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1.  CD47 Blockade Reduces Ischemia Reperfusion Injury and Improves Outcomes in a Rat Kidney Transplant Model 
Transplantation  2014;98(4):394-401.
Ischemia/reperfusion injury (IRI) significantly contributes to delayed graft function and inflammation leading to graft loss. IRI is exacerbated by the thrombospondin-1/CD47 system through inhibition of nitric oxide signaling. We postulate that CD47 blockade and prevention of nitric oxide inhibition reduces IRI in organ transplantation.
We used a syngeneic rat renal transplantation model of IRI with bilaterally nephrectomized recipients to evaluate the effect of a CD47 monoclonal antibody (CD47mAb) on IRI. Donor kidneys were flushed with CD47mAb OX101 or an isotype-matched control immunoglobulin and stored at 4°C in UW solution for 6 hours prior to transplantation.
CD47mAb perfusion of donor kidneys resulted in marked improvement in post-transplant survival, lower levels of serum creatinine, BUN, phosphorus and magnesium and less histologic evidence of injury. In contrast, control groups did not survive more than 5 days, had increased biochemical indicators of renal injury and exhibited severe pathological injury with tubular atrophy and necrosis. Recipients of CD47mAb-treated kidneys showed decreased levels of plasma biomarkers of renal injury including cystatin C, osteopontin, TIMP1, β2-microglobulin, VEGF-A and clusterin compared to the control group. Furthermore, laser Doppler assessment showed higher renal blood flow in the CD47mAb-treated kidneys.
These results provide strong evidence for the use of CD47 antibody-mediated blockade to reduce IRI and improve organ preservation for renal transplantation.
PMCID: PMC4887281  PMID: 24983310
Antibody therapy; Nitric oxide; Ischemia reperfusion injury; Kidney transplant
2.  Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma 
Cancer letters  2015;360(2):302-309.
Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.
PMCID: PMC4886734  PMID: 25721088
CD47; HCC; Antibody therapy; Xenograft models
3.  Delayed-onset cytomegalovirus disease coded during hospital readmission in a multicenter retrospective cohort of liver transplant recipients (Manuscript ID LT-14-570) 
Delayed-onset cytomegalovirus (CMV) disease can occur among liver transplant recipients after stopping CMV prophylaxis. We hypothesized that delayed-onset CMV disease (> 100 days post-transplant) occurs more commonly than early-onset CMV disease and is associated with clinical sepsis and death.
We assembled a large and more representative cohort of 7,229 adult liver transplant recipients from 26 transplant centers using 2004 to 2010 ICD-9-CM billing data from 4 Healthcare Cost and Utilization Project State Inpatient Databases, and identified demographics, comorbidities, CMV disease and clinical sepsis coded during readmission, and inpatient death. Multivariate analysis was performed using Cox proportional hazards models.
Delayed-onset CMV disease occurred in 4.3% (n=309), while early-onset CMV disease occurred in 2% (n=142). Delayed-onset CMV disease was associated with previous transplant failure or rejection (aHR 1.4, 95% CI 1.1-1.7). Clinical sepsis > 100 days post-transplant was associated with previous CMV disease (aHR 1.3, 95% CI 1.0-1.7), previous transplant failure or rejection (aHR 2.1, 95% CI 1.8-2.4), female sex (aHR 1.3, 95% CI 1.1-1.5) and several comorbidities. Death > 100 days post-transplant was associated with delayed-onset CMV disease (aHR 2.0, 95% CI 1.6-2.6), transplant failure or rejection (aHR 4.3, 95% CI 3.4-5.5), increasing age (by decade: aHR 1.1, 95% CI 1.0-1.2) and some comorbidities.
Delayed-onset CMV disease is more common than early-onset CMV disease among liver transplant recipients. Previous CMV disease may be a risk factor for clinical sepsis > 100 days post-transplant, and delayed-onset CMV disease may be a risk factor for death > 100 days post-transplant.
PMCID: PMC4545663  PMID: 25678072
outcomes research; survival analysis; administrative data; opportunistic infections; clinical sepsis
4.  BH3-only Proteins Contribute to Steatotic Liver IR injury 
The Journal of surgical research  2014;194(2):653-658.
Ischemia-reperfusion injury (IRI) to the liver continues to be a source of significant morbidity, especially in patients with hepatic steatosis. This is a growing problem given the increase in nonalcoholic fatty liver disease. BH3-only members of the BCL-2 protein family are known mediators of cellular apoptosis, although their role in hepatic IRI is still emerging. The goal of this study was to investigate the effect of Bim and Bid on warm hepatic IRI in the setting of steatosis.
Study Design
Lean and obese Bim/Bid wild type (WT) and double knock out (DKO) mice underwent 60 minutes of warm hepatic ischemia using a 70% segmental occlusion technique. Obesity and hepatic steatosis were induced using a high fat diet. Hepatocellular injury patterns were compared among lean and steatotic mice following reperfusion. Differences were analyzed using a student’s t-test and reported as mean ± SEM.
DKO mice were protected from IRI relative to WT. A high fat diet created equal degrees of steatosis in both WT and DKO mice. The IRI was increased in steatotic WT livers; however, DKO mice remained protected relative to WT despite hepatic steatosis.
The BH3-only proteins are important mediators of hepatic IRI in both lean and steatotic livers. These mechanisms have been underappreciated in steatotic liver injury and may be leveraged as targets for intervention in clinical scenarios such as transplant and hypovolemic shock.
PMCID: PMC4369674  PMID: 25483735
Ischemia-reperfusion; Steatosis; Apoptosis; BCL-2 proteins
5.  CD47 Blockade Reduces Ischemia/Reperfusion Injury and Improves Survival in a Rat Liver Transplantation Model 
Orthotopic liver transplantation (OLT) remains the standard treatment option for nonresponsive liver failure. Because ischemia/reperfusion injury (IRI) is an important impediment to the success of OLT, new therapeutic strategies are needed to reduce IRI. We investigated whether blocking the CD47/thrombospondin-1 inhibitory action on nitric oxide signaling with a monoclonal antibody specific to CD47 (CD47mAb400) would reduce IRI in liver grafts. Syngeneic OLT was performed with Lewis rats. Control immunoglobulin G or CD47mAb400 was administered to the donor organ at procurement or to both the organ and the recipient at the time of transplant. Serum transaminases, histological changes of the liver, and animal survival were assessed. Oxidative stress, inflammatory responses, and hepatocellular damage were also quantified. A significant survival benefit was not achieved when CD47mAb400 was administered to the donor alone. However, CD47mAb400 administration to both the donor and the recipient increased animal survival afterward. The CD47mAb400-treated group showed lower serum transaminases, bilirubin, oxidative stress, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining, caspase-3 activity, and proinflammatory cytokine expression of tumor necrosis factor α, interleukin-1β, and interleukin-6. Thus, CD47 blockade with CD47mAb400 administered both to the donor and the recipient reduced liver graft IRI in a rat liver transplantation model. This may translate to decreased liver dysfunction and increased survival of liver transplant recipients.
PMCID: PMC4601606  PMID: 25482981
6.  Epidemiology of Bloodstream Infections in a Multicenter Retrospective Cohort of Liver Transplant Recipients 
Transplantation Direct  2016;2(3):e67.
Supplemental digital content is available in the text.
Although some studies have examined the epidemiology of bloodstream infections after liver transplantation, they were based in single centers and did not identify bloodstream infections treated in other hospitals.
We retrospectively examined a cohort of 7912 adult liver transplant recipients from 24 transplant centers using 2004 to 2012 International Classification of Diseases, Ninth Revision, Clinical Modification billing data from 3 State Inpatient Databases, and identified bloodstream infections, inpatient death, and cumulative 1-year hospital costs. Multilevel Cox regression analyses were used to determine factors associated with bloodstream infections and death.
Bloodstream infections were identified in 29% (n = 2326) of liver transplant recipients, with a range of 19% to 40% across transplant centers. Only 63% of bloodstream infections occurring more than 100 days posttransplant were identified at the original transplant center. Bloodstream infections were associated with posttransplant laparotomy (adjusted hazard ratio [aHR], 1.52), prior liver transplant (aHR, 1.42), increasing age (aHR, 1.07/decade), and some comorbidities. Death was associated with bloodstream infections with and without septic shock (aHR, 10.96 and 3.71, respectively), transplant failure or rejection (aHR, 1.41), posttransplant laparotomy (aHR, 1.40), prior solid-organ transplant (aHR, 1.48), increasing age (aHR, 1.15/decade), and hepatitis C cirrhosis (aHR, 1.20). The risk of bloodstream infections and death varied across transplant centers. Median 1-year cumulative hospital costs were higher for patients who developed bloodstream infections within 1 year of transplant compared with patients who were bloodstream infection-free (US $229 806 vs US $111 313; P < 0.001).
Bloodstream infections are common and costly complications after liver transplantation that are associated with a markedly increased risk of death. The incidence and risk of developing bloodstream infections may vary across transplant centers.
PMCID: PMC4946508  PMID: 27458606
7.  Liver Volume in the Cirrhotic Patient: Does Size Matter? 
Digestive diseases and sciences  2014;59(4):886-891.
While it is established that cirrhosis results in a decrease in liver volume (LV), whether LV itself predicts patient survival is unknown. We hypothesize that estimated LV is an important prognostic indicator in patients with cirrhosis.
Data was gathered retrospectively from consecutive patients evaluated for a liver transplant from January 2001 to June 2006. Of 500 patients identified, 323 patients met both inclusion and exclusion criteria. LV per ideal body weight (IBW) was used to correct for body size, and LV/IBW was stratified by median split for survival analyses. Patients were classified into one of three clinical groups: hepatocellular disease (n = 229), cholestatic disease (n = 56), and miscellaneous (n = 38). One of three possible clinical outcomes (survival, liver transplantation, or death) was recorded during the 5-year follow-up, the latter two grouped together as “transplant/death.”
Transplant/death occurred in 283 (88 %) subjects. Overall, there was a significant increase in transplant/death in those with lower LV/IBW (χ2 = 5.27, p = 0.022). When considering the subset with hepatocellular disease, lower LV/IBW was a robust predictor of transplant/death (χ2 = 9.62, p = 0.002). In multivariate analyses, the LV/IBW trended toward predicting transplant/death (ExpB = 0.943, p = 0.053) independent of Model for End stage Liver Disease (MELD) (ExpB = 1.13, p = 0.001).
LV has important predictive value in patients with cirrhosis from hepatocellular disease. This observation appears to be independent of MELD, suggesting LV may impart important prognostic information that is not captured by the MELD score alone. Thus, LV may serve as an important adjunct to the MELD score in patients with hepatocellular disease.
PMCID: PMC4565509  PMID: 24504591
Cirrhosis; Liver volume; Liver transplant; MELD
8.  Current surgical treatment strategies for hepatocellular carcinoma in North America 
World Journal of Gastroenterology : WJG  2014;20(41):15007-15017.
Hepatocellular carcinoma (HCC) is an aggressive tumor that often occurs in the setting of chronic liver disease. Many patients do not initially manifest any symptoms of HCC and present late when cure with surgical resection or transplantation is no longer possible. For this reason, patients at high risk for developing HCC are subjected to frequent screening processes. The surgical management of HCC is complex and requires an inter-disciplinary approach. Hepatic resection is the treatment of choice for HCC in patients without cirrhosis and is indicated in some patients with early cirrhosis (Child-Pugh A). Liver transplantation has emerged in the past decade as the standard of care for patients with cirrhosis and HCC meeting Milan criteria and in select patients with HCC beyond Milan criteria. Loco-regional therapy with transarterial chemoembolization, transarterial embolization, radiofrequency ablation and other similar local treatments can be used as neo-adjuvant therapy to downstage HCC to within Milan criteria or as a bridge to transplantation in patients on transplant wait list.
PMCID: PMC4223234  PMID: 25386049
Hepatocellular carcinoma; Liver transplantation; Liver resection; Transarterial chemoembolization; Radiofrequency ablation
9.  Incorporation of a laser range scanner into image-guided liver surgery: Surface acquisition, registration, and tracking 
Medical physics  2003;30(7):1671-1682.
As image guided surgical procedures become increasingly diverse, there will be more scenarios where point-based fiducials cannot be accurately localized for registration and rigid body assumptions no longer hold. As a result, procedures will rely more frequently on anatomical surfaces for the basis of image alignment and will require intraoperative geometric data to measure and compensate for tissue deformation in the organ. In this paper we outline methods for which a laser range scanner may be used to accomplish these tasks intraoperatively. A laser range scanner based on the optical principle of triangulation acquires a dense set of three-dimensional point data in a very rapid, noncontact fashion. Phantom studies were performed to test the ability to link range scan data with traditional modes of image-guided surgery data through localization, registration, and tracking in physical space. The experiments demonstrate that the scanner is capable of localizing point-based fiducials to within 0.2 mm and capable of achieving point and surface based registrations with target registration error of less than 2.0 mm. Tracking points in physical space with the range scanning system yields an error of 1.4±0.8 mm. Surface deformation studies were performed with the range scanner in order to determine if this device was capable of acquiring enough information for compensation algorithms. In the surface deformation studies, the range scanner was able to detect changes in surface shape due to deformation comparable to those detected by tomographic image studies. Use of the range scanner has been approved for clinical trials, and an initial intraoperative range scan experiment is presented. In all of these studies, the primary source of error in range scan data is deterministically related to the position and orientation of the surface within the scanner’s field of view. However, this systematic error can be corrected, allowing the range scanner to provide a rapid, robust method of acquiring anatomical surfaces intraoperatively.
PMCID: PMC4445740  PMID: 12906184
10.  Novel APC promoter and exon 1B deletion and allelic silencing in three mutation-negative classic familial adenomatous polyposis families 
Genome Medicine  2015;7(1):42.
The overwhelming majority (approximately 80%) of individuals with classic familial adenomatous polyposis (FAP) exhibit mutations in the coding sequence of the adenomatous polyposis coli (APC) tumor suppressor gene. Families without detectable APC mutations are unable to benefit from the use of genetic testing for clinical management of this autosomal dominant syndrome.
We used exome sequencing and linkage analysis, coupled with second-generation sequencing of the APC locus including non-coding regions to investigate three APC mutation-negative classical FAP families.
We identified a novel ~11 kb deletion localized 44 kb upstream of the transcription start site of APC that encompasses the APC 1B promoter and exon. This deletion was present only in affected family members of one kindred with classical FAP. Furthermore, this same deletion with identical breakpoints was found in the probands of two additional APC mutation-negative classical FAP kindreds. Phasing analysis of single nucleotide polymorphisms (SNPs) around the deletion site in the three probands showed evidence of a shared haplotype, suggesting a common founder deletion in the three kindreds. SNP analysis within the coding sequence of APC, revealed that this ~11 kb deletion was accompanied by silencing of one of the APC alleles in blood-derived RNA of affected individuals.
These results support the causal role of a novel promoter deletion in FAP and suggest that non-coding deletions, identifiable using second-generation sequencing methods, may account for a significant fraction of APC mutation-negative classical FAP families.
PMCID: PMC4418073  PMID: 25941542
11.  Novel In Vitro Model for Studying Hepatic Ischemia-Reperfusion Injury Using Liver Cubes 
Surgery  2012;152(2):247-253.
While inflow occlusion techniques have given surgeons the ability to carry out increasingly complex liver resections, ischemia-reperfusion (IR) injury continues to be a source of morbidity. Efforts to ameliorate IR injury have been hindered in absence of adequate pre-clinical models. The goal of the present study was to develop a simple, efficient, and cost-effective means of studying hepatic IR injury.
Liver cubes were procured from normal (C57BL/6) mice. Following hepatectomy, 4 mm punch biopsies were taken for individual placement in culture wells containing hepatocyte media. Experimental cubes underwent hypoxia for 60 minutes, while controls remained normoxic. Supernatants were collected from individual wells following 0, 6 and 12 hours of rediffusion for transaminase and cytokine measurement. Histologic examination was performed on individual cubes.
Extensive histologic injury was seen in the experimental cubes compared to controls with greater staining for activated caspase-3 and TUNEL at 6 and 24 hours, respectively. Changes consistent with ischemic injury occurred more centrally in liver cubes whereas markers for rediffusion injury were appreciated along the periphery. Transaminases were significantly higher at 6 hours following rediffusion in experimental cubes compared to controls, p = 0.02. TNF-α and IL-1β were significantly higher in the media of experimental cubes compared to controls at 12 hours rediffusion, p = 0.05 and 0.03 respectively.
In vitro IR of cubes produces a significant injury whose pattern is reflective of hepatic lobular architecture. This novel technique may open new avenues for uncoupling the mechanisms of IR while facilitating rapid screening of potential therapies.
PMCID: PMC4258692  PMID: 22698934
12.  Aortic Balloon Valvuloplasty Prior to Orthotopic Liver Transplantation: A Novel Approach to Aortic Stenosis and End-Stage Liver Disease 
Case Reports in Cardiology  2014;2014:325136.
The combination of severe aortic stenosis and end-stage liver disease increases the morbidity and mortality of surgical aortic valve replacement or orthotopic liver transplantation resulting in a prohibitive operative risk. We propose a staged approach of balloon aortic valvuloplasty prior to orthotopic liver transplantation as a bridge to definitive aortic valve replacement. Between 2010 and 2012, four patients with severe aortic stenosis and end-stage liver disease underwent staged balloon aortic valvuloplasty followed by orthotopic liver transplantation. All patients had been deemed to be inappropriate candidates for liver transplantation or aortic valve surgery due to their comorbidity. One patient died of complications from a perivalvular abscess. Three patients went on to successful graft implantation and function and surgical recovery. Two of the three patients proceeded to definitive surgical aortic valve replacement with the remainder currently undergoing evaluation. In this case series, we present a novel approach of balloon aortic valvuloplasty prior to liver transplantation as a potential bridge to definitive treatment of severe aortic stenosis in the end-stage liver patient.
PMCID: PMC4241694  PMID: 25431682
13.  Interplay between Immune responses to HLA and Non-HLA self-antigens in allograft rejection 
Human immunology  2013;74(11):10.1016/j.humimm.2013.07.002.
Recent studies strongly suggest an increasing role for immune responses against self-antigens (Ags) which are not encoded by the major histocompatibility complex in the immunopathogenesis of allograft rejection. Although, improved surgical techniques coupled with improved methods to detect and avoid sensitization against donor human leukocyte antigen (HLA) have improved the immediate and short term function of transplanted organs. However, acute and chronic rejection still remains a vexing problem for the long term function of the transplanted organ. Immediately following organ transplantation, several factors both immune and non immune mechanisms lead to the development of local inflammatory milieu which sets the stage for allograft rejection. Traditionally, development of antibodies (Abs) against mismatched donor HLA have been implicated in the development of Ab mediated rejection. However, recent studies from our laboratory and others have demonstrated that development of humoral and cellular immune responses against non-HLA self-Ags may contribute in the pathogenesis of allograft rejection. There are reports demonstrating that immune responses to self-Ags especially Abs to the self-Ags as well as cellular immune responses especially through IL17 has significant pro-fibrotic properties leading to chronic allograft failure. This review summarizes recent studies demonstrating the role for immune responses to self-Ags in allograft immunity leading to rejection as well as present recent evidence suggesting there is interplay between allo- and autoimmunity leading to allograft dysfunction.
PMCID: PMC3813452  PMID: 23876679
Alloimmunity; Autoimmunity; Transplant rejection; Antigen presentation; Interleukin-17
14.  Hepatitis C Virus-Induced Changes in MicroRNA 107 (miRNA-107) and miRNA-449a Modulate CCL2 by Targeting the Interleukin-6 Receptor Complex in Hepatitis 
Journal of Virology  2014;88(7):3733-3743.
Hepatitis C virus (HCV)-mediated liver diseases are one of the major health issues in the United States and worldwide. HCV infection has been reported to modulate microRNAs (miRNAs) that control various cell surface receptors and gene-regulatory complexes involved in hepatic inflammation and liver diseases. We report here that specific downregulation of miRNA-107 and miRNA-449a following HCV infection in patients with HCV-mediated liver diseases modulates expression of CCL2, an inflammatory chemokine upregulated in patients with chronic liver diseases, by targeting components of the interleukin-6 receptor (IL-6R) complex. Computational analysis for DNA-bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBPα, spleen focus-forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBPα, PU.1, and STAT3 interacted with each other physically to cooperatively bind to the promoter and activate CCL2 expression. Analysis of IL-6R and JAK1 expression in HCV patients by quantitative PCR showed significant upregulation when there was impaired miRNA-107 and miRNA-449a expression, along with upregulation of PU.1 and STAT3, but not CEBPα. miRNA-449a and miRNA-107 target expression of IL-6R and JAK1, respectively, in vitro and also inhibit IL-6 signaling and impair STAT3 activation in human hepatocytes. Taken together, our results demonstrate a novel gene-regulatory mechanism in which HCV-induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL-6-mediated signaling cascade, which we propose will result in HCV-mediated induction of inflammatory responses and fibrosis.
IMPORTANCE Hepatitis C virus (HCV)-induced hepatitis is a major health concern worldwide. HCV infection results in modulation of noncoding microRNAs affecting major cellular pathways, including inflammatory responses. In this study, we have identified a microRNA-regulated pathway for the chemokine CCL2 in HCV-induced hepatitis. Understanding microRNA-mediated transcriptional-regulatory pathways will result in development of noninvasive biomarkers for better disease prediction and development of effective therapeutics.
PMCID: PMC3993535  PMID: 24429361
15.  Temporal association between increased virus-specific Th17 response and spontaneous recovery from recurrent hepatitis C in a liver transplant recipient 
Transplantation  2011;92(12):1364-1370.
Spontaneous clearance of Hepatitis C virus (HCV) following liver transplantation (OLT) is a rare occurrence. Here we present detailed immunological analysis of an interferon naive OLT recipient receiving uninterrupted immunosuppression who cleared HCV spontaneously two years after transplantation.
Enzyme linked immunospot assay (ELISpot) analysis of peripheral T-cell IFNγ, IL-10, and IL-17 response to HCV core and non-structural antigen 4 (NS4) and enzyme linked immunosorbent assay (ELISAs) to collagen (Col) subtypes I, II, IV, and V were performed in the index patient at the time of viral clearance and compared to an OLT cohort with persistent viremia matched for time from OLT, immunosuppression, and histology. ELISpot and ELISA analysis were repeated on the patient 4 years after OLT. Transcription-mediated amplification assays were used to confirm viral clearance.
Compared to a cohort of post-OLT and non-transplanted viremic HCV patients, the index patient with HCV clearance demonstrated higher IL-17, IL-10 and lower IFN-γ response to NS4 and core antigen and a higher titer of Abs to Col subtypes I, II, and V during clearance. On follow-up 2 years later, HCV specific IFN-γ was increased in the index patient, with a decline in IL-17 and IL-10 response as well as Col I, II, and V Ab titer.
Virus induced activation of Th-17 cells may contribute to HCV clearance post- OLT. Maintenance of viral suppression may be facilitated by restoration of Th1 (IFNγ) responses. Modulation of Th17 immunity deserves further attention as a therapeutic strategy in the treatment of HCV recurrence post-OLT.
PMCID: PMC3697051  PMID: 22082818
Transplant immunology  2012;26(4):201-206.
Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury.
We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury.
I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06 O.D./min/10μg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs 264.5±30.1 pg/2μg protein, p=0.003) and IL-1β (6.0±0.91 vs. 19.8±5.2 vs 5±1.7 pg/10μg protein, p= 0.02) along with a significant reduction in ALT levels (715±71 vs 3712.5±437.5 vs 606±286 U/L, p=0.01).
These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.
PMCID: PMC3675789  PMID: 22286145
hepatic steatosis; I/R injury; liver transplantation; NFκB; PS-341; bortezomib; obese; marginal graft
17.  Donor Graft Steatosis Influences Immunity to Hepatitis C Virus and Allograft Outcome After Liver Transplantation 
Transplantation  2011;92(11):1259-1268.
Hepatitis C (HCV) recurrence following orthotopic liver transplantation (OLT) is universal, often with accelerated allograft fibrosis. Donor liver steatosis is frequently encountered and often associated with poor early post-operative outcome. The study’s aim was to test the hypothesis that allograft steatosis alters immune responses to HCV and self-antigens promoting allograft fibrosis.
Forty-eight HCV OLT recipients (OLTr) were enrolled and classified based on amount of allograft macrovesicular steatosis at time of OLT. Group 1-No Steatosis (0–5% steatosis, n=21), Group 2 – Mild (5–35% - n=16), Group 3 – moderate (>35%, n=11). Cells secreting IL-17, IL-10, IFN-γ in response to HCV antigens were enumerated by ELISpot. Serum cytokines were measured by Luminex, antibodies (Abs) to Collagen (Col) I, II, III, IV, V by ELISA.
OLTr of moderate steatotic grafts had the highest incidence of advanced fibrosis in protocol one-year post-OLT biopsy (10.8% vs. 15.8% vs. 36.6%, r = 0.157, p<0.05). OLTr from Groups 2 and 3 had increased HCV specific IL-17 (p<0.05) and IL-10 (p<0.05) with reduced IFN-γ (p<0.05) secreting cells when compared to group 1. This was associated with increase in serum IL-17, IL-10, IL-1β, IL-6, IL-5 and decreased IFN-γ. In addition, there was development of Abs to Col I, II, III and V in OLTr with increased steatosis (p<0.05).
The results demonstrate that allograft steatosis influences post-OLT HCV specific immune responses leading to a IL-17 T-helper response and activation of humoral immune responses to liver associated self antigens which may contribute to allograft fibrosis and poor outcome.
PMCID: PMC3223266  PMID: 22011763
Allograft Steatosis; Hepatitis C; Recurrence; Fibrosis; Liver Transplantation
18.  Efficacy of Neoadjuvant Chemoradiation followed by Liver Transplantation for Perihilar Cholangiocarcinoma at 12 US Centers 
Gastroenterology  2012;143(1):10.1053/j.gastro.2012.04.008.
Excellent single-center outcomes of neoadjuvant chemoradiation and liver transplantation (LT) for unresectable perihilar cholangiocarcinoma caused the United Network of Organ Sharing (UNOS) to offer a standardized model of end-stage liver disease (MELD) exception for this disease. We analyzed data from multiple centers to determine the effectiveness of this treatment and the appropriateness of the MELD exception.
We collected and analyzed data from 12 large-volume transplant centers in the US who met the inclusion criteria of treating three or more patients with perihilar cholangiocarcinoma using neoadjuvant therapy followed by liver transplantation from 1993–2010 (n=287 total patients). Center-specific protocols and medical charts were reviewed on-site.
The patients completed external radiation (99%), brachytherapy (75%), radio-sensitizing (98%), and/or maintenance chemotherapy (65%). Seventy-one patients dropped out before liver transplantation (rate of 11.5% in 3 months). Intent-to-treat survival was 68% and 53%, 2 and 5 years after therapy, respectively; post-transplantation, recurrence-free survival rates were 78% and 65%, respectively. Patients outside the UNOS criteria (those with tumor mass >3 cm, trans-peritoneal tumor biopsy, or metastatic disease) or with a prior malignancy had significantly shorter survival times (P<.001). There were no differences in outcomes among patients based on differences in operative staging or brachytherapy. Although most patients came from 1 center (n=193), the other 11 centers had similar survival times after therapy.
Patients with perihilar cholangiocarcinoma who were treated with neoadjuvant therapy followed by liver transplantation at 12 US centers had a 65% rate of recurrence-free survival after 5 years, demonstrating this therapy to be highly effective. An 11.5% dropout rate after 3.5 months of therapy indicates the appropriateness of the MELD exception. Rigorous selection is important for the continued success of this treatment.
PMCID: PMC3846443  PMID: 22504095
liver cancer; biliary; hepatic; treatment efficacy
19.  Concepts and Preliminary Data Toward the Realization of Image-guided Liver Surgery 
Image-guided surgery provides navigational assistance to the surgeon by displaying the surgical probe position on a set of preoperative tomograms in real time. In this study, the feasibility of implementing image-guided surgery concepts into liver surgery was examined during eight hepatic resection procedures. Preoperative tomographic image data were acquired and processed. Accompanying intraoperative data on liver shape and position were obtained through optically tracked probes and laser range scanning technology. The preoperative and intraoperative representations of the liver surface were aligned using the iterative closest point surface matching algorithm. Surface registrations resulted in mean residual errors from 2 to 6 mm, with errors of target surface regions being below a stated goal of 1 cm. Issues affecting registration accuracy include liver motion due to respiration, the quality of the intraoperative surface data, and intraoperative organ deformation. Respiratory motion was quantified during the procedures as cyclical, primarily along the cranial–caudal direction. The resulting registrations were more robust and accurate when using laser range scanning to rapidly acquire thousands of points on the liver surface and when capturing unique geometric regions on the liver surface, such as the inferior edge. Finally, finite element models recovered much of the observed intraoperative deformation, further decreasing errors in the registration. Image-guided liver surgery has shown the potential to provide surgeons with important navigation aids that could increase the accuracy of targeting lesions and the number of patients eligible for surgical resection.
PMCID: PMC3839065  PMID: 17458587
Image-guided surgery; Liver resection; Surface registration; Laser range scanning; Finite element
20.  Organ Surface Deformation Measurement and Analysis in Open Hepatic Surgery: Method and Preliminary Results From 12 Clinical Cases 
IEEE transactions on bio-medical engineering  2011;58(8):10.1109/TBME.2011.2146782.
The incidence of soft tissue deformation has been well documented in neurosurgical procedures and is known to compromise the spatial accuracy of image-guided surgery systems. Within the context of image-guided liver surgery (IGLS), no detailed method to study and analyze the observed organ shape change between preoperative imaging and the intraoperative presentation has been developed. Contrary to the studies of deformation in neurosurgical procedures, the majority of deformation in IGLS is imposed prior to resection and due to laparotomy and mobilization. As such, methods of analyzing the organ shape change must be developed to use the intraoperative data [e.g., laser range scan (LRS) surfaces] acquired with the organ in its fully deformed shape. To achieve this end we use a signed closest point distance deformation metric computed after rigid alignment of the intraoperative LRS data with organ surfaces generated from the preoperative tomograms. The rigid alignment between the intraoperative LRS surfaces and preoperative image data was computed with a feature weighted surface registration algorithm. In order to compare the deformation metrics across patients, an interpatient nonrigid registration of the preoperative CT images was performed. Given the interpatient liver registrations, an analysis was performed to determine the potential similarities in the distribution of measured deformation between patients for which similar procedures had been performed. The results of the deformation measurement and analysis indicate the potential for soft tissue deformation to compromise surgical guidance information and suggests a similarity in imposed deformation among similar procedure types.
PMCID: PMC3819167  PMID: 21521662
Image-guided surgery (IGS); laser range scanning; open hepatic surgery; soft tissue deformation
21.  Hepatitis C Virus Induced miR200c Down Modulates FAP-1, a Negative Regulator of Src Signaling and Promotes Hepatic Fibrosis 
PLoS ONE  2013;8(8):e70744.
Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. 22 miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to growth factors and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of fibroblasts with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and fibroblast growth factor (2.8 and 3.4 fold, p<0.05). Therefore, we propose that HCV induced increased expression of miR200c can down modulate the expression of FAP1, a critical regulator of Src and MAP kinase pathway that play an important role in the production of fibrogenic growth factors and development of fibrosis.
PMCID: PMC3741284  PMID: 23950995
Hepatitis C Virus (HCV) infection and recurrence post-transplant (OLT) is associated with extracellular matrix (ECM) components remodeling, particularly collagen (Col), leading to fibrosis. Our aim was to determine whether development of antibodies (Abs) to self antigen Col in HCV infection correlates with fibrosis stage and peripheral cytokine response. Chronic HCV patients, those with recurrence after OLT undergoing biopsy and healthy control subjects were enrolled. HCV subjects (n=70) were stratified: 1) Non OLT No Fibrosis (Scheuer Stage 0–2) 2) Non OLT Fibrosis (Scheuer Stage 3–4) 3) Post OLT No Fibrosis (Scheuer 0–2) and 4) Post OLT Fibrosis (Scheuer 3–4). Serum was analyzed for Abs against Col-1, 2, 4, 5 and vimentin using ELISA. Serum levels of cytokines were measured using Multiplex Bead immunoassays. Levels of Abs to Col 1 were higher in fibrosis groups compared with no fibrosis groups and control both Non OLT (p<0.0001) and Post OLT (P=0.01). There were increased levels of Abs to Col 2, 4, 5 and vimentin, in fibrotic groups Non OLT (Col 2: p=0.0001, Col 4: p =0.09, Col 5: p<0.0001, vimentin: p=0.36) and Post OLT (Col 2: p=0.006, Col 4: p = 0.1, Col 5: p<0.0001, vimentin: p=0.24) compared with non fibrotic groups. Fibrotic groups non-OLT and post OLT demonstrated significantly higher Th2, Th17 cytokines and lower Th1 cytokines compared to non fibrotic groups. Our results demonstrate that in HCV infection, Abs to ECM Collagen 1, 2, 5 positively correlate with liver fibrosis which is associated with a predominant Th2 and Th17 cytokine profile.
PMCID: PMC3700355  PMID: 21425431
Autoantibodies; IL-17; Fibrosis; HCV Recurrence
23.  Hepatitis C Virus Mediated Changes in miRNA-449a Modulates Inflammatory Biomarker YKL40 through Components of the NOTCH Signaling Pathway 
PLoS ONE  2012;7(11):e50826.
Liver disease due to hepatitis C virus (HCV) infection is an important health problem worldwide. HCV induced changes in microRNAs (miRNA) are shown to mediate inflammation leading to liver fibrosis. Gene expression analyses identified dysregulation of miRNA-449a in HCV patients but not in alcoholic and non-alcoholic liver diseases. By sequence analysis of the promoter for YKL40, an inflammatory marker upregulated in patients with chronic liver diseases with fibrosis, adjacent binding sites for nuclear factor of Kappa B/P65 and CCAAT/enhancer-binding protein alpha (CEBPα) were identified. P65 interacted with CEBPα to co-operatively activate YKL40 expression through sequence specific DNA binding. In vitro analysis demonstrated that tumor necrosis factor alpha (TNFα) mediated YKL40 expression is regulated by miRNA-449a and its target NOTCH1 in human hepatocytes.NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Further, HCV patients demonstrated upregulation of NOTCH1 along with downregulation of miRNA-449a. Taken together it is demonstrated that miRNA-449a plays an important role in modulating expression of YKL40 through targeting the components of the NOTCH signaling pathway following HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammatory responses and fibrosis will be important towards developing strategies to prevent hepatic fibrosis especially following HCV recurrence in liver transplant recipients.
PMCID: PMC3511274  PMID: 23226395
24.  The role of molecular chaperonins in warm ischemia and reperfusion injury in the steatotic liver: A proteomic study 
BMC Biochemistry  2012;13:17.
The molecular basis of the increased susceptibility of steatotic livers to warm ischemia/reperfusion (I/R) injury during transplantation remains undefined. Animal model for warm I/R injury was induced in obese Zucker rats. Lean Zucker rats provided controls. Two dimensional differential gel electrophoresis was performed with liver protein extracts. Protein features with significant abundance ratios (p < 0.01) between the two cohorts were selected and analyzed with HPLC/MS. Proteins were identified by Uniprot database. Interactive protein networks were generated using Ingenuity Pathway Analysis and GRANITE software.
The relative abundance of 105 proteins was observed in warm I/R injury. Functional grouping revealed four categories of importance: molecular chaperones/endoplasmic reticulum (ER) stress, oxidative stress, metabolism, and cell structure. Hypoxia up-regulated 1, calcium binding protein 1, calreticulin, heat shock protein (HSP) 60, HSP-90, and protein disulfide isomerase 3 were chaperonins significantly (p < 0.01) down-regulated and only one chaperonin, HSP-1was significantly upregulated in steatotic liver following I/R.
Down-regulation of the chaperones identified in this analysis may contribute to the increased ER stress and, consequently, apoptosis and necrosis. This study provides an initial platform for future investigation of the role of chaperones and therapeutic targets for increasing the viability of steatotic liver allografts.
PMCID: PMC3445822  PMID: 22962947
Ischemia repurfusion injury; Two dimensional gel electrophoresis; Mass spectrometry; Liver transplantation; Chaperonins; Endoplasmic reticulum (ER) stress
25.  Endoplasmic Reticulum Stress is a Mediator of Post-Transplant Injury in Severely Steatotic Liver Allografts 
Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have an increased susceptibility to cold ischemia and reperfusion (CIR) injury compared to otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance and the metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin-resistant rodents had increased ER stress responses and increased markers of hepatocellular injury following liver transplantation into strain-matched lean recipients. ER stress response components were decreased by the chemical chaperone, TUDCA, resulting in improvement of the allograft injury. TUDCA treatment decreased NF-κB activation, and the pro-inflammatory cytokines IL-6 and IL-1β. However, the predominant response was decreased expression of the ER stress cell death mediator, CHOP. Further, activation of the inflammation-associated caspase 11 was decreased linking ER Stress/CHOP to pro-inflammatory cytokine production following steatotic liver transplantation. These data confirm ER stress in steatotic allografts, and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft.
PMCID: PMC3056557  PMID: 21280192
Liver Transplantation; Endoplasmic Reticulum Stress; Hepatic Steatosis; Ischemia-Reperfusion Injury

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