Exosomes; a Potential Source of Biomarkers, Therapy, and Cure for Type-1 Diabetes.

The scourge of type-1 diabetes (T1D) is the morbidity and mortality it and its complications cause at a younger age. This propels the constant search for better diagnostic, treatment, and management strategies, with the ultimate quest being a cure for T1D. Recently, the therapeutic potential of exosomes has generated a lot of interest. Among the characteristics of exosomes of particular interest are (a) their regenerative capacity, which depends on their "origin", and (b) their "content", which determines the cell communication and crosstalk they influence. Other functional capacities, including paracrine and endocrine homeostatic regulation, pathogenic response ability resulting in insulin secretory defects or ß-cell death under normal metabolic conditions, immunomodulation, and promotion of regeneration, have also garnered significant interest. Exosome "specificity" makes them suitable as biomarkers or predictors, and their "mobility" and "content" lend credence to drug delivery and therapeutic suitability. This review aims to highlight the functional capacities of exosomes and their established as well as novel contributions at various pathways in the onset and progression of T1D. The pathogenesis of T1D involves a complex crosstalk between insulin-secreting pancreatic ß-cells and immune cells, which is partially mediated by exosomes. We also examine the potential implications for type 2 diabetes (T2D), as the link in T2D has guided T1D exploration. The collective landscape presented is expected to help identify how a deeper understanding of exosomes (and their cargo) can provide a framework for actionable solutions to prevent, halt, or change the very course of T1D and its complications.

Stem Cell Therapy for Aging Related Diseases and Joint Diseases in Companion Animals.

Stem cell therapy is an attractive treatment for diseases in companion animals that cannot be treated by conventional veterinary medicine practices. The unique properties of stem cells, particularly the ability to differentiate into specific cell types, makes them a focal point in regenerative medicine treatments. Stem cell transplantation, especially using mesenchymal stem cells, has been proposed as a means to treat a wide range of injuries and ailments, resulting in tissue regeneration or repair. This review aims to summarize the veterinary use of stem cells for treating age-related and joint diseases, which are common conditions in pets. While additional research is necessary and certain limitations exist, the potential of stem cell therapy for companion animals is immense.

A Receptor Story: Insulin Resistance Pathophysiology and Physiologic Insulin Resensitization's Role as a Treatment Modality.

Physiologic insulin secretion consists of an oscillating pattern of secretion followed by distinct trough periods that stimulate ligand and receptor activation. Apart from the large postprandial bolus release of insulin, ß cells also secrete small amounts of insulin every 4-8 min independent of a meal. Insulin resistance is associated with a disruption in the normal cyclical pattern of insulin secretion. In the case of type-2 diabetes, ß-cell mass is reduced due to apoptosis and ß cells secrete insulin asynchronously. When ligand/receptors are constantly exposed to insulin, a negative feedback loop down regulates insulin receptor availability to insulin, creating a relative hyperinsulinemia. The relative excess of insulin leads to insulin resistance (IR) due to decreased receptor availability. Over time, progressive insulin resistance compromises carbohydrate metabolism, and may progress to type-2 diabetes (T2D). In this review, we discuss insulin resistance pathophysiology and the use of dynamic exogenous insulin administration in a manner consistent with more normal insulin secretion periodicity to reverse insulin resistance. Administration of insulin in such a physiologic manner appears to improve insulin sensitivity, lower HgbA1c, and, in some instances, has been associated with the reversal of end-organ damage that leads to complications of diabetes. This review outlines the rationale for how the physiologic secretion of insulin orchestrates glucose metabolism, and how mimicking this secretion profile may serve to improve glycemic control, reduce cellular inflammation, and potentially improve outcomes in patients with diabetes.

Mechanisms of Foreign Body Giant Cell Formation in Response to Implantable Biomaterials.

Long term function of implantable biomaterials are determined by their integration with the host's body. Immune reactions against these implants could impair the function and integration of the implants. Some biomaterial-based implants lead to macrophage fusion and the formation of multinucleated giant cells, also known as foreign body giant cells (FBGCs). FBGCs may compromise the biomaterial performance and may lead to implant rejection and adverse events in some cases. Despite their critical role in response to implants, there is a limited understanding of cellular and molecular mechanisms involved in forming FBGCs. Here, we focused on better understanding the steps and mechanisms triggering macrophage fusion and FBGCs formation, specifically in response to biomaterials. These steps included macrophage adhesion to the biomaterial surface, fusion competency, mechanosensing and mechanotransduction-mediated migration, and the final fusion. We also described some of the key biomarkers and biomolecules involved in these steps. Understanding these steps on a molecular level would lead to enhance biomaterials design and improve their function in the context of cell transplantation, tissue engineering, and drug delivery.

Monitoring of Autoantibodies Following Autologous Hematopoietic Stem Cell Transplantation in 6 Children with Recently Diagnosed Type 1 Diabetes Mellitus.

BACKGROUND Bone marrow stem cells have been shown to be a promising therapeutic strategy for autoimmune diseases. This study aimed to assess the safety and efficacy of autologous hematopoietic stem cell (ABMSC) transplantation without immunoablation used to suppress the autoimmune reaction in 6 children with newly diagnosed autoimmune diabetes mellitus. We monitored the levels of islet cell antibodies (ICA), antibodies against islet antigen-related tyrosine phosphatase 2 (IA2), glutamic acid-decarboxylase (GAD) antibodies, and anti-insulin antibodies (AIA). MATERIAL AND METHODS Between 2018 and 2022, 6 children (age 6-10 years, average 8 years) recently diagnosed with type 1 diabetes mellitus with the presence of ICA, IA2, GAD, AIA and ketoacidosis, were treated with an ABMSC stimulated with Filgrastim, granulocyte colony-stimulating factor (G-CSF), 10 ug/kg/day for 4 days. Bone marrow was harvested on day 5, collected by puncture and identified as mononuclear cells >180×106/kg, CD34+ >0.22%, and transplanted by intravenous (i.v.) infusion. Patients were monitored with ICA, IA2, GAD, AIA, C-peptide, blood glucose, and glycosylated hemoglobin A1c (HbA1C) 6 months after the procedure. RESULTS At 6-month follow-up, we observed a negative value of the ICA, which was previously positive (P<0.001). The IA2 (p=0.037) and GAD (P=0.377) antibodies decreased slowly but were significantly lower. AIA remained high. A decrease in blood glucose and HbA1C levels was observed (P<0.001). No complications occurred during follow-up. CONCLUSIONS Autologous hematopoietic stem cell transplantation without immunoablation was safe and effective in significantly decreasing the production and effect of autoantibodies against ICA, GAD, and IA2, as well as decreasing blood sugar levels and HbA1c.

Successful Islet Transplantation Into a Subcutaneous Polycaprolactone Scaffold in Mice and Pigs.

Islet transplantation is a promising treatment for type 1 diabetes. It has the potential to improve glycemic control, particularly in patients suffering from hypoglycemic unawareness and glycemic instability. As most islet grafts do not function permanently, efforts are needed to create an accessible and replaceable site, for islet grafts or for insulin-producing cells obtained from replenishable sources. To this end, we designed and tested an artificial, polymeric subcutaneous transplantation site that allows repeated transplantation of islets. Methods: In this study, we developed and compared scaffolds made of poly(D,L,-lactide-co-ε-caprolactone) (PDLLCL) and polycaprolactone (PCL). Efficacy was first tested in mice' and then, as a proof of principle for application in a large animal model, the scaffolds were tested in pigs, as their skin structure is similar to that of humans. Results: In mice, islet transplantation in a PCL scaffold expedited return to normoglycemia in comparison to PDLLCL (7.7 ± 3.7 versus 16.8 ± 6.5 d), but it took longer than the kidney capsule control group. PCL also supported porcine functional islet survival in vitro. Subcutaneous implantation of PDLLCL and PCL scaffolds in pigs revealed that PCL scaffolds were more stable and was associated with less infiltration by immune cells than PDLLCL scaffolds. Prevascularized PCL scaffolds were therefore used to demonstrate the functional survival of allogenic islets under the skin of pigs. Conclusions: To conclude, a novel PCL scaffold shows efficacy as a readily accessible and replaceable, subcutaneous transplantation site for islets in mice and demonstrated islet survival after a month in pigs.

Anti-Oxidative Therapy in Islet Cell Transplantation.

Islet cell transplantation has become a favorable therapeutic approach in the treatment of Type 1 Diabetes due to the lower surgical risks and potential complications compared to conventional pancreas transplantation. Despite significant improvements in islet cell transplantation outcomes, several limitations hamper long-term graft survival due to tremendous damage and loss of islet cells during the islet cell transplantation process. Oxidative stress has been identified as an omnipresent stressor that negatively affects both the viability and function of isolated islets. Furthermore, it has been established that at baseline, pancreatic ß cells exhibit reduced antioxidative capacity, rendering them even more susceptible to oxidative stress during metabolic stress. Thus, identifying antioxidants capable of conferring protection against oxidative stressors present throughout the islet transplantation process is a valuable approach to improving the overall outcomes of islet cell transplantation. In this review we discuss the potential application of antioxidative therapy during each step of islet cell transplantation.

Inclusion of extracellular matrix molecules and necrostatin-1 in the intracapsular environment of alginate-based microcapsules synergistically protects pancreatic ß cells against cytokine-induced inflammatory stress.

Immunoisolation of pancreatic islets in alginate-based microcapsules is a promising approach for grafting of islets in absence of immunosuppression. However, loss and damage to the extracellular matrix (ECM) during islet isolation enhance susceptibility of islets for inflammatory stress. In this study, a combined strategy was applied to reduce this stress by incorporating ECM components (collagen type IV/RGD) and necroptosis inhibitor, necrostatin-1 (Nec-1) in alginate-based microcapsules in vitro. To demonstrate efficacy, viability and function of MIN6 ß-cells and human islets in capsules with collagen type IV/RGD and/or Nec-1 was investigated in presence and absence of IL-1ß, IFN-γ and TNF-α. The combination of collagen type IV/RGD and Nec-1 had higher protective effects than the molecules alone. Presence of collagen type IV/RGD and Nec-1 in the intracapsular environment reduced cytokine-induced overproduction of free radical species and unfavorable shifts in mitochondrial dynamics. In addition, the ECM components collagen type IV/RGD prevented a cytokine induced suppression of the FAK/Akt pathway. Our data indicate that the inclusion of collagen type IV/RGD and Nec-1 in the intracapsular environment prevents islet-cell loss when exposed to inflammatory stress, which might contribute to higher survival of ß-cells in the immediate period after transplantation. This approach of inclusion of stress reducing agents in the intracapsular environment of immunoisolating devices may be an effective way to enhance the longevity of encapsulated islet grafts. STATEMENT OF SIGNIFICANCE: Islet-cells in immunoisolated alginate-based microcapsules are very susceptible to inflammatory stress which impacts long-term survival of islet grafts. Here we show that incorporation of ECM components (collagen type IV/RGD) and necrostatin-1 (Nec-1) in the intracapsular environment of alginate-based capsules attenuates this susceptibility and promotes islet-cell survival. This effect induced by collagen type IV/RGD and Nec-1 was probably due to lowering free radical production, preventing mitochondrial dysfunction and by maintaining ECM/integrin/FAK/Akt signaling and Nec-1/RIP1/RIP3 signaling. Our study provides an effective strategy to extend longevity of islet grafts which might be of great potential for future clinical application of immunoisolated cells.

Physiologic Insulin Resensitization as a Treatment Modality for Insulin Resistance Pathophysiology.

Prevalence of type 2 diabetes increased from 2.5% of the US population in 1990 to 10.5% in 2018. This creates a major public health problem, due to increases in long-term complications of diabetes, including neuropathy, retinopathy, nephropathy, skin ulcers, amputations, and atherosclerotic cardiovascular disease. In this review, we evaluated the scientific basis that supports the use of physiologic insulin resensitization. Insulin resistance is the primary cause of type 2 diabetes. Insulin resistance leads to increasing insulin secretion, leading to beta-cell exhaustion or burnout. This triggers a cascade leading to islet cell destruction and the long-term complications of type 2 diabetes. Concurrent with insulin resistance, the regular bursts of insulin from the pancreas become irregular. This has been treated by the precise administration of insulin more physiologically. There is consistent evidence that this treatment modality can reverse the diabetes-associated complications of neuropathy, diabetic ulcers, nephropathy, and retinopathy, and that it lowers HbA1c. In conclusion, physiologic insulin resensitization has a persuasive scientific basis, significant treatment potential, and likely cost benefits.

Applying Immunomodulation to Promote Longevity of Immunoisolated Pancreatic Islet Grafts.

Islet transplantation is a promising therapy for insulin-dependent diabetes, but large-scale application is hampered by the lack of a consistent source of insulin-producing cells and need for lifelong administration of immunosuppressive drugs, which are associated with severe side effects. To avoid chronic immunosuppression, islet grafts can be enveloped in immunoisolating polymeric membranes. These immunoisolating polymeric membranes protect islet grafts from cell-mediated rejection while allowing diffusion of oxygen, nutrients, and insulin. Although clinical trials have shown the safety and feasibility of encapsulated islets to control glucose homeostasis, the strategy does up till now not support long-term graft survival. This partly can be explained by a significant loss of insulin-producing cells in the immediate period after implantation. The loss can be prevented by combining immunoisolation with immunomodulation, such as combined administration of immunomodulating cytokines or coencapsulation of immunomodulating cell types such as regulatory T cells, mesenchymal stem cells, or Sertoli cells. Also, administration of specific antibodies or apoptotic donor leucocytes is considered to create a tolerant microenvironment around immunoisolated grafts. In this review, we describe the outcomes and limitations of these approaches, as well as the recent progress in immunoisolating devices. Impact statement Immunoisolation by enveloping islets in semipermeable membranes allows for successful transplantation of islet grafts in the absence of chronic immunosuppression, but the duration of graft survival is still not permanent. The reasons for long-term final graft failure is not fully understood, but combining immunoisolation with immunomodulation of tissues or host immune system has been proposed to enhance the longevity of grafts. This article reviews the recent progress and challenges of immunoisolation, as well as the benefits and feasibility of combining encapsulation approaches with immunomodulation to promote longevity of encapsulated grafts.

An overview of current advancements in pancreatic islet transplantation into the omentum.

Pancreatic islet transplantation to restore insulin production in Type 1 Diabetes Mellitus patients is commonly performed by infusion of islets into the hepatic portal system. However, the risk of portal vein thrombosis or elevation of portal pressure after transplantation introduces challenges to this procedure. Thus, alternative sites have been investigated, among which the omentum represents an ideal candidate. The surgical site is easily accessible, and the tissue is highly vascularized with a large surface area for metabolic exchange. Furthermore, the ability of the omentum to host large volumes of islets represents an intriguing if not ideal site for encapsulated islet transplantation. Research on the safety and efficacy of the omentum as a transplant site focuses on the utilization of biologic scaffolds or encapsulation of islets in a biocompatible semi-permeable membrane. Currently, more clinical trials are required to better characterize the safety and efficacy of islet transplantation into the omentum.

Comparison of islet isolation result and clinical applicability according to GMP-grade collagenase enzyme blend in adult porcine islet isolation and culture.

BACKGROUND: Porcine islet xenotransplantation is a promising treatment for type 1 diabetes as an alternative to human pancreatic islet transplantation and long-term insulin therapy. Several research groups have explored porcine islets as an alternative to the inconsistent and chronic shortage of pancreases from human organ donors. Studies have confirmed successful transplant of porcine islets into non-human primate models of diabetes; however, in most cases, they require more than one adult porcine donor to achieve sufficient viable islet mass for sustained function. The importance of GMP-grade reagents includes the following: specific enzymes utilized in the pancreatic isolation process were identified as a key factor in successful human clinical islet transplantation trials using cadaveric islets. As xenotransplantation clinical research progresses, isolation reagents and digestion enzymes play a key role in the consistency of the product and ultimately the outcome of the islet xenotransplant. In this study, we evaluated several commercially available enzyme blends that have been used for islet isolation. We evaluated their impact on islet isolation yield and subsequent islet function as part of our plan to bring xenotransplantation into clinical xenotransplantation trials. METHODS: Adult porcine islets were isolated from 16 to 17-month-old Yucatan miniature pigs following standard rapid procurement. Pigs weighed on average 48.71 ± 2.85 kg, and the produced pancreases were 39.51 ± 1.80 grams (mean ± SEM). After ductal cannulation, we evaluated both GMP-grade enzymes (Collagenase AF-1 GMP grade and Liberase MTF C/T GMP grade) and compared with standard non-GMP enzyme blend (Collagenase P). Islet quality control assessments including islet yield, islet size (IEQ), membrane integrity (acridine orange/propidium iodide), and functional viability (GSIS) were evaluated in triplicate on day 1 post-islet isolation culture. RESULTS: Islet yield was highest in the group of adult pigs where Collagenase AF-1 GMP grade was utilized. The mean islet yield was 16 586 ± 1391 IEQ/g vs 8302 ± 986 IEQ/g from pancreases isolated using unpurified crude Collagenase P. The mean islet size was higher in Collagenase AF-1 GMP grade with neutral protease than in Collagenase P and Liberase MTF C/T GMP grade. We observed no significant difference between the experimental groups, but in vitro islet function after overnight tissue culture was significantly higher in Collagenase AF-1 GMP grade with neutral protease and Liberase MTF C/T GMP grade than the crude control enzyme group. As expected, the GMP-grade enzyme has significantly lower endotoxin levels than the crude control enzyme group when measured. CONCLUSIONS: This study validates the importance of using specifically blended GMP grade for adult pig islet isolation for xenotransplantation trials and the ability to isolate a sufficient number of viable islets from one adult pig to provide a sufficient number for islets for a clinical islet transplantation. GMP-grade enzymes are highly efficient in increasing islet yield, size, viability, and function at a lower and acceptable endotoxin level. Ongoing research transplants these islets into animal models of diabetes to validate in vivo function. Also, these defined and reproducible techniques using GMP-grade enzymes allow for continuance of our plan to advance to xenotransplantation of isolated pig islets for the treatment of type 1 diabetes.

Exosome loaded immunomodulatory biomaterials alleviate local immune response in immunocompetent diabetic mice post islet xenotransplantation.

Foreign body response (FBR) to biomaterials compromises the function of implants and leads to medical complications. Here, we report a hybrid alginate microcapsule (AlgXO) that attenuated the immune response after implantation, through releasing exosomes derived from human Umbilical Cord Mesenchymal Stem Cells (XOs). Upon release, XOs suppress the local immune microenvironment, where xenotransplantation of rat islets encapsulated in AlgXO led to >170 days euglycemia in immunocompetent mouse model of Type 1 Diabetes. In vitro analyses revealed that XOs suppressed the proliferation of CD3/CD28 activated splenocytes and CD3+ T cells. Comparing suppressive potency of XOs in purified CD3+ T cells versus splenocytes, we found XOs more profoundly suppressed T cells in the splenocytes co-culture, where a heterogenous cell population is present. XOs also suppressed CD3/CD28 activated human peripheral blood mononuclear cells (PBMCs) and reduced their cytokine secretion including IL-2, IL-6, IL-12p70, IL-22, and TNFα. We further demonstrate that XOs mechanism of action is likely mediated via myeloid cells and XOs suppress both murine and human macrophages partly by interfering with NFκB pathway. We propose that through controlled release of XOs, AlgXO provide a promising new platform that could alleviate the local immune response to implantable biomaterials.

The effects of necrostatin-1 on the in vitro development and function of young porcine islets over 14-day prolonged tissue culture.

BACKGROUND: Necrostatin-1 (Nec-1) supplementation to tissue culture media on day 3 has recently been shown to augment the insulin content, endocrine cellular composition, and insulin release of pre-weaned porcine islets (PPIs); however, its effects were only examined for the first 7 days of tissue culture. The present study examined whether the addition of Nec-1 on day 3 could further enhance the in vitro development and function of PPIs after 14 days of tissue culture. METHODS: PPIs were isolated from 8- to 15-day-old, pre-weaned Yorkshire piglets and cultured in an islet maturation media supplemented with Nec-1 on day 3. The recovery, viability, insulin content, endocrine cellular composition, GLUT2 expression in beta cells, differentiation and proliferation potential, and glucose-stimulated insulin secretion of PPIs were assessed on days 3, 7, and 14 of tissue culture (n = 5 on each day). RESULTS: Compared with day 7 of tissue culture, islets on day 14 had a lower recovery, GLUT2 expression in beta cells, proliferation capacity of endocrine cells, and glucose-induced insulin stimulation index. Prolonging the culture time to 14 days did not affect islet viability, insulin content, proportion of endocrine cells, and differentiation potential. CONCLUSION: The growth-inducing effects of Nec-1 on PPIs were most effective on day 7 of tissue culture when added on day 3. Our findings support existing evidence that the in vitro activities of Nec-1 are short-lived and encourage future studies to explore the use of other novel growth factors during prolonged islet tissue culture.

Toll-like receptor 2-modulating pectin-polymers in alginate-based microcapsules attenuate immune responses and support islet-xenograft survival.

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro, and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo, we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site.

Dose-dependent effects of necrostatin-1 supplementation to tissue culture media of young porcine islets.

Previous studies have shown that necrostatin-1 (Nec-1) supplementation improved the viability of murine islets following exposure to nitric oxide, increased the survival of human islets during hypoxic culture, and augmented the maturation of pre-weaned porcine islets (PPIs) after 7 days of tissue culture. A limitation of these studies is that only one concentration of Nec-1 was used, and no studies have determined the optimal dose of Nec-1 for PPIs. Thus, the present study examined the effects of Nec-1 on PPIs at four different doses-0, 25, 50, 100, and 200 µM-after 7 days of tissue culture when supplemented on day 3. PPIs were isolated from pancreata of pre-weaned Yorkshire piglets (8-15 days old) and cultured in a specific islet maturation media added with Nec-1 on day 3 of tissue culture at 4 different doses-0, 25, 50, 100, and 200 µM (n = 6 for each dose). After 7 days of tissue culture, islets were assessed for recovery, viability, endocrine cellular content, GLUT2 expression in beta cells, and insulin secretion after glucose challenge. Nec-1 did not affect the viability of both intact islets and dissociated islets cells during tissue culture regardless of doses. Islets cultured in media supplemented with Nec-1 at 100 µM, but not 25, 50, or 200 µM, had a significantly higher recovery, composition of endocrine cells, GLUT2 expression in beta cells, and insulin secretion capacity than control islets cultured in media without Nec-1 supplementation. Moreover, culturing islets in 200 µM Nec-1 supplemented media not only failed to improve the insulin release but resulted in a lower glucose-induced insulin stimulation index compared to islets cultured in media added with 100 µM Nec-1. Xenotransplantation using porcine islets continues to demonstrate scientific advances to justify this area of research. Our findings indicate that Nec-1 supplementation at 100 µM was most effective to enhance the in vitro maturation of PPIs during tissue culture.

Optimal Time to Ship Human Islets Post Tissue Culture to Maximize Islet.

Access to functional high-quality pancreatic human islets is critical to advance diabetes research. The Integrated Islet Distribution Program (IIDP), a major source for human islet distribution for over 15 years, conducted a study to evaluate the most advantageous times to ship islets postisolation to maximize islet recovery. For the evaluation, three experienced IIDP Islet Isolation Centers each provided samples from five human islet isolations, shipping 10,000 islet equivalents (IEQ) at four different time periods postislet isolation (no 37°C culture and shipped within 0 to 18 hours; or held in 37°C culture for 18 to 42, 48 to 96, or 144 to 192 hours). A central evaluation center compared samples for islet quantity, quality, and viability for each experimental condition preshipment and postshipment, as well as post 37°C culture 18 to 24 hours after shipment receipt. Additional evaluations included measures of functional potency by static glucose-stimulated insulin release (GSIR), represented as a stimulation index. Comparing the results of the four preshipment holding periods, the greatest IEQ loss postshipment occurred with the shortest preshipment times. Similar patterns emerged when comparing preshipment to postculture losses. In vitro islet function (GSIR) was not adversely impacted by increased tissue culture time. These data indicate that allowing time for islet recovery postisolation, prior to shipping, yields less islet loss during shipment without decreasing islet function.

In Quest of Pathognomonic/Endophenotypic Markers of Attention Deficit Hyperactivity Disorder (ADHD): Potential of EEG-Based Frequency Analysis and ERPs to Better Detect, Prevent and Manage ADHD.

Attention deficit hyperactivity disorder (ADHD) is a chronic heritable developmental delay psychiatric disorder requiring chronic management, characterized by inattention, hyperactivity, hyperkinectivity and impulsivity. Subjective clinical evaluation still remains crucial in its diagnosis. Discussed are two key aspects in the "characterizing ADHD" and on the quest for objective "pathognomonic/endophenotypic diagnostic markers of ADHD". The first aspect briefly revolves around issues related to identification of pathognomonic/endophenotypic diagnostic markers in ADHD. Issues discussed include changes in ADHD definition, remission/persistence and overlapping-symptoms cum shared-heritability with its co-morbid cross-border mental disorders. The second aspect discussed is neurobiological and EEG-based studies on ADHD. Given the neurobiological and temporal aspects of ADHD symptoms the electroencephalograph (EEG) like NeuralScan by Medeia appears as an appropriate tool. The EEGs appropriateness is further enhanced when coupled with suitable behavior/cognitive/motor/psychological tasks/paradigms yielding EEG-based markers like event-related-potential (ERPs like P3 amplitudes and latency), reaction time variability (RTV), Theta:Beta ratio (TBR) and sensorimotor rhythm (SMR). At present, these markers could potentially help in the neurobiological characterization of ADHD and either help in identifying or lay the groundwork for identifying pathognomonic and/or endophenotypic EEG-based markers enabling its diagnosis, treatment and management.

The Effect of a Fast-Releasing Hydrogen Sulfide Donor on Vascularization of Subcutaneous Scaffolds in Immunocompetent and Immunocompromised Mice.

Islet transplantation into subcutaneous polymer scaffolds has shown to successfully induce normoglycemia in type 1 diabetes models. Vascularization of these scaffolds is imperative for optimal control of glucose levels. We studied the effect of the vascular stimulator hydrogen sulfide (H2S) on the degree of vascularization of a scaffold and the role of the immune system in this process. Scaffolds were subcutaneously implanted in immunocompetent C57BL/6 and immunocompromised nude mice. Mice received twice-daily intraperitoneal injections of the fast-releasing H2S donor sodium hydrosulfide (NaHS, 25 or 50 µmol/kg) or saline for 28 days. After 63 days the vascular network was analyzed by histology and gene expression. Here we showed that the vascularization of a subcutaneous scaffold in nude mice was significantly impaired by H2S treatment. Both the CD31 gene and protein expression were reduced in these scaffolds compared to the saline-treated controls. In C57BL/6 mice, the opposite was found, the vascularization of the scaffold was significantly increased by H2S. The mRNA expression of the angiogenesis marker CD105 was significantly increased compared to the controls as well as the number of CD31 positive blood vessels. In conclusion, the immune system plays an important role in the H2S mediated effect on vascularization of subcutaneous scaffolds.

An islet maturation media to improve the development of young porcine islets during in vitro culture.

BACKGROUND: The use of pancreata from pre-weaned piglets has the potential to serve as an unlimited alternative source of islets for clinical xenotransplantation. As pre-weaned porcine islets (PPIs) are immature and require prolonged culture, we developed an islet maturation media (IMM) and evaluated its effect on improving the quantity and quality of PPIs over 14 days of culture. METHODS: PPIs were isolated from the pancreata of pre-weaned Yorkshire piglets (8-15 days old). Each independent islet isolation was divided for culture in either control Ham's F-10 media (n = 5) or IMM (n = 5) for 14 days. On day 3, 7 and 14 of culture, islets were assessed for islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of beta cells, and insulin secretion during glucose stimulation. RESULTS: In comparison to control islets, culturing PPIs in IMM significantly increased islet yield. PPIs cultured in IMM also maintained a stable isolation index and viability throughout 14 days of culture. The insulin content, endocrine cellular composition, and differentiation of beta cells were significantly improved in PPIs cultured in IMM, which subsequently augmented their insulin secretory capacity in response to glucose challenge compared to control islets. CONCLUSIONS: Culturing PPIs in IMM increases islet yield, isolation index, viability, insulin content, endocrine cellular composition, differentiation of endocrine progenitor cells toward beta cells, and insulin secretion. Due to the improved islet quantity and quality after in vitro culture, the use of IMM in the culture of PPIs will assist to advance the outcomes of clinical islet xenotransplantation.