Biomedical subjects

Rita Bottino

Publications and source records attributed to Rita Bottino.

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

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes.

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

Alleles↗

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

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↗

PubMed2005

Gene transfer of constitutively active Akt markedly improves human islet transplant outcomes in diabetic severe combined immunodeficient mice.

Akt is an important intracellular mediator of beta-cell growth and survival in rodents. However, whether constitutive activation of Akt in human beta-cells enhances the survival and function of transplanted islets is unknown. In the current study, we examined the efficacy of constitutive activation of Akt in improving human islet transplant outcomes using a marginal mass model in diabetic severe combined immunodeficient (SCID) mice. Human islets transduced with adenoviruses encoding constitutively active Akt1 (Adv-CA-Akt) displayed increased total and phosphorylated Akt and Akt kinase activity compared with control islets. Expression of CA-Akt in human islets induced a significant increase in beta-cell replication and a significant decrease in beta-cell death induced by serum and glucose deprivation or chronic hyperglycemia. Two control groups of islets (1,500 uninfected or adenovirus LacZ [Adv-LacZ]-transduced human islet equivalents [IEQs]) transplanted under the kidney capsule of streptozotocin-induced diabetic SCID mice were insufficient to correct hyperglycemia. Importantly and in marked contrast to these controls, 1,500 Adv-CA-Akt-transduced IEQs were capable of restoring euglycemia in diabetic SCID mice. Moreover, blood glucose normalization persisted for at least 6 months. Human plasma insulin at day 54 after transplant was 10-fold higher in Adv-CA-Akt islet recipients (2.4 +/- 0.4 ng/ml) compared with those receiving Adv-LacZ islets (0.25 +/- 0.08 ng/ml) (P < 0.05). In summary, expression of CA-Akt in human islets improves islet transplant outcomes in a subcapsular renal graft model in SCID mice. Akt is an attractive target for future strategies aimed at reducing the number of islets required for successful islet transplantation in humans.

Adenoviridae↗

PubMed2005

Multifaceted therapeutic approaches for a multigenic disease.

Diabetes is a severe chronic disease that affects approximately 200 million individuals worldwide, with extremely debilitating effects and considerably high health care costs. The two major classes of diabetes, known as type 1 (previously known as insulin-dependent or juvenile-onset diabetes) and type 2 (non-insulin-dependent diabetes), share common symptoms such as hyperglycemia and the development of long-term complications, but they differ in many aspects, including their etiopathogenesis. New insights suggest that overlapping factors, formerly considered typical hallmarks of each specific type, can coexist in the same diabetic patient, making it difficult to support a sharp distinction between the two classes and, more importantly, to adopt appropriate therapeutic solutions. In type 1 and type 2 diabetic subjects, but even more in patients with combined types, multiple genetic factors play a role in determining susceptibility or resistance to the disease, and perhaps also the time of onset, the severity of the symptoms, the possibility of developing complications and, ultimately, the response to therapy. In this review, the therapeutic treatments currently under investigation, as well as the curative strategies envisioned for future applications, are reanalyzed considering the multifaceted and complex aspects of a continuum that can be just defined as "diabetes."

Animals↗

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

Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes.

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous beta cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

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↗

PubMed2002

Production of alpha 1,3-galactosyltransferase-deficient pigs.

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

Alleles↗

PubMed2002

Islet/pancreas transplantation: challenges for pediatrics.

Beta cell replacement is a valid alternative to exogenous insulin injections to treat type 1 diabetic patients. The rate of success obtained after whole-pancreas transplantation, performed alone or in combination with kidney, and, as shown recently, by islet transplantation, justifies optimism and sets the stage for a larger clinical application of these approaches. Lifetime immunosuppression, however, required to protect the graft against recurrent autoimmune destruction and allorejection, raises serious doubts about the safety of its employment in children. While it is evident that children may be helped even more than adults by the possibility to correct diabetic metabolic disorders without exogenous insulin, and to lower in a more effective way the chance to develop secondary complications, the drawbacks of the currently used immunosuppressive drugs largely overcome the potential benefits. A great step forward for immediate applicability of transplantation to children involves the optimization of tolerogenic protocols and a better understanding of the concept of immune ignorance. Functional tolerance should be sufficient to entail the absence of immune reactivity against self- and graft antigens, while maintaining immune reactivity against other non-self, non-donor antigens. In addition, novel strategies aimed at utilizing surrogate beta cells obtained from non-islet cells, or by genetic manipulation of beta-cell precursors merit consideration as the use of xenogeneic donors. However, much work is still needed for their safe clinical implementation.

Journal Article↗

PubMed2002

Pancreas and islet cell transplantation.

Currently, for the patient with type 1 diabetes, a definitive treatment without resorting to the use of exogenous insulin can be achieved only with pancreas or islet cell transplantation. These means of restoring beta-cell mass can completely maintain essentially normal long-term glucose homeostasis, although the need for powerful immunosuppressive regimens limits their application to only a subgroup of adult patients. Apart from the shortage of donors that has limited all kinds of transplantation, however, the widespread use of beta-cell replacement has been precluded until recently by the drawbacks associated with both organ and islet cell transplantation. Although the study of recurrence of diabetes has generated attention, the fundamental obstacle to pancreas and islet transplantation has been, and remains, the alloimmune response. With a better elucidation of the mechanisms of alloengraftment achieved during the last 3 years, the stage has been set for further advances.

Diabetes Mellitus, Type 1↗