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Biomedical subjects

G Kurtzman

Publications and source records attributed to G Kurtzman.

14 recordsLinked to original sources
PubMed1999

Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations.

Animals↗
PubMed1998

Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Animals↗
PubMed1998

Adeno-associated virus vector containing the herpes simplex virus thymidine kinase gene causes complete regression of intracerebrally implanted human gliomas in mice, in conjunction with ganciclovir administration.

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene therapy because of its wide host range, high transduction efficiency, and lack of cytopathogenicity. In this experiment, we evaluated the efficacy of AAV vector containing the herpes simplex virus thymidine kinase (HSV-tk) gene on human gliomas transplanted into the brain of nude mice. Complete regression of the tumors was observed after multiple AAV-tk injections followed by intraperitoneal ganciclovir (GCV) administration, and the survival of mice treated with AAV-tk vector and GCV administration was markedly prolonged. These results suggest that AAV-tk vectors may be useful for gene therapy against malignant gliomas in humans.

Animals↗
PubMed1997

Gene transfer into vascular cells using adeno-associated virus (AAV) vectors.

OBJECTIVES: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. METHODS: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining. RESULTS: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. CONCLUSIONS: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

Aorta, Thoracic↗
PubMed1996

Gene therapy against an experimental glioma using adeno-associated virus vectors.

The efficacy of gene therapy for glioma was examined using adeno-associated virus (AAV)-based vectors to deliver genes to experimental tumors in mice. Stereotactic injection of 2 x 10(5) U-251SP human glioma cells into the brains of nude mice produced tumors of 19.06 +/- 1.79 mm2 17 days after injection. Employing a high titer preparation of AAV vector containing the gene for beta-galactosidase (AAV-lacZ), dose-dependent transduction of U-251SP cells was seen in vitro. When 1.6 x 10(10) AAV-lacZ particles were directly injected into tumors in vivo, 30-40% of the cells along the needle track expressed beta-galactosidase. Transduction of U-251SP cells in vitro with an AAV vector containing a bicistronic gene encoding both herpes simplex thymidine kinase and human interleukin-2 (AAV-tk-IRES-IL2) rendered them sensitive to the cytocidal effects of ganciclovir (GCV) and IL-2 was produced in a dose-dependent manner. Cocultures of AAV-tk-IRES-IL2 transduced cells and nontransduced cells proved highly sensitive to GCV indicating the contribution of the bystander effect. Stereotactic delivery of 6 x 10(10) AAV-tk-IRES-IL2 particles into day 7 tumors in nude mice followed by administration of GCV for 6 days, resulted in a 35-fold reduction in the mean volume of tumors compared with controls. Normal brains did not suffer from any toxic effect of the administration of AAV-tk-IRES-IL2 and GCV. These results indicate that high titer AAV vector treatment may be safe and effective for in vivo gene therapy of human brain tumors.

Animals↗
PubMed1992

Nosocomial human parvovirus B19 infection: lack of transmission from a chronically infected patient to hospital staff.

OBJECTIVE: To assess the potential for nosocomial spread of parvovirus B19 from a chronically infected patient. DESIGN: Employees exposed to the index case and control (unexposed) employees were evaluated by baseline and follow up parvovirus B19 serologies and hematologic assessments, and completed baseline and follow up epidemiologic questionnaires. SETTING: A chronically infected patient was hospitalized on a hematology ward in a research referral hospital for 3.5 weeks prior to a diagnosis of parvovirus B19 infection and the institution of isolation precautions. METHODS: Sera were screened for parvovirus B19 DNA (dot blot analysis), and IgG and IgM anti-B19 antibodies (capture immunoassay). Hematologic assessment included CBC, differential, and reticulocyte count. RESULTS: The index case had parvovirus B19 DNA at approximately 10(6) genome copies per ml of serum, elevated IgM and low levels of IgG B19 antibodies. Of the 21 exposed staff, 11 (52%) had IgG B19 antibodies and were immune; of the 8 unexposed staff, 6 (75%) had IgG B19 antibodies. No employees developed IgM B19 antibodies, B19 DNA, hematologic abnormalities, or clinical symptoms. CONCLUSIONS: In contrast to reports of documented nosocomial transmission of B19 parvovirus from patients in transient aplastic crisis, nosocomial transmission did not occur--even in the absence of isolation precautions--presumably from the lower level of B19 viremia in our chronically infected (rather than acutely infected) patient.

Antibodies, Viral↗
PubMed1989

Viruses and bone marrow failure.

Some generalizations can be drawn from a review of virus-associated bone marrow failure. The story of B19 parvovirus illustrates that viral infection may be an occult cause of marrow failure. Although the epidemiology of transient aplastic crisis suggested a viral aetiology, the implication of a single virus was surprising; the sporadic appearance of chronic bone marrow failure in immunosuppressed persons has had none of the features of a viral illness. The incrimination of parvovirus in these cases required development of specific immunological and molecular assays. Human and animal retrovirus studies have shown that small changes in the virus genome can have dramatic effects on the biology of the infectious agent and its pathogenicity in infected hosts. In Epstein-Barr virus infection, the host's immune response may play a more important role in mediating disease than virus cytotoxicity. Finally, the association of aplastic anaemia with hepatitis may be underestimated because of the inability to diagnose virus infection without obvious liver disease. The true spectrum of bone marrow disease due to virus infection is not known.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Novel transcription map for the B19 (human) pathogenic parvovirus.

The B19 parvovirus, a small single-stranded DNA virus of 5.4 kilobases, is pathogenic in humans. B19 has remarkable specificity for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 transcription had unusual features in comparison with that in other animal parvoviruses. At least nine overlapping poly(A)+ transcripts were identified in infected cells; all but one contained large introns. B19 differed from other parvoviruses in the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter; the presence of short 5' leader sequences of about 60 bases and very large introns for RNAs encoded by the right side of the genome; two separate transcription termination sites, in contrast to cotermination at the far right side of the genome for other parvoviruses; the probable utilization by three transcripts of a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome; and the abundance of two unique transcripts from the middle of the genome which did not code for capsid proteins. The unusual transcription map of B19 suggests that regulation of the relative abundance of transcripts occurs by splicing and termination-polyadenylation events rather than by promoter strength. In combination with the published nucleotide sequence, the novel transcription map separated the pathogenic B19 virus at a molecular level from other animal parvoviruses and human adeno-associated virus.

Bone Marrow↗
PubMed1987

Productive infection by B19 parvovirus of human erythroid bone marrow cells in vitro.

B19 parvovirus, the cause of fifth disease and transient aplastic crisis, has been successfully propagated in suspension cultures of human erythroid bone marrow cells obtained from patients with sickle cell disease and stimulated by erythropoietin. B19 inoculation in vitro resulted in a marked decline in identifiable erythroid cells over seven to nine days of incubation. Characteristic giant early erythroid cells were seen on Wright's-Giemsa stain of infected cultures. By in situ hybridization, 30% to 40% of erythroblasts were infected at 48 hours; a similar proportion of cells showed B19 capsid protein by immunofluorescence. B19 DNA was present in erythroblasts but not in the leukocyte fraction of bone marrow. B19 replication, as determined by Southern analysis, and B19 encapsidation, as determined by sensitivity of isolated cell fractions to DNase I, were restricted to the nuclei. B19 DNA was detectable in the nuclei from infected cultures beginning at 18 hours and in the supernatant at 32 hours; B19 genome copy number was estimated at about 25,000 to 30,000/infected cell at 48 hours. Recovery of virus depended on the multiplicity of infection (moi); at low moi, approximately 200x input virus was recovered from total cultures and 50x from the culture supernatants. Virus released into the supernatant was as infectious or more infectious than virus obtained from sera of infected patients. Human erythroid bone marrow culture represents a safe in vitro system for the elucidation of the cellular and molecular biology of the pathogenic B19 parvovirus.

Bone Marrow↗
PubMed1986

Replication of the B19 parvovirus in human bone marrow cell cultures.

The B19 parvovirus is responsible for at least three human diseases. The virus was successfully propagated in suspension cultures of human erythroid bone marrow from patients with hemolytic anemias; release of newly synthesized virus into the supernatants of infected cultures was observed. This culture system allowed study at a molecular level of events associated with the B19 life cycle. The B19 parvovirus replicated through high molecular weight intermediate forms, linked through a terminal hairpin structure. B19 replication in vitro was highly dependent on the erythropoietic content of cultures and on addition of the hormone erythropoietin.

Anemia, Hemolytic↗