S. Paraskevas

Structural and functional changes resulting from islet isolation lead to islet cell death

BACKGROUND Islet isolation exposes the islet to a variety of cellular stresses and disrupts the cell-matrix relationship--events known to be associated with apoptosis. The purpose of this study was to determine whether islet isolation leads invariably to islet cell death and to specify the mechanisms involved. METHODS Canine islets were isolated using Liberase CI and purified using a centrifuge. Islets were sampled for up to 5 days in culture and analyzed by routine histology, electron microscopy, immunocytochemistry, and reticulin staining for basement membrane. Apoptosis was assessed by cell death enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated decoxyuridine triphosphate nick and labeling (TUNEL) assay. Activation of the prosurvival ERK1/2 and proapoptotic p38 and JNK were determined by immunoblotting. RESULTS Immediately after isolation, the peri-insular basement membrane was absent, and integrin-alpha 5 expression diminished. DNA fragmentation rose from 2.5 +/- 1.8 (arbitrary units) on the day of isolation to 42.4 +/- 6.7 48 hours later (P < .05), coinciding with the appearance of pyknotic nuclei and apoptotic bodies. The apoptotic index determined by TUNEL assay increased from 5% +/- 1% on the day of isolation to 60% +/- 2% on day 5 (P < .01), and most of the affected cells were beta-cells. Finally, the p38 and JNK activity were elevated relative to ERK1/2. CONCLUSIONS During isolation, islet cells undergo profound changes in structure and function, resulting in beta-cell apoptosis. These findings suggest that strategies directed to the manipulation of the cell-matrix relationship and the modulation of mitogen-activated protein kinase signal transduction may offer a valuable new approach to improving islet transplant outcome.

Rapid Discontinuation of Steroids in Living Donor Kidney Transplantation: A Pilot Study

Steroids are associated with significant postoperative complications (hypertension, cosmetic changes, bone loss, hyperlipidemia, diabetes, and cataracts). Most develop early; in addition, late post‐transplant steroid withdrawal in kidney transplant recipients has been associated with increased acute rejection (AR). To obviate these problems, we studied outcome of a protocol of rapid discontinuation of prednisone (RDS) (steroids stopped on POD6). Between November 1, 1999 and October 31, 2000, 51 adult living donor (LD) first transplant recipients (2 HLA‐id, 28 non‐id relative, 21 LURD) were immunosuppressed with thymoglobulin (1.25 mg/kg intraoperatively and then qdx4); prednisone (P) (500 mg methylprednisolone intraoperatively, 1 mg/kg × 1 day, 0.5 mg/kg × 2 days, 0.25 mg/kg × 2 days, then d/c); MMF, 1 g b.i.d.; and CSA, 4 mg/kg b.i.d. adjusted to achieve levels of 150–200 ng/mL (by HPLC). Exclusion criteria were delayed graft function or 1° disease requiring P. Minimum follow‐up was 5.5 months (range 5.5 to 17.5 months). Outcome was compared vs. previous cohorts of LD recipients immunosuppressed with P/AZA/CSA (n = 171) or P/MMF/CSA (n = 43) (both without antibody induction). Results: For the RDS group, average CSA level (± S.E.) at 3 and 6 months was 190 ± 12 and 180 ± 9; avg. MMF dose, 1.7 ± 0.1 g and 1.7 ± 0.1 g. There was no significant difference in 6‐ and 12‐month actuarial patient survival, graft survival and rejection‐free graft survival between recipients on the RDS protocol vs. historical controls. For RDS recipients, actuarial 6‐ and 12‐month rejection‐free graft survival was 87%. Of the 51 RDS recipients, five (10%) have had AR (at 20 days, 1 month, 3 months, 3 months, and 3.5 months post‐transplant). After treatment, all five were maintained on 5 mg P; there have been no second AR episodes. Two additional recipients were started on 5 mg P due to low white blood count (WBC) and low/no MMF. Of the 51 grafts, one has failed (death with function). Average serum Cr level (± S.E.) at 3 and 6 months for RDS recipients was 1.7 ± 0.5 (NS vs. historical controls). Conclusion: For low‐risk LD recipients, a kidney transplant with an RDS protocol does not increase risk of AR or graft loss. Future studies will need to be done to assess AR rates with an RDS protocol in cadaver transplant recipients and in recipients with delayed graft function.

Guidelines for the Diagnosis of Antibody‐Mediated Rejection in Pancreas Allografts—Updated Banff Grading Schema

The first Banff proposal for the diagnosis of pancreas rejection (Am J Transplant 2008; 8: 237) dealt primarily with the diagnosis of acute T‐cell‐mediated rejection (ACMR), while only tentatively addressing issues pertaining to antibody‐mediated rejection (AMR). This document presents comprehensive guidelines for the diagnosis of AMR, first proposed at the 10th Banff Conference on Allograft Pathology and refined by a broad‐based multidisciplinary panel. Pancreatic AMR is best identified by a combination of serological and immunohistopathological findings consisting of (i) identification of circulating donor‐specific antibodies, and histopathological data including (ii) morphological evidence of microvascular tissue injury and (iii) C4d staining in interacinar capillaries. Acute AMR is diagnosed conclusively if these three elements are present, whereas a diagnosis of suspicious for AMR is rendered if only two elements are identified. The identification of only one diagnostic element is not sufficient for the diagnosis of AMR but should prompt heightened clinical vigilance. AMR and ACMR may coexist, and should be recognized and graded independently. This proposal is based on our current knowledge of the pathogenesis of pancreas rejection and currently available tools for diagnosis. A systematized clinicopathological approach to AMR is essential for the development and assessment of much needed therapeutic interventions.

Clostridium difficile colitis: Increasing incidence, risk factors, and outcomes in solid organ transplant recipients

Background Clostridium difficile-associated diarrhea (CDAD) is an increasingly important diagnosis in solid organ transplant recipients, with rising incidence and mortality. We describe the incidence, risk factors, and outcomes of colectomy for CDAD after solid organ transplantation. Methods Patients with CDAD were identified from a prospective transplant database. Complicated Clostridium difficile colitis (CCDC) was defined as CDAD associated with graft loss, total colectomy, or death. Results From 1999 to 2010, we performed solid organ transplants for 1331 recipients at our institution. The incidence of CDAD was 12.4% (165 patients); it increased from 4.5% (1999) to 21.1% (2005) and finally 9.5% (2010). The peak frequency of CDAD was between 6 and 10 days posttransplantation. Age more than 55 years (hazard ratio [HR]: 1.47, 95% confidence interval [CI]=1.16–1.81), induction with antithymocyte globulin (HR: 1.43, 95% CI=1.075–1.94), and transplant other than kidney alone (liver, heart, pancreas, or combined kidney organ) (HR: 1.41, 95% CI=1.05–1.92) were significant independent risk factors for CDAD. CCDC occurred in 15.8% of CDAD cases. Independent predictors of CCDC were white blood cell count more than 25,000/&mgr;L (HR: 1.08, 95% CI=1.025–1.15) and evidence of pancolitis on computed tomography scan (HR: 2.52, 95% CI=1.195–5.35). Six patients with CCDC underwent colectomy with 83% patient survival and 20% graft loss. Of the medically treated patients with CCDC (n=20), the patient survival was 35% with 100% graft loss. Conclusions We have identified significant risk factors for CDAD and predictors of progression to CCDC. Furthermore, we found that colectomy can be performed with excellent survival in selected patients.

Analysis of Beta-Cell Gene Expression Reveals Inflammatory Signaling and Evidence of Dedifferentiation following Human Islet Isolation and Culture

The stresses encountered during islet isolation and culture may have deleterious effects on beta-cell physiology. However, the biological response of human islet cells to isolation remains poorly characterized. A better understanding of the network of signaling pathways induced by islet isolation and culturing may lead to strategies aimed at improving islet graft survival and function. Laser capture microdissection (LCM) was used to extract beta-cell RNA from 1) intact pancreatic islets, 2) freshly isolated islets, 3) islets cultured for 3 days, and changes in gene expression were examined by microarray analysis. We identified a strong inflammatory response induced by islet isolation that continues during in-vitro culture manifested by upregulation of several cytokines and cytokine-receptors. The most highly upregulated gene, interleukin-8 (IL-8), was induced by 3.6-fold following islet isolation and 56-fold after 3 days in culture. Immunofluorescence studies showed that the majority of IL-8 was produced by beta-cells themselves. We also observed that several pancreas-specific transcription factors were down-regulated in cultured islets. Concordantly, several pancreatic progenitor cell-specific transcription factors like SOX4, SOX9, and ID2 were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. Our findings suggest islet isolation and culture induces an inflammatory response and loss of the mature endocrine cell phenotype. A better understanding of the signals required to maintain a mature beta-cell phenotype may help improve the efficacy of islet transplantation.

Lysine deacetylase inhibition prevents diabetes by chromatin-independent immunoregulation and β-cell protection

Significance Type 1 diabetes is due to immune-mediated pancreatic β-cell destruction. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. The orally active and clinically well-tolerated KDACi vorinostat and givinostat reverted diabetes in a mouse model of type 1 diabetes and counteracted inflammatory target cell damage. Importantly, these effects were achieved with doses that are safe and effective in human inflammatory diseases. Of note, the mechanism of action was compatible with transcription factor—rather than global chromatin—hyperacetylation, causing inhibition of transcription factor binding and reduction of proinflammatory gene expression in leukocytes and β-cells. These data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with Type 1 diabetes. Type 1 diabetes is due to destruction of pancreatic β-cells. Lysine deacetylase inhibitors (KDACi) protect β-cells from inflammatory destruction in vitro and are promising immunomodulators. Here we demonstrate that the clinically well-tolerated KDACi vorinostat and givinostat revert diabetes in the nonobese diabetic (NOD) mouse model of type 1 diabetes and counteract inflammatory target cell damage by a mechanism of action consistent with transcription factor—rather than global chromatin—hyperacetylation. Weaning NOD mice received low doses of vorinostat and givinostat in their drinking water until 100–120 d of age. Diabetes incidence was reduced by 38% and 45%, respectively, there was a 15% increase in the percentage of islets without infiltration, and pancreatic insulin content increased by 200%. Vorinostat treatment increased the frequency of functional regulatory T-cell subsets and their transcription factors Gata3 and FoxP3 in parallel to a decrease in inflammatory dendritic cell subsets and their cytokines IL-6, IL-12, and TNF-α. KDACi also inhibited LPS-induced Cox-2 expression in peritoneal macrophages from C57BL/6 and NOD mice. In insulin-producing β-cells, givinostat did not upregulate expression of the anti-inflammatory genes Socs1-3 or sirtuin-1 but reduced levels of IL-1β + IFN-γ–induced proinflammatory Il1a, Il1b, Tnfα, Fas, Cxcl2, and reduced cytokine-induced ERK phosphorylation. Further, NF-κB genomic iNos promoter binding was reduced by 50%, and NF-κB-dependent mRNA expression was blocked. These effects were associated with NF-κB subunit p65 hyperacetylation. Taken together, these data provide a rationale for clinical trials of safety and efficacy of KDACi in patients with autoimmune disease such as type 1 diabetes.

Recipient age and risk of chronic allograft nephropathy in primary deceased donor kidney transplant

Single center and registry data studies have had conflicting results regarding the impact of recipient age on chronic allograft nephropathy (CAN). We tested the hypothesis that advanced recipient age is a risk factor for graft failure due to CAN. All patients who underwent primary deceased donor kidney transplant between January 1, 1995 and December 31, 2000 recorded in the United Network of Organ Sharing (UNOS) database were analyzed for the occurrence of death censored graft loss and by two different definitions of graft loss due to CAN. Kaplan–Meier analysis based on the recipient age, and Cox proportional hazard regression was used to estimate the independent effect of recipient age on the three endpoints of interest. For all endpoints, after age of 9 years, the risk of graft loss declined with each successive decade increase in age. This pattern of risk was similar for both Caucasian and African‐American recipients, although for any given age the risk of graft loss was always higher in African‐American recipients. Analysis of UNOS data does not support the hypothesis that advanced recipient age is a risk factor for CAN.

Posttransplant lymphoproliferative disorder in pancreas transplantation: a single-center experience.

Background. Posttransplant lymphoproliferative disorder (PTLD) is a rare, serious complication of transplantation. The characteristics and associations of this disease in pancreas recipients have not been extensively studied. Methods. From January 1988 through December 2002, 787 pancreas and 569 kidney-pancreas transplants were performed at our institution. Eighteen pancreas recipients developed polymorphic PTLD or malignant lymphoma. Data on clinical course, organ involvement, molecular characteristics, and association with immunosuppression and recent cytomegalovirus (CMV) infection were compiled from the institutional transplant database. Patient survival was compared to recipients of liver and kidney transplants at the same center by using Kaplan-Meier analysis. Results. The 5-year cumulative incidence of PTLD in simultaneous pancreas-kidney, pancreas after kidney, and pancreas transplant alone recipients was 2.5%, 1.2%, and 1.0%, respectively (P = 0.23). A noticeably, but not significantly, higher cumulative incidence was seen in the more recent era since 1995 (2.1% vs. 0.9%, P = 0.15). PTLD in pancreas recipients carried a worse prognosis than in liver or kidney for recipients B-cell, early-onset, and Epstein Barr virus-positive lesions. PTLD was more aggressive in pancreas recipients, with a higher stage at presentation and a trend to more bone marrow involvement. There appeared to be a tendency toward association with recent CMV infection. Since 1995, PTLD recipients have had a lower exposure to antilymphocyte preparations (25 ± 5 vs. 10 ± 0.8)(P < 0.05). Conclusions. PTLD in pancreas recipients remains a rare but aggressive disease, and carries a worse prognosis in comparison to other transplant recipients. These heavily immunosuppressed patients, who often face multiple transplants, may be at greater risk; CMV infection may play an antecedent role.

Autocrine insulin action activates Akt and increases survival of isolated human islets

Aims/hypothesisThe phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt.MethodsIn order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period.ResultsWe demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation.Conclusions/interpretationOur findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.

Factors Associated with Improvement in Deceased Donor Renal Allograft Function in the 1990s

The decade of the 1990s saw an improvement in cadaveric renal graft function and dramatic reduction in the acute rejection (AR) rate. The purpose of this study was to determine whether the reduction in rejection rate was the primary cause of the improvement in graft function seen and whether this improved long-term graft survival. All adult patients who received a cadaver renal transplant between 1991 and 2000 and had graft survival of at least 6 mo and complete data for creatinine at 6 mo, HLA mismatch, delayed graft function, and acute rejection (AR) were identified in the United Network for Organ Sharing database. A total of 40,164 cases that met the inclusion criteria were identified. The mean Modification of Diet in Renal Disease GFR at 6 mo improved from 49.94 ml/min per 1.73 m2 in 1991 to 54.59 ml/min per 1.73 m2 in 2000 (P < 0.001). The improvement in GFR was not gradual but occurred over a 4-yr period between 1994 and 1997, coinciding with the introduction of new immunosuppressive agents mycophenolate mofetil and tacrolimus into maintenance immunosuppression regimens. The improvement was seen in all subgroups of patients, even patients without clinical AR or delayed graft function. The magnitude of improvement in patients without clinical AR was similar to that seen in patients with AR. The drop in clinical AR rate accounted for a minority of the improvement in graft function in the 1990s. Other factors, such as reduced drug toxicity and improved control of subclinical rejection, seem to account for the majority of the improvement. This improvement in graft function at 6 mo did not translate into improved long-term graft survival, however.