PDF file - 56K, HDIL-2 administration does not result in liver and idney dysfunction in mice.

PDF file - 428K, Supplemental Figure 1. Administration of rIL-2 inhibits tumor growth in murine hepatic metastasis model in a dose dependent fashion. Supplemental Figure 2. HDIL-2 alone or combined with CQ significantly inhibits Panc02 tumor growth, prolonging survival time in a hepatic metastasis model. Supplemental Figure 3. HDIL-2 combined with CQ inhibits Renca tumor growth in an hepatic metastasis model. Supplemental Figure 4. CQ treatment results in mitochondrial morphologic changes in MC38 cells. Supplemental Figure 5. Murine tumor cells are dependent on glycolysis. Supplemental Figure 6. Ethyl pyruvate alters tumor cell metabolism. Supplemental Figure 7. Proposed mechanisms of autophagy inhibition during HDIL-2 immunotherapy promoting long term tumor regression.

PDF file - 86K, Transmission electron microscopy (TEM), ATP Quantification, and XF Bioenergetic Assay.

The desmoplastic and complex tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) has presented tremendous challenges for developing effective therapeutic strategies. Strategies targeting tumor stroma, albeit with great potential, have met with limited success due to the lack of knowledge on the molecular dynamics within the tumor microenvironment (TME). In pursuit of a better understanding of the influence of miRNAs on TME reprogramming and to explore circulating miRNAs as diagnostic and prognostic biomarkers for PDAC, using RNA-seq, miRNA-seq, and single-cell RNA-seq (scRNA-seq), we investigated the dysregulated signaling pathways in PDAC TME modulated by miRNAs from plasma and tumor tissue. Our bulk RNA-seq in PDAC tumor tissue identified 1445 significantly differentially expressed genes with extracellular matrix and structure organization as the top enriched pathways. Our miRNA-seq identified 322 and 49 abnormally expressed miRNAs in PDAC patient plasma and tumor tissue, respectively. We found many of the TME signaling pathways were targeted by those dysregulated miRNAs in PDAC plasma. Combined with scRNA-seq from patient PDAC tumor, our results revealed that these dysregulated miRNAs were closely associated with extracellular matrix (ECM) remodeling, cell-ECM communication, epithelial-mesenchymal transition, as well as immunosuppression orchestrated by different cellular components of TME. The findings of this study could assist the development of miRNA-based stromal targeting biomarkers or therapy for PDAC patients.

Purpose: Southern CA is at the epicenter of the Covid-19 pandemic. We reviewed outcomes of our center's liver, kidney, and pancreas transplant patients stricken with Covid-19 infection. Methods: Retrospective review of 161 post-transplant patients with Covid-19 infection. Results: From March 2020 to January 2021, 43 liver, 107 kidney, 6 liver/kidney, and 4 kidney/pancreas patients came down with Covid-19 (TABLE). Transplants were performed from August 2000 to December 2020. Mean age was 54±1 yrs. Median time of infection was 27 months post-transplant (range 15 days to 21 years). Frequency of symptoms were: shortness of breath (55%), fever (52%), muscle aches (48%), diarrhea (36%), headaches (34%), loss of taste or smell (27%). Only 10 (6%) pts were asymptomatic. Overall mortality rate was 20% (33/161) and severe Covid-19 (hospitalization and death) occurred in 90/161 (56%) patients. Mortality risk factors included older age (62±2 vs 52±1 yrs, p<0.01), hospitalization (32/33, 97% vs 59/128, 46% p<0.01), mechanical ventilation (30/33, 91% vs 4/128, 3% p<0.01);there was no difference in gender (p=0.5), race (p=0.88), presence of diabetes (p=0.26), hypertension (p=0.06), or obesity (p=0.83). Liver/kidney recipients had the highest mortality rate (Table). Risk factors for severe Covid-19 included age (56±1 vs 51±2 yrs, P=0.01) and presence of diabetes (54/90, 60% vs 29/71, 41% p=0.02);there was no difference in gender (p=0.97), race (p=0.39), presence of hypertension (p=0.09), or obesity (p=0.82). Kidney patients had more severe Covid-19 than the other organ recipients. Kidney/pancreas patients were younger and tended to have mild infection and had no mortality. 156 (97%) patients were on tacrolimus (2 were on CyA, 2 on belatacept), 113 (70%) on MMF, and 127 (79%) on prednisone. 101 (63%) of patients were on triple immunosuppression. MMF was the most common agent to be adjusted (48/113, 42%) followed by tacrolimus (9/156, 6%). Of the 90 pts hospitalized, 70 (78%) received steroids, 30 (33%) received Remdesivir, and 53 (59%) were anticoagulated. 5 patients received convalescent plasma. Peak C-reactive protein levels when measured were significantly higher in patients who died (53.7±15.8 vs 19.8±5.3 mg/L, p=0.02). Conclusions: Covid-19 infection inflicts a high mortality rate in liver, kidney, and pancreas transplant recipients. (Table Presented).

Objective We evaluated the effects of the B-cell activating factor (BAFF)-targeting antibody Belimumab on human nonmemory B-cell pools. Human B-cell pools were identified using surface markers adapted from mouse studies that specifically assessed reductions in immature B cells due to BAFF depletion. Patients with systemic lupus erythematosus (SLE) have high levels of both BAFF and immature B cells. Mechanistic mouse studies provide a framework for understanding human responses to therapies that target B cells. Methods Peripheral blood mononuclear cells were isolated from healthy donors and SLE patients on Belimumab or standard-of-care therapy (SCT). Cells were stained for flow cytometry to identify B-cell subsets based on CD21/CD24. Differences in subset proportions were determined by one-way ANOVA and Tukey’s post hoc test. Results Patients treated with Belimumab show alterations in the nonmemory B-cell pool characterized by a decrease in the Transitional 2 (T2) subset (p = 0.002), and an increase in the proportion of Transitional 1 (T1) cells (p = 0.005) as compared with healthy donors and SCT patients. The naïve B-cell compartment showed no significant differences between the groups (p = 0.293). Conclusion Using a translational approach, we show that Belimumab-mediated BAFF depletion reduces the T2 subset in patients, similar to observations in mouse models with BAFF depletion.

Objectives. The purpose of this study was to identify risk factors that may predict heart failure with reduced ejection fraction (HFrEF) following orthotopic liver transplantation (OLT) and associated mortality. Background. HFrEF following OLT is a poorly understood phenomenon, reported in 3% to 7% of transplanted patients. Methods. This is a retrospective analysis of 176 consecutive patients who underwent OLT from 2010 to 2017. Multivariate logistic regression was used to identify associations between cardiovascular risk factors and perioperative variables with post-OLT HFrEF, defined as reduction in left ventricular ejection fraction of at least 10% and left ventricular ejection fraction less than or equal to 40% with acute heart failure symptoms. Multivariate cox proportional hazards regression (with inverse probability weighting by propensity scores) was used to evaluate effects of HFrEF on 1-year mortality. Results. Of the 176 patients, 14% developed HFrEF with a median of 5 days. History of heart failure (OR 10.99, 2.15-56.09; P = .04) and intraoperative transfusion of greater than 11 units of packed red blood cells (OR 3.377, 1.025-11.13; P = .045) were associated with increased incidence of HFrEF. Pre-transplant hemoglobin greater than 8.5 g/dL (OR 0.252, CI 0.0954-0.665; P = .05) was protective against HFrEF. Thirty-three percent of HFrEF group died within 1 year (HR 7.36, 2.57-21.12; P < .001). Conclusions. The incidence of acute HFrEF post-OLT is 14% and is associated with a 7-fold increase in 1-year mortality. Cirrhotic cardiomyopathy and stress-induced cardiomyopathy maybe the underlying mechanisms. Our study identified risk factors associated with post-OLT HFrEF and should provide additional guidance for risk stratification of patients undergoing OLT.

Anecdotal reports have suggested that transplantation of hepatitis C virus (HCV) antibody positive (Ab+)/nucleic acid test negative (NAT−) donor kidneys into HCV negative recipients is not associated with HCV transmission. We reviewed our center's outcomes of 32 HCV negative patients who received kidney allografts from 25 donors who were HCV Ab+/NAT−. The mean recipient age was 56.9 ± 12.1 years and the mean donor age was 41.5 ± 14 years, with a median Kidney Donor Profile Index (KDPI) of 68%. Twelve donors (48%) met Public Health Service (PHS) increased risk status. All patients received antithymocyte globulin induction followed by tacrolimus, mycophenolate mofetil, and steroid maintenance immunosuppression. With a mean follow‐up posttransplant of 10 ± 2.7 months, 1‐ and 3‐ month serum creatinine levels were 1.7 ± 0.8 and 1.3 ± 0.4, respectively, and patient and graft survival rates were 100% and 97%, respectively. Fourteen patients (44%) seroconverted and became HCV Ab+ posttransplant. However, all 32 patients were HCV RNA negative at 1‐ and 3‐ months posttransplant, and 27 and 8 patients tested at 6‐ and 12‐months posttransplant, respectively, remain HCV RNA negative. In conclusion, transplantation of HCV Ab+/NAT− kidneys to HCV negative recipients frequently causes HCV Ab seroconversion but not HCV viremia.

The Banff Working Group on Liver Allograft Pathology reviewed and discussed literature evidence regarding antibody-mediated liver allograft rejection at the 11th (Paris, France, June 5-10, 2011), 12th (Comandatuba, Brazil, August 19-23, 2013), and 13th (Vancouver, British Columbia, Canada, October 5-10, 2015) meetings of the Banff Conference on Allograft Pathology. Discussion continued online. The primary goal was to introduce guidelines and consensus criteria for the diagnosis of liver allograft antibody-mediated rejection and provide a comprehensive update of all Banff Schema recommendations. Included are new recommendations for complement component 4d tissue staining and interpretation, staging liver allograft fibrosis, and findings related to immunosuppression minimization. In an effort to create a single reference document, previous unchanged criteria are also included.

Purpose: To describe results of a planned interim analysis of a prospective, randomized clinical trial developed to compare treatment outcomes among patients with newly diagnosed hepatocellular carcinoma (HCC).Methods and Materials: Eligible subjects had either clinical or pathologic diagnosis of HCC and met either Milan or San Francisco transplant criteria. Patients were randomly assigned to transarterial chemoembolization (TACE) or to proton beam radiation therapy. Patients randomized to TACE received at least 1 TACE with additional TACE for persistent disease. Proton beam radiation therapy was delivered to all areas of gross disease to a total dose of 70.2 Gy in 15 daily fractions over 3 weeks. The primary endpoint was progression-free survival, with secondary endpoints of overall survival, local tumor control, and treatment-related toxicities as represented by posttreatment days of hospitalization.Results: At the time of this analysis 69 subjects were available for analysis. Of these, 36 were randomized to TACE and 33 to proton. Total days of hospitalization within 30 days of TACE/proton was 166 and 24 days, respectively (P<.001). Ten TACE and 12 proton patients underwent liver transplantation after treatment. Viable tumor identified in the explanted livers after TACE/proton averaged 2.4 and 0.9 cm, respectively. Pathologic complete response after TACE/proton was 10%/25% (P=.38). The 2-year overall survival for all patients was 59%, with no difference between treatment groups. Median survival time was 30 months (95% confidence interval 20.7-39.3 months). There was a trend toward improved 2-year local tumor control (88% vs 45%, P=.06) and progression-free survival (48% vs 31%, P=.06) favoring the proton beam treatment group.Conclusions: This interim analysis indicates similar overall survival rates for proton beam radiation therapy and TACE. There is a trend toward improved local tumor control and progression-free survival with proton beam. There are significantly fewer hospitalization days after proton treatment, which may indicate reduced toxicity with proton beam therapy. (C) 2016 Elsevier Inc. All rights reserved.

Hepatic epithelioid hemangioendothelioma (HEHE) is an infrequent vascular tumor of endothelial origin that primarily occurs in women in the mid-fifth decade of life without underlying chronic liver disease or cirrhosis. Liver transplant should be the first-line of therapy in patients with large or diffuse unresectable tumors even in the presence of metastatic disease due to the favorable long-term outcome. We report the case of a 48-year-old female who complained of abdominal pain and weight loss. She has a history of cirrhosis secondary to chronic hepatitis C (HCV) and was treated with interferon and ribavirin with sustained virological response. Her work-up revealed multiple confluent infiltrating bilobar liver masses diagnosed as HEHE. She underwent a successful liver transplant without evidence of recurrent HCV infection. She developed cervical spine (C4-C6) HEHE metastases 4 years after transplant. She underwent surgical resection and local radiotherapy after resection with good clinical response. To the best of our knowledge, this is the first report of HEHE that developed in a patient with HCV cirrhosis successfully treated with antiviral therapy before transplant and liver transplant with good allograft function without evidence of recurrent liver tumor or HCV infection but developed metastases to the cervical spine 4 years after transplant.

PBRT has been shown to be an effective treatment against several types of malignancies. In this study, we reviewed the results of our long-term experience with the use of PBRT for the treatment of HCC prior to LT.

The purpose of this study is to compare the outcome of pediatric recipients of kidneys procured using a hand‐assisted laparoscopic ( HALDN group) to an open technique ( ODN group). Twenty‐eight patients ≤18 yr old ( HALDN group) were compared with 17 patients ( ODN group). The serum creatinine for HALDN and ODN groups at discharge were 0.93 ± 0.48 and 0.94 ± 0.54 mg/ dL (p = 0.917), respectively. The serum creatinine for HALDN and ODN groups at six and 12 months was 1.01 ± 0.44 and 1.11 ± 0.55, and 1.04 ± 0.52 and 1.14 ± 0.46 mg/ dL (p = 0.516, p = 0.554), respectively. The eGFR for HALDN and ODN groups at discharge was 108.66 ± 37.23 and 106.1 ± 50.55 mL/min/1.73 m 2 (p = 0.845), respectively. The eGFR for HALDN and ODN groups at six and 12 months was 97.77 ± 28.25 and 81.73 ± 27.46, and 94.56 ± 28.3 and 85.74 ± 30.1 mL/min/1.73 m2 (p = 0.085, p = 0.344), respectively. The patient and graft survival for both groups were 100% at 12 months post‐transplant. In conclusion, the short‐term outcome of recipients of kidneys procured via HALDN is comparable to that of kidneys procured via ODN in pediatric patients.

Abstract Administration of high-dose interleukin-2 (HDIL-2) has durable antitumor effects in 5% to 10% of patients with melanoma and renal cell carcinoma. However, treatment is often limited by side effects, including reversible, multiorgan dysfunction characterized by a cytokine-induced systemic autophagic syndrome. Here, we hypothesized that the autophagy inhibitor chloroquine would enhance IL-2 immunotherapeutic efficacy and limit toxicity. In an advanced murine metastatic liver tumor model, IL-2 inhibited tumor growth in a dose-dependent fashion. These antitumor effects were significantly enhanced upon addition of chloroquine. The combination of IL-2 with chloroquine increased long-term survival, decreased toxicity associated with vascular leakage, and enhanced immune cell proliferation and infiltration in the liver and spleen. HDIL-2 alone increased serum levels of HMGB1, IFN-γ, IL-6, and IL-18 and also induced autophagy within the liver and translocation of HMGB1 from the nucleus to the cytosol in hepatocytes, effects that were inhibited by combined administration with chloroquine. In tumor cells, chloroquine increased autophagic vacuoles and LC3-II levels inhibited oxidative phosphorylation and ATP production and promoted apoptosis, which was associated with increased Annexin-V+/propidium iodide (PI)− cells, cleaved PARP, cleaved caspase-3, and cytochrome c release from mitochondria. Taken together, our findings provide a novel clinical strategy to enhance the efficacy of HDIL-2 immunotherapy for patients with cancer. Cancer Res; 72(11); 2791–801. ©2012 AACR.

1Department of Surgery, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh PA, 15213 2Center for Biologic Imaging, University of Pittsburgh 3Department of Immunology 4UPCI Molecular and Cellular Biology Program. University of Pittsburgh Running Title: Inhibition of IL-2 Induced Autophagy Prolongs Survival Precis: The preclinical study shows how the inexpensive autophagy inhibitor chloroquine might be repositioned to greatly enhance the antitumor effects of IL2, a central growth and survival factor for T cells used for melanoma treatment, while limiting the toxicity that has its restricted its broad use in clinic.

C4d-assisted recognition of antibody-mediated rejection (AMR) in formalin-fixed paraffin-embedded tissues (FFPE) from donor-specific antibody-positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO- incompatibility status, histopathology and clinical-serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO-incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection-related causes of allograft dysfunction in DSA- and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA- recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

Biliary complications remain a cause of morbidity after liver transplantation. The aim of this study was to determine whether changes in clinical practice in the era of the Model for End-Stage Liver Disease (MELD) has affected biliary complications after liver transplantation. We retrospectively reviewed all deceased donor liver transplants at a single center. Patients were categorized as pre- or post-MELD (transplant before or after February 28, 2002). A total of 1798 recipients underwent deceased donor liver transplants. Biliary stricture was more common in the post-MELD era (15.4% versus 6.4%, P < 0.001). The strongest risk factors for stricture development were donor age (odds ratio [OR] = 1.01), presence of a prior bile leak (OR = 2.24), use of choledochocholedochostomy (OR = 2.22), and the post-MELD era (OR = 2.30). Bile leak was more common in the pre-MELD era (7.5% versus 4.9%, P = 0.02), with use of a T-tube as the strongest risk factor (OR = 3.38). Surgical factors did not influence the biliary complication rate. In conclusion, even when employing multivariate analysis to allow for factors that may influence biliary strictures, transplant in the post-MELD era was an independent predictor for stricture development. Further studies are warranted to determine the etiology of this increase.