Publications by authors named "Anthony J Demetris"

136 Publications

Clinicopathologic Analysis of Uterine Allografts Including Proposed Scoring of Ischemia-reperfusion Injury and T Cell-mediated Rejection-Dallas UtErus Transplant Study: A Pilot Study.

Transplantation 2021 Jan 20. Epub 2021 Jan 20.

Department of Pathology, Baylor University Medical Center, Dallas, TX Annette C. and Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, TX Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA.

Background: Uterus transplantation (UTx) enables pregnancy in infertile women. This study describes the histopathological changes of ischemia reperfusion injury and mostly acute T cell-mediated rejection (TCMR) in UTx and proposes modification toward a working formulation grading system with associated treatments.

Methods: Protocol and indication biopsies from 11 living and 2 deceased donor UTx recipients were analyzed. Serving as a control were 49 age-matched nontransplanted uteri. All posttransplant histopathological specimens were evaluated in a blinded fashion by 3 pathologists. Response to treatment was assessed by follow-up biopsies. Serial serum donor-specific antibody (DSA) responses were also recorded.

Results: Changes attributed to ischemia reperfusion resolved within 2 weeks of UTx in most of the patients. For TCMR grading, perivascular inflammation, focal capillary disruption, and interstitial hemorrhage were added to interface inflammation, intercellular edema, stromal inflammation, and epithelial apoptotic bodies. Of the 173 protocol biopsies, 98 were classified as negative for TCMR; 34, indeterminate-borderline; 35, mild; 3, moderate; and 3, severe, 1 of which occurred in a DSA-positive recipient and also showed microvascular injury. Corticosteroids successfully treated all moderate to severe TCMR episodes. Mild TCMR was treated by increasing existing baseline immunosuppression. Indeterminate-borderline episodes were not treated. Neither ischemia reperfusion injury nor TCMR with DSA adversely affected embryo transfer.

Conclusion: Relying on a modified histopathological grading system, we developed a treatment strategy resulting in resolution of TCMR and successful pregnancies.
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http://dx.doi.org/10.1097/TP.0000000000003633DOI Listing
January 2021

Efficacy and Safety of Immunosuppression Withdrawal in Pediatric Liver Transplant Recipients: Moving Towards Personalized Management.

Hepatology 2020 Aug 12. Epub 2020 Aug 12.

Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.

Background And Aims: Tolerance is transplantation's holy grail as it denotes allograft health without immunosuppression and its toxicities. Our aim was to determine, among stable long-term pediatric liver transplant recipients, the efficacy and safety of immunosuppression withdrawal to identify operational tolerance.

Approach And Results: We conducted a multi-center, single-arm trial of immunosuppression withdrawal over 36-48 weeks. Liver tests were monitored biweekly (year 1), monthly (year 2) and bimonthly (years 3-4). For-cause biopsies were done at investigators' discretion but mandated when alanine aminotransferase or gamma glutamyl transferase exceeded 100U/L. All subjects underwent final liver biopsy at trial end. The primary efficacy endpoint was operational tolerance, defined by strict biochemical and histological criteria 1 year after stopping immunosuppression. Among 88 subjects (median age 11 years; 39 boys; 57 deceased donor grafts), 33 (37.5%; 95%CI 27.4%, 48.5%) were operationally tolerant, 16 were non-tolerant by histology (met biochemical but failed histological criteria) and 39 were non-tolerant by rejection. Rejection, predicted by subtle liver inflammation in trial entry biopsies, typically (n=32) occurred at ≤32% of the trial entry immunosuppression dose and was treated with corticosteroids (n=32) and/or tacrolimus (n=38) with resolution (liver tests within 1.5X baseline) for all but 1 subject. No death, graft loss, or chronic, severe, or refractory rejection occurred. Neither fibrosis stage nor the expression level of a rejection gene set increased over 4 years for either tolerant or non-tolerant subjects.

Conclusions: Immunosuppression withdrawal showed that 37.5% of selected pediatric liver transplant recipients were operationally tolerant. Allograft histology did not deteriorate for either tolerant or non-tolerant subjects. The timing and reversibility of failed withdrawal justifies future trials exploring the efficacy, safety, and potential benefits of immunosuppression minimization.
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http://dx.doi.org/10.1002/hep.31520DOI Listing
August 2020

A three-tier Rescue stent improves outcomes over balloon occlusion in a porcine model of noncompressible hemorrhage.

J Trauma Acute Care Surg 2020 08;89(2):320-328

From the Division of Vascular Surgery (C.G., B.W.T.), University of Pittsburgh Medical Center; McGowan Institute for Regenerative Medicine (C.G., B.W.T.), Industrial Engineering (Y.E., Y.C.), Swanson School of Engineering, University of Pittsburgh; Department of Neurosurgery (P.D.T.), University of Pittsburgh Medical Center; Mechanical Engineering and Materials Science (W.W.C., S.K.C.), Swanson School of Engineering, University of Pittsburgh; Starzl Transplantation Institute (A.J.D.), Department of Pathology (A.J.D.), and Department of Surgery (B.W.T.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania.

Background: Noncompressible hemorrhage remains a high-mortality injury, and aortic balloon occlusion poses limitations in terms of distal ischemic injury. Our hypothesis was that a retrievable Rescue stent would confer improved outcome over aortic balloon occlusion.

Methods: A three-tier, retrievable stent graft was laser welded from nitinol and polytetrafluoroethylene to provide rapid thoracic and abdominal coverage with an interval bare metal segment to preserve visceral flow. Anesthetized swine had injury of the thoracic or abdominal aorta followed by balloon occlusion or a Rescue stent. A 1-hour long damage-control phase with blood repletion was used to simulate the prolonged interval between injury and repair, especially in the battlefield setting. Following the damage-control phase, the balloon or stent were retrieved followed by vascular repair and recovery to 48 hours. Animals were compared in terms of hemodynamics, blood loss, neurophysiologic spinal cord ischemia, ischemic organ injury, and survival.

Results: Despite antegrade hemorrhage control, balloon occlusion averaged 3.5 L of retrograde hemorrhage, loss of visceral perfusion, and permanent spinal cord ischemia by neurophysiology in six of seven animals. After permanent repair, all balloon occlusion animals died with only a single short term (5 hours) survivor. Conversely, Rescue stent animals revealed rapid hemorrhage control (in under 2 minutes) whether the injury was thoracic or abdominal with improved hemodynamics, preserved visceral flow, reduced spinal cord ischemia, negligible histologic organ injury and survival to end of study in all abdominal injured animals (n = 6) and four of six thoracic injured animals, with two deaths related to arrhythmia.

Conclusion: Compared with aortic balloon occlusion, a Rescue stent offers superior hemorrhage control and survival by virtue of reduced ischemic injury and direct control of the hemorrhagic injury. The Rescue stent may become a useful tool for damage control, especially on the battlefield where definitive repair presents logistical challenges.
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http://dx.doi.org/10.1097/TA.0000000000002715DOI Listing
August 2020

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection.

J Clin Invest 2020 10;130(10):5397-5412

Department of Surgery and.

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.
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http://dx.doi.org/10.1172/JCI133008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524467PMC
October 2020

The Fourth International Workshop on Clinical Transplant Tolerance.

Am J Transplant 2021 01 22;21(1):21-31. Epub 2020 Jul 22.

Department of Surgery, Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.
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http://dx.doi.org/10.1111/ajt.16139DOI Listing
January 2021

Precision transplant pathology.

Curr Opin Organ Transplant 2020 08;25(4):412-419

Thomas E. Starzl Transplantation Institute, University of Pittsburgh.

Purpose Of Review: Transplant pathology contributes substantially to personalized treatment of organ allograft recipients. Rapidly advancing next-generation human leukocyte antigen (HLA) sequencing and pathology are enhancing the abilities to improve donor/recipient matching and allograft monitoring.

Recent Findings: The present review summarizes the workflow of a prototypical patient through a pathology practice, highlighting histocompatibility assessment and pathologic review of tissues as areas that are evolving to incorporate next-generation technologies while emphasizing critical needs of the field.

Summary: Successful organ transplantation starts with the most precise pratical donor-recipient histocompatibility matching. Next-generation sequencing provides the highest resolution donor-recipient matching and enables eplet mismatch scores and more precise monitoring of donor-specific antibodies (DSAs) that may arise after transplant. Multiplex labeling combined with hand-crafted machine learning is transforming traditional histopathology. The combination of traditional blood/body fluid laboratory tests, eplet and DSA analysis, traditional and next-generation histopathology, and -omics-based platforms enables risk stratification and identification of early subclinical molecular-based changes that precede a decline in allograft function. Needs include software integration of data derived from diverse platforms that can render the most accurate assessment of allograft health and needs for immunosuppression adjustments.
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http://dx.doi.org/10.1097/MOT.0000000000000772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737245PMC
August 2020

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell- and antibody-mediated rejection.

Am J Transplant 2020 09 28;20(9):2318-2331. Epub 2020 May 28.

Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.

The XV. Banff conference for allograft pathology was held in conjunction with the annual meeting of the American Society for Histocompatibility and Immunogenetics in Pittsburgh, PA (USA) and focused on refining recent updates to the classification, advances from the Banff working groups, and standardization of molecular diagnostics. This report on kidney transplant pathology details clarifications and refinements to the criteria for chronic active (CA) T cell-mediated rejection (TCMR), borderline, and antibody-mediated rejection (ABMR). The main focus of kidney sessions was on how to address biopsies meeting criteria for CA TCMR plus borderline or acute TCMR. Recent studies on the clinical impact of borderline infiltrates were also presented to clarify whether the threshold for interstitial inflammation in diagnosis of borderline should be i0 or i1. Sessions on ABMR focused on biopsies showing microvascular inflammation in the absence of C4d staining or detectable donor-specific antibodies; the potential value of molecular diagnostics in such cases and recommendations for use of the latter in the setting of solid organ transplantation are presented in the accompanying meeting report. Finally, several speakers discussed the capabilities of artificial intelligence and the potential for use of machine learning algorithms in diagnosis and personalized therapeutics in solid organ transplantation.
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http://dx.doi.org/10.1111/ajt.15898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496245PMC
September 2020

Host tissue response to floating microelectrode arrays chronically implanted in the feline spinal nerve.

J Neural Eng 2020 07 10;17(4):046012. Epub 2020 Jul 10.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States of America. Center for the Neural Basis of Cognition, University of Pittsburgh and Carnegic Mellon University, Pittsburgh, PA, United States of America. McGowan Institute for Regenerative Medicine, Pittsburgh, PA, United States of America. Systems Neuroscience Center, Pittsburgh, PA, United States of America. Live Like Lou Center for ALS Research, Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, United States of America.

Objective: Neural interfacing technologies could significantly improve quality of life for people living with the loss of a limb. Both motor commands and sensory feedback must be considered; these complementary systems are segregated from one another in the spinal nerve.

Approach: The dorsal root ganglion-ventral root (DRG-VR) complex was targeted chronically with floating microelectrode arrays designed to record from motor neuron axons in the VR or stimulate sensory neurons in the DRG. Hematoxylin and eosin and Nissl/Luxol fast blue staining were performed. Characterization of the tissue response in regions of interest and pixel-based image analyses were used to quantify MAC387 (monocytes/macrophages), NF200 (axons), S100 (Schwann cells), vimentin (fibroblasts, endothelial cells, astrocytes), and GLUT1 (glucose transport proteins) reactivity. Implanted roots were compared to non-implanted roots and differences between the VR and DRG examined.

Main Results: The tissue response associated with chronic array implantation in this peripheral location is similar to that observed in central nervous system locations. Markers of inflammation were increased in implanted roots relative to control roots with MAC387 positive cells distributed throughout the region corresponding to the device footprint. Significant decreases in neuronal density and myelination were observed in both the VR, which contains only neuronal axons, and the DRG, which contains both neuronal axons and cell bodies. Notably, decreases in NF200 in the VR were observed only at implant times less than ten weeks. Observations related to the blood-nerve barrier and tissue integrity suggest that tissue remodeling occurs, particularly in the VR.

Significance: This study was designed to assess the viability of the DRG-VR complex as a site for neural interfacing applications and suggests that continued efforts to mitigate the tissue response will be critical to achieve the overall goal of a long-term, reliable neural interface.
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http://dx.doi.org/10.1088/1741-2552/ab94d7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768891PMC
July 2020

Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation.

Am J Transplant 2020 09 27;20(9):2305-2317. Epub 2020 Jun 27.

Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

This meeting report from the XV Banff conference describes the creation of a multiorgan transplant gene panel by the Banff Molecular Diagnostics Working Group (MDWG). This Banff Human Organ Transplant (B-HOT) panel is the culmination of previous work by the MDWG to identify a broadly useful gene panel based on whole transcriptome technology. A data-driven process distilled a gene list from peer-reviewed comprehensive microarray studies that discovered and validated their use in kidney, liver, heart, and lung transplant biopsies. These were supplemented by genes that define relevant cellular pathways and cell types plus 12 reference genes used for normalization. The 770 gene B-HOT panel includes the most pertinent genes related to rejection, tolerance, viral infections, and innate and adaptive immune responses. This commercially available panel uses the NanoString platform, which can quantitate transcripts from formalin-fixed paraffin-embedded samples. The B-HOT panel will facilitate multicenter collaborative clinical research using archival samples and permit the development of an open source large database of standardized analyses, thereby expediting clinical validation studies. The MDWG believes that a pathogenesis and pathway based molecular approach will be valuable for investigators and promote therapeutic decision-making and clinical trials.
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http://dx.doi.org/10.1111/ajt.16059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496585PMC
September 2020

Discovery and validation of a novel blood-based molecular biomarker of rejection following liver transplantation.

Am J Transplant 2020 08 25;20(8):2173-2183. Epub 2020 May 25.

University of Arizona College of Medicine, Tucson, Arizona.

Noninvasive biomarker profiles of acute rejection (AR) could affect the management of liver transplant (LT) recipients. Peripheral blood was collected following LT for discovery (Northwestern University [NU]) and validation (National Institute of Allergy and Infectious Diseases Clinical Trials in Organ Transplantation [CTOT]-14 study). Blood gene profiling was paired with biopsies showing AR or ADNR (acute dysfunction no rejection) as well as stable graft function samples (Transplant eXcellent-TX). CTOT-14 subjects had serial collections prior to AR, ADNR, TX, and after AR treatment. NU cohort gene expression (46 AR, 45 TX) was analyzed using random forest models to generate a classifier training set (36 gene probe) distinguishing AR vs TX (area under the curve 0.92). The algorithm and threshold were locked and tested on the CTOT-14 validation cohort (14 AR, 50 TX), yielding an accuracy of 0.77, sensitivity 0.57, specificity 0.82, positive predictive value (PPV) 0.47, and negative predictive value (NPV) 0.87 for AR vs TX. The probability score line slopes were positive preceding AR, and negative preceding TX and non-AR (TX + ADNR) (P ≤ .001) and following AR treatment. In conclusion, we have developed a blood biomarker diagnostic for AR that can be detected prior to AR-associated graft injury as well a normal graft function (non-AR). Further studies are needed to evaluate its utility in precision-guided immunosuppression optimization following LT.
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http://dx.doi.org/10.1111/ajt.15953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496674PMC
August 2020

Banff Digital Pathology Working Group: Going digital in transplant pathology.

Am J Transplant 2020 09 19;20(9):2392-2399. Epub 2020 Apr 19.

University of Alberta, Edmonton, Canada.

The Banff Digital Pathology Working Group (DPWG) was formed in the time leading up to and during the joint American Society for Histocompatibility and Immunogenetics/Banff Meeting, September 23-27, 2019, held in Pittsburgh, Pennsylvania. At the meeting, the 14th Banff Conference, presentations directly and peripherally related to the topic of "digital pathology" were presented; and discussions before, during, and after the meeting have resulted in a list of issues to address for the DPWG. Included are practice standardization, integrative approaches for study classification, scoring of histologic parameters (eg, interstitial fibrosis and tubular atrophy and inflammation), algorithm classification, and precision diagnosis (eg, molecular pathways and therapeutics). Since the meeting, a survey with international participation of mostly pathologists (81%) was conducted, showing that whole slide imaging is available at the majority of centers (71%) but that artificial intelligence (AI)/machine learning was only used in ≈12% of centers, with a wide variety of programs/algorithms employed. Digitalization is not just an end in itself. It also is a necessary precondition for AI and other approaches. Discussions at the meeting and the survey highlight the unmet need for a Banff DPWG and point the way toward future contributions that can be made.
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http://dx.doi.org/10.1111/ajt.15850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496838PMC
September 2020

IgG4 donor-specific HLA antibody profile is associated with subclinical rejection in stable pediatric liver recipients.

Am J Transplant 2020 02 1;20(2):513-524. Epub 2019 Nov 1.

Department of Surgery, University of California San Francisco, San Francisco, California, USA.

The impact of donor-specific HLA antibody (DSA) following liver transplantation remains controversial. We hypothesized DSA IgG subclass characteristics, compared to total DSA IgG, might correlate with specific histopathological phenotype(s) of subclinical graft injury. We therefore studied 129 stable, arguably "clinically ideal," pediatric liver recipients at the time of a screening biopsy to enter an immunosuppression withdrawal trial. Sixty-five (50%) subjects tested positive for class II DSA. IgG subclass profile was characterized by mean fluorescence intensity (MFI) and normalized subclass composition (>5%). A prominent IgG4 DSA profile was strongly correlated with greater HLA mismatch, a histopathological phenotype characterized by the presence of interface activity (with variable degrees of fibrosis), and a transcriptional profile of attenuated T cell-mediated rejection. Specifically, compared to those without class II DSA, those with IgG4 class II DSA MFI sum >2000 exhibited an odds ratio (OR) of 20.79 (95% confidence interval [CI] 4.38-98.69) and IgG4 subclass composition >5% exhibited an OR of 8.99 (95% CI 2.70-29.9). Our data suggest that IgG4 DSA may serve as a useful biomarker to identify, among clinically and biochemically stable liver transplant recipients, a subset with histological and transcriptional features indicative of an active, suboptimally controlled alloimmune response.
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http://dx.doi.org/10.1111/ajt.15621DOI Listing
February 2020

CD24CD38 and CD24CD27 Human Regulatory B Cells Display Common and Distinct Functional Characteristics.

J Immunol 2019 10 11;203(8):2110-2120. Epub 2019 Sep 11.

Department of Immunology, Mayo Clinic, Scottsdale, AZ 85259;

Although IL-10-producing regulatory B cells (Bregs) play important roles in immune regulation, their surface phenotypes and functional characteristics have not been fully investigated. In this study, we report that the frequency of IL-10-producing Bregs in human peripheral blood, spleens, and tonsils is similar, but they display heterogenous surface phenotypes. Nonetheless, CD24CD38 transitional B cells (TBs) and CD24CD27 B cells (human equivalent of murine B10 cells) are the major IL-10-producing B cells. They both suppress CD4 T cell proliferation as well as IFN-γ/IL-17 expression. However, CD24CD27 B cells were more efficient than TBs at suppressing CD4 T cell proliferation and IFN-γ/IL-17 expression, whereas they both coexpress IL-10 and TNF-α. TGF-β1 and granzyme B expression were also enriched within CD24CD27 B cells, when compared with TBs. Additionally, CD24CD27 B cells expressed increased levels of surface integrins (CD11a, CD11b, α1, α4, and β1) and CD39 (an ecto-ATPase), suggesting that the in vivo mechanisms of action of the two Breg subsets are not the same. Lastly, we also report that liver allograft recipients with plasma cell hepatitis had significant decreases of both Breg subsets.
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http://dx.doi.org/10.4049/jimmunol.1900488DOI Listing
October 2019

Enhancing the Value of Histopathological Assessment of Allograft Biopsy Monitoring.

Transplantation 2019 07;103(7):1306-1322

Division of Transplant Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Traditional histopathological allograft biopsy evaluation provides, within hours, diagnoses, prognostic information, and mechanistic insights into disease processes. However, proponents of an array of alternative monitoring platforms, broadly classified as "invasive" or "noninvasive" depending on whether allograft tissue is needed, question the value proposition of tissue histopathology. The authors explore the pros and cons of current analytical methods relative to the value of traditional and illustrate advancements of next-generation histopathological evaluation of tissue biopsies. We describe the continuing value of traditional histopathological tissue assessment and "next-generation pathology (NGP)," broadly defined as staining/labeling techniques coupled with digital imaging and automated image analysis. Noninvasive imaging and fluid (blood and urine) analyses promote low-risk, global organ assessment, and "molecular" data output, respectively; invasive alternatives promote objective, "mechanistic" insights by creating gene lists with variably increased/decreased expression compared with steady state/baseline. Proponents of alternative approaches contrast their preferred methods with traditional histopathology and: (1) fail to cite the main value of traditional and NGP-retention of spatial and inferred temporal context available for innumerable objective analyses and (2) belie an unfamiliarity with the impact of advances in imaging and software-guided analytics on emerging histopathology practices. Illustrative NGP examples demonstrate the value of multidimensional data that preserve tissue-based spatial and temporal contexts. We outline a path forward for clinical NGP implementation where "software-assisted sign-out" will enable pathologists to conduct objective analyses that can be incorporated into their final reports and improve patient care.
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http://dx.doi.org/10.1097/TP.0000000000002656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594872PMC
July 2019

Outcomes of immunosuppression minimization and withdrawal early after liver transplantation.

Am J Transplant 2019 05 31;19(5):1397-1409. Epub 2018 Dec 31.

Immune Tolerance Network, San Francisco, California.

The Immune Tolerance Network ITN030ST A-WISH assessed immunosuppression withdrawal in liver transplant recipients with hepatitis C or nonimmune nonviral liver disease. Of 275 recipients enrolled before transplantation, 95 were randomly assigned 4:1 to withdrawal (n = 77) or maintenance (n = 18) 1- to 2-years posttransplant. Randomization eligibility criteria included stable immunosuppression monotherapy; adequate liver and kidney function; ≤Stage 2 Ishak fibrosis; and absence of rejection on biopsy. Immunosuppression withdrawal followed an 8-step reduction algorithm with ≥8 weeks per level. Fifty-two of 77 subjects (67.5%) reduced to ≤50% of baseline dose, and 10 of 77 (13.0%) discontinued all immunosuppression for ≥1 year. Acute rejection and/or abnormal liver tests were treated with increased immunosuppression; 5 of 32 rejection episodes required a methylprednisolone bolus. The composite end point (death or graft loss; grade 4 secondary malignancy or opportunistic infection; Ishak stage ≥3; or >25% decrease in glomerular filtration rate within 24 months of randomization) occurred in 12 of 66 (18%) and 4 of 13 (31%) subjects in the withdrawal and maintenance groups. Early immunosuppression minimization is feasible in selected liver recipients, while complete withdrawal is successful in only a small proportion. The composite end point comparison was inconclusive for noninferiority of the withdrawal to the maintenance group.
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http://dx.doi.org/10.1111/ajt.15205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482056PMC
May 2019

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants.

Gastroenterology 2018 12 23;155(6):1838-1851.e7. Epub 2018 Aug 23.

Institute of Liver Studies, King's College, London, London, United Kingdom.

Background & Aims: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles.

Methods: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data.

Results: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3.

Conclusion: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.
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http://dx.doi.org/10.1053/j.gastro.2018.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279538PMC
December 2018

Study rationale, design, and pretransplantation alloantibody status: A first report of Clinical Trials in Organ Transplantation in Children-04 (CTOTC-04) in pediatric heart transplantation.

Am J Transplant 2018 09 23;18(9):2135-2147. Epub 2018 Mar 23.

Division of Pediatric Cardiology, Monroe Carrell Jr. Children's Hospital at Vanderbilt, Nashville, TN, USA.

Anti-HLA donor-specific antibodies are associated with worse outcomes after organ transplantation. Among sensitized pediatric heart candidates, requirement for negative donor-specific cytotoxicity crossmatch increases wait times and mortality. However, transplantation with positive crossmatch may increase posttransplantation morbidity and mortality. We address this clinical challenge in a prospective, multicenter, observational cohort study of children listed for heart transplantation (Clinical Trials in Organ Transplantation in Children-04 [CTOTC-04]). Outcomes were compared among sensitized recipients who underwent transplantation with positive crossmatch, nonsensitized recipients, and sensitized recipients without positive crossmatch. Positive crossmatch recipients received antibody removal and augmented immunosuppression, while other recipients received standard immunosuppression with corticosteroid avoidance. This first CTOTC-04 report summarizes study rationale and design and relates pretransplantation sensitization status using solid-phase technology. Risk factors for sensitization were explored. Of 317 screened patients, 290 were enrolled and 240 underwent transplantation. Core laboratory evaluation demonstrated that more than half of patients were anti-HLA sensitized. Greater than 80% of sensitized patients had class I (with or without class II) HLA antibodies, and one-third of sensitized patients had at least 1 HLA antibody with median fluorescence intensity of ≥8000. Logistic regression models demonstrated male sex, weight, congenital heart disease history, prior allograft, and ventricular assist device are independent risk factors for sensitization.
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http://dx.doi.org/10.1111/ajt.14695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093810PMC
September 2018

Global quality assessment of liver allograft C4d staining during acute antibody-mediated rejection in formalin-fixed, paraffin-embedded tissue.

Hum Pathol 2018 03 27;73:144-155. Epub 2017 Dec 27.

Department of Pathology, University of Pittsburgh Medical Center, UPMC Montefiore University Hospital, Pittsburgh, PA 15213, USA. Electronic address:

Discussion of liver antibody-mediated rejection during the 2011, 2013, and 2015 Banff liver sessions raised concerns over reliability of complement fragment 4d (C4d) staining, precipitating a global survey followed by a tissue microarray staining quality assessment study among centers on formalin-fixed, paraffin-embedded tissue. Tissue microarray sections containing tissue plugs of resected native and allograft (with acute antibody-mediated rejection) liver, heart, and kidney (n = 33 total cores) were sent to 31 centers for C4d staining using local method(s) and pathologist scoring. Digital whole-slide images (n = 40) were then semiquantitatively scored by 7 experts for background, distribution, and intensity of portal vein and capillary, hepatic artery, sinusoidal, and central vein endothelia and portal and central stromal staining. Results showed that strong and diffuse portal vein and capillary C4d staining, as determined by both local and central pathologists, clearly distinguished allografts showing acute antibody-mediated rejection from native livers and from those with evidence of weaker donor-specific antibody. Downstream vascular endothelial cell C4d staining and assessment were more variable and difficult to identify. C4d staining in the majority of laboratories reliably detects acute liver allograft antibody-mediated rejection in formalin-fixed, paraffin-embedded tissues. Assessment should focus on portal veins and capillaries, sinusoids, and central veins present in peripheral core needle biopsies. C4d staining in one organ does not always translate to staining in another.
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http://dx.doi.org/10.1016/j.humpath.2017.12.007DOI Listing
March 2018

PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-versus-Host Disease.

J Immunol 2018 01 6;200(2):834-846. Epub 2017 Dec 6.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520;

Effector memory T cells (T) are less capable of inducing graft-versus-host disease (GVHD) compared with naive T cells (T). Previously, in the TS1 TCR transgenic model of GVHD, wherein TS1 CD4 cells specific for a model minor histocompatibility Ag (miHA) induce GVHD in miHA-positive recipients, we found that cell-intrinsic properties of TS1 T reduced their GVHD potency relative to TS1 T Posttransplant, TS1 T progeny expressed higher levels of PD-1 than did TS1 T progeny, leading us to test the hypothesis that T induce less GVHD because of increased sensitivity to PD-ligands. In this study, we tested this hypothesis and found that indeed TS1 T induced more severe skin and liver GVHD in the absence of PD-ligands. However, lack of PD-ligands did not result in early weight loss and colon GVHD comparable to that induced by TS1 T, indicating that additional pathways restrain alloreactive T TS1 T also caused more severe GVHD without PD-ligands. The absence of PD-ligands on donor bone marrow was sufficient to augment GVHD caused by either T or T, indicating that donor PD-ligand-expressing APCs critically regulate GVHD. In the absence of PD-ligands, both TS1 T and T induced late-onset myocarditis. Surprisingly, this was an autoimmune manifestation, because its development required non-TS1 polyclonal CD8 T cells. Myocarditis development also required donor bone marrow to be PD-ligand deficient, demonstrating the importance of donor APC regulatory function. In summary, PD-ligands suppress both miHA-directed GVHD and the development of alloimmunity-induced autoimmunity after allogeneic hematopoietic transplantation.
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http://dx.doi.org/10.4049/jimmunol.1701076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760452PMC
January 2018

Late graft dysfunction after pediatric heart transplantation is associated with fibrosis and microvasculopathy by automated, digital whole-slide analysis.

J Heart Lung Transplant 2017 Dec 29;36(12):1336-1343. Epub 2017 Sep 29.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; Division of Transplant Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

Background: Histopathologic features of late graft dysfunction (LGD) in endomyocardial biopsies (EMBs) after pediatric heart transplantation (HT) have been incompletely described and rarely quantified. We employed automated, morphometric analysis of whole-slide EMB images to objectively quantify fibrosis and microvasculopathy after pediatric HT.

Methods: Nine recipients with clinical LGD were matched with controls on age, listing diagnosis, crossmatch and time since HT. Fibrosis was quantified as percent tissue area with fibrosis and capillary density as capillaries per unit area, number of capillary "neighbors" within 30 μm of each myocyte and myocyte-to-nearest-capillary diffusion distance. Clinical data, including all EMB reports, were also reviewed.

Results: The groups were well matched for age at HT (median 4.0 vs 3.1 years), listing diagnosis (50% congenital heart disease for each), positive crossmatch (11% each) and days post-HT (2,628 vs 2,894, p = 0.69). Despite a similar number of previous EMBs (median 23 each, p = 0.43), areas occupied by fibrosis were greater in LGD cases (44.5% vs 23.2%, p = 0.012). Capillary number/area data were not statistically different between LGD cases and controls (378/mm vs 559/mm, p = 0.57), but LGD cases more commonly had zero capillary neighbors (35% vs 20%, p = 0.02) and greater myocyte-to-nearest-capillary distances (27.1 μm vs 18.7 μm, p = 0.005). Cumulative rejection history correlated with fibrosis (r = 0.49, p = 0.039) and myocyte-to-nearest-capillary distance (r = 0.5, p = 0.036).

Conclusions: LGD after pediatric HT is associated with previous rejection and characterized histologically by fibrosis and microvasculopathy, which are not readily appreciated by traditional semi-quantitative EMB analysis. Software-assisted EMB analysis may enable greater pathophysiologic understanding of LGD and identification of targets for future study and intervention.
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http://dx.doi.org/10.1016/j.healun.2017.09.012DOI Listing
December 2017

Longterm outcome of the liver graft: The pathologist's perspective.

Liver Transpl 2017 10;23(S1):S70-S75

Division of Transplant Pathology, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA.

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http://dx.doi.org/10.1002/lt.24851DOI Listing
October 2017

Non-HLA Antibodies Impact on C4d Staining, Stellate Cell Activation and Fibrosis in Liver Allografts.

Transplantation 2017 10;101(10):2399-2409

1 Annette C. & Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, TX. 2 Department of Pathology, University of Pittsburgh, Pittsburg, PA. 3 Department of Nephrology and Critical Care Medicine, Charité, Berlin, Germany. 4 Terasaki Foundation Laboratory, Los Angeles, CA. 5 Celltrend, Luckenwalde, Germany.

Background: Recent data have shown an increased risk for rejection, fibrosis progression, and death in liver transplantation (LT) recipients with preformed or de novo HLA donor-specific alloantibodies (DSA). However, the role of non-HLA autoantibodies and the interaction between HLA DSA and non-HLA autoantibodies remains uncharacterized.

Methods: We analyzed 1269 primary LT recipients from 1 of 2000 to 4 of 2009 with known HLA DSA status for angiotensin II type-1 receptor and endothelin-1 type A receptor autoantibodies pre-LT, and year 1 post-LT.

Results: Preformed non-HLA autoantibodies alone did not impact outcomes. In multivariable modeling, the combination of preformed non-HLA autoantibodies and HLA-DSA were associated with an increased risk for death (hazard ratio [HR], 1.66; P = 0.02) especially if the HLA DSA was of the IgG3 subclass (HR, 2.28; P = 0.01). A single de novo non-HLA autoantibody was associated with an increased risk for T cell-mediated rejection or antibody-mediated rejection (68% vs 41%, P = 0.01) and fibrosis progression (HR, 1.84; P = 0.02). Biopsies with de novo non-HLA autoantibodies revealed a new sinusoidal C4d staining pattern when compared with HLA DSA (71% vs 3%; P < 0.001). Liver sinusoidal endothelial cell activation and stellate cell activation was increased in patients with non-HLA autoantibodies in the location of C4d positivity.

Conclusions: A non-HLA autoantibody combined with a preformed HLA DSA is associated with an increased mortality risk. Isolated de novo anti-angiotensin II type-1 receptor and anti-endothelin-1 type A receptor autoantibodies are associated with an increased risk of rejection and fibrosis progression. The novel location of C4d staining in proximity to liver sinusoidal endothelial cell capillarization and stellate cell activation demonstrates allograft injury in proximity to non-HLA autoantibody binding.
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http://dx.doi.org/10.1097/TP.0000000000001853DOI Listing
October 2017

NK1.1 cells promote sustained tissue injury and inflammation after trauma with hemorrhagic shock.

J Leukoc Biol 2017 07 17;102(1):127-134. Epub 2017 May 17.

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;

Various cell populations expressing NK1.1 contribute to innate host defense and systemic inflammatory responses, but their role in hemorrhagic shock and trauma remains uncertain. NK1.1 cells were depleted by i.p. administration of anti-NK1.1 (or isotype control) on two consecutive days, followed by hemorrhagic shock with resuscitation and peripheral tissue trauma (HS/T). The plasma levels of IL-6, MCP-1, alanine transaminase (ALT), and aspartate aminotransferase (AST) were measured at 6 and 24 h. Histology in liver and gut were examined at 6 and 24 h. The number of NK cells, NKT cells, neutrophils, and macrophages in liver, as well as intracellular staining for TNF-α, IFN-γ, and MCP-1 in liver cell populations were determined by flow cytometry. Control mice subjected to HS/T exhibited end organ damage manifested by marked increases in circulating ALT, AST, and MCP-1 levels, as well as histologic evidence of hepatic necrosis and gut injury. Although NK1.1 cell-depleted mice exhibited a similar degree of organ damage as nondepleted animals at 6 h, NK1.1 cell depletion resulted in marked suppression of both liver and gut injury by 24 h after HS/T. These findings indicate that NK1.1 cells contribute to the persistence of inflammation leading to end organ damage in the liver and gut.
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http://dx.doi.org/10.1189/jlb.3A0716-333RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608053PMC
July 2017

Chronic AMR in Liver Transplant: Validation of the 1-Year cAMR Score's Ability to Determine Long-term Outcome.

Transplantation 2017 09;101(9):2062-2070

1 Annette C. & Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas TX. 2 Terasaki Foundation Laboratory, Los Angeles, CA. 3 Department of Pathology, University of Pittsburgh, Pittsburg, PA.

Background: A proposed chronic antibody-mediated rejection (AMR) score has recently predicted 50%10-year death-censored allograft loss in patients with donor-specific alloantibodies (DSA) mean florescence intensity (MFI) greater than 10 000 and requires confirmation in patients with lower MFI (1000-10 000).

Methods: All patients who underwent liver transplantation from January 2000 to April 2009, had DSA (MFI ≥1000) in serum 10 to 14 months postliver transplantation, and had a protocolized liver biopsy were evaluated (n = 230). The previously proposed chronic AMR (cAMR) score was used to risk-stratify putative chronic AMR in DSA+ patients with MFI from 1000 to 10 000.

Results: The MFI distribution of DSA+ recipients were as follows: 66% had MFI 1000 to 4999, 14% had MFI 5000 to 10 000, and 20% had MFI greater than 10 000. The cAMR score distribution on 1-year protocol liver biopsy found that 41% had a score less than 13; 27% a score of 13 to 27.5, and 32% a score greater than 27.5. MFI correlated with 1-year cAMR category (<13, 46% vs 21% and >27.5, 29% vs 42% when MFI was 1000-10 000 vs MFI >10 000; P = 0.047). In patients with a cAMR score less than 13, 10-year death-censored allograft survival was 96% to 100% regardless of MFI (P = NS). The risk of allograft loss increased in patients with a cAMR score greater than 13 (P = 0.004) in DSA+ patients with MFI 1000 to 10 000. DSA MFI greater than 10 000 versus MFI 1000 to 10 000 at 1 year was also more likely to persist at 5 years (95% vs 68%; P < 0.0001).

Conclusions: Validation of the previously proposed cAMR score in a separate cohort predicts death-censored long-term allograft failure in DSA+ patients regardless of MFI, and higher MFI at 1 year predicts DSA persistence at 5 years.
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http://dx.doi.org/10.1097/TP.0000000000001802DOI Listing
September 2017

Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R.

Hepatology 2017 02 27;65(2):647-660. Epub 2016 Jul 27.

Immune Tolerance Network, Bethesda, MD.

Pediatric liver transplant recipients arguably have the most to gain and the most to lose from discontinuing immunosuppression (IS). Whereas IS undoubtedly exerts a cumulative toll, there is concern that insufficient or no IS may contribute to allograft deterioration. Twelve pediatric recipients of parental living donor liver grafts, identified as operationally tolerant through complete IS withdrawal (WISP-R; NCT00320606), were followed for a total of 5 years (1 year of IS withdrawal and 4 years off IS) with serial liver tests and autoantibody and alloantibody assessments. Liver biopsies were performed 2 and 4 years off IS, and, at these time points, immunoglobulin G (IgG) subclass and C1q binding activity for donor-specific antibodies (DSAs) were determined. There were no cases of chronic rejection, graft loss, or death. Allografts did not exhibit progressive increase in inflammation or fibrosis. Smooth-muscle actin expression by stellate cells and CD34 expression by liver sinusoidal endothelial cells remained stable, consistent with the absence of progressive graft injury. Three subjects never exhibited DSA. However, 3 subjects showed intermittent de novo class I DSA, 4 subjects showed persistent de novo class II DSA, and 5 subjects showed persistent preexisting class II DSA. Class II DSA was predominantly against donor DQ antigens, often of high mean fluorescence intensity, rarely of the IgG3 subclass, and often capable of binding C1q.

Conclusion: Operationally tolerant pediatric liver transplant recipients maintain generally stable allograft histology in spite of apparently active humoral allo-immune responses. The absence of increased inflammation or progressive fibrosis suggests that a subset of liver allografts seem resistant to the chronic injury that is characteristic of antibody-mediated damage. (Hepatology 2017;65:647-660).
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http://dx.doi.org/10.1002/hep.28681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159322PMC
February 2017

Prevention and treatment of liver allograft antibody-mediated rejection and the role of the 'two-hit hypothesis'.

Curr Opin Organ Transplant 2016 Apr;21(2):209-18

aAnnette C. and Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, Texas bDepartment of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Purpose Of Review: The review outlines the diagnosis, prevention strategies, and possible treatment options for acute and chronic antibody-mediated rejection (AMR).

Recent Findings: Although rare, severe acute AMR (aAMR) usually occurs in patients with high mean fluorescence intensity despite serial dilutions or high-titer preformed class I donor-specific alloantibodies (DSA). The diagnosis is suspected when allograft dysfunction occurs with DSA, diffuse C4d staining, and a microvascular injury, and may be aided by the aAMR score. However, the incidence of and treatment approach to combined T-cell-mediated rejection (TCMR) with DSA present and some but not all features of AMR is yet to be determined. Chronic liver allograft AMR is characterized by low-grade chronic inflammation and progressive fibrosis with DSA, the chronic AMR (cAMR) score may facilitate diagnosis. The 'two-hit' hypothesis, whereby a coexistent insult upregulates human leukocyte antigen class II target antigens on the microvascular endothelium, may explain why suboptimal donors with lower sensitization levels might suffer from acute AMR and those with chronic complications (e.g., recurrent original disease) might be more susceptible to chronic AMR. Although treatment algorithms are needed, prevention is preferable and at a minimum includes transfusion minimization, and medication adherence.

Summary: Severe acute AMR is rare but diagnosable, and there is need to determine the incidence of and optimal therapy for less severe combined AMR and TCMR. Chronic AMR is likely more common and of significant relevance to long-term allograft survival improvement. The two-hit hypothesis may help to explain the rarity of both findings and shed insight onto future prevention and treatment strategies.
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http://dx.doi.org/10.1097/MOT.0000000000000275DOI Listing
April 2016

A pilot study of operational tolerance with a regulatory T-cell-based cell therapy in living donor liver transplantation.

Hepatology 2016 08 10;64(2):632-43. Epub 2016 Mar 10.

Center for Allergy and Immunology, Juntendo University School of Medicine, Tokyo, Japan.

Unlabelled: Potent immunosuppressive drugs have significantly improved early patient survival after liver transplantation (LT). However, long-term results remain unsatisfactory because of adverse events that are largely associated with lifelong immunosuppression. To solve this problem, different strategies have been undertaken to induce operational tolerance, for example, maintenance of normal graft function and histology without immunosuppressive therapy, but have achieved limited success. In this pilot study, we aimed to induce tolerance using a novel regulatory T-cell-based cell therapy in living donor LT. Adoptive transfer of an ex vivo-generated regulatory T-cell-enriched cell product was conducted in 10 consecutive adult patients early post-LT. Cells were generated using a 2-week coculture of recipient lymphocytes with irradiated donor cells in the presence of anti-CD80/86 monoclonal antibodies. Immunosuppressive agents were tapered from 6 months, reduced every 3 months, and completely discontinued by 18 months. After the culture, the generated cells displayed cell-number-dependent donor-specific inhibition in the mixed lymphocyte reaction. Infusion of these cells caused no significant adverse events. Currently, all patients are well with normal graft function and histology. Seven patients have completed successful weaning and cessation of immunosuppressive agents. At present, they have been drug free for 16-33 months; 4 patients have been drug free for more than 24 months. The other 3 recipients with autoimmune liver diseases developed mild rejection during weaning and then resumed conventional low-dose immunotherapy.

Conclusions: A cell therapy using an ex vivo-generated regulatory T-cell-enriched cell product is safe and effective for drug minimization and operational tolerance induction in living donor liver recipients with nonimmunological liver diseases. (Hepatology 2016;64:632-643).
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http://dx.doi.org/10.1002/hep.28459DOI Listing
August 2016

IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production.

J Immunol 2015 Jun 11;194(12):6045-56. Epub 2015 May 11.

Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; Liver Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA 15261

Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx. IRF-1 was induced in liver grafts immediately after reperfusion in both human and mouse LTx. IRF-1 contributed significantly to I/R injury because IRF-1-knockout (KO) grafts displayed much less damage as assessed by serum alanine aminotransferase and histology. In vitro, IRF-1 regulated both constitutive and induced expression of IL-15, as well as IL-15Rα mRNA expression in murine hepatocytes and liver dendritic cells. Specific knockdown of IRF-1 in human primary hepatocytes gave similar results. In addition, we identified hepatocytes as the major producer of soluble IL-15/IL-15Rα complexes in the liver. IRF-1-KO livers had significantly reduced NK, NKT, and CD8(+) T cell numbers, whereas rIL-15/IL-15Rα restored these immune cells, augmented cytotoxic effector molecules, promoted systemic inflammatory responses, and exacerbated liver injury in IRF-1-KO graft recipients. These results indicate that IRF-1 promotes LTx I/R injury via hepatocyte IL-15/IL-15Rα production and suggest that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.
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http://dx.doi.org/10.4049/jimmunol.1402505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458432PMC
June 2015

ABO-compatible liver allograft antibody-mediated rejection: an update.

Curr Opin Organ Transplant 2015 Jun;20(3):314-24

aDivision of Transplantation, Department of Pathology, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania bAnnette C. & Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, Texas, USA.

Purpose Of Review: Liver allograft antibody-mediated rejection (AMR) studies have lagged behind parallel efforts in kidney and heart because of a comparative inherent hepatic resistance to AMR. Three developments, however, have increased interest: first, solid phase antibody testing enabled more precise antibody characterization; second, increased expectations for long-term, morbidity-free survival; and third, immunosuppression minimization trials.

Recent Findings: Two overlapping liver allograft AMR phenotypic expressions are beginning to emerge: acute and chronic AMR. Acute AMR usually occurs within the several weeks after transplantation and characterized clinically by donor-specific antibodies (DSA) persistence, allograft dysfunction, thrombocytopenia, and hypocomplementemia. Acute AMR appears histopathologically similar to acute AMR in other organs: diffuse microvascular endothelial cell hypertrophy, C4d deposits, neutrophilic, eosinophilic, and macrophag-mediated microvasculitis/capillaritis, along with liver-specific ductular reaction, centrilobular hepatocyte swelling, and hepatocanalicular cholestasis often combined with T-cell-mediated rejection (TCMR). Chronic AMR is less well defined, but strongly linked to serum class II DSA and associated with late-onset acute TCMR, fibrosis, chronic rejection, and decreased survival. Unlike acute AMR, chronic AMR is a slowly evolving insult with a number of potential manifestations, but most commonly appears as low-grade lymphoplasmacytic portal and perivenular inflammation accompanied by unusual fibrosis patterns and variable microvascular C4d deposition; capillaritis can be more difficult to identify than in acute AMR.

Summary: More precise DSA characterization, increasing expectations for long-term survival, and immunosuppression weaning precipitated a re-emergence of liver allograft AMR interest. Pathophysiological similarities exist between heart, kidney, and liver allografts, but liver-specific considerations may prove critical to our ultimate understanding of all solid organ AMR.
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http://dx.doi.org/10.1097/MOT.0000000000000194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646419PMC
June 2015

Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia.

J Hepatol 2015 Aug 12;63(2):399-407. Epub 2015 Mar 12.

Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, PA 15260, USA; The Department of Pathology, Division of Liver and Transplantation Pathology, University of Pittsburgh Medical Center, PA 15260, USA. Electronic address:

Background & Aims: Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers.

Methods: Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a.

Results: BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers.

Conclusion: BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.
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http://dx.doi.org/10.1016/j.jhep.2015.02.047DOI Listing
August 2015