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Engineered fibril solutions from whey protein beta-lactoglobulin are known for their excellent foaming capacity. These amyloid aggregates solutions (AAS) usually contain a polydisperse mixture of different protein structures. An optimized ratio of amyloid (AF, fibrils) and non-amyloid fractions (n-AF) in AAS may improve foaming, particularly by interactions at the air-water interface. Foamability, surface activity, and monolayer phase behavior at the air-water interface of isolated AF and n-AF as well as AAS with different AF/n-AF-ratios were investigated using drop tensiometry, Langmuir trough and foam analysis. N-AF exhibited faster migration, twice as fast adsorption and thus faster spreading at the air-water interface than the fibrils (AF). N-AF required less energy to assemble in a liquid-expanded phase in a monolayer, i.e., they were more compressible in the monolayer than AF. This resulted in rapid stabilization of lamellae in foam. High surface hydrophobicity of AF results in faster adsorption and formation of capillary forces between adsorbed fibrils, improving the attraction for additional fibrils. Orientation of semi-flexible and larger fibrils in AF consumes high energy. In combination with n-AF, the energy needed for orientation and assembly of fibrils is equivalent, however, the yield of AF in AAS was only 20 %, indicating the interplay of amyloid and non-amyloid proteins at the air-water interface. N-AF can be incorporated into a fibrillar film, which increase the network's density and stiffness and the interfacial film stability. Consequently, in AAS fibrils and non-amyloid material acted synergistically at the air-water interface, whereby only small amounts of amyloid aggregates are required to stabilize foams.

Recent studies report the boron-dipyrromethene (BODIPY) moiety to be interesting for caging applications in photopharmacology based on its response to irradiation with wavelengths in the biooptical window. Thus, in a model study, we investigated the meso-methyl-BODIPY caged CDK2 inhibitor AZD5438 and aimed to assess the usability of BODIPY as a photoremovable protecting group in photoresponsive kinase inhibitor applications. Photochemical analysis and biological characterisation in vitro revealed significant limitations of the BODIPY-caged inhibitor concept regarding solubility and uncaging in aqueous solution. Notably, we provide evidence for BODIPY-caged compounds generating singlet oxygen/radicals upon irradiation, followed by photodegradation of the caged compound system. Consequently, instead of caging, a non-specific induction of necrosis in cells suggests the potential usage of BODIPY derivatives for photodynamic approaches.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. Absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. Absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH/NAD+ ratio at reoxygenation. Rather, absence of NDAs led to elevated ROS production while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress. This article is protected by copyright. All rights reserved.

The increasing knowledge about natural functional fibrils has triggered the interest in synthetic or engineered fibrils. Naturally occurring amyloid fibrils (functional and pathogenic) have been analyzed for many years at different structural levels. Engineered fibrils are structurally similar to natural fibrils and the main sub-structural feature of amyloids is characterized by cross-beta structure stabilizing the fibril formation. However, a number of peculiarities exist comparing natural and engineered fibrils that may affect their analysis, especially in spectroscopic and scattering methods. For this reason, several methods that are commonly used for natural fibril analysis are presented and particularities for their application in the characterization of engineered fibrils are described. In addition, the understanding about structure–function relation of fibrils studied in the different research areas may mutually improve when using the same analytical approaches for natural and engineered fibrils.

When carrier systems like solid lipid nanoparticles (SLN) are added to a protein rich food matrix adsorption of protein to the particles alters the surface properties of SLN which in turn can alter the properties of the whole system. Thus, the effect of the SLN composition on the protein adlayer (protein corona) is important to understand. The adsorption of β-lactoglobulin (βLG) to unloaded SLN and SLN loaded with ferulic acid or tocopherol was studied at pH 5.7 and particle:protein ratios of 2:1 to 10:1 (w/w) by centrifugation, AF4 and ultrafiltration (UF). Up to 10% of the βLG was strongly bound to SLN whereas 40–80% of the βLG formed a loose adlayer. The amount of bound βLG was increased by the presence of tocopherol and decreased by the presence of ferulic acid. The adsorbed protein layer thus depends on SLN characteristics governed by encapsulated compounds.

Site-directed spin labeling (SDSL) of natural β-lactoglobulin (β-lg) was established. Combined electron paramagnetic resonance (EPR) and mass spectrometric analysis following tryptic digestion demonstrated that spin labels bind site-specifically but are not directed to all five cysteine residues to various preferred and reproducible extents. MTSSL and iodoacetamido-proxyl spin label (IPSL) were 80 and 60% reliably bound to the H strand, respectively, and combined in one spectral component and buried in the protein core. After heat incubation at pH 2 and fractionation, all labeled side chains (peptides) were part of the amyloid and non-amyloid fractions, even if they could not detect amyloid structures. It was assumed that the IPSL-labeled side chains of peptides with Cys160 from random coil were incorporated into small non-amyloid aggregates in non-polar environments. After heating at pH 3.5, a rearrangement of the previous α-helix was assumed to shift from the autonomous folding domain during partial unfolding, which improved the accessibility of β-sheets to the water/DMSO-environment. β-sheets were likely densely packed by the accumulation of intermolecular β-sheets, which suggests that amyloid-like structures can be formed from building blocks of the entire primary β-lg structure. Double electron-electron resonance (DEER) confirmed that the spatial distribution of labels within the amyloid-like fraction in a one-dimensional arrangement of the entire protein aggregates was similar to a string of pearls. Thus, SDSL of proteins containing several cysteine residues can be used to gain deep insights into the aggregation mechanism of proteins under food processing conditions.

Site-directed spin labeling (SDSL) of the natural food protein β-lactoglobulin (β-lg) was established with the aim of characterizing amyloid aggregation while explicitly avoiding the usual manipulation of primary protein structure. For its successful application, spin labels must not alter secondary protein structure or the formation of β-lg amyloid aggregates. The two spin labels—the MTSSL (flexible S–S binding) and Iodoacetamido-proxyl spin label (IPSL) (more rigid C–S binding)—were used for amyloid aggregation at pH 2 and pH 3.5. At pH 3.5, IPSL caused minor changes in the secondary protein structure, where it reduced intra- and intermolecular β-sheets as determined by ATR-FTIR. Analysis of the extent of amyloid aggregation using thioflavin T fluorescence indicated that the spin probes interfered with the binding of the fluorescent probes to the β-sheets. Non-amyloid and amyloid fractions were obtained from the amyloid aggregated system by ultrafiltration (300 kDa), which also proved to be equivalent and independent of the spin labeling process. Atomic force microscopy and size-exclusion chromatography results suggest the same building blocks and morphologies between unlabeled and spin-labeled proteins; therefore, substantial changes in the behavior of β-lg during amyloid aggregation can be excluded. Ultimately, 10–17% of the β-lg molecules were labeled so that the SDSL approach can be used to label the natural food protein at these low concentrations without affecting protein conformation or the formation of amyloid aggregates, which may subsequently provide deep insights into the aggregation mechanism of food proteins under processing conditions.

The mechanical stability of interfacial protein films is limited through the intermolecular interaction of the adsorbed proteins. Since the intermolecular interactions vary in dependence of the protein structure, the investigation of intentionally induced structural modifications (i.e., through high hydrostatic pressure) of interfacial active proteins is required to mechanistically understand their structure-function-relationship. The aim of this study was to link the structural analysis of pressure-treated whey protein β-lactoglobulin at the oil/water-interface with the formation of the interfacial protein film and the resulting film stability against mechanical stress. For this purpose, we treated β-lactoglobulin in water at pH 7 with hydrostatic pressures of 600 MPa for 10 min at 20 °C and analyzed the protein structure in oil/water-emulsion with Fourier-transform-infrared-spectroscopy, extrinsic fluorescence, and ζ-potential. We characterized the molecular density and interfacial film properties via Langmuir trough analysis, electron-paramagnetic-resonance-spectroscopy, interfacial shear and dilatational rheology. The structural flexibility of pressure-treated β-lactoglobulin favored its unfolding and a fast interfacial protein film formation that resulted in pronounced intermolecular interactions, including van der Waals interactions, hydrophobic associations and disulfide bonds. The high interfacial molecule density combined with many and strong intermolecular interactions stabilized the pressure-treated protein film against shear deformation and small dilatational deformation. However, against high dilatational deformation, the pressure-treated β-lactoglobulin film showed a lower stability due to a slow migration towards unoccupied interfacial area. Hence, our research contributes to the control of the mechanical protein film stability, and thus the colloidal stability of emulsions in dependence of the protein structure at the oil/water-interface.

Background: Depending on environmental conditions, almost all proteins can form amyloid and amyloid-like aggregates that have unique functional properties. This opens numerous applications for designed aggregates in materials, medical and food applications. However, it is poorly understood how the amyloid (-like) aggregation and their resulting morphology is induced or influenced by various environmental and processing conditions. Scope and approach: We identified and summarized conditions under which amyloid (-like) aggregates are formed and their impact on aggregate morphology. The focus is on β-lactoglobulin, but generic effects on other proteins are discussed, in order to elucidate common mechanistic properties. Key findings and conclusions: The flexibility of linear aggregates can be evaluated by comparing the persistence (Lp) and contour length (i.e., length when completely stretched; Lc). Shorter and more flexible amyloid-like aggregates (Lp < Lc) usually occur from a relatively fast assembly (e.g., low repulsion, high concentration, solvents). Longer, semi-flexible amyloid aggregates (Lp ∼ Lc) based on fibrillization-prone peptides that are slowly formed and assembled (e.g., high repulsion, low concentration). In either case, the aggregation kinetics increases through protein destabilization (e.g., heating, zinc addition, solvent and hydrolysis effects) and decreases through stabilization (e.g., glycerol addition). Post-processing (e.g., mechanical or interfacial stress) fragments aggregates into stiffer rods (Lp > Lc). Semi-flexible morphologies can align in liquid crystalline phases or interact with linear polysaccharides; while flexible aggregates can entangle. This allows for various possibilities to build higher order fibril or hybrid networks for various applications, such as bundles, coatings/films, or gels. This knowledge is crucial to produce specific morphologies for applications and to draw conclusions about how morphologies will be affected during processing (e.g., shearing).

Edible solid lipid nanoparticles (SLN) stabilized by a mixture of food grade emulsifiers (soy bean lecithin, Tween 20, sucrose stearate) were added to o/w emulsions previously stabilized by anionic SDS, non-ionic Tween 20 or cationic CTAB. The aim of the study was to understand the fate of both SLN and oil droplets in these mixtures focusing on the impact of the surfactant used to stabilize the emulsion. The presence of SLN in emulsions led to increased emulsion stability as reflected by droplet size measurements and accelerated creaming experiments. This could be attributed to an increase in the viscosity of the sample, but also to changed properties of the o/w interface. Zeta potential measurements revealed that the surfactant composition at the o/w interface had changed in SDS and CTAB stabilized but not in Tween 20 stabilized emulsions. SLN remained detectable in the continuous phase of each emulsion system over three weeks of storage but were not detected at the o/w interface of oil droplets. The particle size of the SLN remained unchanged whereas their zeta potential increased in SDS and CTAB stabilized emulsions to a similar magnitude (+/− 68 mV) but opposite signs. The melting temperature and melting enthalpy of SLN decreased in emulsions indicating that part of the lipid matrix was dissolved by oil from the emulsion. Accordingly, a time-dependent transfer of crystalline triglycerides originating from the SLN into the oil phase of separated model emulsion systems was verified.

The release and partitioning of encapsulated compounds from solid lipid nanoparticles (SLN) to other matrix constituents was investigated in o/w emulsions stabilized by different surfactants (CTAB, SDS, Tween 20) by confocal laser scanning microscopy (CLSM) and electron paramagnetic resonance spectroscopy (EPR) spectroscopy. CLSM images revealed that the encapsulated probe Coumarin 6 (C6) was located in the oil droplets after SLN were mixed with the different emulsions. The EPR spin probe TEMPOL‐benzoate (TB) showed different dynamics as a function of its specific solubilization sites in SLN. Four different populations of the spin probe could be separated giving the smallest population in the aqueous environment and the major population at the SLN interface. Two further populations were associated with differently crystalized lipid environments. This implies that both loosely structured regions with α- and β’-polymorphs and environments of β-polymorphs are present in SLN. In separated model emulsion systems, a prolonged release of TB from SLN was evident which predominately originated from the SLN interface. The released TB partitioned into the oil phase (25%) and into the o/w interface (30–45%), regardless of the presence or absence of additional surfactant micelles. Hence, these edible SLN may be used as delivery system for bioactive compounds in food emulsions with low impact of the surfactant used to stabilize the emulsion. The partial redistribution of the encapsulated compounds should be taken into account, but can also offer benefits when a prolonged release of lipophilic additives is desired.

Photosynthetic electron flow, driven by photosystem I and II, provides chemical energy for carbon fixation. In addition to a linear mode a second cyclic route exists, which only involves photosystem I. The exact contributions of linear and cyclic transport are still a matter of debate. Here, we describe the development of a method that allows quantification of electron flow in absolute terms through photosystem I in a photosynthetic organism for the first time. Specific in-vivo protocols allowed to discern the redox states of plastocyanin, P700 and the FeS-clusters including ferredoxin at the acceptor site of PSI in the cyanobacterium Synechocystis sp. PCC 6803 with the near-infrared spectrometer Dual-KLAS/NIR. P700 absorbance changes determined with the Dual-KLAS/NIR correlated linearly with direct determinations of PSI concentrations using EPR. Dark-interval relaxation kinetics measurements (DIRKPSI) were applied to determine electron flow through PSI. Counting electrons from hydrogen oxidation as electron donor to photosystem I in parallel to DIRKPSI measurements confirmed the validity of the method. Electron flow determination by classical PSI yield measurements overestimates electron flow at low light intensities and saturates earlier compared to DIRKPSI. Combination of DIRKPSI with oxygen evolution measurements yielded a proportion of 35% of surplus electrons passing PSI compared to PSII. We attribute these electrons to cyclic electron transport, which is twice as high as assumed for plants. Counting electrons flowing through the photosystems allowed determination of the number of quanta required for photosynthesis to 11 per oxygen produced, which is close to published values.

The formation of short-lived and stable radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy and compared with hydroperoxides and hexanal in complex starch-protein-lipid model systems, as well as in corn extrudates. Stable radicals were detected directly in ground samples. Short-lived lipid radicals were measured ex situ in ethyl acetate extracts of model systems and extrudates by the use of the spin trap PBN. Significant adduct formation was found after 30 min at 50 °C. During storage, lipid radicals (PBN adducts) increased in model systems. Simulation of EPR spectra from bulk oil demonstrated that mainly alkoxyl radical adducts were detected, to which rapidly decomposing peroxyl radical adducts also contributed. Stable radicals in extrudates were attributed to protein radicals based on g-value of 2.00467 compared with 2.00474 found in model system prepared with zein. The signal intensity of the stable radical remained constant during storage, but increased during extrusion.

Solvent-induced fibrillar aggregates of beta-lactoglobulin occur only in a certain balance between hydrophobic forces and electrostatic interactions. We hypothesize, that different hydrophobic solvent molecules as well as rising temperatures influence this equilibrium and thus the optimum to produce amyloid aggregates. Dimethyl sulfoxide (DMSO), methanol and ethanol all resulted in polydisperse solutions with worm-like and spherical-aggregates, albeit to different degrees: the volume fraction required for aggregation was DMSO (50%) > methanol (40%) > ethanol (30%) which does not reflect their hydrophobicity. Further solvent addition decreased the fibrillar aggregation again. Increasing the temperature by 10–20 K decreased the solvent concentration needed to induce amyloid-like aggregates. A targeted production of solvent-based amyloid-like aggregates is therefore not only dependent on the hydrophobicity of the solvents, but also on their direct interaction with the protein (denaturation).

Significance Flooding due to extreme weather events can be highly detrimental to plant development and yield. Speedy recovery following stress removal is an important determinant of tolerance, yet mechanisms regulating this remain largely uncharacterized. We identified a regulatory network in Arabidopsis thaliana that controls water loss and senescence to influence recovery from prolonged submergence. Targeted control of the molecular mechanisms facilitating stress recovery identified here could potentially improve performance of crops in flood-prone areas., Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

Abiotic stresses in plants are often transient and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG (RBOHD), SENESCENCE-ASSOCIATED GENE113 (SAG113) and ORESARA1 (ORE1/NAC6) which function in a regulatory network involving a reactive oxygen species (ROS) burst upon de-submergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to pre-stress homeostasis.Significance statementFlooding due to extreme weather events can be highly detrimental to plant development and yield. Speedy recovery following stress removal is an important determinant of tolerance, yet mechanisms regulating this remain largely uncharacterized. We identified a regulatory network in Arabidopsis thaliana that controls water loss and senescence to influence recovery from prolonged submergence. Targeted control of the molecular mechanisms facilitating stress recovery identified here can potentially improve performance of crops in flood-prone areas.

In this study valorization of cocoa bean husk (Theobroma cacao L.), common poppy (Papaver somniferum L.) and industrial hemp (Cannabis sativa L.) as frequent waste and under-utilised raw materials was proposed by a recovery process of their macrostructural and bioactive compounds and their utilization in the formulation of alginate-based hydrogel particulate delivery systems. Compositional analysis of the raw materials was performed and a simple routine for simultaneous recovery of both macrostructural and bioactive fractions developed by sequential, solvent extraction and alkaline extraction-isoelectric precipitation at two different extraction temperatures (20 °C and 70 °C). An innovative approach to utilization of both recovered macrostructural and bioactive isolates in combination with alginate was examined, aiming to formulate particulate delivery systems constituted from the macrostructural fractions as the carrier adjuncts and encapsulating the produced bioactive extracts of used raw materials. Macrostructural isolates obtained at higher extraction temperature (70 °C) were characterized by better functional properties, i.e. higher recovery yields (

International audience; Allyl isothiocyanate (AITC) is a small electrophilic molecule which can be found in cabbage after degradation of glucosinolates. The covalent attachment of AITC to whey protein isolate (WPI) was previously reported to increase their hydrophobicity and structural flexibility at acidic pH values. It is thus hypothesized, that the o/w interface adsorption behaviour and interfacial structure will be altered. To further understand the effect of the AITC-modification on the emulsifying capacity, adsorption kinetic and interfacial properties of unmodified and modified WPI were investigated at the o/w interface. The WPI-modification resulted in a significantly increased surface adsorption kinetic and a lower equilibrium interfacial tension at acidic pH values. The ratio of α-lactalbumin (ALA) and β-lactoglobulin (BLG) at the oil droplet surface differed between unmodified and modified WPI (modBLG > ALA+modALA). Several layers of loosely attached proteins were evident on the oil droplet surface in all modified WPI emulsions. The hyperfine coupling (a(N)) of the EPR spin probe TB residing at the oil droplet surface reflected an increased hydrophobicity of the modified proteins. A lower order parameter (S) in the lipid phase of the modified WPI emulsions gave evidence of an altered alignment of the modified proteins at the interface, probably sticking into the oil phase. In conclusion, the present results indicate that the increased flexibility and hydrophobicity of the modified whey proteins, especially of modified BLG, surpass the surface activity of unmodified ALA in acidic pH values.

Liposomes are suitable for encapsulating lipophilic bioactive compounds, enhancing compound solubility, stability and bioavailability. To enhance physical stability of liposomes in food-like matrices they were coated with positively charged whey protein isolate (WPI). WPI concentration, for a successful coating, was optimised by dynamic light scattering (DLS) and zeta potential measurements. Membrane properties of coated and uncoated vesicles were investigated by electron paramagnetic resonance (EPR) with site-directed and non-site-directed spin probes. Coexistence of two or three simulated spin probe populations indicated a less fluid membrane and higher concentration of water molecules in the phosphate/glycerol moiety with WPI coating. This relies on the insertion of WPI into the membrane, which is favoured by the molten globule state under investigated acidic conditions. Physical stability of liposomes benefits from WPI coating, as indicated by prolonged shelf-life, cancellation of osmotic effects in the presence of salts or sugars and a lower sensitivity towards low pH values during in vitro gastric digestion.

Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies during the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit widespread application of these strategies. In this manuscript, we present an experimental study using a bioartificial pancreas device for the transplantation of xenogeneic islet without affecting the immune system in nonhuman primates. We could demonstrate stable graft function and adequate glucose-regulated insulin secretion without the need for immunosuppressive medication. This strategy opens up new avenues for more widespread and safe application of various cell-based therapies.

For the majority of patients with type 1 diabetes intensive insulin therapy is effective and safe for maintaining glycemia and minimizing diabetes-associated complications. However, a rare number of patients show highly labile metabolic control and experience repeated and unpredictable hypoglycemic episodes. Such condition is often caused by defective counterregulatory mechanisms and autonomous neuropathy. Patients are at high risk for severe acute and chronic complications, and quality of life is considerably impaired. For this small subset of patients, restoration of endogenous insulin secretion can substantially improve metabolic control and quality of life. In our experience, this is irrespective of insulin independency. Here, we report on our 5 years' experience with implementing islet transplantation as a potential treatment option for type 1 diabetes. All patients were treated by long-term insulin pump therapy prior to enrolment. The main indication was severely unstable diabetes and repeated hypoglycemia. From 2008 to 2013, 10 patients have been transplanted with single islet infusion; mean follow-up time was 35 months. All patients show persistent graft function, stable glycemic control with a reduction in HbA1c in the absence of hypoglycemia. All patients are kept on minimal exogenous insulin. In conclusion, islet transplantation can be an excellent therapy for selected patients. Key prerequisite for success is a strict indication. The primary goal for islet transplantation should be stabile glycemia and prevention of hypoglycemia rather than insulin independence. In fact, maintaining minimal exogenous insulin may protect the islet graft from metabolic stress and even prolong islet graft function.

Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies over the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit a widespread application of these strategies. In this paper we present the clinical success of using a bioartificial pancreas for the transplantation of insulin-producing islets without affecting the immune system. In a patient with long-standing type-1 diabetes we could demonstrate persistent graft function and regulated insulin secretion without the need for immune-modulating medication. This strategy opens up avenues for more widespread and safe application of various cell-based therapies.

BACKGROUND The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT). METHODS Goettingen minipigs of younger age (1 year) and retired breeder animals (3.5 years) were studied. Pancreata were harvested according to the standards of human organ retrieval including in situ cold perfusion with either Custodiol$^{®}$ -HTK or Belzer$^{®}$ UW solution. Pancreatic tissue was characterized by quantification of apoptotic cells. Islet isolations were performed according to a modified Ricordi method, and isolation outcome was assessed by determining islet particle numbers (IP), islet equivalents (IEQ), and isolation factor (IF). Isolated islets were cultured for 24 and 48 h for the assessment of in vitro survival. RESULTS Islet viability was significantly higher in Custodiol$^{®}$ -HTK preserved pancreas organs compared to Belzer$^{®}$ UW. Furthermore, organs harvested from retired breeder preserved in Custodiol$^{®}$ -HTK resulted in stable islet isolation yields even after prolonged CIT and showed superior survival rates of islets in vitro compared to the Belzer$^{®}$ UW group. Younger porcine donor organs resulted generally in lower islet yield and survival rates. CONCLUSIONS In summary, Custodiol$^{®}$ -HTK solution should be preferred over Belzer$^{®}$ UW solution for the preservation of pancreata from porcine origin. Custodiol$^{®}$ -HTK allows for maintaining islet viability and promotes reproducible isolation outcome and survival even after longer CIT. The usage of retired breeder animals over young animals for islet isolation is highly advisable to yield high quality and quantity.

Quillaja saponin extract comprises both, surfactants and phenolic compounds, which makes it interesting, in particular, for the formulation of sensitive functional food ingredients and its protection against oxidation. The aim of this study was to investigate the antioxidant effect of Quillaja saponin extract in oil/water emulsions. Emulsions stabilised by Quillaja saponin showed decreased oxidation stability due to naturally occurring metals but stability increased to a great extent when a chelating agent was added. Antioxidant efficiency of the saponin extract was determined photometrically by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay and by the use of electron paramagnetic resonance spectroscopy (EPR). EPR spectroscopy applying stable hydrophilic and hydrophobic radicals is advantageous, especially for characterisation of antioxidant efficiency at the interface. The extract showed antioxidant activity towards radicals in both environments, aqueous and hydrophobic, indicating the importance of phenolic compounds for the antioxidant properties of Quillaja saponin extract and their presence at the interface facilitated by saponin molecules.

slet cell hormone release is modulated by signals from endothelial and endocrine cells within the islet. However, models of intra-islet vascularization and paracrine cell signaling are mostly based on rodent pancreas. We here assessed for the first time the architecture and endocrine cell interaction of the vascular network in unperturbed human islets in situ and their potential to re-establish their endogenous vascular network after transplantation in vivo. We prepared pancreas tissue slices of fresh tissue obtained from non-diabetic patients undergoing partial pancreatectomy. In addition, we transplanted human donor islets into the anterior chamber of the mouse eye. Next, we performed three-dimensional in situ and in vivo imaging of islet cell and vessel architecture at cellular resolution and compared our findings to mouse and porcine islets. Our data reveal a significantly different vascular architecture with decreased vessel diameter, reduced vessel branching and shortened total vessel network in human compared to mouse islets. Together with the distinct cellular arrangement in human islets this limits beta-endothelial cell interactions, facilitates connection of alpha and beta cells and promotes the formation of independent beta cell clusters within islets. Furthermore, our results show that the endogenous vascular network of islets is significantly altered after transplantation in a donor-age related mechanism. Thus, our study provides new insight into vascular architecture and cellular arrangement of human islets with apparent consequences for intercellular islet signaling. Moreover, our findings suggest that human islet engraftment after transplantation can be improved by the use of alternative, less mature islet cell sources.

Background Safe and reliable diabetes models are a key prerequisite for advanced preclinical studies on diabetes. Chemical induction is the standard model of diabetes in rodents and also widely used in large animal models of non-human primates and minipigs. However, uncertain efficacy, the potential of beta-cell regeneration, and relevant side effects are debatable aspects particularly in large animals. Therefore, we aimed to evaluate a surgical approach of total pancreatectomy combined with splenectomy for diabetes induction in an exploratory study in Goettingen minipigs. Methods Total pancreatectomy was performed in Goettingen minipigs (n = 4) under general anesthesia and endotracheal intubation. Prior to surgery, a central venous line was established for drug application and blood sampling. After median laparotomy, splenectomy was performed and the lobular pancreas was carefully dissected with particular attention to the duodenal vascular arcade. Close monitoring of blood glucose was initiated immediately after surgery by standard glucometer measurement or continuous glucose monitoring systems (CGMS). Exogenous insulin was given by multiple daily subcutaneous (s.c.) injections or via insulin pump systems (CSII). Complete endogenous insulin deficiency was confirmed by intravenous glucose tolerance test (ivGTT) and measurement of c-peptide. For establishing a suitable regimen for diabetes management, the animals were followed for 4-6 weeks. Results Following pancreatectomy and splenectomy, the animals showed a quick recovery from surgery and initial analgetic medication and volume substitution could be terminated within 24 h. A rapid increase in blood glucose was observed immediately following pancreatectomy necessitating insulin therapy. The induced exocrine insufficiency did not cause any clinical symptoms. Complete insulin deficiency could be confirmed in all animals by determination of negative c-peptide during glucose challenge. The two regimen of insulin treatment (multiple daily injections (MDI) and continuous subcutaneous insulin infusion (CSII)) were both feasible with respect to acceptable glycemic control whereas CSII was considerably advantageous in comfort and popularity for both animals and care takers. Conclusions Surgical pancreatectomy in combination with splenectomy to facilitate access to the pancreas is a feasible model for efficient diabetes induction in minipigs. The procedure itself and postoperative animal care could be performed without complications in this exploratory study. Nevertheless, this approach requires well-equipped infrastructure, experienced and skilled surgeons and anesthesiologists and dedicated animal care takers. The impact of total pancreatectomy in combination with splenectomy on the digestive and immune system must be considered in the design and definition of end points of experimental diabetes and transplantation studies.

SummaryPlatelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood–islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.Supplementary Material to this article is available online at www.thrombosis-online.com.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing β-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11β-HSD) isoforms; 11β-HSD–type-1 and 11β-HSD–type-2. We demonstrated expression of mRNA for 11β-HSD-1 and 11β-HSD-2 and protein for 11β-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11β-HSD-1 and up-regulating 11β-HSD-2. The 11β-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11β-HSD-1 enzyme activity or increased 11β-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen-permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma-derived cells (GOS-3) and rat pheochromacytoma cells (PC12) were cultured on the gas-permeable PTFE foil as well as a conventional non oxygen-permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

Islet transplantation as a biological β-cell replacement therapy has emerged as a promising option for achieving restoration of metabolic control in type 1 diabetes patients. However, partial or complete loss of islet graft function occurs in relatively short time (months to few years) after implantation. The high rate of early transplant dysfunction has been attributed to poorly viable and/or functional islets and is mediated by innate inflammatory response at the intravascular (hepatic) transplant site and critical lack of initial nutrient/oxygen supply prior to islet engraftment. In addition, the diabetogenic effect of mandatory immunosuppressive agents, limited control of alloimmunity, and the recurrence of autoimmunity limit the long-term success of islet transplantation. In order to abrogate instant blood-mediated inflammatory reaction and to provide oxygen supply for the islet graft, we have developed an extravascular (subcutaneous) transplant macrochamber (the 'βAir' device). This device contains islets immobilized in alginate, protected from the immune system by a thin hydrophilized teflon membrane impregnated with alginate and supplied with oxygen by daily refueling with oxygen-CO (2) mixture. We have demonstrated successful utilization of the oxygen-refueling macrochamber for sustained islet viability and function as well as immunoprotection after allogeneic subcutaneous transplantation in healthy minipigs. Considering the current limitations of intraportal islet engraftment and the restricted indication for islet transplantation mainly due to necessary immunosuppressive therapy, this work could very likely lead to remarkable improvements in the procedure and moreover opens up further strategies for porcine islet cell xenotransplantation.

Whole organ pancreas and pancreatic islet transplantation are currently the only forms of clinically available β-cell replacement. Both therapeutic options can provide good glycemic control and prevention or stabilization of diabetic complications, but at the price of permanent immunosuppression. Therefore, the indication for transplantation of type 1 diabetes patients must be balanced carefully and should be restricted to a subgroup of patients with extreme lability of metabolic control and frequent hypoglycemia despite optimal medical therapy.

Aims The present study was designed to test the hypothesis that NADPH oxidase inhibition with apocynin would lower blood pressure and improve endothelial function in spontaneously hypertensive rats (SHRs). Although apocyin effectively dilated arterial segments in vitro , it failed to lower blood pressure or improve endothelial function. Further experiments were performed in normotensive rats and in NADPH oxidase subunit knock-out mice to test if apocynin-induced vasodilation depends on NADPH oxidase inhibition at all. Methods and results SHRs were treated with apocynin orally or i.v. Arterial pressure was recorded directly. Rat and mouse arterial function was investigated in vitro by small vessel wire myography. NADPH oxidase activity was measured in human granulocytes and in rat vascular preparations. Rho kinase activity was determined by Western blot analysis. Apocynin did not reduce arterial pressure acutely in SHR when given at 50, 100, or 150 mg kg−1 day−1 orally over 1-week intervals or when given i.v. Apocynin potently inhibited granulocyte NADPH oxidase but not vascular NADPH-oxidase-dependent oxygen radical formation unless exogenous peroxidase was added to vascular preparations. Apocynin dilated rat intrarenal and coronary arteries independently of pharmacological interventions that reduce vascular superoxide radical abundance and actions. Aortic rings from p47phox−/− mice were more sensitive to apocynin-induced dilation than wild-type aortic rings. Rho kinase inhibition reduced or prevented the inhibitory effect of apocynin on agonist-induced vasoconstriction and apocynin inhibited the phosphorylation of Rho kinase substrates. Conclusion Apocynin per se does not inhibit vascular NADPH-oxidase-dependent superoxide formation. Its in vitro vasodilator actions are not due to NADPH oxidase inhibition but may be explained at least in part by inhibition of Rho kinase activity. The discrepancy between apocynin-induced vasodilation in vitro and the failure of apocynin to lower arterial pressure in SHR suggests opposing effects on arterial pressure-regulating systems in vivo . Its use as a pharmacological tool to investigate vascular NADPH oxidase should be discontinued.

We have developed a luminol-based assay using intact islets, which allows for quantification of reactive oxygen species (ROS). In addition, an index capable of characterizing metabolic and mitochondrial integrity prior to transplantation was created based on the capacity of islets to respond to high glucose and rotenone (mitochondrial respiratory chain complex I inhibitor) by production of ROS. To validate this assay, lipid peroxidation and antioxidative defense capacity were evaluated by detection of malondialdehyde (MDA) levels and glutathione peroxidase activity (GPx), respectively. Also, flow cytometric analyses of ROS (dihydroethidine), apoptosis (Annexin V, active caspases), necrosis (Topro3), and mitochondrial membrane potential (JC-1) were done in parallel to correlate with changes in luminol-measured ROS. ATP/ADP ratios were quantified by HPLC and the predictive value of ROS measurement on islet functional potency was correlated with capacity to reverse diabetes in a streptozotocin-induced diabetic NOD.scid mouse model as well as in human transplant recipients. Our data demonstrate that levels of ROS in islets correlate with the percentage of apoptotic cells and their functional potency in vivo. The ROS indices following glucose and rotenone exposure are indicative of metabolic potency and mitochondrial integrity and can be used as surrogate markers to evaluate the quality of islets prior to transplantation.

Hypothesis The partitioning behavior of paramagnetic nitroxides in dispersed systems can be determined by deconvolution of electron paramagnetic resonance (EPR) spectra giving equivalent results with the validated methods of ultrafiltration techniques (UF) and pulsed-field gradient nuclear magnetic resonance spectroscopy (PFG-NMR). Experiments The partitioning behavior of nitroxides with increasing lipophilicity was investigated in anionic, cationic and nonionic micellar systems and 10 wt% o/w emulsions. Apart from EPR spectra deconvolution, the PFG-NMR was used in micellar solutions as a non-destructive approach, while UF based on separation of very small volume of the aqueous phase. Findings As a function of their substituent and lipophilicity, the proportions of nitroxides that were solubilized in the micellar or emulsion interface increased with increasing nitroxide lipophilicity for all emulsifier used. Comparing the different approaches, EPR deconvolution and UF revealed comparable nitroxide proportions that were solubilized in the interfaces. Those proportions were higher than found with PFG-NMR. For PFG-NMR self-diffusion experiments the reduced nitroxides were used revealing a high dynamic of hydroxylamines and emulsifiers. Deconvolution of EPR spectra turned out to be the preferred method for measuring the partitioning behavior of paramagnetic molecules as it enables distinguishing between several populations at their individual solubilization sites.

Unsaturated soy phosphatidyl choline (PC) liposomes were systematically analyzed for chemical and physical stability and for influence on membrane fluidity, when quercetin and fish oil were encapsulated. The physical stability of liposomes was lowered with loading, which is mainly due to fish oil leakage. While fish oil did not induce oxidative acceleration, quercetin did not reduce lipid-derived radical formation but it did inhibit hexanal formation. It also showed no relevant effects on membrane fluidity, polarity or partitioning of the spin probe TEMPOL-benzoate (TB), as proved by EPR measurements and simulation. However, increasing concentration of fish oil in a membrane might increase the acyl chain dynamics and therefore apply a more attractive environment for TB. In contrast to the encapsulates increasing fluidity of saturated membranes by disturbing the lipid packing, membrane properties of unsaturated systems with a Tm below 0 °C were not influenced by encapsulation of quercetin or fish oil.

VEGF-C and VEGF-D are lymphangiogenic factors thatbind to and activate VEGFR-3, a fms-like tyrosine kinasereceptor whose expression is limited almost exclusively tolymphatic endothelium in the adult. Processed forms ofVEGF-C and VEGF-D can also activate VEGFR-2, a keyplayer in the regulation of angiogenesis. There is increasingevidence to show that these receptor–ligand interactionsplay a pivotal role in a number of pathological situations.Inhibition of receptor activation by VEGF-C and VEGF-Dcould therefore be pharmaceutically useful. Furthermore,to understand the different roles of VEGF-C, VEGF-D,VEGFR-2 and VEGFR-3 in pathological situations it willbe necessary to dissect the complex interactions of theseligands and their receptors. To facilitate such studies wecloned, sequenced and characterized the expression ofrat VEGF-C and VEGF-D. We showed that Cys152!Sermutants of processed rat VEGF-C can activate VEGFR-3but not VEGFR-2, while the corresponding mutation inrat VEGF-D inhibits its ability to activate both VEGFR-2and VEGFR-3. We also synthesized and characterizedindolinones that differentially block VEGF-C- and VEGF-D-induced VEGFR-3 kinase activity compared to thatof VEGFR-2. These tools should be useful in analysingthe different activities and roles of VEGF-C, VEGF-D andtheir ligands, and in blocking VEGFR-3-mediatedlymphangiogenesis.Keywords: VEGFR-3; VEGF-C; VEGF-D; lymphangio-genesis; inhibitors.The new growth of lymphatic endothelium (LE) is termedlymphangiogenesis. This occurs after tissue injury [1,2] orobstruction or damage of lymphatic vessels [3] and serves toreduce the increased tissue pressure associated with edemaand inflammation [4,5]. It has recently emerged that a fms-like tyrosine kinase receptor called VEGFR-3/Flt4 and itsligands are key players in the molecular regulation oflymphangiogenesis [6].In the adult, VEGFR-3 is expressed virtually exclusivelyon LE [7,8], and therefore serves as a useful marker for thesecells.VEGF-CandVEGF-D,theknownligandsforVEGFR-3[9–12], are members of the VEGF/PDGF family, and arevirtually identical in their molecular properties. They areprogressively processed during their biosynthesis to removethe N- and C-terminal ends of the protein, leaving the centralVEGF homology domain (VHD) [13,14]. This processingprogressively increases the affinity of VEGF-C and VEGF-Dfor VEGFR-3. After binding to VEGF-C and VEGF-D,VEGFR-3 is capable of transducing signals that can triggerthe proliferation of VEGFR-3-expressing cells in vitro and invivo [11,15–17]. Blocking of ligand-induced VEGFR-3activation inhibits the formation of lymphatic vessels in thedeveloping embryo [18]. These data demonstrate a role forVEGFR-3 and its ligands in regulating lymphangiogenesis.In addition to stimulating lymphangiogenesis, VEGF-Cand VEGF-D can also contribute to angiogenesis. The fullyprocessed forms of VEGF-C and VEGF-D have been demon-strated to activate VEGFR-2, although they are less potentactivators of VEGFR-2 than they are of VEGFR-3 [12–14].For example, transgenic expression of VEGF-C and VEGF-Din the skin of mice stimulated proliferation of LE in thedermis, but did not result in new blood vessel growth [15,17].Moreover, when recombinant VEGF-C was placed on thechick chorioallantoic membrane only lymphangiogenesis wasstimulated [16]. In contrast, VEGF-C induces angiogenesis inthe context of tissue ischaemia [19], in the cornea [20] and inan in vitro angiogenesis assay [21]. VEGF-D also inducesangiogenesis [22]. Thus the context of expression of VEGF-Cand VEGF-D may be critical in determining the angiogenicresponse.

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.