The American Journal of Pathology

BackgroundNeonatal sepsis is a major cause of morbidity and mortality, particularly in low- and middle-income countries such as Senegal, where incidence is 78-104 per 1,000 live births and mortality exceeds 20 per 1,000, with case fatality rates around 36%. Diagnosis is difficult due to non-specific clinical signs and lack of molecular biomarkers, highlighting the need for improved early diagnostic molecular markers that could be applied even outside of hospital settings. ObjectivesCompare neonatal and adult serum proteomes to establish a reference and identify serum protein biomarkers of neonatal sepsis. MethodsSerum samples from Senegalese neonates and adults were analyzed using data-independent acquisition (DIA) proteomics on neat serum (Evosep-timsTOF HT platform). The cohort comprised 6 neonates with non-confirmed sepsis (NCS), 22 with confirmed sepsis (CS), 17 healthy newborn controls (HC), 6 unclassified and 20 healthy adults. Downstream analyses included differential protein abundance testing, unsupervised clustering, weighted gene co-expression network analysis (WGCNA), and correlation analyses with clinical parameters. ResultsWe identified 979{+/-}20 proteins in newborns versus 718{+/-}40 in adults. Newborns showed reduced immune-response proteins, a narrower dynamic range, and increased structural proteins such as collagens, consistent with immune immaturity and tissue development. Unsupervised WGCNA analysis led to a 53-protein cluster discriminated CS from NCS/HC. Some of these dysregulated proteins identified have already been reported in independent studies using different approaches in neonatal and/or adult sepsis. Our larger panel however of identified markers maps to three major biological processes involved in sepsis: (i) pathogen sensing (LBP, CD14), and acute-phase inflammation (e.g. CRP, SAA1/2, ORM1/2); (ii) innate immune activation and leukocyte recruitment (e.g., FCGR3A, CSF1R, CD163, CD206) and final platelet exhaustion and metabolic dysregulation, (e.g., PF4, PPBP, THBS1, GP5); (iii) endothelial injury and microvascular dysfunction with tissue remodeling (e.g., ICAM1, VCAM1, VWF, SPARC) and loss of protective lipoproteins and serpins (e.g., APOA1, APOA2, APOM, SERPINA4, SERPINA5) ConclusionThis study provides a very comprehensive neonatal serum proteome characterization and identifies, for the first time, a protein panel of proteins mapped to three major processes in sepsis.

Background & AimsPrimary sclerosing cholangitis (PSC) is a progressive cholangiopathy characterized by ductular remodeling, inflammation, and periportal fibrosis, for which effective medical therapies remain limited. The Hippo pathway effector TEAD1 has been implicated in liver regeneration and fibrogenesis; however, its role in cholestatic injury remains poorly defined. We investigated whether hepatocyte TEAD1 regulates injury-associated remodeling in a PSC-mimicking model and whether this mechanism is conserved in human PSC liver. MethodsHepatocyte-specific TEAD1 knockout mice (Alb-TEAD1-/-) and littermate controls were subjected to DDC-induced cholestatic injury. Ductular reaction, fibrosis, inflammation, and bile acid-related gene programs were assessed by histology, immunostaining, and gene expression analyses. Translational relevance was evaluated using bulk and single-cell transcriptomic datasets from human PSC liver. ResultsHepatocyte TEAD1 deletion attenuated DDC-induced fibrosis, ductular expansion, and inflammatory cell accumulation, while preserving hepatocyte proliferative responses. TEAD1-deficient livers exhibited reduced expression of profibrotic mediators, including Spp1, Ctgf, and Cyr61, with decreased extracellular matrix deposition. In contrast, canonical transcriptional adaptations to cholestatic stress, including suppression of bile acid uptake, induction of efflux pathways, and repression of bile acid synthesis genes, were preserved in the absence of TEAD1. Analysis of human PSC datasets demonstrated coordinated upregulation of TEAD1 and TEAD-associated target genes. Single-cell transcriptomic analysis further revealed hepatocyte-enriched TEAD1 expression and activation of a TEAD1 target gene program across all hepatic zones in PSC, with effect sizes exceeding those observed in non-parenchymal populations. TEAD1 activation was accompanied by co-expression of profibrotic mediators and downregulation of hepatocyte differentiation markers, consistent with a maladaptive hepatocyte state. ConclusionsHepatocyte TEAD1 drives ductular, inflammatory, and fibrogenic remodeling during cholestatic injury without disrupting bile acid metabolic adaptation. These findings identify TEAD1 as a hepatocyte-intrinsic regulator of epithelial-stromal crosstalk and establish conserved activation of this pathway in human PSC, supporting TEAD-directed signaling as a therapeutic target.

Oxidative stress is proposed to be a driver of age-related diseases. Age-related macular degeneration is one such disease, where the retinal pigment epithelium (RPE) is affected early in the disease. Vasculature damage also occurs, sometimes preceding RPE damage. To model some aspects of dry AMD, we used the NaIO3 mouse model of oxidative damage. Disruption of the deep retinal vascular plexus, disorganization and death of capillaries within the choriocapillaris, and marked electroretinographic decline were observed. AAV overexpressing the transcription factor, NRF2, which induces anti-oxidation enzymes and represses inflammation, was tested for protection of damage. The BEST1 promoter limited expression to the RPE. The RPE, photoreceptors, and vascular architecture in both retinal and choroidal compartments were protected. Conditioned medium from RPE-choroid explants, infected by AAV8/BEST1-NRF2, was sufficient to transfer partial protection in vivo, indicating that NRF2 induces a protective secreted factor(s). Analysis of RNA-seq data identified growth differentiation factor 15 (GDF15) as a candidate downstream mediator. Injection of recombinant GDF15 reproduced key protective phenotypes in vivo, whereas Gdf15-deficiency attenuated NRF2-mediated rescue. Pharmacologic inhibition of TGF-{beta} receptor signaling diminished NRF2 associated protection, supporting involvement of this signaling pathway. In a laser-induced choroidal neovascularization model, intravitreal GDF15 injection reduced fluorescein leakage and lesion size. These findings support a model in which NRF2 activation in the RPE induces expression of GDF15, which is capable of protecting the RPE, photoreceptors, and the retinal and choroidal vasculature. NRF2 and GDF15 have therapeutic potential for ocular diseases, as well as for other diseases with vascular pathology.

Albino individuals are clinically recognized to exhibit heightened susceptibility to light-induced retinal injury, yet the cellular and metabolic mechanisms underlying this vulnerability remain poorly defined. Here, we investigated whether retinal pigment epithelium (RPE) pigmentation governs mitochondrial structure, metabolism, and inflammatory responses that ultimately determine retinal resilience to blue light stress. Using pigmented (C57BL/6J) and albino (Balb/c) mice, we demonstrate that albino animals exhibit markedly increased retinal phototoxicity following blue light exposure, manifested by fundus lesions, outer nuclear layer (ONL) disruption, and structural degeneration evident by OCT. Primary RPE cultures derived from albino mice exhibited profound difference in mitochondrial morphology, characterized by increased mitochondrial number, reduced size, and enhanced fragmentation, accompanied by elevated mitochondrial DNA copy number. These structural changes correlated with transcriptional skewing toward mitochondrial fission (increased Drp1) and suppression of mitochondrial fusion (Mfn1, Mfn2, OPA1). Functionally, albino and depigmented RPE displayed impaired oxidative phosphorylation, reduced ATP production, and diminished reliance on mitochondrial pyruvate carrier (MPC)-dependent metabolism. In parallel, albino RPE demonstrated cell-cycle accumulation at G2/M and heightened basal and blue light-induced secretion of pro-inflammatory cytokines, particularly IFN-{beta}1, IL-6, and TNF-. Importantly, exogenous melanin supplementation partially restored mitochondrial fusion gene expression, pyruvate-dependent respiration, and inflammatory restraint. Together, these findings identify melanin as a critical regulator of RPE mitochondrial architecture, metabolic substrate utilization, and inflammatory signaling, providing a mechanistic framework to explain enhanced photo-vulnerability in the albino retina. These insights establish pigmentation-dependent mitochondrial metabolism as a determinant of retinal resilience and suggest mitochondrial bioenergetics as a therapeutic target.

Clear cell renal cell carcinoma (ccRCC) is characterized by transcriptional reprogramming driven by hypoxia signaling, metabolic rewiring, and immune modulation. While gene-level analyses have defined key features of ccRCC biology, they do not capture isoform-level variation arising from alternative splicing. Differential transcript usage (DTU) represents an additional regulatory layer that may influence protein function, pathway activity, and clinical outcomes, yet its role in ccRCC biology and prognosis remains incompletely understood. We assessed differential expression in 127 ccRCC tumors and 33 normal-adjacent tissues from the Dartmouth Cancer Center cohort, with external validation in 94 CPTAC tumors, adjusting for cell-type proportions. DTU was identified using DRIMSeq/stageR, followed by limmavoom modeling with clinical and tumor microenvironment covariates. Transcript-based consensus clustering defined tumor subgroups, and Cox proportional hazards modeling integrated transcript-level features with clinical variables. In tumor versus normal comparisons, 1,170 transcripts exhibited significant differential usage, mapping to canonical ccRCC pathways with distinct patterns across functional and non-functional transcript classes. Consensus clustering based on transcript us-age identified two subgroups with distinct angiogenic profiles and significant survival differences. Cluster-level analysis revealed DTU in genes involved in cytoskeletal organization (ACTB), immune processes (B2M), extracellular matrix organization (FN1, APLP2), and iron metabolism (FTH1) with protein domain alterations, including the loss of actin-associated domains in ACTB and immunoglobulin-like domains in B2M. Prognostic modeling identified twelve transcripts consistently retained across bootstraps, improving risk stratification over clinical variables alone. External validation confirmed overlapping prognostic transcripts, including FGFR1 and NUCB1. Isoform-level features may serve as biomarkers and therapeutic targets in ccRCC. Statement of significanceTranscript-level analysis uncovers potential regulatory pathways in ccRCC missed by gene-level approaches, revealing isoform-specific alterations that define survival sub-groups and offer potential biomarkers and therapeutic targets.

CD239, also known as Lutheran blood group glycoprotein (Lu) or basal cell adhesion molecule (B-CAM), is a transmembrane protein belonging to the immunoglobulin superfamily (IgSF). CD239 serves as a specific receptor for laminin 5, a subunit of laminin-511/-521, which are major components of renal basement membranes. A previous study of another group demonstrated that CD239-null mice are healthy and develop normally. Although no alteration in renal function was observed, most glomeruli in the mutant kidneys exhibited morphological abnormalities. In this study, we investigated the role of CD239 in renal tubules. We examined the distribution of CD239 using renal tubule-specific markers. CD239 was localized to the Henles loop, distal tubule, and collecting duct, but not to the proximal tubule. Next, we analyzed the localization of renal tubular molecules in CD239-null mice. The localization of uromodulin (UMOD) and Na-K-Cl cotransporter (NKCC2) was disrupted in the distal tubules lacking CD239, suggesting that CD239 plays a role in maintaining the polarity of renal epithelial cells. Furthermore, to examine the stability of the distal tubules, CD239-null mice were subjected to chronic kidney disease (CKD) using an adenine-rich diet. Blood analysis revealed that CD239-null mice fed an adenine diet readily developed CKD. Adenine-fed null mice exhibited more marked histological injury along the distal tubules compared to that by controls. These results indicate that CD239 is essential not only for maintaining cellular polarity but also for ensuring the stability of the distal tubules. Although CD239-null humans exhibit no obvious associated pathology, it could be a predisposition to CKD.

Secondary degeneration following optic nerve crush (ONC) is driven in part by mitochondrial dysfunction and microglial activation. Inspired by hibernation, where reduced succinate oxidation limits reactive oxygen species (ROS) production, we tested whether pharmacological inhibition of this pathway confers neuroprotection. Using in vivo ONC models and in vitro microglial assays, we evaluated the effects of dimethyl malonate (DMM), an inhibitor of succinate dehydrogenase, and a cell-permeable succinate analog (succinate-NV). Succinate-NV increased pro-inflammatory cytokine expression (IL-1{beta}) and reduced anti-inflammatory IL-10, whereas non-permeable succinate had no effect, indicating that intracellular succinate can drive microglial activation. In hibernating animals, succinate-NV disrupted neuroprotection and reduced retinal ganglion cell (RGC) survival following optic nerve injury. Although DMM partially reduced select inflammatory cytokines, it failed to normalize IL-1{beta} or IL-10 and suppressed microglial phagocytosis while exhibiting cytotoxic effects. In vivo, DMM-treated animals showed reduced IBA1{square} microglia but increased CD68{square} activation and accumulation of DAPI{square} cells at 7 days post-injury at the crush site. RGC somas persisted but were Caspase3+ consistent with impaired clearance. Astrocyte reactivity increased at lesion borders, while reduced and fragmented GFAP at the lesion site indicated localized astrocyte loss. Collectively, these findings demonstrate that inhibition of succinate oxidation alone is insufficient for neuroprotection and underscore the need for coordinated metabolic and immune regulation that cannot be achieved through single-pathway pharmacological intervention.

In clinical trials for ulcerative colitis (UC), pathologists assess disease severity through standardized histological indices, including the Geboes Score, Robarts Histopathology Index (RHI), and Nancy Histologic Index (NHI). Despite strong associations with clinical outcomes, histologic scoring suffers from inter- and intra-reader variability, and consensus criteria for histologic remission remain uncertain. Through a consortium approach, we developed an artificial intelligence-based measurement (AIM) tool for scoring histology in UC mucosal biopsies (AIM-HI UC). This model, trained on a large dataset of UC biopsies (N=10,230), utilizes additive multiple instance learning models leveraging PLUTO, a pathology foundation model, that predict each of the Geboes subgrades, from which the Geboes grade-level score, RHI, and NHI can be calculated. Evaluation of this model on a standalone verification set including clinical trial specimens established algorithm non-inferiority and/or superiority relative to standard qualified pathologists through comparison of algorithm-consensus and pathologist-consensus agreement metrics (non-inferior if difference >-0.1, superior if difference >0, inclusive of confidence intervals). AIM-HI UC was determined to be non-inferior to pathologists (N=3) for the prediction of all seven Geboes subgrades, grade-level Geboes, RHI, NHI, histologic improvement (GS<3.1), 2A histologic remission (GS<2A.0), and 2B histologic remission (GS<2B.0). AIM-HI UC was superior to pathologists for several Geboes subgrades (GS 0, GS 1, GS 2B, and GS 5), as well as grade-level Geboes, RHI, and positive percent agreement of 2A histologic remission. The model was shown to be greater than 99% repeatable for all histologic scoring metrics examined. Model-derived scores were shown to strongly correlate with canonical histologic features of inflammation, including the proportion of total epithelium that is inflamed (Spearman r=0.83; p<0.01), the proportion of neutrophils localized within crypt epithelium (Spearman r=0.83, p<0.01), and the amount of mucosal area classified as erosion or ulceration (Spearman r=0.80, p<0.01). Overall, these results suggest that AIM-HI UC has the potential to improve consistency of UC histology interpretation, providing a path toward standardization of UC histology scoring in clinical trials.

Background. Balanced (1:1:1) transfusion of red blood cells (RBCs), plasma, and platelets is the standard of care in trauma-induced massive haemorrhage, where early coagulopathy is a defining feature. In gastrointestinal (GI) haemorrhage this physiology is non-prominent, and whether plasma and platelets provide benefit when [&ge;] 10 RBC units are required within 24 hours is unknown. Objective. To test whether a red-blood-cell-only (RBC-only) transfusion strategy is non-inferior to a balanced (Balanced) strategy for in-hospital mortality in adults meeting massive-transfusion criteria for GI haemorrhage. Design. Single-centre retrospective cohort of 559 adult massive-transfusion encounters (536 patients; 2021-2025) with a primary admitting diagnosis of upper, lower, or unspecified GI haemorrhage. Exposures were RBC-only versus Balanced (RBCs with any plasma and/or platelets). The primary outcome was in-hospital mortality, with a pre-specified 5-percentage-point (pp) non-inferiority margin on the absolute risk difference and a 3-pp sensitivity margin. Analysis used augmented inverse-probability-of-treatment weighting (AIPTW) with bootstrap inference (2,000 resamples by patient). Five pre-specified sensitivity analyses were performed. Results. 505 encounters (90.3%) received RBC-only and 54 (9.7%) received Balanced transfusion. The AIPTW risk difference for in-hospital mortality (RBC-only - Balanced) was -19.8 pp (95% CI -68.1 - -2.2 pp). Non-inferiority was demonstrated at both the primary 5-pp and the more stringent 3-pp margins. Five pre-specified sensitivity analyses, (1) a propensity-score matched cohort, (2) a complete-case model incorporating INR, (3) a broader GI diagnosis set (n = 749), (4) a first encounter per patient restriction, and (5) E-value bound analysis were concordant with the primary estimate. Conclusion. In this propensity-score-weighted cohort of adults with massive GI haemorrhage, an RBC-only transfusion strategy was non-inferior to a balanced strategy for in-hospital mortality at both 5-pp and 3-pp margins. The findings support individualized use of plasma and platelets in GI haemorrhage rather than reflexive application of the 1:1:1 trauma protocol; prospective confirmation is warranted.

The Microphthalmia-associated transcription factor (MITF) plays a critical role in retinal pigment epithelium (RPE) development and function. Dysfunctional autophagy and lysosomal degradation in the RPE have been implicated in age-related retinal degeneration, yet the contribution of MITF to these pathways remains incompletely understood. Here, we show that reduced Mitf expression impairs autophagy in mouse and human RPE cells. Primary RPE cells from Mitfmi-vga9/+ heterozygotes mice displayed altered autophagic flux characterized by accumulation of LC3B-II and p62, while MITF knockdown in human ARPE-19 cells promoted autophagosome accumulation. Ultrastructural analysis further revealed age-dependent accumulation of autolysosomes and lipofuscin-like granules in mutant RPE cells. In addition, expression of autophagy-related genes was altered in mutant RPE tissue, supporting disrupted lysosomal-autophagic homeostasis. Together, our findings identify MITF as an important regulator of autophagy in the RPE and suggest that impaired MITF-dependent homeostasis may contribute to retinal degeneration.

BackgroundHyaluronan (HA) is a major extracellular matrix glycosaminoglycan that regulates vascular integrity and immune signaling in the lung. Its biological effects are strongly size-dependent, with high-molecular-weight HA (HMW-HA) generally protective and low-molecular-weight HA (LMW-HA) pro-inflammatory. However, how different HA sizes and concentrations globally remodel endothelial cell signaling remains poorly understood. MethodsHuman lung microvascular endothelial cells (HULEC-5a) were treated with physiologic (200 ng/mL) or supraphysiologic (1 {micro}g/mL) concentrations of LMW-, medium-molecular-weight (MMW-), or HMW-HA. Cell viability was confirmed by LDH assay. Quantitative proteomics with downstream Ingenuity Pathway Analysis (IPA) was used to profile HA-induced signaling networks. ResultsProteomic analysis revealed a conserved HA-response signature across all conditions involving cell cycle regulation, senescence, and immune modulation, with distinct size-and dose-dependent differences. At supraphysiologic concentrations, HMW-HA suppressed proliferative and inflammatory pathways, consistent with a protective, quiescent phenotype. LMW-HA induced the broadest stress-associated proteomic changes, consistent with its role as a damage-associated molecular pattern. Unexpectedly, physiologic MMW-HA elicited the strongest responses, driving metabolic and cytoskeletal pathways including insulin signaling and Rho GTPase activity. Network analysis highlighted 176 overlapping pathways across HA treatments, with unique contributions of LMW- and HMW-HA to stress- versus barrier-stabilizing signaling, respectively. ConclusionHA is not a passive structural molecule but an active regulator of endothelial signaling, with effects shaped by both molecular weight and concentration. Our findings identify a distinct role for MMW-HA at physiologic levels and highlight how HA fragmentation and accumulation may contribute to endothelial dysfunction in lung injury, with implications for targeted HA-based therapies.

Background Coeliac disease affects approximately 1% of the global population and remains substantially underdiagnosed. Histopathological assessment of duodenal biopsies is the diagnostic gold standard but is subject to approximately 20% inter-observer disagreement. While machine learning approaches show promise, most prior work relies on black-box models with limited interpretability, restricting clinical adoption. Methods We present an interpretable pipeline that follows established histopathological criteria by extracting clinically meaningful morphological features from H&E-stained whole-slide images. Five sequential stages perform pre-processing, semantic segmentation of villi, crypts, intraepithelial lymphocytes (IELs) and enterocytes, crypt morphometry, villus length estimation via a novel polyline-based keypoint model, and coeliac disease classification using three quantitative features: IEL-to-enterocyte ratio, villus-to-crypt area ratio, and villus-length-to-crypt-depth ratio. Training and validation used data from four institutions; independent testing used 1,357 WSIs from two further institutions including one with a previously unseen scanner manufacturer, spanning five diagnostic categories: coeliac disease, normal mucosa, chronic inflammation, gastric metaplasia, and gastric heterotopia. Results Semantic segmentation achieved villus and crypt precision and recall of 87-90%. Villus length estimation correlated strongly with expert annotations (Pearson's r=0.85, mean relative error 13.5% post-calibration). All three morphological features significantly separated coeliac disease from all non-coeliac diagnostic groups across internal and external datasets (p<0.01 in all comparisons). On the test set the diagnostic classifier achieved accuracy 94.5%, PPV 92.9%, NPV 94.7%, and AUC 0.982. Conclusions This interpretable framework achieves strong multi-centre diagnostic performance while producing quantitative morphological outputs, villus length, crypt depth, and IEL-to-enterocyte ratios, that directly reflect established histopathological criteria, representing a meaningful step towards standardised AI-assisted coeliac disease diagnosis.

The choroid plexus epithelial cells (CPECs) at the blood-cerebrospinal fluid (CSF) interface possess an exceptionally high mitochondrial content to support CNS homeostasis. Oncocytic CPECs (O-CPECs), characterized by enlarged and granular eosinophilic cytoplasm composed of excessive abnormal mitochondria, likely contribute to an energetic failure of this energy-demanding tissue. The relationship between O-CPECs and other CPEC pathologies in humans, such as Biondi body (BB) amyloid inclusions, remains poorly defined. In the present study, using H&E-stained sections from 68 postmortem cases, we classified O-CPECs by quantitative size criteria and cytological features, and found an increase in the prevalence of O-CPECs with age after adjusting for sex and tissue source. After excluding two influential control cases, there was evidence for a further increase associated with Alzheimers disease. Using antibodies to ATP synthase beta chain to classify O-CPECs, and thioflavin-S to identify BBs, we revealed an increased prevalence of BBs in O-CPECs compared to neighboring non-oncocytic cells. Small multiple BB inclusions were responsible for the increase in O-CPECs, while the prevalence of larger inclusions was decreased in O-CPECs. Together, our data support a clear age-associated oncocytic transformation of CPECs and implicate mitochondrial dysfunction-amyloid interactions.

Mammalian single-stranded DNA binding protein 1 (SSB1) has been established as an essential component of genome stability in both human cells and mice. Moreover, SSB1 was recently implicated in cytoplasmic stress response by its involvement in Ras GTPase-activating protein-binding protein 1 (G3BP1)-containing cytoplasmic stress granules (SGs) upon various forms of stress. Here, we generated and analyzed human cellular knockout and rodent ischemia-reperfusion (I/R) models to define SSB1s roles in cytoplasmic stress response. Analysis of wild-type as well as SSB1 and G3BP1 knockout human retinal pigment epithelial (RPE-1) cells shows stress-specific incorporation of SSB1 into SGs and a negative regulator role for SSB1 in SG dynamics under sublethal stress conditions. We find that SSB1 knockout measurably increases cellular sensitivity to oxidative stress but does not alter cell proliferation following mild acute stress. Moreover, we detect SSB1 efflux from the nucleus upon stress that is dependent upon the presence of G3BP1 in a stress-specific manner. In addition, using mouse and rat models we observe significant upregulation and robust cytoplasmic granulation of SSB1 upon renal ischemia-reperfusion stress, establishing SSB1s involvement in complex organismal stress response in vivo. Together, our data demonstrate active involvement of SSB1 in cytoplasmic response to cellular stress and acute kidney injury, with implications for targeting stress response functions in cancerous versus non-cancerous contexts. HIGHLIGHTSO_LISSB1 is incorporated into cytoplasmic stress granules and negatively regulates stress granule assembly under sublethal stress conditions C_LIO_LISSB1 shows stress- and G3BP1-dependent nuclear efflux C_LIO_LISSB1 is upregulated and undergoes apical granulation in renal epithelial cells during renal ischemia-reperfusion injury C_LI

Aortic dissection is life-threatening due to continued loss of medial integrity that may culminate in secondary rupture within hours to days. While pre-existing defects or hemodynamic loads compound structural deterioration of the aorta, pathological progression from symptomatic dissection channel to lethal transmural tear is poorly understood. We examined the structure of referent and acutely dissected ascending aortas by microscopy. Elastic, collagen, and cellular components of non-dissected media were intricately interconnected. Medial damage in dissection lesions was traced from ingress to central to peripheral areas. Entry tears broke cleanly through successive laminae leading to cavernous false lumens in which medial structure was destroyed. Nearby laminae with widening between flanking elastic lamellae (termed minor delaminations) were filled with blood and showed severe medial damage. Farther laminae without delamination but containing red blood cells (termed blood extravasation) displayed moderate medial damage. More distant, non-delaminated laminae with accumulation of albumin but not red blood cells (termed plasma extravasation) exhibited mild medial damage. Varying medial hemorrhage with scattered sloughing of laminae was observed along the entire false lumen. We conclude that hydraulic fracturing of residual dissected media by pressurized blood via communications from the false lumen contributes to further structural weakening of the aortic wall.

BACKGROUNDPatients with inflammatory bowel disease (IBD) are at increased risk of cardiovascular disease, yet the mechanisms linking chronic intestinal inflammation to cardiac dysfunction remain poorly understood. IBD is characterized by profound gut microbiota dysbiosis, which we hypothesize drives systemic immune dysregulation and contributes to cardiac dysfunction. METHODSA chronic colitis mouse model was used to assess gut microbiota dysbiosis, systemic immune cell metabolism, and cardiac remodeling. Cardiac outcomes were evaluated by echocardiography, histology, and molecular analyses. Mechanisms were examined using fecal microbiota transplantation, immune cell depletion, exosome transfer, bone marrow chimeras, RNA-seq, co-immunoprecipitation, confocal microscopy, and siRNA-mediated gene silencing. RESULTSChronic DSS colitis induced cardiac dysfunction, hypertrophy, and fibrosis in mice. These changes were accompanied by sustained gut microbiota dysbiosis, metabolic reprogramming, and mitochondrial dysfunction in circulating immune cells. Fecal microbiota transfer experiments demonstrated that colitis-associated microbiota were sufficient to reprogram systemic immune cells and promote cardiac dysfunction. Immune cell depletion studies identified macrophages as key mediators of colitis-associated cardiac injury. Colitis increased systemic lipopolysaccharide (LPS) translocation, bone marrow chimera experiments demonstrated that hematopoietic TLR4 signaling was required for immune cell metabolic remodeling and cardiac dysfunction during chronic colitis. Transcriptomic analysis identified guanylate-binding protein 2b (GBP2b/GBP1, hereafter referred to as GBP1) as a key downstream effector of LPS-TLR4 signaling. Upon LPS stimulation, GBP1 localized to mitochondria, where it interacted with DRP1 and FIS1 to promote mitochondrial fission, oxidative stress, and enhanced immune cell migration into the heart. In addition, GBP1 was secreted via exosomes, which were taken up by cardiomyocytes and contributed to hypertrophic remodeling, and cardiac dysfunction. CONCLUSIONSThese findings establish the LPS-TLR4-GBP1 axis as a key driver of colitis-associated cardiovascular dysfunction and highlight this pathway as a promising therapeutic target for reducing cardiovascular risk in patients with IBD. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIPatients with inflammatory bowel disease have an increased risk of cardiovascular dysfunction that cannot be fully explained by traditional cardiovascular risk factors. C_LIO_LIGut microbiota dysbiosis and chronic innate immune activation are hallmarks of inflammatory bowel disease, but their direct contribution to cardiac remodeling remains unclear. C_LI What New Information Does This Article Contribute?O_LIChronic colitis-associated gut microbiota dysbiosis induces systemic immune cell metabolic and mitochondrial reprogramming that is sufficient to drive cardiomyocyte hypertrophy and cardiac dysfunction. C_LIO_LIHematopoietic Toll-like receptor 4 signaling links colitis associated gut microbiota to immune metabolic dysfunction and cardiac impairment, establishing a causal gut-immune-heart axis. C_LIO_LIGuanylate-binding protein 2b (GBP2b/GBP1) is identified as a critical downstream effector that promotes mitochondrial fission, oxidative stress, immune cell cardiac infiltration, and exosome-mediated cardiac remodeling. C_LI

Background Coeliac disease (CD) diagnosis on duodenal biopsies is limited by interobserver variability. We have previously demonstrated pathologist-level performance with our artificial intelligence (AI) model for the histopathological diagnosis of adult CD, but not in paediatric practice. As paediatric CD screening programmes expand internationally, accurate and scalable diagnostic tools are needed. We investigated whether an AI model trained exclusively on adult whole-slide images (WSIs) can generalise to paediatric CD diagnosis across independent centres. Methods A training and validation dataset of 9,958 WSIs from 8,421 adult patients (961 CD) from five centres was used to develop an ensemble of multiple-instance learning models using features from a foundation model. Testing was performed on 708 consecutive paediatric patients (86 CD) from two centres (Edinburgh and Southampton) not included in training. Model calibration was assessed, and probability outputs were grouped into clinically interpretable categories. Findings In adult cross-validation, the AI model achieved an area under the receiver operating characteristic curve (AUC) of 98.7%, sensitivity of 84.9%, specificity of 99.0%, and negative predictive value (NPV) of 98.1%. On testing (paediatric) datasets, performance remained high (AUC 98.8%, sensitivity 80.2%, specificity 98.4%, NPV 97.3%). Restricting analysis to predictions outside the intermediate-probability range (predicted CD probability <10% or [&ge;]65%; 85.3% of cases) improved sensitivity to 100% and specificity to 98.7%. No misclassifications were observed among high-confidence predictions (<2% or [&ge;]85%; 66.0% of cases). The expected calibration error was 0.03. Performance improved significantly when biopsies from both duodenal sites (bulb [D1] and descending [D2/3]) were considered. Interpretation Our AI model, trained on adult biopsies, generalises to paediatric CD diagnosis across centres and scanner platforms. Well-calibrated probability outputs provide clinically interpretable measures of diagnostic confidence and could support safe identification of CD-negative biopsies within defined thresholds. These findings demonstrate the feasibility of applying adult-derived AI models in paediatric populations and reinforce the importance of multi-site (D1 & D2) biopsy sampling.

BackgroundTumor-associated macrophages (TAMs) are key drivers of the immunosuppressive tumor microenvironment (TME), supporting tumor progression through diverse functions. However, mechanistic studies of TAM polarization remain limited by the lack of physiologically relevant human model systems that capture stromal-immune interactions and macrophage heterogeneity. MethodsWe established advanced human co-culture systems that integrate healthy donor-derived macrophages with patient-derived organoids and tumoroids (PDOs and PDTs), as well as matched normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). These multicellular models enabled the investigation of interactions among stromal, epithelial, and immune cells within tumor and adjacent normal tissue environments. ResultsThe co-culture systems recapitulated distinct macrophage states associated with tumor and adjacent normal environments and identified fibroblasts as major regulators of macrophage phenotypes. CAFs promoted macrophage metabolic remodeling characterized by altered lipid handling and enrichment of TAM-like signatures. Mechanistically, we identified thrombospondin 1 (TSP1) as a CAF-secreted factor linked to metabolic priming. Recombinant TSP1 induced transient lipid accumulation followed by mitochondrial remodeling. In tumor co-culture conditions, CD36 inhibition reduced lipid accumulation in macrophages, supporting a role for TSP1-linked lipid crosstalk in stromal-immune interactions. ConclusionOur study establishes advanced patient-derived co-culture models as a platform to investigate human TAM biology and stromal-immune interactions in CRC. Using these systems, we identify a fibroblast-associated TSP1-lipid axis linked to macrophage metabolic remodeling and TAM-like polarization, highlighting stromal metabolic communication as a potential targetable feature of the CRC microenvironment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/728363v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@16d60aborg.highwire.dtl.DTLVardef@1e593e7org.highwire.dtl.DTLVardef@15124b0org.highwire.dtl.DTLVardef@3fca92_HPS_FORMAT_FIGEXP M_FIG C_FIG

MicroRNAs (miRNAs) have been widely implicated in cancer initiation and progression, yet examination of the effects of global miRNA disruption on these processes has been limited. We developed novel genetically engineered mouse models of Kras-driven pulmonary adenocarcinoma (LUAD) with cell-type-specific disruption of miRNA biosynthesis via Dicer1 allele deletion, which exhibit significant differences in tumor progression rates and expected survival. Dicer1 is an RNase III enzyme that is required for the biogenesis of mature, functional miRNAs. Lung tumor progression was accelerated, and expected survival was decreased only when we initiated tumors and deleted one allele of Dicer1 in club cells and mutated Dicer1 in alveolar type 2 (AT2) cells. Reversing the cell types by inducing tumorigenesis, deleting one Dicer1 allele in AT2 cells, and mutating Dicer1 in club cells modestly accelerated tumor progression and had no effect on expected survival. Collectively, our results demonstrate that Dicer1 disruption accelerates lung cancer progression in a cell-type-dependent and non-cell-autonomous manner, and our mice represent tools for investigating the roles of miRNAs and miRNA-mediated intercellular communication in tumor progression. SummaryKras-driven mouse models show that Dicer1 mutations accelerate lung adenocarcinoma (LUAD) progression in a cell-type-dependent manner and suggest that the influence of miRNA-mediated intercellular communication is unidirectional and non-cell-autonomous.

Xenotransplantation using genetically engineered pig organs offers a promising solution to the shortage of donor organs for life-saving transplants. However, human preformed antibodies against unknown pig xenoantigens remain a significant barrier to successful xenotransplantation. Current methods for characterizing these antibodies or xenoantigens are limited to cellular-level crossmatch assays. In this study, we developed a novel approach to identify pig xenoantigens, including peptide and glycopeptide epitopes that react with human preformed antibodies. First, human preformed antibodies against xenoantigens were enriched from plasma using immobilized pig kidney proteins. The enriched antibodies were then immobilized and used to isolate pig kidney proteins, peptides, and intact glycopeptides, followed by liquid chromatography-tandem mass spectrometry analysis. This dual-level approach identified 221 peptides corresponding to 153 proteins, with a significant enrichment of plasma membrane and extracellular proteins. Notably, 11 peptides were unique to pig sequences, suggesting their potential role in driving xenogeneic immune responses. Glycoproteomic analysis identified 122 intact glycopeptides, predominantly complex/hybrid glycoforms and Neu5Gc-containing glycans. Our method effectively identifies peptides and intact glycopeptides reactive to human preformed antibodies, providing critical insights for discovering xenoantigens. These findings could guide genetic engineering strategies and enhance recipient candidate screening for xenotransplantation, ultimately increasing the feasibility and success of xenogeneic organ transplantation.