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R J Gregory

Publications and source records attributed to R J Gregory.

At least 19 recordsLinked to original sources
PubMed1997

Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles.

The expanding use of adenoviral vectors for gene therapy has brought about the need for new analytical tools. We have developed an anion-exchange high-performance liquid chromatography method to analyze recombinant adenovirus serotype 5 samples. Before this assay, available analytical methods consisted of either long-term biological assays or required highly purified test articles. These methods were inadequate for optimizing adenovirus production and purification. This assay can quantitate viral particles in either crude lysates or highly pure samples. It can be used to assess particles in both dilute and concentrated samples over a wide dynamic range. Moreover, the population of viral particles eluted in the peak contains most of the infectious virions. This assay is a sensitive technique that overcomes the limitations of previous methods. It provides an essential tool to accomplish process optimization.

Adenoviridae↗
PubMed1997

Effect of the E4 region on the persistence of transgene expression from adenovirus vectors.

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.

Adenoviridae↗
PubMed1996

Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo.

Alterations in the p53 tumor-suppressor gene occur in 35-60% of human glioblastomas, and re-introduction of p53 can suppress neoplastic growth. To evaluate the potential for p53 gene therapy of glioblastoma, we have analyzed the response of human glioblastoma cell lines in vitro and in vivo to experimental therapy with replication-deficient recombinant adenoviruses encoding wild-type p53 (rAd-p53). Western blot analyses showed high-level expression of p53 protein after treatment with rAd-p53, and transgene expression was dependent on promoter strength. A p53-specific dose-dependent inhibition of in vitro cellular proliferation was observed in 5 of 6 cell lines, and growth inhibition corresponded to adenovirus-mediated gene transfer and expression. p53-specific cell death was quantitated by release of the lactate dehydrogenase enzyme. Fragmentation of DNA into nucleosomal oligomers and the occurrence of a hypodiploid cell population detected by flow cytometry provided evidence for apoptosis. Studies in nude mice demonstrated that ex vivo infection with rAd-p53 suppressed the tumorigenic potential of human glioblastoma cells. Furthermore, direct injection of rAd-p53 into established s.c. xenografts inhibited tumor growth. Our observations suggest that re-introduction of wild-type p53 may have potential clinical utility for gene therapy of glioblastoma.

Adenoviridae↗
PubMed1996

Inhalant use patterns among Eskimo school children in western Alaska.

An inhalant use survey was administered to 376 school children in 14 isolated villages. Questions in the survey elicited gender and ethnic distribution, frequency and duration of use, age of onset, reasons for using and inhalant of choice. These characteristics were further examined by dividing users into 2 groups based on use frequency and duration, i.e., Light Users and Heavy Users. Lifetime prevalence was 48%. Most inhalant use appeared to be sporadic, transient and occurring in social context. Heavy Users differed from Light Users in that they were more likely to be male, have an earlier age of onset, have different ethnic distribution and use inhalants in response to affect. These children may represent an inhalant dependent subgroup. This finding of user heterogeneity has important implications for the development of effective prevention and treatment programs.

Adhesives↗
PubMed1996

Information exchange as empowerment: a case study.

In July 1994 a newspaper was created to share information in and among the community of people with disabilities in Palmerston North, New Zealand. The project became, in only a few months, not only economically viable, but extremely successful as a social and community experiment, and an endeavour by people with disabilities to inform each other and the larger community. This article describes the origin, design and development, and the current status of the newspaper, and advances the recommendation that people with disability elsewhere, and those professionally employed in the rehabilitation field, consider similar initiatives to inform, and thereby empower, people with disabilities.

Consumer Advocacy↗
PubMed1996

Rehabilitation interventions: ideas based on a South Pacific example.

The South Pacific represents a unique area of the world where people with disabilities do need and want rehabilitation interventions. Cross-cultural work, however, carries inherent misunderstandings. Some ideas and examples illustrate how rehabilitation interventions can take place if and when deeper insight into cultural differences is available.

Adult↗
PubMed1995

Characterization of an adenovirus gene transfer vector containing an E4 deletion.

We describe the construction and characterization of an adenovirus type 2 vector, Ad2E4ORF6, which has been modified in the E4 region to contain only open reading frame 6. When assayed in cultured cells, Ad2E4ORF6 virus replication is slightly delayed but viral DNA synthesis, host-cell protein synthesis shut-off, and virus yield are indistinguishable from wild type. Late protein synthesis is normal with the exception of fiber synthesis, which is reduced approximately 10-fold. Despite the reduced fiber synthesis, Ad2E4ORF6 viral particles appear to contain a full complement of fiber protein. Virus replication in cotton rats indicates that Ad2E4ORF6 is replication defective in vivo. This may have safety implications for the development adenovirus vectors in that virus arising by recombination in the E1 region of an Ad2E4ORF6-based vector would be defective for growth in vivo. The deletion of E4 open reading frames that are not required for virus growth in vitro increases the cloning capacity of adenovirus vectors by 1.9 kb and may be generally useful for the construction of adenovirus vectors containing large cDNA inserts and/or regulatory elements. We describe the inclusion of the A2E4ORF6 modification in a recombinant adenovirus vector, Ad2/CFTR-2, for gene transfer of the human cystic fibrosis transmembrane regulator (CFTR).

Adenoviridae↗
PubMed1995

Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene.

We have investigated whether adenovirally mediated gene transfer of the herpes simplex thymidine kinase gene to human hepatocellular carcinoma (HCC) cell lines can sensitize these cells to the prodrug ganciclovir and thereby provide a therapeutic option for this intractable cancer. Two replication-deficient adenoviruses encoding for the herpes simplex virus type-1 (HSV) thymidine kinase (TK) gene were generated in which expression of TK is under the control of either the human cytomegalovirus immediate early promoter (CMV) or the human alpha-fetoprotein (AFP) promoter/enhancer. We demonstrate that the combination of adenovirally mediated TK gene transfer and ganciclovir treatment effectively inhibits proliferation and causes cell death of HCC cells in vitro and that in vivo TK gene transfer and ganciclovir treatment inhibits hepatocellular tumor growth in a mouse model of this cancer. Furthermore, we show that expression of the TK gene can be restricted to those HCCs that express the tumor marker AFP through the incorporation of the AFP enhancer/promoter within an adenoviral vector.

Animals↗
PubMed1994

The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of proteins implicated in the transport of ions, proteins, and hydrophobic substances. Recent studies have demonstrated that CFTR is a protein kinase A-sensitive anion channel regulated by ATP. In the present study, patch-clamp techniques were used to assess the role of CFTR in the transport of Cl- and ATP. The stable transfection of mouse mammary carcinoma cells, C127i, with the cDNA for human CFTR resulted in the appearance of a diphenylamine-2-carboxylate-inhibitable Cl- channel, which was activated by cAMP under whole-cell and cell-attached conditions and by protein kinase A plus ATP under excised, inside-out conditions. CFTR expression was also associated with the electrodiffusional movement of ATP as indicated by the cAMP activation of ATP currents measured under whole-cell conditions. In excised, inside-out patches, it was demonstrated that ATP currents were mediated by ATP-conductive channels, which were also activated by protein kinase A and blocked by the Cl- channel blocker diphenylamine-2-carboxylate under excised, inside-out conditions. Single-channel currents observed in the presence of asymmetrical Cl-/ATP concentrations indicated that the same conductive pathway was responsible for both ATP and Cl- movement. Thus, CFTR is a multifunctional protein with more than one anion transport capability and may modify signal transduction pathways for Cl- or other secretory processes by the selective delivery of nucleotides to the extracellular domain.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

External ATP and its analogs activate the cystic fibrosis transmembrane conductance regulator by a cyclic AMP-independent mechanism.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel activated by protein kinase A and regulated by ATP in a complex manner. We have applied patch-clamp techniques to C127i mouse mammary carcinoma cells transfected with human CFTR to assess the role of external ATP in the modulation of CFTR function. Extracellular ATP was sufficient to activate non-rectifying, Cl(-)-selective whole-cell currents in CFTR-transfected, but not mock-transfected cells. The ATP-mediated activation of CFTR was independent of protein kinase A since channel activation by ATP was preserved in cells that were (a) depleted of intracellular ATP, (b) incubated with the cAMP antagonist Rp-cAMPS, or (c) exposed to the protein kinase A inhibitor, 5-24 amide. In each of these conditions, 8-Br-cAMP was no longer capable of activating CFTR. The possibility that the extracellular ATP activation of Cl- currents in CFTR-expressing C127i cells was mediated by a P2-type purinergic receptor was supported by studies in which the effect of external ATP on the Cl- currents was mimicked by the ATP analogs, ATP gamma S and beta,gamma-methylene ATP, but not the uridine nucleotide, UTP. Single-channel analysis of ATP-activated Cl -currents under both cell-attached and excised, inside-out patch-clamp configurations indicated that this channel is only present in CFTR-transfected cells and indistinguishable from CFTR. External ATP also activated ATP currents in CFTR-transfected cells, a novel function of CFTR. These findings are consistent with the presence of a purinergic receptor signal transduction mechanism in C127i cells whose activation by external ATP is linked to the activation of CFTR in a cAMP-independent manner. The data provide additional support for the use of ATP and its analogs as alternative therapies in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats.

Gene therapy for cystic fibrosis (CF) will require the safe transfer of CFTR cDNA to airway epithelia in vivo. We showed previously that a recombinant adenovirus, Ad2/CFTR-1, expresses CFTR in vitro. As adenovirus rarely integrates, treatment will require repeated vector administration. We applied Ad2/CFTR-1 to intrapulmonary airway epithelia of cotton rats and nasal epithelia of Rhesus monkeys. In both species we detected CFTR mRNA and protein after repeated administration and in monkeys, protein was detected six weeks after repeat administration. The vector did not replicate and was rapidly cleared. Despite an antibody response, there was no evidence of a local or systemic inflammatory response after repeat administration. These data indicate that repetitive administration of Ad2/CFTR-1 is both safe and efficacious.

Adenoviridae↗
PubMed1994

Grief and loss among Eskimos attempting suicide in western Alaska.

Data were collected prospectively from psychiatric evaluations performed on 53 consecutive Eskimos in the Bering Strait region who attempted suicide. Depressive diagnoses were common (N = 49). Thirty-seven (70%) of the attempts were preceded by a recent interpersonal loss. Sixty percent of the patients had lost a parent during childhood. Poor affective relatedness, especially around issues of loss, was noted in most of the patients. Thus, both multiple losses and limited grieving mechanisms may be important risk factors for attempted suicide in this population.

Adult↗
PubMed1994

The Zung Self-Rating Depression Scale as a potential screening tool for use with Eskimos.

OBJECTIVE: The study assessed the utility of a modified Zung Self-Rated Depression Scale (SDS) in detecting major depression among Eskimo patients in the Bering Straits region of Alaska. METHODS: The modified SDS was administered to Eskimo patients referred for psychiatric evaluation at the community mental health center in Nome. Diagnoses were assigned by a staff psychiatrist based on DSM-III-R criteria. The SDS scores of 25 consecutive patients with a diagnosis of major depression were compared with the scores of 25 consecutive patients assigned other psychiatric diagnoses. RESULTS: The modified SDS was readily understood by patients and took between three and four minutes to complete. The mean SDS score of patients with a diagnosis of major depression was significantly different from that of patients with other diagnoses. The SDS had a sensitivity of 80 percent and specificity of 88 percent for detecting patients with major depression. CONCLUSIONS: The SDS is easy to administer and can differentiate between patients with major depression and those with other psychiatric diagnoses. It can be modified to fit local idiom in an Eskimo community. The SDS shows promise as a clinically relevant screening tool for major depression in populations with limited access to psychiatric care.

Adolescent↗
PubMed1994

After traumatic brain injury: a discussion of consequences.

Traumatic brain injury is followed by many changes in cognition, emotion, social interaction and functioning, and communication. These changes may range from severe to subtle. They create complex difficulties for the individual and his or her network of support. Presented here is a review of the literature on the changes and their impact. The review comprises Part I, on brain injury and its effects, and Part II, on social interaction research.

Adolescent↗