Chitinase 3-like-1 promotes intrahepatic activation of coagulation through induction of tissue factor in mice.

Coagulation is a critical component in the progression of liver disease. Identification of key molecules involved in the intrahepatic activation of coagulation (IAOC) will be instrumental in the development of effective therapies against liver disease. Using a mouse model of concanavalin A (ConA)-induced hepatitis, in which IAOC plays an essential role in causing liver injury, we uncovered a procoagulant function of chitinase 3-like 1 (Chi3l1). Chi3l1 expression is dramatically elevated after ConA challenge, which is dependent on ConA-induced T cell activation and the resulting interferon γ and tumor necrosis factor α productions. Compared with wild-type mice, Chi3l1-/- mice show less IAOC, reduced tissue factor (TF) expression, and attenuated liver injury. Reconstituting Chi3l1-/- mice with recombinant TF triggers IAOC and augments liver injury. CONCLUSION: Our data demonstrate that Chi3l1, through induction of TF via mitogen-activated protein kinase activation, promotes IAOC and tissue injury. (Hepatology 2018;67:2384-2396).

Ischaemia reperfusion injury in liver transplantation: Cellular and molecular mechanisms.

Liver disease causing end organ failure is a growing cause of mortality. In most cases, the only therapy is liver transplantation. However, liver transplantation is a complex undertaking and its success is dependent on a number of factors. In particular, liver transplantation is subject to the risks of ischaemia-reperfusion injury (IRI). Liver IRI has significant effects on the function of a liver after transplantation. The cellular and molecular mechanisms governing IRI in liver transplantation are numerous. They involve multiple cells types such as liver sinusoidal endothelial cells, hepatocytes, Kupffer cells, neutrophils and platelets acting via an interconnected network of molecular pathways such as activation of toll-like receptor signalling, alterations in micro-RNA expression, production of ROS, regulation of autophagy and activation of hypoxia-inducible factors. Interestingly, the cellular and molecular events in liver IRI can be correlated with clinical risk factors for IRI in liver transplantation such as donor organ steatosis, ischaemic times, donor age, and donor and recipient coagulopathy. Thus, understanding the relationship of the clinical risk factors for liver IRI to the cellular and molecular mechanisms that govern it is critical to higher levels of success after liver transplantation. This in turn will help in the discovery of therapeutics for IRI in liver transplantation - a process that will lead to improved outcomes for patients suffering from end-stage liver disease.

Biologics in organ transplantation.

The last two decades have witnessed a pandemic in antibody development, with over 600 entering clinical studies and a total of 28 approved by the FDA and European Union. The incorporation of biologics in transplantation has made a significant impact on allograft survival. Herein, we review the armamentarium of clinical and preclinical biologics used for organ transplantation--with the exception of belatacept--from depleting and IL-2R targeting induction agents to costimulation blockade, B-cell therapeutics, BAFF and complement inhibition, anti-adhesion, and anti-cytokine approaches. While individual agents may be insufficient for tolerance induction, they provide possibilities for reduction of steroid or calcineurin inhibitor use, alternatives to rejection episodes refractory to conventional therapies, and specialized immunosuppression for highly sensitized patients.

The Impact of Health Literacy on Kidney Transplant Listing.

Introduction: Limited health literacy has been associated with poor health outcomes in the general population, but there have been few studies investigating the association between functional health literacy and kidney transplant listing. The primary objective of this study was to determine if functional health literacy was associated with kidney transplant listing after controlling for demographic, psychosocial, and medical variables, which were secondarily examined for correlation with transplant listing. Design: We retrospectively reviewed 423 kidney transplant candidates who were prospectively administered the Test of Functional Health Literacy in Adults during their transplant evaluation. Results: The functional health literacy scores were found to correlate with transplant listing (P = 0.013). Unexpectedly, a subset of patients (n = 14 out of 36) who had scores < 59 was still able to obtain approval for listing. The probability of approval decreased when functional health literacy scores ranged from 0 to 59 and increased when functional health literacy scores varied between 60 to 100. Multivariable analysis found transplant listing to also be associated with substance use (OR = 0.15, P < 0.001), ESKD etiology other than diabetes or hypertension (OR = 2.62, P < 0.001), time on dialysis (P = 0.012), and pace of transplant evaluation (P < 0.001). Conclusion: Functional health literacy was associated with kidney transplant listing. Programmatic interventions that can help overcome the impact of functional health literacy and improve access to transplantation should be explored.

Hypoxia-inducible factor-1α-dependent induction of miR122 enhances hepatic ischemia tolerance.

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation.

Quantitative assessment of liver fibrosis by digital image analysis reveals correlation with qualitative clinical fibrosis staging in liver transplant patients.

Technologies for digitizing tissues provide important quantitative data for liver histopathology investigation. We aimed to assess liver fibrosis degree with quantitative morphometric measurements of histopathological sections utilizing digital image analysis (DIA) and to further investigate if a correlation with histopathologic scoring (Scheuer staging) exists. A retrospective study of patients with at least two post-liver transplant biopsies having a Scheuer stage of ≤ 2 at baseline were gathered. Portal tract fibrotic percentage (%) and size (µm2) were measured by DIA, while clinical fibrosis score was measured by the Scheuer system. Correlations between DIA measurements and Scheuer scores were computed by Spearman correlation analysis. Differences between mean levels of fibrosis (score, size, and percentage) at baseline versus second visit were computed by Student's t-test. P values < 0.05 were considered significant. Of 22 patients who met the study criteria, 54 biopsies were included for analysis. Average levels ±standard error [S.E.] of portal tract fibrotic percentage (%) and size (µm2) progressed from 46.5 ± 3.6% at baseline to 61.8 ± 3.8% at the second visit (P = 0.005 by Student's t-test), and from 28,075 ± 3,232 µm2 at base line to 67,146 ± 10,639 µm2 at the second visit (P = 0.002 by Student's t-test), respectively. Average levels of Scheuer fibrosis scores progressed from 0.55±0.19 at baseline to 1.14±0.26 at the second visit (P = 0.02 by Student's t-test). Portal tract fibrotic percentage (%) and portal tract fibrotic size were directly correlated with clinical Scheuer fibrosis stage, with Spearman correlation coefficient and P value computed as r = 0.70, P < 0.0001 and r = 0.41, P = 0.002, respectively. Digital quantitative assessment of portal triad size and fibrosis percentage demonstrates a strong correlation with visually assessed histologic stage of liver fibrosis and complements the standard assessment for allograft monitoring, suggesting the utility of future WSI analysis.

Mst1/2 kinases restrain transformation in a novel transgenic model of Ras driven non-small cell lung cancer.

Non-small cell lung cancer remains a highly lethal malignancy. Using the tamoxifen inducible Hnf1b:CreERT2 (H) transgenic mouse crossed to the LsL-KrasG12D (K) transgenic mouse, we recently discovered that an Hnf1b positive cell type in the lung is sensitive to adenoma formation when expressing a mutant KrasG12D allele. In these mice, we observe adenoma formation over a time frame of three to six months. To study specificity of the inducible Hnf1b:CreERT2 in the lung, we employed lineage tracing using an mTmG (G) reporter allele. This technique revealed recombined, GFP+ cells were predominantly SPC+. We further employed this technique in HKG mice to determine Hnf1b+ cells give rise to adenomas that express SPC and TTF1. Review of murine lung tissue confirmed a diagnosis of adenoma and early adenocarcinoma, a pathologic subtype of non-small cell lung cancer. Our expanded mouse model revealed loss of Mst1/2 promotes aggressive lung adenocarcinoma and large-scale proteomic analysis revealed upregulation of PKM2 in the lungs of mice with genetic deletion of Mst1/2. PKM2 is a known metabolic regulator in proliferating cells and cancer. Using a human lung adenocarcinoma cell line, we show pharmacologic inhibition of Mst1/2 increases the abundance of PKM2, indicating genetic loss or pharmacologic inhibition of Mst1/2 directly modulates the abundance of PKM2. In conclusion, here we report a novel model of non-small cell lung cancer driven by a mutation in Kras and deletion of Mst1/2 kinases. Tumor development is restricted to a subset of alveolar type II cells expressing Hnf1b. Our data show loss of Mst1/2 regulates levels of a potent metabolic regulator, PKM2.

Exploration of the Stanford Integrated Psychosocial Assessment for Transplantation With Psychosocial and Medical Outcomes in Kidney and Kidney-Pancreas Transplant Recipients.

INTRODUCTION: The Stanford Integrated Psychosocial Assessment for Transplantation (SIPAT) is a psychometric instrument designed to assess patient risk for transplant. We investigated the association between SIPAT scores and demographic data with psychosocial and medical outcomes within a diverse kidney/kidney-pancreas transplant population. DESIGN: The SIPAT was administered to all pretransplant candidates. A retrospective review of transplanted patients who had at least 6 months of follow-up was completed. RESULTS: The sample included 136 patients: male (n = 77 [57%]) with a mean age of 47 years old. Thirty-eight percent were black (n = 51), 55% had less than a high school education (n = 74), and 65% had low socioeconomic status (n = 89). Statistical difference was found among SIPAT scores and substance use and support system instability (P = .035, P = .012). Females (P = .012) and patients with a history of psychopathology (P = .002) developed or had a relapse of psychopathology following transplant. Patients with more than a high school education (P = .025) and who were less than 30 years (P = .026) had higher rejection incidence rates. Risk factors for rehospitalizations included Hispanic race, diabetes, and low socioeconomic status (P = .036, P = .038, P = .014). African American/Black and male patients had higher incidence of infection events (P = .032, P = .049). Mortality and treatment nonadherence were not significantly associated with SIPAT scores or demographic variables. CONCLUSION: The SIPAT was associated with posttransplant substance use and support system instability, while demographic variables were associated with the development and/or relapse of psychopathology, graft loss, rejection, infection events, and medical rehospitalizations. Revision of the SIPAT to include additional demographic components may lend to improved prediction of transplant outcomes.

Seroprevalence of Strongyloides stercoralis and Evaluation of Universal Screening in Kidney Transplant Candidates: A Single-Center Experience in Houston (2012-2017).

BACKGROUND: Disseminated strongyloidiasis in solid organ transplant recipients is a rare but devastating infection. In our center, we implemented a universal screening of all candidates for kidney transplantation. We assessed the seroprevalence and utility of universal screening for strongyloidiasis in our center. METHODS: Patients were identified from our transplant referral list (from July 2012 to June 2017). Demographics, pretransplant laboratory, and serological screenings were retrospectively collected. For Strongyloides-seropositive (SSp) patients, data on travel history, symptoms, treatment, and stool ova and parasite examinations were extracted. Logistic regression and multiple imputation for missing data were performed. RESULTS: A total of 1689 patients underwent serological screening, of whom 168 (9.9%) were SSp. Univariate analysis revealed that SSp patients had higher rates of eosinophilia, diabetes mellitus, latent tuberculosis and were likely to be either Hispanic or Asian (P < .05). In multivariate analysis, eosinophilia (P = .01), diabetes mellitus (P = .02), and Asian race (P = .03) were associated with being SSp, but 45 (27%) of the SSp patients did not have any of these 3 factors, and 18 SSp patients (11%) had no epidemiological risk factors. All patients received ivermectin, and none developed disseminated strongyloidiasis. Of patients who underwent serological screening on multiple occasions, 6.8% seroconverted while waiting for kidney transplantation. CONCLUSIONS: We found a high rate of Strongyloides seropositivity among our kidney transplantation candidates. No epidemiological risk factors effectively predicted SSp status in our population, and universal screening identified a large number of patients without such factors. Serial screening should be considered when a long wait time is expected before transplantation.

Mutant p53R175H promotes cancer initiation in the pancreas by stabilizing HSP70.

Pancreatic cancer remains a highly lethal malignancy. We have recently shown that simultaneous expression of Kras and mutant Tp53R175H promotes invasive ductal adenocarcinoma from pancreatic ductal cells. We hypothesized specific mutations in TP53 have divergent mechanisms of transforming ductal cells. In order to understand the role of mutant TP53 in transforming pancreatic ductal cells, we used a lentiviral system to express mutant TP53R175H and TP53R273H, two of the most frequently mutated TP53 alleles in pancreatic cancer patients, in immortalized, but not transformed, pancreatic ductal epithelial cells carrying a KRAS mutation (HPNE:KRASG12D). Mutant TP53 expression enhanced colony formation and an RPPA assay results revealed TP53R175H uniquely induced HSP70 expression in HPNE:KRASG12D cells. In the context of TP53R175H expression; we observed nuclear localization of HSP70. We performed immunoprecipitation experiments to show mutant p53R175H binds to HSP70. We also provide evidence mutant p53R175H is important for HSP70 stability and, more importantly, HSP70 is required for mutant p53 stability. These data are critical in the context of events leading to cellular transformation in the pancreas.

The New Kidney Donor Allocation System and Implications for Anesthesiologists.

Given potential disparity and limited allocation of deceased donor kidneys for transplantation, a new federal kidney allocation system was implemented in 2014. Donor organ function and estimated recipient survival in this system has implications for perioperative management of kidney transplant recipients. Early analysis suggests that many of the anticipated goals are being attained. For anesthesiologists, implications of increased dialysis duration and burdens of end-stage renal disease include increased cardiopulmonary disease, challenging fluid, hemodynamic management, and central vein access. With no recent evidence to guide anesthesia care within this new system, we describe the kidney allocation system, summarize initial data, and briefly review organ systems of interest to anesthesiologists. As additional invasive and echocardiographic monitoring may be indicated, one consideration may be development of a dedicated anesthesiology team experienced in management and monitoring of complex patients, in a similar manner as has been done for liver transplant recipients.

Retrograde Flushing of Living Donor Renal Allografts via the Renal Vein: A Simple, Effective Technique.

BACKGROUND: Prograde flushing (PF) of living donor renal allografts with preservation solution via the renal artery or arteries is standard practice. PF may be difficult and potentially injurious to the donor kidney, especially in grafts with small or multiple arteries. In this report, we present our experience with retrograde flushing (RF) of 7 living donor kidneys via the renal vein. METHODS: Retrospective review of 7 consecutive living donor renal transplants performed using the RF technique was performed. The 7 preceding living donor renal transplants performed using the standard arterial PF technique served as a control group. RESULTS: All 7 recipients of RF kidneys experienced immediate graft function. At postoperative days 3 and 30, there was no difference in estimated glomerular filtration rate between the RF study group and PF controls. CONCLUSIONS: The RF technique is simple and safe, with results equivalent to the PF technique. The RF technique may be especially useful after recovering kidneys with small and/or multiple arteries.

Tolerogenic therapies in transplantation.

Since the concept of immunologic tolerance was discovered in the 1940s, the pursuit of tolerance induction in human transplantation has led to a rapid development of pharmacologic and biologic agents. Short-term graft survival remains an all-time high, but successful withdrawal of immunosuppression to achieve operational tolerance rarely occurs outside of liver transplantation. Collaborative efforts through the NIH sponsored Immune Tolerance Network and the European Commission sponsored Reprogramming the Immune System for Establishment of Tolerance consortia have afforded researchers opportunity to evaluate the safety and efficacy of tolerogenic strategies, investigate mechanisms of tolerance, and identify molecular and genetic markers that distinguish the tolerance phenotype. In this article, we review traditional and novel approaches to inducing tolerance for organ transplantation, with an emphasis on their translation into clinical trials.

CXCR3-mediated T-cell chemotaxis involves ZAP-70 and is regulated by signalling through the T-cell receptor.

The chemokine receptor CXCR3 is critical for the function of activated T cells. We studied the molecular mechanisms of CXCR3 signalling. The addition of CXCR3 ligands to normal human T cells expressing CXCR3 led to the tyrosine phosphorylation of multiple proteins. Addition of the same ligands to Jurkat T cells engineered to express CXCR3 induced tyrosine phosphorylation of proteins with molecular weights similar to those in normal cells. Immunoblotting with phosphotyrosine-specific antibodies identified Zeta-associated protein of 70,000 molecular weight (ZAP-70), linker for the activation of T cells (LAT), and phospholipase-C-gamma1 (PLCgamma1) to be among the proteins that become phosphorylated upon CXCR3 activation. ZAP-70 was phosphorylated on tyrosine 319, LAT on tyrosines 171 and 191, and PLCgamma1 on tyrosine 783. The ZAP-70 inhibitor piceatannol reduced CXCR3-mediated tyrosine phosphorylation of ZAP-70, LAT, PLCgamma1 and mitogen-activated protein kinase Erk and it reduced CXCL10-mediated chemotaxis of both CXCR3-transfected Jurkat T cells and normal T cells expressing CXCR3. These results are consistent with the involvement of ZAP-70 in CXCR3-mediated protein tyrosine phosphorylation and CXCR3-induced T-cell chemotaxis. Studies with the Lck-deficient Jurkat T-cell line, JCAM1.6, demonstrated that phosphorylation of ZAP-70 after CXCR3 activation is a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function.