Oncostatin M Plays a Critical Role in Survival after Acute Intestinal Ischemia: Reperfusion Injury.

Background: Acute intestinal ischemia-reperfusion injury (AIIRI) is a devastating clinical condition relevant to multiple diseases processes, including sepsis, trauma, transplantation, and burns. An AIIRI is a contributor to the development of multiple organ dysfunction syndrome (MODS). Oncostatin M (OSM)/oncostatin M receptor (OSMR) signaling is an unrecognized and novel candidate pathway for the mediation of MODS. In this study, we hypothesized that OSM mediates the injury mechanism of AIIRI leading to MODS. Methods: Wild-type (WT) and OSMR-knockout (OSMR-/-) C57BL/6 mice underwent AIIRI using a well-established model of selective occlusion of the superior mesenteric artery (SMA). Serum cytokine concentrations were measured using a multiplex detection system. Further tissue analysis was conducted with polymerase chain reaction, enzyme-linked immunosorbent assay, Western blots, and histologic review. Results: Survival was significantly higher in WT than in OSMR-/- groups at 30 minutes of ischemia with 2 hours of reperfusion (100% versus 42.9%; P = 0.015). No significant differences in the degree of local intestinal injury was seen in the two groups. In contrast, the degree of lung injury, as evidenced by myeloperixodase activity, was lower in OSMR-/- animals in the early AIIRI groups. There was a greater degree of renal dysfunction in OSMR-/- mice. Oncostatin M mediated interleukin (IL)-10 upregulation, with WT animals having significantly lower IL-10 concentrations (52.04 ± 23.06 pg/mL versus 324.37 ± 140.35 pg/mL; P = 0.046). Conclusion: Oncostatin M signalling is essential during acute intestinal ischemia-reperfusion injury. An OSMR deficiency results in decreased early lung injury but increased renal dysfunction. There was a significantly increased mortality rate after AIIRI in mice with OSMR deficiency. Augmentation of OSM may be a novel immunomodulatory strategy for AIIRI.

Clinicopathologic predictors of renal outcomes in light chain cast nephropathy: a multicenter retrospective study.

Light chain cast nephropathy (LCCN) in multiple myeloma often leads to severe and poorly reversible acute kidney injury. Severe renal impairment influences the allocation of chemotherapy and its tolerability; it also affects patient survival. Whether renal biopsy findings add to the clinical assessment in predicting renal and patient outcomes in LCCN is uncertain. We retrospectively reviewed clinical presentation, chemotherapy regimens, hematologic response, and renal and patient outcomes in 178 patients with biopsy-proven LCCN from 10 centers in Europe and North America. A detailed pathology review, including assessment of the extent of cast formation, was performed to study correlations with initial presentation and outcomes. Patients presented with a mean estimated glomerular filtration rate (eGFR) of 13 ± 11 mL/min/1.73 m2, and 82% had stage 3 acute kidney injury. The mean number of casts was 3.2/mm2 in the cortex. Tubulointerstitial lesions were frequent: acute tubular injury (94%), tubulitis (82%), tubular rupture (62%), giant cell reaction (60%), and cortical and medullary inflammation (95% and 75%, respectively). Medullary inflammation, giant cell reaction, and the extent of cast formation correlated with eGFR value at LCCN diagnosis. During a median follow-up of 22 months, mean eGFR increased to 43 ± 30 mL/min/1.73 m2. Age, ß2-microglobulin, best hematologic response, number of cortical casts per square millimeter, and degree of interstitial fibrosis/tubular atrophy (IFTA) were independently associated with a higher eGFR during follow-up. This eGFR value correlated with overall survival, independently of the hematologic response. This study shows that extent of cast formation and IFTA in LCCN predicts the quality of renal response, which, in turn, is associated with overall survival.

Apelin directs endothelial cell differentiation and vascular repair following immune-mediated injury.

Sustained, indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized, maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes, associated with expression of apelin. In the adult, the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo, loss of donor heart expression of apelin facilitated graft immune cell infiltration, blunted vascular repair, and worsened occlusive vasculopathy in mice. In vitro, an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus, apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together, these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature.

Archetype Analysis Identifies Distinct Profiles in Renal Transplant Recipients with Transplant Glomerulopathy Associated with Allograft Survival.

BACKGROUND: Transplant glomerulopathy, a common glomerular lesion observed after kidney transplant that is associated with poor prognosis, is not a specific entity but rather the end stage of overlapping disease pathways. Its heterogeneity has not been precisely characterized to date. METHODS: Our study included consecutive kidney transplant recipients from three centers in France and one in Canada who presented with a diagnosis of transplant glomerulopathy (Banff cg score ≥1 by light microscopy), on the basis of biopsies performed from January of 2004 through December of 2014. We used an unsupervised archetype analysis of comprehensive pathology findings and clinical, immunologic, and outcome data to identify distinct groups of patients. RESULTS: Among the 8207 post-transplant allograft biopsies performed during the inclusion period, we identified 552 biopsy samples (from 385 patients) with transplant glomerulopathy (incidence of 6.7%). The median time from transplant to transplant glomerulopathy diagnosis was 33.18 months. Kidney allograft survival rates at 3, 5, 7, and 10 years after diagnosis were 69.4%, 57.1%, 43.3%, and 25.5%, respectively. An unsupervised learning method integrating clinical, functional, immunologic, and histologic parameters revealed five transplant glomerulopathy archetypes characterized by distinct functional, immunologic, and histologic features and associated causes and distinct allograft survival profiles. These archetypes showed significant differences in allograft outcomes, with allograft survival rates 5 years after diagnosis ranging from 88% to 22%. Based on those results, we built an online application, which can be used in clinical practice on the basis of real patients. CONCLUSIONS: A probabilistic data-driven archetype analysis approach applied in a large, well defined multicenter cohort refines the diagnostic and prognostic features associated with cases of transplant glomerulopathy. Reducing heterogeneity among such cases can improve disease characterization, enable patient-specific risk stratification, and open new avenues for archetype-based treatment strategies and clinical trials optimization.

Antibody-Mediated Rejection in a Blood Group A-Transgenic Mouse Model of ABO-Incompatible Heart Transplantation.

BACKGROUND: ABO-incompatible (ABOi) organ transplantation is performed owing to unremitting donor shortages. Defining mechanisms of antibody-mediated rejection, accommodation, and tolerance of ABOi grafts is limited by lack of a suitable animal model. We report generation and characterization of a murine model to enable study of immunobiology in the setting of ABOi transplantation. METHODS: Transgenesis of a construct containing human A1- and H-transferases under control of the ICAM-2 promoter was performed in C57BL/6 (B6) mice. A-transgenic (A-Tg) mice were assessed for A-antigen expression by histology and flow cytometry. B6 wild-type (WT) mice were sensitized with blood group A-human erythrocytes; others received passive anti-A monoclonal antibody and complement after heart transplant. Serum anti-A antibodies were assessed by hemagglutination. "A-into-O" transplantation (major histocompatibility complex syngeneic) was modeled by transplanting hearts from A-Tg mice into sensitized or nonsensitized WT mice. Antibody-mediated rejection was assessed by morphology/immunohistochemistry. RESULTS: A-Tg mice expressed A-antigen on vascular endothelium and other cells including erythrocytes. Antibody-mediated rejection was evident in 15/17 A-Tg grafts in sensitized WT recipients (median titer, 1:512), with 2 showing hyperacute rejection and rapid cessation of graft pulsation. Hyperacute rejection was observed in 8/8 A-Tg grafts after passive transfer of anti-A antibody and complement into nonsensitized recipients. Antibody-mediated rejection was not observed in A-Tg grafts transplanted into nonsensitized mice. CONCLUSIONS: A-Tg heart grafts transplanted into WT mice with abundant anti-A antibody manifests characteristic features of antibody-mediated rejection. These findings demonstrate an effective murine model to facilitate study of immunologic features of ABOi transplantation and to improve potential diagnostic and therapeutic strategies.

Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology.

Impact of combination antiretroviral therapy in the NOD.c3c4 mouse model of autoimmune biliary disease.

BACKGROUND & AIMS: The NOD.c3c4 mouse model develops autoimmune biliary disease characterized by spontaneous granulomatous cholangitis, antimitochondrial antibodies and liver failure. This model for primary biliary cirrhosis (PBC) has evidence of biliary infection with mouse mammary tumour virus (MMTV), suggesting that the virus may have a role in cholangitis development and progression of liver disease in this mouse model. We tested the hypothesis that MMTV infection is associated with cholangitis in the NOD.c3c4 mouse model by investigating whether antiretroviral therapy impacts on viral levels and liver disease. METHODS: NOD.c3c4 mice were treated with combination antiretroviral therapy. Response to treatment was studied by measuring MMTV RNA in the liver, liver enzyme levels in serum and liver histology using a modified Ishak score. RESULTS: Combination therapy with the reverse transcriptase inhibitors, tenofovir and emtricitabine, resulted in a significant reduction in serum liver enzyme levels, attenuation of cholangitis and decreased MMTV levels in the livers of NOD.c3c4 mice. Furthermore, treatment with the retroviral protease inhibitors, lopinavir and ritonavir, in addition to the reverse transcriptase inhibitors, resulted in further decrease in MMTV levels and attenuation of liver disease in this model. CONCLUSIONS: The attenuation of cholangitis with regimens containing the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the protease inhibitors, lopinavir and ritonavir, suggests that retroviral infection may play a role in the development of cholangitis in this model.

Isolated endarteritis and kidney transplant survival: a multicenter collaborative study.

Isolated endarteritis of kidney transplants is increasingly recognized. Notably, microarray studies revealed absence of immunologic signatures of rejection in most isolated endarteritis biopsy samples. We investigated if isolated endarteritis responds to rejection treatment and affects kidney transplant survival. We retrospectively enrolled recipients of kidney transplant who underwent biopsies between 1999 and 2011 at seven American and Canadian centers. Exclusion criteria were recipients were blood group-incompatible or crossmatch-positive or had C4d-positive biopsy samples. After biopsy confirmation, patients were divided into three groups: isolated endarteritis (n=103), positive controls (type I acute T cell-mediated rejection with endarteritis; n=101), and negative controls (no diagnostic rejection; n=103). Primary end points were improved kidney function after rejection treatment and transplant failure. Mean decrease in serum creatinine from biopsy to 1 month after rejection treatment was 132.6 µmol/L (95% confidence interval [95% CI], 78.7 to 186.5) in patients with isolated endarteritis, 96.4 µmol/L (95% CI, 48.6 to 143.2) in positive controls (P=0.32), and 18.6 µmol/L (95% CI, 1.8 to 35.4) in untreated negative controls (P<0.001). Functional improvement after rejection treatment occurred in 80% of patients with isolated endarteritis and 81% of positive controls (P=0.72). Over the median 3.2-year follow-up period, kidney transplant survival rates were 79% in patients with isolated endarteritis, 79% in positive controls, and 91% in negative controls (P=0.01). In multivariate analysis, isolated endarteritis was associated with an adjusted 3.51-fold (95% CI, 1.16 to 10.67; P=0.03) risk for transplant failure. These data indicate that isolated endarteritis is an independent risk factor for kidney transplant failure.

A systematic review of the role of C4d in the diagnosis of acute antibody-mediated rejection.

In this study, we conducted a systematic review of the literature to re-evaluate the role of C4d in the diagnosis of acute antibody-mediated rejection of kidney allografts. Electronic databases were searched until September 2013. Eligible studies allowed derivation of diagnostic tables for the performance of C4d by immunofluorescence or immunohistochemistry with comparison to histopathological features of acute antibody-mediated rejection and/or donor-specific antibody (DSA) assays. Of 3492 unique abstracts, 29 studies encompassing 3485 indication and 868 surveillance biopsies were identified. Assessment of C4d by immunofluorescence and immunohistochemistry exhibited slight to moderate agreement with glomerulitis, peritubular capillaritis, solid-phase DSA assays, DSA with glomerulitis, and DSA with peritubular capillaritis. The sensitivity and specificity of C4d varied as a function of C4d and comparator test thresholds. Prognostically, the presence of C4d was associated with inferior allograft survival compared with DSA or histopathology alone. Thus, our findings support the presence of complement-dependent and -independent phenotypes of acute antibody-mediated rejection. Whether the presence of C4d in combination with histopathology or DSA should be considered for the diagnosis of acute antibody-mediated rejection warrants further study.

Pathologic basis of antibody-mediated organ transplant rejection: from pathogenesis to diagnosis.

PURPOSE OF REVIEW: Although antibody-mediated rejection of clinical organ transplants has been recognized more than a half-century ago, our understanding of its pathological/clinical phenotypes has dramatically increased over the past decade. This review highlights the pathological/clinical spectrum of ABMR and discusses its microscopic pathology in relationship with pathogenesis. RECENT FINDINGS: Newly recognized pathological manifestations of ABMR are: (1) C4d-negative active ABMR, which cannot be definitely diagnosed by current diagnostic systems and often remains underdetected. Novel molecular diagnostic tests can fill this diagnostic gap but these new tests are yet to be prepared for routine application; (2) antibody-mediated vascular rejection, which is misclassified by the current Banff Classification, is therefore inadequately treated and has a high risk for transplant failure; and (3) subclinical (insidious) microvascular inflammation, which can be with or without complement activation, predicts progression to chronic rejection, transplant dysfunction, and failure. SUMMARY: A major progress has been made in understanding of ABMR of clinical transplants in the last 5 years. New pathology types of ABMR are not appropriately classified and updates to the Banff diagnostic criteria are required. Better diagnosis would help develop effective antiantibody treatment strategies and improve long-term outcomes for patients.

Molecular transplantation pathology: the interface between molecules and histopathology.

PURPOSE OF REVIEW: In the last decade, high-throughput molecular screening methods have revolutionized the transplantation research. This article reviews the new knowledge that has emerged from transplant patient sample-derived 'omics data by examining the interface between molecular signals and allograft pathology. RECENT FINDINGS: State-of-the-art molecular studies have shed light on the biology of organ transplant diseases and provided several potential molecular tests with diagnostic, prognostic, and theranostic applications for the implementation of personalized medicine in transplantation. By comprehensive molecular profiling of patient samples, we have learned numerous new insights into the effector mechanisms and parenchymal response during allograft diseases. It has become evident that molecular profiles are coordinated and move in patterns similar to histopathology lesions, and therefore lack qualitative specificity. However, molecular tests can empower precision diagnosis and prognostication through their objective and quantitative manner when they are integrated in a holistic approach with histopathology and clinical factors of patients. SUMMARY: Despite clever science and large amounts of public money invested in transplant 'omics studies, multiparametric molecular testing has not yet been translated to patient care. There are serious challenges in the implementation of transplant molecular diagnostics that have increased frustration in transplant community. We appeal for a full collaboration between pathologists and researchers to accelerate transition from research to clinical practice in transplantation.

Advances in the understanding of transplant glomerulopathy.

Transplant glomerulopathy is a sign of chronic kidney allograft damage. It has poor survival and no effective therapies. This entity develops as a maladaptive repair/remodeling response to sustained endothelial injury and is characterized by duplication/multilamination of capillary basement membranes. This review provides up-to-date information for transplant glomerulopathy, including new insights into underlying causes and mechanisms, and highlights unmet needs in diagnostics. Transplant glomerulopathy is widely accepted as the principal manifestation of chronic antibody-mediated rejection, mostly with HLA antigen class II antibodies. However, recent data suggest that at least in some patients, there also is an association with hepatitis C virus infection, autoimmunity, and late thrombotic microangiopathy. Furthermore, intragraft molecular studies reveal nonresolving inflammation after sustained endothelial injury as a key mechanism and therapeutic target. Unfortunately, current international criteria rely heavily on light microscopy and miss patients at early stages, when they likely are treatable. Therefore, better tools, such as electron microscopy or molecular probes, are needed to detect patients when kidney injury is in an early active phase. Better understanding of causes and effector mechanisms coupled with early diagnosis can lead to the development of new therapeutics for transplant glomerulopathy and improved kidney outcomes.

Endothelial molecules decipher the mechanisms and functional pathways in antibody-mediated rejection.

Microvascular endothelium is the main target of injury in antibody-mediated rejection of human organ transplants. Hence, antibody-mediated rejection histologically presents with microvascular inflammation (pulmonary or myocardial or peritubular capillaritis, glomerulitis), thrombosis, and endothelial remodeling (duplication and/or multilayering of glomerular and capillary basement membranes). We previously observed upregulation of several endothelial genes in kidney transplant biopsies from patients with donor specific antibodies, indicating active antibody-mediated rejection and poor graft survival. Furthermore, endothelial molecular signals discovered a previously unknown clinical phenotype: C4d negative antibody-mediated rejection. With the recognition of C4d negative antibody-mediated rejection, data from multiple transplant centers now show that antibody-mediated rejection is the most common cause of late kidney transplant failure. This paper reviews the current understanding of endothelial cell biology in antibody-mediated rejection, emphasizing recent advances and pending questions. Furthermore, the paper discusses functionally active pathways in human antibody-mediated rejection, which include aspects of endothelial activation with increased endothelial adhesive and pro-coagulant signals facilitating leukocyte trafficking and attachment, cell-to-basement membrane interactions, platelet activation, coagulation, and endothelial repair responses. To understand effector mechanisms of antibody-mediated rejection and quantify the degree of antibody-mediated tissue injury in clinical transplants, endothelial cells provide a useful read-out.

Phenotypes of antibody-mediated rejection in organ transplants.

Antibody-mediated hyperacute rejection was the first rejection phenotype observed in human organ transplants. This devastating phenotype was eliminated by reliable crossmatch technologies. Since then, the focus was on T-cell-mediated rejection and de novo donor-specific antibodies were considered an epiphenomenon of cognate T-cell activation. The immune theory was that controlling the T-cell response would entail elimination of antibody-mediated rejection (ABMR). With modern immunosuppressive drugs, T-cell-mediated rejection is essentially treatable. However, this did not prevent ABMR from emerging as a significant phenotype in all types of organ transplants. It became obvious that both rejection types require distinct treatment and thus reliable diagnosis. This is the current challenge. ABMR, depending on stage, grade, time course, organ type or prior treatment, can present with a wide spectrum of phenotypes. This review summarizes the current diagnostic consensus for ABMR, describes unmet needs and challenges in diagnostics, and proposes new approaches for consideration.

Molecular phenotypes of acute kidney injury in kidney transplants.

Little is known regarding the molecular phenotype of kidneys with AKI because biopsies are performed infrequently. However, all kidney transplants experience acute injury, making early kidney transplants an excellent model of acute injury, provided the absence of rejection, because donor kidneys should not have CKD, post-transplant biopsies occur relatively frequently, and follow-up is excellent typically. Here, we used histopathology and microarrays to compare indication biopsies from 26 transplants with acute injury with 11 pristine protocol biopsies of stable transplants. Kidneys with acute injury showed increased expression of 394 transcripts associated with the repair response to injury, including many epithelium-like injury molecules tissue, remodeling molecules, and inflammation molecules. Many other genes also predicted the phenotype, including the acute injury biomarkers HAVCR1 and IL18. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score in kidneys with acute injury correlated with reduced graft function, future renal recovery, brain death, and need for dialysis, but not with future graft loss. In contrast, histologic features of acute tubular injury did not correlate with function or with the molecular changes. Thus, the transcripts associated with repair of injury suggest a massive coordinated response of the kidney parenchyma to acute injury, providing both an objective measure for assessing the severity of injury in kidney biopsies and validation for many biomarkers of AKI.

Pros and cons for C4d as a biomarker.

The introduction of C4d in daily clinical practice in the late nineties aroused an ever-increasing interest in the role of antibody-mediated mechanisms in allograft rejection. As a marker of classical complement activation, C4d made it possible to visualize the direct link between anti-donor antibodies and tissue injury at sites of antibody binding in a graft. With the expanding use of C4d worldwide several limitations of C4d were identified. For instance, in ABO-incompatible transplantations C4d is present in the majority of grafts but this seems to point at 'graft accommodation' rather than antibody-mediated rejection. C4d is now increasingly recognized as a potential biomarker in other fields where antibodies can cause tissue damage, such as systemic autoimmune diseases and pregnancy. In all these fields, C4d holds promise to detect patients at risk for the consequences of antibody-mediated disease. Moreover, the emergence of new therapeutics that block complement activation makes C4d a marker with potential to identify patients who may possibly benefit from these drugs. This review provides an overview of the past, present, and future perspectives of C4d as a biomarker, focusing on its use in solid organ transplantation and discussing its possible new roles in autoimmunity and pregnancy.