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.

Molecular and cellular key players in human islet transplantation.

Human islet transplantation could represent an attractive alternative to insulin injections for the treatment of diabetes type 1. However, such an approach requires a better understanding of the molecular and cellular switches controlling ?-cell function in general as well as after transplantation into the liver. Although much research has been done into the suitability of stem or progenitor cells to generate a limitless supply of human ?-cells, a reproducible and efficient protocol for the differentiation of such cells into stably insulin-secreting ?-cells suitable for transplantation has yet to be reported. Fueled by recent findings showing that mature ?-cells are able to regenerate, many efforts have been undertaken to expand this cell pool. Unfortunately, also these approaches had problems to yield sufficiently differentiated human islet cells. The aim of this review is to summarize recent findings describing some of the molecular and cellular key players of islet biology. A more complete understanding of their orchestration and the use of new methods such as real time confocal imaging for the assessment of islet quality may yield the necessary advancements for more successful human islet transplantation.

Dickkopf-3 is expressed in a subset of adult human pancreatic beta cells.

The Dickkopf (Dkk) gene family of secretory modulators of canonical Wnt/beta catenin signals is involved in the control of stem cell proliferation, homeostasis and differentiation. Bioinformatic data on dkk-1/3 gene expression, indicating high expression levels in the human pancreas, led us to analyze these two proteins in adult human pancreatic tissue. Dkk-1/3 mRNA levels and protein distribution were analyzed in isolated human islets vs. the exocrine/ductal pancreatic cells and in paraffin sections of adult human pancreata. Using real time PCR only lowest amounts of dkk-1 mRNA were detectable in the endocrine fractions. Immunohistochemistry did not reveal any Dkk-1 protein in adult human pancreatic tissue. Interestingly, Dkk-3 mRNA and protein were clearly present in adult human pancreatic islets. Messenger RNA levels for Dkk-3 were significantly higher in isolated islets as compared to the exocrine/ductal fraction. Co-staining with an antibody against insulin identified the beta cells of the pancreas as the Dkk-3-positive cells. Notably, only a subset of beta cells contained Dkk-3. As shown by western blot analysis Dkk-3 seems to be proteolytically processed in beta cells. To our knowledge, this is the first study describing a molecule with which the pool of pancreatic beta cells can be further subdivided. Future studies will show whether this sub-classification of beta cells translates into functional differences.

Tetrahydrobiopterin attenuates microvascular reperfusion injury following murine pancreas transplantation.

In this study we investigated the effect of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases, on ischemia-reperfusion injury (IRI) following murine pancreas transplantation. Pancreatic grafts were exposed to prolonged cold ischemia times (CIT) and different treatment regimens: normal saline (S), S + 16 h CIT, BH4 50 mg/kg + 16 h CIT. Nontransplanted animals served as controls. Graft microcirculation was analyzed by means of functional capillary density (FCD) and capillary diameters (CD) after 2 h reperfusion using intravital microscopy. Quantification of inflammatory responses (mononuclear infiltration) and endothelial disintegration (edema formation) was done by histology (hematoxylin and eosin), and peroxynitrite formation assessed by nitrotyrosine immunostaining. FCD was significantly reduced after prolonged CIT, paralleled by increased peroxynitrite formation as compared with controls (all p < 0.05). Microcirculatory changes correlated significantly with intragraft peroxynitrite generation (Spearman: r = -0.56; p < 0.01). Pancreatic grafts treated with BH4 displayed markedly higher FCD values (p < 0.01) and abrogated nitrotyrosine staining (p = 0.03). CD were not significantly different in any group. Histology showed increased inflammation, interstitial edema, hemorrhage, acinar vacuolization and focal areas of necrosis after 16 h CIT, which was diminished by BH4 administration (p < 0.01). BH4 treatment significantly reduces post-ischemic deterioration of microcirculation as well as histologic damage and might be a promising novel strategy in attenuating IRI following pancreas transplantation.

Islet isolation and GMP, ISO 9001:2000: what do we need--a 3-year experience.

Pancreatic islet cell isolation and transplantation has been performed for many years at several institutions. Although all institutions aim to produce high-quality islets, applied standards widely deviate from standards in the pharmaceutical industry. The legal situation within the European Union has changed requirements for setting up and running such a laboratory. The process is now clearly defined as a production of a pharmaceutical and therefore must be licensed by federal authorities. Analysis of workload for establishing an islet isolation program that fulfil GMP and ISO 9001 criteria including an estimation of costs and the impact of such a system on the isolation process. The definition of quality parameters and documentation is a central issue of all islet isolation laboratories. Therefore, GMP and ISO 9001:2000 do not add additional work per se. On the other hand, clear guidelines, a clear policy, working place descriptions, forms, checklists, and, particularly standard operating procedures, are instrumental for smooth functioning within the department. Collection of data such as errors, improvement measures, and preventive measures reduces subsequent costs. A clear definition of responsibilities minimizes organizational problems. Steering of inspection devices prevents bias errors and validating the processes clearly points out incorrect assumptions. Documentation helps to prove the correctness of the production at any time and is of use also for scientific evaluations. We strongly feel that GMP criteria are mandatory and together with an ISO 9001:2000 quality management system offers significant advantages for the process of islet isolation and a continuous improvement process.

"Real-time" assessment of human islet preparations with confocal live cell imaging.

Pancreatic islet cell transplantation is a promising approach to restoring normoglycemia in diabetic patients. The outcome of islet transplantation is uncertain for two reasons: The quality of isolated islets is still poorly defined and the functional potential of transplanted islets is difficult to predict. Therefore, one of the primary challenges in islet transplantation is to identify and understand the changes taking place in islets after isolation and culture. Description of such changes in living islet cells offers insights not achievable by use of fixed-cell techniques. Three fluorescent dyes, dichlorodihydrofluorescein diacetate, tetramethylrhodamine methyl ester perchlorate (TMRM), and fluorescent wheat germ agglutinin, were used to assess either overall oxidative stress, time-dependent mitochondrial membrane potentials, or localization of oligosaccharides. Confocal microscopy was performed with a microlens-enhanced Nipkow disk-based confocal system mounted on an inverse microscope. We were able to show differences in the amount of oligosaccharides on the cell surface between endocrine and exocrine cells in freshly isolated human islet preparations. The study of the mitochondrial membrane potential via TMRM proved to be useful to early identification of damaged or stressed cells. Thus a combination of fluorescent dyes as subcellular markers, with a powerful live confocal imaging system may be of great value to isolation and culture.

In the search of potential human islet stem cells: is tetranectin showing us the way?

Pancreatic islet cell transplantation is a promising approach to restore the required mass of functional beta cells in diabetic patients as a means to achieve long-term normoglycemia. This therapy is, however, not yet widely used, in part because of the shortage of human islet cells. Gaining detailed knowledge of the physiological basis governing the processes of differentiation of pancreatic stem or progenitor cells and the mechanisms and molecules necessary for a successful engraftment of the transplanted cells into the liver is instrumental for the ambitious goal of engineering new pancreatic islets to cure type I diabetes. We describe the in vivo and in vitro localisation of tetranectin (TN) in human and murine islet cells. Similar to human islets, murine islets stain positive for tetranectin. The amount and localization of TN is influenced by different culture conditions. The ability of TN to bind plasminogen indicates that it may have a role in regulating pericellular proteolysis and proteolytic activation of latent forms of metalloproteinases and growth factors. Tetranectin may thereby play an important role in the survival of islets in the liver after islet transplantation. TN-positive cells can be isolated and maintained in culture after human islet isolation, thereby providing the possibility to further clarify its role and function in vivo as well as in the course of islet transplantation.

A cluster of rotavirus enteritis in adult transplant recipients.

Diarrhea following solid organ transplantation is a common side effect of some immunosuppressive agents but can also be caused by many pathogens. An outbreak of rotavirus (RV) enteritis presenting with severe diarrhea in four solid organ recipients was analyzed. The first case was diagnosed in a 6-month-old liver recipient who was prehospitalized on a pediatric ward. Within 1 month, three adult patients (two liver, one renal recipient) presented with enteritis. During diarrhea a significant rise in tacrolimus levels was observed. One patient developed toxic megacolon with ulcerative colitis. Infections were self-limiting but led to secondary infectious complications and prolonged hospitalization. This is the first reported outbreak of RV enteritis in a multiorgan transplant unit involving adult patients. Although no fingerprinting or subtyping of the virus was performed we assume the child was the primary source. In transplant recipients presenting with diarrhea RV infection should be considered.

Successful [correction of Succesful] transplantation of human islets in recipients bearing a kidney graft.

AIMS/HYPOTHESIS: Islet transplantation is a minimally invasive approach to curing Type I (insulin-dependent) diabetes mellitus. Success has recently been reported in patients receiving solitary islet transplants but the outcome in patients receiving islets together with, or after, kidney transplants has been limited and unpredictable. METHODS: Here we report successful islet transplantation in a cohort of 15 patients with Type I diabetes who were followed for at least 1 year after islet transplantation, after having already received kidney allografts because of end-stage nephropathy. RESULTS: C-peptide after transplantation was higher than 0.17 nmol/l in all 15 recipients, reflecting the absence of primary non-function. Insulin requirement was reduced by over 50 % in all but one patient, and insulin independence was achieved in 10 (66 %) recipients, five of whom now have stable, prolonged insulin independence, well controlled fasting glycaemia, a substantial first-phase and normal second-phase response to glucose, normal insulin sensitivity (HOMA analyses) and HbA1 c of under 6.2 % (33, 26, 18, 13 and 12 months after transplantation respectively). Of importance for patient management, an assessment of fasting blood glucose and proinsulin values following overnight withdrawal of insulin administration one month after transplantation was a potent predictor of insulin independence, and could be used to decide patients who should have further islet preparations. CONCLUSION/INTERPRETATION: These findings support the use of islet transplantation as a cure for Type I diabetes in patients with severe complications.

Influence of irradiation on neutrophilic granulocyte function.

BACKGROUND: Neoadjuvant radiotherapy is a common treatment modality for patients with Stage II and III rectal carcinoma but, after surgery, often is complicated by local infections. To define a possible influence of radiotherapy on neutrophilic granulocytes in the neighborhood of tumor cells, the authors investigated their function in vitro. METHODS: Density gradient-purified granulocytes from healthy donors were used for all tests. These cells were cocultured with the colon carcinoma cell line HRT-18 and irradiated. Their function was assessed by measuring luminol-enhanced chemiluminescence and migration against the chemoattractant formyl-methionyl-leucine-phenylalanine. RESULTS: Although irradiation decreased, the addition of tumor cells increased reactive-oxygen species release in granulocytes, which was enhanced further by phorbol myristate acid (PMA), even after several hours. All contacts with tumor cells, however, caused immediate radical release that was inversely proportional to the radiation dose. Naïve and irradiated cells were stimulated further by PMA. Migration of granulocytes clearly was inhibited by tumor cells and irradiation, whereas the depth of invasion was enhanced by higher doses of radiation. CONCLUSIONS: The current data show clearly that the influence of radiotherapy on local defense against colorectal carcinoma is limited and cannot explain the increased rate of infectious complications.

Bactericidal activity of micromolar N-chlorotaurine: evidence for its antimicrobial function in the human defense system.

N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time. N-Chlorotaurine (12.5 to 50 microM) demonstrated a bactericidal effect i.e., a 2 to 4 log(10) reduction in viable counts, after incubation at 37 degrees C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5. 0), with a 3 to 4 log(10) reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated in N-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function of N-chlorotaurine and other chloramines during inflammation.

Polymorphonuclear leukocyte functions as predictive markers for infections after organ transplantation.

Infectious complications are still a major cause of morbidity and mortality after organ transplantation, and early therapy would certainly reduce the risk associated with severe infections. We therefore investigated the significance of polymorphonuclear leukocyte (PMN) functional tests as predictive markers for infection in transplant patients under immunosuppressive therapy in a longitudinal study. In 41 patients, blood PMN migration and reactive oxygen species release, the blood levels of PMN elastase, malondialdehyde, neopterin, sICAM-1 and sVCAM-1, and urine neopterine were measured in 3- and 4-day intervals after liver-, kidney-, kidney-pancreas-, and heart and lung transplantation. PMN migration was determined in whole blood and estimated by the amount of PMNs to penetrate into a membrane filter upon FMLP stimulation. Three groups of patients were formed according to their postoperative course. Group I patients (n = 23) had no or only minor local infection, group II patients (n = 11) had infections with distinct systemic involvement, and group III patients (n = 7) developed sepsis. A first elastase-level of over 100 mg/L after surgery, followed by a drop in the amount of blood PMNs ready to migrate, on FMLP stimulation, to below 12 %, turned out to be a marker for impending infection, whereas all other parameters tested were not predictive. In six of seven group III patients, this marker became positive (sensitivity 85.7 %) up to 15 days before clinical manifestation of sepsis. In group I (largely uneventful recovery) only one of 23 patients was positive (specificity 95.6 % ), whereas group II patients were in between (4 of 11 positive). By this method it seems possible to diagnose severe infections in the pre-clinical phase, which may help prevent them if treatment is begun promptly.

Improved technique for infrahepatic vena cava reconstruction in guinea pig-to-rat liver transplantation.

Guinea pig-to-rat orthotopic liver transplantation is associated with serious technical problems contributing to impaired graft perfusion and primary graft failure. In order to shorten the procurement procedure and thereby minimize liver damage before flushing, a simplified technique for infrahepatic caval reconstruction was developed. Dissection of the infrahepatic vena cava (IHVC) from adrenal glands and renal and lumbar veins represents the most difficult and time-consuming part of the donor operation, which is often not well tolerated by the animal; we avoided this step by using an isogeneic vena cava interposition graft (VCIG) following in situ perfusion. This graft is connected with the IHVC transsected just below the liver with a cuff technique. Donor operations lasted 15 to 20 minutes with the new technique (n = 7) compared to 52 to 76 minutes with conventional technique (n = 7). Reduced operating time was associated with markedly improved graft perfusion and significantly better graft survival. This modification of the donor procedure for the guinea pig-to-rat liver xenograft using a VCIG significantly reduces operating time and improves reperfusion and recipient survival.

Cytomegalovirus infections after treatment with daclizumab, an anti IL-2 receptor antibody, for prevention of renal allograft rejection. Roche Study Group.

Daclizumab is a newly developed humanized anti-IL-2 receptor monoclonal antibody. We describe the effect of adding daclizumab to conventional dual or triple cyclosporine A immunosuppressive therapy on the incidence and nature of cytomegalovirus (CMV) infections in patients receiving a first cadaveric renal graft. In the triple therapy study there was no evidence of any difference in CMV rate or course of disease between the two treatment arms, although in the dual therapy study a decrease in the incidence of CMV infection was observed in the patients treated with daclizumab. The onset of CMV disease was markedly delayed in the daclizumab groups in both studies. Daclizumab can effectively reduce the risk of acute rejection without causing a concomitant increase in opportunistic infections, and by decreasing the need for antirejection therapy may also have a beneficial effect on CMV infection rates.

Optimization of phagocyte chemiluminescence measurements using microplates and vials.

In order to cope with large amounts of samples for chemiluminescence (CL), vials were replaced with microplates. Although various types of plates have been commercially available for quite some time and the free-plate mode is advocated by the producer of the counter, little is known about their impact on the outcome of CL measurements. We tested two different 24-well microplates and six different 96-well microplates in two different luminometers, and results were compared with those achieved with vials. Before these comparative tests, we attempted to optimize measurement conditions. CL sensitivity was highest with luminol concentrations of 0.8-3.3 micromol/L, PMA concentrations of 0.06-80 micromol/L, a pH value of 10 and a temperature of 20 degrees C. An indirect correlation was found between fluid volume and yield in counts: the lower the volume, the higher the counts. With regard to sensitivity and cross-talk, the 96-well Isoplatetrade mark was superior to all other plates tested. While all white plates tested gave acceptable results, usage of the black 96-well plates resulted in an extremely low sensitivity. Plates designed for cell culturing gave even lower counts and a cross-talk of up to 31%. All attempts to reduce cross-talk and improve sensitivity, such as aluminium foil or grids, irrespective of the position of the photomultiplier, did not give results comparable to the original 96-well isoplate. Our results suggest that, with the exception of black 96-well microplates and cell culture plates, all other plates tested have a sufficient sensitivity when compared to vials and acceptable cross-talk, the 96-well Isoplatetrade mark being the best. Both types of luminometers used gave reproducible results, Wallac having a somewhat higher sensitivity, Canberra Packard somewhat less cross-talk.

The postantibiotic effect of N-chlorotaurine on Staphylococcus aureus. Application in the mouse peritonitis model.

This study was designed to investigate the delay of regrowth (postantibiotic effect) in the presence of N-chlorotaurine (NCT), an endogenous active N-chlorine compound, of Staphylococcus aureus, strain Smith diffuse. The low reactivity of NCT enabled clear temporal separation of the postantibiotic and killing effect to be defined. Delay of regrowth proved to be dependent both on concentration of NCT, and incubation time. The maximum delay was 3 h. Using the model of lethal staphylococcal peritonitis in mice, in-vivo delay of regrowth of bacteria pretreated with N-chlorotaurine could be demonstrated to correlate with survival. It is concluded that the postantibiotic effect of N-chlorotaurine could be an important factor on decreasing virulence of bacteria. This effect was observed after relatively short incubation times.

Expression of stress proteins, adhesion molecules, and interleukin-8 in endothelial cells after preservation and reoxygenation.

Endothelial activation is a central feature of preservation-induced allograft injury. The present study aims at a quantitative assessment of stress proteins, adhesion molecules, and interleukin-8 in a cell culture-based model of organ preservation. Human umbilical vein endothelial cells were exposed to cold, hypoxic storage in University of Wisconsin (UW), histidine-tryptophane-ketoglutarate (HTK), and EuroCollins solutions for 8 h with subsequent rewarming/reoxygenation (rew/reox) for 1 and 4 h. A cell-based ELISA was designed for detection of heat shock proteins (HSP) 60 and 70, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1). Immunohistochemical staining was performed for comparison. Interleukin-8 was quantified by ELISA. HSP 70 was expressed after cold storage in HTK and EuroCollins solution and after rew/reox in all groups. A constitutive expression of HSP 60 was observed with further upregulation after rew/reox following cold storage in all experimental groups. ICAM-1 was clearly upregulated, but VCAM-1 showed only weak expression after cold storage and rew/reox. ELAM-1 was detectable in minimal amounts after cold storage but was considerably upregulated after 4 h of rew/reox. A significant increase of interleukin-8 release could be found after 4 h of rew/reox following storage in EuroCollins solution. Expression of stress proteins can be considered as a new parameter of preservation-associated endothelial activation. Apart from possible protective effects, allograft vasculopathy could be in part a consequence of the antigeneic potential of heat shock proteins connected with effects caused by adhesion molecules and inflammatory cytokines.

Myocardial fatty acid metabolism during acute cardiac allograft rejection.

Fatty acids are promptly taken up, metabolised and eliminated by healthy cardiomyocytes. Cardiomyopathy, coronary heart disease and chronic rejection are known to be associated with an impaired fatty acid metabolism. It was the aim of this study to investigate fatty acid metabolism in a rat heart transplant model and to correlate scintigraphic findings with histological changes. After right-side nephrectomy of Lewis recipients Brown Norway cardiac allografts were anastomosed to the renal vessels. Animals were given no immunosuppression. The metabolism of carrier-free 17-123 jodo-heptadecanoic acid (123J-HDA) with a specific activity of > 2 x 10(17) Bq/ml was scintigraphically measured between days 1 and 11. An increase in the grade of rejection was observed over time. Fifty-six frames of 30 s duration each were recorded. For the region of interest (native heart, transplanted heart, left kidney) frames 10-56 were superimposed, time-activity curves generated and monoexponentially fitted. Furthermore, elimination half-life and intercepts were calculated. Following scintigraphic evaluation the animals were killed and graft as well as native hearts excised for histological examination. The uptake of the tracer identified severe grades of rejection. Elimination half-life of the tracer was twice as long from hearts with mild rejection and more than 14 times as long in severe rejection compared with no rejection. Elimination half-life and amplitude did not permit discrimination between grades 1, 2 and 3 a, but significantly decreased in groups 3 b and 4. This method therefore seems to be a valuable tool for the noninvasive detection of severe acute cardiac allograft rejection. Since fatty acid metabolism is clearly stress-dependent it remains to be seen whether this method allows detection of earlier rejection in loaded hearts.