Background: Urinary cellular biomarkers detected by flow cytometry have emerged as promising non-invasive tools for the diagnosis, prognosis, and monitoring of renal and urological diseases. However, a comprehensive synthesis of their clinical applicability is lacking. Objectives: This review aimed to systematically evaluate and summarize the evidence of urinary cellular biomarkers identified through flow cytometry in human populations with renal or urological diseases. Methods: A systematic search of PubMed, Scopus and Web of Science was conducted from inception to January 2025. Observational studies analyzing urinary samples by flow cytometry were included, whereas animal studies, genetic-only analyses and studies with incomplete data were excluded. Data extraction and risk of bias assessment were performed independently by two reviewers using a standardized form and the Newcastle-Ottawa Scale. Due to heterogeneity in study populations, designs, and cytometry methods, results were synthesized narratively. Results: Of 3938 records screened, 23 studies met the inclusion criteria. All studies applied flow cytometry to characterize urinary cellular biomarkers across renal diseases. Several studies reported promising diagnostic and monitoring applications, but substantial heterogeneity in study design, cytometry protocols, and marker panels limited comparability. Longitudinal analyses and robust prognostic validation were infrequently reported. Conclusions: Urinary cellular biomarkers assessed by flow cytometry represent a promising non-invasive approach for evaluating renal and urological diseases. However, clinical implementation remains constrained by heterogeneity and insufficient validation. Future research should focus on standardized methodologies and large prospective studies to establish their diagnostic and prognostic utility.

This study aimed to characterize human intestinal conventional dendritic cells (cDCs) in health and inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD). Three cDC subsets (CD103- cDC2, CD103+ cDC2, and cDC1) were identified from lamina propria mononuclear cells. Their phenotype and function were analyzed in healthy and IBD-inflamed gut tissues. In the healthy gut, cDC2 predominated over cDC1, with CD103+ cDC2 dominating the duodenum and CD103- cDC2 prevalent in the ileum and colon. CD103+ cDC2 expressed higher PD-L1 and produced more IL-10. In culture, CD103+ cDC2 increased proportionally unless inhibited by LPS. All subsets induced IL-10+ helper T-cell differentiation, with ileal cDCs being more stimulatory than colonic ones. In IBD, cDCs showed constitutively lower SIRPα expression across conditions. Notably, UC-inflamed colon exhibited reduced cDC1 and CD103+ cDC2, while CD-inflamed colon maintained these subsets but showed increased T-cell stimulation and IL-17+ T-cell priming. Intestinal cDC subsets prime IL-10+ helper T-cells in health. In UC, reduced cDC1 and CD103+ cDC2 in inflamed mucosa contrast with CD, suggesting distinct pathogenic mechanisms that could inform targeted therapies.

Long COVID is a complex condition characterized by a broad spectrum of persistent, multisystemic symptoms that often impair daily activities and cause disability with significant socioeconomic impact. Although several hypotheses have been proposed to explain its pathophysiology—including immune dysregulation—the underlying mechanisms remain poorly understood. Indeed, there are no current treatments for this condition. Therefore, we aimed to characterize immune alterations induced by SARS-CoV-2 during the acute phase of infection and three months post-hospital discharge in a cohort of patients who later developed Long COVID, referred to a matched cohort who did not. We performed high-dimensional immune profiling of peripheral blood mononuclear cells using a 40-marker mass cytometry panel. Data analysis combined classical hierarchical gating strategies with unsupervised computational approaches. Our results revealed distinct immune signatures between Long COVID and non-Long COVID patients, affecting both the innate and adaptive immune compartments. Notably, individuals who subsequently developed Long COVID exhibited early abnormalities at infection in innate immunity, particularly involving dendritic cells and γδ T cells. Moreover, three months post-discharge, persistent alterations were observed in several adaptive immune cell subsets in patients who next developed Long COVID over time. In summary, our findings suggest that early immune dysregulation following viral infection may predispose individuals to persistent post-viral pathology.

Acute pancreatitis (AP) patients can be classified into mild or severe outcome based on the presence of organ failure days during hospitalization. However, there are no biomarkers that can predict severity outcome. This study aim is to perform an unbiased characterization of the circulating immunome of AP patients at hospital admission aiming to identify novel biomarkers which could predict disease outcome prior to the onset of organ damage and clinical deterioration. Peripheral blood mononuclear cells (PBMC) were collected from newly hospitalized AP patients. Based on their evolution, patients were classified following the Modified Atlanta classification into mild acute pancreatitis (mAP) (n = 20) as well as moderately and/or severe acute pancreatitis (ms-sAP) (n = 20). PBMC were analysed by high-dimensional spectral cytometry with unsupervised dimensionality reduction and clustering algorithms based on the subsequent outcome of the patients. Results were further validated by classical gating and functional approaches with a smaller cohort (n = 8 mAP and n = 8 ms-sAP). A total of 120 different immune cell clusters were identified and characterized in AP patients at hospitalization. Following computational analysis and classical gating validation approaches, circulating CD2+CD8dim NKT-Like cells were found to be specifically reduced in ms-sAP patients. Moreover, NKT-Like cell subsets from these patients displayed an IL-15 induced expansion of NKG2a and NKp30 referred to mAP. Patients with AP at hospitalization with subsequent ms-sAP display a specific reduction of NKT-Like cells coupled with an expanded function. Hence, quantifying the phenotype and/or function of circulating NKT-Like cells during hospital admission could serve as a predictive biomarker for AP severity, aiding in early stratification and personalized treatment strategies.

e14529 Background: Bladder urothelial cancer is a common neoplasm with a significant proportion of patients who progress to advanced disease despite optimal local and systemic therapy. Newly immune related therapies have improved patients’ outcome; however, only certain patients achieve durable responses. Hence, there is a need for non-invasive biomarkers for early response prediction. Methods: Peripheral Blood Mononuclear Cells (PBMCs) from advanced bladder cancer patients treated with avelumab (n=11) were obtained prior to immunotherapy administration. One year follow up revealed treatment response, which allowed a cohort division for immune cell signature comparison. A 40-plex spectral cytometry panel was applied, and further computational analysis performed. Results: Unsupervised spectral cytometry analysis revealed a highly heterogeneous circulating immunome landscape, capable of identifying 78 distinct immune clusters. Further analysis discriminated 10 clusters that significantly differed between responders and non-responders, 8 clusters from T, NK & innate lymphoid cells lineage and 2 clusters from Myeloids & antigen presenting cells lineage (Figure 2). Further hierarchical validation confirmed an expansion of effector CD4+ T cells and CD8+ TEMRA cells, an among non-responder patients before therapy. On the contrary, those with subsequent response displayed an overrepresentation of CD2⁻ innate lymphoid cells as well as CD25⁺ classical monocytes, supporting a blood-based immune signature associated with therapeutic benefit. Conclusions: We have demonstrated that spectral cytometry analysis is capable of revealing distinct immune fingerprint among bladder cancer patients in response to immunotherapy. The biomarkers found in this study could therefore be of use for anticipating clinical outcome in these patients, improving disease management.

While cytokine-targeted therapies have significantly transformed the treatment landscape of inflammatory bowel disease (IBD), a substantial proportion of patients remain refractory or lose responsiveness over time. Moreover, cytokine blockade alone may be insufficient to control the highly complex and heterogeneous immune dysregulation that characterizes intestinal inflammation. This has prompted the development of alternative biologic and small-molecule therapies targeting non-cytokine pathways implicated in intestinal inflammation, acting at distinct regulatory levels including immune cell trafficking, intracellular signalling, and lymphocyte recirculation. Here, we review emerging and approved non-cytokine-targeted therapies in IBD, focusing on three major mechanistic categories: anti-integrin agents (vedolizumab, natalizumab, etrolizumab) that selectively block leukocyte trafficking to the intestinal mucosa; Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway inhibitors (tofacitinib, upadacitinib, filgotinib) that interrupt multiple cytokine-mediated intracellular signalling cascades; and sphingosine-1-phosphate (S1P) receptor modulators (ozanimod, etrasimod) that sequester lymphocytes within lymphoid organs by functionally antagonizing S1P1 receptors. For each therapeutic class, we summarize the mechanistic rationale, clinical efficacy, safety profiles, and positioning within current treatment algorithms. Non-cytokine-based therapies represent a critical advance in the personalized management of IBD. By targeting complementary immunological mechanisms beyond cytokine inhibition, they offer new therapeutic options for patients with treatment-refractory disease and expand the arsenal for combination strategies. Furthermore, they highlight the need for precision medicine approaches guided by immune phenotyping, pharmacokinetic monitoring, and biomarker development to optimize therapeutic selection and improve long-term outcomes.

Patients with inflammatory bowel disease (IBD) frequently fail to achieve protective immunity after hepatitis B vaccination, even with intensified vaccination schedules. In this observational real-world study, 18 patients with IBD who were seronegative for hepatitis B virus (HBV) received three standard doses of the Engerix-B® vaccine (at 0, 1, and 6 months). After immunisation, patients were classified into responders and non-responders according to their serological response. Blood samples were collected before the first dose and after completion of the vaccination schedule. Responders activated pathways that supported durable protection, including conventional dendritic cells type 1 mobilisation, expansion of IgG plasmablasts, and preservation of B- and T-cell memory. In contrast, non-responders displayed a more inflammatory innate profile, characterised by enrichment of CCR2+ monocytes. They also showed higher baseline Treg frequencies, which may suppress effective effector responses, together with impaired natural killer (NK) activation and progressive loss of memory potential. This study shows that hepatitis B vaccine failure in inflammatory bowel disease reflects a convergence of excessive immune regulation, inflammatory activation, and loss of memory potential, underscoring that no single pathway can explain the impaired response.

Celiac disease (CD) is markedly underdiagnosed, with significant diagnostic delays, while long-term follow-up is often inconsistent or incomplete. Current CD care, marked by heterogeneity and limited standardization, fails to address these challenges. The Spanish Society for Celiac Disease aimed to define quality standards to support the design, implementation, and evaluation of comprehensive care units for CD. A structured consensus process was conducted using the Delphi methodology, with collaboration from the Spanish Association of Gastroenterology, the Spanish Society of Digestive Pathology, and the Spanish Society of Pediatric Gastroenterology, Hepatology and Nutrition, and patients and caregivers nominated by Spain’s three main celiac associations. This process defined 18 structural, 16 process, and 5 scientific-educational standards, providing guidance on human resources, infrastructure, diagnostic and therapeutic procedures, research, and continuous professional development. All but one standard reached a very high consensus, with a mean coefficient of variation of 9.1%. These standards provide a framework for centers of excellence in CD care, promoting multidisciplinary management and patient-centered outcomes. By integrating institutional expertise and patient perspectives, they ensure relevance, feasibility, and alignment with current clinical challenges, serving as a potential model for other healthcare systems and as a foundation for future accreditation processes.

BACKGROUND:Colorectal cancer is the third most common malignancy worldwide. Although current screening relies on faecal occult blood testing (FOBT), its sensitivity for advanced adenomas (key precursor lesions) remains limited. Indeed, colonoscopy remains the gold standard for definitive diagnosis. Hence, development of novel blood-based screening tools in FOBT-positive patients is needed to optimize colonoscopy referral. METHODS:We prospectively recruited 104 FOBT-positive patients undergoing colonoscopy. Based on endoscopic and histopathological assessment, patients were classified into no polyps (NP, n = 46), non-advanced polyps (NA, n = 33) and advanced polyps (AP, n = 25). Seventy-five immune cell subsets and their homing, activation and exhaustion profiles were characterized by spectral cytometry, yielding 900 variables. Differentially expressed variables were used to train five supervised machine learning models including random forest, decision tree, multinomial regression, polynomial kernel support vector machine (SVM) and Kernel K-nearest neighbours. RESULTS:Among 91 differentially expressed immune variables, Boruta-based feature selection identified seven key cell populations. Th1-like cells emerged as the dominant predictive variable. The decision tree model achieved the best overall performance, with total classification of AP patients (AUC=1.0, 100% sensitivity and specificity). Global model accuracy reached 75% (p < 0.01 vs. no-information rate), with a macro-AUC of 0.85. CONCLUSIONS:This study demonstrates that spectral-cytometry immunotyping of the circulating immunome, combined with machine learning, can identify patients harbouring advanced colorectal adenomas from a blood sample. Hence, Th1-like cells emerge not just as key cells, but also as a promising biomarker that could complement current FOBT screening to prioritize colonoscopy in patients at highest risk of pre-malignant lesions.

Background: Ulcerative colitis (UC) is an inflammatory disorder of the gastrointestinal tract. Although Tofacitinib, which inhibits the JAK1 and JAK3 signalling pathway, is approved to treat patients with UC, its specific mechanism of action remain elusive. Given the central role that conventional dendritic cells (cDC) elicit in gut homeostasis, we hypothesised that Tofacitinib acts modulating cDC function in UC. Methods: Human biopsies were obtained from colon of controls, and patients with UC (active and quiescent). Lamina propria mononuclear cells (LPMC) were ex-vivo cultured in the presence/absence of Tofacitinib. The specific effect elicited over human intestinal cDC, monocytes and macrophages was assessed by flow cytometry. cDC were also enriched following Tofacitinib conditioning in order to assess its effect over naïve T-cells. Results: Several human intestinal cDC, monocyte and macrophage subsets can be found in the human colon, with these cells being more similar between controls and patients with qUC referred to patients with aUC. Following ex-vivo culture, Tofacitinib downregulated JAK1 expression on intestinal monocytes from patients with both active and quiescent UC. As for macrophages, JAK1 was decreased on patients with active UC while JAK was downregulated on macrophages from patients with quiescent disease. Tofacitinib did not modulate the phenotype or function of human intestinal cDC. Conclussion: Tofacitinib does not modulate the phenotype and function of human intestinal cDC in UC. On the contrary, it displays a differential capacity to modulate intestinal monocyte and macrophage phenotype. Future studies should address whether it also translates into a differential function of these cells.

Abstract Background Crohn's disease (CD) is a chronic inflammatory condition of the gastrointestinal tract, with a globally rising incidence. It may result from a complex interaction among genetic predisposition, environmental influences, and alterations in gut microbiota, leading to dysregulation of both innate and adaptive immune responses. Despite advances in understanding CD, the precise immunological mechanisms underlying this disease remain unclear. Currently, biological therapies, such as vedolizumab, are among the most effective options for inducing clinical remission and controlling inflammation in CD. However, approximately two-thirds of patients do not respond adequately to these treatments. Consequently, it is essential to identify immune cell populations that can predict clinical responses to vedolizumab, facilitating more personalized treatment selection for each patient. Methods We characterized peripheral blood mononuclear cells (PBMCs) using spectral flow cytometry with a panel of 40 markers in patients with CD, both before and after 14 weeks of treatment with vedolizumab. Patients were categorized as responders or non-responders based on their SES-CD. Results At baseline Hierarchical phenotypic analysis revealed a total of 39 unique subsets of immune cells within PBMCs (Figure 1). The main populations included T cells (66%), monocytes (0.25%), B cells (12%), and natural killer cells (11%). Within the T cell group, CD4+ cells were twice as prevalent as CD8+ cells. In our evaluation of conventional dendritic cells percentages, we noted an increase in this subset within the responder group, contrasting with the trend observed in macrophages. Furthermore, an unsupervised analysis applying dimensionality reduction algorithms identified 59 clusters (Figure 2), with 37 of them showing differences when comparing responder and non-responder patients. Interestingly, no differences were observed between pre-treatment and post-treatment samples. Conclusion This study presents the first immunophenotyping analysis in CD patients, comparing responders and non-responders to vedolizumab based on SES-CD and highlighting differences between these groups. For the first time, these results reveal circulating immune populations as potential predictors of response to vedolizumab. Although these findings should be validated in a larger cohort to assess their clinical significance in identifying patients most likely to benefit from each therapeutic approach, they open the possibility for changes in clinical practice.

Long-term consequences of SARS-CoV-2 infection are unknown since recovered individuals can experience symptoms and latent viral reactivation for months. Indeed, acute post-infection sequelae have also been observed in other respiratory viral infections, including influenza. To characterize post-COVID-19 and post-influenza induced alterations to the cellular immunome, peripheral blood mononuclear cells (PBMCs) were obtained from patients 3 months after recovery from COVID-19 (n = 93) or influenza (n = 25), and from pre-pandemic healthy controls (n = 25). PBMCs were characterized using a 40-plex mass cytometry panel. Principal component analysis (PCA), classification models, and K-means clustering were subsequently applied. PCA identified distinct immune profiles between cohorts, with both post-COVID and post-flu patients displaying an altered chemokine receptor expression compared to pre-pandemic healthy controls. These alterations were more prominent in post-COVID patients since they exhibited highly increased expression of chemokine receptors CXCR3 and CCR6 by various lymphoid populations, while post-influenza patients mainly showed a decrease in CCR4 expression by naïve T cells, monocytes, and conventional dendritic cells. Classification models using immunophenotyping data confirm the three groups, while K-means clustering revealed two subgroups among post-COVID patients, with younger patients showing more pronounced immune alterations in the chemokine receptor profile, independently of long COVID symptoms. In conclusion, post-COVID and post-influenza patients exhibit distinct and unique persistent immune alterations. Understanding these altered immune profiles can guide targeted therapies for post-COVID syndrome and highlight differences in immune recovery from various respiratory infections.

In the past decade, a major goal in biomedical research has been to understand why individuals differ in disease susceptibility, disease dynamics, and progression. In many pathologies, this variability stems from evolved immune mechanisms that resist inflammatory stress from various diseases that have been encountered throughout life. These may provide advantages against other diseases, reduce comorbidities, and enhance longevity. This study evaluates prior immunity as a prognostic factor in COVID-19 patients, crucial for understanding plasmatic signaling cascades in different disease stages and their impact on disease progression. COVID-19, caused by SARS-CoV-2, primarily affects the respiratory system and presents a wide range of symptoms, posing significant challenges to medicine. This study systematically analyzed prior immunity and inflammation in two independent cohorts of infected patients. A serological profile is determined by protein microarrays, which identify IgM and IgG responses against 37 prevalent microbial pathogens and provide a comprehensive plasma analysis of 21 acute-phase proteins. Our results reveal distinct serological profiles correlating with disease severity, indicating that immune system dysregulation in COVID-19 patients is linked to existing immunity. These findings highlight the relevance of prior immunity for monitoring disease progression, particularly in infections and vaccine failure, and underscore the importance of functional proteomics in determining prognostic biomarkers.

Abstract Background Human intestinal conventional dendritic cells (cDC) maintain the mechanisms of immune tolerance in health. However, their specific contribution to the development of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn´s disease (CD) remains elusive Methods Human intestinal cDC were identified by flow cytometry in the human intestinal mucosal, and divided into CD103- cDC2, CD103+ cDC2 and cDC1. Their phenotype was determined in health and IBD, together with their stimulatory capacity over naïve T-cells. Results cDC1 (minority) and cDC2 (majority) did not change their proportion throughout the healthy gut. However, CD103+ cDC2 were the main subset in the duodenum as opposed to CD103- cDC2 which were predominant in the colon and ileum. CD103+ cDC2 displayed higher levels of PD-L1 together with an enhanced production of IL-10. CD103+ cDC2 also increased their proportion following culture, referred to their CD103- cDC2 precursors, being this process abrogated in the presence of LPS. All human intestinal cDC subsets primed the generation of IL-10+ helper T-cells, being ileal cDC more stimulatory than their colonic counterparts. In IBD, cDC had a constitutive lower expression of SIRPα irrespectively of IBD type (CD/UC) or condition (inflamed/not-inflamed). Of note, the inflamed colon from UC patients, but not from CD, displayed a specific reduction of both cDC1 and CD103+ cDC2, while colonic cDC from CD patients were more stimulatory and primed IL-17+ T-cells in the inflamed tissue. Conclusion All human intestinal cDC subsets prime the generation of IL-10+ helper T-cells. The specific reduction of cDC1 and CD103+ cDC2 in the inflamed mucosa from UC, but not CD, suggests the presence of different pathogenic mechanisms occurring in IBD.

The clinical manifestation of COVID-19 after SARS-CoV-2 infection varies greatly, with many patients requiring intensive care due to complications like acute respiratory distress syndrome, reduced respiratory system compliance, and altered iron metabolism, which can be mistaken for worsening Influenza A infection. This highlights the need to study the humoral immune response to better understand the pathophysiology of viral respiratory infections and improve treatments and diagnostics. This study analyzed autoantibody and acute-phase reactant profiles in patients infected with SARS-CoV-2 and Influenza A using customized protein microarrays for sensitive and reproducible results. The findings revealed a significant increase in autoantibodies in SARS-CoV-2 patients, including those targeting calcitonin-related polypeptides, hepatocyte growth factor, or interleukin 8, compared to Influenza A patients, who showed elevated levels of selectin E and surfactant protein D. Additionally, most acute-phase reactants were higher in SARS-CoV-2 patients. The serological profile showed that the wild-type SARS-CoV-2 strain induced both IgM and IgG responses to all viral proteins, while other strains triggered an IgG response only at a later stage. Multiomics factor analysis identified key factors driving variation in COVID-19's heterogeneous disease presentation. These insights offer valuable information for developing vaccines and therapeutic strategies against SARS-CoV-2.

Inflammatory bowel disease is associated with complex changes in the intestinal microbiota. While bacterial dysbiosis has been widely studied, the role of fungal communities and their interaction with bacteria remains less explored. This study aims to characterize both bacterial and fungal populations in patients with Crohn's disease and ulcerative colitis, identifying potential microbial biomarkers and inter-kingdom interactions related to disease activity. We analysed paired intestinal tissue and faecal samples from patients with inflammatory bowel disease. Bacterial, fungal and viral composition were assessed by 16S, ITS DNA and shotgun sequencing, respectively. Bioinformatics analyses were done using Qiime2 and R studio platforms. PERMANOVA and ANOSIM analyses revealed significant compositional differences between faecal and mucosal samples in most groups. Several genera were consistently shared across tissues, while others showed tissue- or disease-specific distributions. Exploratory correlations between bacteria and fungi revealed that Wallemia could play a role in balancing the presence of potentially beneficial and pathogenic bacteria. Importantly, Prevotella was associated with active disease, Fusobacterium with active Crohn's disease, and Roseburia with remission in ulcerative colitis, supporting their potential as biomarkers. All these hypotheses should be further tested in future studies. Our findings reveal distinct microbial signatures in inflammatory bowel disease that could promote the intestinal dysbiosis hypotheses perpetuate chronic intestinal inflammation. The identification of faecal biomarkers may complement approaches for noninvasive monitoring of disease activity. These results underscore the need for further research into bacteria-fungi interactions and their role in gut inflammation.

The high prevalence of disorders of the gut-brain interaction (DGBI), the availability of breath tests for the diagnosis of small intestine bacterial overgrowth (SIBO) together with some confusion about the concept of SIBO are leading to an increase in the number of SIBO diagnoses. We aimed to analyze the factors associated with the severity of gastrointestinal symptoms in patients undergoing a SIBO breath test. A cross-sectional observational study including 70 patients who underwent a SIBO test with lactitol and completed questionnaires including the ROME IV criteria for Irritable Bowel Syndrome (IBS), the Irritable Bowel Syndrome Severity Score (IBSSS), and the HAD anxiety and depression scales. Additionally, blood levels of histamine, citrulline, ghrelin, Intestinal-Fatty Acid Binding Protein (I-FABP) and Transient Receptor Potential Cation Channel Subfamily V, Member 1 (TRPV-1) were measured. The mean age was 45±16 years and 70% were women. Abdominal pain and/or abdominal distension were present in 85% of patients. 44% met IBS Rome IV criteria. IBSSS total score had a correlation with age (-0.354, p<0.001), HAD-A (0.391, p<0.001) and HAD-D (0.409, p<0.001) scores, and histamine levels (0.279, p=0.019). Abdominal pain correlated with levels of histamine (0.320, p<0.05; 0.282, p<0.05) and ghrelin (0.252, p<0.05, 0.347, p<0.05), while abdominal distension correlated with I-FABP levels (0.314, p<0.05). The SIBO test was positive in 75% but did not correlate with symptom severity. We hereby unveiled some factors associated with the severity of abdominal pain and distension such as age, auto-perceived anxiety and depression and some biomarkers but not SIBO test result.

The proteomic analysis of serum extracellular vesicles (EVs) could be a useful tool for studying the pathophysiology of Crohn’s disease (CD) and ulcerative colitis (UC), as well as for biomarker discovery. To characterize the proteomic composition of serum EVs in patients with CD and UC to identify biomarkers and molecular pathways associated with pathogenesis and activity. Methods: Serum EVs were enriched and analyzed in patients with quiescent CD, active CD (aCD), quiescent UC, active UC (aUC), and healthy controls (HCs) (n = 30 per group). All groups were matched for age and sex. Disease activity was assessed by ileocolonoscopy and categorized based on the SES-CD (CD) and the endoscopic sub-score of the Mayo Score (UC). EVs were enriched by ul-tracentrifugation, and their size and concentration were determined by nanoparticle tracking analysis. The expression of CD63, CD81, and CD9 was determined using West-ern blotting. Proteomic analysis was performed by label-free nano-LC MS/MS. A total of 324 proteins were identified; 60 showed differential abundance in CD-HC, 34 in UC-HC, and 21 in CD-UC. Regarding disease activity, the abundance of 58 and 32 proteins was altered in aCD-HC and aUC-HC, respectively. Functional analyses revealed that proteins associated with aCD were involved in immune regulation, whereas those linked to aUC were enriched in oxidative stress. We have identified expressed proteins between EVs from patients with CD and UC, depending on the presence of disease, the disease type, and the disease activity. These proteins are potential candidates as disease biomarkers and open new research avenues to better understand these conditions.

INTRODUCTION:Although celiac disease (CD) current and only treatment is a life-long strict gluten-free diet (GFD), some patients suffer from persistent duodenal lesions despite years into the diet. Hence, we aimed to study the effect that the GFD elicits on the mucosal immune infiltrate from these patients. METHOD:To that end, duodenal biopsies were collected from non-celiac controls and CD patients, both at diagnosis and after at least one year into the GFD. The profile of duodenal intraepithelial lymphocytes (lymphogram) and the lamina propria immune infiltrate were determined by spectral cytometry. RESULTS:At diagnosis, all CD patients had mucosal atrophy, a compatible lymphogram, and an expansion of lamina propria NK cells, innate lymphoid cells, B-cells, Treg and Tγδ cells, all of them expressing high levels of Fas, and Integrins α4 and β7. However, despite all GFD-treated patients had negative serology, 68.4% of them displayed persistent villous atrophy (Marsh score ≥ 3), while 73.3% had a compatible lymphogram. Nevertheless, despite such persistent atrophy, the lamina propria mucosal immune infiltrate was normalized in all GFD-treated patients. Besides, time on the GFD, but not the persistence of mucosal atrophy, correlated with an increased expression of gut-homing migration markers on lamina propria effector T-cells from these patients. CONCLUSION:Hence, we hereby have proved how the lamina propria immune infiltrate, as opposed to intraepithelial lymphocytes, is normalized in GFD-treated CD patients despite persistent villous atrophy, suggesting that the epithelial layer may be the driver of such paradoxical persistent mucosal inflammation.