FAMIN Is a Multifunctional Purine Enzyme Enabling the Purine Nucleotide Cycle.

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Evaluation of Intestinal Fibrosis with 68Ga-FAPI PET/MR Enterography in Crohn Disease.

Background In Crohn disease, differentiation between active intestinal inflammation and fibrosis has implications for treatment, but current imaging modalities are not reliably accurate. Purpose To evaluate the predictive value of gallium 68 (68Ga)-labeled fibroblast activation protein inhibitor (FAPI) PET/MR enterography for the assessment of bowel wall fibrosis in Crohn disease. Materials and Methods In this prospective single-center study, consecutive participants with Crohn disease and obstructive symptoms underwent preoperative 68Ga-FAPI PET/MR enterography from May 2021 to January 2022. Histopathologic analysis of resected bowel segments was performed to grade active inflammation (A0-A2) and fibrosis (F0-F2), which served as the reference standard. The fibroblast activation protein (FAP) expression in bowel wall layers was analyzed immunohistochemically for each layer. 68Ga-FAPI-derived maximum standardized uptake value (SUVmax) was compared with histopathologic results by using mixed-model analysis of variance and Bonferroni-corrected post hoc tests. Results In 14 participants (mean age, 45 years ± 9 [SD]; 10 men), fibrosis was diagnosed histopathologically in 28 of 51 bowel segments (grade F1, n = 14; grade F2, n = 14). Mean SUVmax was higher in segments with fibrosis than without (7.6 vs 2.0; P < .001). In severe fibrosis, mean SUVmax was higher than in mild to moderate fibrosis (8.9 ± 0.9 vs 6.2 ± 0.9; P = .045). Bowel segments with isolated active inflammation had lower mean 68Ga-FAPI uptake than segments with combined active inflammation and fibrosis (SUVmax, 3.2 ± 0.4 vs 8.1 ± 0.1; P = .005). With an SUVmax cutoff value of 3.5, the area under the receiver operating characteristic curve for the prediction of fibrosis was 0.94 (95% CI: 0.9, 1.0), with sensitivity of 26 of 28 segments (93%) and specificity of five of six segments (83%). 68Ga-FAPI-derived SUVmax correlated with FAP expression across all bowel layers (R2 = 0.50, P < .001). Conclusion Higher gallium 68 fibroblast activation protein inhibitor uptake at PET/MR enterography was associated with histopathologically assessed bowel wall fibrosis in participants with Crohn disease, suggesting diagnostic potential for treatment decisions. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by O'Shea in this issue.

In vivo Treg expansion under costimulation blockade targets early rejection and improves long-term outcome.

CTLA4Ig has been shown to improve kidney allograft function, but an increased frequency of early rejection episodes poses a major obstacle for more widespread clinical use. The deleterious effect of CTLA4Ig on Treg numbers provides a possible explanation for graft injury. Therefore, we aimed at improving CTLA4Ig's efficacy by therapeutically increasing the number of Tregs. Murine cardiac allograft transplantation (BALB/c  to B6) was performed under CTLA4Ig therapy modeled after the clinically approved dosing regimen and Tregs were transferred early or late after transplant. Neither early nor late Treg transfer prolonged allograft survival. Transferred Tregs were traceable in various lymphoid compartments but only modestly increased overall Treg numbers. Next, we augmented Treg numbers in vivo by means of IL2 complexes. A short course of IL2/anti-IL2-complexes administered before transplantation reversed the CTLA4Ig-mediated decline in Tregs. Of note, the addition of IL2/anti-IL2-complexes to CTLA4Ig therapy substantially prolonged heart allograft survival and significantly improved graft histology on day 100. The depletion of Tregs abrogated this effect and resulted in a significantly diminished allograft survival. The increase in Treg numbers upon IL2 treatment was associated with a decreased expression of B7 on dendritic cells. These results demonstrate that therapy with IL2 complexes improves the efficacy of CTLA4Ig by counterbalancing its unfavorable effect on Tregs.

Distinct roles for major and minor antigen barriers in chimerism-based tolerance under irradiation-free conditions.

Eliminating cytoreductive conditioning from chimerism-based tolerance protocols would facilitate clinical translation. Here we investigated the impact of major histocompatibility complex (MHC) and minor histocompatibility antigen (MiHA) barriers on mechanisms of tolerance and rejection in this setting. Transient depletion of natural killer (NK) cells at the time of bone marrow (BM) transplantation (BMT) (20 × 106 BALB/c BM cells → C57BL/6 recipients under costimulation blockade [CB] and rapamycin) prevented BM rejection. Despite persistent levels of mixed chimerism, BMT recipients gradually rejected skin grafts from the same donor strain. Extending NK cell depletion did not improve skin graft survival. However, F1 (C57BL/6×BALB/c) donors, which do not elicit NK cell-mediated rejection, induced durable chimerism and tolerance. In contrast, if F1 donors with BALB/c background only were used (BALB/c×BALB.B), no tolerance was observed. In the absence of MiHA disparities (B10.D2 donors, MHC-mismatch only), temporal NK cell depletion established stable chimerism and tolerance. Conversely, MHC identical BM (BALB.B donors, MiHA mismatch only) readily engrafted without NK cell depletion but no skin graft tolerance ensued. Therefore, we conclude that under CB and rapamycin, MHC disparities provoke NK cell-mediated BM rejection in nonirradiated recipients whereas MiHA disparities do not prevent BM engraftment but impede skin graft tolerance in established mixed chimeras.

Spatially Resolved Analysis of Urban Thermal Environments Based on a Three-Dimensional Sampling Algorithm and UAV-Based Radiometric Measurements.

A new method and workflow to assess outdoor thermal comfort and thermal stress in urban areas is developed. The new methodology is applied to a case of an urban quarter in the city of Graz. The method recognises the significance of detailed and accurate spatially resolved determination of mean radiant temperatures taking into account all relevant radiative components, comprising thermal radiation, as well as global radiation. The method relies on radiometric imaging data that are mapped onto a three-dimensional model. The image data are acquired by means of drones (UAVs) equipped with multispectral and thermographic cameras to capture short- and long-wave radiation. Pre-existing city models and a Monte Carlo raytracing algorithm to perform anisotropic sampling based on a 3D model with human topology are used to determine local radiation temperatures with high spatial resolution. Along with spot measurements carried out on the ground simultaneously, the spatially resolved and three-dimensionally determined mean radiation temperatures are used to calculate thermal comfort indicator maps using UTCI and PMV calculation. Additional ground measurements are further used to validate the detection, as well as the entire evaluation process.

Hepatic Steatosis in Lean Patients: Risk Factors and Impact on Mortality.

BACKGROUND: The prognostic impact of liver steatosis in obese patients is well established. Limited data on the risk factors for and impact of hepatic steatosis in lean patients are available. AIMS: Assess risk factors for liver steatosis in lean patients and investigate its impact on survival. METHODS: Patients without viral hepatitis and with a BMI ≤ 25 kg/m2 undergoing liver stiffness measurement (LSM) and controlled attenuation parameter (CAP) by transient elastography were retrospectively identified. Clinical characteristics and laboratory test results were obtained at the time of LSM/CAP measurement. National death registry data were obtained in order to assess survival. RESULTS: Among n = 218 lean patients, n = 97 (34.5%) showed significant liver steatosis (CAP ≥ 268 dB/m), while n = 184 (65.5%) had no or just mild steatosis (CAP < 268 dB/m). Patients with steatosis had higher GGT (238.0(± 450.3) vs. 112.1(± 180.0) IU/mL; p = 0.013), AST (63(± 67.4) vs. 38.5(± 32.9) IU/mL; p = 0.001), ALT (59.1(± 58.8) vs. 44.3(± 52.7) IU/mL; p = 0.048) and triglyceride levels (120.1(± 80.3) vs. 96.1(± 58.2) mg/dL; p = 0.014), and showed a trend toward more severe fibrosis (LSM 15.6(± 19.5) vs. 12.0(± 15.7) kPa; p = 0.115). In multivariate binary logistic regression analysis, only serum uric acid levels were independently associated with liver steatosis (odds ratio 1.43 per unit mg/dL; 95% CI 1.001-2.054; p = 0.049). During a mean follow-up of 38.9(± 10.6) months, n = 14 patients (5.0%) died. In the absence of advanced fibrosis, survival after 1 year was similar in patients without (98.7%) and with (98.6%) significant steatosis. Patients with advanced fibrosis had worse 1-year survival without concomitant significant steatosis (84.8%) than patients with steatosis (95.8%; log-rank p < 0.001). CONCLUSIONS: High serum uric acid levels increase the risk of liver steatosis in lean patients. Liver fibrosis but not hepatic steatosis is a risk factor for impaired survival in lean patients.

Allograft rejection is associated with development of functional IgE specific for donor MHC antigens.

BACKGROUND: Donor-specific antibodies of the IgG isotype are measured routinely for diagnostic purposes in renal transplant recipients and are associated with antibody-mediated rejection and long-term graft loss. OBJECTIVE: This study aimed to investigate whether MHC-specific antibodies of the IgE isotype are induced during allograft rejection. METHODS: Anti-MHC/HLA IgE levels were measured in sera of mice grafted with skin or heart transplants from various donor strains and in sera of kidney transplant patients with high levels of HLA IgG. Mediator release was triggered in vitro by stimulating basophils that were coated with murine or human IgE-positive serum, respectively, with specific recombinant MHC/HLA antigens. Kidney tissue samples obtained from organ donors were analyzed by using flow cytometry for cells expressing the high-affinity receptor for IgE (FcεRI). RESULTS: Donor MHC class I- and MHC class II-specific IgE was found on acute rejection of skin and heart grafts in several murine strain combinations, as well as during chronic antibody-mediated heart graft rejection. Anti-HLA IgE, including donor HLA class I and II specificities, was identified in a group of sensitized transplant recipients. Murine and human anti-MHC/HLA IgE triggered mediator release in coated basophils on stimulation with specific MHC/HLA antigens. HLA-specific IgE was not linked to atopy, and allergen-specific IgE present in allergic patients did not cross-react with HLA antigens. FcεRI+ cells were found in the human renal cortex and medulla and provide targets for HLA-specific IgE. CONCLUSION: These results demonstrate that MHC/HLA-specific IgE develops during an alloresponse and is functional in mediating effector mechanisms.

Controlled attenuation parameter does not predict hepatic decompensation in patients with advanced chronic liver disease.

BACKGROUND & AIMS: Assessment of hepatic steatosis by transient elastography (TE)-based controlled attenuation parameter (CAP) might predict hepatic decompensation. Therefore, we aimed to evaluate the prognostic value of CAP in patients with compensated advanced chronic liver disease (cACLD) and decompensated cirrhosis (DC). METHODS: A total of 430 patients who underwent TE (liver stiffness ≥10 kPa) and CAP measurements were included in this retrospective analysis. Half of patients (n = 189) underwent simultaneous HVPG measurement. In cACLD patients, first hepatic decompensation was defined by new onset of ascites, hepatic encephalopathy or variceal bleeding. In patients with DC, the following events were considered as further hepatic decompensation: requirement of paracentesis, admission for/development of grade 3/4 hepatic encephalopathy, variceal (re-)bleeding or liver-related death. RESULTS: First hepatic decompensation occurred in 25 of 292 (9%) cACLD patients, while 46 of 138 (33%) DC patients developed further hepatic decompensation during a median follow-up of 22 and 12 months respectively. CAP was not predictive of first (cACLD; per 10 dB/m; hazard ratio [HR]: 0.97, 95% confidence interval [95% CI]: 0.91-1.03, P = 0.321) or further hepatic decompensation (DC; HR: 0.99, 95% CI: 0.94-1.03, P = 0.554) in adjusted analysis. Using the well-established CAP cut-off of ≥248 dB/m for hepatic steatosis, the incidence of first (cACLD; P = 0.065) and further hepatic decompensation (DC; P = 0.578) was similar in patients with hepatic steatosis or without. Serum albumin levels (per mg/dL; HR: 0.83, 95% CI: 0.77-0.89, P < 0.001) and MELD-Na (per point; HR: 1.15, 95% CI: 1.04-1.28, P = 0.006) in cACLD and MELD-Na (per point; HR: 1.12, 95% CI: 1.05-1.19, P < 0.0001) in DC patients were the only parameters independently associated with first and further hepatic decompensation, respectively. CONCLUSION: Controlled attenuation parameter does not predict the development of first (cACLD)/further (DC) hepatic decompensation, while serum albumin levels and MELD-Na are of prognostic value.

Outcomes after placement of a SX-ELLA oesophageal stent for refractory variceal bleeding-A national multicentre study.

BACKGROUND: Current guidelines favour the use of bleeding stents over balloon tamponade (BT) for refractory variceal bleeding (VB) from oesophageal varices. However, data on the efficacy and safety of self-expandable metal SX-ELLA Danis stents (SEMS) are limited. METHODS: Cirrhotic patients receiving SEMS for VB at four tertiary care centres were included in this retrospective multicentre study. Rates of failure-to-control bleeding (within 5 days) and bleeding-related mortality (6 weeks) were assessed. RESULTS: SEMS controlled VB in 79.4% (27/34) of patients. In the rest of patients, other rescue treatments including endoscopic band ligation (EBL, n = 3), SEMS renewed (n = 2) or Linton (n = 2) were applied; however, VB was only controlled in one patient. Early rebleeding within six weeks occurred in 17.6% (6/34) patients. Median SEMS dwell time was three (IQR:6) days. Overall n = 13/34 (38.2%) patients died with SEMS in situ. After SEMS removal, rebleeding and bleeding-related death occurred in n = 7 (35%) and n = 5 (14.7%) patients respectively. Only 32.4% (10/34) patients did not experience any rebleeding within six weeks after SEMS removal. Bleeding-related mortality was 47.1% (n = 16/34) and the median survival after SEMS placement was 2.1 months. Notably, no patient received an early transjugular intrahepatic portosystemic shunt (TIPS). The most common adverse events were stent dislocations (n = 13; 38.2%), while ulcers/necrosis of the oesophageal mucosa was seen in only four (11.8%) patients. CONCLUSION: SEMS controlled refractory VB in most patients. However, bleeding-related mortality remained high. While SEMS dislocations were frequent, ulcers/necrosis of the oesophagus was rare. Further studies should investigate whether the wider use of early TIPS reduces bleeding-related mortality after SEMS placement.

Management of portal hypertension before and after liver transplantation.

Orthotopic liver transplantation (OLT) represents a curative treatment option for end-stage liver disease (ESLD). Although epidemiology of ESLD has recently changed due to the rising prevalence of nonalcoholic fatty liver disease and the decreased burden of hepatitis C virus infections due to highly effective antiviral regimens, the management of portal hypertension (PHT) remains a clinical challenge in the pre- and post-OLT setting. The measurement of the hepatic venous pressure gradient represents the most reliable but invasive tool for assessment of the severity of PHT. Although novel liver ultrasound and magnetic resonance-based elastography methods have been developed, their value to screen for liver fibrosis and PHT in transplanted patients remains to be established. Nonselective beta-blockers represent the cornerstone of medical treatment of PHT, but more studies on their effects on clinical endpoints after OLT are needed. Statins are widely used to treat hyperlipidemia, which is a common condition after OLT. Although a growing body of evidence suggests that statins decrease portal pressure and PHT-related complications in ESLD, studies on potential benefits of statins after OLT are lacking. Finally, transjugular intrahepatic portosystemic shunts (TIPS) are effective in decreasing PHT and seem to decrease mortality on the OLT waiting list. Moreover, TIPS does not have an impact on liver function nor complicate the transplant surgical procedures. TIPS may also be used after OLT, but the evidence is limited. In conclusion, whereas the management of PHT in patients with ESLD is based on strong evidence, further data on the value of noninvasive monitoring tools as well as on medical and invasive treatment options in the post-OLT setting are needed to improve management strategies in patients with recurrent PHT after liver transplantation. Liver Transplantation 24 112-121 2018 AASLD.

An intra-individual surgical wound comparison shows that octenidine-based hydrogel wound dressing ameliorates scar appearance following abdominoplasty.

Hypertrophic scar formation because of surgical procedures is associated with higher levels of pain, a lower quality of life, and poor cosmetic outcome and requires more resources in follow-up management. An octenidine-based hydrogel has been shown to modulate immunological function in an in vitro wound model, suggesting an improved scar formation. In this prospective, randomised, observer-blinded, and intra-patient-controlled study, 45 patients who underwent abdominoplasty or mastectomy with transverse rectus abdominis muscle (TRAM) flap reconstruction were given both a standard postoperative wound dressing on one wound side and an octenidine-based hydrogel with transparent film dressing, covered with standard postoperative dressing on the other side. Four instances of hypertrophia were reported in the gel side versus 12 in the standard dressing side. Visual Analogue Scale (VAS) pain scores taken during postoperative dressing changes showed reduced scores on the gel side at all time points. Vancouver Scar Scale (VSS) scores showed improvement in the gel side at 3, 6, and 12 months postoperatively. Skin distensibility measured using a cutometer showed significantly improved measures in gel-treated wounds, similar to measures of healthy skin. Trans-epidermal water loss (TEWL), measured using a tewameter, showed improved values on the gel side soon after surgery, with both the control and the gel side normalising after approximately 6 months. The octenidine-based wound dressing demonstrates improved wound healing associated with a lower incidence of hypertrophic scar formation.

Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients.

While costimulation blockade-based mixed chimerism protocols work well for inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficulties is the high frequency of alloreactive memory T cells (Tmem) found in the (pre)clinical setting as opposed to laboratory mice. In the present study, we therefore developed a murine bone marrow transplantation (BMT) model employing recipients harboring polyclonal donor-reactive Tmem without concomitant humoral sensitization. This model was then used to identify strategies to overcome this additional immune barrier. We found that B6 recipients that were enriched with 3 × 10(7) T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with anti-CD40L and CTLA4Ig (while recipients not enriched developed chimerism). Adjunctive short-term treatment of sensitized BMT recipients with rapamycin or anti-LFA-1 mAb was demonstrated to be effective in controlling Tmem in this model, leading to long-term mixed chimerism and donor-specific tolerance. Thus, rapamycin and anti-LFA-1 mAb are effective in overcoming the potent barrier that donor-reactive Tmem pose to the induction of mixed chimerism and tolerance despite costimulation blockade.

Circulating caspase-cleaved cytokeratin 18 correlates with tumour burden and response to therapy in patients with colorectal cancer liver metastasis.

BACKGROUND AND AIMS: Systemic chemotherapy followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CRCLM) but reliable biomarkers predicting response to therapy are needed. Spontaneous apoptosis of single tumour cells is common in CRCLM. We explored the potential of circulating apoptosis markers to predict treatment response. MATERIALS AND METHODS: Fifty-eight patients with CRCLM or hepatocellular carcinoma (HCC) were included in this study. Tumour tissue and blood samples were obtained before and after initiation of chemotherapy. Immunohistochemistry and ELISA assays were utilized to quantify the apoptosis marker caspase-cleaved cytokeratin 18 (M30) in tissue and circulation. RESULTS: CRCLM tissues showed more apoptotic tumour cells than HCC, or healthy liver. This was associated with elevated levels of circulating M30 (median = 244 U/l vs. 37 U/l in healthy controls, p = 0.009) which correlated with tumour volume (r2 = 0.92). Patients with progressive disease during chemotherapy showed higher M30 levels before therapy than responders (745 U/l vs. 136 U/l, p = 0.016). The predictive potential of M30 was higher than that of the tumour markers CA19-9 or CEA (AUC: 0.93, 0.63, and 0.78, respectively). CONCLUSIONS: Apoptotic tumour cells release cellular debris into the circulation, which provides information about tumour size and vitality.

The predictive value of comorbidities on postoperative complication rates and overall survival in left-sided oncological colorectal resections: a multicentre cohort study.

INTRODUCTION: Surgical- and nonsurgical complications significantly worsen postoperative outcomes, and identification of patients at risk is crucial to improve care. This study investigated whether comorbidities, graded by the Charlson Comorbidity Index (CCI), impact complication rates and impair long-term outcome in a cohort of left-sided colorectal resections. METHODS: Retrospective analysis of patients undergoing oncological left-sided colorectal resections due to colorectal cancer between 01/2015 and 12/2020 in two referral centers in Austria using electronic medical records and national statistical bureau survival data. Patients with recurrent disease, peritoneal carcinomatosis, and emergency surgeries were excluded. Comorbidities were assessed using the CCI, and complication severity was defined by the Clavien-Dindo classification (CDC). Logistic regression analysis was performed to identify factors influencing the risk for postoperative complications, and overall survival was assessed using data from the national statistics bureau. RESULTS: A total of 471 patients were analyzed. Multinominal logistic regression analysis identified a CCI greater than or equal to 6 ( P =0.049; OR 1.59, 95% CI: 1.10-2.54) and male sex ( P =0.022; OR 1.47, 95% CI: 1.21-2.98) as independent risk factors for major complications. While patients with a high CCI had the worst postoperative survival rates, perioperative complications only impacted on overall survival in patients with low CCIs, but not in patients with high CCIs. CONCLUSION: Although a high CCI is a risk factor for major postoperative complications, the presence of comorbidities should not result in withholding surgery.

GLP-2 Improves Hepatic Inflammation and Fibrosis in Mdr2-/- Mice Via Activation of NR4a1/Nur77 in Hepatic Stellate Cells and Intestinal FXR Signaling.

BACKGROUND & AIMS: Glucagon-like peptide (GLP)-2 may exert antifibrotic effects on hepatic stellate cells (HSCs). Thus, we aimed to test whether application of the GLP-2 analogue teduglutide has hepatoprotective and antifibrotic effects in the Mdr2/Abcb4-/- mouse model of sclerosing cholangitis displaying hepatic inflammation and fibrosis. METHODS: Mdr2-/- mice were injected daily for 4 weeks with teduglutide followed by gene expression profiling (bulk liver; isolated HSCs) and immunohistochemistry. Activated HSCs (LX2 cells) and immortalized human hepatocytes and human intestinal organoids were treated with GLP-2. mRNA profiling by reverse transcription polymerase chain reaction and electrophoretic mobility shift assay using cytosolic and nuclear protein extracts was performed. RESULTS: Hepatic inflammation, fibrosis, and reactive cholangiocyte phenotype were improved in GLP-2-treated Mdr2-/- mice. Primary HSCs isolated from Mdr2-/- mice and LX2 cells exposed to GLP-2 in vitro displayed significantly increased mRNA expression levels of NR4a1/Nur77 (P < .05). Electrophoretic mobility shift assay revealed an increased nuclear NR4a1 binding after GLP-2 treatment in LX2 cells. Moreover, GLP-2 alleviated the Tgfß-mediated reduction of NR4a1 nuclear binding activity. In vivo, GLP-2 treatment of Mdr2-/- mice resulted in increased intrahepatic levels of muricholic acids (accordingly Cyp2c70 mRNA expression was significantly increased), and in reduced mRNA levels of Cyp7a1 and FXR. Serum Fgf15 levels were increased in Mdr2-/- mice treated with GLP-2. Accordingly, GLP-2 treatment of human intestinal organoids activated their FXR-FGF19 signaling axis. CONCLUSIONS: GLP-2 treatment increased NR4a1/Nur77 activation in HSCs, subsequently attenuating their activation. GLP-2 promoted intestinal Fxr-Fgf15/19 signaling resulting in reduced Cyp7a1 and increased Cyp2c70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/- mouse model.

Multiomics-empowered Deep Phenotyping of Ulcerative Colitis Identifies Biomarker Signatures Reporting Functional Remission States.

INTRODUCTION: Ulcerative colitis [UC] is a chronic disease with rising incidence and unclear aetiology. Deep molecular phenotyping by multiomics analyses may provide novel insights into disease processes and characteristic features of remission states. METHODS: UC pathomechanisms were assessed by proteome profiling of human tissue specimens, obtained from five distinct colon locations for each of the 12 patients included in the study. Systemic disease-associated alterations were evaluated thanks to a cross-sectional setting of mass spectrometry-based multiomics analyses comprising proteins, metabolites, and eicosanoids of plasma obtained from UC patients during acute episodes and upon remission, in comparison with healthy controls. RESULTS: Tissue proteome profiling indicated colitis-associated activation of neutrophils, macrophages, B and T cells, fibroblasts, endothelial cells and platelets, and hypoxic stress, and suggested a general downregulation of mitochondrial proteins accompanying the establishment of apparent wound healing-promoting activities including scar formation. Whereas pro-inflammatory proteins were apparently upregulated by immune cells, the colitis-associated epithelial cells, fibroblasts, endothelial cells, and platelets seemed to predominantly contribute anti-inflammatory and wound healing-promoting proteins. Blood plasma proteomics indicated chronic inflammation and platelet activation, whereas plasma metabolomics identified disease-associated deregulations of gut and gut microbiome-derived metabolites. Upon remission several, but not all, molecular candidate biomarker levels recovered back to normal. CONCLUSION: The findings may indicate that microvascular damage and platelet deregulation hardly resolve upon remission, but apparently persist as disease-associated molecular signatures. This study presents local and systemic molecular alterations integrated in a model for UC pathomechanisms, potentially supporting the assessment of disease and remission states in UC patients.

Chronic CD40L blockade is required for long-term cardiac allograft survival with a clinically relevant CTLA4-Ig dosing regimen.

Introduction: In de-novo kidney transplantation, the CTLA4-Ig fusion protein belatacept is associated with improved graft function but also an increased risk of acute rejection compared to calcineurin inhibitor therapy. The combination with a second costimulation blocker could potentially improve outcome while avoiding calcineurin inhibitor toxicity. The aim of this study was to define the conditions under which the combination of CTLA4-Ig and CD40L blockade leads to rejection-free permanent graft survival in a stringent murine heart transplantation model. Methods: Naïve wild-type or CD40L (CD154) knock-out mice received a fully mismatched BALB/c cardiac allograft. Selected induction and maintenance protocols for CTLA4-Ig and blocking αCD40L monoclonal antibodies (mAB) were investigated. Graft survival, rejection severity and donor-specific antibody (DSA) formation were assessed during a 100-day follow-up period. Results and Discussion: Administering αCD40L mAb as monotherapy at the time of transplantation significantly prolonged heart allograft survival but did not further improve the outcome when given in addition to chronic CTLA4-Ig therapy (which prolongs graft survival to a median of 22 days). Likewise, chronic αCD40L mAb therapy (0.5mg) combined with perioperative CTLA4-Ig led to rejection in a proportion of mice and extensive histological damage, despite abrogating DSA formation. Only the permanent interruption of CD40-CD40L signaling by using CD40L-/- recipient mice or by chronic αCD40L administration synergized with chronic CTLA4-Ig to achieve long-term allograft survival with preserved histological graft integrity in all recipients without DSA formation. The combination of α-CD40L and CTLA4-Ig works most effectively when both therapeutics are administered chronically.