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Intestinal ischemia-reperfusion injury (IR) is implicated in various clinical conditions and causes damage to the intestinal epithelium resulting in intestinal barrier loss. This presents a substantial clinical challenge, emphasizing the importance of gaining a comprehensive understanding of molecular events to aid in the identification of novel therapeutic targets. This review systematically explores the extent to which omics technologies-transcriptomics, proteomics, metabolomics, and metagenomics-have already contributed to deciphering the molecular mechanisms contributing to intestinal IR injury, in in vivo and in vitro animal and human models, and in clinical samples. Recent breakthroughs involve applying omics methodologies on exosomes, organoids, and single cells, shedding light on promising avenues and valuable targets to reduce intestinal IR injury. Future directions aimed at expediting clinical translation are discussed as well and include multi-omics data integration to facilitate the identification of key regulatory nodes driving intestinal IR injury and advancing human organoid models based on the novel insights by single-cell omics technologies, offering hope for clinical application of therapeutic strategies in the years to come., (© 2024 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

Introduction: Diagnosing non-occlusive mesenteric ischaemia (NOMI) in patients is complicated, due to poor signs and symptoms and non-specific laboratory tests, leading to a high mortality rate. This case study presents the rare case of a patient who developed mesenteric ischaemia after an emergency thoracic endovascular aneurysm repair (TEVAR) for a type B aortic dissection (TBAD) and peri-operative cardiogenic shock. Study outcomes revealed that intestinal fatty acid binding protein (I-FABP) identified early mucosal damage two days before the clinical presentation., Report: A 43 year old male patient was admitted to the emergency department with an acute TBAD and a dissection of the superior mesenteric artery (SMA), for which TEVAR was performed with additional stent placement in the SMA. Peri-operatively, the patient went into cardiogenic shock with a sustained period of hypotension. Post-operatively, the plasma I-FABP levels were measured prospectively, revealing an initial increase on post-operative day five (551.1 pg/mL), which continued beyond day six (610.3 pg/mL). On post-operative day seven, the patient developed a fever and demonstrated signs of peritonitis and bowel perforation. He underwent an emergency laparotomy, followed by an ileocaecal resection (<100 cm) with a transverse ileostomy. Pathological analysis confirmed the diagnosis of mesenteric ischaemia., Discussion: The diagnosis of NOMI in critically ill patients is often complicated, and the currently available diagnostic markers lack the specificity and sensitivity to detect early intestinal injury. This case report highlights that elevated I-FABP in plasma levels may indicate the presence of early mesenteric injury. Further research needs to be conducted before I-FABP can be applied in daily practice., (© 2024 The Authors.)

Local intraperitoneal drug administration is considered a challenging drug delivery route. The therapeutic efficiency is low, mainly due to rapid clearance of drugs. To increase the intraperitoneal retention time of specific drugs, a pH-sensitive supramolecular hydrogel that can act as a drug delivery vehicle is developed. To establish the optimal formulation of the hydrogel and to study its feasibility, safety, and tissue compatibility, in vitro, postmortem, and in vivo experiments are performed. In vitro tests reveal that a hydrogelator formulation with pH ≥ 9 results in a constant viscosity of 0.1 Pa·s. After administration postmortem, the hydrogel covers the parietal and visceral peritoneum with a thin, soft layer. In the subsequent in vivo experiments, 14 healthy rats are subjected to intraperitoneal injection with the hydrogel. Fourteen and 28 days after implantation, the animals are euthanized. Intraperitoneal exposure to the hydrogel is not resulted in significant weight loss or discomfort. Moreover, no macroscopic adverse effects or signs of organ damage are detected. In several intra-abdominal tissues, vacuolated macrophages are found indicating a physiological degradation of the synthetic hydrogel. This study demonstrates that the supramolecular hydrogel is safe for intraperitoneal application and that the hydrogel shows good tissue compatibility in rats., (© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H] - ( m / z 742.5) and PI 38:4 [M - H] - ( m / z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H] - ( m / z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

The prognosis of colorectal cancer patients with peritoneal metastases is very poor. Intraperitoneal drug delivery systems, like supramolecular hydrogels, are being developed to improve local delivery and intraperitoneal residence time of a cytostatic such as mitomycin C (MMC). In this study, we evaluate the effect of intraperitoneal hydrogel administration on anastomotic healing. Forty-two healthy Wistar rats received a colonic end-to-end anastomosis, after which 6 animals received an intraperitoneal injection with saline, 18 with unloaded hydrogel and 18 with MMC-loaded hydrogel. After 7 days, animals were euthanized, and the anastomotic adhesion and leakage score were measured as primary outcome. Secondary outcomes were bursting pressure, histological anastomosis evaluation and body weight changes. Twenty-two rats completed the follow-up period (saline: n = 6, unloaded hydrogel: n = 10, MMC-loaded hydrogel: n = 6) and were included in the analysis. A trend towards significance was found for anastomotic leakage score between the rats receiving saline and unloaded hydrogel after multiple-comparison correction ( p = 0.020, α = 0.0167). No significant differences were found for all other outcomes. The main reason for drop-out in this study was intestinal blood loss. Although the preliminary results suggest that MMC-loaded or unloaded hydrogel does not influence anastomotic healing, the intestinal blood loss observed in a considerable number of animals receiving unloaded and MMC-loaded hydrogel implies that the injection of the hydrogel under the studied conditions is not safe in the current rodent model and warrants further optimalisation of the hydrogel.

Important considerations in the choice of future sustainable protein sources for human application are tolerance, nutritional quality, and potential health benefits. We evaluated, in a double-blind cross-over intervention trial, tolerance, nutritional quality, and potential health effects of two sustainable protein sources. Thirty-six apparently healthy older adults (age 62.3 ± 7.2yrs, BMI 25 ± 3 kg/m2) received 40 g/day bovine-plasma protein (BP), corn protein (CP) or, as a benchmark, whey protein (WP) for one week with a washout period of one week in-between. In 12 participants, we also determined postprandial amino acid (PAA) uptake kinetics upon consumption of 20 g BP, CP, or WP. Changes in self-reported gastrointestinal complaints and intestinal permeability assessed using a multi-sugar acetylsalicylic acid test did not differ between the interventions. Clear differences in PAA responses were observed after consumption of the different proteins, but clear essential amino acid responses were observed for all proteins. BP consumption resulted in a small but significant increase in blood pressure outcomes, and CP consumption resulted in a small but significant decrease in insulin levels when compared to the other interventions. In conclusion, alternative protein concentrates and isolates studied here can be consumed in relative high quantities without experiencing unwanted GI complaints or gut barrier dysfunction and they can be a good source of essential amino acids. The rise in blood pressure observed during the BP intervention, potentially linked to the elevated salt content of the BP, constitutes a potential health issue. Future studies with longer intervention periods might however be recommended., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (© 2023 The Authors.)

Introduction: Acute mesenteric ischaemia (AMI) is a life-threatening condition with short-term mortality of up to 80%. The diagnosis of AMI has remained troublesome due to the non-specific clinical presentation, symptoms and laboratory findings. Early unambiguous diagnosis of AMI is critical to prevent progression from reversible to irreversible transmural intestinal damage, thereby decreasing morbidity and improving survival. The present study aims to validate a panel of plasma biomarkers and investigate volatile organic compound (VOC) profiles in exhaled air as a tool to timely and accurately diagnose AMI., Methods and Analysis: In this international multicentre prospective observational study, 120 patients (>18 years of age) will be recruited with clinical suspicion of AMI. Clinical suspicion is based on: (1) clinical manifestation, (2) physical examination, (3) laboratory measurements and (4) the physician's consideration to perform a CT scan. The patient's characteristics, repetitive blood samples and exhaled air will be prospectively collected. Plasma levels of mucosal damage markers intestinal fatty acid-binding protein and villin-1, as well as transmural damage marker smooth muscle protein 22-alpha, will be assessed by ELISA. Analysis of VOCs in exhaled air will be performed by gas chromatography time-of-flight mass spectrometry. Diagnosis of AMI will be based on CT, endovascular and surgical reports, clinical findings, and (if applicable) verified by histopathological examination., Ethics and Dissemination: The study protocol was approved by the Medical Research Ethics Committee (METC) of Maastricht University Medical Centre+ and Maastricht University (METC azM/UM), the Netherlands (METC19-010) and the Ethics Committee Research UZ/KU Leuven, Belgium (S63500). Executive boards and local METCs of other Dutch participating centres Gelre Ziekenhuizen (Apeldoorn), Medisch Spectrum Twente (Enschede), and University Medical Centre Groningen have granted permission to carry out this study. Study results will be disseminated via open-access peer-reviewed scientific journals and national/international conferences., Trial Registration Number: NCT05194527., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Patients with peritoneal metastases (PM) of colorectal cancer have a very poor outcome. Intraperitoneal delivery of chemotherapy is the preferred route for PM treatment. The main limitation of the treatment options is the short residence time of the cytostatic, with subsequent short exposure of the cancer cells. To address this, a supramolecular hydrogel has been developed that allows both local and slow release of its encapsulated drug, mitomycin C (MMC) or cholesterol-conjugated MMC (cMMC), respectively. This experimental study investigates if drug delivery using this hydrogel improves the therapeutic efficacy against PM. PM was induced in WAG/Rij rats (n = 72) by intraperitoneally injecting syngeneic colon carcinoma cells (CC531) expressing luciferase. After seven days, animals received a single intraperitoneal injection with saline (n = 8), unloaded hydrogel (n = 12), free MMC (n = 13), free cMMC (n = 13), MMC-loaded hydrogel (n = 13), or cMMC-loaded hydrogel (n = 13). Primary outcome was overall survival with a maximum follow-up of 120 days. Intraperitoneal tumor development was non-invasive monitored via bioluminescence imaging. Sixty-one rats successfully underwent all study procedures and were included to assess therapeutic efficacy. After 120 days, the overall survival in the MMC-loaded hydrogel and free MMC group was 78% and 38%, respectively. A trend toward significance was found when comparing the survival curves of the MMC-loaded hydrogel and free MMC (p = 0.087). No survival benefit was found for the cMMC-loaded hydrogel compared to free cMMC. Treating PM with our MMC-loaded hydrogel, exhibiting prolonged MMC exposure, seems effective in improving survival compared to treatment with free MMC., (© 2023. The Author(s).)

Background: Cancer cachexia is a multifactorial syndrome characterized by body weight loss and systemic inflammation. The characterization of the inflammatory response in patients with cachexia is still limited. Lipocalin-2, a protein abundant in neutrophils, has recently been implicated in appetite suppression in preclinical models of pancreatic cancer cachexia. We hypothesized that lipocalin-2 levels could be associated with neutrophil activation and nutritional status of pancreatic ductal adenocarcinoma (PDAC) patients., Methods: Plasma levels of neutrophil activation markers calprotectin, myeloperoxidase, elastase, and bactericidal/permeability-increasing protein (BPI) were compared between non-cachectic PDAC patients (n=13) and cachectic PDAC patients with high (≥26.9 ng/mL, n =34) or low (<26.9 ng/mL, n =34) circulating lipocalin-2 levels. Patients' nutritional status was assessed by the patient-generated subjective global assessment (PG-SGA) and through body composition analysis using CT-scan slices at the L3 level., Results: Circulating lipocalin-2 levels did not differ between cachectic and non-cachectic PDAC patients (median 26.7 (IQR 19.7-34.8) vs . 24.8 (16.6-29.4) ng/mL, p =0.141). Cachectic patients with high systemic lipocalin-2 levels had higher concentrations of calprotectin, myeloperoxidase, and elastase than non-cachectic patients or cachectic patients with low lipocalin-2 levels (calprotectin: 542.3 (355.8-724.9) vs . 457.5 (213.3-606.9), p =0.448 vs . 366.5 (294.5-478.5) ng/mL, p =0.009; myeloperoxidase: 30.3 (22.1-37.9) vs . 16.3 (12.0-27.5), p =0.021 vs . 20.2 (15.0-29.2) ng/mL, p =0.011; elastase: 137.1 (90.8-253.2) vs . 97.2 (28.8-215.7), p =0.410 vs . 95.0 (72.2-113.6) ng/mL, p =0.006; respectively). The CRP/albumin ratio was also higher in cachectic patients with high lipocalin-2 levels (2.3 (1.3-6.0) as compared to non-cachectic patients (1.0 (0.7-4.2), p =0.041). Lipocalin-2 concentrations correlated with those of calprotectin ( r s =0.36, p <0.001), myeloperoxidase ( r s =0.48, p <0.001), elastase ( r s =0.50, p <0.001), and BPI ( r s =0.22, p =0.048). Whereas no significant correlations with weight loss, BMI, or L3 skeletal muscle index were observed, lipocalin-2 concentrations were associated with subcutaneous adipose tissue index ( r s =-0.25, p =0.034). Moreover, lipocalin-2 tended to be elevated in severely malnourished patients compared with well-nourished patients (27.2 (20.3-37.2) vs . 19.9 (13.4-26.4) ng/mL, p =0.058)., Conclusions: These data suggest that lipocalin-2 levels are associated with neutrophil activation in patients with pancreatic cancer cachexia and that it may contribute to their poor nutritional status., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Deng, Aberle, van Bijnen, van der Kroft, Lenaerts, Neumann, Wiltberger, Schaap, Olde Damink and Rensen.)

Background: A dysregulated 'gut-kidney axis' may contribute to immunoglobulin A nephropathy (IgAN). We studied whether IgAN patients have disturbed intestinal permeability., Methods: In a prospective, cross sectional, pilot study we assessed intestinal permeability in 35 IgAN patients, 18 patients with non-IgAN glomerulonephritides (GNs) and 19 healthy controls. After an overnight fast, trial participants ingested a multi-sugar solution and samples were obtained from 0 to 2, 2 to 5- and 5 to 24-h urine portions. Urinary sugar concentrations were quantified using isocratic ion-exchange high performance liquid chromatography. Indices of small intestinal permeability (0-2-h lactulose/L-rhamnose (L/R) ratio), distal small intestinal and proximal colonic permeability (2-5-h sucralose/erythritol (S/E) ratio) and colonic permeability (5-24-h sucralose/erythritol (S/E) ratio) were evaluated. Associations between groups and indices of intestinal permeability were investigated by a linear mixed model., Results: Small intestinal permeability (0-2 h L/R-ratio) was significantly increased in patients with glomerular diseases versus healthy controls. More precisely, increased small intestinal permeability was exclusively noted in non-IgAN GN patients, whereas IgAN patients exhibited a trend towards elevated small intestinal permeability. In total, 54% of patients with IgAN and 67% of non-IgAN GN patients had increased small intestinal permeability. Neither distal small intestinal and proximal colonic permeability nor colonic gut permeability indices (i.e., 2-5 h and 5-24 h S/E ratios) were significantly different between controls and any of the GN patient groups., Conclusion: The present single center pilot study suggests that disturbed intestinal permeability is common in patients with glomerular diseases and is not specific for IgAN., Trial Registration Number: German Clinical Trials Register DRKS00021533, Date: 24.04.2020., (© 2022. The Author(s).)

For peritoneal metastases (PM), there are few curative treatment options, and they are only available for a select patient group. Recently, new therapies have been developed to deliver intraperitoneal chemotherapy for a prolonged period, suitable for a larger patient group. These drug delivery systems (DDSs) seem promising in the experimental setting. Many types of DDSs have been explored in a variety of animal models, using different cytostatics. This review aimed to provide an overview of animal studies using DDSs containing cytostatics for the treatment of gastro-intestinal PM and identify the most promising therapeutic combinations. The review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) guidelines. The 35 studies included revealed similar results: using a cytostatic-loaded DDS to treat PM resulted in a higher median survival time (MST) and a lower intraperitoneal tumor load compared to no treatment or treatment with a 'free' cytostatic or an unloaded DDS. In 65% of the studies, the MST was significantly longer and in 24% the tumor load was significantly lower in the animals treated with cytostatic-loaded DDS. The large variety of experimental setups made it impossible to identify the most promising DDS-cytostatic combination. In most studies, the risk of bias was unclear due to poor reporting. Future studies should focus more on improving the clinical relevance of the experiments, standardizing the experimental study setup, and improving their methodological quality and reporting., (© 2022. The Author(s).)

Intestinal helminths are highly prevalent in low-SES children and could contribute to poor health outcomes either directly or via alteration of the gut microbiome and gut barrier function. We analysed parasitic infections and gut microbiota composition in 325 children attending high- and low-SES schools in Makassar, Indonesia before and after albendazole treatment. Lactulose/Mannitol Ratio (LMR, a marker of gut permeability); I-FABP (a surrogate marker of intestinal damage) as well as inflammatory markers (LBP) were measured. Helminth infections were highly prevalent (65.6%) in low-SES children. LMR and I-FABP levels were higher in low-SES children (geomean (95%CI): 4.03 (3.67-4.42) vs. 3.22 (2.91-3.57); p. adj < 0.001; and 1.57 (1.42-1.74) vs. 1.25 (1.13-1.38); p. adj = 0.02, respectively) while LBP levels were lower compared to the high-SES (19.39 (17.09-22.01) vs. 22.74 (20.07-26.12); p.adj = 0.01). Albendazole reduced helminth infections in low-SES and also decreased LMR with 11% reduction but only in helminth-uninfected children (estimated treatment effect: 0.89; p.adj = 0.01). Following treatment, I-FABP decreased in high- (0.91, p.adj < 0.001) but increased (1.12, p.adj = 0.004) in low-SES children. Albendazole did not alter the levels of LBP. Microbiota analysis showed no contribution from specific bacterial-taxa to the changes observed. Intestinal permeability and epithelial damage are higher while peripheral blood inflammatory marker is lower in children of low-SES in Indonesia. Furthermore, treatment decreased LMR in helminth-uninfected only., (© 2022. The Author(s).)

Intestinal ischemia-reperfusion (IR) injury is a severe clinical condition, and unraveling its pathophysiology is crucial to improve therapeutic strategies and reduce the high morbidity and mortality rates. Here, we studied the dynamic proteome and phosphoproteome in the human intestine during ischemia and reperfusion, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to gain quantitative information of thousands of proteins and phosphorylation sites, as well as mass spectrometry imaging (MSI) to obtain spatial information. We identified a significant decrease in abundance of proteins related to intestinal absorption, microvillus, and cell junction, whereas proteins involved in innate immunity, in particular the complement cascade, and extracellular matrix organization increased in abundance after IR. Differentially phosphorylated proteins were involved in RNA splicing events and cytoskeletal and cell junction organization. In addition, our analysis points to mitogen-activated protein kinase (MAPK) and cyclin-dependent kinase (CDK) families to be active kinases during IR. Finally, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MSI presented peptide alterations in abundance and distribution, which resulted, in combination with Fourier-transform ion cyclotron resonance (FTICR) MSI and LC-MS/MS, in the annotation of proteins related to RNA splicing, the complement cascade, and extracellular matrix organization. This study expanded our understanding of the molecular changes that occur during IR in the human intestine and highlights the value of the complementary use of different MS-based methodologies.

Background & Aims: Intestinal ischemia-reperfusion injury is a serious and life-threatening condition. A better understanding of molecular mechanisms related to intestinal ischemia-reperfusion injury in human beings is imperative to find therapeutic targets and improve patient outcome., Methods: First, the in vivo dynamic modulation of mucosal gene expression of the ischemia-reperfusion-injured human small intestine was studied. Based on functional enrichment analysis of the changing transcriptome, one of the predominantly regulated pathways was selected for further investigation in an in vitro human intestinal organoid model., Results: Ischemia-reperfusion massively changed the transcriptional landscape of the human small intestine. Functional enrichment analysis based on gene ontology and pathways pointed to the response to unfolded protein as a predominantly regulated process. In addition, regulatory network analysis identified hypoxia-inducing factor 1A as one of the key mediators of ischemia-reperfusion-induced changes, including the unfolded protein response (UPR). Differential expression of genes involved in the UPR was confirmed using quantitative polymerase chain reaction analysis. Electron microscopy showed signs of endoplasmic reticulum stress. Collectively, these findings point to a critical role for unfolded protein stress in intestinal ischemia-reperfusion injury in human beings. In a human intestinal organoid model exposed to hypoxia-reoxygenation, attenuation of UPR activation with integrated stress response inhibitor strongly reduced pro-apoptotic activating transcription factor 4 (ATF4)-CCAAT/enhancer-binding protein homologous protein (CHOP) signaling., Conclusions: Transcriptome analysis showed a crucial role for unfolded protein stress in the response to ischemia-reperfusion in human small intestine. UPR inhibition during hypoxia-reoxygenation in an intestinal organoid model suggests that downstream protein kinase R-like ER kinase (PERK) signaling may be a promising target to reduce intestinal ischemia-reperfusion injury. Microarray data are available in GEO (https://www.ncbi.nlm.nih.gov/gds, accession number GSE37013)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We explored whether metabolic health is linked to intestinal permeability, using a multi-sugar (MS) permeability test, and whether intestinal permeability is correlated with the leaky gut-related markers (LGM) zonulin, LBP, and sCD14. Metabolically healthy ( n = 15) and unhealthy subjects ( n = 15) were recruited based on waist circumference, fasting glucose, and high-density lipoprotein cholesterol levels. Participants underwent an MS permeability test that assessed site-specific permeabilities of the gastroduodenum and small and large intestines. The test was performed with/without an acetylsalicylic acid challenge to measure and correlate the gut permeability, LGM, and metabolic health. At baseline, metabolic health showed no correlation with gut permeability. Significant correlations were found between the metabolic health parameters and LGM. In the acetylsalicylic acid challenged MS permeability test, low-density lipoprotein cholesterol was correlated with the sucralose/erythritol ratio, reflecting the whole intestinal permeability. Correlations between most metabolic health parameters and LGM during the acetylsalicylic acid challenge were less pronounced than at baseline. In both MS permeability tests, no significant correlations were found between LGM (plasma and serum) and gut permeability. Thus, correlations between LGM and metabolic health might not be linked with paracellular gut permeability. Transcellular translocation and/or lipoprotein-related transportation is a more likely mechanism underlying the association between LGM and metabolic health.