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

Assessment of Mitochondrial Respiration During Hypothermic Storage of Liver Biopsies Following Normothermic Machine Perfusion

Organ quality can be assessed prior to transplantation, during normothermic machine perfusion (NMP) of the liver. Evaluation of mitochondrial function by high-resolution respirometry (HRR) may serve as a viability assessment concept in this setting. Freshly collected tissue is considered as optimal sample for HRR, but due to technical and personnel requirements, more flexible and schedulable measurements are needed. However, the impact of cold storage following NMP before processing biopsy samples for mitochondrial analysis remains unknown. We aimed at establishing an appropriate storage protocol of liver biopsies for HRR. Wedge biopsies of 5 human livers during NMP were obtained and assessed by HRR. Analysis was performed after 0, 4, 8, and 12 h of hypothermic storage (HTS) in HTK organ preservation solution at 4°C. With HTS up to 4 h, mitochondrial performance did not decrease in HTS samples compared with 0 h (OXPHOS, 44.62 [34.75–60.15] pmol·s−1·mg wet mass−1 vs. 43.73 [40.69–57.71], median [IQR], p > 0.999). However, at HTS beyond 4 h, mitochondrial respiration decreased. We conclude that HTS can be safely applied for extending the biopsy measurement window for up to 4 h to determine organ quality, but also that human liver respiration degrades beyond 4 h HTS following NMP

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (−0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation

Strategies to improve outcome following the transplantation of organs from brain-dead donors

Es gibt klinische aber auch experimentelle Evidenz, dass der Spenderhirntod die Organqualität nach solider Organtransplantation negativ beeinflusst. In der vorliegenden Arbeit untersuchten wir in einem experimentellen Pankreastransplantationmodell den Einfluss des Spenderhintodes und des Ischämie Reperfusions Schadens auf die frühe Organfunktion. Weiter untersuchten wir inwieweit sich durch eine Behandlung mit Tetrahydrobiopterin die Organqualität verbessern lässt.Brain death (BD) has been shown to exacerbate ischemia reperfusion injury (IRI) in the context of solid organ transplantation and has also been seen as a phenomenon thought to immunologically prime the graft. In several already published studies we were able to demonstrate that tetrahydrobiopterin (BH4), an essential nitric oxide synthase co-factor, diminishes IRI following syngeneic transplantation. The aim of the present study was in a first step to study the impact of donor BD and IRI in a murine model of syngeneic pancreas transplantation. In a second step we assessed the therapeutic potential of BH4 in this clinically relevant setting. Analysis of mRNA expression by PCR revealed when compared to sham-operated controls that donor BD resulted in intrapancreatic inammation as reected by induction of IL-1ß, IL-6, VCAM-1, and P-selectin. Similar results were obtained from the analysis of microcirculation after transplantation using fluorescence microscopy. In contrast, pretreatment of the BD donor with BH4 improved microcirculation following reperfusion. Moreover, BD exerted a dramatic impact on cell viability, whereas BH4-treated grafts showed a signicantly greater percentage of viable cells in biopsies taken after reperfusion. Histopathological damage in pancreatic grafts was signicantly more pronounced in organs from BD donors than from sham or non-BD donors. However, BH4 pretreatment signicantly abrogated tissue damage in BD organs. Importantly, donor treatment with BH4 resulted in signicantly prolonged recipient survival. In conclusion, our data underscore the negative synergistic impact of donor BD and IRI on pancreatic isografts. Moreover, the protective effect of donor pretreatment using BH4 was shown.Oberhuber Rupert, MD1 Artikeln beigebundenMedical University of Innsbruck, Dissertation, 2016OeBB(VLID)127443

Analisis Pohon Berstruktur Menggunakan Metode CHAID pada Data Respons Ordinal

Eksplorasi data merupakan suatu hal yang penting dilakukan sebelum menganalisa data dengan metode lain. Salah satu metode eksplorasi untuk data respons kategorik adalah metode CHAID (Chi-square Automatic Interaction Detection). Tulisan ini berisi tentang kajian teori metode CHAID dan penerapannya yaitu menganalisis faktor-faktor yang berpengaruh pada peningkatan omset USAha anggota koperasi simpan pinjam (KSP) sebagai respons ordinal. Hasil kajian menunjukkan bahwa metode CHAID tidak hanya dapat digunakan sebagai metode eksplorasi tetapi juga mampu menggunakan struktur hubungan antara variabel respons kategorik dengan serangkaian variabel penjelas serta interaksi antar variabel penjelas. Struktur hubungan ini digambarkan dengan suatu pohon berstruktur

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

<div><p>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.</p></div

Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy

While both living donation and donation after circulatory death provide alternative opportunities for organ transplantation, donation after donor brain death (BD) still represents the major source for solid transplants. Unfortunately, the irreversible loss of brain function is known to induce multiple pathophysiological changes, including hemodynamic as well as hormonal modifications, finally leading to a systemic inflammatory response. Models that allow a systematic investigation of these effects in vivo are scarce. We present a murine model of BD induction, which could aid investigations into the devastating effects of BD on allograft quality. After implementing intra-arterial blood pressure measurement via the common carotid artery and reliable ventilation via a tracheostomy, BD is induced by steadily increasing intracranial pressure using a balloon catheter. Four hours after BD induction, organs may be harvested for analysis or for further transplantation procedures. Our strategy enables the comprehensive analysis of donor BD in a murine model, therefore allowing an in-depth understanding of BD-related effects in solid organ transplantation and potentially paving the way to optimized organ preconditioning

Lymphocytes as an Indicator for Initial Kidney Function: A Single Center Analysis of Outcome after Alemtuzumab or Basiliximab Induction

Alemtuzumab, an anti-CD52 T-cell and B-cell depleting monoclonal antibody, is established for induction therapy in renal transplantation (KTx). We herein provide a comparative analysis between alemtuzumab and basiliximab induction therapy and correlate lymphocyte depletion and recovery with the clinical course after KTx. This is a single center retrospective analysis of 225 patients/consecutive kidney transplantations treated with alemtuzumab for lymphocyte depletion and 205 recipients treated with basiliximab. Mean lymphocyte counts were 22.8 ± 9.41% before Tx and 2.61 ± 3.11% between week 1 and week 3 in the alemtuzumab group and 23.77 ± 10.42% before Tx and 13.92 ± 8.20% in the basiliximab group. Delayed graft function (DGF), cytomegalovirus (CMV) status, and recipient age showed a significant correlation with lymphocyte counts in the alemtuzumab group only. The outcome was read in reference to the velocity of lymphocyte recovery and in comparison to the control group. Lymphocyte counts early after transplantation, following alemtuzumab treatment, could be identified as a predictive factor for kidney function early after transplantation. A detailed analysis of phenotype and function of lymphocytes after alemtuzumab induction together with a correlation with the clinical course is warranted

Murine cervical heart allotransplantation.

<p>BALB/c huCD3Ɛ were injected i.v with ATG or control rabbit Ig. Cervical heart transplantation was performed and graft function was evaluated by daily regular heart palpation. (A) Cardiac graft function and (B) graft survival (days). Graft function was expressed as the beating score, 0 no organ function, 1: fibrillation, only visible through magnification, 2: poor or partial organ function, 3: impairment in frequency or intensity of heart beating, 4: physiological organ function). Four to five mice per group, two independent experiments were performed. Data are shown as means ± SEM. Mann-Whitney statistical test was used, *p<0.05, ** p<0.001.</p

Ex Vivo Mesenchymal Stem Cell Therapy to Regenerate Machine Perfused Organs

Transplantation represents the treatment of choice for many end-stage diseases but is limited by the shortage of healthy donor organs. Ex situ normothermic machine perfusion (NMP) has the potential to extend the donor pool by facilitating the use of marginal quality organs such as those from donors after cardiac death (DCD) and extended criteria donors (ECD). NMP provides a platform for organ quality assessment but also offers the opportunity to treat and eventually regenerate organs during the perfusion process prior to transplantation. Due to their anti-inflammatory, immunomodulatory and regenerative capacity, mesenchymal stem cells (MSCs) are considered as an interesting tool in this model system. Only a limited number of studies have reported on the use of MSCs during ex situ machine perfusion so far with a focus on feasibility and safety aspects. At this point, no clinical benefits have been conclusively demonstrated, and studies with controlled transplantation set-ups are urgently warranted to elucidate favorable effects of MSCs in order to improve organs during ex situ machine perfusion