Stephan Schneider

Publications of Stephan Schneider

• Long-term graft function of cryostored alginate encapsulated rat islets.

  Authors: Stephan Schneider, H H Klein

  European journal of medical research. 09/2011; 16(9):396-400.

  Microencapsulation of pancreatic islets before transplantation is a promising approach to enable graft function in an immunocompetent recipient without immunosuppression. However, the insufficientMicroencapsulation of pancreatic islets before transplantation is a promising approach to enable graft function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of encapsulated allogenic islets. Recently, we reported a gentle cryopreservation protocol for rat islets encapsulated in an alginate-based microcapsule system. Here, we report for the first time long-term transplantation data of these cryopreserved microencapsulated islets. We detected a stable graft function for more than 12 month (experiments still continuing) after transplantation of 2500 cryopreserved microencapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Moreover, the glucose clearance rate during an IPGTT was well preserved up to 56 weeks after transplantation. In addition, hyperglycemic blood glucose levels after removal of rat islet grafts 12 and 56 weeks after transplantation confirmed the efficacy of the encapsulated islets. Finally, the retrieved encapsulated rat islets responded well with a 7-fold increase of insulin secretion to a glucose stimulus (12 and 56 weeks). In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function for a very long time.

• Protection from diabetes development by single-chain antibody-mediated delivery of a NF-κB inhibitor specifically to β-cells in vivo.

  Authors: Sandra Ueberberg, Timo Deutschbein, Harald H Klein, Johannes W Dietrich, Sara Akinturk, Nora Prochnow, Ralph Schirrmacher, Stephan Schneider

  American journal of physiology. Endocrinology and metabolism. 04/2011; 301(1):E83-90.

  Recently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human β-cells. Our current report describes the generation of a fusion protein of one of theseRecently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human β-cells. Our current report describes the generation of a fusion protein of one of these SCAs (SCA B1) with a NF-κB essential modifier (NEMO)-binding domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in β-cells. The SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in bacteria, and purified by metal affinity chromatography; the newly generated complex was then administered intravenously to rodents and evaluated for its ability to protect β-cells against cytokines in vitro and diabetogenic agents in vivo. First, it was shown clearly that our SCA B1-NBD fusion protein binds highly selective to CD rat β-cells in vivo. Second, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1β + IFNγ- and TNFα + IFNγ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin in CD mice not only induced a striking resistance to diabetes development but also preserved β-cell mass. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects β-cells against cytokines in vitro and allows protection from diabetes development in CD mice in vivo.

• In vitro and initial in vivo evaluation of (68)Ga-labeled transferrin receptor (TfR) binding peptides as potential carriers for enhanced drug transport into TfR expressing cells.

  Authors: Carmen Wängler, Dina Nada, Georg Höfner, Simone Maschauer, Björn Wängler, Stephan Schneider, Esther Schirrmacher, Klaus T Wanner, Ralf Schirrmacher, Olaf Prante

  Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging. 04/2011; 13(2):332-41.

  The transferrin receptor (TfR) is one of the most attractive targets to overcome the blood-brain barrier (BBB). It has recently been shown that THRPPMWSPVWP binds to the TfR and is subsequentlyThe transferrin receptor (TfR) is one of the most attractive targets to overcome the blood-brain barrier (BBB). It has recently been shown that THRPPMWSPVWP binds to the TfR and is subsequently internalized into TfR-expressing cells. Here, we evaluated the ability of THRPPMWSPVWP to become internalized into human TfR-expressing cells via endocytosis to determine its potential to act as a carrier system for the transport of small molecules across the BBB. To validate the underlying concept of a conjugate consisting of a small brain imaging tracer and a large peptidic carrier molecule, a conjugate of the high affinity D2 receptor ligand fallypride and the TfR targeting peptide THRPPMWSPVWP has been synthesized. Furthermore, two derivatives of THRPPMWSPVWP were labeled with (68)Ga in high radiochemical yields (>96%) and a radiochemical purity of 96-98% and evaluated in vitro and in vivo. The fallypride-THRPPMWSPVWP conjugate still displayed a K(i) of 27 nM. The uptake of the (68)Ga-labeled peptides into TfR-bearing cells was investigated using U87MG and HT-29 cells to assess the capability of the peptide to act as a carrier molecule targeting the TfR. The in vitro binding studies revealed negligible uptake of the tested (68)Ga-labeled conjugates ranging from 0.08% to 0.66% after 60 min incubation at 37°C. Initial in vivo experiments with (68)Ga-DOTA-S-maleimido-THRPPMWSPVWP in two healthy rats showed a mean brain uptake of 0.037% injected dose per gram, confirming the results obtained in vitro. These results suggest that the accumulation of the (68)Ga-radiolabeled conjugates of the TfR-binding peptide THRPPMWSPVWP into TFR expressing human cell lines is nonspecific and too low to render this peptide suitable as a possible carrier molecule for a receptor-mediated transport of compounds across the BBB.

• Diminished glucagon suppression after β-cell reduction is due to impaired α-cell function rather than an expansion of α-cell mass.

  Authors: Juris J Meier, Sandra Ueberberg, Simone Korbas, Stephan Schneider

  American journal of physiology. Endocrinology and metabolism. 02/2011; 300(4):E717-23.

  Impaired suppression of glucagon levels after oral glucose or meal ingestion is a hallmark of type 2 diabetes. Whether hyperglucagonemia after a β-cell loss results from a functional upregulation ofImpaired suppression of glucagon levels after oral glucose or meal ingestion is a hallmark of type 2 diabetes. Whether hyperglucagonemia after a β-cell loss results from a functional upregulation of glucagon secretion or an increase in α-cell mass is yet unclear. CD-1 mice were treated with streptozotocin (STZ) or saline. Pancreatic tissue was collected after 14, 21, and 28 days and examined for α- and β-cell mass and turnover. Intraperitoneal (ip) glucose tolerance tests were performed at day 28 as well as after 12 days of subcutaneous insulin treatment, and glucose, insulin, and glucagon levels were determined. STZ treatment led to fasting and post-challenge hyperglycemia (P < 0.001 vs. controls). Insulin levels increased after glucose injection in controls (P < 0.001) but were unchanged in STZ mice (P = 0.36). Intraperitoneal glucose elicited a 63.1 ± 4.1% glucagon suppression in control mice (P < 0.001), whereas the glucagon suppression was absent in STZ mice (P = 0.47). Insulin treatment failed to normalize glucagon levels. There was a significant inverse association between insulin and glucagon levels after ip glucose ingestion (r(2) = 0.99). β-Cell mass was reduced by ∼75% in STZ mice compared with controls (P < 0.001), whereas α-cell mass remained unchanged (P > 0.05). α-Cell apoptosis (TUNEL) and replication (Ki67) were rather infrequently noticed, with no significant differences between the groups. These studies underline the importance of endogenous insulin for the glucose-induced suppression of glucagon secretion and suggest that the insufficient decline in glucagon levels after glucose administration in diabetes is primarily due to a functional loss of intraislet inhibition of α-cell function rather than an expansion of α-cell mass.

• Preserved insulin secretion capacity and graft function of cryostored encapsulated rat islets.

  Authors: Stephan Schneider, Harald H Klein

  Regulatory peptides. 10/2010; 166(1-3):135-8.

  Encapsulation of pancreatic islets before transplantation enables survival and function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenicEncapsulation of pancreatic islets before transplantation enables survival and function in an immunocompetent recipient without immunosuppression. However, the insufficient availability of allogenic islet tissue is a major problem. One concept to overcome these shortcomings is the cryopreservation of microencapsulated allogenic islets, to allow their unlimited collection and use on demand. Therefore, this report outlines the development of a cryopreservation protocol for CD rat islets encapsulated in an alginate-based microcapsule-system. We determined RPMI-medium plus 10% FCS as freezing medium, equilibration at 0°C for 15 min with the cryoprotectant dimethyl sulfoxide (DMSO; final concentration 2.0M), and a stepwise removal of DMSO by sucrose dilution after thawing, as best protocol for cryopreservation of encapsulated islets. Importantly, the cryopreserved encapsulated islets showed post thawing in vitro an insulin increase upon a glucose challenge comparable to that of non-cryopreserved encapsulated islets. Moreover, a stable graft function without the need of immunosuppression was detected after transplantation of 2500 cryopreserved encapsulated CD rat islets in streptozotocin-diabetic Wistar rats. Finally, the glucose clearance rate during an IPGTT 4 weeks after transplantation was comparable to that of rats transplanted with non-cryopreserved encapsulated islets. In conclusion, our study demonstrates for the first time that cryopreservation of encapsulated rat islets is possible without substantial losses on graft function. Future studies will now have to carry on this approach to human islets, aiming to apply such a bioartificial pancreas consisting of cryopreserved encapsulated islets in humans.

• Generation of novel single-chain antibodies by phage-display technology to direct imaging agents highly selective to pancreatic {beta}- or {alpha}-cells in vivo.

  Authors: Sandra Ueberberg, Juris J Meier, Carmen Waengler, Wolfgang Schechinger, Johannes W Dietrich, Andrea Tannapfel, Inge Schmitz, Ralf Schirrmacher, Manfred Köller, Harald H Klein, Stephan Schneider

  Diabetes. 08/2009;

  Objective: Non-invasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell specific imaging agents. This report outlines an approach for theObjective: Non-invasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet-cells in vivo. Research Design and Methods: In order to generate agents specifically binding to pancreatic islets, a phage-library was screened for single-chain-antibodies (SCAs) on rat islets using two different approaches: (1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 minutes. (2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. Results: We report the generation of SCAs that bind highly selective to either beta- or alpha-cells. These SCAs are internalised by target cells, disappear rapidly from the vasculature and exert no toxicity in vivo. Specific binding to beta- or alpha-cells was detected in cell-lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [(125)I]-labeled SCAs after i.v. administration in rats strongly predicted the beta-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. Conclusions: Our data provide strong evidence that the presented SCAs are highly specific for pancreatic beta-cells and enable imaging and quantification in vivo.

• Effect of fenofibrate on microcirculation and wound healing in healthy and diabetic mice.

  Authors: S Valentin, J Rudolph, O Goertz, N Botteck, S Langer, Stephan Schneider

  European journal of medical research. 03/2009; 14(2):65-70.

  Objective: Disturbances in wound healing in patients with hyperglycaemic blood sugar values are a common clinical problem. Recent studies identified PPARalpha-ligands as potential skin therapeuticObjective: Disturbances in wound healing in patients with hyperglycaemic blood sugar values are a common clinical problem. Recent studies identified PPARalpha-ligands as potential skin therapeutic agents. The aim of this study was to investigate the effects of oral fenofibrate treatment on dermal wound healing and microcirculatory parameters in diabetic mice. - Methods: Dermal wounds were created in CD-1 mice. Mice were randomized into four treatment groups: diabetic mice treated (dbf) or not-treated with fenofibrate (dbnf). As controls served non-diabetic mice treated (ndf) or not-treated with fenofibrate (ndnf). At various points in time microcirculation was analyzed by intravital fluorescent microscopy to determine wound surface area, vessel diameter, plasma leakage, functional capillary density, and leukocyte/endothelium interaction. - Results: The dbf-mice showed a significantly increased diameter of the venules and the arterioles up to 3 days after wound creation compared to dbnf-mice. However, wound healing was not improved in dbf-compared to dbnf-mice. Surprisingly, all microcirculatory parameter (vessel diameter, plasma leakage and functional capillary density) were not deteriorated in dbnf- compared to ndnf-mice. - Conclusion: We confirm that high blood sugar values lead to a delayed wound healing, but this could not traced back to altered microcirculatory patterns. Furthermore, in dbf-mice an improved vasodilatatory function of small vessels could be detected, but with no substantial effect on wound healing. Further studies are needed to clarify, if topical application of fenofibrate might be beneficial.

• [Endosonography -- an additional diagnostic possibility in the differentiation between the two common types of primary hyperaldosteronism]

  Authors: Daniela Roggenland, Stephan Schneider, Harald H Klein, Peter H Kann

  Medizinische Klinik (Munich, Germany : 1983). 02/2006; 101(1):65-8.

  BACKGROUND: Primary aldosteronism is an important and one of the few potentially curable forms of secondary hypertension. The distinction between aldosterone-producing adenoma (APA) and idiopathicBACKGROUND: Primary aldosteronism is an important and one of the few potentially curable forms of secondary hypertension. The distinction between aldosterone-producing adenoma (APA) and idiopathic adrenal hyperplasia (IHA) may be difficult, but establishing the correct diagnosis is essential because surgery is only effective in patients with adrenal adenoma. CASE REPORT: The case of a 65-year-old man with long-term hypertension due to an APA is reported. The routine laboratory tests displayed an elevated plasma aldosterone/renin quotient as well as an elevated 24-h urinary excretion of aldosterone and its metabolites. The serum aldosterone concentration did not decrease normally in the saline suppression test. The posture testing demonstrated an increase in aldosterone. These facts might lead to the conclusion of an IHA. Magnetic resonance imaging (MRI) of the adrenal glands revealed no abnormalities. Because of the unusual combination of laboratory findings and radiologic results an endosonographic examination of the adrenal glands was performed which yielded a unilateral adrenal adenoma. With establishing this diagnosis, curative surgery became possible. CONCLUSION: This case demonstrates that in the differential diagnosis of primary aldosteronism, endosonography is more important than previously discussed. It may be helpful in the differentiation of an unusual constellation of laboratory and radiologic findings.

• Inosine improves islet xenograft survival in immunocompetent diabetic mice.

  Authors: Stephan Schneider, H H Klein

  European journal of medical research. 08/2005; 10(7):283-6.

  BACKGROUND: Inflammation is likely to be one of the factors responsible for early islet xenograft rejection. In this study, we have used a novel anti-inflammatory compound, inosine, to investigateBACKGROUND: Inflammation is likely to be one of the factors responsible for early islet xenograft rejection. In this study, we have used a novel anti-inflammatory compound, inosine, to investigate the role of anti-inflammatory blockade in protecting rat pancreatic islets from xenograft rejection in diabetic balb/c mice. Inosine is a safe, naturally occurring purine, which appears to be nontoxic to humans, that has recently been shown to be an immunomodulator and anti-inflammatory agent. METHODS: For the transplantation model rat islets were placed under the renal capsule of streptozotocin diabetic balb/c mice and inosine -- (200 mg kg (-1) d (-1) or vehicle- treatment started the day of islet transplantation. RESULTS: In vehicle-treated mice rat islet xenografts were rejected within 2 - 11 days after transplantation. In contrast, in inosine-treated mice graft survival was markedly prolonged for up to 3 weeks. Furthermore, immunohistochemistry and morphometric analyses revealed that early islet graft infiltration by leukocytes (CD45(+)) is significantly reduced in the grafts from inosine-treated diabetic mice compared to vehicle-treated mice. As suggested from the graft survival data, inosine-treated mice had also a significantly better preserved beta-cell mass. CONCLUSIONS: These results clearly demonstrate that inosine treatment markedly reduced the early inflammatory reaction elicited by an islet xenograft and substantially prolongs the graft survival. Therefore, such an approach may form an important future component of therapeutic regimens applied in clinical islet xenotransplantation.

• Long-term graft function of adult rat and human islets encapsulated in novel alginate-based microcapsules after transplantation in immunocompetent diabetic mice.

  Authors: Stephan Schneider, Peter J Feilen, Frank Brunnenmeier, Timo Minnemann, Heiko Zimmermann, Ulrich Zimmermann, Matthias M Weber

  Diabetes. 03/2005; 54(3):687-93.

  We describe the results of the first study to show that adult rat and human islets can be protected against xenogenic rejection in immunocompetent diabetic mice by encapsulating them in a novelWe describe the results of the first study to show that adult rat and human islets can be protected against xenogenic rejection in immunocompetent diabetic mice by encapsulating them in a novel alginate-based microcapsule system with no additional permselective membrane. Nonencapsulated islets lost function within 4-8 days after being transplanted into diabetic Balb/c mice, whereas transplanted encapsulated adult rat or human islets resulted in normoglycemia for >7 months. When rat islet grafts were removed 10 and 36 weeks after transplantation, the mice became immediately hyperglycemic, thus demonstrating the efficacy of the encapsulated islets. The explanted capsules showed only a mild cellular reaction on their surface and a viability of >85%, and responded to a glucose stimulus with a 10-fold increase in insulin secretion. Furthermore, transplanted mice showed a slight decrease in the glucose clearance rate in response to intraperitoneal glucose tolerance tests 3-16 weeks after transplantation; after 16 weeks, the rate remained stable. Similar results were obtained for encapsulated human islets. Thus we provide the first evidence of successful transplantation of microencapsulated human islets. In conclusion, we have developed a novel microcapsule system that enables survival and function of adult rat and human islets in immunocompetent mice without immunosuppression for >7 months.

• Synthesis and in vitro evaluation of (S)-2-([11C]methoxy)-4-[3-methyl-1-(2-piperidine-1-yl-phenyl)-butyl-carbamoyl]-benzoic acid ([11C]methoxy-repaglinide): a potential beta-cell imaging agent.

  Authors: Björn Wängler, Carmen Beck, Chyng Yann Shiue, Stephan Schneider, Christina Schwanstecher, Mathias Schwanstecher, Peter Johannes Feilen, Abass Alavi, Frank Rösch, Ralf Schirrmacher

  Bioorganic & medicinal chemistry letters. 11/2004; 14(20):5205-9.

  The 11C-labeled sulfonylurea receptor 1 (SUR1) ligand (S)-2-([11C]methoxy)-4-[3-methyl-1-(2-piperidine-1-yl-phenyl)-butyl-carbamoyl]-benzoic acid ([11C]methoxy-repaglinide) was synthesized in anThe 11C-labeled sulfonylurea receptor 1 (SUR1) ligand (S)-2-([11C]methoxy)-4-[3-methyl-1-(2-piperidine-1-yl-phenyl)-butyl-carbamoyl]-benzoic acid ([11C]methoxy-repaglinide) was synthesized in an overall radiochemical yield of 35% after 55 min with a radiochemical purity higher than 99%. This compound is considered for the noninvasive investigation of the SUR1 receptor status of pancreatic beta-cells by positron emission tomography (PET) in the context of type 1 and type 2 diabetes. The specific activity was 40-70 GBq/micromol. In vitro testing of the nonradioactive methoxy-repaglinide was performed to characterize the affinity for binding to the human SUR1 isoform. Methoxy-repaglinide induced a complete monophasic inhibition curve with a Hill coefficient close to 1 (1.03) yielding a dissociation constant (KD) of 83 nM and an IC50 of 163 nM. Insulin secretion experiments on isolated rat islets were performed to prove biological activity, which was determined to be in the same range as that of original repaglinide.

• Intraportal transplantation of allogenic pancreatic islets encapsulated in barium alginate beads in diabetic rats.

  Authors: Stephan Schneider, Marc-Alexander von Mach, Oliver Kraus, Peter Kann, Peter Johannes Feilen

  Artificial organs. 12/2003; 27(11):1053-6.

  The survival of microencapsulated islets transplanted into the unmodified peritoneal cavity is limited, even if capsular overgrowth is restricted to a minimum, due to an insufficient oxygen supply toThe survival of microencapsulated islets transplanted into the unmodified peritoneal cavity is limited, even if capsular overgrowth is restricted to a minimum, due to an insufficient oxygen supply to the islets. Therefore, research efforts should focus on finding or creating a transplantation site, which permits a closer contact between the encapsulated islets and the blood. For this reason, the liver could be an interesting candidate. The aim of the present study was to test the hypothesis that the intraportal transplantation of allogenic islets encapsulated in small-sized barium alginate beads is safe and succeeds to induce normoglycemia in diabetic rats. The intraportal transplantation of 1,500 islets encapsulated in barium alginate beads leads within 10 h and up to 24 h to blood sugar concentrations below 40 mg/dL, most likely due to an acute cell lysis of the graft. Afterwards, the reappearance of the diabetic state could be detected in these animals. Most likely these findings are induced by a sudden hypoxia to the islets. We believe that the occlusion of small- and medium-sized portal venules by the alginate beads is responsible for this effect. Therefore, in forthcoming studies, barium alginate beads, with a diameter below 350 micro m, stabilized with medical approved additives should be used.

• Biocompatibility of alginates for grafting: impact of alginate molecular weight.

  Authors: Stephan Schneider, Peter J Feilen, Oliver Kraus, Tanja Haase, Tolga A Sagban, Hans-Anton Lehr, J Beyer, Rainer Pommersheim, Mathias M Weber

  Artificial cells, blood substitutes, and immobilization biotechnology. 12/2003; 31(4):383-94.

  Optimising microencapsulation technology towards the effective clinical transplantation has created the need for highly biocompatible alginates. Therefore, in this study the biocompatibility ofOptimising microencapsulation technology towards the effective clinical transplantation has created the need for highly biocompatible alginates. Therefore, in this study the biocompatibility of different beads prepared from alginates with varying average molecular weight was examined. In some experiments the beads were covered with a multilayer membrane surrounded by an alginate layer. First of all, we found that beads made of a lower weight average alginate elicted a much stronger fibrotic response compared to beads made of a higher weight average alginate (LV-alginate > MV-alginate). The results were confirmed by the observation that the extent of tissue fibrosis was significantly increased in multilayer capsules made of an alginate with a lower weight average (core and surface LV-alginate, Mw 0.7-1 * 10(6) g/mol, viscosity of a 0.1% solution 1-2.5 mPa s(-1)) compared to multilayer capsules made of an alginate with a higher weight average (core and surface MV-alginate; Mw 1.2-1.3 * 10(6) g/mol, viscosity of a 0.1% solution 5-7 mPa s(-1)). It should be stressed, that the pro-fibrotic effect of the LV-alginate alginate in the core was only partially reversed by a MV-alginate on the surface of the multilayer capsules. On the basis of the raised data, it can be assumed that the molecular weight average of the alginates have an decisive effect on the biocompatibility. Therefore, it seems to be recommendable to reduce the low molecular weight fractions of the alginate during the purification process to improve the biocompatibility.

• Cryopreservation of islets of Langerhans

  Authors: Jürgen Beyer, Peter J Feilen, Jan G Hengstler, Peter Kann, Marc-Alexander von Mach, Michael Ringel, Stephan Schneider, Josef Schlosser, M. M. Weber, Marcus Weiland, L. Sacha Weilemann