Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation.

Intrahepatic islet transplantation for type 1 diabetes is limited by the need for multiple infusions and poor islet viability posttransplantation. The development of alternative transplantation sites is necessary to improve islet survival and facilitate monitoring and retrieval. We tested a clinically proven biodegradable temporizing matrix (BTM), a polyurethane-based scaffold, to generate a well-vascularized intracutaneous "neodermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and it facilitated diabetes cure for over 150 days. Furthermore, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is nontoxic for islets. Two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days after BTM implantation. In a preclinical porcine skin model, BTM implants created a highly vascularized intracutaneous site by day 7 postimplantation. When syngeneic neonatal porcine islets were transplanted intracutaneously, the islets remained differentiated as insulin-producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here, we show that BTM facilitates formation of an islet-supportive intracutaneous neodermis in a porcine preclinical model, as an alternative islet-transplant site. ARTICLE HIGHLIGHTS: Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

An overview on small molecule-induced differentiation of mesenchymal stem cells into beta cells for diabetic therapy.

The field of regenerative medicine provides enormous opportunities for generating beta cells from different stem cell sources for cellular therapy. Even though insulin-secreting cells can be generated from a variety of stem cell types like pluripotent stem cells and embryonic stem cells, the ideal functional cells should be generated from patients' own cells and expanded to considerable levels by non-integrative culture techniques. In terms of the ease of isolation, plasticity, and clinical translation to generate autologous cells, mesenchymal stem cell stands superior. Furthermore, small molecules offer a great advantage in terms of generating functional beta cells from stem cells. Research suggests that most of the mesenchymal stem cell-based protocols to generate pancreatic beta cells have small molecules in their cocktail. However, most of the protocols generate cells that mimic the characteristics of human beta cells, thereby generating "beta cell-like cells" as opposed to mature beta cells. Diabetic therapy becomes feasible only when there are robust, functional, and safe cells for replacing the damaged or lost beta cells. In this review, we discuss the current protocols used to generate beta cells from mesenchymal cells, with emphasis on small molecule-mediated conversion into insulin-producing beta cell-like cells. Our data and the data presented from the references within this review would suggest that although mesenchymal stem cells are an attractive cell type for cell therapy they are not readily converted into functional mature beta cells.

Oxygen-permeable microwell device maintains islet mass and integrity during shipping.

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20-40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping.

Viral RNA in the influenza vaccine may have contributed to the development of ANCA-associated vasculitis in a patient following immunisation.

A number of large studies have demonstrated influenza vaccinations to be safe and effective. However, there have been some sporadic case reports, describing a temporal association of influenza vaccination with onset or relapse of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. The nature of this association, beyond time of occurrence, remains unknown. The presentation of a previously healthy patient who developed ANCA-associated vasculitis (AAV) shortly after influenza vaccination provided us with the rare opportunity to study the possible mechanisms behind this observation. We tested the ability of different types and batches of influenza vaccines to stimulate proteinase-3 ANCA (PR3-ANCA) production in vitro. We found that only some influenza vaccines stimulated PR3-ANCA production in this patient. We demonstrated that this unusual response was associated with those vaccines that contained viral ribonucleic acid (RNA), the natural ligand for Toll-like receptor-7. Exome sequencing of the patient's DNA did not show any mutation in any of the molecules associated with Toll-like receptor signalling. We propose that hyper-reaction to viral RNA in the influenza vaccine may have contributed to the development of AAV following influenza vaccination in this patient.

CpG oligodeoxynucleotide stimulates production of anti-neutrophil cytoplasmic antibodies in ANCA associated vasculitis.

BACKGROUND: Wegener's Granulomatosis and Microscopic Polyangiitis are life-threatening systemic necrotizing vasculitides of unknown aetiology. The appearance of circulating antibodies to neutrophil cytoplasmic antigens (ANCA) is strongly associated with the development of the disease. A link between infection and disease has long been suspected, and the appearance of ANCA antibodies has been reported following bacterial and viral infections. The depletion of circulating B cells with monoclonal antibody therapy can induce remission, and this observation suggests a pathogenic role for B cells in this disease. As bacterial DNA is known to induce B cell proliferation and antibody production via TLR-9 stimulation, we have explored the possibility that unmethylated CpG oligodeoxynucleotide, as found in bacterial and viral DNA, may play a role in stimulating circulating autoreactive B cells to produce ANCA in patients with vasculitis. RESULTS: We have confirmed that unmethylated CpG oligonucleotide is a potent stimulator of antibody production by PBMC in vitro. The stimulation of PBMC with CpG oligonucleutides resulted in the production of similar amounts of IgG in both ANCA+ patients and normal controls. In spite of this, PR3 ANCA+ patients synthesised significantly higher amount of IgG ANCA than normal controls. In MPO ANCA+ patients, there was a tendency for patients to produce higher amount of ANCA than controls, however, the difference did not reach significance. Furthermore, we were able to detect circulating MPO-reactive B cells by ELISpot assay from the peripheral blood of 2 MPO+ ANCA vasculitis patients. Together, this indicates that circulating anti-neutrophil autoreactive B cells are present in ANCA+ vasculitis patients, and they are capable of producing antibodies in response to CpG stimulation. Of note, CpG also induced the production of the relevant autoantibodies in patients with other types of autoimmune diseases. CONCLUSION: Circulating ANCA autoreactive B cells are present in patients with ANCA+ vasculitis. The production of ANCA from these cells in response to unmethylated CpG stimulation lead us to propose that stimulation of these cells by immunostimulatory DNA sequences such as CpG oligodeoxynucleotide during infection may provide a link between infection and ANCA associated vasculitis. This phenomenon may also apply to other antibody mediated autoimmune diseases.

Effect of pravastatin on markers of endothelial activation in dialysis patients.

AIM: The aim of this pilot study was to test the effect of pravastatin on serum levels of high-sensitivity CRP (hs-CRP), IL-6 and the soluble adhesion molecules sVCAM-1, sICAM-1 and sE-selectin in chronic dialysis patients. METHODS: At the commencement of the study, serum levels of lipids, liver function tests and endothelial markers (CRP, IL-6, sICAM-1, sVCAM-1, sE-selectin) were measured. Patients then commenced 1 month of 10 mg of pravastatin per day, and if tolerated, then 4 months of 40 mg of pravastatin per day. Serum levels of lipids, liver function tests and endothelial markers were repeated after the total of 5 months of pravastatin therapy. RESULTS: Thirty-nine patients were enrolled, and 25 (male/female 17/8; 21 haemodialysis, 4 peritoneal dialysis) patients completed the study. Pravastatin therapy significantly improved the patients' lipid profiles, but had no significant effect on the levels of CRP, IL-6, sICAM-1, sVCAM-1, or sE-selectin. CONCLUSION: Short-term (5 months) treatment with pravastatin in patients receiving chronic dialysis improved their lipid profile, but had no significant effect on surrogate markers of endothelial activation.