Journal of Cystic Fibrosis

AimCystic fibrosis (CF) care has been transformed by CFTR modulator therapies, yet most efficacy data arise from clinical trials with restrictive eligibility criteria. Real-world registry data can capture treatment outcomes in broader, more diverse patient populations. We used the Cystic Fibrosis Foundation Patient Registry (CFFPR) data to evaluate longitudinal clinical outcomes, and care benchmarking at a single Adult CF Program Center over a decade. MethodsA retrospective, descriptive analysis of CFFPR data (2011-2022) was performed to assess trends in modulator use, lung function (ppFEV1), body mass index (BMI), respiratory microbiology, and pulmonary exacerbations (PEx). Comparative Effectiveness Research (CER) methods were applied to compare outcomes across peak modulator eras: pre-modulator (2011), ivacaftor (2015), mixed-modulator (2019), and elexacaftor/tezacaftor/ivacaftor (ELE/TEZ/IVA) (2021). Program-level outcomes were benchmarked against national network metrics to assess adherence to guideline-based care. ResultsOver ten years, median ppFEV1 improved from 63.4% (2011) to 78.8% (2021), and BMI increased from 22.3 to 24.8 kg/m2. The proportion of adults experiencing more than one PEx annually declined from 39.7% to 19.5%, while Pseudomonas aeruginosa (P.a.) culture positivity decreased from 79% to 47%. ELE/TEZ/IVA therapy was associated with greatest clinical improvements. Program-level performance remained comparable to national network benchmarks, reflecting high adherence to standard care metrics. ConclusionRegistry-based CER provides valuable real-world insights into CF care effectiveness and quality improvement. This decade-long analysis demonstrates significant clinical gains associated with modulator therapies and highlights the importance of patient registries in monitoring outcomes, benchmarking care, and informing global CF care models and standards for rare disease management. Key Messages{square} Real-world registry data enables decade-long evaluation of CFTR modulator effectiveness. {square}Elexacaftor/tezacaftor/ivacaftor demonstrates the greatest clinical benefit among modulator therapies. {square}Benchmarking Adult CF Program performance against the Network of Adult CF Programs facilitates quality improvement and standard care guideline adherence. {square}Patient registries provide insights for personalized care, program-level decision-making, and international standards for rare disease management.

BackgroundCystic fibrosis (CF) is an autosomal recessive genetic disorder that leads to chronic infection and mucus retention in the lungs, with lung function gradually deteriorating through recurrent pulmonary exacerbations (PEx). Virulence factors (VFs) of Pseudomonas aeruginosa and Staphylococcus aureus are thought to contribute to pulmonary exacerbations. Our study objective was to identify VF genes related to PEx, high Pseudomonas abundance, and high Staphylococcus abundance in persons with CF (pwCF). MethodsThis was an ancillary study of pwCF treated with IV antibiotics for PEx between 2016-2020 at Childrens National Hospital. Using shotgun metagenomics and ShortBRED, we identified bacterial VF genes and used DESeq2 to determine differential expression of VF genes across comparators. ResultsTwenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years, 41% female, 59% white, 36% Hispanic, and 45% had an F508del homozygous CFTR mutation. Minimal differences in VF gene abundance were identified across clinical state. The most differentially increased VF genes found in Pseudomonas high samples were associated with an aminotransferase (log2FC 25.9), flagellar biosynthesis (log2FC 8.3), and type VI secretion systems (log2FC 8.2). The most differentially increased VF genes found in Staphylococcus high samples were an exotoxin (log2FC 26.7), macrolide phosphotransferase (log2FC 25.8), pathogenicity island proteins (log2FC 25.2 and 24.7), and VOC family proteins (log2FC 24.8). ConclusionsThese findings demonstrate that specific VFs associated with immune modulation, motility secretion systems, bacterial motility, and antibiotic resistance are related to P. aeruginosa and S. aureus abundance, providing potential targets for more personalized antimicrobial interventions.

Staphylococcus aureus is the most common bacterial pathogen affecting pediatric patients with Cystic fibrosis (CF), a genetic disorder that causes thick mucus buildup in the lungs, providing a scaffold for chronic infections. Antibiotic treatment is typically guided by standard in vitro antimicrobial susceptibility testing (AST) in Mueller-Hinton broth (MHB), which does not represent the infection site in CF lungs. Notably, discordances between AST predictions and antibiotic therapeutic outcomes were reported in up to 50% of CF cases. To address this gap, we conducted ASTs against methicillin-resistant S. aureus (MRSA) in CF sputum-mimetic media compared with MHB, demonstrating [≥]4-fold discordances across four of nine antibiotic classes. Most significantly, we observed unexpected {beta}-lactam sensitization of MRSA strains (up to 128-fold) in CF sputum-like media, crossing the CLSI clinical breakpoint, suggesting this shift may alter therapeutic outcomes. Genome-wide screens and follow-up assays revealed underlying cell envelope remodelling and alterations to cell envelope stress responses. On the other hand, mucin binding to daptomycin may have led to an apparent 8-fold increase in resistance to this antibiotic in one of the CF sputum-like media. Overall, our AST results in CF sputum-mimetic conditions provide insights into bacterial responses during CF infections. Importantly, they suggest {beta}-lactams may be effective in treating MRSA infections in CF patients, warranting further investigation in relevant in vivo systems.

More than 1,200 variants of the cystic fibrosis transmembrane conductance regulator gene (CFTR) are associated with cystic fibrosis (CF), an autosomal recessive pulmonary disease affecting over 100,000 people. Most people with CF bear a common CFTR variant (F508del) that can be treated with therapeutics containing "correctors" that suppress the misfolding of the CFTR chloride channel. However, the pharmacological responsiveness of other rare CF variants can vary tremendously. The approval of VX-121, a VX-445 analog that serves as a key component of Alyftrek, potentially provides a new therapeutic option for those with rare CF variants. Nevertheless, it remains unclear whether VX-121 offers superior rescue across the entire spectrum of rare CF variants. In this work, we use deep mutational scanning (DMS) to survey the impact of VX-121 on the plasma membrane expression of 232 rare CF variants. Our results show that VX-121 generally enhances CF variant expression more than VX-445 and is most potent towards variants with mutations in the first membrane spanning domain (MSD1). However, we identify one variant (Y1032C) with diminished proteostatic and functional selectivity for VX-121 relative to VX-445. Computational docking suggests that the native Y1032 side chain forms favorable interactions with VX-121 that are disrupted by this mutation in a manner that alters its coordination. Finally, using photo-crosslinking, we show that VX-121 avoids a key off-target interaction of VX-445. Together, our findings provide new insights into the similarities and differences between current approved CF therapeutics.

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a pulmotype detected in [~]34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF. IMPORTANCECystic fibrosis (CF) is a genetic disease characterized by chronic polymicrobial lung infections that, if untreated, are one of the primary causes of death in CF. Elexacaftor/Tezacaftor/Ivacaftor (ETI) has many positive clinical outcomes, but it does not eliminate chronic polymicrobial lung infections or inflammation. Using a new biologically relevant co-culture model, we have demonstrated that bacteria secrete vesicles (bEVs) that contain proteins and RNAs. We observed that these RNA-loaded bEVs are predicted to promote the pathogenesis of chronic CF lung infections by enhancing bacterial virulence and biofilm formation, as well as upregulating the pro-inflammatory response in lung cells. ETI does not ameliorate the response of lung cells to bEVs. Our research will facilitate the development of more effective approaches to eliminate infection and inflammation in CF and other lung diseases characterized by chronic polymicrobial infections and excessive inflammation.

Collagens are key components of the extracellular matrix (ECM) that play a crucial role in maintaining structure, strength, and function of the lungs. Fibrillar collagens are crosslinked by enzymes such as lysyl oxidases and transglutaminases and organized into networks by proteoglycans and glycoproteins. Collagens are the main load-bearing components and along with elastin may impart a non-linear strain hardening behavior to the lung. In disease, collagen crosslinking and organization can be disrupted, possibly due to abnormal levels of enzymes or ECM components. Few studies have examined collagen crosslinking and organization in healthy and diseased human lungs. In this study, alterations in collagen crosslinking and organization were investigated in human lung control, fibrotic and chronic obstructive pulmonary disease (COPD) tissue sections. Ultra-performance liquid chromatography and second harmonic generation microscopy measured pyridinoline crosslinks and the distribution of mature and immature collagens within the decellularized scaffolds, respectively. Fibrotic scaffolds had higher total collagen but less crosslinking per mole of collagen compared with COPD donors. Image analysis by second harmonic generation microscopy showed mature collagens populated airway or blood vessel walls in all three groups and in the parenchyma of fibrotic scaffolds. Immature collagens, on the other hand, were mainly localized to parenchymal regions in control and COPD scaffolds, with fewer immature collagens in fibrotic parenchyma. Additionally, quantification of the mature to immature collagen ratio in defined regions of control and diseased scaffolds showed increased organized collagen in fibrotic tissue. Our study shows that collagen crosslinking and organization are disrupted in fibrotic and COPD lungs and these changes may be compartment specific and can contribute to aberrant mechanical properties of diseased lungs. Our findings highlight that along with total collagen content, collagen crosslinking and organization are equally important while investigating collagen-mediated pathological changes in lung tissue. These changes may have implications for developing ECM-based therapeutics for patients with lung diseases.

The increasing prevalence of antibiotic-resistant bacterial infections highlights the need for physiologically relevant in vitro models that recapitulate host-pathogen interactions. Pseudomonas aeruginosa is a clinically important opportunistic pathogen associated with hospital-acquired infections and chronic airway diseases, including cystic fibrosis, where dysregulated inflammatory responses contribute to disease progression. While air-liquid interface (ALI) models have advanced the study of airway epithelium, most of these modes lack immune components, limiting their ability to capture immune-epithelial interactions. Here, we expanded a previously established dual-cell ALI model incorporating human monocyte-derived macrophages to investigate how immune context, bacterial dose, and time influence early infection dynamics. Standard BCi-NS1.1 epithelial monocultures and macrophage co-cultures were infected with P. aeruginosa (PAO1) at low (100 colony-forming units (CFU) and high (1000 CFU) inoculum and analyzed over 10, 16, and 24 h post-infection (hpi). Macrophage presence did not significantly alter total bacterial burden but markedly influenced cytokine responses and bacterial spatial organization. Pro-inflammatory cytokines (interleukin (IL)-1, IL-1{beta}, Tumor Necrosis Factor (TNF)-) were enhanced in dual-cell culture models, while IL-6 exhibited a threshold-dependent response detectable only at higher bacterial loads. Confocal imaging revealed that macrophages altered bacterial distribution, promoting a more dispersed pattern compared to the clustered organization observed in epithelial monocultures. These effects were most pronounced at lower bacterial inocula. Together, our findings demonstrate that macrophages reshape early infection dynamics by modulating inflammatory signaling and bacterial spatial organization without affecting overall bacterial burden. This study highlights the importance of incorporating immune cells into in vitro airway models.

BackgroundEmerging evidence suggests that the oral microbiome may contribute to aberrant gut immune responses in Inflammatory Bowel Disease (IBD). MethodsWe performed a comprehensive, harmonised analysis of aggregated oral microbiome 16S rRNA datasets across multiple cohorts. Data were processed using a unified bioinformatics pipeline including DADA2 for taxonomic assignment, PICRUSt2 for functional prediction, MaAsLin2 for multivariable modelling, and machine learning. ResultsAcross 25 studies (n = 1,136 IBD; n = 759 controls), meta-analysis showed significantly reduced oral microbial Shannon diversity in IBD (standardised mean difference -0.31, p = 0.007). Secondary bioinformatics analysis of six datasets plus in-house data confirmed this reduction (Shannon diversity; Hedges SMD = -0.372, p < 0.001), driven primarily by Crohns disease (CD). Beta diversity demonstrated global compositional shifts, with CD demonstrating greater divergence from controls than ulcerative colitis (UC). Multivariable modelling identified reproducible taxa enriched in IBD, including Corynebacterium, Serratia and Streptoccocus, while Porphyromonas and Ruminococcaceae.G1 were enriched in controls. Functional pathway prediction indicated reduced butyrate metabolism in IBD sub-types and increased aromatic amino acid and related metabolite degradation pathways. Machine learning classifiers achieved modest discrimination (mean AUC [~]0.67), supporting the potential of saliva-based microbiome profiling to study dysbiosis in IBD. ConclusionsThese findings demonstrate that the oral microbiome in IBD is characterised by reduced diversity and reproducible structural community reorganisation. Together, these data support a contributory role for the oral-gut axis in CD pathogenesis and provide a rationale for targeted mechanistic and longitudinal studies to define causal links between oral dysbiosis and intestinal inflammation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/26351936v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@57306corg.highwire.dtl.DTLVardef@2c0ef0org.highwire.dtl.DTLVardef@88b0b3org.highwire.dtl.DTLVardef@8ed62_HPS_FORMAT_FIGEXP M_FIG C_FIG

RationaleBronchopulmonary dysplasia (BPD), the lung disease associated with premature birth, is a significant health problem, often with long-term respiratory consequences. Recent research has highlighted the potential role of the lung and gut microbiome in the development and progression of BPD, yet it is unclear what aspects of the microbiome may contribute to BPD susceptibility. ObjectivesTo comprehensively characterize the lung and gut microbiomes of preterm infants and identify shared microbial taxa that are associated with BPD development. MethodsTracheal aspirate and stool samples were collected from 39 premature infants over the first month of life. To assess the taxonomic microbial composition of the lung and gut, samples were analyzed using shotgun metagenomic sequencing. BPD classification was determined using the National Institute of Child Health and Human Development severity-based definition at 36 weeks postmenstrual age. Measurements and Main ResultsMicrobial communities of the lung and gut were significantly different between infants who went on to develop BPD and those who did not, with an enrichment of skin-associated microbial genera such as Staphylococcus, Corynebacterium, and Cutibacterium in infants who developed BPD. Specifically, Staphylococcus epidermidis was enriched in premature infants who developed BPD and was the most prominent species shared between lung and gut communities. Temporal changes in gut microbial communities co-occurred with feeding practices and antibiotic exposure, suggesting an influence of external factors on microbiome composition. ConclusionsOur findings provide evidence that certain microbial colonization patterns among premature infants are closely associated with the pathogenesis and progression of BPD.

Objectives: Patients with Cystic fibrosis (CF) often receive aminoglycosides (AGs) to manage recurrent pulmonary infections, placing them at risk for ototoxicity. Chronic AG use can lead to complex cochlear damage affecting inner and outer hair cells, the stria vascularis, and spiral ganglion neurons. The greatest damage is typically in the basal cochlear region, which encodes high-frequency hearing, with additional involvement of more apical regions. While extended-high-frequency (EHF) hearing loss (EHFHL; 9-16 kHz) is often the earliest sign of AG ototoxicity, speech in noise (SiN) effects are rarely studied. Our overall hypothesis is that SiN perception difficulties in individuals with CF, treated with AGs, are related to combined cochlear and neural damage, primarily in the EHF range but also in the standard frequency (SF; 0.25-8 kHz) range. Three mechanisms that contribute to SiN perception were evaluated in children and young adults: 1) a primary effect of reduced EHF sensitivity, measured by pure-tone audiometry (PTA) and transient-evoked otoacoustic emissions (TEOAEs); 2) a secondary effect of subclinical damage in the SF range, measured by PTA and TEOAEs; and 3) additional neural effects, measured by middle ear muscle reflex (MEMR) threshold (afferent) and growth functions (efferent).Design:A total of 185 participants were enrolled; 101 individuals with CF treated with intravenous AGs and 84 age and sex-matched Controls without hearing concerns or CF. Assessments included EHF and SF PTA; the Bamford-Kowal-Bench (BKB)-SIN test for SiN perception; double-evoked TEOAEs with chirp stimuli from 0.71 to 14.7 kHz; and ipsilateral and contralateral wideband MEMR thresholds and growth functions using broadband stimuli. Results: Reduced sensitivity at EHFs (PTA, TEOAEs) was not associated with impaired SiN perception in the CF group. SF hearing, regardless of EHF status, was the primary predictor of SiN performance in the CF group. Increased MEMR growth was also significantly associated with poorer SiN in the CF group. Conclusions: In CF, impaired SiN perception was primarily predicted by SF hearing impairment, with additional involvement of the efferent auditory pathway through increased MEMR growth. These results build on prior evidence for efferent neural effects due to ototoxic exposures, supporting both sensory (afferent) and neural (efferent) mechanisms that contribute to listening difficulties in CF. Thus, preventive and intervention strategies should consider these combined mechanisms in people with AG ototoxicity to address their SiN problems.

Background Unplanned hospital admissions in Inflammatory Bowel Diseases (IBD) account for nearly three-quarters of IBD inpatient stays in the United Kingdom. Although costly to services and distressing for patients, research exploring experiences and potential drivers of admissions is limited. We undertook a qualitative study to explore the healthcare experiences and access needs of people with IBD who had unplanned admissions, along with their caregivers and clinicians. Methods Semi-structured interviews with 25 participants from a single tertiary IBD service in England (17 people with IBD, 3 informal caregivers, 5 clinicians) were conducted. We applied thematic framework analysis, guided by the Candidacy Framework, and worked with 2 patient and public contributors to generate final themes. Results We identified four themes: 1) Difficulties in Identifying flares and asserting severity before admission, summarised the prevailing uncertainty in identifying a flare and access to timely IBD care. 2) Navigating a disjointed healthcare system, highlighted how lack of care plans and systemic barriers can delay access. 2) Emergency care access challenges highlighted the gaps in emergency and inpatient care during flares. Whilst 4) fighting for care and individual advocacy needs, described the persistent assertion for care that may disproportionally impact access to vulnerable groups, also highlighting the importance of positive interpersonal relationships. Conclusions Individual, interpersonal and healthcare factors across the patient pathway were perceived to shape access to care in unplanned IBD admissions. Potentially reducing admissions requires proactive strategies, including the integration of patient education, monitoring tools, establishment of specialist rapid-access pathways, and formal psychological support to address barriers to access.

Bronchopulmonary dysplasia (BPD) in infancy is a risk factor for obstructive lung disease in adults. We hypothesized that adults born preterm and diagnosed with BPD have an altered lung architecture which is correlated to lung function impairment. Individuals from the LUNAPRE cohort (clinicaltrials.gov/ct2/show/NCT02923648) were included: preterm born (gestational week <32) with (n=24) or without (n=23) a previous diagnosis of BPD, full term born with allergic asthma (n=22) and healthy volunteers (n=24). Inspiratory and expiratory HRCT scans were performed and interpreted by two expert reviewers in a blinded manner. Structural changes were scored and quantitative density measurements were analysed automatically using a dedicated post-processing workstation. The HRCT scores were significantly higher in the BPD group compared to the other groups (p<0.001) and had highest numbers in subjects diagnosed with severe BPD. Most common HRCT changes were small peripheral opacities. Hypoattenuation during inspiration was only observed in the BPD group. Architectural distortion was observed in 6/24 BPD and 2/23 premature without BPD. HRCT scores correlated to FEV1 in a negative manner for preterm (p<0.001) and BPD (p<0.05) groups. Oxygen supplementation during the neonatal period correlated with HRCT score in a positive manner for preterm group (p<0.001). No differences in lung density were observed between the groups. Young adults previously diagnosed with BPD have structural changes on CT which correlate with airway obstruction. Severity of BPD at the diagnosis was associated with CT abnormalities in adulthood. HRCT changes in adults with BPD were correlated with spirometry findings.

ObjectiveTo establish a fetal lamb model of complex gastroschisis and characterize the impact on the intestines over time. Summary Background DataGastroschisis is a congenital abdominal wall defect and in its complex form is associated with serious morbidity. Robust large-animal models may help understanding are lacking. MethodsAt gestational day 75, gastroschisis was induced by creating a 1-cm abdominal wall defect reinforced by a silicone ring. Fetuses were assessed either at term or at mid-gestation (13-21 days post-induction). The primary outcome was complex gastroschisis occurrence, defined by bowel stenosis, atresia, volvulus, perforation or necrosis; otherwise classified as simple. At mid-gestation, occurrence was compared between early (13-16 days) and late (17-21 days) intervals. Secondary outcomes included prenatal ultrasound findings, in vivo bowel motility and morphology, ex-vivo bowel contractility, amniotic fluid composition, and histology across complex, simple, and normal groups. ResultsGastroschisis was induced in 32 fetuses. At term (n=14), all survivors (7/14; 50%) had complex gastroschisis, with impaired bowel motility, altered enteric neural contractile responses and smooth muscle remodeling. At mid-gestation (n=18), complex gastroschisis occurred more frequently in the late than in the early group (71% vs. 11%; p=0.035). Mid-gestation gastroschisis fetuses showed greater intra-abdominal bowel dilatation on ultrasound and higher amniotic fluid digestive enzyme levels compared with non-operated littermates, with the greatest dilation observed in complex gastroschisis. ConclusionsThis model consistently reproduces complex gastroschisis in term survivors. After induction, complex gastroschisis occurrence increases with disease duration and is accompanied by structural and functional bowel changes.

RationaleHeterogeneous alveolar collapse is prevalent in inflammatory lung conditions such as chronic obstructive pulmonary disease, acute respiratory distress syndrome, and pneumonia. Although neutrophil-released proteases contribute to the tissue remodeling that leads to alveolar collapse, how this altered mechanical environment in turn affects neutrophil migration remains largely unexplored. ObjectivesIn this study, we investigate how alveolar collapse and stretch influence neutrophil migration and identify the mechanical and biochemical factors that govern regional migration differences. MethodsWe developed a novel precision-cut lung slice platform that generates collapsed vs non-collapsed regions within the same slice. Neutrophils in both regions were longitudinally imaged for up to 5 hours to quantify motility behavior. Migration mechanisms were probed using migration-related inhibitors, collagenase, and cigarette smoke extract. A crystal ribcage system, which preserves intact alveolar shape and the air-liquid interface, was also used to assess the effects of ventilation on neutrophil migration. ResultsNeutrophil migration was faster in the collapsed region compared to not-collapsed regions. This regional difference was eliminated by Rho-associated protein kinase (ROCK) inhibition, which selectively increased migration speed in the non-collapsed region. The regional difference persisted with the addition of collagenase and cigarette smoke extract, both of which significantly increased the migration speed in both regions. In the crystal ribcage, the preserved air-liquid interface and ventilation together enhanced neutrophil migration compared with a collapsed lung. ConclusionsAlveolar collapse and stretch facilitate neutrophil migration, indicating the role of localized tissue remodeling in driving neutrophil activity and further disease progression.

BackgroundTryptophan (Trp) metabolism is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. While individual Trp metabolites have been associated with disease severity and treatment response, systems-level frameworks to define metabolic subtypes in IBD are lacking. ObjectiveTo identify reproducible Trp-related metabolic subtypes ("metabotypes") in IBD and assess their association with disease activity, clinical outcomes, and early disease development. DesignWe applied unsupervised clustering to serum concentrations of 16 Trp-related metabolites in a discovery cohort of patients with IBD undergoing biologic induction therapy (n=134). Metabotypes were validated in three independent IBD cohorts (total n>2,800), a healthy reference population, and a prospective cohort of first-degree relatives at risk for Crohns disease. Associations with disease activity, longitudinal outcomes, and metabolic pathways were assessed using multivariable regression and survival analysis. ResultsFour reproducible metabotypes with distinct metabolite profiles were identified across cohorts: Low Kyna, High Kyna, High Quin, and Balanced. Low Kyna and High Quin metabotypes were consistently associated with increased inflammatory activity and adverse clinical outcomes, including increased risk of treatment escalation and disease progression. Pathway-level analyses revealed alterations in NAD-related, lipid, and amino acid pathways between inflammatory metabotypes. A metabotype resembling inflammatory disease states was enriched in individuals who later developed Crohns disease in a prospective pre-disease cohort. ConclusionTrp-linked metabotypes define reproducible immunometabolic states in IBD that associate with disease activity and clinical outcomes and may precede disease onset. These findings provide a framework for metabolic stratification and biomarker-guided clinical trials targeting immunometabolic pathways. What is already known on this topicTryptophan metabolism through the kynurenine pathway is a central immunometabolic axis in inflammatory bowel disease (IBD) and has been linked to inflammatory activity and immune regulation. Individual tryptophan metabolites have been associated with disease severity and treatment response, but their clinical utility for patient stratification remains limited. Systems-level approaches to define clinically meaningful metabolic subtypes in IBD are lacking. What this study addsWe identify four reproducible tryptophan-related metabolic subtypes ("metabotypes") that are consistently associated with disease activity across multiple independent IBD cohorts. Inflammation-associated metabotypes show distinct pathway-level alterations, including differences in NAD-related metabolism and broader metabolic programs. A metabotype resembling inflammatory disease states is detectable before clinical diagnosis in individuals who later develop Crohns disease. How this study might affect research, practice or policyMetabotype-based classification provides a framework for molecular stratification of patients in mechanistic studies and clinical trials targeting immunometabolic pathways. This approach may support biomarker-guided monitoring of disease activity and disease progression in IBD. Identification of preclinical metabolic states highlights the potential of metabolomics for early disease detection and prevention-oriented research strategies.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory airway disease associated with impaired mucociliary clearance and persistent inflammation. While prior work has focused on inflammatory and molecular pathways, the physicochemical properties of mucus itself remain poorly characterized. This study aimed to define compositional and biophysical features of CRS mucus that may contribute to dysfunction. Methods: A prospective cross-sectional study was conducted in 15 adults undergoing endoscopic sinus surgery (11 CRS, 4 controls). Mucus was collected from the middle meatus. Hydration was measured by lyophilization. Ionic composition was quantified using mass spectrometry. Viscoelasticity was assessed via oscillatory shear rheology. Total protein, total carbohydrate, sialic acid (Sia) and fucose (Fuc) content were quantified using enzymatic and chemical assays. Statistical comparisons were performed using nonparametric tests. Results: CRS mucus exhibited significantly higher Ca2+; and Mg2+; concentrations (approximately two-fold; p<0.05) and increased variability in hydration and ion content compared to controls. Rheology showed greater heterogeneity and a non-significant trend toward increased viscoelasticity in CRS. Total protein and carbohydrate content were not significantly different; however, the carbohydrate-to-protein ratio was significantly reduced in CRS (p=0.04). Sia content and Sia-to-carbohydrate ratio were significantly elevated in CRS (p=0.04 and p=0.002), particularly in CRS with nasal polyps. Fuc content did not differ between groups. Conclusions: CRS mucus demonstrates coordinated alterations in ionic composition and glycosylation, characterized by increased cation content, hypersialylation, and reduced carbohydrate-to-protein ratios. These changes may contribute to altered mucus properties and impaired mucociliary clearance, highlighting mucus composition as a potential therapeutic target in CRS.

Background Iron deficiency (ID) is a readily treatable condition once identified. Ferritin is the primary diagnostic marker, but cut-offs vary and inflammation complicates interpretation in patients with long-term conditions (LTCs). Aim To describe ferritin distribution and the prevalence of threshold-defined low ferritin in adults with and without LTCs in primary care. Design and setting Cross-sectional observational study using routinely collected electronic health records from a national primary care database in England (1st January 2015 to 31st December 2021). Method Adults with >1 ferritin test in Clinical Practice Research Datalink (CPRD) Aurum were included. LTCs were identified using validated primary-care code lists. Outcomes included ferritin distribution and threshold-defined ID prevalence using World Health Organization (WHO) (<15 ug/L; <70 ug/L if inflammation) and National Institute for Health and Care Excellence (NICE) (<30 ug/L) cut-offs, stratified by sex and, in women, by age <50 versus >=50 as a proxy for menopausal status. Results 4,489,594 individuals were included; 55% (n=2,469,882) had >1 LTC. Ferritin was lowest in women <50 and in LTCs characterised by impaired absorption or blood loss (coeliac disease, inflammatory bowel disease). Among women <50 with an LTC, 80% had ferritin <70 ug/L versus 47% <30 ug/L, leaving 33% in the 30 to 70 ug/L range potentially missed by standard cut-offs; equivalent figures were 28% in women >=50 and 17% in men. Conclusion Threshold-defined low ferritin is very common across LTCs and disproportionately affects women, particularly those under 50. Condition-specific, inflammation-adjusted ferritin thresholds may improve detection, management, and equity in primary care.

Background & Aims: Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the leading cause of chronic liver disease in children. However, accurate, noninvasive diagnostic tools remain limited. Current screening methods are invasive or lack sensitivity. Breath-based volatile organic compound (VOC) analysis offers a simple approach with potential for point of care screening. This study aimed to identify and validate breath VOC signatures of pediatric MASLD. Approach & Results: We conducted a prospective IRB approved cohort study at the Childrens Hospital of Philadelphia (CHOP). Children aged between 7 and 20 years with MASLD (n=22), as defined by hepatic steatosis either by liver biopsy or imaging and 1 cardiometabolic risk factor, and a control group without MASLD (n=20) were enrolled. Breath samples were collected using a standardized protocol and analyzed by untargeted comprehensive two-dimensional gas chromatography-mass spectrometry (GCGCMS). Machine learning and unsupervised clustering were applied to identify discriminatory VOCs and assess heterogeneity. Untargeted GCGCMS analysis identified a distinct breath VOC signature in children with MASLD compared with non MASLD controls. A Random Forest model achieved a sensitivity of 73% and specificity of 65%, with AUC of 0.84. The VOC 2,4-dimethyl-1-heptene demonstrated strong diagnostic performance in the discovery cohort with a sensitivity of 85%, specificity of 77% and an AUC of 0.81. Unsupervised clustering revealed four MASLD subgroups with distinct volatile phenotypes associated with differences in liver enzymes and metabolic parameters. External validation in a second pediatric cohort confirmed reproducible reductions in o/p-xylene in subjects with MASLD. Conclusions: Pediatric MASLD is associated with a reproducible breath VOC signature identified by untargeted GCGCMS. These findings support breath analysis as a scalable, noninvasive screening and stratification tool for pediatric MASLD and warrant validation in larger, longitudinal studies.

Background and Aims: Diet plays a critical role in managing Crohns disease (CD) inflammation. We assessed whether dietary replacement of animal protein (AnimalP) by soy-pea protein (SoyP) decreases the pro-inflammatory potential of gut microbiota and intestinal inflammation in CD patients. Design: In an open-label, randomized controlled feeding trial at University Hospitals Cleveland Medical Center, CD participants and healthy controls were randomized (1:1) to a soy-pea or animal protein diet for 7-days. Primary outcomes were the absolute difference (d7-d0) in; Crohns Disease Activity Index (CDAI) score and fecal myeloperoxidase (MPO). Secondary outcomes included fecal calprotectin (FC) and high-sensitivity C-reactive protein (hsCRP). Murine fecal transplantation experiments were performed to determine the inflammatory potential of diet-altered gut microbiota. Results: The study randomized 66 participants and 60 were included in the final analysis (n=31 CD, n=29 HC). After 7 days, CD-SoyP participants were more likely than CD-AnimalP to show reductions in HBI (RR=4.68, 95% CI: 1.22-17.98, P=0.009) and fecal MPO (RR=2.30, 95% CI: 1.04-4.85, P=0.032), with a similar directional trend for CDAI (RR=1.52, 95% CI: 0.89-2.58, P=0.135). No participants experienced worsening of CDAI. The rank-based composite CDAI-MPO score was lower in the CD-SoyP vs CD-AnimalP group (median [IQR]: 5 [4-6] vs 8 [7-9]; P=0.012). Stratified analyses showed significant reductions in fecal MPO among CD participants with lower baseline disease activity (CDAI <150; P<0.0001), but not in those with higher activity (P=0.799) Conclusion: Short-term addition of plant-based soy-pea protein within a controlled diet exerted a beneficial, anti-inflammatory effect in CD, with evidence of greater effects among participants with lower baseline disease activity. ClinicalTrials.gov, Number NCT04065048.

STRUCTURED ABSTRACTO_ST_ABSObjectiveC_ST_ABSTo characterize intestinal transcriptional profiles in gastroschisis, their temporal evolution, and response to fetal intervention. Summary Background DataGastroschisis causes significant intestinal dysfunction, with intra-abdominal bowel dilation clinically shown to correlate with worse outcomes. While inflammation and neurovascular impairment have been implicated, genome-wide transcriptional characterization of disease severity remains lacking. MethodsUsing a fetal ovine model of complex gastroschisis, in which all gastroschisis animals demonstrated significant intra-abdominal bowel dilation at term, bulk RNA sequencing was performed on proximal small intestinal tissue from mid-gestation and term fetuses across three groups: normal, gastroschisis, and prenatally repaired gastroschisis. Differential gene expression (FDR [&le;] .05, |log2 fold change| [&ge;] 1.5) and pathway enrichment analyses were performed, with targeted interrogation of extracellular matrix (ECM), enteric nervous system (ENS), angiogenic, and inflammatory pathways. ResultsAt mid-gestation, gastroschisis intestine showed minimal transcriptional differences (150 differentially expressed genes [DEGs]) and some bowel dilation. By term, dysregulation was substantial (2,423 DEGs) alongside significant dilation. Normal ontogenetic intestinal maturation patterns were altered, with fewer expected developmental gene changes and discordant pathway regulation. ECM pathway aberrations emerged early and persisted, while ENS, angiogenic, and inflammatory pathways were only dysregulated at term. Fetal repair was associated with normalization of gene expression at term (29 DEGs vs controls). ConclusionIntestinal transcriptional changes in experimental gastroschisis parallel progressive bowel dilation, consistent with a mechanical stress contribution to intestinal injury. Prenatal repair normalizes both dilation and gene expression, indicating a dynamic and potentially modifiable transcriptional program that supports the rationale for early fetal intervention. Mini AbstractIn a fetal ovine model, progressive bowel dilation in gastroschisis parallels transcriptomic dysregulation of ECM remodeling, neurovascular impairment, and inflammation which is normalized by prenatal repair.