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S E Raper

Publications and source records attributed to S E Raper.

At least 37 records · Page 2Linked to original sources
PubMed1997

Engineering tissue-specific expression of a recombinant adenovirus: selective transgene transcription in the pancreas using the amylase promoter.

Recombinant adenovirus accomplishes highly efficient gene transfer in vivo. Adenoviral vectors would be more attractive vehicles for gene therapy if transgene expression was inducible and restricted to the target tissue. In these studies, we hypothesized that selective transgene expression of a recombinant adenovirus could be accomplished by using a tissue-specific promoter of transcription. A replication-defective adenoviral vector was engineered to express the lacZ marker gene under control of the murine pancreatic amylase promoter. Expression of this vector occurred exclusively in the pancreas in neonatal and adult mice, while a similar vector with a constitutive promoter accomplished transgene expression in several organs. Within the adenoviral construct, the amylase promoter retained its ability to be induced by dexamethasone and insulin. This model will serve as a paradigm for selective and inducible adenoviral transgene expression.

Adenoviridae↗
PubMed1997

Efficient adenoviral-mediated murine neonatal small intestinal gene transfer is dependent on alpha(v) integrin expression.

BACKGROUND/PURPOSE: Clinical application of gene therapy for patients who have inflammatory bowel disease or short bowel syndrome will require the development of new strategies to improve the efficiency of small intestinal gene transfer. Previously, the authors developed a method for adenoviral-mediated small intestinal gene transfer in vivo in neonatal and adult mice. The present study evaluates the hypothesis that the integrins alpha(v)beta3 and alpha(v)beta5, the secondary receptors for adenoviral internalization, play a facilitative role in neonatal murine adenoviral-mediated small intestinal gene transfer. METHODS: Immunohistochemical techniques identified the integrin alpha(v)beta3 and the integrin subcomponents alpha(v), beta3, and beta5 in neonatal and adult small intestine. The effects of integrin receptor antagonists on transgene expression was also studied in our neonatal model of adenoviral-mediated small intestinal gene transfer in vivo. RESULTS: Gene transfer was significantly decreased by the addition of integrin receptor antagonists versus control peptide. Integrin alpha(v)beta3 and integrin subcomponent alpha(v), beta3, and beta5 are expressed in neonatal and adult small intestine. Integrin antagonists administered simultaneously blocked efficient adenoviral-mediated neonatal small intestinal gene transfer in vivo compared with control peptide. CONCLUSION: Strategies to upregulate integrin expression may improve adenoviral-mediated small intestinal gene transfer.

Adenoviruses, Human↗
PubMed1997

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Animals↗
PubMed1997

Effect of adenoviral early genes and the host immune system on in vivo pancreatic gene transfer in the mouse.

Gene transfer technology may provide a novel approach to treatment for pancreatic diseases. Recombinant adenovirus achieves efficient gene transfer in vivo. In this study, a murine model of adenoviral-mediated pancreatic gene transfer was developed, and the factors responsible for adenoviral elimination were investigated. Three days after direct pancreatic injection of a replication-defective adenovirus containing the lacZ transgene, a high proportion (76.8 +/- 6.7%) of pancreatic cells expressed beta-galactosidase, the gene product. Gene expression was absent by 28 days posttransduction. In immunodeficient mice, beta-galactosidase expression persisted with 20.0 +/- 6.0% of pancreatic cells staining positive 60 days after viral transduction. To test whether early viral proteins are the antigenic components responsible for the potent antiviral immune response, normal mice were injected with different adenoviral vectors containing early gene deletions. Vectors containing deletions in early region 2 or 4 expressed beta-galactosidase at 28 days. Presently available adenoviral vectors engineered to avoid this response offer minimal improvements in transgene duration. Further vector modifications or alternative strategies are needed to achieve stable pancreatic adenoviral transgene expression.

Adenoviridae↗
PubMed1996

Targeted retroviral gene transfer into the rat biliary tract.

The ability to induce proliferation by temporary duct ligation suggested an hypothesis that retrovirus-mediated gene transfer into cells of the biliary tract could be accomplished. The time course of histologic changes, incorporation of 3H-thymidine and immunofluorescent staining with a monoclonal antibody to cytokeratin-19 (a marker for differentiated bile ducts) was studied in male Fischer F344 rats. A recombinant Gibbon ape leukemia virus (GALV), containing a gene encoding Escherichia coli beta-galactosidase was next introduced into 24 hr obstructed bile ducts. Gene transfer was maximal when virus was exposed to the obstructed duct for 12 hr (approximately 0.1%). The majority of X-gal positive cells were in cytokeratin-19 negative peribiliary tissues, which had the appearance of newly forming bile ducts. The data suggest that cells targeted by retroviral infection of the obstructed rat bile duct may be a precursor of mature, fully differentiated biliary epithelium.

Animals↗
PubMed1996

Liver-directed gene therapy.

Liver-directed gene therapy represents a promising new modality for the treatment of inherited and acquired liver diseases. Clinical trials of liver-directed gene therapy are underway for diseases such as FH, OTC deficiency, and cancer. The main obstacles to effective gene therapy are the limitations of present gene delivery technology to express a desired gene safely and stably at therapeutic levels. With improved gene delivery technology and refinements in the ex vivo and in vivo approaches, a truly useful clinical tool will emerge.

Ethics, Medical↗
PubMed1996

Safety and feasibility of liver-directed ex vivo gene therapy for homozygous familial hypercholesterolemia.

OBJECTIVE: The purpose of this report was to provide detailed information on the safety and feasibility of surgical procedures associated with the first ex vivo liver-directed gene therapy trial for the treatment of vivo gene therapy for homozygous familial hypercholesterolemia (FH). SUMMARY BACKGROUND DATA: Familial hypercholesterolemia is an autosomal dominant disease in which the gene encoding the low density lipoprotein receptor is defective. Patients homozygous for this mutation have extraordinarily high levels of cholesterol and accelerated atherosclerosis and die prematurely of myocardial infarction. The concept of liver-directed gene therapy was based on the report of normalization of cholesterol levels by orthotopic cardiac/liver transplant in a child with homozygous FH. METHODS: Five patients with homozygous FH were selected for inclusion in this trial. The patients underwent hepatic resection and placement of a portal venous catheter. Primary hepatocytes cultures were prepared from the resected liver and transduced with a recombinant retrovirus encoding the gene for the human low density lipoprotein receptor. The genetically modified cells were then transplanted into the liver through the portal venous catheter. RESULTS: Numerous clinical, laboratory, and radiologic parameters were analyzed. Elevations of the hepatic transaminases and leukocyte counts and a decline in hematocrit count were noted. Transient elevations of the portal pressure were observed during cell infusion. No major perioperative morbidity--specifically, myocardial infarct, perioperative hemorrhage, or portal vein thrombosis--or death occurred as a result of this protocol. CONCLUSION: Liver-directed ex vivo gene therapy can be accomplished safely in humans and is appropriate for selected patients.

Adult↗
PubMed1996

Adenovirus-mediated in vivo gene transfer and expression in normal rat pancreas.

Gene transfer into the pancreas would be useful for the treatment of a variety of disorders, including cystic fibrosis, diabetes, cancer, and immunomodulation of pancreatic allografts. A hypothesis that various cell populations in the pancreas could be targeted by recombinant adenoviruses was developed and tested. Gene transfer into the rat ductal epithelium, acinar cells, and islets of Langerhans was accomplished with a recombinant adenovirus containing bacterial beta-galactosidase by retrograde delivery of adenovirus into the pancreaticobiliary duct. Maximal gene expression was observed at 3 days and correlated with DNA blot analysis. Histologic analysis of sections from pancreatic tissue in the adenovirus-treated rats demonstrated severe pancreatitis. Immunophenotyping of the inflammatory infiltrate with rat lymphocyte-specific markers showed CD45-, CD8-, and CD4-positive cells. Tissue injury resolved as gene expression was lost, with both features absent by 21 days. Pancreatic regeneration was documented by the presence of 5-bromo-2'-deoxyuridine-positive staining cells. Pancreatic gene transfer with first-generation recombinant adenoviruses can be accomplished by techniques applicable to clinical situations. The use of first-generation recombinant adenoviruses for pancreas-directed gene transfer is limited by the development of inflammation and transient expression.

Adenoviridae↗
PubMed1996

Prolongation of adenoviral transgene expression in mouse liver by T lymphocyte subset depletion.

Although first generation recombinant adenoviruses are efficient vehicles for gene transfer, their immunogenicity precludes long-term persistence. We show that adenoviral transgene expression in the liver of normal mice is prolonged from a baseline of less than 2 weeks to 7 weeks by depleting CD4+ T lymphocytes with thymectomy and a 3-day course of anti-CD4 monoclonal antibody or by nonselectively depleting T cells with a single dose of anti-thymocyte serum (ATS). Transgene expression persisted despite the development of an antiviral humoral immune response by 3 weeks after virus administration. In vitro assays of T lymphocyte function revealed an initial diminished capacity to proliferate in the presence of adenoviral antigens in animals depleted of CD4+ T cells or given anti-thymocyte serum. Eventual loss of recombinant gene expression coincided with the development of adenovirus-specific cytotoxic T lymphocyte activity in vitro. Immunosuppression provides a useful experimental tool to elucidate the immunobiology of recombinant adenoviruses and may have clinical application to adenovirus-mediated gene therapy.

Adenoviruses, Human↗
PubMed1995

Preferential suppression of insulin-stimulated proliferation of cultured hepatocytes by somatostatin: evidence for receptor-mediated growth regulation.

The role of somatostatin (SS-14) in the regulation of rat liver regeneration was examined by using thymidine incorporation into hepatocyte DNA labeled with tritiated thymidine, a nuclear-labeling index, and the binding of 125I-tyr11-SS-14 to hepatocytes isolated at various times after partial hepatectomy. The data demonstrated no suppressive effect of SS-14 on insulin and glucagon-stimulated thymidine incorporation into hepatocyte DNA as early as 2 h after partial hepatectomy. These data were substantiated by a nuclear labeling index studies. At 2 h, 125I-tyr11-SS-14 binding to its specific sites on isolated hepatocytes was undetectable. There was a time-dependent increase in binding of 125I-tyr11-SS-14 to hepatocytes obtained at various times after partial hepatectomy. There was a significant decrease in the number of binding sites after partial hepatectomy as determined by Scatchard analysis. The data were supported by autoradiography analysis of affinity labeled 125I-tyr11-SS-14-binding protein complex followed by SDS-PAGE. SS-14 also inhibited intracellular cAMP in hepatocytes obtained at 18 h after hepatectomy. The data are consistent with the hypothesis that SS-14 participates via its own receptor in the regulation of the liver regeneration.

Animals↗
PubMed1995

Impaired phorbol ester-induced hepatocyte proliferation in cirrhosis.

Cirrhotic livers are considered to regenerate less actively than normal livers after hepatic resection. Little is known about the mechanisms responsible for impaired capacity of regeneration in cirrhotic liver. In the present study, we investigated the effect of phorbol ester on hepatocyte proliferation in healthy and cirrhotic hepatocytes, using one of the phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which has a direct effect on activation of protein kinase C (PKC). Cirrhosis was established by the administration of carbon tetrachloride and phenobarbital to rats. Healthy and cirrhotic hepatocytes were isolated from Wistar male rats by a two-step collagenase perfusion technique. DNA synthesis was estimated by [3H]thymidine incorporation into DNA and by autoradiographic nuclear labeling index. [3H]Thymidine incorporation was measured 24 hr after hepatocytes were stimulated by appropriate reagents. TPA (50 nM) stimulated [3H]thymidine incorporation in healthy hepatocytes (control vs TPA, 991 +/- 247 vs 2569 +/- 766 mean +/- SEM cpm/microgram DNA; P < 0.05), whereas TPA (50 nM) failed to stimulate in cirrhotic hepatocytes (control vs TPA, 1144 +/- 184 vs 1304 +/- 187 cpm/microgram DNA; NS). Staurosporine, a specific PKC inhibitor, suppressed [3H]thymidine incorporation in TPA-stimulated healthy hepatocytes (806 +/- 263 cpm/microgram DNA; P < 0.05); however, it had no effect on cirrhotic hepatocytes (1295 +/- 180 cpm/microgram DNA; NS). An autoradiographic nuclear labeling index exhibited the same results with [3H]thymidine incorporation. We conclude that TPA stimulates hepatocyte proliferation in healthy rat hepatocytes but has no effect on cirrhotic hepatocytes.

Alkaloids↗
PubMed1995

A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia.

The outcome of the first pilot study of liver-directed gene therapy is reported here. Five patients with homozygous familial hypercholesterolaemia (FH) ranging in age from 7 to 41 years were enrolled; each patient tolerated the procedure well without significant complications. Transgene expression was detected in a limited number of hepatocytes of liver tissue harvested four months after gene transfer from all five patients. Significant and prolonged reductions in low density lipoprotein (LDL) cholesterol were demonstrated in three of five patients; in vivo LDL catabolism was increased 53% following gene therapy in a receptor negative patient, who realized a reduction in serum LDL equal to approximately 150 mg dl-1. This study demonstrates the feasibility of engrafting limited numbers of retrovirus-transduced hepatocytes without morbidity and achieving persistent gene expression lasting at least four months after gene therapy. The variable metabolic responses observed following low-level genetic reconstitution in the five patients studied precludes a broader application of liver-directed gene therapy without modifications that consistently effect substantially greater gene transfer.

Adult↗
PubMed1995

Gene transfer to the thymus. A means of abrogating the immune response to recombinant adenovirus.

OBJECTIVE: The authors investigated whether adenoviral gene transfer to the thymus could be accomplished in vivo and whether immunologic unresponsiveness to recombinant adenovirus could be induced by intrathymic inoculation. SUMMARY BACKGROUND DATA: A major barrier to the clinical application of adenovirus-mediated gene therapy for diseases requiring long-lasting gene expression is the immunogenicity of adenoviral vectors, which limits the duration of its effects. In other experimental models, intrathymic inoculation of foreign proteins or cells has proven to be an effective means to induce immunologic tolerance. METHODS: The efficiency of gene transfer to the mouse thymus after direct inoculation of recombinant adenovirus was compared with that of several other vectors. Animals inoculated with adenovirus-infected pancreatic islets into the thymus were tested for unresponsiveness to the virus with a subsequent challenge of adenovirus administered into the liver by intravenous injection. RESULTS: Adenovirus accomplished highly efficient gene transfer to the thymus, unlike plasmid DNA, DNA-liposome complexes, retrovirus, and adeno-associated virus. Adenoviral transgene expression was transient in the thymus of immunocompetent mice but persistent in CD8+ T-cell-deficient and severe combined immunodeficiency (SCID) mice, implicating the role of cytotoxic T lymphocytes in viral clearance. Intrathymic transplantation of syngeneic pancreatic islet cells infected with adenovirus impaired the normal antiviral cytotoxic T-lymphocyte response and prolonged hepatic transgene expression after an intravenous challenge with adenovirus. CONCLUSIONS: Recombinant adenovirus accomplishes highly efficient gene transfer to the thymus in vivo. Intrathymic inoculation of adenovirus-infected islets can be used to induce immunologic unresponsiveness to the adenoviral vector and, potentially, to other proteins that it might be engineered to encode.

Adenoviridae↗
PubMed1995

Are traditional prognostic criteria useful in pancreatic abscess?

Pancreatic abscess remains a potentially lethal disease. Efforts to relate outcome to the severity of associated pancreatitis or the type of surgical drainage employed have yielded conflicting results. This study was designed to test the validity of traditional prognostic criteria in the clinical setting of pancreatic abscess and to determine whether the technique of surgical drainage employed correlated with survival. The records of 40 consecutive patients with pancreatic abscess were reviewed. In each case the diagnosis was confirmed by operation. Prognostic factors analyzed included number of Ranson criteria, etiology, type, and number of microorganisms isolated, extent of abscess, time to diagnosis and operation, and technique of surgical drainage. Of the 11 Ranson criteria evaluated, only an elevation in blood urea nitrogen > 5 mg/dl correlated with decreased survival (p < 0.001). Polymicrobial abscesses (three or more organisms) resulted in a higher mortality than abscesses where fewer than three organisms were isolated (45.4 vs 13.8%; p < 0.05). Intraperitoneal extension of the abscess was associated with an increased mortality rate compared to those confined to the retroperitoneum (57.1 vs 15.2%; p < 0.01). In patients requiring unplanned reexploration, mortality was significantly increased (42.9 vs 11.5%; p < 0.05). The technique of surgical drainage employed (open versus closed) did not influence overall mortality (23.5 vs 21.7%; p = NS). Extent of disease at operation, polymicrobial abscess, reexploration for persistent or recurrent disease, and deterioration in renal function were all predictive of increased mortality in cases of pancreatic abscess.(ABSTRACT TRUNCATED AT 250 WORDS)

Abscess↗