Intestinal Endothelium Research Articles

P073 Enhancing the MFSD2A protein for the treatment of colitis-associated cancer: novel insights for pathogenesis and treatment

Abstract Background The intrinsic connection between inflammation and tumor promotion is well characterized and is a key pathogenic event in patients with colorectal cancer (CRC). A small fraction of patients with CRC suffers from genetic predisposition. However chronic inflammation, such as ulcerative colitis (UC), increases the risk of intestinal carcinogenesis, thus strengthening the connection between these conditions. A few years ago, our laboratory described that the intestinal endothelium isolated from actively inflamed UC patients displayed defective production of inflammation-resolving DHA-derived metabolites, by comparison with healthy controls and tissues in remission. We also reported that the Major Facilitator Superfamily Domain containing 2A (MFSD2A) participated in the production of the inflammation-resolving DHA-derived metabolites by the intestinal endothelium of patients with UC in remission, ameliorating colonic inflammation. Therefore, we hypothesized that the endothelial MFSD2A also has a role in preventing and/or counteracting cancer-associated inflammation. Methods Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with either MFSD2A protein-encoding lentiviral vectors (MFSD2A) or GFP as control, or MFSD2A targeting shRNA or its scramble, and were analyzed by lipidomics. Furthermore, Caco-2 cells co-cultured with HIMEC-MFSD2A were analyzed by FACS, evaluating their proliferation. Moreover, an orthotopic model of CRC was made intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and HUVEC overexpressing MFSD2A or GFP as control. Mice were gavaged with DHA-ethyl-ester or PBS. FACS analysis was performed on tumor and colonic samples. Results Lipidomic analysis revealed that the loss of MFSD2A in CRC is detrimental because there is a reduced production of pro-resolving lipids confirming that, as for UC, MFSD2A is important for the maintenance of an adequate balance between pro-resolving and pro-inflammatory phenotype. Moreover, enhancing MFSD2A expression at endothelial level limits Caco-2 cell proliferation, supporting its beneficial role. In addition, DHA administration in mice ameliorated clinical symptoms of inflammation upon intestinal endothelial MFSD2A overexpression, supporting the beneficial role of MFSD2A in vivo. Conclusion Tumor-associated inflammation remains a crucial hallmark of CRC. Our study seeks to identify the role of MFSD2A in the resolution phase of inflammation, pointing out alternative therapies for CRC treatment. These data suggested that MFSD2A might contrast the tumor-associated inflammation and ensure the correct balance between pro-inflammatory and pro-resolving milieu.

P655 MadCAM1 Negativity of Lamina Propria Endothelial Cells is Associated with Non-Response to Vedolizumab in Inflammatory Bowel Disease Patients

Abstract Background Vedolizumab (VDZ), a monoclonal antibody against α4β7 integrin has been shown to be effective in inducing and maintaining remission in inflammatory bowel disease (IBD). By blocking α4β7, it is preventing the homing of lymphocytes through binding to mucosal vascular adressin cell adhesion molecule 1 (MadCAM1) localised on the intestinal endothelial cells. We have previously reported that MadCAM1 is expressed to various extent on lamina propria endothelial cells of IBD patients and that this expression is stable over time. The aim of the present study was to analyse whether expression of MadCAM1 was related to the response to VDZ in IBD patients. Methods All IBD patients treated with VDZ in one referral IBD centre were included. The biopsies or resection specimen from the inflamed intestinal tissue were stained by immunohistochemistry for MadCAM1 expression. Clinical response to vedolizumab was assessed in patients with the minimal treatment duration of 10 weeks and the response rate between patients with positive and negative MadCAM1 were analyzed by Fisher`s exact test. Results In total, 92 IBD patients were treated with VDZ; for 83 of them intestinal biopsies or surgical specimen were available for assessment of MadCAM1 expression. In four patients (5%) no expression of MadCAM1 was detected, remaining 79 patients expressed MadCAM1 to various extent. Overal, 30 patients (36%) were non-responders to VDZ treatment. With regards to MadCAM1 expression, 67% of patients expressing MadCAM1 responded to VDZ (53 patients) while none of the patients negative for MadCAM1 responded to treatment (p=0.0171). Conclusion Lack of MadCAM1 expression in intestinal endothelium is associated with clinical non-response to vedolizumab.

ENTERAL NUTRITION IN PATIENTS WITH SEVERE ACUTE PANCREATITIS

Objective: Acute pancreatitis is a common disease that accounts for 5-10% of urgent pathology of the abdominal cavity and ranks third (25%) place, yielding to the incidence of acute cholecystitis (28%) and acute appendicitis (26%) [1]. In the general structure of the disease, severe acute pancreatitis occurs in 20% of cases, requires treatment in the intensive care unit, is accompanied by a high risk of complications (up to 50%) and death (40-70%) [2]. According to the literature, even in the early period of severe acute pancreatitis there are changes in microcirculation and damage to the intestinal endothelium, leading to an increase in toxic products, mediators of inflammation and translocation of intestinal microflora into the bloodstream and surrounding tissues [3]. In turn, early use of enteral nutrition in patients with severe acute pancreatitis significantly improves the condition of the intestinal wall and the course of the disease as a whole, reducing the number of complications and mortality.

New In Vitro Coculture Model for Evaluating Intestinal Absorption of Different Lipid Nanocapsules.

Standard models used for evaluating the absorption of nanoparticles like Caco-2 ignore the presence of vascular endothelium, which is a part of the intestinal multi-layered barrier structure. Therefore, a coculture between the Caco-2 epithelium and HMEC-1 (Human Microvascular Endothelial Cell type 1) on a Transwell® insert has been developed. The model has been validated for (a) membrane morphology by transmission electron microscope (TEM); (b) ZO-1 and β-catenin expression by immunoassay; (c) membrane integrity by trans-epithelial electrical resistance (TEER) measurement; and (d) apparent permeability of drugs from different biopharmaceutical classification system (BCS) classes. Lipid nanocapsules (LNCs) were formulated with different sizes (55 and 85 nm) and surface modifications (DSPE-mPEG (2000) and stearylamine). Nanocapsule integrity and particle concentration were monitored using the Förster resonance energy transfer (FRET) technique. The result showed that surface modification by DSPE-mPEG (2000) increased the absorption of 55-nm LNCs in the coculture model but not in the Caco-2. Summarily, the coculture model was validated as a tool for evaluating the intestinal absorption of drugs and nanoparticles. The new coculture model has a different LNCs absorption mechanism suggesting the importance of intestinal endothelium and reveals that the surface modification of LNCs can modify the in vitro oral absorption.

Abstract P087: Mitochondrial Reactive Oxygen Species Mediate Endothelial Glycocalyx Damage And Vascular Dysfunction In Hemorrhagic Shock And Resuscitation

The endothelial glycocalyx forms an anti-thrombotic layer on the apical surface of endothelial cells and maintains the selective permeability barrier of blood vessels. Ischemia reperfusion injury like hemorrhagic shock is shown to cause glycocalyx damage. We have previously shown that mitochondrial reactive oxygen species (mitoROS) mediate glycocalyx damage in cultured endothelial cells. Of note, angiotensin II elevates endothelial mitoROS, suggesting a possible exacerbation of glycocalyx damage in hypertensive patients. It is unknown, however, whether mitoROS mediate glycocalyx damage in vivo . We hypothesize that mitoROS mediate glycocalyx damage in a rat model of hemorrhagic shock.We investigated the effect of mitochondrial ROS on the endothelial glycocalyx in vivo , in a rat model of hemorrhagic shock. In anesthetized rats, mean arterial pressure was reduced to 40 mmHg by withdrawing blood from the femoral artery and kept at 40 mmHg for 30 minutes. The rats were then resuscitated with IV (jugular vein) Ringer’s lactate solution for 30 minutes more. Sham rats received the vascular lines only. Syndecan-1 in the plasma was increased after 30 minutes of resuscitation with LR in hemorrhage/resuscitation (H/R) rats (p=0.02) but was not elevated in Sham rats (p=0.52). Resuscitation with IV mitoROS scavenger mitoTEMPOL significantly blunted this increase (5.9 pg/ml vs. 8.7 pg/ml, p=0.03). At the organ level, the glycocalyx was decreased in the endothelium of muscle (p=0.0444) and intestine (P=5.47 * 10 -7 ) vascular beds in H/R rats vs. Sham rats. Our findings show that mitoROS mediate the glycocalyx damage after H/R. This mechanism suggests possible therapies that target mitoROS generation. Future work will investigate whether preexisting hypertension increases glycocalyx damage during H/R due to exacerbated mitoROS levels.

FABP1 and FABP2 as markers of diabetic nephropathy.

Background: Diabetes mellitus is the leading cause of diabetic nephropathy and a major public health issue worldwide. Approximately 20-30% of patients with type 2 diabetes mellitus (T2DM) have renal impairment. Fatty acid-binding protein 1 (FABP1) is expressed in renal proximal tubule cells and released into urine in response to hypoxia caused by decreased peritubular capillary blood flow, and FABP2 is responsible for the transport of free fatty acids in the intestinal endothelium cells. There is increasing evidence that FABP1 and FABP 2 play a role in the development and progression of chronic kidney disease. The aim of this study was to investigate the relation of circulating FABP1 and FABP2 levels to nephropathy in patients with T2DM.Methods: For this study, 268 subjects with T2DM who were enrolled in a disease management program were stratified according to urinary microalbumin and serum creatinine measurements. The plasma FABP1 and FABP2 concentrations were examined by enzyme-linked immunosorbent assay. Demographic and potential metabolic confounding factors were analyzed with logistic regression to calculate the effects of FABP1 and FABP2 levels on diabetic nephropathy.Results: The FABP1 and FABP2 levels increased in parallel with the advancement of diabetic nephropathy. Increasing concentrations of FABP1 and FABP2 were independently and significantly associated with diabetic nephropathy. Multiple logistic regression analysis revealed FABP1 and FABP2 as an independent association factor for diabetic nephropathy, even after full adjustment of known biomarkers. Furthermore, receiver operating characteristic curve analysis showed that a FABP1 level of >33.8 ng/mL and a FABP2 level of >2.8 ng/mL were associated with diabetic nephropathy.Conclusion: Our results suggest that FABP1 and FABP2 may be novel biomarkers of diabetic nephropathy.

Unfractionated heparin attenuates histone-mediated cytotoxicity in vitro and prevents intestinal microcirculatory dysfunction in histone-infused rats.

Extracellular histones are major mediators of organ dysfunction and death in sepsis, and they may cause microcirculatory dysfunction. Heparins have beneficial effects in sepsis and have been reported to bind to histones and neutralize their cytotoxicity. The aim of this study was to investigate the impact of histones on intestinal microcirculation and the intestinal endothelium and to discuss the protective effect of unfractionated heparin (UFH) on the endothelial cytotoxicity and microcirculatory dysfunction induced by histones. Anesthetized rats were infused with 30 mg/kg calf thymus histones, and UFH was administered intravenously at a concentration of 100 IU/kg per hour. The intestinal microcirculation was visualized and measured with incident dark field microscope. Plasma von Willebrand factor (vWF) and soluble thrombomodulin were detected, and structural changes in the rat intestinal microvascular endothelium were examined. The effects of histones and UFH on cell survival rates, vWF release and calcium influx were investigated in human intestinal microvascular endothelial cells (HIMECs). Histone infusion caused severe intestinal microcirculatory dysfunction in the absence of obvious hemodynamic changes, and UFH protected intestinal microcirculation in histone-infused rats. Concentrations of the plasma endothelial injury markers vWF and soluble thrombomodulin were elevated, and structural abnormalities were found in the intestinal microvascular endothelium in the histone-infused rats. These events were attenuated by UFH. In vitro, UFH significantly reduced the histone-induced cytotoxicity of HIMECs, reduced the release of vWF from the cytoplasm into the culture medium, and inhibited calcium influx into HIMECs. Histones induce intestinal microcirculatory dysfunction followed by direct injury to the endothelial cells; UFH protects the intestinal microcirculation partly by antagonizing the endothelial toxicity of histones.

Adrenomedullin as a Protein with Multifunctional Behavior and Effects in Various Organs and Tissues

In literature, it has been reported that adrenomedullin, which is generally thought to have vasodilator, natriuretic and diuretic effects, is synthesized in almost all body, especially CNS, vascular muscles and endothelium, heart, liver, lung, kidney, gastric mocosa, intestinal endothelium and various blood cells. It has been found that the possible effects of adrenomedullin can be demonstrated directly or indirectly by means of active mediators, neuropeptides, enzymes and hormones. It is also suggested that it regulates the endocrine system by affecting the hypothalamic-pituitary axis. It increases in heart failure, acute coronary syndromes, hypertensive conditions, cerebrovascular accessory, chronic renal failure and periodontitis and decreases in peptic ulcer and intestinal diseases. However, it is still not clear whether increase/decrease in adrenomedullin level is a cause of a disease or is a result of damage due to an illness. This peptide, which could be thought to multifunctional, should be considered as a molecule with genetic coding that may have different effects on different tissues and conditions. For all these reasons, we aimed to review the multifonctional behavior of adrenomedullin in the light of the current literature to pioneer new hypotheses and discuss possible mechanisms.

SILDENAFIL IS NOT PROTECTIVE TO THE BOWEL FOLLOWING INTESTINAL ISCHEMIA AND REPERFUSION INJURY

Background and Hypothesis: 
 Acute Mesenteric Ischemia (AMI) occurs when blood supply to the intestine is decreased. This can lead to intestinal ischemia, cellular damage, necrosis, and if corrected, subsequent reperfusion injury. There are currently no medical treatments to help reverse ischemia and reperfusion injury (I/R) and, therefore novel treatments are necessary. Sildenafil, a compound that increases endogenous nitric oxide (NO) by blocking the phosphodiesterase-5 induced breakdown of cGMP, may function as a potent mesenteric vasodilator. We therefore hypothesized that sildenafil would improve mesenteric perfusion during a mouse model of intestinal I/R. 
 Experimental Design or Project Methods: 
 Adult male C57Bl6J mice were anesthetized with isoflurane and a midline laparotomy performed. The base of the superior mesenteric artery was occluded with a non-crushing vascular clamp for 60 minutes. At the end of ischemia, sildenafil (1mg/kg, 10mg/kg, or 100mg/kg) or a PBS vehicle control were administered via intraperitoneal injection. Animals were then allowed to recover. Twenty-four hours after ischemia, animals underwent assessment of mesenteric perfusion by Laser-Doppler imaging. Animals were then euthanized. Perfusion was expressed as a percentage of baseline, depicted as mean +/- SEM, and analyzed by ANOVA. P<0.05 was significant. 
 Results: 
 There were no significant differences in mesenteric perfusion between the vehicle group or any of the therapeutic groups. (PBS: 53.03±11.35%; Sildenafil-low: 59.59±7.55%; Sildenafil-medium: 70.51±8.49; Sildenafil-high: 66.4±10.2, p=0.61). 
 Conclusion: 
 Sildenafil does not appear to be an effective treatment for improving mesenteric perfusion following intestinal ischemia. Further studies are required to determine the reasons for the ineffectiveness of sildenafil. One possible explanation for these observations could be a lack of PGE5 in the intestinal vascular endothelium.

The role of atenolol in the modulation of the expression of genes encoding pro- (caspase-1) and anti- (Bcl2L1) apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats.

To investigate the role of atenolol in the gene expression of caspase 1 (Casp1) and Bcl2L1 on vascular endothelium of rat intestine after ischemia and reperfusion (IR). Eighteen adult male Wistar rats were randomly divided into 3 groups (n=6): SG (Sham group): no clamping of the superior mesenteric artery; IRG: IR plus saline group: IRG+At: IR plus Atenolol group. Rats from IRG and IRG+At were subjected to 60 min of intestinal ischemia and 120 min of reperfusion. Atenolol (2mg/kg) or saline were injected in the femoral vein 5 min before ischemia, 5 min and 55 min after reperfusion. Thereafter, intestinal segments were appropriately removed and processed for Endothelial Cell Biology Rat RT2 Profiler PCR Array. the anti-apoptotic Bcl2L1 gene expression was significantly down-regulated (-1.10) in the IRG and significantly up-regulated in the IRG+At (+14.15). Meanwhile, despite Casp1 gene expression was upregulated in both groups, it was significantly higher in the IRG (+35.06) than the IRG+At (+6.68). Atenolol presents antiapoptotic effects on rat intestine subjected to IR partly by the up-regulation of the anti-apoptotic Bcl2L1 gene expression. Moreover, atenolol can mitigate the pro-apoptotic and pro-inflammatory effects of Casp1 gene on rat intestine after IR.

The expression of IL10RA in colorectal cancer and its correlation with the proliferation index and the clinical stage of the disease

IntroductionDespite the widely described role of IL10 in immune response regulation during carcinogenesis, there is no established model describing the role of its receptor. The aim of this study is to elucidate the relationship between the subunit alpha of IL10 receptor (IL10RA) in the pathogenesis of colorectal cancer (CRC). MethodsThe study was conducted on archived paraffin blocks of 125 CRC patients, from which tissue microarrays (TMA) were made. These were subsequently used for immunohistochemistry to assess the expression of IL10RA, IL10, phosphorylated STAT3 (pSTAT3) and the Ki67 proliferation index. The intensity of both reactions was assessed by independent researchers using two approaches: digital image analysis and the Remmele and Stegner score (IRS). To assess the possible correlations between the two investigated markers and the clinical stage of CRC, the Pearson correlation coefficient was calculated. The expression of aforementioned proteins was assessed in tumor samples, healthy surgical margins and healthy control samples, obtained from cadavers during autopsy from the Department of Forensic Medicine. Statistical analysis was conducted using Statistica ver. 13.05 software. ResultsThe final analysis included 105 CRC patients with complete clinical and pathological data, for whom the expressions of IL10RA, IL10, pSTAT3 and Ki67 were assessed using two independent methods. There was a positive correlation between the IL10RA expression and Ki-67 proliferation index (R = 0.63, p < 0.001) and a negative correlation between the IL10RA expression and the clinical stage of CRC (R = −0.21, p = 0.022). IL10RA correlated positively with pSTAT3 and IL10 in neoplastic tissue and tumor margin (with p < 0.01 for all correlations).We also observed a significantly higher expression of IL10RA in healthy surgical margins when compared to the actual tumor (p = 0.023, the paired t-test). The expression of IL10 was significantly higher in tumors than in healthy intestinal endothelium from control group. ConclusionsThe correlations between the expression of IL10RA and the proliferation index or the clinical stage of CRC seem to confirm the importance of IL10RA in the pathogenesis of CRC. The higher expression of IL10RA in healthy surgical margins than in the tumor itself may suggest that IL10RA plays a role in regulating immune response to the neoplasm.

Exercise Effects on Gutdysbiosis, Intestinal Permeability and Systemic Inflammation in Patients with Type 2 Diabetes: A Pilot Study

Exercise plays a significant role in the prevention of the diabetes. Recent data propose that dysbiosis of intestinal microbiota composition contributes to development of Type 2 diabetes (T2D). Moreover, dysbiosis alters intestinal endothelium permeability causing the “Leaky gut syndrome†(LGS). We measured in 15 selected patients with standard medical cures for sable T2D the effects of 6 months of endurance, resistance and flexibility training on the gut microbiota composition and intestinal permeability.

P339 MadCAM1 expression in intestinal lamina propria endothelium varies among inflammatory bowel disease patients

Background: Vedolizumab, a monoclonal antibody against α4β7 integrin has been shown to be effective in inducing and maintaining remission in inflammatory bowel disease (IBD). By blocking α4β7, it is preventing the homing of lymphocytes through binding to mucosal vascular adressin cell adhesion molecule 1 (MadCAM1) localised on the intestinal endothelial cells. It is unclear to which extent the gut homing of lymphocytes is subject to redundant biological process involving other pathways than α4β7-MadCAM1 interaction. There are no data on the interindividual variability of MadCAM1 expression that might influence the effect of vedolizumab. The aim of our study was thus first, to determine the expression of MadCAM1 on the intestinal endothelial cells of IBD patients. Second, we aimed to assess the relationship of MadCAM1 expression with the clinical response to vedolizumab. Methods: All IBD patients referred for the treatment with vedolizumab to one referral center were included. The biopsies or resection specimen from the inflamed intestinal tissue were stained by immunohistochemistry for MadCAM1 expression. Clinical response to vedolizumab was assessed in patients with the minimal treatment duration of 10 weeks and the differences in MadCAM1 expression between responders and non-responders were analyzed. Results: In total, 34 IBD patients were referred for vedolizumab treatment; for 21 of them intestinal biopsies or surgical specimen could have been retrieved and stained for MadCAM1 expression. In three out of these 21 patients (14%) no MadCAM1 expression in intestinal endothelium was detected, remaining 18 patients expressed MadCAM1 to various extent (from 30 to 100% of all vessels). Ten patients had the minimal follow-up of 10 weeks of treatment, 4 out of these 10 patients had clinical response to vedolizumab. The only two patients not expressing MadCAM1 in this group were both no responders with the respective follow-up of 16 and 14 weeks of treatment. Conclusions: MadCAM1 expression in intestinal endothelium varies among IBD patients. Other gut homing mechanisms of lymphocytes might thus prevail in some IBD patients and limit the anti-inflammatory effect of vedolizumab.

Targeting Integrin a4b7-Expressing T-Cells in Steroid Refractory Intestinal GvHD

IntroductionGrade III-IV GvHD is associated with poor outcome. Severe steroid refractory (SR) intestinal manifestations are difficult to treat, and current treatment modalities augment the immunodeficiency and often lead to opportunistic infections, multiorgan failure (MOF) and death. 2nd and 3rd line treatments are less than optimally documented and often with erratic responses.Vedolizumab (Vedo), a monoclonal antibody targeting the homing of T-cells to the intestinal endothelium through inhibition of the binding of integrin a4b7 to MadCAM, is effective in inflammatory bowel disease (IBD) refractory to TNF-a inhibitors.Encouraged by the responses reported in IBD, we used Vedo in six patients with intestinal SR GvHD.MethodsPatient characteristics are provided in table 1. Patients 1 and 2 had been through 2nd and 3rd line therapy without response or resolution of the GvHD. Patients 3 to 6 were given Vedo as second line therapy after steroid failure. Patient 4 already had MOF with gastrointestinal GvHD before receiveing Vedo, and died of MOF.Vedo was delivered as prescribed in IBD; 300 mg iv without premedication week 0, 2 and 6, followed by infusions every 8 weeks on clinical indication.ResultsAll patients exhibited clinical responses within 7 - 10 days after start of treatment with decrease in abdominal pain and watery diarrhoea. Serial endoscopies were performed in patients 1,2,3,5 and 6. These revealed gradual macroscopic and histologic improvement; the upper GI-tract showing a more rapid improvement than the colon. Figure 1 shows colon histology before (a, b, c) and after 3 doses of Vedo (d, e, f) in patient 1, 2 and 3.After 3 doses of Vedo, most patients could taper systemic corticosteroid therapy. All patients, except patient 4, were on oral medication including immunosuppressants. Concomitant immunosuppressive therapy was administered as oral cyclosporine or MMF. There were no or sparse clinical symptoms of acute intestinal GvHDPatient 1 is in continued clinical remission of GvHD 8 months after start of therapy. Patient 2 was asymptomatic for 5 months, then a molecular relapse of acute promyelocytic leukemia was diagnosed and intestinal GvHD recurred when all immunosuppression was stopped. In patient 3, the intestinal GvHD resolved after three doses of Vedo, but he developed skin GvHD after cessation of IS and died of an opportunistic infection following treatment with high dose methyl-prednisolone. Patient 4 had MOF prior to start of treatment with Vedo and died in MOF. Patient 5 and 6 have no clinical signs of intestinal GvHD after 2 and 3 doses of Vedo.Five out of six patients could be discharged from hospital after treatment with Vedo.Immunophenotyping of peripheral blood revealed high levels of CD25+ Treg cells in 4 out of 5 evaluable patients prior to treatment and the numbers decreased to normal levels after start of therapy and with signs of clinical effect. Patient 2 stopped IS after a molecular relapse of APL and had a recurrence of GvHD. This recurrence coincided with an increase in Treg percentage.DiscussionThis case series provides the first proof of concept that targeting integrin a4b7 T-cells is feasible, safe and may provide clinical improvement in intestinal SR GvHD. The mechanism of action is not known, but may be due to the inhibition of the homing of a4b7 expressing allo-reactive T-cells to recipient MadCAM expressing intestinal endothelial cells. The mechanism behind the Treg patterns is unclear. One might speculate that the initial high levels of Tregs are part of the physiologic reaction to the alloreactive inflammation in the intestinal epithelium and that the subsequent normalization occurs as the inflammation subsides.Table 1Patient123456DiagnosisAMLAMLNHLAMLNHLAMLSexmalemalemalefemalemalemaleAge at transplant465842445062Lines of therapy after steroid refractoriness210000Other manifestations12,3,41Steroid dose (mg) at last FUN/AN/A2 mg/kg1,7 mg/kg100Number of Vedolizumab doses at last FU553123Immunosuppression at last FUNoCyACyA + steroidsMMF + steroidsCyA + steroidsNowatery stools at last FUNoYesNoNoNoNoTreg % before start of Vedo (reference: 2,5 - 5,8)10,229,641,7336,5Treg % follow up 15,7152,113,313Treg % follow up 26,26Treg % follow up 34,66,6Treg % follow up 44,9Treg % follow up 58,5**IS cessation due to molecular relapse followed by relapse of GvHD1CMV2renal failure3hepatic failure4multi-organ GvHD [Display omitted] DisclosuresOff Label Use: Vedolizumab is approved for the treatment of inflammatory bowel disease. The present study describes its effect on steroid-refractory acute intestinal GvHD. Lundin:Takeda: Consultancy, Honoraria.

The protective effects of hydrocortisone on glycocalyx in the intestinal capillary endothelium after trauma-hemorrhagic shock

Objective To study the protective effects of hydrocortisone on glycocalyx in the vascular endothelium after trauma-hemorrhagic shock (T/HS) because the role of glycocalyx in maintaining the permeability of vascular endothelium intact, and in turn to identify the hydrocortisone protecting intestinal microcirculation. Methods Studies were carried out, in vivo, on a model of rats with induced T/HS. Intestinal perfusion and changes in endothelial glycocalyx and the associated molecular mechanism were assessed by using laser-Doppler velocimetry and electron microscopy, and the measurements of heparan sulfate, syndacan-1, and TNF-α in the superior mesenteric vein (SMV) with ELISA and Western-blot, and the expression of NF-κB in the vascular endothelium. Protective effects of hydrocortisone on the intestinal microcirculation after T/HS were evaluated. Results Degradation of the glycocalyx in intestinal vascular endothelium occurred 1 -3 hours after T/HS in rats (P <0.05) . By 3 hours later, significant reduction in intestinal perfusion was observed (P <0.05) . The level of TNF-α in the SMV and the expression of NF-κB in the vascular endothelium increased. With the use of hydrocortisone, intestinal perfusion was improved, and the degradation of glycocalyx was attenuated. Conclusions The degradation of glycocalyx is associated with the malfunction of intestinal microcirculation after T/HS. The NF-κB/TNF-α system in vascular endothelium participates in this process of glycocalyx degradation. Hydrocortisone may be a good agent to interrupt the course of glycocalx degradation. Key words: Traumatic hemorrhage shock; Capillary leak syndrome; Glycocalyx; Intestinal mucosal barrier; Hydrocortisone; TNF-α

Memory B cells with gut-homing potential are generated by a prototype anti-enterotoxigenic E. coli vaccine given by intradermal immunization with LT(R192G) as an adjuvant (MUC3P.945)

Abstract Lymphocytes that express α4β7 integrin can home to the gut mucosa through interactions with MAdCAM-1 addressin on the intestinal endothelium. α4β7+ Ag-specific plasmocytes and memory B (BM) cells can be found after infection or experimental challenge with enteric pathogens as well as mucosal immunization, yet gut-homing B cells have not been reported in high frequencies in humans after parenteral immunization. Intradermal (ID) immunization is an attractive delivery route for enteric vaccines due to a potential connection between the skin and gut. Mucosal responses may be further enhanced by the addition of adjuvants such as LT(R192G), an attenuated form of the enterotoxigenic E. coli (ETEC) LT toxin. We analyzed BM cell generation in volunteers with two prototype vaccines based on the ETEC class 5a fimbriae tip adhesin CfaE with LT(R192G) by ID delivery. We observed both anti-CfaE and -LTB IgG and IgA BM cells, with a higher prevalence of Ag-specific IgG BM cells. Interestingly, anti-LTB IgA and anti-CfaE IgG BM cells were elicited at frequencies similar to those reported after ETEC infection, whereas anti-LTB IgG BM cells were found in higher frequencies after ID immunization compared to infection. The frequencies of anti-CfaE and -LTB IgG BM cells were similar in α4β7+ and α4β7- fractions, while the majority of anti-CfaE and -LTB IgA BM cells were α4β7-. These data suggest that the adjuvant LT(R192G) may be used to enhance mucosal BM responses following ID immunization.

Are intact peptides absorbed from the healthy gut in the adult human?

For over 100 years it was believed that dietary protein must be completely hydrolysed before its constituent amino acids could be absorbed via specific amino acid transport systems. It is now known that the uptake of di- and tripeptides into the enterocyte is considerable, being transported across the intestinal endothelium by the PepT1 H+/peptide co-transporter. There is also evidence that some di- and tripeptides may survive cytosolic hydrolysis and be transported intact across the basolateral membrane. However, other than antigen sampling, the transport of larger intact macromolecules across the intestinal endothelium of the healthy adult human remains a controversial issue as there is little unequivocal in vivo evidence to support this postulation. The aim of the present review was to critically evaluate the scientific evidence that peptides/proteins are absorbed by healthy intestinal epithelia and pass intact into the hepatic portal system. The question of the absorption of oliogopeptides is paramount to the emerging science of food-derived bioactive peptides, their mode of action and physiological effects. Overall, we conclude that there is little unequivocal evidence that dietary bioactive peptides, other than di- and tripeptides, can cross the gut wall intact and enter the hepatic portal system in physiologically relevant concentrations.

Effects of HM30181, a P‐glycoprotein inhibitor, on the pharmacokinetics and pharmacodynamics of loperamide in healthy volunteers

HM30181 is a third generation P-glycoprotein (P-gp) inhibitor currently under development. The objectives of this study were to evaluate the effects of a single dose of HM30181 on the pharmacodynamics and pharmacokinetics of loperamide, a P-gp substrate, and to compare them with those of quinidine. Eighteen healthy male subjects were administered loperamide alone (period 1) or with loperamide plus quinidine or HM30181 in period 2 or 3, respectively. In period 3, subjects randomly received one of three HM30181 doses: 15, 60 or 180 mg. Changes in pupil size, alertness, oxygen saturation and the oral bioavailability of loperamide were assessed in each period. In addition, the pharmacokinetics of HM30181 were determined. Pupil size, alertness and oxygen saturation did not change over time when loperamide alone or loperamide plus HM30181 was administered while HM30181 significantly increased the systemic exposure to loperamide, i.e. the geometric mean ratio (90% confidence interval) of AUC(0,tlast ) for loperamide with and without HM30181 was 1.48 (1.08, 2.02). Co-administered quinidine significantly increased the systemic exposure to loperamide 2.2-fold (1.53, 3.18), which also markedly reduced pupil size, resulting in a decrease of 24.7 mm h in the area under the effect curve of pupil size change from baseline compared with loperamide alone. HM30181 inhibits P-gp mainly in the intestinal endothelium, which can be beneficial because pan-inhibition of P-gp, particularly in the brain, could lead to detrimental adverse events. Further studies are warranted to investigate adequately the dose-exposure relationship of HM30181, along with its duration of effect.

Combination of dehydroepiandrosterone and orthovanadate administration reduces intestinal leukocyte recruitment in models of experimental sepsis

BackgroundDehydroepiandrosterone (DHEA) was shown to improve the immune function and survival in experimental sepsis. This study examined the effect of DHEA on intestinal leukocyte recruitment during experimental sepsis, considering factors of gender (male, female and ovariectomized female animals) and combined treatment using orthovanadate (OV) in two models of sepsis. Methodology/findingsMale rats underwent colon ascendens stent peritonitis (CASP) or endotoxemia. DHEA was administered after induction of experimental sepsis. Changes in leukocyte adherence and capillary perfusion (measured as intestinal functional capillary density — FCD) were assessed using intravital microscopy. While DHEA increased baseline leukocyte adherence in control animals, DHEA reduced leukocyte adherence and increased FCD in male animals with CASP. These effects were also observed in DHEA-treated ovariectomized female rats with CASP. Similarly, the administration of DHEA reduced the number of adherent leukocytes to intestinal venules by 30% in the endotoxemia model. The combined treatment of DHEA and OV significantly reduced adherence of leukocytes to intestinal venules and improved FCD. ConclusionsOur results indicate that DHEA is able to reduce intestinal leukocyte recruitment induced by experimental sepsis. Combination of DHEA with OV inhibits leukocyte adherence to intestinal endothelium, similar to what is achieved by the single administration of DHEA but with significantly improved FCD. These findings suggest a potential role for DHEA and OV in clinical sepsis.

PTK6 mediates intestinal endothelial barrier dysfunction in response to TNFα (695.1)

A key event in the progression of systemic inflammation resulting from severe trauma or shock involves hyperpermeability of the intestinal endothelium. The signaling events leading to increased endothelial permeability in these disease states are incompletely understood. Although the role of protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in mechanisms underlying metastasis and cancer, it has also been shown that PTK6 participates in mediating colonic epithelial barrier dysfunction. In this study, we hypothesized that PTK6 is 1‐ expressed in intestinal endothelial cells, and 2‐ contributes to endothelial hyperpermeability in response to inflammation. To this end, we investigated the role of PTK6 in mediating the disruption of intestinal endothelial barrier function. Results showed that PTK6 was detected in intestinal endothelial cells at the level of protein and mRNA. In addition, PTK6 knockdown attenuated decrease in transendothelial electric resistance (TER) as measured by electric cell‐substrate impedance sensing (ECIS) of intestinal endothelial monolayers in response to inflammatory factors including TNFα. Furthermore, we showed that TNFα increased activity of PTK6 as evidenced by increased phosphorylation at Y342, supporting the hypothesis that this kinase plays a role in endothelial inflammation. We further studied the potential targets of PTK6 including junctional structure proteins to determine the molecular mechanism of its role in regulating endothelial barrier function. Our study explores a potential implication that targeting PTK6 may serve as a novel therapeutic strategy in treating diseases characterized by hyperpermeability of the intestinal endothelium.Grant Funding Source: Supported by VA Merit Review 5101BX000799, the NIH grants HL96640, HL61507 and HL70752