Papers by William Osborne
Physiological Genomics, 2010
Rodents homozygous for autosomal leptin receptor gene mutations not only become obese, insulin re... more Rodents homozygous for autosomal leptin receptor gene mutations not only become obese, insulin resistant, and hyperleptinemic but also develop a dysregulated immune system. Using marker-assisted breeding to introgress the Koletsky rat leptin receptor mutant ( lepr−/lepr−), we developed a novel congenic BBDR. lepr−/lepr− rat line to study the development of obesity and type 2 diabetes (T2D) in the BioBreeding (BB) diabetes-resistant (DR) rat. While heterozygous lepr (−/+) or homozygous (+/+) BBDR rats remained lean and metabolically normal, at 3 wk of age all BBDR. lepr−/lepr− rats were obese without hyperglycemia. Between 45 and 70 days of age, male but not female obese rats developed T2D. We had previously developed congenic BBDR. Gimap5−/Gimap5− rats, which carry an autosomal frameshift mutation in the Gimap5 gene linked to lymphopenia and spontaneous development of type 1 diabetes (T1D) without sex differences. Because the autoimmune-mediated destruction of pancreatic islet β-cel...
The Journal of Gene Medicine, 2010
Background Type 1 diabetes (T1D) in both humans and BioBreeding (BB) rats is an autoimmune diseas... more Background Type 1 diabetes (T1D) in both humans and BioBreeding (BB) rats is an autoimmune disease that results in complete destruction of islets and insulin dependency for life. Glucagon-like peptide 1 (GLP-1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. We hypothesized that the expression of GLP-1 before disease onset would increase islet mass, delay diabetes and prolong survival of BB rats. Methods Vascular smooth muscle cells retrovirally transduced to secrete GLP-1 were seeded into TheraCyte encapsulation devices, implanted subcutaneously, and rats were monitored for diabetes. In untreated control rats, plasma GLP-1 levels were 34.5-39.5 pmol/l, whereas, in treated rats, plasma levels were elevated, in the range 90-250.4 pmol/l. Hypoglycemia was not detected and this was anticipated from the glucose-regulated action of GLP-1. Diabetes onset (mean ± SEM) in untreated rats occurred at 56.5 ± 0.6 days (n = 6) and, in GLP-1-treated rats, was delayed until 76.4 ± 3.3 days (n = 5) (p < 0.001). After disease onset, untreated control rats showed a rapid weight loss and elevated blood glucose (>650 mg/dl) and did not survive beyond 11 days. At 5 days after diabetes onset, insulin-secreting islets were absent in untreated rats. By contrast, treated rats maintained weight for up to 143 days of age and showed insulin-secreting β cells. Conclusions Sustained GLP-1 expression delivered by encapsulated cells before diabetes onset in BB rats showed an improved clinical outcome, suggesting the potential for treating patients using long lasting GLP-1 analogs.
Journal of Cellular and Molecular Medicine, 2008
Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to ... more Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte™ immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose >350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte™ devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.
Journal of Bioscience and Bioengineering, 2011
Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major pub... more Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyte TM encapsulation devices, implanted subcutaneously and rats monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3±10.2 pM that was significantly elevated over control values of 32.4±2.9 pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9±2.3 ng/ml that were significantly increased over control levels of 7.3±1.5 ng/ ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with βcell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of β-cells and increased islet mass. These data suggest encapsulated transduced cells may offer a potential long term treatment of patients.
Glucose-Regulated Insulin Expression in Diabetic Rats
Human Gene Therapy, 2001
Retroviral vectors encoding glucose-responsive promoters driving furin expression may provide an ... more Retroviral vectors encoding glucose-responsive promoters driving furin expression may provide an amplified, glucose-regulated secretion of insulin. We constructed LhI*TFSN virus to encode a glucose-regulatable transforming growth factor alpha promoter controlling furin expression with a viral LTR promoter driving constitutive expression of furin-cleavable human proinsulin. Autologous BB rat vascular smooth muscle cells transduced with LhI*TFSN virus and cultured in 1.7 and 16.7 mM glucose secreted 50.7 +/- 3.2 and 136.0 +/- 11.0 microU (mean +/- SD) of insulin per 10(6) cells per day, respectively. After the onset of diabetes spontaneously diabetic congenic DR lyp/lyp BB rats received stomach implants containing 2 x 10(6) LhI*TFSN-transduced primary rat vascular smooth muscle cells. In eight treated rats there was a major reduction in insulin requirement to as low as 25% of pretreatment level for up to 3 months and one rat became insulin free without hypoglycemia. Intraperitoneal glucose tolerance tests (IPGTTs) in diabetic rats receiving control implants did not show the characteristic decline in blood glucose of normal rats after glucose administration. In contrast, diabetic rats receiving LhI*TFSN-transduced cells showed significant clearances of blood glucose. These data suggest clinically significant levels of glucose-regulated insulin delivery from implanted vascular smooth muscle cells transduced with LhI*TFSN vector.
Real time imaging of intracellular hydrogen peroxide in pancreatic islets
The Biochemical journal, Dec 11, 2016
A real time method to measure intracellular H2O2 would be very impactful in characterizing rapid ... more A real time method to measure intracellular H2O2 would be very impactful in characterizing rapid changes that occur in physiologic and pathophysiologic states. Current methods do not provide the sensitivity, specificity and spatiotemporal resolution needed for such experiments on intact cells. We developed the use of HyPer, a genetic indicator for H2O2 that can be expressed in the cytosol (cyto-HyPer) or the mitochondria (mito-HyPer) of live cells. INS-1 cells or islets were permeabilized and the cytosolic HyPer signal was a linear function of extracellular H2O2, allowing fluorescent cyto-HyPer signals to be converted to H2O2 concentrations. Glucose increased cytosolic H2O2, an effect that was suppressed by overexpression of catalase. Large perturbations in pH can influence the HyPer signal, but inclusion of HEPES in the perfusate prevented pH changes, but did not affect glucose-induced cyto-HyPer signals suggesting this effect is largely pH-independent. Using the assay, two fundame...
Archives of Biochemistry and Biophysics, Apr 30, 1984
Studies were undertaken on the heat denaturation and proteolytic degradation by cY-chymotrypsin o... more Studies were undertaken on the heat denaturation and proteolytic degradation by cY-chymotrypsin of the normal red cell carbonic anhydrase isozyme, CA II, and two electrophoretic variants of carbonic anhydrase I, CA Ia and CA Ib, of the pigtail macaque. The heat degradation results showed a difference of about 40-fold in the rate constants between CA Ia and CA Ib, which is due to the marked thermostability of CA Ib compared to CA Ia. The enthalpies and entropies of activation were calculated from the heat denaturation constants. These values were compared, on enthalpy-entropy compensation plots, with those values previously determined for the human CA I and CA II isozymes. They were highly correlated and clearly fell into two distinct clusters, separated by about 200 kJ mol-'; one group comprising the macaque and human CA I isozymes and the other the CA II isozymes. The proteolytic degradation results showed that CA Ia is degraded about 2.5 times more rapidly than CA Ib by cY-chymotrypsin. Thus, the characteristic 3/l ratio of CA IWCA Ia in mature red cells could be accounted for by the greater susceptibility of CA Ia to degradation at some stage in red cell development.
Long-Term Expression of Human Adenosine Deaminase in Mice after Transplantation of Bone Marrow Infected with Amphotropic Retroviral Vectors
Http Dx Doi Org 10 1089 Hum 1990 1 1 31, Jun 12, 2008
Page 1. HUMAN GENE THERAPY 1:31-41 (1990) Mary Ann Liebert, Inc., Publishers Long-Term Expression... more Page 1. HUMAN GENE THERAPY 1:31-41 (1990) Mary Ann Liebert, Inc., Publishers Long-Term Expression of Human Adenosine Deaminase in Mice after Transplantation of Bone Marrow Infected with Amphotropic Retroviral Vectors ...
Inhibtion of the enzyme purine nucleoside phosphorylase (PNP) reduces refractioriness to transfused platelets in a dog model
British Journal of Haematology, 1990
Background/Aims: Lentiviral vectors were designed to obtain efficient transduction of primary qui... more Background/Aims: Lentiviral vectors were designed to obtain efficient transduction of primary quiescent hepatocytes. Methods: A hepatitis B virus (HBV) fragment containing enhancers and posttranscriptional regulatory element was used to increase expression levels. The human immunodeficiency virus (HIV) central polypurine tract (PPT) was used to increase transduction of quiescent cells. HBV elements were incorporated downstream and the HIV PPT was incorporated upstream of green fluorescent protein expression cassettes in third generation self inactivating lentiviral vectors. Results: The HBV fragment increased mean fluorescence of transduced HepG2 hepatoma cells 4.3^1.7-fold and 2.3-6.0-fold in various other cell types. A role of HBV £ £ £ protein in the function of the HBV element was excluded. The HBV element increased the number of transducing units per pg of HIV p24 twofold. The unmodified lentiviral vector transduced 5^1% of cultured quiescent primary rat hepatocytes, HBV elements increased transduction to 54^13% and increased fluorescence 2.8^0.6-fold. The PPT increased transduction to 47^11% and increased fluorescence 2.3^0.4-fold. The vector with PPT and HBV elements transduced 68^10% of hepatocytes and increased fluorescence synergistically, 17^6 fold. Conclusions: This study shows that HBV elements or HIV PPT are required for efficient transduction of primary hepatocytes.
Gene Transfer in Baboons Using Prosthetic Vascular Grafts Seeded with Retrovirally Transduced Smooth Muscle Cells: A Model for Local and Systemic Gene Therapy
Http Dx Doi Org 10 1089 Hum 1994 5 10 1211, Mar 19, 2008
Prosthetic vascular grafts containing retrovirally transduced autologous vascular smooth muscle c... more Prosthetic vascular grafts containing retrovirally transduced autologous vascular smooth muscle cells were studied as a model for introduction of human genes into baboons. Retroviral vectors encoding beta-galactosidase (beta-Gal) (LNPoZ) or human purine nucleoside phosphorylase (LPNSN-2), a control gene, were used for ex vivo transduction of autologous baboon smooth muscle cells obtained from vein biopsies. Transduced cells were placed into a collagen solution and seeded into the interstices of polytetrafluoroethylene vascular grafts. Endothelial cells were then seeded onto the luminal surface of the grafts to reduce thrombus formation. One LNPoZ-seeded graft and one LPNSN-2-seeded control graft were implanted bilaterally into the aorto-iliac circulation of each of 4 animals. All grafts remained patent until they were removed after 3-5 weeks and examined histochemically for vector-expressing cells. All histological cross-sections from the beta-Gal vector seeded grafts contained cells staining blue with the X-Gal chromogen. For the four grafts, the mean fraction of LNPoZ expressing cells was 10%, with a range of 2-20%, while no sections from the control grafts contained stainable cells. Smooth muscle cells expressing the reporter gene were localized within the graft wall but not in the newly forming intima or outer capsule of fibrous tissue. Implantation of transduced cells within this type of vascular graft may provide a useful approach for long-term local and systemic gene therapy.
Transfected Cells and Methods for Treating Diabetes
Life Sci, 2007
In this report we describe development and characterization of four human cell lines that are abl... more In this report we describe development and characterization of four human cell lines that are able to secrete insulin and C-peptide in response to higher concentrations of glucose. These cell lines have been developed by stably and constitutively expressing human proinsulin with a furincleavable site, whereas expression of furin is regulated by glucose concentration. These cell lines have been cloned and, therefore, the transgene in each cell is located in an identical location of the genome leading to a uniform expression. Cloning has also allowed us to identify cell lines with more desirable properties such as higher basal insulin secretion and/or better glucose responsiveness. We have further shown that the insulin produced by these cells is biologically active and induces normoglycemia when injected in diabetic animals. Our objective in initiating these studies was to identify a cell line that could serve as a surrogate beta cell line for therapeutic intervention in type I diabetic patients.
Cap-independent multicistronic retroviral vectors
R-Region cDNA Inserts in Retroviral Vectors Are Compatible with Virus Replication and High-Level Protein Synthesis from the Insert
Http Dx Doi Org 10 1089 Hum 1995 6 9 1169, Mar 19, 2008
Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by ... more Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.
Cyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is chara... more Cyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is characterized by recurrent severe neutropenia leading to bacterial infections and shortened life expectancy. Daily injections of recombinant granulocyte colony-stimulating factor (rG-CSF) are effective in shortening the period of severe neutropenia and reducing infections. After demonstrating that rG-CSF induced elevated neutrophil production in an affected dog, cytokine administration was stopped and 10 9 infectious units (IUs) of a lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding canine G-CSF cDNA was administered intramuscularly. Serial blood cell counts showed elevated neutrophil production for longer than 17 months. Although neutrophil counts continued to cycle, the range at nadirs was from 3710 to 5300 cells/L, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts ؎ SD were 12 460 ؎ 4240 cells/ L, significantly increased over both pretreatment values of 3040 ؎ 2540 cells/L (P < .0001) and neutrophil counts during G-CSF administration of 10 290 ؎ 4860 cells/L (P < .007). The changes in blood counts from lentivirus injection were associated with absence of clinical signs of infection and fever. The gray collie continued to gain weight and was no longer housed in a pathogen-free environment. Genomic DNA from muscle at injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, leukocytes, and noninjected muscle samples were all negative for provirus. Thus, intramuscular administration of lentivirus encoding G-CSF provided sustained therapeutic levels of neutrophils, suggesting this approach may be applied for long-term treatment of patients with cyclic and other neutropenias. (Blood.
Purine Nucleoside Phosphorylase Deficiency: A Molecular Model for Selective Loss of T Cell Function1
The Journal of Immunology, Jun 1, 1979
... UTE H. OCHS, SHI-HAN CHEN, HANS D. OCHS, 2 WILLIAM RA OSBORNE, AND C. RONALD SCOTT From the D... more ... UTE H. OCHS, SHI-HAN CHEN, HANS D. OCHS, 2 WILLIAM RA OSBORNE, AND C. RONALD SCOTT From the Department of Pediatrics, University of Washington, School of Medicine, Seattle, Washington 98195 Absence ...
An adult dog with cyclic neutropenia treated by lentivirus- mediated delivery of granulocyte colony-stimulating factor
Http Dx Doi Org 10 1089 Hum 2006 17 464, Apr 1, 2006
Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropen... more Cyclic neutropenia occurs in humans and gray collie dogs, is characterized by recurrent neutropenia, and is treated by daily injections of recombinant granulocyte colony-stimulating factor (G-CSF). After showing that canine recombinant G-CSF increased neutrophil counts in an affected dog, we administered intramuscularly 2 x 10(9) infectious units (IU) of a lentiviral vector encoding canine G-CSF cDNA. Elevated, therapeutic neutrophil production was obtained for nearly 18 months. Lentiviral vector treatment provided a mean neutrophil count of 29,230 +/- 12,930 cells/microl, which was significantly increased over both the pretreatment value (5,240 +/- 4,800 cells/microl; p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001) and the neutrophil count during G-CSF administration (17,820 +/- 11,100 cells/microl; p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001). By systemic infusion of recombinant G-CSF to normal dogs we estimated that 2 x 10(9) IU of lentivirus delivered 3.5 microg of G-CSF per kilogram per day. After lentiviral vector treatment the gray collie gained weight, showed no clinical signs of infection and fever, and no longer needed housing in a pathogen-free environment. Genomic DNA harvested from muscle at the injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, and noninjected muscle samples were negative. These studies show that an adult animal is responsive long-term to lentivirus-mediated G-CSF delivery, suggesting this approach may be applied for treatment of adult patients with cyclic and other neutropenias.
Evolution of carbonic anhydrase isozymes
Isozymes, 1975
Uploads
Papers by William Osborne