Stephan Schneider

Multilayer capsules: a promising microencapsulation system for transplantation of pancreatic islets

In 1980, Lim and Sun introduced a microcapsule coated with an alginate/polylysine complex for encapsulation of pancreatic islets. Characteristic to this type of capsule is, that it consists of a plain membrane which is formed during a single procedural step. With such a simple process it is difficult to obtain instantly a membrane optimized with respect to all the properties requested for islet transplantation. To overcome these difficulties, it is recommended to build up the membrane in several consecutive steps, each optimized for a certain property. In this study, we have analysed such a multilayer microcapsule for the encapsulation of pancreatic islets. Therefore, empty and islet containing alginate beads were coated with alternating layers of polyethyleneimine, polyacrylacid or carboxymethylcellulose and alginate. By scanning electron microscopy the thickness of the covering multilayer-membrane was estimated to be less than 800 nm by comparison with an apparatus scale. Ellipsometric measurements showed that the membrane thickness is in the range of 145 nm. Neither the encapsulation procedure, nor the membrane-forming step did impede the stimulatory response of the islets. The encapsulation even lead to a significantly better stimulatory response of the encapsulated islets during week three and five of cell culture. Furthermore, the multilayer-membrane did not deteriorate the biocompatibility of the transplanted microcapsules, allowing an easy tuning of the molecular cut-off and the mechanical stability depending on the polycation-polyanion combination used. The multilayer membrane capsule has obvious advantages compared to a one-step encapsulation procedure. These beads guarantee a high biocompatibility, a precisely adjusted cut-off, an optimal insulin-response and high mechanical stability although the membrane is only 145 nm thick.

Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

OBJECTIVE Noninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-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 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 min. 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 β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-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 [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-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 β-cells and enable imaging and quantification in vivo.

A morphometric comparison of dissimilar early development in sibling species of Platynereis (Annelida, Polychaeta)

SummaryEarly development of Platynereis massiliensis was studied in serial sections of fixed embryos and in living or fixed embryos whose nuclei had been made visible with a fluorescent label. The unfertilized egg is an ellipsoid with three axes of differing length. The longest axis corresponds to the dorsoventral axis of the developing embryo. Egg volume is ten times that in the sibling species, P. dumerilii, mainly due to increased yolk content. The timing and spatial pattern of cleavage were observed from first cleavage to the 62-cell stage. Volumes of the blastomeres, their nuclei, their yolk-free cytoplasm and their yolk were determined from serial sections up to the 29-cell stage. In the P. massiliensis embryo, cell cycles are on average 3.7 times longer than in P. dumerilii; volume proportions among the blastomeres also differ and the macromeres containing the bulk of yolk are particularly large, but otherwise the cleavage patterns, differential segregation of yolk and yolk-free cytoplasm, and the histogenetic fates of the blastomeres are the same as in P. dumerilii. This equivalence of cell lineage and of cytoplasmic segregation mechanisms in both species, maintained in spite of the different appearance of the embryos, suggests functional importance of and selective constraint on these developmental features. The relatively accelerated divisions of the 2d cell line in P. massiliensis may be interpreted as the precocious development of cell lines which give rise to adult structures. Several structures, obviously functional in developing P. dumerilii, have lost their function in P. massiliensis: the egg contains few cortical granules, giving rise to only a moderate egg jelly layer in the zygote; prototroch cells develop cilia, but the heavy embryo is unable to swim; the larva develops three pairs of parapodia but, unlike the corresponding stage in P. dumerilii, is not capable of coordinate locomotion. This loss of motility is related to the brooding habit of the species developing inside the parental tube and is explained as the result of a switch from pelagic to benthic, protected reproduction in P. massiliensis.

Size of pancreatic islets of Langerhans: a key parameter for viability after cryopreservation.

Abstract.Large amounts and excellent viabilities of pancreatic islets are prerequisites for recent advances in islet transplantation. Cryopreservation has been shown to enlarge transplanted cell mass, but has been accompanied by reduced viability. In this study rat pancreatic islets were differentiated into small (<200 µm), medium (200–400 µm) and large (>400 µm) categories and their susceptibilities to different freezing conditions were evaluated: concentration of cryoprotectant (0.7–3.1 M), equilibration (15 vs. 45 min, 22° C vs. on ice) and post-thaw removal of cryoprotectant (15 vs. 30 min, stepwise vs. one-step). The most prominent finding was a negative correlation between islet size and viability observed in non-frozen islets to a minor degree (r=-0.44) and significantly enhanced after cryopreservation (r<-0.8). The concentration of cryoprotectant showed the most significant influence on viability affecting small, medium and large islets. Different techniques of equilibration with the cryoprotectant resulted in significant changes of islet viability of medium islets, whereas small and large islets were unaffected. For different techniques of removal of the cryoprotectant, no significant influence on viabilities was found. We conclude that large islets represented a highly susceptible population concerning damage due to cryopreservation.

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

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 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.

Biocompatibility of Alginates for Grafting: Impact of Alginate Molecular Weight

Optimising 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*106 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*106 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.

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

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 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.

Differential expression of cell-cycle regulators in human beta-cells derived from insulinoma tissue.

INTRODUCTION The low frequency of beta-cell replication in the adult human pancreas limits beta-cell regeneration. A better understanding of the regulation of human beta-cell proliferation is crucial to develop therapeutic strategies aiming to enhance beta-cell mass. METHODS To identify factors that control beta-cell proliferation, cell-cycle regulation was examined in human insulinomas as a model of increased beta-cell proliferation (n=11) and healthy pancreatic tissue from patients with benign pancreatic tumors (n=9). Tissue sections were co-stained for insulin and cell-cycle proteins. Transcript levels of selected cell-cycle factors in beta-cells were determined by qRT-PCR after performing laser-capture microdissection. RESULTS The frequency of beta-cell replication was 3.74±0.92% in the insulinomas and 0.11±0.04% in controls (p=0.0016). p21 expression was higher in insulinomas (p=0.0058), and Rb expression was higher by trend (p=0.085), whereas p16 (p<0.0001), Cyclin C (p<0.0001), and p57 (p=0.018) expression levels were lower. The abundance of Cyclin D3 (p=0.62) and p27 (p=0.68) was not different between the groups. The reduced expression of p16 (p<0.0001) and p57 (p=0.012) in insulinomas and the unchanged expression of Cyclin D3 (p=0.77) and p27 (p=0.55) were confirmed using qRT-PCR. CONCLUSIONS The expression of certain cell-cycle factors in beta-cells derived from insulinomas and healthy adults differs markedly. Targeting such differentially regulated cell-cycle proteins may evolve as a future strategy to enhance beta-cell regeneration.

Endosonographie—eine zusätzliche diagnostische Möglichkeit bei der Differenzierung der beiden häufigsten Formen des primären Hyperaldosteronismus@@@Endosonography—an Additional Diagnostic Possibility in the Differentiation between the Two Common Types of Primary Hyperaldosteronism

ZusammenfassungHintergrund:Der primäre Hyperaldosteronismus hat in der Abklärung sekundärer Hypertonieursachen eine bedeutende Rolle. Bei der Diagnostik ist die Unterscheidung zwischen den beiden häufigsten Ursachen einer Mineralokortikoidhypertonie, dem aldosteronproduzierenden Adenom (APA) und dem idiopathischen Hyperaldosteronismus (beidseitige Nebennierenrindenhyperplasie; IHA), wichtig, da beide Formen prinzipiell unterschiedlich behandelt werden.Fallbeschreibung:Berichtet wird über den Fall eines 65-jährigen Patienten mit langjährig bekannter arterieller Hypertonie, bei dem eine suffiziente Blutdruckeinstellung nicht gelang und daher nach sekundären Hypertonieursachen gesucht wurde. In der Diagnostik fielen ein deutlich erhöhter Aldosteron/Renin-Quotient im Serum und erhöhte Werte für Aldosteron und dessen Metaboliten im 24-h-Sammelurin auf. Der daraufhin durchgeführte Salzbelastungstest ergab eine nicht ausreichende Suppression des Aldosterons, der zur differentialdiagnostischen Abklärung durchgeführte Orthostasetest zeigte einen Anstieg des Aldosterons, so dass von einem IHA ausgegangen werden musste. In der Bildgebung mittels MRT ließ sich kein pathologischer Befund der Nebennieren darstellen. Aufgrund der unklaren Konstellation der laborchemischen Tests und der Bildgebung wurde eine Endosonographie durchgeführt, mit welcher sich ein einseitiges Adenom darstellen ließ. Daraufhin konnte der Patient kurativ adrenalektomiert werden.Schlussfolgerung:Die Endosonographie spielt in der Differentialdiagnostik der beiden häufigsten Formen des primären Hyperaldosteronismus eine bedeutendere Rolle als bislang angenommen. Sie bietet eine Hilfestellung bei einer unklaren Konstellation von laborchemischen Tests und der Bildgebung mittels CT oder MRT. Anhand des Fallberichts wird das gegenwärtige diagnostische Vorgehen dargestellt.AbstractBackground: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.