Walter Mark

T cell independence of macrophage and natural killer cell infiltration, cytokine production, and endothelial activation during delayed xenograft rejection

Rejection of guinea pig cardiac grafts in rats depleted of complement takes place in 3-4 days and involves progressive mononuclear cell infiltration and cytokine expression, fibrin and antibody deposition, and endothelial cell up-regulation of adhesion and procoagulant molecules, a process termed delayed xenograft rejection (DXR). The relative contribution of each effector mechanism and the role of T cells in this complex process are unknown, although small numbers of interleukin (IL) 2 receptor-positive T cells are present at the time of rejection. We investigated the importance of T cells in DXR by comparing discordant xenograft responses of nude rats, which lack T cell receptor (TCR)-alpha/beta+ cells, with those of normal Lewis rats. Nude or Lewis rats receiving guinea pig cardiac grafts were assigned to one of three groups: no therapy, daily administration of cobra venom factor (CVF), or splenectomy plus daily CVF. All untreated rats rejected their xenografts within 10-15 min, whereas grafts in complement-depleted recipients survived a further 3-4 days; splenectomy had no significant additional effect upon graft survival. Immunohistologic analysis in CVF-treated nude recipients with or without splenectomy showed: (1) considerable leukocyte infiltration of xenografts (mean +/- SD, 76+/-14 and 71+/-16 leukocytes/field, respectively, at 72 hr, compared with 68+/-17 in Lewis rats), consisting largely of macrophages (>75% of total leukocytes) plus small numbers of natural killer cells (10-20%) with no detectable B or T cells (TCR-alpha/beta or TCR-gamma/delta); (2) at least 10-fold lower levels of intragraft IgM or IgG deposition than in corresponding Lewis recipients; and (3) considerable cytokine expression by intragraft macrophages (IL-12, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, IL-1beta, IL-6, IL-7, IL-12) and natural killer cells (interferon-gamma), as well as up-regulation of tissue factor expression and dense fibrin deposition. Analysis of recipient sera of both control and nude rats by ELISA, for the binding of IgG or IgM to guinea pig platelets, showed a rapid rise after transplantation in the titers of IgM and IgG antibodies, which was abrogated by prior splenectomy; i.e., data from splenectomized xenograft recipients reflect the presence of only basal levels of IgM and IgG. Thus, our data in nude rats show rejection times and intragraft features of DXR comparable to those in immunocompetent Lewis recipients, despite a lack of detectable host T cells, and, in the case of splenectomized rats, only about one tenth of normal xenoreactive antibody levels. Our data document a new model in which to analyze the immunopathogenesis of DXR.

Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation.

Although chronological donor age is the most potent predictor of long‐term outcome after renal transplantation, it does not incorporate individual differences of the aging‐process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre‐implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.

Extensive Surveillance Promotes Early Diagnosis and Improved Survival of De Novo Malignancies in Liver Transplant Recipients

The aim of our study was to examine whether an extensive surveillance protocol will promote early diagnosis and improved survival in patients with de novo cancer following liver transplantation (LT). Of 779 consecutive LT recipients, 96 (12.3%) developed 105 malignancies. The cumulative risk for the development of de novo cancer was 10%, 24%, 32% and 42% at 5, 10, 15 and 20 years after LT, respectively. The most frequent tumor types were skin (17%), lung (16%), oropharyngeal (11%) and prostate cancer (11%). The overall standard incidence ratio as compared to that of the general population was 1.9 (95% CI: 1.5–2.3). The median survival of patients with de novo non‐skin cancers was 3.1 years after diagnosis. Only patients with skin cancers and solid tumors, diagnosed at early stages, showed an excellent outcome. After introducing an intensified surveillance protocol, the detection rate of de novo cancers increased from 4.9% to 13% and more de novo malignancies were diagnosed in earlier stages. For non‐skin cancers, the median tumor‐related survival significantly improved from 1.2 to 3.3 years as well as the median overall survival post‐LT. This study indicates that an extensive tumor surveillance program is highly recommendable in LT recipients.

Status 5 Years after Bilateral Hand Transplantation

Graft survival and function early after hand transplantation is good. It remains unknown, however, whether long‐term survival is limited by chronic rejection. We here describe the clinical course and the status 5 years after bilateral hand transplantation with emphasis on immunosuppression (IS), function, morphology and graft vascular changes.

A prospective randomized multicenter trial comparing histidine-tryptophane-ketoglutarate versus University of Wisconsin perfusion solution in clinical pancreas transplantation

We aimed to evaluate early pancreas transplant graft function after histidine–tryptophan–ketoglutarate (HTK) versus University of Wisconsin (UW) perfusion. Prospective randomized multicenter study including 68 pancreas transplantations stratified according to preservation fluid used (27 HTK vs. 41 UW). Primary endpoint was pancreas graft survival at 6 months. Serum α‐amylase, lipase, C‐peptide, HbA1C and exogenous insulin requirement were compared at several time points. Mean pancreas cold ischemia time was 10.8 ± 3.7 (HTK) vs. 11.8 ± 3.4 h (UW) (P = 0.247). Simultaneous pancreas–kidney transplantation was performed in 95.6% of the patients, pancreas transplantation alone in 2.9%, and pancreas after kidney transplantation in 1.5%. Six months graft survival was 85.2% (HTK) vs. 90.2% (UW) (P = 0.703). Serum amylase and lipase values did not differ between both the groups during the observation period. C‐peptide levels were elevated in both the groups without significant differences at each time point. Higher exogenous insulin requirement early after transplantation in the UW group had resolved at 3 months. Six month patient survival was 96.3% (HTK) vs. 100% (UW) (P = 0.397). With a mean cold ischemia time of 10 h in this study, HTK and UW solutions appear to be equally suitable for perfusion and organ preservation in clinical pancreas transplantation.

Immunomodulatory effects of the alkaloid sinomenine in the high responder ACI-to-Lewis cardiac allograft model.

Extracts of the plant Sinomenium acutum have been used safely since ancient times in Chinese medicine for treatment of rheumatic diseases, and the purified alkaloid, sinomenine, was recently shown to have anti-inflammatory and antirheumatic effects. This study describes the effects of sinomenine in the high responder ACI-->Lewis cardiac transplant model in which allograft rejection occurred at 5 days posttransplant. Treatment with sinomenine (15-30 mg/kg/day i.p.) or a subtherapeutic dose of cyclosporine (CsA, 1.5 mg/kg/day, i.m.) prolonged allograft survival only marginally (mean survival of 5.4 and 7.8 days, respectively). In contrast, the combination of sinomenine and CsA had a statistically significant synergistic effect, with a mean survival of 42.2 days (P < 0.001). Allografts harvested at day 5 from recipients treated with either sinomenine or CsA showed dense mononuclear cell infiltrates with widespread subepicardial infarcts, edema, and microvascular platelet and fibrin deposition. Immunohistologic analysis showed that intragraft leukocytes consisted of >75% macrophages with approximately 10-20% T cells and <5% B or NK cells. Mononuclear cell activation was shown by expression of IL-2R (CD25, 10-20%) and labeling for IL-2 (approximately 10%), and IFN-gamma (10-20%), as well as TNF-alpha (>50%) and iNOS (>50%), but only low levels of IL-4 or IL-10 (<5%). Intragraft endothelial cells were activated, as shown by upregulation of MHC class II antigen and ICAM-1 (CD54) compared with only basal levels in normal donors hearts. Combined sinomenine/CsA therapy significantly enhanced graft morphology, resulting in only mild mononuclear cell infiltration, and an absence of infarcts, platelets, or fibrin deposition. Though residual intragraft mononuclear cells at day 5, as in control grafts, consisted primarily of macrophages plus small numbers of IL-2R+ T cells, these cells lacked expression of IL-2, had only low levels of IFN-gamma, but showed dense labeling for IL-4 and IL-10. In addition, TNF-alpha and iNOS were reduced to basal levels and no endothelial cell activation was observed, despite high titers of endothelium-bound IgM, IgG, and C3. Mitogen-induced in vitro proliferation of rat thymocytes was also more effectively decreased by the sinomenine/CsA combination than by either agent alone. These studies demonstrate the therapeutic value of sinomenine in transplantation, and indicate that this agent has novel and interesting antimacrophage, T cell, and endothelial effects that warrant further evaluation.

Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection

Background. Chronic rejection (CR) with graft vasculopathy is recognized as a major cause of graft loss over time. Sinomenine (SN) has anti-inflammatory, antirheumatic, and immunomodulatory effects. Previously, we demonstrated antimacrophage and anti-T cell effects of SN in acute rejection. In the current study, we investigated the effect of SN in a rat cardiac allograft model of CR. Materials and Methods. After a brief course of cyclosporine A (CsA), Lewis recipients of F344 hearts were treated with SN alone, CsA alone, or a combination of both drugs. Grafts were analyzed morphometrically and by immunohistochemistry. Expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and endothelin 1 was assessed by reverse transcription-polymerase chain reaction. Antidonor IgM formation was investigated by FACS. Results. Cardiac grafts from SN-treated rats showed less pronounced vasculopathy in comparison with untreated rats or CsA-treated recipients. After treatment with a combination of both drugs, rats had significantly less graft vasculopathy than rats receiving either drug alone. Treatment with CsA alone led to a decrease in bFGF expression, whereas SN alone did not affect gene expression. SN in combination with CsA, however, markedly reduced expression of bFGF, vascular endothelial growth factor, and endothelin 1. SN alone did not inhibit antidonor antibody formation. Conclusion. These studies demonstrate for the first time the therapeutic value of SN in a model of chronic cardiac allograft rejection. SN in combination with low-dose T cell–targeted immunosuppression is effective in controlling tissue remodeling in the context of CR and is associated with inhibition of intragraft expression of mediators involved in angiogenesis, vascular tone, and tissue remodeling.

Nocardiosis following solid organ transplantation: a single-centre experience

Nocardiosis is a localized or disseminated bacterial infection caused by aerobic Actinomyces that commonly affects immunocompromised hosts. The aim of this study was to retrospectively review clinical course and outcome of nocardiosis in solid organ recipients at our centre. Five cases of nocardiosis were identified in a series of more than 4000 consecutive solid organ transplants performed at Innsbruck university hospital during a 25‐year period. Of the five patients with nocardiosis, two had undergone multivisceral, one liver, one kidney and one lung transplantation. Three patients with Nocardia asteroides infection were treated successfully and recovered from their infectious disease, however, one lost his renal graft following withdrawal of immunosuppression. The lung recipient recovered from nocardiosis but died later on from Pseudomonas pneumonia. One multivisceral recipient died from Nocardia farcinica‐disseminated infection. Nocardiosis is a rare, difficult‐to‐diagnose‐and‐treat complication following solid organ transplantation. Intestinal recipients might be at increased risk to develop this infection.

Malignancies of the colorectum and anus in solid organ recipients.

Patients undergoing solid organ transplantation (SOT) are at increased risk for developing malignancies due to the long term immunosuppression. Data on malignancies of the large intestine after various types of SOT are rare. A total of 3595 SOTs were performed between 1986 and 2005 at our center and retrospectively analyzed with regard to the incidence and course of malignancies of the colon, rectum, and anus. Standard immunosuppression consisted of calcineurin inhibitors in combination with azathioprine or mycophenolate mofetil and steroids with or without antithymocyte globulin or IL‐2 receptor antagonist induction. A total of 206 patients (5.7%) developed malignancies. Colorectal adenocarcinoma was diagnosed in nine patients (0.25%; mean age at diagnosis 65 years) at a mean of 5.3 years after transplantation. Five patients (55%) died 7.2 years post‐transplant due to cardiovascular disease (n = 4) and tumor progression (n = 1). Four patients developed anal neoplasia (0.11%) 7 years post‐transplant with 100% 1‐year survival. Five patients showed post‐transplant lymphoproliferative disorders (PTLD) with intestinal involvement. The incidence of anal but not of colorectal cancers in our transplant recipients differed from that of immunocompetent individuals of corresponding age (0.11% vs. 0.002% and 0.25% vs. 0.3%). PTLD may involve the colon.

Early viral load and recipient interleukin‐28B rs12979860 genotype are predictors of the progression of hepatitis C after liver transplantation

There have been few detailed studies of viral kinetics after liver transplantation (LT), and conflicting data have been reported on viral loads and the severity of recurrent hepatitis C virus (HCV) disease. This long‐term study aimed to examine (1) the impact of HCV RNA levels at specific points in time within the first year and (2) the influence of interleukin‐28B (IL‐28B) genotypes on patient outcomes and the severity of recurrent HCV disease. The viral loads were measured 2, 4, 12, 24, and 48 weeks after LT, and the recipient/donor IL‐28B genotypes of 164 patients were determined. A Cox regression analysis showed that the viral load at week 2 was an independent negative predictor of recipient outcomes. A week 2 viral load ≥ 6.0 log10 IU/mL was significantly associated with reduced patient survival. After a mean follow‐up of 6.5 years, 21 of 164 patients (12.8%) developed a cholestatic type of HCV recurrence and/or rapidly progressed to cirrhosis within 1 year. A multivariate binary regression analysis showed that HCV viremia at week 2 and a non‐C/C recipient IL‐28B genotype were independent risk factors for cholestatic recurrent HCV. No predictive factors could be found for the occurrence of recurrent liver cirrhosis 5 and 10 years after LT. Our study shows that the HCV RNA level at week 2 and the recipient IL‐28B genotype are independent, statistically significant risk factors for post‐LT cholestatic HCV, and it emphasizes the importance of viral load monitoring and IL‐28B genotyping for identifying HCV recipients at risk for severe HCV recurrence. Liver Transpl 18:671–679, 2012.