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Biomedical subjects

A N Balamurugan

Publications and source records attributed to A N Balamurugan.

At least 19 recordsLinked to original sources
PubMed2007

Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies.

INTRODUCTION: Pig islets have been proposed as an alternative to human islets for clinical use, but their use is limited by rejection. The availability of genetically modified pigs devoid of alpha1,3-galactosyltransferase might provide islets more suitable for xenotransplantation. To limit the costs involved in the logistics and health care of pigs for clinical xenotransplantation, we have studied whether younger, rather than older, pigs that are typically preferred can be used as islet donors. METHODS: We utilized pancreases from Yorkshire and White Landrace wild-type pigs and alpha1,3-galactosyltransferase gene-knockout pigs of three main different age and size groups: (i) <6 months, (ii) 6 to 12 months, and (iii) >2 yr of age, inclusive of retired breeders. We compared isolation yield and in vitro and in vivo function of islet cells obtained from these groups. RESULTS: Islets from adult pigs (>2 yr) offered not only higher islet yields, but retained the ability to preserve intact morphology during the isolation process and culture, in association with high functional properties after transplantation. Following isolation, islet cells from young (<6 m) and young-adult (6 to 12 m) pigs dissociated into small aggregates and single cells, and exhibited inferior functional properties than adult islets both in vitro and in vivo. CONCLUSIONS: These data support the conclusion that, in view of the large number of islets needed to maintain normoglycemia after xenotransplantation, organ-source pigs need to reach adult age (>2 yr) before being considered optimal islet donors, in spite of the higher costs involved.

Aging↗
PubMed2006

Suitability of human juvenile pancreatic islets for clinical use.

AIMS/HYPOTHESIS: The limited availability of deceased donor pancreases suitable for pancreas and islet transplantation calls for a broader utilisation of donor tissue for transplantation purposes. Young donors, representing, fortunately, a minor but significant pool of individuals, have been largely under-employed, mainly because of anatomical and functional incompatibilities with potential recipients. For islet transplantation, the isolation of pancreatic islets from young donors rarely occurs, because of technical problems. As a result of the peculiar characteristics of young donor pancreases, the standard isolation procedure does not allow efficient separation of the islets from the surrounding exocrine tissue, and favours the generation of mantled islets. Nonetheless, young donor islets offer high qualitative and clinically appealing characteristics. SUBJECTS AND METHODS: We standardised a modified methodology to obtain purified and mantle-free human islets from young donors. This method principally involves efficient delivery of isolation enzyme with reduced mechanical disruption of the pancreas combined with additional filtration steps. RESULTS: We were able to obtain purified and mantle-free human islets from donors as young as 6 months of age with good morphological and functional properties. The good qualitative characteristics of the islets, evidenced in vitro, were proven in vivo, as they were qualitatively superior to islets of older donors in transplantation studies. CONCLUSIONS/INTERPRETATION: This study justifies the utilisation of islets derived from young donors for islet transplantation.

Adolescent↗
PubMed2006

Facilitating physiologic self-regeneration: a step beyond islet cell replacement.

Type 1 diabetes (T1D) is an autoimmune disease, the clinical onset of which most frequently presents in children and adolescents who are genetically predisposed. T1D is characterized by specific insulin-producing beta cell destruction. The well-differentiated and specialized islet beta cells seem to physiologically retain the ability to compensate for the cells lost by reproducing themselves, whereas undifferentiated cell sources may help in generating new ones, even while the autoimmune process takes place. Diabetes clinical onset, i.e., establishment of a detectable, chronic hyperglycemia, occurs at a critical stage when autoimmunity, having acted for a while, supersedes the regenerative effort and reduces the number of beta cells below the physiologic threshold at which the produced insulin becomes insufficient for the body's needs. Clinical solutions aimed at avoiding cumbersome daily insulin administrations by the reestablishment of physiologic insulin production, like whole pancreas or pancreatic islet allotransplantation, are limited by the scarcity of pancreas donors and by the toxic effects of the immunosuppressive drugs administered to prevent rejection. However, new accumulating evidence suggests that, once autoimmunity is abrogated, the endocrine pancreas properties may be sufficient to allow the physiological regenerative process to restore endogenous insulin production, even after the disease has become clinically manifest. Knowledge of these properties of the endocrine pancreas suggests the testing of reliable and clinically translatable protocols for obliterating autoimmunity, thus allowing the regeneration of the patient's own endocrine cells. The safe induction of an autoimmunity-free status might become a new promising therapy for T1D.

Animals↗
PubMed2006

Prospective and challenges of islet transplantation for the therapy of autoimmune diabetes.

Pancreatic islet cell transplantation is an attractive treatment of type 1 diabetes (T1D). The success enhanced by the Edmonton protocol has fostered phenomenal progress in the field of clinical islet transplantation in the past 5 years, with 1-year rates of insulin independence after transplantation near 80%. Long-term function of the transplanted islets, however, even under the Edmonton protocol, seems difficult to accomplish, with only 10% of patients maintaining insulin independence 5 years after transplantation. These results differ from the higher metabolic performance achieved by whole pancreas allotransplantation, and autologous islet cell transplantation, and form the basis for a limited applicability of islet allografts to selected adult patients. Candidate problems in islet allotransplantation deal with alloimmunity, autoimmunity, and the need for larger islet cell masses. Employment of animal islets and stem cells, as alternative sources of insulin production, will be considered to face the problem of human tissue shortage. Emerging evidence of the ability to reestablish endogenous insulin production in the pancreas even after the diabetic damage occurs envisions the exogenous supplementation of islets to patients also as a temporary therapeutic aid, useful to buy time toward a possible self-healing process of the pancreatic islets. All together, islet cell transplantation is moving forward.

Autoimmunity↗
PubMed2006

Induction of diabetes in cynomolgus monkeys with high-dose streptozotocin: adverse effects and early responses.

OBJECTIVES: Streptozotocin (STZ) has been widely used to induce diabetes in nonhuman primates, although it has been found difficult to achieve complete diabetes without serious adverse effects. We have investigated different types and dosages of STZ to find a way to safely induce complete diabetes in cynomolgus monkeys. METHODS: After adequate hydration, 10 monkeys received STZ. Five monkeys received conventional STZ (Sigma) at a dosage of 1250 mg/m ("high dose"; n = 4) or 60 mg/kg ("low dose"; n = 1; Group 1). Five monkeys received Zanosar STZ (Sicor Pharmaceuticals, Irvine, CA) at 150 mg/kg (high dose; n = 5; Group 2). RESULTS: High-dose Group 1 monkeys became completely diabetic (n = 4), but a protein-losing nephropathy was observed in 3 of the 4 monkeys. The monkey that received 60 mg/kg STZ failed to become fully diabetic (C-peptide, > 1.86 ng/mL). Group 2 (high-dose Zanosar-treated) monkeys became completely diabetic but with no apparent adverse effects. A triphasic blood glucose response to STZ was documented in all the high-dose STZ-treated monkeys. Low-dose STZ failed to result in a triphasic response. CONCLUSIONS: (1) High-dose Zanosar STZ induced diabetes safely in cynomolgus monkeys without adverse effects. (2) A triphasic blood glucose response suggested the complete induction of diabetes.

Animals↗
PubMed2006

Sirolimus is associated with reduced islet engraftment and impaired beta-cell function.

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a fraction of transplanted islets can survive and become engrafted, and yet the underlying mechanism remains unclear. In this study, we examined the effect of sirolimus, a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function. To distinguish the effect of sirolimus on immune rejection from its effect on islet engraftment, we used a syngeneic model. Diabetic mice were transplanted with 250 islets under the renal capsule, followed by treatment with sirolimus or vehicle for 14 days. Thirty days posttransplantation, islet grafts were retrieved for the determination of insulin content and vascular density. Compared with mock-treated controls, diabetic recipient mice receiving sirolimus exhibited impaired blood glucose profiles and reduced glucose-stimulated insulin secretion, correlating with reduced intragraft insulin content and decreased vascular density. Islets exposed to sirolimus for 24 h in culture displayed significantly diminished glucose-stimulated insulin release, coinciding with decreased pancreas duodenum homeobox-1 and GLUT2 expression in cultured islets. Furthermore, sirolimus-treated diabetic recipient mice, as opposed to mock-treated controls, were associated with dyslipidemia. These data suggest that sirolimus, administered in the early posttransplantation phase, is a confounding factor for reduced islet engraftment and impaired beta-cell function in transplants.

Animals↗
PubMed2005

Efficient gene delivery to human and rodent islets with double-stranded (ds) AAV-based vectors.

Transplantation of allogeneic pancreatic islets is an effective approach to treat type 1 diabetes. To bypass the need for systemic administration of immunosuppression drugs following transplantation, approaches to genetically modify allogeneic islets to express anti-inflammatory, immunosuppressive, or antiapoptotic proteins prior to transplantation are being developed. Adeno-associated viral (AAV) based vectors have been used for gene transfer to islets, but the efficiency of functional transduction is low. Recently, double-stranded (ds) or double-copy (dc) based AAV vectors have been developed that allow for more rapid and efficient AAV-mediated transgene expression following transduction. Here we demonstrate that intact human and murine islets can be transduced with dsAAV2-eGFP efficiently compared to single-stranded AAV2-eGFP. Furthermore, our results demonstrate that murine islets transduced with dsAAV2-eGFP have normal islet glucose responsiveness, viability, and islet insulin content. Transplantation of the dsAAV2-eGFP transduced islet restored normal glycemia in diabetic mice without eliciting an immune response. Significant dsAAV2-mediated eGFP expression was observed in the islet grafts for at least 6 months post-transplant. Finally, we demonstrated that dsAAV serotypes 2, 6, and 8 infect human islets efficiently. Taken together, these results suggest that dsAAV based vectors are highly appropriate for gene transfer to islets to facilitate transplantation.

Animals↗
PubMed2005

Harmful delayed effects of exogenous isolation enzymes on isolated human islets: relevance to clinical transplantation.

The isolation process exposes human pancreatic islets to exogenous isolation enzymes. Exposure to these enzymes, as a result of intraductal injection in the pancreas or simple contact of islets with enzyme components, causes internalization into the islet cells of enzymes and their by-products. Human islets exposed to Liberase-HI exhibit a decreased insulin secretory ability that correlates with the time of exposure. This phenomenon is paralleled by increased expression of adhesion molecules (CD106 and CD62p) and activation of apoptotic pathways (Bax and Bcl-2) in islet cells. Increased functional impairment is also observed after islet transplantation in diabetic immunodeficient mice. Experimental exposure of islet grafts to exogenous isolation enzymes causes intense inflammation (CD11b positive cells) at the transplant site and it was associated with sickness behavior and eventually death of mouse recipients. The extent of these adverse effects likely deceives the standard qualitative protocols currently in use to assess islet quality in vitro. Reducing the secondary effects of exogenous isolation enzymes on isolated human islets may be crucial to enhance the quality of islets as tissue grafts.

Animals↗
PubMed2004

Insulin secretory characteristics of monkey pancreatic islets: a simple method of islet isolation and the effect of various density gradients on separation.

We describe a simple stationary digestion method of islet isolation and separation by various density gradients from monkey pancreas (Macaca radiata radiata). Effective method, different types and concentrations of collagenase were standardized. Sigma type XI collagenase yielded >1000 islets/gram pancreas at the concentration of 4 mg/ml and 3 ml Hank's/gram pancreas. Slow digestion with less concentration of collagenase was suitable for monkey islet isolation. Discontinuous density gradients of bovine serum albumin (BSA) and dextran were compared with standard Ficoll for separation of islets. Islet yield (1038 +/- 81), insulin secretory response (stimulation index, S.I.11) and histological examination revealed dextran gradients were more appropriate for monkey islets when compared to BSA and Ficoll. Insulin secretory characteristics of monkey islets were studied by exposing them to low and high concentrations glucose (S.I.11.5), arginine (S.I.4.2), leucine (S.I.2.3) and tolbutamide (S.I.1.7). The results indicated that the magnitude of glucose induced insulin secretion of monkey islet is about half as that of rat and mouse islets. However, it is higher than that of porcine and bovine islets. In conclusion, the knowledge of insulin secretory ability of Indian bonnet monkey islets together with the techniques of isolation and separation are useful tool for diabetic research especially islet transplantation.

Amino Acids↗
PubMed2004

Prolongation of islet allograft survival following ex vivo transduction with adenovirus encoding a soluble type 1 TNF receptor-Ig fusion decoy.

Islet transplantation is a viable long-term therapeutic alternative to daily insulin replacement for type I diabetes. The allogeneic nature of the transplants poses immunological challenges for routine clinical utility. Gene transfer of immunoregulatory molecules and those that improve insulin release kinetics provides rational approaches to facilitate allogeneic islet transplantation as a potential therapy. We have examined the efficacy of a soluble type 1 tumor necrosis factor receptor (TNFR) immunoglobulin-Fc fusion transgene (TNFR-Ig) to protect human islets from cytokine-induced apoptosis in culture, as well as in facilitating allogeneic islet transplants in diabetic mice. Cultured human islets were transduced with an adenoviral vector encoding human TNFR-Ig (Ad-TNFR-Ig). TNFR-Ig protein was secreted by cultured islets, as well as by transduced mouse islet transplants recovered from mouse recipients. Glucose-induced insulin release kinetics were comparable among untransduced, Ad-TNFR-Ig-infected human islets and vector-transduced islets exposed to cytokines. In parallel, Ad-TNFR-Ig-infected islets were protected from cytokine-induced apoptosis activation. Finally, diabetic mice transplanted with allogeneic islets expressing TNFR-Ig returned to and maintained normoglycemia significantly longer than untransduced islet recipients. These data support the potential utility of TNFR-Ig gene transfer to islets as a means of facilitating allogeneic islet transplantation.

Adenoviridae↗
PubMed2004

Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.

Adenoviridae↗
PubMed2004

Response of human islets to isolation stress and the effect of antioxidant treatment.

The process of human islet isolation triggers a cascade of stressful events in the islets of Langerhans involving activation of apoptosis and necrosis and the production of proinflammatory molecules that negatively influence islet yield and function and may produce detrimental effects after islet transplantation. In this study, we showed that activation of nuclear factor-kappaB (NF-kappaB) and poly(ADP-ribose) polymerase (PARP), two of the major pathways responsible for cellular responses to stress, already occurs in pancreatic cells during the isolation procedure. NF-kappaB-dependent reactions, such as production and release of interleukin-6 and -8 and macrophage chemoattractant protein 1, were observed days after the isolation procedure in isolated purified islets. Under culture conditions specially designed to mimic isolation stress, islet proinflammatory responses were even more pronounced and correlated with higher islet cell loss and impaired secretory function. Here we present novel evidence that early interventions aimed at reducing oxidative stress of pancreatic cells and islets through the use of the catalytic antioxidant probe AEOL10150 (manganese [III] 5,10,15,20-tetrakis [1,3,-diethyl-2imidazoyl] manganese-porphyrin pentachloride [TDE-2,5-IP]) effectively reduces NF-kappaB binding to DNA, the release of cytokines and chemokines, and PARP activation in islet cells, resulting in higher survival and better insulin release. These findings support the concept that the isolation process predisposes islets to subsequent damage and functional impairment. Blocking oxidative stress can be beneficial in reducing islet vulnerability and can potentially have a significant impact on transplantation outcome.

Active Transport, Cell Nucleus↗
PubMed2003

Protection of islets by in situ peptide-mediated transduction of the Ikappa B kinase inhibitor Nemo-binding domain peptide.

We have previously demonstrated that adenoviral gene transfer of the NF-kappaB inhibitor IkappaB to human islets results in protection from interleukin (IL)-1beta-mediated dysfunction and apoptosis. Here we report that human and mouse islets can be efficiently transduced by a cationic peptide transduction domain (PTD-5) without impairment of islet function. PTD mediated delivery of a peptide inhibitor of the IL-1beta-induced IkappaB kinase (IKK), derived from IKKbeta (NBD; Nemo-binding domain), and completely blocked the detrimental effects of IL-1beta on islet function and NF-kappaB activity, in a similar manner to Ad-IkappaB. We also demonstrate that mouse islets can be transduced in situ by infusion of the transduction peptide through the bile duct prior to isolation, resulting in 40% peptide transduction of the beta-cells. Delivery of the IKK inhibitor transduction fusion peptide (PTD-5-NBD) in situ to mouse islets resulted in improved islet function and viability after isolation. These results demonstrate the feasibility of using PTD-mediated delivery to transiently modify islets in situ to improve their viability and function during isolation, prior to transplantation.

Adenoviridae↗
PubMed2003

Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata.

Worldwide growing interest in reproducing the result of the Edmonton protocol in islet transplantation trials poses the problem of paucity of donors to supply sufficient amount of islets for clinical use. Improved outcomes include finding better ways to obtain higher yields from every donor organ processed and the possibility of extending islet isolation processing to glands of suboptimal quality. In order to optimize enzymatic digestion of marginal donor organs, we have modified the technique of tissue collection following enzymatic digestion of human pancreatic organs, allowing for reduced time of exposure of free islets to warm Liberase trade mark solution. Our results indicate that better controlled exposure to enzyme yields: (i) higher islet numbers; (ii) complete dissociation of all parts of pancreatic tissue; (iii) successful islet harvest from organs otherwise excluded. We also show that by limiting the exposure of free islets to enzyme solution, islet fragmentation and loss of insulin content are reduced. We further support evidence that enzymatic digestion may contribute to impairment of insulin secretory capacity of the islets in vitro during culture.

Adult↗
PubMed2003

Bioartificial pancreas transplantation at prevascularized intermuscular space: effect of angiogenesis induction on islet survival.

INTRODUCTION: Bioartificial pancreas (BAP) transplantation offers a potential treatment of diabetes mellitus. The optimal site for BAP transplantation has not yet been established. AIM: To monitor the effect of induction of neovascularization at the intermuscular space on islet survival after allogenic transplantation of BAP. METHODOLOGY: Angiogenesis was induced at the intermuscular space of diabetic Lewis rats by implanting a polyethylene terephthalate (PET) mesh bag, which enclosed a collagen sponge and biodegradable gelatin microspheres containing basic fibroblast growth factor. After confirmation of angiogenesis, BAP was prepared by mixing of 5% agarose with approximately 2,800 isolated rat (Sprague-Dawley) islets and transplanted into the prevascularized PET mesh bag. RESULTS: Neovascularization was observed in and around the PET mesh bag within 10 days after implantation as confirmed by macroscopic and microscopic examinations. In the presence of a collagen sponge, new blood vessels penetrated into the PET mesh bag and formed a vascular bed. After transplantation, normoglycemia was achieved in the rats within 3 days and maintained for >35 days. The rats gradually gained body weight, and the results of intravenous glucose tolerance test showed normal patterns of blood glucose clearance 1 month after transplantation. CONCLUSION: It can be concluded that the prevascularized PET mesh bag enabled transplanted BAP to survive and maintain function, thus indicating a potential site for BAP transplantation.

Animals↗
PubMed2003

Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein.

Type 1 diabetes results from the selective destruction of insulin-producing beta cells in the islets of Langerhans, and autoimmune T cells are thought to be the mediators of this destruction. T cells are also responsible for allorejection once the islets are transplanted into a patient to reduce the negative consequences of a lack of insulin. To better understand these processes, we have developed a transgenic mouse expressing proinsulin II tagged with a live-cell fluorescent reporter protein, Timer. Timer protein is unique because it changes color from green to red in the first 24 h after synthesis. With this marker, insulin synthesis can be carefully monitored through fluorescent changes over time. To complement this new biotechnological research tool, we designed a body window to allow for in vivo imaging over time of the islets transplanted under the kidney capsule. The window device, which is sutured to replace the underlying skin and body wall over the site of islet transplantation, may be used to simultaneously observe beta cells and T cells that have been labeled with a fluorochrome distinguishable from Timer. The imaging of both insulin-producing cells and T cells may be carried out repeatedly for a week or more with no need for repeated surgery, while preserving the life of the studied animal.

Animals↗