Sjögren's syndrome-like disease of C57BL/6.NOD-Aec1 Aec2 mice: gender differences in keratoconjunctivitis sicca defined by a cross-over in the chromosome 3 Aec1 locus.

Sjögren's syndrome (SjS) is a systemic autoimmune disease in which an immunological attack primarily against the salivary and lacrimal glands results in loss of acinar cell tissue and function leading to stomatitis sicca and keratoconjunctivitis sicca. In recent years, the NOD mouse has become an accepted model of SjS, exhibiting a spontaneously developing disease that strongly mimics the human condition. Two genetic regions, one on chromosome 1 (designated Aec2) and the second on chromosome 3 (designated Aec1) of NOD mice, have been shown to be necessary and sufficient to recapitulate SjS-like disease in non-susceptible C57BL/6 mice. Here we describe a newly derived strain, C57BL/6.NOD-Aec1R1Aec2, in which a recombination in Aec1 has resulted in reducing this genetic region to less than 20 cM from 48.5 cM. Profiling of this recombinant inbred strain has revealed that male mice maintain a full SjS-like disease, whereas female mice exhibit stomatitis sicca in the absence of detectable keratoconjunctivitis sicca. These data suggest SjS-like disease in the NOD mouse shows gender-specific regulation determined by autosomal genes.

Use of in vitro-generated, stem cell-derived islets to cure type 1 diabetes: how close are we?

Recent successes in treating type 1 diabetic patients with islet transplantation portends a future need for an increase in available islets. Ductal structures of the adult pancreas contain multipotent stem cells that, under the proper in vitro conditions, can both self-renew and differentiate into functional islets of Langerhans. In vitro-generated islets exhibit temporal changes in mRNA transcripts for islet-associated markers as well as regulated insulin responses following glucose challenge. When implanted into diabetic mice, in vitro-generated islets induce neovascularization and reverse insulin-dependent diabetes. The possibility of growing functional endocrine pancreas from stem cells provides new opportunities to produce large numbers of islets, even autologous islets, for use as implants.

Pancreatic stem cells: building blocks for a better surrogate islet to treat type 1 diabetes.

Type 1, insulin-dependent, diabetes is one of the more costly chronic diseases of children, adolescents and adults in Europe and North America. While routine insulin injections currently provide diabetic patients with their daily insulin requirements, blood glucose excursions are common, leading eventually to microvascular and macrovascular complications and early death. A 'cure' for Type 1 diabetes relies on replacement of the beta-cell mass which, today, is accomplished by pancreas transplants or islets of Langerhans implants. Recent advances in the isolation of stem cells that possess the capacity to differentiate to functional endocrine pancreas provide new opportunities to produce large numbers of islets, even autologous islets, that can be used as implants. We discuss briefly this new technology and its meaning for diabetes.

Pancreatic stem cells: a therapeutic agent that may offer the best approach for curing type 1 diabetes.

Type 1 (insulin-dependent) diabetes is one of the most costly chronic diseases of children and adolescents in North America and Europe. It occurs in genetically predisposed individuals when the immune system attacks and destroys specifically the insulin-producing beta cells of the pancreatic islets of Langerhans. Although routine insulin injections can provide diabetic patients their daily insulin requirements, non-compliance commonly results in blood glucose excursions that eventually lead to microvascular and macrovascular complications and early death. The only real 'cure' for type 1 diabetes is replacement of the beta-cell mass which, today, is either an ectopancreatic transplant or an islet of Langerhans implant. Two new developments may offer additional options: surrogate, non-endocrine cells genetically modified to secrete insulin in response to high blood glucose levels; and stem cells that possess the capacity to differentiate to endocrine pancreas. In this short review, we discuss the efforts currently being made to regulate pancreatic stem cell growth in order to produce large numbers of functional islets that can be used as implants. Hopefully, autologous stem cell-derived islet cell implants without lifelong immunosuppressive therapy may one day be realized.

Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.

Ductal structures of the adult pancreas contain stem cells that differentiate into islets of Langerhans. Here, we grew pancreatic ductal epithelial cells isolated from prediabetic adult non-obese diabetic mice in long-term cultures, where they were induced to produce functioning islets containing alpha, beta and delta cells. These in vitro-generated islets showed temporal changes in mRNA transcripts for islet cell-associated differentiation markers, responded in vitro to glucose challenge, and reversed insulin-dependent diabetes after being implanted into diabetic non-obese diabetic mice. The ability to control growth and differentiation of islet stem cells provides an abundant islet source for beta-cell reconstitution in type I diabetes.

Direct correlation between hyperoxaluria/oxalate stone disease and the absence of the gastrointestinal tract-dwelling bacterium Oxalobacter formigenes: possible prevention by gut recolonization or enzyme replacement therapy.

Oxalobacter formigenes is a specific oxalate-degrading, anaerobic bacterium inhabiting the gastrointestinal tracts of vertebrates, including humans. This bacterium maintains an important symbiotic relationship with its host by regulating oxalate homeostasis, primarily by preventing enteric absorption. Increased absorption of oxalate can lead to multiple complications associated with hyperoxaluria, especially recurrent calcium oxalate urolithiasis. Detection of O. formigenes in the gastrointestinal tract has attracted attention because the absence of this bacterium appears to be a risk factor for development of hyperoxaluria and/or recurrent calcium oxalate kidney stone disease. In the present study, epidemiologic studies with patients at high risk for calcium oxalate urolithiasis showed a direct correlation between the number of recurrent kidney stone episodes and the lack of O. formigenes colonization. As expected, the lack of O. formigenes revealed a clear association with prophylactic antibiotic therapy. To confirm the importance of O. formigenes in regulating hyperoxaluria, laboratory rats known to be noncolonized were colonized with live bacteria or treated with a preparation of oxalate-degrading enzymes derived from O. formigenes to determine any subsequent increased resistance to high oxalate challenge. Rats receiving either bacteria or enzyme replacement therapy excreted far lower levels of oxalate, did not develop the crystalluria observed with control rats, and resisted the formation of calcium oxalate crystals in their nephrons. These observations, taken together, support the concept that O. formigenes is important in maintaining oxalate homeostasis, that its absence from the gut increases the risk for hyperoxaluria and recurrent kidney stone disease, and that replacement therapy is an efficient procedure to prevent hyperoxaluria and its complications.

In vitro-generation of islets in long-term cultures of pluripotent stem cells from adult mouse pancreas.

Pancreatic islets of Langerhans exhibit an architecture and cellular organization ideal for rapid, yet finely controlled, responses to changes in blood glucose levels. In type I, insulin-dependent diabetes (IDD), this organization is lost as a result of the progressive autoimmune response which selectively destroys the insulin-producing pancreatic beta cells. Since beta cells are perceived as end-stage differentiated cells having limited capacity for regeneration in situ, individuals with IDD resulting from beta cell loss or dysfunction require life-long insulin therapy. Efforts to produce islet neogenesis or initiate islet growth in vitro from either fetal or adult tissue have had minimal success. We now report that pancreatic-derived, pluripotent islet-producing stem cells (IPSCs), isolated from prediabetic mice, can be grown in long-term cultures and differentiated into immature functional islet-like structures containing cells which express low levels of insulin, glucagon and/or somatostatin. When such in vitro grown islets were implanted into clinically diabetic NOD mice, the implanted mice were successfully weaned from insulin long-term (>50 days) without ill effects. The implanted mice maintained blood glucose levels just above euglycemic (180-220 mg/dl) and showed no signs of disease. Thus, this technical breakthrough provides new therapeutic approaches to diabetes as an alternative to insulin therapy.

A new method for isolation of cardiac myocytes by percutaneous endomyocardial biopsy.

The objective of this study was to demonstrate the feasibility of isolating viable canine cardiac myocytes from percutaneous right ventricular endomyocardial biopsy specimens. Although histologic data can be obtained from percutaneous endomyocardial biopsies, this approach has not been used as a source of viable cells for evaluating pathological conditions. Study of isolated viable myocytes may provide insight into the electrical, biochemical, and physiologic functions of the heart. Using a standard 8F sheath, a 5F bioptome was introduced via the right femoral vein and advanced to the right ventricle, where 85 biopsies were obtained from 8 mongrel dogs. An average of six biopsy specimens were pooled for processing to provide adequate tissue substrate. This resulted in 14 groups of specimens which were then processed to isolate individual myocytes. Viable myocytes were striated, rod-shaped, and excluded trypan blue dye. Nonviable myocytes were rounded, had no cross-striations, and did stain with trypan blue. Partially-injured myocytes contracted spontaneously and had a region of loss of cross-striations. The average number of viable cells recovered per group of pooled specimens was 1.8 x 10(4) (1.8 x 10(3) cells/mg of tissue). The greatest yield of viable myocytes recovered was 8.0 x 10(4), which represented a viability of 90% by trypan blue dye exclusion and morphological criteria. Percutaneous right ventricular endomyocardial biopsy is a novel method for obtaining viable cardiac myocytes. Its feasibility and utility in humans warrant further investigation.

Autoimmune diabetes-prone NOD mice express the Lyt2 alpha (Lyt2.1) and Lyt3 alpha (Lyt3.1) alleles of CD8.

Predisposition to Type I insulin-dependent diabetes (IDD) has a strong underlying genetic basis involving class II major histocompatibility complex (MHC) genes as well as several non-MHC genetic systems. In the non-obese diabetic (NOD) mouse, a model for human IDD, genes associated with the appearance of immune cell infiltrates in the pancreatic islets (insulitis) and/or overt IDD have been mapped to chromosomes 1, 3, 6, 11, and 17. A recent report has suggested that CD8+ lymphocytes of the NOD mouse might be deficient in the expression of the CD8 beta molecule, a protein encoded by a gene on chromosome 6. The CD8 beta molecule is a T-cell surface marker, the lack of which could affect selection in the thymus, possibly permitting auto-reactive T-cell clones to populate the peripheral lymphoid tissues. For this reason, we examined the expression of the CD8 molecule by lymphocytes in the NOD mouse. Results indicate that the NOD mouse is not deficient in its transcription of detectable mRNA encoding either the CD8 alpha or beta subunits. However, the NOD mouse expresses the Lyt2 alpha and Lyt3 alpha alleles, suggesting that a portion of chromosome 6 centromeric to the diabetes-susceptibility genetic region is derived from an ancestry common to AKR and, like AKR, the CD8 alpha and CD8 beta 3.1 (but not CD8 beta 3.2) subunits are detected on the cell surface of T lymphocytes of the NOD mouse. Interestingly, though, the CD8 beta 3.1 molecule may not be expressed in the NOD mouse to the same extent as it is expressed in the AKR/J mouse, suggesting the possibility that the NOD mouse possesses a defect somewhere between transcription and cell surface expression of the CD8 beta molecule.

Antioxidant enzyme activities in IDD-prone and IDD-resistant mice: a comparative study.

Insulin-dependent diabetes (IDD) in the nonobese diabetic (NOD) mouse is believed to result from the specific autoimmune destruction of pancreatic beta cells. The frequency of diabetes in the NOD mouse is sex-dependent, with approximately 90% of females and 40% of males developing clinical diabetes by 40 weeks of age. Recently, attention has focused on determining possible mechanisms for beta cell destruction. One potential mechanism is the toxic effect of free oxygen radicals produced as a result of the influx of inflammatory cells into the pancreas. A deficiency in available antioxidant enzymes could form a basis for diabetes susceptibility. To test the feasibility of this idea, we have compared the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in isolated islets, pancreas, and other tissues of age- and sex-matched NOD, BALB/c, C57BL/10, and B10.GD mice. Enzyme profiles revealed that female NOD mice do not differ significantly in antioxidant enzyme activity from females of the other inbred strains. However, antioxidant enzyme activity in females was generally lower than in males regardless of mouse strain. While isolated islet cells exhibited somewhat lower levels of enzyme activity than other tissues, the islets of NOD mice proved to be no more deficient than those of BALB/c mice. Therefore, it is unlikely that any toxic effect of free oxygen radicals on the beta cells of NOD mice results directly or solely from an antioxidant enzyme deficiency. Nevertheless, one possible explanation for the lower incidence of diabetes in NOD males versus females may be the inherently higher male antioxidant enzyme activities.

Insulin-dependent diabetes in the NOD mouse model. I. Detection and characterization of autoantibody bound to the surface of pancreatic beta cells prior to development of the insulitis lesion in prediabetic NOD mice.

Type I, insulin-dependent diabetes (IDD) results from an autoimmune response against the insulin producing pancreatic beta cells. This autoimmune reaction involves both humoral and cell-mediated factors; nevertheless, the relative role of each remains unresolved. Furthermore, while adoptive transfer experiments have provided evidence for the role of T cells in beta cell destruction, the specific events which initiate leukocyte migration into the islets (insulitis) are unknown. Earlier studies indicated that NOD pancreatic beta cells may bind small amounts of autoantibody. Because of the possible importance of an early humoral response to the initiation of insulitis and subsequent disease, we have investigated a number of aspects of this phenomenon to determine the nature and specificity of the early autoantibodies as well as the time at which autoantibody binds to beta cells. Results of this study demonstrate that NOD/Uf mice are sensitized to islet-cell associated antigens, including GAD, prior to the first appearance of insulitis; that a small percentage of the beta cells of NOD/Uf mice have autoantibody bound to their surface prior to insulitis; that sera collected from preinsulitis NOD/Uf mice contain autoantibodies which will bind to beta cells of both IDD-prone and IDD-resistant mice; and that the autoantibodies which bind pancreatic beta cells are predominantly IgM with lesser amounts of IgG and IgA. These findings suggest that, in the natural course of IDD, insulitis may develop in response to an initial autoantibody-mediated injury of beta cells.

Insulin-dependent diabetes in the NOD mouse model. II. Beta cell destruction in autoimmune diabetes is a TH2 and not a TH1 mediated event.

Type I, insulin-dependent diabetes (IDD) in both man and animals results from a specific autoimmune destruction of the pancreatic beta cells involving both humoral and cellular immune mechanisms. The pathognomonic histologic lesion, termed insulitis, is an inflammatory and immune cell infiltrate of the pancreatic islet cells. While recent histological and flow cytometric analyses have identified the cell composition of the infiltrate, the presence of a cell population may not reflect the functional reactivities important for beta cell destruction. In the present study, we have investigated the possible functional reactivities of islet-infiltrating mononuclear cell populations by measuring increased cytokine mRNA usage. Results indicate that 1) cytokine mRNA profiles exhibited by islet-infiltrating cells of female and male NOD mice were quite similar with the exception of IL-6 expression and the marked differences in the levels of IL-2 receptor and IL-1 alpha mRNA, 2) CD4+ T lymphocytes expressed IL-4, presumably IL-5, and occasionally IL-10 mRNA but no detectable IL-2 mRNA, 3) CD8+ T lymphocytes exhibited TNF-beta, perforin and high levels of IFN-gamma, and 4) IL-7 was expressed in the islet at very high levels. These findings, together with our earlier flow cytometric analyses of the islet-infiltrating cells, have permitted construction of a detailed model for the natural history of autoimmune diabetes. Interestingly, this model, based on a TH2- and not a TH1-mediated scheme, questions the more popular concepts currently thought to form the bases of the autoimmune reactions underlying IDD.

Cloning and expression of the oxalyl-CoA decarboxylase gene from the bacterium, Oxalobacter formigenes: prospects for gene therapy to control Ca-oxalate kidney stone formation.

Evidence suggests that the formation of calcium-oxalate stones in the urine is dependent on the saturation levels of both calcium and oxalate; thus, management of one or both of these ions in individuals susceptible to urolithiasis appears important. Since there are no known naturally occurring enzymes in vertebrates capable of degrading oxalate, we have initiated a study to insert a plant-derived oxalate degrading enzyme gene into human cells as a means of lowering plasma and urinary oxalate concentrations. We present here the cloning of the oxalyl-CoA decarboxylase gene from the bacterium Oxalobacter formigenes and its subsequent expression in a foreign environment. These results provide the basis for eventual transfer of an oxalate decarboxylase gene into mammalian cells.

Isolation of a low molecular weight inhibitor of lymphocyte proliferation from tumorous ascites.

Intraperitoneal growth of P-815 mastocytoma cells in syngeneic DBA/2 mice produces ascites fluid which strongly inhibits mitogen-stimulated lymphocyte proliferation. The less than 10,000 m.w. fraction from gel filtration chromatography of tumorous ascites on Sephadex G-150 showed no inhibition of proliferation when eluted under physiologic conditions but was inhibitory when eluted with a high ionic strength, acidic buffer. The organic phase of a chloroform/methanol extract of the low m.w. fraction contained all the inhibitory activity. Purification of the inhibitor to relative homogeneity was achieved by reverse phase, HPLC with a gradient of acetonitrile in dilute acetate buffer. Inhibitory activity eluted between 30 and 35% acetonitrile. The active fraction contained less than 30 pg/ml PGE by RIA which was insufficient to inhibit proliferation and may actually have been stimulatory. Inhibition comparable to that produced by the ascites fraction required greater than 300 pg/ml of PGE. This low m.w. (less than 10,000), lipid-like inhibitor of lymphocyte proliferation is acid stable, not sensitive to proteolytic enzymes, soluble in both aqueous and organic solvents and occurs normally bound to a higher m.w. carrier molecule.