated HIV-1 trans infection of CD4+ T cells, particularly in secondary lymphoid organs (SLO) where B and CD4+ T cells interact frequently in and around B cell follicles, represents an efficient pathway for establishing and maintaining the latent HIV-1 reservoir. The molecular events involved in HIV-1 binding to B cells and transfer to CD4+ T cells are poorly understood. B cells are exposed to various activation signals in SLO including CD40 ligand (CD40L), interleukin-4 (IL-4), interferon-g (IFN-g), and B cell activating factor (BAFF). Here, we treated B cells with these different signals, or combination of signals, to identify those that facilitate HIV-1 binding to B cells and trans-infection of CD4+ T cells, and the mechanisms involved. We found that CD40L/IL-4 stimulated B cells are highly efficient mediators of HIV-1 trans infection of CD4+ T cells due to their enhanced capacity to bind HIV-1. Single cell RNA sequencing of differentially stimulated B cell populations revealed that CD40L/IL-4 stimulation significantly induced expression of the C-type lectin CD205. Confocal microscopy revealed that HIV-1 and CD205 co-localized on CD40L/IL-4 stimulated B cells, and antibody blocking of CD205 on these cells significantly reduced HIV-1 binding. Taken together, this study identifies CD205 as a critical receptor on B cells that facilitates HIV-1 binding and the transfer of virus to CD4+ T cells. Insight into the role of B cell mediated HIV-1 trans infection of CD4+ T cells is critical to optimizing the effectiveness of HIV-1 therapies in SLO.

Myeloproliferative neoplasms (MPN) are chronic hematologic malignancies characterized by clonal myeloid expansion, inflammation, oxidative stress, and progressive bone marrow (BM) remodeling that may culminate in fibrosis and secondary acute leukemia. Here, we evaluated the therapeutic efficacy and the underlying mechanisms of melatonin (MT) and liposomal melatonin (nano-MT) in preclinical MPN models. MT selectively inhibited clonogenic growth of patient-derived peripheral blood mononuclear cells and induced pluripotent stem cell-derived CD34 hematopoietic stem and progenitor cells in comparison to healthy controls. This effect was associated with increased apoptosis, reduced reactive oxygen species (ROS), and decreased glucose uptake, independently of MT receptor signaling. Transcriptomic profiling of primary MPN CD34 cells revealed suppression of MYC targets, G2M checkpoint signaling, ROS, and glycolysis pathways. In co-culture models, MT reduced stromal -smooth muscle actin and phosphorylated SMAD2/3, indicating inhibition of TGF-{beta}-driven mesenchymal stromal cell-to-myofibroblast formation. In tamoxifen-inducible SclCreER;JAK2V617F mice, nano-MT achieved efficient spleen and BM targeting. Therapeutically, nano-MT reduced erythrocytosis, myeloid progenitor expansion, and BM IL-1{beta} levels. Longitudinal micro-computed tomography and histological analyses demonstrated normalization of BM architecture, reduced osteosclerotic remodeling and splenomegaly, decreased reticulin deposition and megakaryocyte numbers. In a dose-escalation study, nano-MT restored erythrocyte, hematocrit, and platelet counts and normalized megakaryocyte-erythroid progenitors. Combination treatment with ruxolitinib further reduced leukocytosis, neutrophilia, and monocytosis. Collectively, these findings demonstrate that (nano-)MT attenuates MPN and BM remodeling by targeting metabolic, inflammatory, and fibrotic pathways. This study provides the first evidence for a therapeutic benefit of nano-MT in MPN and establishes a rationale for further translational evaluation.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

Abstract Calcific deposits in the arterial media have been associated with a number of metabolic and genetic disorders including diabetes, chronic kidney disease and generalized arterial calcification of infancy. While medial calcification and physiologic hard tissue mineralization in the skeleton are both regulated by several common determinants, emerging data suggest that there might be fundamental differences in the mechanisms underlying these two processes. Objective: We previously demonstrated that elastin haploinsufficiency delays medial calcification in MGP-deficient mice. Here, using mice in which a human ELN transgene rescues mouse elastin deficiency, we investigated whether the origin and abundance of arterial elastin differentially affect the initiation and progression of medial calcification. Approach and Results: We pursued a transgenic approach to alter the arterial elastin scaffold in MGP-deficient mice. Our analyses of a humanized MGP-deficient model with 40% reduction of medial elastin content showed a complete absence of the early-stage vascular calcification. Additionally, we showed that mouse and human elastin orthologues affect vascular calcification in a comparable manner. Conclusion: Arterial elastin abundance, rather than orthologue origin, modulates the initiation and progression of medial calcification in MGP-deficient mice. A further reduction in arterial elastin beyond that achieved by elastin haploinsufficiency profoundly delays mineral deposition and maturation, whereas restoration of elastin abundance through transgenic human ELN expression restores arterial calcification.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

Background Microbiome-derived metabolites regulate host physiology, yet bacterial gaseous metabolites remain largely overlooked. Traditionally regarded as fermentation end-products, bacterial gases may act as biologically active mediators of host-microbiome communication. We hypothesized that bile acids regulate bacterial gaseous metabolism and influence host epithelial responses. Methods A high gas-producing clinical Escherichia coli isolate from a patient with moderately severe acute pancreatitis was cultured with selected primary and secondary bile acids. Gas production was assessed by pressure measurements, GC-TCD and GC-MS. Biological activity was evaluated by indirect exposure of Caco-2 and PANC-1 epithelial cells, followed by apoptosis/necrosis assays and whole-transcriptome RNA sequencing. Results Bile acids markedly reshaped bacterial gaseous metabolism. Cholic acid and deoxycholic acid promoted intense gas production, whereas chenodeoxycholic acid almost completely abolished it. Despite minimal apoptosis and necrosis, bacterial gaseous metabolites induced extensive transcriptional remodeling. Caco-2 cells showed stronger responses than PANC-1 cells, particularly to deoxycholic acid-derived gases, involving inflammatory signaling, extracellular matrix remodeling, epithelial plasticity, stress responses, and cancer-associated genes including PTGS2, MMP1, PLAUR, NR4A2, and SERPINE1. PANC-1 cells exhibited a more restricted response involving oxidative stress, proteostasis, and autophagy-associated pathways. Conclusions Our findings indicate that bacterial gases are a previously underrecognized class of microbiome-derived signaling molecules capable of modulating host gene expression independently of direct bacterial contact. We identify a gas-producing microbiome phenotype regulated by bile acid composition, linking microbial metabolism with epithelial signaling. These findings expand the concept of host-microbiome communication and provide a framework for investigating bacterial gaseous metabolites in intestinal and pancreatic diseases.

Introduction: Polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS), is a common endocrine disorder affecting women of reproductive age. Besides reproductive and metabolic disturbances, PMOS negatively impacts psychological well-being and quality of life. Despite available treatment options, there remains a need for safe and effective therapies that improve both clinical symptoms and fertility outcomes. Aim: To compare the efficacy of VAMHA and MYRHA tablet combination therapy with standard non-hormonal therapy in restoring regular menstruation. Secondary objectives included assessment of ovulation, menstrual symptoms, polycystic ovarian morphology, hormonal and metabolic parameters, anthropometric measures, and skin manifestations. Study Design: Open-label, randomized, multicentre, prospective comparative clinical study. Methods: Seventy-one women with PMOS were randomized to Group A (n=37) or Group B (n=34). Group A received VAMHA and MYRHA tablets (2 tablets each), while Group B received Metformin 500 mg plus Myoinositol 600 mg (1 tablet), twice daily for 180 days. Data were recorded in Case Report Forms. Statistical Analysis: Continuous variables were summarized using mean and standard deviation, while categorical variables were expressed as frequencies and percentages. Appropriate statistical tests, including Chi-square, were used. A p-value [&le;]0.05 was considered significant. Results: Significantly more participants in Group A achieved regular menstrual cycles than Group B (31 vs. 22; p<0.05). Ovulation occurred in 16 participants in Group A compared with 6 in Group B (p<0.05). Both groups showed significant improvement in menstrual irregularity and related symptoms. Significant reductions in Anti-Mullerian Hormone (AMH), fasting insulin, and body mass index (BMI) were observed in both groups (p<0.05). Resolution of polycystic ovarian morphology occurred in 13 participants (38.23%) in Group A and 10 (33.33%) in Group B. Both treatments were well tolerated with no major safety concerns. Conclusions: VAMHA and MYRHA combination therapy was superior to standard non-hormonal therapy in improving menstrual regularity and ovulation. It also produced favourable metabolic, hormonal, and ultrasonographic outcomes, suggesting its potential as a safe and effective option for comprehensive PMOS management and fertility enhancement.

Purpose: To evaluate the safety, procedural performance, sample recovery, and surgeon preference of an ophthalmic needle designed specifically for anterior chamber (AC) paracentesis. Methods: In this multicenter study, AC paracentesis was performed in clinic and operating-room settings using a 32-gauge x 4-mm needle with low dead space. The procedure was evaluated using a standardized physician survey. Prespecified outcomes included procedure-related adverse events (primary outcome), needle entry and handling, aspiration and sample recovery, comparative performance versus a 30-gauge needle, and physician preference for future use. Results: A total of 110 needle uses by eight surgeons were included. No ocular complications occurred, including lens or iris injury, hyphema, AC collapse, wound leak, hypotony, infection, or retinal complication, and no procedure required needle exchange or conversion to another device. Two technical events without ocular sequelae were noted, in which needle entry was partial thickness and did not reach the AC (1.8%; exact 95% CI, 0.2%-6.4%). Physicians rated needle entry, handling and sample recovery as good or excellent. Compared with a 30-gauge needle, the study needle was rated as at least comparable across all assessed domains. All surgeons rated it better or much better for intra-procedural safety and preferred it for future AC taps. Conclusions and Relevance: This short, 32-gauge low-dead-space ophthalmic needle demonstrated a favorable safety profile and was preferred over a 30-gauge needle by all surgeons. As aqueous humor liquid biopsy expands in clinical diagnostics and trials, an ophthalmic-specific needle design may help improve the consistency and safety of aqueous humor collection for molecular analysis and broader clinical use. Keywords: Anterior chamber paracentesis; Aqueous humor; Liquid biopsy; Low dead space; Ophthalmic needle

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [&ge;]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.

Importance: Evidence on GLP-1/GIP therapies is largely derived from trials enrolling selected populations or medical records that miss utilization outside healthcare channels. No nationally representative cohort has characterized real-world uptake, indications, and access. Objective: To characterize GLP-1/GIP prevalence, indication, clinical profile, and access. Design: Prospective cohort study with three GLP-1/GIP surveillance waves (March 2024, December 2024, October 2025). Setting: The Understanding America Study, an address-based, nationally representative panel of approximately 15,000 US adults aged 18+ years initiated in 2014. Participants: UAS participants responding to at least one surveillance wave (n=9150). Exposures: GLP-1/GIP use status (never vs any use, comprising current and former use), self-reported primary indication (diabetes, weight loss, or other), and access pathway (traditional vs non-traditional). Main Outcomes and Measures: Survey-weighted prevalence of GLP-1/GIP use, overall and by indication and access pathway; sociodemographic, cardiometabolic, treatment, and access characteristics; and smartwatch-derived resting heart rate, heart rate variability, maximum activity heart rate, step count, and sleep duration and variability. Results: Among n=9150 adults (1274 with any use; 60.9% female; median age 53 years), weighted prevalence increased 46%, from 8.2% (March 2024) to 12.0% (October 2025) representing 32 million. Weight-loss indications grew, reaching nearly half of use (4.1% to 5.6%); diabetes-indicated use was stable (5.3% to 5.4%). Users carried high cardiometabolic burden (obesity, 68.2%; diabetes, 53.6%) but diverged by indication: diabetes-indicated users were older (median, 59 vs 49 years), whereas weight-loss-indicated users were more often female (69.9% vs 51.3%) and healthier. One in three users (~9 million) had non-traditional access, especially in weight-loss-indicated users, of whom 33% had no conventional prescription; 41% used compounding, online, or foreign pharmacies; and, 43% lacked coverage. Non-traditional users were five times as likely to report an unlisted, likely compounded formulation (19.8% vs 4.1%). All p<0.05. Conclusions and Relevance: Real-world GLP-1/GIP use has grown rapidly and diversified substantially in indication, access, and population profile. One in 3 users obtained treatment through nontraditional channels largely invisible to claims data, raising long-term safety, efficacy, and coverage questions. GLIMMER provides a public, nationally representative longitudinal evidence base for future payer and provider decisions.

Objectives To evaluate whether access to a certified large language model (LLM)-based clinical decision support system improves physician diagnostic performance in rheumatology compared with conventional diagnostic resources alone. Methods In this multicentre, open-label, randomised controlled trial, 82 physicians from seven hospitals in two countries were randomised 1:1 to conventional diagnostic resources plus Prof. Valmed or conventional resources alone. Participants assessed three rheumatology vignettes before and after assistance. The primary outcome was top-1 diagnostic accuracy. Secondary outcomes included top-3 accuracy, diagnostic reasoning, confidence, case-processing time and perceived support quality. Results Top-1 accuracy increased from 22.2% to 33.3% in the intervention group and from 23.3% to 35.0% in the control group, with no between-group difference in improvement (adjusted OR 0.99, 95% CI 0.45 to 2.19; p=0.979). Differences in top-3 accuracy, diagnostic reasoning and confidence were also not significant. Assisted case-processing time was substantially shorter with LLM support (94 vs 206 s; adjusted mean difference -112 s, 95% CI -141 to -83; p<0.001). Information timeliness and perceived diagnostic support quality were rated significantly higher in the intervention group. Exploratory analyses showed persistent overconfidence and substantial AI over-reliance. Conclusions Certified LLM-based diagnostic support did not improve diagnostic accuracy compared with conventional resources, but substantially reduced case-processing time and improved perceived support quality. These findings suggest potential workflow benefits while highlighting overconfidence and over-reliance as important safety considerations.

Muscle architecture shapes muscle function and changes with age, growth, training and disease, yet quantifying three-dimensional (3D) muscle architecture in vivo remains challenging. We introduce a hybrid fascicle tractography approach for freehand 3D ultrasound data that accurately reconstructs 3D muscle fascicles with respect to an objective, anatomically relevant coordinate system defined by the muscle's central aponeurosis. The hybrid approach combines Hessian-based fascicle detection with wavelet-based refinement to generate volumetric fascicle orientations. In a synthetic dataset with known ground truth, fascicle orientations and lengths were estimated with errors of [&le;]2{degrees} and ~1.5%, respectively. In vivo, the approach detected physiologically plausible fascicle lengthening in the human tibialis anterior following a passive plantar flexion rotation, whereas diffusion tensor imaging of the same muscle did not. The proposed method enables anatomically relevant, objective and non-invasive quantification of 3D muscle architecture in vivo, providing a practical framework for applications in clinical and applied muscle physiology.

Background. Videolaryngoscopy still requires adjuncts or hyperangulated rescue in a clinically important minority, and bedside screening discriminates modestly. Point-of-care ultrasound (POCUS) of the anterior airway is a promising alternative, but existing prediction models are opaque or assume a pre-specified functional form. We developed and internally validated a parsimonious, fully disclosed POCUS risk equation whose form is recovered from data and whose structural properties are machine-checked by formal proof - to our knowledge the first formally verified clinical risk predictor - following TRIPOD+AI 2024. Methods. In a prospective single-centre, single-operator cohort of 259 adults undergoing elective videolaryngoscopy (no-Easy airway 68/259, 26.3%), Sequentially Thresholded Least Squares with bootstrap stability selection (B=300) screened a 71-term library of nine POCUS features and retained a seven-term logistic equation; a two-term bootstrap-stable model was pre-specified as robustness analysis. Internal validation used 5x10 repeated cross-validation plus temporal and device hold-outs, with pre-specified overfitting and optimism assessments. Five behavioural properties of the deployed equation were machine-checked in Lean 4. Results. Two interactions met the |c|/sigma_c>2 stability criterion: skin-to-epiglottis x skin-to-hyoid-bone distance and tongue volume x sagittal tongue area. The seven-term equation reached a 5x10 cross-validated C-statistic of 0.966 (optimism-corrected 0.968) and held across temporal and device hold-outs (0.94-0.97). Calibration-in-the-large matched prevalence, with cross-validated slope 0.90 attenuating to 0.625 out-of-time; standard recalibration restored 0.92 without loss of discrimination. The pre-specified two-term robustness model reproduced this performance (C-statistic 0.964-0.968; events-per-parameter 34; shrinkage 0.99), confirming the result is not an artefact of the screening stage. Net benefit over a clinical baseline was positive across 10-50% thresholds. All five Lean 4 theorems compiled without sorry. Conclusions. A sparse, formally verified POCUS equation predicts difficult videolaryngoscopy with high internally validated discrimination and quantified, modest overfitting. Because the equation was developed in a single-operator cohort and its inputs are operator-dependent, external validation requires prior harmonisation of the measurement protocol and operator credentialing.