The Expanding Role of Natural Killer Cells in Type 1 Diabetes and Immunotherapy.

Treatments for autoimmune diseases including type 1 diabetes (T1D) are aimed at resetting the immune system, especially its adaptive arm. The innate immune system is often ignored in the design of novel immune-based therapies. There is increasing evidence for multiple natural killer (NK) subpopulations, but their role is poorly understood in autoimmunity and likely is contributing to the controversial role reported for NKs. In this review, we will summarize NK subsets and their roles in tolerance, autoimmune diabetes, and immunotherapy.

A physiological pattern of oxygenation using perfluorocarbon-based culture devices maximizes pancreatic islet viability and enhances ß-cell function.

Conventional culture vessels are not designed for physiological oxygen (O2) delivery. Both hyperoxia and hypoxia-commonly observed when culturing cells in regular plasticware-have been linked to reduced cellular function and death. Pancreatic islets, used for the clinical treatment of diabetes, are especially sensitive to sub- and supraphysiological O2 concentrations. A result of current culture standards is that a high percentage of islet preparations are never transplanted because of cell death and loss of function in the 24-48 h postisolation. Here, we describe a new culture system designed to provide quasiphysiological oxygenation to islets in culture. The use of dishes where islets rest atop a perfluorocarbon (PFC)-based membrane, coupled with a careful adjustment of environmental O2 concentration to target the islet physiological pO2 range, resulted in dramatic gains in viability and function. These observations underline the importance of approximating culture conditions as closely as possible to those of the native microenvironment, and fill a widely acknowledged gap in our ability to preserve islet functionality in vitro. As stem cell-derived insulin-producing cells are likely to suffer from the same limitations as those observed in real islets, our findings are especially timely in the context of current efforts to define renewable sources for transplantation.

Quantitative assessment of islet cell products: estimating the accuracy of the existing protocol and accounting for islet size distribution.

The ability to consistently and reliably assess the total number and the size distribution of isolated pancreatic islet cells from a small sample is of crucial relevance for the adequate characterization of islet cell preparations used for research or transplantation purposes. Here, data from a large number of isolations were used to establish a continuous probability density function describing the size distribution of human pancreatic islets. This function was then used to generate a polymeric microsphere mixture with a composition resembling those of isolated islets, which, in turn, was used to quantitatively assess the accuracy, reliability, and operator-dependent variability of the currently utilized manual standard procedure of quantification of islet cell preparation. Furthermore, on the basis of the best fit probability density function, which corresponds to a Weibull distribution, a slightly modified scale of islet equivalent number (IEQ) conversion factors is proposed that incorporates the size distribution of islets and accounts for the decreasing probability of finding larger islets within each size group. Compared to the current calculation method, these factors introduce a 4-8% downward correction of the total IEQ estimate, but they reflect a statistically more accurate contribution of differently sized islets.

The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: oxygen consumption as a potential measure of islet potency.

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding ("dO2 slope"). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized "dO2 slope" in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.

The use of multiparametric monitoring during islet cell isolation and culture: a potential tool for in-process corrections of critical physiological factors.

Variables such as pH, pCO2, and PO2 have been established in the literature as critical factors that could affect the outcome of the islet cell processing and, therefore, the quality of the cells that could be transplanted. This report describes a highly accurate continuous multiparametric monitoring system and its evaluation for continuous monitoring of physiological variables during critical steps of the islet isolation procedure as well as during in vitro culture of the insulin-producing cells. Close monitoring of these variables could be of assistance to improve the outcome of islet cell processing, allowing to identify as soon as possible problems that could be corrected during the procedure, as well as during in vitro preservation, or shipment to remote sites.

Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution.

Last year, from the approximately 6,000 organ donors, only approximately 1,500 pancreata were used for clinical transplantation. Factors that contribute to this poor pancreas use include strict donor selection criteria and the requirement for short cold-ischemia time (CIT). Numerous pancreata have not been used because of long ischemia times postprocurement. Given the oxygen-rich environment of the islets in the native pancreas, it is conceivable that islets are highly susceptible to irreversible damage following prolonged ischemia. The use of continuously oxygenated perfluorohydrocarbons (PFCs), known for their high oxygen-solubility coefficients, in a two-layer culture with standard University of Wisconsin preservation media, has extended the acceptable range CIT, and, furthermore, there has been no evidence of adverse effects from PFCs on the outcome of transplanted cells, whereas they often enhance islet cell function. The purpose of this study was to use the two-layer culture method to improve donor-organ use from marginal donors. Fifteen organs were procured using the two-layer method, and 18 without using it, from donors greater than 50 years of age. Despite nonsignificant differences in age, weight of the donors, weight of the organ and CIT, the PFC group yielded an average of twofold more islet equivalents than those harvested from the control group. As a result, from the control group, only 2 of 18 organs were used for clinical islet transplantation, whereas 8 of 15 were used from the PFC group. To this end, the two-layer method may help clinicians overcome the problem of organ underuse.

Neonatal porcine pancreatic cell clusters as a potential source for transplantation in humans: characterization of proliferation, apoptosis, xenoantigen expression and gene delivery with recombinant AAV.

Neonatal porcine islets are characterized by reproducible isolation success and high yields, sizable advantages over adult islets. In this work we have analyzed selected phenotypic and functional characteristics of porcine neonatal islets relevant to their possible use for transplant in humans. We show that porcine islet cells proliferate in culture, and synthesize and store islet-specific hormones. Proliferating beta cells can be easily identified. Implant of cultured neonatal islets in immunodeficient rodents results in the reversal of diabetes, albeit with delay. We also show that measurable apoptosis occurs in cultured neonatal porcine islets. Further, antigens recognized by human natural antibodies are expressed in a dynamic fashion over the culture period analyzed and are not limited to the alpha-Gal epitope. Lastly, we demonstrate that a recombinant Adeno-Associated virus can be used to efficiently deliver a reporter gene in porcine islets. This characterization might be helpful in the definition of the potential use of neonatal porcine islets for human transplantation.