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Biomedical subjects

R A Morgan

Publications and source records attributed to R A Morgan.

At least 73 records · Page 4Linked to original sources
PubMed1994

Inhibition of human immunodeficiency virus type-1 by retroviral vectors expressing antisense-TAR.

The human immunodeficiency virus type-1 (HIV-1) Tat activation response (TAR) region is essential for Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR). The TAR element is present on the 5' and 3' ends of all HIV-1 transcripts and is relatively conserved among different HIV-1 isolates. These properties make it an attractive target for anti-HIV-1 gene therapy strategies. We have constructed a Moloney murine leukemia-based retroviral vector that expresses a chimeric tRNA(iMet)-antisense TAR fusion transcript complementary to the HIV-1 TAR region. The potential of this anti-TAR retroviral vector to inhibit HIV-1 was initially tested by transient transfections with an HIV-1-LTR-Tat expression plasmid into HeLa-CAT cells. Anti-TAR inhibited Tat-mediated HIV-1 LTR-driven CAT reporter gene expression in a dose-dependent fashion. The antisense-TAR vector was then used to transduce the human SupT1 T cell line. Cotransfection of these SupT1 cells with a Tat expression plasmid plus an HIV-1 LTR-CAT reporter plasmid resulted in decreased CAT gene expression in comparison to control transduced SupT1 cells. The antisense-TAR engineered SupT1 cell line was then challenged with HIV-1MN.HIV-1 viral production was inhibited in SupT1 cells transduced with the antisense-TAR retroviral vector. Greater inhibition of HIV-1 was observed with antisense-TAR as compared to antisense-Tat expressing retroviral vector. These observations suggest that antisense-TAR retroviral vectors are potentially useful for clinical anti-HIV-1 gene therapy.

Blotting, Southern↗
PubMed1994

Retroviral vectors containing chimeric promoter/enhancer elements exhibit cell-type-specific gene expression.

Retroviral vectors were constructed in which the U3 promoter/enhancer of Moloney murine leukemia (Mo-MLV) was replaced by the corresponding region from five related murine retroviruses--AKR murine leukemia virus (AKV), Harvey murine sarcoma virus (HaMSV), myeloproliferative sarcoma virus (MPSV), SL3-3, and the NZB-xenotropic virus (Xeno). In these vectors the chimeric long terminal repeat (chLTR) drives the expression of the chloramphenicol acetyl transferase (CAT) reporter gene that is followed by an internal SV40 virus early region promoter linked to the neomycin phosphotransferase II (NEO) gene. As an initial measure of the relative promoter/enhancer strength of the chLTR vectors, the murine NIH-3T3 cell line and the human JURKAT cell lines were transfected and assayed for CAT reporter activity. Relative to the MoMLV vector, the HaMSV construct was the most active in NIH-3T3 cells whereas the SL3-3 vector displayed the greatest activity in JURKAT cells. Retroviral vector producer cell populations and cell clones were established for each chLTR vector, and all were capable of yielding high vector titers (> 10(5) G418R cfu/ml on NIH-3T3). Supernatant from these cells was used to transduce both mouse and human cell lines and primary cells. In NIH-3T3 cells and two murine fibrosarcoma cell lines, the HaMSV chLTR vector was slightly more active than the MoMLV chLTR vector. In the human HepG2 and HeLa cell lines, the MPSV chLTR vector was the most active. Data from the human JURKAT T-cell line and a T cell line derived from an ADA-deficient severe combined immunodeficiency (SCID) patient demonstrate that the SL3-3 chLTR is the most active in these lymphoid cell lines. The greatest difference in the comparison of the different chLTR vectors was observed in primary human umbilical vein endothelial cells, where the MoMLV vector produced up to 100 times more CAT activity than the SL3-3 vector. These data suggest that the use of specific promoter/enhancer elements may lead to higher levels of gene expression following retroviral-mediated gene transfer into specific cell types and these observations may be useful in the design of human gene therapy experiments.

Animals↗
PubMed1993

In vitro infection of human bone marrow by feline leukemia viruses.

Four subgroup feline leukemia viruses (FeLV) were tested for their ability to infect primary cultures of human bone marrow in vitro. Using three sequential exposures to both concentrated and unconcentrated virus, FeLV infection of human bone marrow was documented by indirect immunofluorescence, ELISA, and polymerase chain reaction (PCR) assays. Based on comparison to known standards, it was estimated that between 5 and 10% of the bulk human marrow culture could be infected by FeLV. Further evidence for FeLV reverse transcription, integration, and expression was obtained using specific PCR assays.

Bone Marrow↗
PubMed1993

Allastair B. Karmody Award. Improved recovery of limb function with ATP/MgCl2 in an ischemic canine hind limb.

The ability of a solution of low-vanadium-content (less than 1 ppm) adenosine triphosphate and magnesium chloride (ATP/MgCl2) versus normal saline to improve recovery of function and reduce necrosis of skeletal muscle after severe ischemia was investigated in an in situ autoperfused canine hind limb model. The study consisted of 12 dogs divided into 3 study groups: nonischemic control (NIL) (n = 7 limbs), ischemic (IL) (n = 7 limbs), and ischemic ATP/MgCl2-treated (IATP) (n = 7 limbs). In groups IL and IATP, the limb was reperfused for 3 hours following 4 hours of complete ischemia. In IATP limbs, 200 mumol/kg of ATP/MgCl2 was infused upon reperfusion of the limbs, whereas IL limbs received a similar volume of normal saline at the time of reperfusion. Function was determined by stimulating the deep peroneal nerve and anterior tibial muscle and measuring the resultant isometric twitch contractile force of paw dorsiflexion. Muscle necrosis was evaluated by photographic analysis of sectioned anterior tibial muscle stained with nitroblue tetrazolium dye. ATP/MgCl2 significantly increased functional recovery (p < 0.01) and significantly reduced skeletal muscle necrosis (p < 0.01). This study suggests that ATP/MgCl2 may be useful in reducing the clinical sequelae of severe limb ischemia and reperfusion.

Adenosine Triphosphate↗
PubMed1993

Detection of pneumothorax with lateral shoot-through digital radiography.

The aim of this study was to compare the performance of digital frontal radiographs vs digital lateral shoot-through radiographs for the diagnosis of pneumothoraces in supine patients. A total of 146 pairs of radiographs were performed on 32 ventilated patients on the Intensive Care Unit. Each radiograph was independently assessed by three observers for the presence or absence of a pneumothorax. A degree of confidence was assigned to each observation and an image quality score was given to each radiograph. At least two out of three observers positively diagnosed a pneumothorax in 13/146 (8.9%) of the frontal radiographs compared to 43/146 (29.4%) of the lateral radiographs (P < 0.0001), but suboptimal images were obtained more often with lateral shoot-through radiographs than with frontal radiographs. We conclude that digital lateral shoot-through radiographs are significantly more sensitive than digital frontal radiographs for the diagnosis of pneumothoraces in supine patients.

Humans↗
PubMed1993

Infection of human cells with murine amphotropic replication-competent retroviruses.

Replication of the murine wild-type 4070A amphotropic retrovirus and a recombinant amphotropic replication-competent retrovirus arising from the PA12 packaging cell line varied considerably among the primate cell types tested. Medium from infected primate fibroblasts and endothelial cells contained the highest viral titers [10(4)-10(5) focus-forming units (ffu)/ml], while most hematopoietic cell lines, such as K562 and MOLT4, were associated with viral titers in the range of 10(3)-10(4) ffu/ml. Interestingly, HTLV-1-transformed T cell lines (TJF-2 and HM) and primary tumor infiltrating lymphocytes (TIL) had very low viral titer (0-10(1) ffu/ml). The low production of virus was not due to low infectivity and, in contrast to the virus, retroviral vectors were expressed without difficulty. Because screening for replication-competent retrovirus (RCR) is an important component of human retroviral-mediated gene therapy clinical protocols, a variety of assays were tested for their ability to detect RCR in virus-exposed cell lines. A biologic assay (3T3 amplification) and polymerase chain reaction (PCR) for the 4070A viral envelope are effective screening methods for RCR, even in cell lines associated with low virus production.

Animals↗
PubMed1993

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.

Animals↗
PubMed1993

Analysis of the functional and host range-determining regions of the murine ectropic and amphotropic retrovirus envelope proteins.

A series of Moloney murine leukemia virus (Mo-MuLV) envelope gene constructs were analyzed for biological activity. Three classes of recombinant envelopes were examined: insertions, deletions, and chimeras. Insertion (4 to 5 amino acids) and deletion (31 to 62 amino acids) mutants spanned most of the SU (gp70)-coding region and were all biologically inactive. Radioimmunoprecipitation demonstrated that the mutant envelope proteins were incorrectly processed. The Pr80env envelope precursor proteins failed to obtain the proper posttranslational modifications and were not cleaved into SU (gp70) and TM (p15E), suggesting that disruption of Pr80env structure prevents intracellular transport and processing. To analyze the functional domains of the SU portion of the Env protein, we assembled several chimeric constructs. In these constructs, portions of the ecotropic Mo-MuLV envelope gene were replaced with corresponding sequences from the 4070A amphotropic MuLV envelope. Using a retroviral vector pseudotyping assay, 5 of 12 chimeric envelope proteins were shown to be biologically active. Host range was determined by retroviral vector transduction of the appropriate cell, by viral interference studies, and by the productive infection of Chinese hamster ovary cells expressing the murine ecotropic receptor. These results permit assignment of the amino acids responsible for host range determination. Ecotropic host range is determined by the first 88 amino acids of the Mo-MuLV SU, while the amphotropic host range-determining region spans the first 157 amino acids of the 4070A SU.

3T3 Cells↗
PubMed1993

Human gene therapy.

Explore the source record for details and available documents.

Adenosine Deaminase↗
PubMed1992

Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy.

Recombinant retroviral vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.

Adenosine Deaminase↗
PubMed1992

Pancreatic and renal mobility.

One hundred and forty-five patients undergoing ultrasound examination for unrelated symptomatology were examined with respect to pancreatic position. Patients likely to have distorted upper abdominal anatomy were excluded. All patients were examined in the supine and left posterior oblique positions in the sagittal and transverse planes. The position of the head of the pancreas was noted in the following positions: pre-caval, pre-aortic and to the left of the aorta. The pancreas was not visualized in both positions in 25 patients, and these were excluded from the study. The pancreas changed position with patient movement in 45 patients. In eight females a change in position of the right kidney was also noted. This study demonstrates that pancreatic and to a lesser extent renal mobility is common, particularly in women. Attention is drawn to this finding which may cause distortion of the biliary and pancreatic anatomy. The altered landmarks may cause confusion in the interpretation of normal and abnormal structures.

Adolescent↗
PubMed1992

PCR and other test systems in human gene therapy.

Three protocols using retroviral-mediated gene transfer are currently undergoing clinical trials. The uniqueness of these protocols, which were the first approved experiments in which genetically engineered cells were administered to human beings, necessitated the development of novel biological and physical safety testing procedures. In addition to standard testing for contamination with exogenous pathogens and toxins, specific tests were developed to assay for the presence of recombinant helper virus in both retroviral vector preparations and in the engineered human cells. Two new assays were developed, an enhanced S+/L-virus assay called 3T3 amplification, and a specific polymerase chain reaction protocol of great sensitivity. The results of these safety tests, along with pre-clinical primate safety studies, and the accumulating clinical data, suggest that retroviral-mediated gene transfer does not pose any serious short-term safety problems for patients enrolled in gene therapy protocols.

Animals↗
PubMed1991

Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow.

A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony-forming unit granulocyte-macrophage after more than 200 days. Two of the four cats unexpectedly developed diabetes mellitus 90 days posttransplantation. The expression of a foreign gene in cats, albeit at a low level, demonstrates that retroviral vectors can be used for gene transfer in noninbred large animal species and may be useful for gene therapy of humans. The development of diabetes mellitus in two of the subjects emphasizes the value of animal models for the study of possible deleterious effects of retroviral vector-mediated gene transfer.

Animals↗
PubMed1991

The ultrasound appearance of asbestos-related pleural plaques.

Twenty-five patients with a history of asbestos exposure and radiological evidence of pleural plaques were examined by ultrasound (US). Non-calcified pleural plaque appeared as a regular area of echo-poor tissue which was well defined adjacent to the diaphragm or lateral chest wall, and varied in thickness from 5 mm to 12 mm. Calcified plaques had echogenic and irregular anterior margins with acoustic shadowing beyond. They were associated with characteristic comet tail and straight line artefact.

Asbestos↗