Current status and prospects for gene and cell therapeutics for type 1 diabetes mellitus.
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Animals↗
Biomedical subjects
Massimo Trucco
Publications and source records attributed to Massimo Trucco.
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↗
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↗
Gene therapy technology applied to disorders of glucose metabolism: promise, achievements, and prospects.
Gene transfer technology has spawned an entire realm of clinical investigation, collectively referred to as "gene therapy." The feasibility and achievements of gene therapy to prevent and treat glucose homeostasis disorders, with particular emphasis on diabetes mellitus, are evaluated in this review. While a considerable amount of effort has yielded gene delivery vectors based on adenoviral, retroviral, and herpes simplex virus DNA, the number of successful clinical applications has not been as impressive. Despite the number of successes in vitro and in animal models, preliminary safety trials in humans have not yet been attempted. The current state of this science, outlined here, underlines the necessity of marrying gene transfer technology with cell therapy. The ex vivo transfer of gene combinations into a variety of cell types will likely prove more therapeutically feasible than direct in vivo vector transfer. Current efforts aimed at assessing the future of gene therapy for diabetes must, at the very least, take into account the importance of moving successful methods into human safety trials.
Diabetes Mellitus↗
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↗
Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes.
Islet transplantation is a promising cure for diabetes. However, inflammation, allorejection, and recurrent autoimmune damage all may contribute to early graft loss. Pancreatic islets express lower levels of antioxidant genes than most other tissues of the body, and beta-cells in particular are sensitive to oxidative damage. Therefore, damage from oxidative stress may pose a major obstacle to islet replacement therapy in that both the islet isolation and transplantation processes generate oxygen radicals. To determine whether antioxidant gene overexpression in isolated pancreatic islets can prevent oxidative damage and prolong islet function after transplantation, we used the NOD mouse model to study oxidative stress encountered during both transplantation and autoimmune attack. We transferred an antioxidant gene, manganese superoxide dismutase (MnSOD), by adenoviral infection into isolated islets that were transplanted into streptozotocin-treated NODscid recipient mice. Functioning islet grafts were subsequently exposed to diabetogenic spleen cells and monitored until graft failure. The results show that islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts. This significant prolongation of graft function suggests that the antioxidant activity of MnSOD is beneficial to transplanted islet survival and may be used in combination with other strategies aimed at islet graft protection.
Adenoviridae↗
Prevention of diabetes in NOD mice by administration of dendritic cells deficient in nuclear transcription factor-kappaB activity.
Abnormalities of dendritic cells (DCs) have been identified in type 1 diabetic patients and in nonobese diabetic (NOD) mice that are associated with augmented nuclear transcription factor (NF)-kappaB activity. An imbalance that favors development of the immunogenic DCs may predispose to the disease, and restoration of the balance by administration of DCs deficient in NF-kappaB activity may prevent diabetes. DCs propagated from NOD mouse bone marrow and treated with NF-kappaB-specific oligodeoxyribonucleotide (ODN) in vitro (NF-kappaB ODN DC) were assessed for efficacy in prevention of diabetes development in vivo. Gel shift assay with DC nuclear extracts confirmed specific inhibition of NF-kappaB DNA binding by NF-kappaB ODN. The costimulatory molecule expression, interleukin (IL)-12 production, and immunostimulatory capacity in presenting allo- and islet-associated antigens by NF-kappaB ODN DC were significantly suppressed. NF-kappaB ODN renders DCs resistant to lipopolysaccharide stimulation. Administration of 2 x 10(6) NF-kappaB ODN DCs into NOD mice aged 6-7 weeks effectively prevented the onset of diabetes. T-cells from pancreatic lymph nodes of NF-kappaB ODN DC-treated animals exhibited hyporesponsiveness to islet antigens with low production of interferon-gamma and IL-2. These findings provide novel insights into the mechanisms of autoimmune diabetes and may lead to development of novel preventive strategies.
Animals↗
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↗
Alternative core promoters regulate tissue-specific transcription from the autoimmune diabetes-related ICA1 (ICA69) gene locus.
Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5'-regulatory region of ICA1. 5'-RNA ligase-mediated rapid amplification of cDNA ends evaluation of ICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5'-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1 exon promoters reciprocally stimulate luciferase expression within islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1 exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease.
5' Untranslated Regions↗
Immunosuppressive effects of glucosamine.
Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitro as well as allogeneic mixed leukocyte reactivity in a dose-dependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.
Adenosine Triphosphate↗
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↗
Cellular environment and freedom of gene expression.
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Animals↗
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↗
Gene therapy strategies to prevent autoimmune disorders.
Autoimmunity accounts for a significant percentage of human disease and remains a challenging syndrome to treat. While systemic immunosuppression can be beneficial, the associated toxicity of the pharmacologic agents necessitates an antigen-specific approach to silence, eradicate or prevent the genesis of autoreactive immune cells. Gene therapy offers the possibility of providing precise antigen-targeted therapies, thereby sparing the patient the significant toxicity associated with lifelong commitment to chemical immunosuppressives. Gene-based therapies could include, but are not limited to the manipulation of immune networks of tolerance by antigen presenting cell engineering, pro-inflammatory cytokine blockade using soluble antagonists expressed from viral vectors as well as modulation of immune regulatory networks. The potential utility of gene therapy strategies promoting tolerance in two model autoimmune disorders, type I diabetes mellitus and rheumatoid arthritis are discussed in this review.
Arthritis, Rheumatoid↗
Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes.
Indoleamine 2,3-dioxygenase (IDO) catalyzes the breakdown of the amino acid tryptophan into kyneurenine. It has been shown that IDO production by placental trophoblasts prevents the attack of maternal T-cells activated in response to the paternal HLA alleles expressed by the tissues of the fetus. In this article, we show that adenoviral gene transfer of IDO to pancreatic islets can sufficiently deplete culture media of tryptophan and consequently inhibit the proliferation of T-cells in vitro. Experiments in vivo have also demonstrated that transplantation of IDO-expressing islets from prediabetic NOD mouse donors into NODscid recipient mice is associated with a prolongation in islet graft survival after adoptive transfer of NOD diabetogenic T-cells. This protection is attributed to the depletion of tryptophan at the transplantation site beneath the kidney capsule. These results suggest that local modulation of tryptophan catabolism may be a means of facilitating islet transplantation as a therapy for type 1 diabetes.
Adenoviridae↗
Preservation of human islet cell functional mass by anti-oxidative action of a novel SOD mimic compound.
The most commonly used technical approach to isolate human pancreatic islets intended for allotransplants generates a product that is hampered by mechanical and chemical insults, which dramatically reduce the mass of viable and functional transplantable cells. We tested a novel class of antioxidant chemical compounds (SOD mimics: AEOL10113 and AEOL10150) to protect human islets from oxidative stress in order to improve the preservation of the isolated tissue. Addition of SOD mimic in culture, after isolation, allowed for the survival of a significantly higher islet cell mass. Functional behavior and phenotypic cell characteristics of the SOD-treated islet preparations were preserved, as was the capacity to normalize diabetic mice, even when a marginal mass of islets was transplanted. The addition of SOD mimic during isolation, before culture, further reduced early cell loss. These results indicate that prompt interventions aimed at blocking oxidative stress can improve human islet survival, preserving a functional islet mass two- to threefold larger than the one usually obtained without adding any antioxidant compound. The ability to preserve functional islets without a dramatic loss represents a major advantage considering the scarce availability of islet tissue for clinical transplantation.
Adolescent↗
Genes and engineered cells as drugs for type I and type II diabetes mellitus therapy and prevention.
Despite the manageability of diabetes mellitus, complications associated with the disorder necessitate novel approaches to prevent immune-mediated impairment and destruction in type 1 diabetes, as well as the pancreatic insufficiency and peripheral resistance to insulin in type 2 diabetes. Islet transplantation is evolving into a clinical reality to treat type 1 diabetics and novel uses of gene engineering technology promise to result in tolerance to auto-, allo- and xenoantigens as well as microenvironment-specific immunosuppression. Through the use of a variety of gene delivery vehides, an increasing number of studies demonstrate the feasibility of shielding islet transplants and surrogate beta cells from immune rejection by the local secretion of immunosuppressive soluble molecules and anti-apoptotic factors. Although the achievements of gene and cell therapy in type 2 diabetes mellitus are less clear, seminal studies demonstrate the relevance of this approach to the treatment and perhaps prevention of the underlying causes of the disease, including obesity and insulin resistance. In this review, we attempt to illustrate pivotal studies demonstrating the suitability of genes and cells as drugs in type 1 and type 2 diabetes mellitus, and also provide some other targets that may be suitable for clinical utility.
Animals↗
Distinct characteristics and features of allogeneic chimerism in the NOD mouse model of autoimmune diabetes.
The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.
Animals↗