Very Early Pouchitis Is Associated with an Increased Likelihood of Chronic Inflammatory Conditions of the Pouch.

INTRODUCTION: Chronic inflammatory conditions of the pouch are common after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC). AIMS: We aimed to investigate the relationship between acute pouchitis within 180 days of the final stage of IPAA surgery (very early pouchitis) and the future development of chronic antibiotic dependent pouchitis (CADP) and Crohn's-like disease of the pouch (CLDP). METHODS: We performed a retrospective cohort study, evaluating patients who underwent proctocolectomy with IPAA between January 1, 2004 and December 31, 2016. Multivariable logistic regression was used to evaluate the relationship between very early pouchitis and the development of CADP and CLDP. RESULTS: Among 626 patients undergoing IPAA for UC, 137 (22%) developed very early pouchitis, 75 (12%) developed CADP, and 59 (9%) developed CLDP in a median follow-up of 5.18 years (interquartile range 0.94-10.8 years). Very early pouchitis was associated with a significant increase in the odds of developing CADP (adjusted odds ratio [aOR3.65, 95% CI 2.19-6.10) as was primary sclerosing cholangitis (aOR 3.97, 95% CI 1.44-11.0). Very early pouchitis was associated with increased odds for developing CLDP (aOR 2.77, 95% CI 1.54-4.98) along with a family history of inflammatory bowel disease (aOR 2.10, 95% CI 1.11-3.96). CONCLUSION: In this cohort, very early pouchitis was associated with an increased risk of developing CADP and CLDP. These findings highlight very early pouchitis as a unique risk factor for chronic inflammatory conditions of the pouch and the need for future studies evaluating potential strategies for secondary prophylaxis strategies in this population.

Modulating donor mitochondrial fusion/fission delivers immunoprotective effects in cardiac transplantation.

Early insults associated with cardiac transplantation increase the immunogenicity of donor microvascular endothelial cells (ECs), which interact with recipient alloreactive memory T cells and promote responses leading to allograft rejection. Thus, modulating EC immunogenicity could potentially alter T cell responses. Recent studies have shown modulating mitochondrial fusion/fission alters immune cell phenotype. Here, we assess whether modulating mitochondrial fusion/fission reduces EC immunogenicity and alters EC-T cell interactions. By knocking down DRP1, a mitochondrial fission protein, or by using the small molecules M1, a fusion promoter, and Mdivi1, a fission inhibitor, we demonstrate that promoting mitochondrial fusion reduced EC immunogenicity to allogeneic CD8+ T cells, shown by decreased T cell cytotoxic proteins, decreased EC VCAM-1, MHC-I expression, and increased PD-L1 expression. Co-cultured T cells also displayed decreased memory frequencies and Ki-67 proliferative index. For in vivo significance, we used a novel murine brain-dead donor transplant model. Balb/c hearts pretreated with M1/Mdivi1 after brain-death induction were heterotopically transplanted into C57BL/6 recipients. We demonstrate that, in line with our in vitro studies, M1/Mdivi1 pretreatment protected cardiac allografts from injury, decreased infiltrating T cell production of cytotoxic proteins, and prolonged allograft survival. Collectively, our data show promoting mitochondrial fusion in donor ECs mitigates recipient T cell responses and leads to significantly improved cardiac transplant survival.

Predictors and Outcomes of Ustekinumab Dose Intensification in Ulcerative Colitis: A Multicenter Cohort Study.

Ustekinumab has been shown to be effective for the treatment of ulcerative colitis (UC); however, >40% of patients have suboptimal clinical response after induction and maintenance dosing every 8 weeks.1,2 The best management approach for these patients is unclear. Many undergo empiric dose intensification to every 4 weeks or every 6 weeks, a nonstandardized decision because of limited data supporting therapeutic drug monitoring of ustekinumab.3 In Crohn's disease, approximately 50% of patients undergo ustekinumab dose intensification, which seems to be effective based on prior work from our group and others.4-8 However, similar data in UC are lacking. In this real-world multicenter cohort study, we sought to identify predictors and outcomes of ustekinumab dose intensification in UC.

Relationship Between Stages of Ileal Pouch-Anal Anastomosis, Timing of Restoration of Fecal Continuity, and Pouchitis.

BACKGROUND: The most common complication following ileal pouch-anal anastomosis (IPAA) in patients with ulcerative colitis (UC) is pouchitis. AIMS: We aimed to investigate whether a shorter period between pouch creation and restoration of fecal flow through an IPAA was associated with an increased risk of development of pouchitis within the first 2 years after IPAA. METHODS: We performed a retrospective cohort study evaluating patients undergoing colectomy with IPAA for UC between January 1, 2004 and December 31, 2016. We used Kaplan-Meier testing and Cox Proportional Hazards Modeling to evaluate the relationship between the time between restoration of fecal continuity and time to subsequent development of pouchitis, adjusting for other clinical and demographic factors. RESULTS: We identified 624 patients who underwent proctocolectomy with IPAA for UC, of whom 246 (39%) developed pouchitis within the first 2 years after IPAA. There was no difference when comparing the median time to restoration of continuity among those patients who developed pouchitis and those who did not (49 days vs. 49 days, p = 0.85) or in multivariable analysis. Primary sclerosing cholangitis (Hazard Ratio [HR] 2.14, 95% CI 1.12-4.08), family history of inflammatory bowel disease (HR 1.49, 95% CI 1.08-2.06), and delayed pouch creation (HR 0.75, 95% CI 0.57-1.00) were significantly associated with time to development of pouchitis. CONCLUSION: Although a staged approach to IPAA may have benefits in the surgical management of UC, the timing interval between pouch creation and restoration of continuity did not impact the subsequent development of early pouchitis in this cohort.

Emphysema-associated Autoreactive Antibodies Exacerbate Post-Lung Transplant Ischemia-Reperfusion Injury.

Chronic obstructive pulmonary disease-associated chronic inflammation has been shown to lead to an autoimmune phenotype characterized in part by the presence of lung autoreactive antibodies. We hypothesized that ischemia-reperfusion injury (IRI) liberates epitopes that would facilitate preexisting autoantibody binding, thereby exacerbating lung injury after transplant. We induced emphysema in C57BL/6 mice through 6 months of cigarette smoke (CS) exposure. Mice with CS exposure had significantly elevated serum autoantibodies compared with non-smoke-exposed age-matched (NS) mice. To determine the impact of a full preexisting autoantibody repertoire on IRI, we transplanted BALB/c donor lungs into NS or CS recipients and analyzed grafts 48 hours after transplant. CS recipients had significantly increased lung injury and immune cell infiltration after transplant. Immunofluorescence staining revealed increased IgM, IgG, and C3d deposition in CS recipients. To exclude confounding alloreactivity and confirm the role of preexisting autoantibodies in IRI, syngeneic Rag1-/- (recombination-activating protein 1-knockout) transplants were performed in which recipients were reconstituted with pooled serum from CS or NS mice. Serum from CS-exposed mice significantly increased IRI compared with control mice, with trends in antibody and C3d deposition similar to those seen in allografts. These data demonstrate that pretransplant CS exposure is associated with increased IgM/IgG autoantibodies, which, upon transplant, bind to the donor lung, activate complement, and exacerbate post-transplant IRI.

Dietary Saturated Fat Promotes Development of Hepatic Inflammation Through Toll-Like Receptor 4 in Mice.

Nonalcoholic steatohepatitis (NASH) is currently the third most common cause of end stage liver disease necessitating transplantation. The question remains how inflammation and NASH develop in the setting of nonalcoholic fatty liver disease (NAFLD) and steatosis. Understand the roles of toll-like receptor 4 (TLR4) and dietary fats in the development of hepatic inflammation. Wild-type and TLR4 KO mice were fed a standard high fat diet (LD), a high saturated fat diet (MD), or an isocaloric control diet (CD). Sera and tissue were analyzed for development of hepatic steatosis, inflammation, and injury. MD induced features of hepatic steatosis and inflammation in wild-type, but not in TLR4 KO, mice. TLR4 KO prevented MD induced increases in NAFLD activity scores, serum alanine aminotransferase levels, and inflammatory cytokine expression. Inflammatory cell infiltration and cytokine expression were also lower in the TLR4 KO mice livers than wild-type mice fed MD. Hepatic expression of Collagen I transcripts and collagen deposition were also decreased in the TLR4 KO MD animals. Results show that TLR4 plays a critical role in the effects of dietary fat composition on the development of hepatic steatosis, inflammation, and injury consistent with nonalcoholic steatohepatitis. J. Cell. Biochem. 117: 1613-1621, 2016. © 2015 Wiley Periodicals, Inc.

A simplified cuff technique for abdominal aortic transplantation in mice.

BACKGROUND: Allograft arteriopathy is still a leading cause of late organ failure. The aortic allograft model in mice has been used to study chronic rejection and has given useful information in the development of graft arteriosclerosis. However, the technical difficulties of small vessel anastomoses still continue to limit its widespread use. We introduce a new simple method for aortic transplantation in mice. METHODS: The descending aorta or infrarenal aorta from the donor mouse was anastomosed to the infrarenal aorta using a cuff technique. Aortic transplantation was performed in 30 mice, 10 isografts and 20 allografts. No immunosuppression was administered, and the recipients were sacrificed at day 28. The grafts were histologically analyzed. RESULTS: Implantation of grafts could be completed in an average of 23 min. There was no technical failure in all 60 anastomoses. The overall survival rate was 93.3%. Histology of aortas revealed typical aspects of chronic rejection in the allografts at day 28. No significant lesion was observed in isografts. CONCLUSIONS: We have developed an innovative, stable, and simple aortic transplantation model in mice, which is useful for vascular research in transplantation and beyond.

Extraintestinal Manifestations and Family History of Inflammatory Bowel Disease Increase the Risk of Pouchitis in a State-Level Epidemiology Study.

INTRODUCTION: Our understanding of the epidemiology of inflammatory conditions of the pouch and effectiveness of treatment is largely based on selected populations. We created a state-level registry to evaluate the incidence of pouchitis and the effectiveness of treatments used in an initial episode of pouchitis. METHODS: In a state-level retrospective cohort of all patients undergoing proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis between January 1, 2018, and December 31, 2020, we evaluated the incidence of pouchitis and compared the proportion of patients developing recurrent pouchitis and chronic antibiotic-dependent pouchitis according to initial antibiotic therapy. RESULTS: A total of 177 patients underwent surgery with 49 (28%) developing pouchitis within the 12 months after the final stage of IPAA. Patients with extraintestinal manifestations of inflammatory bowel disease (IBD) were significantly more likely to develop pouchitis within the first 12 months after IPAA (adjusted odds ratio 2.45, 95% confidence interval 1.03-5.81) after adjusting for family history of IBD (adjusted odds ratio 3.50, 95% 1.50-8.18). When comparing the proportion of patients who developed recurrent pouchitis or chronic antibiotic-dependent pouchitis with those who experienced an isolated episode of pouchitis, there were no significant differences among the initial antibiotic regimens used. DISCUSSION: In a state-level examination of outcomes after IPAA for ulcerative colitis, patients with extraintestinal manifestations of IBD were more likely to develop pouchitis; however, the initial antibiotic regimen chosen did not seem to affect long-term outcomes.

Extensive Colitis and Smoking Are Associated With Postoperative Complications Within 30 Days of Ileal Pouch-Anal Anastomosis.

BACKGROUND: Our understanding of outcomes after proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) is largely based on analyses of selected populations. We created a state-level registry to evaluate the epidemiology of IPAA surgery and pouch-related outcomes across the major healthcare systems performing these surgeries in our state. METHODS: We created a retrospective cohort of all patients undergoing restorative proctocolectomy with IPAA for UC at 1 of 4 centers between January 1, 2018, and December 31, 2020. The primary outcomes of this study were the rate of complications and all-cause readmissions within the first 30 days of the final stage of IPAA surgery. RESULTS: During the study period, 177 patients underwent IPAA surgery with 66 (37%) experiencing a complication within 30 days. After adjusting for the number of stages in IPAA surgery, patients with extensive UC (odds ratio, 3.61; 95% confidence interval, 1.39-9.33) and current or former smokers (odds ratio, 2.98; 95% confidence interval, 1.38-6.45) were more likely to experience a complication. Among all patients, 57 (32%) required readmission within 30 days. The most common reasons for readmission were ileus/small bowel obstruction (22%), peripouch abscess (19%), and dehydration (16%). CONCLUSION: In this first state-level examination of the epidemiology of IPAA for UC, we demonstrated that the complication rate after IPAA for UC was 37%, with one-third of patients being readmitted within 30 days. Extensive disease at the time of colectomy appears to be an indicator of more severe disease and may portend a worse prognosis after IPAA.

Transmural Inflammation, Ileitis, and Granulomas at the Time of Proctocolectomy in Patients with Ulcerative Colitis Do Not Predict Future Development of Pouchitis.

BACKGROUND: The most common complication following ileal pouch-anal anastomosis (IPAA) in patients with ulcerative colitis (UC) is pouchitis. Our study aimed to investigate the relationship between histopathologic findings of ileitis, granuloma, or transmural inflammation on the colectomy specimen of patients with clinically and endoscopically diagnosed UC and the development of pouchitis within the first 2 years after IPAA. METHODS: We performed a retrospective cohort study evaluating patients undergoing colectomy with IPAA for UC between January 1, 2004 and December 31, 2016. Bivariate analyses were conducted to evaluate the relationship between clinical factors and the development of pouchitis. We performed multivariate logistic regression to evaluate the relationship between histologic, clinical, and demographic factors at the time of colectomy and subsequent development of pouchitis. RESULTS: Among 626 patients, pouchitis occurred in 246 (39%). Patients with primary sclerosing cholangitis were more likely to develop pouchitis (adjusted odds ratio [aOR] 2.81, 95% confidence interval [CI] 1.02-7.72), as were patients with a family history of inflammatory bowel disease (aOR 1.75, 95% CI 1.11-2.77). Histologic findings of ileitis, granuloma, or transmural inflammation were not associated with an increased odds of developing pouchitis (aOR 0.70, 95% CI 0.45-1.08). DISCUSSION/CONCLUSION: Patients with ileitis, granulomas, or transmural inflammation at the time of colectomy were not at greater risk for development of pouchitis in the 2 years after IPAA. These pathological findings should not preclude IPAA for UC.

Impact of Mitochondrial Permeability on Endothelial Cell Immunogenicity in Transplantation.

BACKGROUND: Microvascular endothelial cells (ECs) are central to an allograft's immunogenicity. Cold ischemia and reperfusion injury associated with static cold storage and warm reperfusion activates ECs and increases the immunogenicity of the allograft. After reperfusion, mitochondrial permeability transition pore (mPTP) opening contributes to mitochondrial dysfunction in the allograft, which correlates to alloimmune rejection. Current understanding of this relationship, however, centers on the whole allograft instead of ECs. This study aimed to elucidate the relationship between EC mPTP opening and their immunophenotype. METHODS: Mitochondrial metabolic fitness and glycolysis in ECs were assessed in parallel with metabolic gene microarray postreperfusion. NIM811 was used to inhibit mPTP opening to rescue mitochondrial fitness. The immunogenicity of NIM811-treated ECs was determined via levels of EC's proinflammatory cytokines and allogeneic CD8 T cell cocultures. Finally, EC surface expression of adhesion, costimulatory, coinhibitory, MHC-I molecules, and MHC-I machinery protein levels were characterized. RESULTS: Genes for glycolysis, tricarboxylic acid cycle, fatty acid synthesis, gluconeogenesis were upregulated at 6 hours postreperfusion but either normalized or downregulated at 24 hours postreperfusion. As mitochondrial fitness was reduced, glycolysis increased during the first 6 hours postreperfusion. Endothelial cell treatment with NIM811 during the early postreperfusion period rescued mitochondrial fitness and reduced EC immunogenicity by decreasing CCL2, KC release, and VCAM-1, MHC-I, TAP1 expression. CONCLUSIONS: Static cold storage and warm reperfusion leads to a reduction in mitochondrial fitness in microvascular ECs due to mPTP opening. Further, mPTP opening promotes increased EC immunogenicity that can be prevented by NIM811 treatment.

Organ preservation with targeted rapamycin nanoparticles: a pre-treatment strategy preventing chronic rejection in vivo.

Hypothermic preservation is the standard of care for storing organs prior to transplantation. Endothelial and epithelial injury associated with hypothermic storage causes downstream graft injury and, as such, the choice of an ideal donor organ preservation solution remains controversial. Cold storage solutions, by design, minimize cellular necrosis and optimize cellular osmotic potential, but do little to assuage immunological cell activation or immune cell priming post transplantation. Thus, here we explore the efficacy of our previously described novel Targeted Rapamycin Micelles (TRaM) as an additive to standard-of-care University of Wisconsin preservation solution as a means to alter the immunological microenvironment post transplantation using in vivo models of tracheal and aortic allograft transplantation. In all models of transplantation, grafts pre-treated with 100 ng mL-1 of TRaM augmented preservation solution ex vivo showed a significant inhibition of chronic rejection post-transplantation, as compared to UW augmented with free rapamycin at a ten-fold higher dose. Here, for the first time, we present a novel method of organ pretreatment using a nanotherapeutic-based cellular targeted delivery system that enables donor administration of rapamycin, at a ten-fold decreased dose during cold storage. Clinically, these pretreatment strategies may positively impact post-transplant outcomes and can be readily translated to clinical scenarios.

Immunosuppressive nano-therapeutic micelles downregulate endothelial cell inflammation and immunogenicity.

In this study, we developed a stable, nontoxic novel micelle nanoparticle to attenuate responses of endothelial cell (EC) inflammation when subjected to oxidative stress, such as observed in organ transplantation. Targeted Rapamycin Micelles (TRaM) were synthesized using PEG-PE-amine and N-palmitoyl homocysteine (PHC) with further tailoring of the micelle using targeting peptides (cRGD) and labeling with far-red fluorescent dye for tracking during cellular uptake studies. Our results revealed that the TRaM was approximately 10 nm in diameter and underwent successful internalization in Human Umbilical Vein EC (HUVEC) lines. Uptake efficiency of TRaM nanoparticles was improved with the addition of a targeting moiety. In addition, our TRaM therapy was able to downregulate both mouse cardiac endothelial cell (MCEC) and HUVEC production and release of the pro-inflammatory cytokines, IL-6 and IL-8 in normal oxygen tension and hypoxic conditions. We were also able to demonstrate a dose-dependent uptake of TRaM therapy into biologic tissues ex vivo. Taken together, these data demonstrate the feasibility of targeted drug delivery in transplantation, which has the potential for conferring local immunosuppressive effects without systemic consequences while also dampening endothelial cell injury responses.