Benno Cardini

Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions.

Atherosclerosis is a multifactorial chronic inflammatory disease characterized by the presence of T-cells, macrophages, and dendritic cells in the arterial intima. Classical risk factors lead to over-expression of stress proteins, especially heat shock protein 60 (HSP60). HSP60 on the surface of arterial endothelial cells (ECs) then becomes a target for pre-existing adaptive anti-HSP60 immunity resulting in infiltration of the intima by mononuclear cells. In the present study, T-cells derived from early, clinically still inapparent human atherosclerotic lesions were analyzed phenotypically and for their reactivity against HSP60 and HSP60-derived peptides. HSP60 was detected in ECs and CD40- and HLA Class II-positive cells within the intima. Effector memory CD4+ T-cells producing high amounts of interferon-γ and low levels of interleukin-4 were the dominant subpopulation. T-cells derived from late lesions displayed a more restricted T-cell receptor repertoire to HSP60-derived peptides than those isolated from early lesions. Increased levels of soluble HSP60 and circulating anti-human HSP60 autoantibodies were found in donors with late but not early lesions. This is the first functional study of T-cells derived from early human atherosclerotic lesions that supports the previously proposed concept that HSP60-reactive T-cells initiate atherosclerosis by recognition of atherogenic HSP60 epitopes.

The faster the better: anastomosis time influences patient survival after deceased donor kidney transplantation

Despite a continuously growing knowledge of the impact of factors on kidney graft function, such as donor age, body mass index, and cold ischemia time, few data are available regarding anastomosis time (AT) and its impact on long‐term results. We investigated whether surgical AT correlates with patient and graft survival after kidney transplantation performing a retrospective analysis of 1245 consecutive deceased donor kidney transplantations between 01/2000 and 12/2010 at Innsbruck Medical University. Kaplan–Meier and log‐rank analyses were carried out for 1‐ and 5‐year patient and graft survival. AT was defined as time from anastomosis start until reperfusion. Median AT was 30 min. Five‐year survival of allografts with an AT >30 min was 76.6% compared with 80.6% in the group with AT <30 min (P = 0.027). Patient survival in the group with higher AT similarly was inferior with 85.7% after 5 years compared with 89.6% (P < 0.0001) [Correction added on February 18, 2015, after first online publication: the percentage value for patient survival was previously incorrect and have now been changed to 89.6%]. Cox regression analysis revealed AT as an independent significant factor for patient survival (HR 1.021 per minute; 95% CI 1.006–1.037; P = 0.006). As longer AT closely correlates with inferior long‐term patient survival, it has to be considered as a major risk factor for inferior long‐term results after deceased donor kidney transplantation.

Donor Pretreatment with Tetrahydrobiopterin Saves Pancreatic Isografts from Ischemia Reperfusion Injury in a Mouse Model

Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B‐substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 μM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI‐related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia‐induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.

Prevention of lethal murine pancreas ischemia reperfusion injury is specific for tetrahydrobiopterin.

Tetrahydrobiopterin has been shown to efficiently abrogate ischemia reperfusion injury (IRI). However, it is unclear, whether its beneficial action relies on cofactor activity of one of the five known tetrahydrobiopterin‐dependent reactions or on its antioxidative capacity. We therefore compared tetrahydrobiopterin with the pterin derivate tetrahydroneopterin (similar biochemical properties, but no nitric oxide synthase cofactor activity) and the antioxidants vitamin C and 5‐methyltetrahydrofolate. Donor mice were pretreated with tetrahydrobiopterin, tetrahydroneopterin, vitamin C, or 5‐methyltetrahydrofolate. Pancreatic grafts were subjected to 16‐h cold ischemia time and implanted in syngeneic recipients. Untreated and nontransplanted animals served as controls. Following 2‐h reperfusion, microcirculation was analyzed by intravital fluorescence microscopy. Graft damage was assessed by histology and nitrotyrosine immunostaining, and tetrahydrobiopterin levels were determined by HPLC. Recipient survival served as ultimate readout. Prolonged cold ischemia time resulted in microcirculatory breakdown. Only tetrahydrobiopterin pretreatment succeeded to preserve the capillary net, whereas all other compounds showed no beneficial effects. Along with increased intragraft tetrahydrobiopterin levels during recovery and implantation, only tetrahydrobiopterin pretreatment led to significant reduction of IRI‐related parenchymal damage enabling recipient survival. These results show a striking superiority of tetrahydrobiopterin in preventing lethal IRI compared with related compounds and suggest nitric oxide synthases as treatment target.

Murine cervical heart transplantation model using a modified cuff technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice(1-3). This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates(4). Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient(5). Herein, we provide additional details of this technique to supplement the video.

Differential depletion of total T cells and regulatory T cells and prolonged allotransplant survival in CD3Ɛ humanized mice treated with polyclonal anti human thymocyte globulin.

Thymoglobulin (ATG) is a polyclonal rabbit antibody against human thymocytes used as a T cell-depleting agent to prevent or treat allotransplant rejection. The aim of the present study was to investigate the effect of low dose ATG treatment exclusively on T cells using a humanized BALB/c human CD3Ɛ transgenic mouse model expressing both human and murine T cell receptors (TCR). Mice received a single intravenous (i.v.) injection of ATG. Blood and peripheral lymphoid organs were obtained after different time points. We found a significant T cell depletion in this mouse model. In addition, regulatory T cells (Tregs) proved to be less sensitive to depletion than the rest of T cells and the Treg:non-Treg ratio was therefore increased. Finally, we also investigated the effect of ATG in a heterotopic allogenic murine model of heart transplantation. Survival and transplant function were significantly prolonged in ATG-treated mice. In conclusion, we showed (a) an immunosuppressive effect of ATG in this humanized mouse model which is exclusively mediated by reactivity against human CD3Ɛ; (b) provided evidence for a relative resistance of Tregs against this regimen; and (c) demonstrated the immunomodulatory effect of ATG under these experimental circumstances by prolongation of heart allograft survival.

Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model

Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV.

Crucial Role for Neuronal Nitric Oxide Synthase in Early Microcirculatory Derangement and Recipient Survival following Murine Pancreas Transplantation

Objective Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation. Background Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined. Methods Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days. Results Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin. Conclusion We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.

Liver transplantation for hilar cholangiocarcinoma (h-CCA): is it the right time?

Liver transplantation (LT) is the therapy of choice in selected patients with hepatocellular carcinoma (HCC). In light of the overall excellent results achieved for HCC, LT has been successfully adapted as a therapeutic option for several non-hepatocellular malignancies (1) including unresectable hepatic epithelioid hemangioendothelioma (HEHE) (2), neuroendocrine tumors (NET) (3) or even colorectal metastases in case of liver-only disease (4).

Graft Pre-conditioning by Peri-Operative Perfusion of Kidney Allografts With Rabbit Anti-human T-lymphocyte Globulin Results in Improved Kidney Graft Function in the Early Post-transplantation Period—a Prospective, Randomized Placebo-Controlled Trial

Introduction: Although prone to a higher degree of ischemia reperfusion injury (IRI), the use of extended criteria donor (ECD) organs has become reality in transplantation. We therefore postulated that peri-operative perfusion of renal transplants with anti-human T-lymphocyte globulin (ATLG) ameliorates IRI and results in improved graft function. Methods: We performed a randomized, single-blinded, placebo-controlled trial involving 50 kidneys (KTx). Prior to implantation organs were perfused and incubated with ATLG (AP) (n = 24 kidney). Control organs (CP) were perfused with saline only (n = 26 kidney). Primary endpoint was defined as graft function reflected by serum creatinine at day 7 post transplantation (post-tx). Results: AP-KTx recipients illustrated significantly better graft function at day 7 post-tx as reflected by lower creatinine levels, whereas no treatment effect was observed after 12 months surveillance. During the early hospitalization phase, 16 of the 26 CP-KTx patients required dialysis during the first 7 days post-tx, whereas only 10 of the 24 AP-KTx patients underwent dialysis. No treatment-specific differences were detected for various lymphocytes subsets in the peripheral blood of patients. Additionally, mRNA analysis of 0-h biopsies post incubation with ATLG revealed no changes of intragraft inflammatory expression patterns between AP and CP organs. Conclusion: We here present the first clinical study on peri-operative organ perfusion with ATLG illustrating improved graft function in the early period post kidney transplantation. Clinical Trial Registration: www.ClinicalTrials.gov, NCT03377283