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Biomedical subjects

S M Freeman

Publications and source records attributed to S M Freeman.

51 records · Page 3Linked to original sources
PubMed1991

Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells.

Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice. Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later. These results indicate that stable recombinant gene expression can be achieved in vivo in the mammary gland without altering the growth properties of normal mammary epithelium.

3T3 Cells↗
PubMed1990

Recombinant gene expression in human umbilical vein endothelial cells transduced by retroviral vectors.

Endothelial cells are attractive targets for gene transfer because of their immediate contact with the bloodstream, and, therefore, they might serve as vehicles for therapeutic drug delivery. Recently, we and others reported that endothelial cells of animal origin efficiently express both secretory and nonsecretory recombinant proteins. We now show that human endothelial cells are also capable of expressing a recombinant gene following transduction with retroviral vectors. Human umbilical vein endothelial cells were transduced with either the N2 or the SAX vector. Following selection with G418, cells transduced by both vectors were found to express neophosphotransferase activity, the product of the neomycin resistance gene. The fact that a recombinant gene can be readily inserted and efficiently expressed into human endothelial cells suggests that these cells may be able to serve a role in human gene therapy.

Anti-Bacterial Agents↗
PubMed1990

Analysis of retroviral packaging lines for generation of replication-competent virus.

Amplification of retroviral vector sequences occurs in cocultures of ecotropic and amphotropic packaging cell lines. Mixed packaging line cocultures were used to determine both the host range and time of appearance of replication-competent virus after introduction of a retroviral vector. Replication-competent virus was generated at a characteristic time for a given ecotropic and amphotropic packaging line combination. The time required to generate replication-competent virus in a packaging line varied with the number of recombination events necessary to generate replication-competent virus from the retroviral sequences present in the line. The psi 2 packaging line generated replication-competent virus within 10 days after transfection of the N2 vector into a mixture of psi 2 (ecotropic) and PA317 (amphotropic) packaging cells. Under the same conditions, it took only 3 days to develop replication-competent virus in psi 2/PA12 cocultures. The host range of replication-competent virus was used to identify the packaging line that initially generates virus. Each packaging line combination generated replication-competent virus at a characteristic time and this time period can be used as a measure of the "safety" of the packaging line and vector combination.

Animals↗
PubMed1989

High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors.

By virtue of its immediate contact with the circulating blood, the endothelium provides an attractive target for retroviral vector transduction for the purpose of gene therapy. To see whether efficient gene transfer and expression was feasible, rabbit aortic endothelial cells were infected with three Moloney murine leukemia virus-derived retroviral vectors. Two of these vectors carry genes encoding products that are not secreted: N2, containing only the selectable marker gene neoR, and SAX, containing both neoR gene and an SV40-promoted adenosine deaminase (ADA) gene. The third vector, G2N, contains a secretory rat growth hormone (rGH) gene and an SV40-promoted neoR gene. Infection with all three vectors resulted in expression of the respective genes. A high level of human ADA expression was observed in infected endothelial cell populations both before and after selection in G418. G2N-infected rabbit aortic endothelial cells that were grown on a synthetic vascular graft continued to secrete rGH into the culture medium. These studies suggest that endothelial cells may serve as vehicles for the introduction in vivo of functioning recombinant genes.

Adenosine Deaminase↗
PubMed1989

Seeding of intravascular stents with genetically engineered endothelial cells.

The use of intravascular stents may be limited by both local thrombosis and restenosis due to intimal proliferation. In an effort to provide solutions to these problems, we seeded stents with genetically engineered endothelial cells in vitro. Using retroviral-mediated gene transfer, we inserted the gene for either bacterial beta-galactosidase or human tissue-type plasminogen activator (t-PA) into cultured sheep endothelial cells. The endothelial cells were seeded onto stainless steel stents and grown until the stents were covered. Expression of intracellular beta-galactosidase and high level secretion of t-PA were demonstrated both before and after the transduced cells were seeded onto the stents. Eight stents were expanded by in vitro balloon inflation, with observation of the seeded endothelial layer both prior to and after expansion. Most of the endothelial cells remained on the stents after balloon inflation. We conclude that intravascular stents can be coated with a layer of genetically engineered endothelial cells that can be either specifically labeled or made to secrete high levels of a therapeutic protein. Much of the layer of genetically engineered cells remains after the expansion of the stent in vitro. In vivo implantation of stents coated with genetically engineered endothelial cells may allow 1) introduction of genetically engineered endothelial cells directly into the vascular wall and 2) improvement of stent function through localized delivery of anticoagulant, thrombolytic, or antiproliferative molecules.

Animals↗
PubMed1988

Oligonucleotide probing. Applications to HLA typing.

Human leukocyte antigen typing is currently being done by both serologic and cellular techniques. It is this methodology that forms the basis for organ transplantation, since HLA antigens are the major proteins involved in graft rejection. Matching organ graft donors with recipients at these polymorphic HLA loci clearly increases graft survival in most transplant systems. However, the serologic and cellular techniques have shortcomings, including the problem of standardizing antisera, the need for technologists with expertise in T-cell cloning, and the failure to work successfully with some cells (eg, leukemic blasts). We have begun to develop a more rapid, sensitive typing method using molecular biological techniques to tissue-type cells. Using oligonucleotide probes, we can now identify HLA-D region alleles (which may differ by only a single nucleotide) that can only be differentiated by a highly technical, lengthy cellular technique. Oligonucleotide probing not only provides the basis for a rapid method of HLA typing, which in all likelihood will become generally applicable as additional sequence information becomes available, but also allows definition of a gene transcript from an individual locus rather than the phenotypic results provided by current HLA typing techniques.

Alleles↗
PubMed1987

Determination of DR beta 1 alleles of DR4/Dw subtypes by oligonucleotide probing.

The extensive Dw polymorphism of HLA-D has, to date, been defined using cumbersome and lengthy techniques involving T-cell proliferative reactions. These techniques have not necessarily provided information about the genotype at any single locus of HLA-D because the products of more than a single locus of HLA-D (e.g., DR beta 1 and DR beta 2 of the DR2 haplotypes and in some cases DQ) can contribute to T-cell proliferation and thus to assignment of Dw phenotype. We have recently reported that DR beta 1 alleles relating to the Dw polymorphism can vary by only a single, or a very few, nucleotide differences; the products of this locus are recognized in allo- and restricted responses. Given the importance of defining this polymorphism, we have used specific oligonucleotides as hybridization probes to differentiate between these very closely related alleles. This approach provides the basis for a rapid method for HLA-D typing which, as additional sequence information becomes available, will likely be generally applicable. In addition, oligonucleotide probing allows definition of a gene transcript from an individual locus of HLA-D rather than the phenotype results provided by Dw typing.

Alleles↗
PubMed1987

Comparison of DR beta 1 alleles from diabetic and normal individuals.

Insulin-dependent diabetes (IDD) is positively associated with HLA-D proteins. A critical question is whether or not sequence differences within the HLA-D coding region are the same or different in diabetics and normal individuals of the same haplotype. We have isolated both DR beta 1 alleles from a Dw4/LD MN2 cDNA library and compared them to DR beta 1 genes isolated from normal individuals of the same Dw phenotype. We found no nucleotide differences in the coding region between the normal and diabetic alleles of DR beta 1 suggesting to us that DNA differences other than the DR beta 1 coding region may account for the observed association of HLA-D and diabetes.

Alleles↗
PubMedDate not supplied

Recombinant interferon alpha2a synergistically enhances ganciclovir-mediated tumor cell killing in the herpes simplex virus thymidine kinase system.

The herpes simplex virus thymidine kinase (HSV-TK) gene is being developed in the treatment of many different types of tumors. The HSV-TK gene sensitizes tumor cells to the antiviral drug ganciclovir (GCV) and mediates the bystander effect in which unmodified tumor cells are killed as well. Although this approach has shown a significant antitumor effect, the need to potentiate this therapy exists. The results of this study indicate that recombinant interferon alpha2a (1FNalpha2a) acts synergistically with GCV to kill HSV-TK-expressing PA1 human ovarian tumor cells. Furthermore, it enhances the bystander killing of nearby unmodified tumor cells that do not express the HSV-TK gene. Previous studies have suggested that in vitro and in vivo bystander effects may be mediated by different mechanisms. However, IFNalpha2a enhanced bystander killing in both systems, with the survival of mice bearing preexisting tumors being significantly prolonged when they were treated with IFNalpha2a and HSV-TK/GCV compared with either treatment alone. Mechanism studies have shown that treatment with IFNalpha2a and GCV caused an increase in cells in S phase 24 hours after therapy in the HSV-TK-expressing cells, but the mechanism of action of IFNalpha2a does not seem to be related to an increase in DNA damage, because GCV incorporation was not increased after treatment with IFNalpha2a. These findings suggest that IFNalpha2a may be a useful adjunctive therapy for the HSV-TK/GCV system.

Animals↗
PubMedDate not supplied

Tests used to assess the cognitive abilities of aged rats: their relation to each other and to hippocampal morphology and neurotrophin expression.

BACKGROUND: Aged rodents have proven to be a useful tool in studying age-related cognitive decline, particularly with regard to hippocampal function. A number of maze tests have been developed to evaluate hippocampal function in aged rodents, including the eight-arm radial maze, Barnes circular platform maze and Morris water maze. To some extent, these mazes have been used interchangeably to evaluate aged animals. Few researchers, however, have examined how performance of individual, aged animals compares in these three mazes. OBJECTIVE: The purpose of this study was to compare the performances in the three mazes and to examine how such performances are related to each other, to hippocampal morphology and to neurotrophin gene expression. METHODS: We screened groups of young and old Fisher 344 x Brown Norway rats for general health and physical abilities, tested the animals in the three mazes and examined correlations among performances in the mazes and in screening tests. Hippocampal neuron density and expression of hippocampal neurotrophin mRNAs were also examined and compared with behavior in the three mazes. RESULTS: Aged animals were found to be impaired in all three mazes and to have lower hippocampal neuron densities compared with young animals, with poor learning behavior significantly correlating with reduced hippocampal neuron density. Differences were observed between performance in the different mazes, but in general the Morris water maze and Barnes circular platform maze were found to give similar results.

Aging↗
PubMedDate not supplied

DNA ploidy and proliferating cell nuclear antigen image analysis of peritoneal and pleural effusions. A possible diagnostic role.

OBJECTIVE: To determine the role of DNA and proliferating cell nuclear antigen (PCNA) image analysis (IA) in enhancing the diagnostic sensitivity of conventional cytology (CC). STUDY DESIGN: The histopathologic and clinical data on 87 consecutive pleural and peritoneal effusions were used to evaluate the accuracy of CC and DNA IA results. RESULTS: CC showed a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 65%, 100%, 100% and 62%, respectively. Aneuploidy peaks were seen in 49 cases; 47 of them were true positives. Thirty of 38 diploid cases were true negatives. The sensitivity, specificity, PPV and NPV were 85%, 94%, 96% and 80%, respectively. There were positive correlations between DNA ploidy profile and PCNA proliferative index (PI), (R = .697) and significant differences in PCNA PI between malignant and benign effusions (P < .001). CONCLUSION: The DNA IA PI by PCNA can be used as a complementary diagnostic tool with CC in cytologically inconclusive cases.

Adult↗
PubMedDate not supplied

Oncogene elements within an endogenous retrovirus.

The human genome contains a large number of endogenous retroviral-related sequences. While the function of these sequences is unknown, they may contribute to disease processes through their regions of homology with infectious retroviruses. We have been further characterizing a recently reported HTLV-1 related endogenous retroviral sequence cloned from T lymphocytes isolated from a patient with essential cryoglobulinemia. We here report further detailed transcriptional analysis of the sequence for tissue and cell-cycle specificity and a novel finding of an association between the endogenous retrovirus and a ras-related gene.

Base Sequence↗
PubMedDate not supplied

Expression of costimulatory molecules: B7 and ICAM up-regulation after treatment with a suicide gene.

The herpes simplex virus thymidine kinase gene (HSV-TK) in combination with ganciclovir (GCV), is currently being used in gene therapy-based clinical trials for cancer treatment. Its therapeutic effect is based on a "bystander effect" whereby HSV-TK gene-modified tumor cells are toxic to nearby unmodified tumor cells when exposed to the antiviral drug GCV. We have recently hypothesized that the in vivo mechanism of this bystander effect is due to alterations in the tumor microenvironment in response to release of cytokines and an infiltration of leukocytes after treatment with HSV-TK gene-modified tumor cells and GCV, which results in tumor regression. Expression of B7, a recently identified costimulatory molecule that is important for T-cell stimulation, has been shown to be modulated by stimulatory cytokines interferon-gamma, tumor necrosis factor-alpha, and inhibited by interleukin-10. In the present study, we investigated whether the cytokines released after HSV-TK and GCV treatment could include the expression of the costimulatory molecules B7-1 and B7-2 and the adhesion molecule (ICAM)-1 in the tumor. Furthermore, we investigated whether this altered environment affected the antitumor properties of host lymphocytes. An in vitro model was developed to establish the effects of HSV-TK gene-modified tumor cells and GCV on tumor infiltrating cells. The murine macrophage cell line (IC21) was exposed to either supernatants or cell lysates collected from a mixture of HSV-TK-transduced (KBALB-STK) and non-transduced (KBALB) murine fibrosarcoma tumor cells previously exposed to GCV (experimental). Immunohistochemical analysis showed a significant expression (P < .0001) of B7-1 and B7-2 post exposure of IC21 cells to either supernatant or lysate. In contrast, the level of expression in IC21 cells exposed to the control lysate or supernatant remained unchanged for B7-1 and B7-2. In vivo analysis for B7-1 and B7-2 expression by immunohistochemistry in tumor tissues from experimental mice receiving HSV-TK gene-modified tumor cells and GCV treatment showed a significant expression of B7.1 (35%, P < .0001) and B7.2 (38.2%, P < .0001) on tumor-infiltrating mononuclear cells. In contrast, tumor-bearing control animals showed low levels of B7-2 expression (5.8%), whereas B7-1 was undetectable, as confirmed by reverse-transcriptase polymerase chain reaction. In addition, a significant up-regulation of ICAM expression (50%) on tumor tissues was observed in the experimental group (P = .0317) as compared with the control group (25%). Furthermore, T cells isolated from experimental mice showed a significant in vitro proliferative response (p = .0202) when exposed to syngeneic tumor cells as compared with the control group. These data demonstrated that the use of HSV-TK gene-modified tumor cells and GCV as a suicide gene in the treatment of an intraperitoneal tumor resulted in the expression of the B7 costimulatory molecules and ICAM-1 adhesion molecule and enhanced proliferative response of host T cells. These findings help to understand the mechanism of tumor cell killing in vivo using HSV-TK gene-modified tumor cells.

Animals↗