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Background To prevent transmission of zoonotic microorganisms from pig transplants to human recipients when performing xenotransplantation using pig cells, tissues, or organs, donor pigs have to be carefully characterized. Göttingen minipigs (Gö MP) are often used for various biomedical investigations and are well characterized concerning the presence of numerous bacteria, fungi, viruses, and parasites. Recently, we studied the prevalence and expression of porcine endogenous retroviruses and the prevalence of hepatitis E virus ( HEV) in Gö MP. Here, we studied the presence of the porcine cytomegalovirus ( PCMV) and porcine lymphotropic herpesviruses ( PLHV) and extended testing for hepatitis E virus ( HEV). Methods PCR, nested PCR, real-time PCR, real-time RT- PCR, and Western blot analyses were used to estimate the prevalence of PCMV, PLHV-1, PLHV-2, PLHV-3, and HEV. Results Using different PCR methods, and different source materials, PCMV was found in 10 of 26 adult Gö MP, which had been derived originally by cesarean section and kept under specified pathogen-free conditions. Only highly sensitive methods gave positive results, not methods of lower sensitivity. The virus load in all positive animals was low (<100-200 copies per mL). PLHV-1, PLHV-2, and PLHV-3 were not detected by PCR; however, an anti- PLHV immune response was found in one of 10 animals tested by Western blot analyses. HEV was detected by RT- PCR in two of nine tested animals, but no anti- HEV immune response was observed. Conclusion Using highly sensitive methods, PCMV, HEV, and PLHV were found in some Gö MP, suggesting that these viruses may be introduced through the placenta. The results show that highly sensitive methods are required to characterize pigs to be used for xenotransplantation to prevent virus transmission. [ABSTRACT FROM AUTHOR]

Background Xenotransplantation using pig cells, tissues or organs may be associated with the transmission of porcine zoonotic micro-organisms. Hepatitis E virus ( HEV), porcine cytomegalovirus ( PCMV) and porcine endogenous retroviruses ( PERVs) are potentially zoonotic micro-organisms which do not show clinical symptoms in pigs and which are due to the low expression level difficult to detect. Göttingen Minipigs (Gö MP) are often used for biomedical investigations and they are well characterized concerning the presence of numerous bacteria, fungi, viruses and parasites and therefore may be used for islet cell transplantation. Methods Islet cells derived from three Gö MP were transplanted into four healthy, non-diabetic cynomolgus monkeys using a macroencapsulation device. PCR, nested PCR, real-time PCR, real-time RT- PCR and Western blot analyses were used to estimate the presence of PERV, PCMV and HEV in the donors and recipients. Results Using sensitive detection methods, no HEV was found in the donor pigs and in the pig islet cell preparations. Antibodies against PERV, PCMV and HEV were not found in all cynomolgus monkeys with exception of one monkey showing an immune response against HEV. Using real-time PCR, no PCMV and HEV were found in the sera of all monkeys. Conclusion Although the donor islet cells and the recipients were negative for HEV using PCR and Western blot analysis, in one recipient, antibodies against HEV were found, indicating infection in a single case. All recipients were negative for antibodies against PERV, and all were negative for PCMV, indicating absence of infection. As HEV was not detected in the donor pig before transplantation, a more complex and regular screening of the animals using highly sensitive methods is required to avoid virus transmission. [ABSTRACT FROM AUTHOR]

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or organs. To reduce the xenotransplantation-associated risk of infection of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of microorganisms was examined in Göttingen Minipigs by PCR methods. Since zoonotic transmission of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly established. In addition, sera were examined by Western blot analysis using two recombinant capsid proteins of HEV as antigens. HEV RNA was not detected in pigs older than one year including gilts, but it was detected in the sera of three of ten animals younger than 1 year. Furthermore, HEV was also detected in the sera of three sows six days after delivery and their offspring, indicating vertical transmission of the virus. PCR amplicons were cloned, sequenced and the viruses were found to belong to the HEV genotype (gt) 3/4. Anti-HEV immunoglobulins G were detected in one sow and maternal antibodies in her six day old piglet. Since Göttingen minipigs were negative for many xenotransplantation-relevant microorganisms, they can now be classified as safe. HEV may be eliminated from the Ellegaard herd by selection of negative animals and/or by treatment of the animals. [ABSTRACT FROM AUTHOR]

Background Xenogeneic transplantation of islets of Langerhans into type I diabetes patients imposes a major challenge. This is even more exaggerated when the target is set as xenotransplantation of islets in relatively oxygen poor environment without the use of immunosuppressive therapy. To overcome these hurdles, adequate encapsulation technique and active oxygen supply should be employed. Methods A two-article device was developed (the βAir). The first contains an implantable bioreactor module comprising of islets of Langerhans encapsulated in alginate hydrogel and protected from the host immune system by a complex membrane. The second is an external oxygen injection device for replenishing of the islet biomass in the implantable device once a day. Results Both small and large diabetic animal models (rats and pigs) were experimented. The βAir device proved: (i) ability to maintain near normal glycemic control in rats transplanted with either isogeneic or allogeneic islets for a period of over 6 months and in pigs transplanted with rat islets using marginal dose of islets (~6,500 IEQ/kg) for a period of up to 75 days; (ii) ability to maintain adequate oxygen tension required to nourish the islets graft for a period of 24 h while maintaining optimal islet's function. (iii) long term separation of transplanted graft from host immune system thus maintaining normal islets function in rat allotype, in rat-to-pig and in human-to-rat transplantation systems and (iv) high level of biocompatibility and tolerability of the device. Conclusion The βAir device proved to offer functional solution to islets transplantation in allogeneic and xenogeneic setting. It may provide a solution to transplantation of porcine islets to diabetic patients. References [1] Barkai U, Weir GC, Colton CK et al. Enhanced oxygen supply improves islet viability in a new bioartificial pancreas. Cell Transplant. 2013; 22: 1463-1476. [2] Neufeld T, Ludwig B, Barkai U et al. The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs. PLoS One. 2013; 8(8): e70150. [ABSTRACT FROM AUTHOR]

Introduction Pancreatic islet transplantation is currently restricted to patients with critical metabolic lability due to long-term need for immunosuppression and a persistent shortage of donor organs [1-3]. To overcome these obstacles we have developed a strategy for islet macroencapsulation that provides sufficient immune-isolation whereas regulated islet graft function is maintained [4-8]. Case Report and Methods A 63 year old patient with type 1 diabetes and severe metabolic lability was transplanted with isolated islets (2,000 islets/kgBW) encapsulated in an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers. Via a small abdominal incision, a pre-peritoneal pocket for the chamber was dissected, connected oxygen ports were implanted subcutaneously. No immunosuppressive therapy was applied. Results The procedure was surgically straightforward and without complications. We could demonstrate persistent graft function by detection of endogenous insulin and c-peptide secretion proving islet viability and function. This observation was accompanied by persistent lowering in HbA1c despite reduction in insulin requirement. For oxygenation of the non-vascularized and therefore immune-shielded islet graft, the chamber-integrated gas reservoir was replenished daily via the implanted ports without complications. Conclusion This encapsulation strategy was for the first time applied to allogeneic human islet transplantation in man. We demonstrated a persistent graft function with regulated insulin secretion without any immunosuppressive therapy. This novel concept may allow for future widespread application for cell-based therapies. References [1] 2007 update on allogeneic islet transplantation from the Collaborative Islet Transplant Registry (CITR). Cell Transplant 2009; 18: 753-767. [2] Ludwig, B., Ludwig, S., Steffen, A., Saeger, H.D., Bornstein, S.R. Islet versus pancreas transplantation in type 1 diabetes: competitive or complementary? Curr Diab Rep 2010; 10: 506-511. [3] Mccall, M., James Shapiro, A.M. Update on islet transplantation. Cold Spring Harb Perspect Med 2012; 2: a007823. [4] Barkai, U., Weir G.C., Colton C.K. et al. Enhanced Oxygen Supply Improves Islet Viability in a New Bioartificial Pancreas. Cell Transplant 2013; 22(8): 1463-1476 [5] Ludwig, B., Rotem A., Schmid J. et al. Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist. Proc Natl Acad Sci U S A 2012; 109: 5022-5027. [6] Ludwig, B., Zimmermann B., Steffen A. et al. A novel device for islet transplantation providing immune protection and oxygen supply. Horm Metab Res 2010; 42: 918-922. [7] Neufeld, T., Ludwig B., Barkai U. et al. The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs. PLoS One 2013; 8: e70150. [8] Ludwig, B., Reichel A, Steffen A et al. Transplantation of human islets without immunosuppression. PNAS 2013; 110: 19054-19058. [ABSTRACT FROM AUTHOR]

The red marine alga Gracilaria sp. (collected on Israel's Mediterranean shore) was groget in the laboratory under controlled conditions. The effects of the medium (natural seawater or enriched seawater) and of irradiance on the following parameters were studied: growth rate, dry weight, content of pigments, EO[sub2] fixation rate, starch content, and the quality and quantity of agar. The total agar content was higher in algae grown in natural seawater than in those grown in enriched seawater and decreased only slightly with increasing irradiance. The agar composition was also different in algae grown in the two media. The starch content was higher in algae grown in natural seawater than in algae grown in enriched seawater, and in algae in both media it increased with increasing irradiance. Since the starch content in Gracilaria can reach 21% on a dry weight basis, it is important to correct for it when determining the agar content. [ABSTRACT FROM AUTHOR]

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO 2. Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO 2 to a supraphysiological level that maintains all islets viable and functional. These levels were determined from measurements of pO 2 profiles in islet-alginate slabs. Encapsulated islets implanted with surface density as high as 4,800 islet equivalents/cm 3 in diabetic rats maintained normoglycemia for more than 7 months and provided near-normal intravenous glucose tolerance tests. Nearly 90% of the original viable tissue was recovered after device explantation. Damaged islets failed after progressively shorter times. The required values of gas chamber pO 2 were predictable from a mathematical model of oxygen consumption and diffusion in the device. These results demonstrate feasibility of developing retrievable macroencapsulated devices small enough for clinical use and provide a firm basis for design of devices for testing in large animals and humans.

Islet transplantation effectively treats diabetes but relies on immune suppression and is practically limited by the number of cadaveric islets available. An alternative cellular source is insulin-producing cells derived from pluripotent cell sources. Three animal cohorts were used in the current study to evaluate whether an oxygen-providing macro-encapsulation device, 'βAIR', could function in conjunction with human embryonic stem cells (hESCs) and their derivatives. The first cohort received macro-encapsulated undifferentiated hESCs, a second cohort received hESCs differentiated to a pancreatic progenitor state with limited endocrine differentiation. A reference cohort received human islets. Macro-encapsulation devices were implanted subcutaneously and monitored for up to 4 months. Undifferentiated pluripotent stem cells did not form teratoma but underwent cell death following implantation. Human C-peptide (hC- peptide) was detectable in host serum one week after implantation for both other cohorts. hC-peptide levels decreasing over time but remained detectable up to the end of the study. Key factors associated with mature endocrine cells were observed in grafts recovered from cohorts containing islets and hESC-derivatives including C-peptide, insulin, glucagon and urocortin 3. We conclude that the 'βAIR' macroencapsulation device is compatible with both human islets and pluripotent derivatives, but has a limited capability of sustaining undifferentiated pluripotent cells.

At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, especially when using large retrievable macro-capsules implanted in poorly oxygenated sites like the subcutis. Notably, islets can survive under low oxygen tension and are physiologically active at > 40 Torr. Surprisingly, when densely crowded, islets are fully functional under hyperoxic pressure of up to 500 Torr (> 300% of atmospheric oxygen tension). The review also addresses an additional category of requirements for optimal performance of transplanted islets, named auxiliary technologies. These include control of inflammation, apoptosis, angiogenesis, and the intra-capsular environment. The review highlights that curing diabetes with a functional bio-artificial pancreas requires optimizing all of these aspects, and that significant advances have already been made in many of them.

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.

The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)-the "β-Air"-that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immuno-protective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.