DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1

In between the period of transplantation and revascularization, pancreatic islets are exposed to low-oxygen and low-nutrient conditions. In the present study we mimicked those conditions in vitro to study the involvement of different cell death processes, release of danger-associated molecular patterns (DAMP), and associated in vitro immune activation. Under low-oxygen and low-nutrient conditions, apoptosis, autophagy and necroptosis occur in human islets. Necroptosis is responsible for DAMP-release such as dsDNA, uric acid, and HMGB1. The sensors of the innate immune system able to recognize these DAMPs are mainly TLR, NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule MyD88, we were able to show that the islet-derived DAMPs signal mainly via TLR. Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules. Another effective strategy was suppressing necroptosis using the inhibitor nec-1. Although the effect on cell-survival was minor, nec-1 was able to reduce the release of HMGB1 and its associated immune activation. Our data demonstrate that in the immediate post-transplant period islets release DAMPs that in vitro enhance responses of innate immune cells. DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells

The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-kappa B dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy

Recent progress in the use and tracking of transplanted islets as a personalized treatment for type 1 diabetes

Introduction: Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which the pancreas produces insufficient amounts of insulin. T1DM patients require exogenous sources of insulin to maintain euglycemia. Transplantation of naked or microencapsulated pancreatic islets represents an alternative paradigm to obtain an autonomous regulation of blood glucose levels in a controlled and personalized fashion. However, once transplanted, the fate of these personalized cellular therapeutics is largely unknown, justifying the development of non-invasive tracking techniques. Areas covered: In vivo imaging of naked pancreatic islet transplantation, monitoring of microencapsulated islet transplantation, visualizing pancreatic inflammation, imaging of molecular-genetic therapeutics, imaging of beta cell function. Expert commentary: There are still several hurdles to overcome before (microencapsulated) islet cell transplantation will become a mainstay therapy. Non-invasive imaging methods that can track graft volume, graft rejection, graft function (insulin secretion), microcapsule engraftment, microcapsule rupture, and pancreatic inflammation are currently being developed to design the best experimental transplantation paradigms

The role of pathogen-associated molecular patterns in inflammatory responses against alginate based microcapsules

<p>Alginate-based microcapsules are used for immunoisolation of cells to release therapeutics on aminute-to-minute basis. Unfortunately, alginate-based microcapsules are suffering from varying degrees of success, which is usually attributed to differences in tissue responses. This results in failure of the therapeutic cells. In the present study we show that commercial, crude alginates may contain pathogen-associated molecular patterns (PAMPs), which are recognized by the sensors of the innate immune system. Known sensors are Toll-like receptors (TLRs), NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule essential in Toll-like receptor signaling, i.e. MyD88, we were able to show that alginates signal mainly via MyD88. This was found for low-G, intermediate-G, and high-G alginates applied in calcium-beads, barium-beads as well as in alginate-PLL-alginate capsules. These alginates did stimulate TLRs 2, 5, 8, and 9 but not TLR4 (LPS receptor). Upon implantation in rats these alginates provoked a strong inflammatory response resulting in fibrosis of the capsules. Analysis demonstrated that commercial alginates contain the PAMPs peptidoglycan, lipoteichoic acid, and flagellin. By applying purification procedures, these PAMPs were largely removed. This was associated with deletion of the inflammatory tissue responses as confirmed by an implantation experiment in rats. Our data also show that alginate itself does not provoke TLR mediated responses. We were able to unravel the sensor mechanism by which contaminants in alginates may provoke inflammatory responses. (C) 2013 Elsevier B.V. All rights reserved.</p>

Confocal microscopy images of alginate-PLL-PEG-b-PLL microcapsules after the addition of a) dextran of 110 kg/mol and b) dextran of 150 kg/mol.

<p>Confocal microscopy images of alginate-PLL-PEG-b-PLL microcapsules after the addition of a) dextran of 110 kg/mol and b) dextran of 150 kg/mol.</p

Permeability of the alginate-PLL₁₀₀, alginate-PLL₁₀₀-PEG₄₅₄-b-PLL₅₀ and alginate-PLL₁₀₀-PEG₄₅₄-b-PLL₁₀₀ capsules determined using dextran-<i>f</i> samples.

<p>Permeability of the alginate-PLL₁₀₀, alginate-PLL₁₀₀-PEG₄₅₄-b-PLL₅₀ and alginate-PLL₁₀₀-PEG₄₅₄-b-PLL₁₀₀ capsules determined using dextran-<i>f</i> samples.</p

Alginate-PEG₄₅₄-b-PLL₁₀₀ capsules a) before implantation and b) at one month after implantation.

<p>GMA-embedded histological sections, Romanovsky-Giemsa staining, original magnification ×10.</p

Kinetics of adsorption of the PEG₄₅₄-b-PLL₅₀ (•) and PEG₄₅₄-b-PLL₁₀₀ (▴) diblock copolymer on a) the alginate gel and b) the alginate gel pretreated for 10 minutes with PLL₁₀₀.

<p>Kinetics of adsorption of the PEG₄₅₄-b-PLL₅₀ (•) and PEG₄₅₄-b-PLL₁₀₀ (▴) diblock copolymer on a) the alginate gel and b) the alginate gel pretreated for 10 minutes with PLL₁₀₀.</p

Elemental surface compositions of alginate-PLL₁₀₀ and alginate-PLL₁₀₀-PEG₄₅₄-b-PLL_y (y = 50 or 100) microcapsules and theoretical atom % of PLL₁₀₀ homopolymer and PEG₄₅₄-b-PLL_y (y = 50 or 100) diblock copolymers.

<p>Elemental surface compositions of alginate-PLL₁₀₀ and alginate-PLL₁₀₀-PEG₄₅₄-b-PLL_y (y = 50 or 100) microcapsules and theoretical atom % of PLL₁₀₀ homopolymer and PEG₄₅₄-b-PLL_y (y = 50 or 100) diblock copolymers.</p

Confocal microscopy images after staining of the PEG blocks.

<p><b>a) Alginate-PLL₁₀₀ capsules, b) alginate-PLL₁₀₀-PEG₄₅₄-b-PLL₅₀ capsules and c) alginate-PLL₁₀₀-PEG₄₅₄-b-PLL₁₀₀ microcapsules.</b> Original magnification 10×.</p