Mathias D. Brendel

Assessment of intracellular insulin content during all steps of human islet isolation procedure.

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

Transduction of non-dividing adult human pancreatic beta cells by an integrating lentiviral vector

Summary Pancreatic islet cells are terminally differentiated endocrine cells and are refractory to stable infection by retroviral vectors, which require the breakdown of the nuclear membrane during cell division in order to insert the transgene into the host cell genome. Thus, attempts to render beta-cell allografts less immunogenic have had to rely on stable transfection of surrogate cells. Similarly, this problem has precluded the development of conditionally immortalized human beta cells for clinical allotransplantation. In this report, we demonstrate that adult human islet beta cells can be transduced by a new three-plasmid integrating lentiviral vector with an efficiency of 62 ± 1.8 % at a multiplicity of infection (MOI) of 2.5 in vitro. This work makes genetic engineering of adult human pancreatic beta cells possible for the first time, allowing strategies to render beta-cell allografts non-immunogenic to be optimized and to creating conditionally immortalized human beta cells for clinical transplantation. [Diabetalogia (1998) 41: 736–739]

Percutaneous transhepatic catheterization of the portal vein: A combined CT- and fluoroscopy-guided technique.

Combined CT- and fluoroscopy-guided transhepatic portal vein catheterization was performed in 44 patients selected for pancreatic islet cell transplantation. The method allowed catheterization with a single puncture attempt in 39 patients. In four patients two attempts and in one patient four attempts were necessary. One minor hematoma of the liver capsule occurred that required no further treatment. Compared with other methods the average number of puncture attempts was reduced.

Evidence for a significant correlation of donor pancreas morphology and the yield of isolated purified human islets

In clinical islet transplantation to patients with type 1 diabetes mellitus, the number of isolated and purified islet has been identified as a key determinant for functional success of the islet graft [1]. With improved isolation methods based on the original procedure published by Ricordi et al. [2] yield and function of isolated islets were considerably enhanced. However, there is still a large variance in the number, purity, viability and secretory capacity of islets isolated from brain-dead human donor pancreata, significantly hampering utilization of human islet preparations derived from a single donor for one diabetic recipient. The reasons for the limited success in islet isolation and purification have not been clarified in detail yet. Recent studies have indicated, that donor preconditions, and a number of technical factors during organ procurement and the islet isolation process itself are critical to successful islet isolation [3, 4]. This study aimed at identifying distinct morphological and histopathological characteristics of the donor pancreas as determinants for the outcome of human islet isolation and purification.

Successful islet auto- and allotransplantation in diabetic pigs

BACKGROUND Because of its anatomical and physiological similarities to humans, the pig appears to be a suitable large animal model for preclinical studies of islet transplantation. The aim of this study was to investigate islet auto- and allotransplantation in a pig model with diabetes induced by total pancreatectomy. METHODS Porcine islets were isolated by a continuous digestion-filtration device at 32 degrees C and purified by a discontinuous iso-osmolar Ficoll-sodium-diatrizoate gradient on a Cobe 2991. The purified islets were autografted into the liver or the renal subcapsular space. The liver appears to be a more suitable site for the islet grafts than the renal subcapsular space, and the minimal amount of islets for reversal of diabetes is >5 microl/kg of body weight. RESULTS Persistent normoglycemia (fasting blood glucose level: 72.4+/-44.38 mg/dl) with a normal insulin secretion response to glucose stimulation was successfully achieved in five of six diabetic pigs by implanting a sufficient islet mass into the liver. Triple-drug immunosuppressive therapy with cyclosporine, azathioprine, and prednisolone did not prevent porcine islet allografts from experiencing early failure. However, the addition of 15-deoxyspergualin to the triple-drug immunosuppressive regimen significantly prolonged the function of the islet allografts. When antithymocyte globulin was added to the above-mentioned immunosuppressive drug regimen, the normoglycemic period was prolonged to more than 1 month (fasting blood glucose level: 75.4+/-17 mg/dl). CONCLUSION We conclude that autotransplantation with a sufficient islet mass can induce normoglycemia with a normal insulin secretion response to glucose stimulation in pancreatectomized diabetic pigs and that allotransplantation can be successfully achieved when 15-deoxyspergualin and antithymocyte globulin are combined with the triple-drug immunosuppression described above. However, this immunosuppressive protocol results in a high rate of infectious complications.

Study on pancreatic islet adaptation and gene expression during pregnancy in rats

During pregnancy, the pancreatic islets undergo major structural and functional changes in response to increased peripheral resistance to insulin. In this study, we investigated the adaptive changes of the pancreatic islet beta-cell mass during pregnancy in rats, and explored profiles of islet gene expression at various stages of pregnancy. Some differentially expressed genes were verified by RT-PCR and Real-time PCR. Our results showed that compared with the non-pregnant control group, insulin synthesis, glucose-stimulated insulin secretion, islet beta-cell proliferation, and islet size were all increased in pregnant rats. The study also demonstrated that expression of several-hundred islet genes were changed during pregnancy, especially at day 14.5. The differentially expressed genes identified were distributed into eight main categories according to their biological functions: (1) genes involved in apoptosis or tumor; (2) genes related to binding; (3) genes involved in metabolism; (4) genes related to cell cycle; (5) genes for signal transducer activity; (6) genes related to structural molecule activity; (7) genes involved in transcription regulator activity; (8) genes for transporter activity. Among these genes, regenerating islet-derived 3 alpha (Reg3a) was remarkably increased during pregnancy. We hypothesize that differentially expressed genes may play an important role in adaptation of pancreatic islets during pregnancy in rats. In addition, the markedly increased expression of gene Reg3a is probably related to islet regeneration.

Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43°C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1β, IFN-γ, or TNF-α was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.

New-Onset Diabetes Mellitus After Renal Transplantation

BACKGROUND New-onset diabetes mellitus after organ transplantation (PTDM) significantly impairs patient and organ survival. Published rates of PTDM range from 2% to 54%, depending on the definition. OBJECTIVES To analyze incidence of PTDM after renal transplantation according to recent guidelines and to evaluate implementation of a prospective standardized screening protocol. PATIENTS AND METHODS Data for all consecutive patients who underwent transplantation from 2000 to 2006 were analyzed retrospectively for PTDM. In a prospective pilot trial all candidates for living related donor transplantation underwent a 75-g oral glucose tolerance test at evaluation prior to renal transplantation and at 3, 6, and 12 months thereafter. RESULTS Data for 181 out of 271 consecutive patients were analyzed. Of these patients, 36 (19.9%) developed PTDM. Age, body mass index, pretransplantation fasting glucose concentration, and number of HLA mismatches were significant predictive risk factors. Posttransplantation diabetes mellitus occurred more frequently in patients receiving a cadaver organ compared with a living donor organ and in those receiving tacrolimus therapy vs cyclosporine therapy. Preliminary results demonstrated a 55.5% incidence of PTDM at 3 months in patients who received a living donor organ, much higher than expected. CONCLUSIONS With an incidence of approximately 20%, PTDM is a frequent complication of transplantation. Prospective screening using oral glucose tolerance testing is a more sensitive method for detection of impaired glucose metabolism and PTDM. Relevance and therapeutic consequences must be determined in large-scale prospective studies.

Circulating cytokines are associated with human islet graft function in type 1 diabetes.

Islet cell transplantation has considerable potential as a cure for type 1 diabetes, but recurrent autoimmunity and allograft rejection in which both cytokines play an important role are major obstacles. Using a new approach considering confounders by regression analysis, we investigated circulating cytokines and their association with graft function in type 1 diabetes patients who underwent either simultaneous islet kidney (SIK) or islet after kidney (IAK) transplantation. After transplantation, interleukin (IL)-10 was lower in SIK recipients with subsequent loss of graft function in comparison to recipients maintaining graft function. Before transplantation, high IL-13 and IL-18 concentrations were prospectively associated for subsequent loss of graft function in IAK recipients, whereas in SIK recipients, high macrophage migration inhibitory factor (MIF) concentrations were associated with subsequent loss of graft function. Circulating cytokines are associated with islet graft function in patients with long-standing type 1 diabetes when considering confounders.

Monitoring of Insulin Content During Human Islet Isolation

SUCCESS or failure of islet isolations are usually determined by the absolute number of isolated islets or islet equivalents (IEQ) obtained after digestion and subsequent purification. Determination of the variability of the insulin content of the native endocrine pancreas may serve as a basis for the analysis of the enormous variability of human islet isolation outcome. Although some efforts have been made to estimate the number of islets of the human pancreas using different morphometrical methods or insulin extraction, little is known about the recovery of the initial islet mass from the native human pancreas by islet isolation procedures. Therefore, the present study aimed to prove the efficiency of our standard islet isolation procedure (digestion-filtration), purification technique (continuous ficoll gradient), and 22°C culture (CMRL 1 10% FCS) to recover the native insulin content of human pancreatic tissue.