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Biomedical subjects

R W Paul

Publications and source records attributed to R W Paul.

16 recordsLinked to original sources
PubMed2000

E1A-mediated paclitaxel sensitization in HER-2/neu-overexpressing ovarian cancer SKOV3.ip1 through apoptosis involving the caspase-3 pathway.

HER-2/neu-overexpressing breast cancer cells are more resistant to the chemotherapeutic agent paclitaxel (Taxol) than low-HER-2/neu-expressing breast cancer cells, and the adenoviral type 5 EIA can down-regulate HER-2/neu overexpression. Therefore, in this study, we asked (a) whether EIA might sensitize response to paclitaxel in human HER-2/neu-overexpressing ovarian cancer cells, and, if so, what is the mechanism responsible; and (b) whether this enhanced chemosensitivity would translate into a therapeutic effect in an ovarian cancer xenograft model. Consequently, we demonstrated that: (a) adenovirus type 5 E1A could enhance the sensitivity of paclitaxel in paclitaxel-resistant HER-2/neu-overexpressing human ovarian cancer cells in vitro by inducing apoptosis, (b) this induction was heavily dependent on activation of the caspase-3 pathway, and (c) nude mice bearing i.p. HER-2/neu-overexpressing human ovarian cancer cells and treated with both paclitaxel and E1A gene therapy survived significantly longer than did mice treated only with paclitaxel or E1A gene therapy. Thus, we concluded that the E1A gene enhanced both the in vitro and in vivo sensitivity of paclitaxel in paclitaxel-resistant HER-2/ neu-overexpressing ovarian cancer SKOV3.ipl cells. Because a Phase I clinical trial using E1A gene targeted to HER-2/neu down-regulation has recently been completed, the current study also provided a scientific basis to further develop a novel therapy that combines paclitaxel and E1A gene therapy and its testing in a Phase II trial.

Adenovirus E1A Proteins↗
PubMed1997

Gene transfer using a novel fusion protein, GAL4/invasin.

The delivery of DNA to target cells using simple, defined, nonviral systems has become an area of intense interest in gene therapy. We describe here the development and characterization of one such novel system. A recombinant, bifunctional, fusion protein was expressed and purified from Escherichia coli. This protein consists of the DNA-binding domain of the yeast transcription factor GAL4 fused to the cell binding, internalization domain of the Yersinia pseudotuberculosis inv gene product, invasin. This protein, GAL4/Inv, together with poly-L-lysine, formed complexes with a chloramphenicol acetyltransferase (CAT) reporter plasmid that contains eight repeats of the GAL4 consensus recognition sequence. These complexes were shown to transfect target cells in an invasin receptor-dependent manner, resulting in transient CAT expression. A simple, targeted DNA delivery vehicle, as we describe here, represents a viable approach to nonviral gene delivery.

Adhesins, Bacterial↗
PubMed1997

Multicomponent gene therapy vaccines for lung cancer: effective eradication of established murine tumors in vivo with interleukin-7/herpes simplex thymidine kinase-transduced autologous tumor and ex vivo activated dendritic cells.

Multiple antitumor modalities may be necessary to overcome lung tumor-mediated immunosuppression and effectively treat non-small cell lung cancer (NSCLC). To evaluate a multimodality gene therapy approach for control of local tumor growth, a weakly immunogenic murine alveolar cell carcinoma, L1C2, was transduced with either the interleukin-7/hygromycin-herpes simplex thymidine kinase (IL-7/HyHSVtk) internal ribosome entry site (IRES) retroviral vector or a vector containing the HyHSVtk, but not the IL-7 gene. Of the many cytokines available for gene transfer, IL-7 was chosen for these studies because it both stimulates CTL responses and down-regulates tumor production of the immunosuppressive peptide TGF-beta. Following selection in hygromycin, IL-7 transduction was confirmed by ELISA. Clones produced 1.25 to 10 ng of IL-7/ml/10(6) cells per 24 h. In vitro, genetically modified tumor cells were significantly more sensitive to ganciclovir (GCV) than unmodified parental tumor cells. The in vivo growth of ex vivo modified L1C2 cells was evaluated. There was a dose-response relationship between the amount of IL-7 secreted in vitro and the growth of genetically modified murine tumor in vivo. Transduced tumor cells regressed in mice following GCV therapy. Although ex vivo gene modification of tumor cells led to complete resolution of the tumor following implantation in vivo, IL-7 and HSVtk gene modified tumor cells were not effective in treating established parental tumors. However when 5 x 10(5) bone marrow-derived, in vitro activated dendritic cells (DC) were administered in combination with transduced tumor and GCV, 5 day old established tumors were eradicated in 80% of mice. These studies suggest that multicomponent vaccines may facilitate improved host responses by replacing host immune deficits and thus could have a role in adjuvant therapy and local control of NSCLC.

Animals↗
PubMed1996

GM-CSF secretion in primary cultures of normal and cancerous human renal cells.

Rates of secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) were measured in 50 primary cell cultures derived from cancerous and normal human kidneys. Mean rates of GM-CSF secretion measured by TF-1 cell proliferation assay (N = 21) and by ELISA (N = 31) were 2.5 and 7.8 ng/10(6) cells/24 hr, respectively. There was no significant difference between the mean rates of GM-CSF secretion by cancerous and normal renal cells. GM-CSF was also secreted by primary renal cell cultures grown in serum-free medium and by renal cell lines. GM-CSF mRNA was detected by RT-PCR in cultured renal cells, but not in undissociated kidney tissue. Rates of GM-CSF secretion were reduced up to 99% under conditions where the cellular density or substratum more closely resembled the in vivo environment. Some cultured human renal carcinoma cells (RCC) secreted GM-CSF at levels that occasionally overlapped the levels produced by the GM-CSF gene-modified human RCC vaccine now in phase I trial. The data indicate that GM-CSF is not expressed in vivo, and that stable GM-CSF secretion is induced by the dissociation and culture of human renal cells.

Adenocarcinoma↗
PubMed1995

Critical thinking and intuitive nursing practice.

In this paper, we analyse how critical thinking and intuitive practice are related to expertise in nursing. We examine how intuitive practice, when performed automatically without care, vigilance, and criticism, can result in prejudice and patterns of practice which are misinformed. We will show that the student nurse develops intuitive, skillful performance in nursing by reasoning about nursing knowledge and applying reflective, critical thought in practice situations, thereby gaining increasing expertise in reasoning as a reliable professional ensuring quality client care. The elements of thought necessary for sound reasoning in clinical practice will be explored. Finally, we suggest educational strategies and tactics which promote the development of the intellectual capacities of student nurses as independent critical thinkers.

Clinical Competence↗
PubMed1993

Increased viral titer through concentration of viral harvests from retroviral packaging lines.

Dependent on the viral vector and the specific assay used, viral titers produced from commonly used retroviral packaging cell lines have an upper limit in the range of 10(5) to 10(7) infectious units/ml. We have developed a generally applicable method, using hollow-fiber filtration technology, which allows for the concentration of infectious virus derived from packaging lines. This method resulted in a reproducible 10- to 30-fold increase in viral titer and can readily be scaled to accommodate larger input volumes. Over 80% of the input virus is recovered in an infectious form in the concentrate. Concentrated virus containing media was seen to produce higher infection frequencies in Jurkat T cells as compared to unconcentrated virus containing media; however, this was not proportional to the differences in viral titer observed by limiting dilution analysis on NIH-3T3 cells. These results are discussed in relation to the importance of factors other than viral titer in determining transduction frequencies.

3T3 Cells↗
PubMed1990

Reovirus binds to multiple plasma membrane proteins of mouse L fibroblasts.

Plasma membranes from mouse L fibroblasts were isolated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were electroblotted to nitrocellulose paper and probed with 125I-labeled type 3 (T3) reovirus. Multiple protein bands with molecular weights ranging from 26 to 200 kDa were consistently recognized by the virus. Such binding was specific since it was blocked in the presence of unlabeled virus. That these proteins were exposed on the cell surface was confirmed by their susceptibility to sulfo-NHS-LC-biotin labeling of intact cells prior to membrane purification. Blots probed with wheat germ agglutinin (WGA)-gold showed a similar pattern of protein bands. These findings are consistent with the ability of WGA to block reovirus binding to L cells, and with our recent demonstration that the alpha-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus (R. W. Paul, A. H. C. Choi, and P. W. K. Lee, Virology 172, 382-385, 1989). Both type 1 and type 3 reoviruses were found to recognize the same set of multiple proteins on the blot, which is again consistent with the previous observation that the two serotypes compete with each other for binding to intact L cells.

Animals↗
PubMed1989

The alpha-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus.

A series of synthetic sialosides were evaluated for their ability to interact with reovirus serotype 3. It was found that sialosides with terminal N-acetylneuraminic acid (NeuNAc) linked in either an alpha 2,3 or alpha 2,6 configuration effectively blocked the binding of reovirus to mouse L fibroblasts, in contrast to a monosaccharide mixture containing the oligosaccharide constituents. Direct binding of reovirus to the sialosides was also demonstrable using sialosides conjugated to bovine serum albumin as ligands in a solid phase binding system. Of particular significance was the finding that the conjugate containing alpha-sialic acid alone (linked to bovine serum albumin) was capable of being recognized by reovirus at a level comparable to that of the other sialoside conjugates. Virus binding was abrogated by pretreating such conjugates with neuraminidase. These results suggest that the alpha-anomeric form of sialic acid is the minimal receptor determinant for reovirus recognition.

Carbohydrate Sequence↗
PubMed1987

Glycophorin is the reovirus receptor on human erythrocytes.

Purified glycophorin (predominantly type A) from human erythrocytes was found to effectively inhibit reovirus hemagglutination (HA) in contrast to other glycoproteins such as fetuin or ovalbumin. Glycophorin was also a potent inhibitor of reovirus and protein sigma 1 binding to mouse L fibroblasts. Glycophorin pretreated with neuraminidase lost these inhibitory properties. Using a solid phase binding assay, it was demonstrated that reovirus as well as protein sigma 1 could specifically bind to glycophorin immobilized on polystyrene plates. This binding was inhibited by wheat germ agglutinin (WGA) but not by other lectins such as peanut agglutinin (PA), Maclura pomifera agglutinin (MPA), Bauhinia purpurea agglutinin (BPA), or concanavalin A (Con A). Binding of reovirus to glycophorin was also partially inhibited by a monoclonal antibody (10F7) (W. L. Bigbee, R. G. Langlois, M. Vanderlaan, and R. H. Jensen, 1984, J. Immunol. 133, 3149-3155), which recognizes a determinant common to the M and N forms of glycophorin, but not by N-specific monoclonal antibodies NN4 and NN5 or an M-specific monoclonal antibody, 6A7. Taken together, these results clearly indicate that the M, N blood group antigen, glycophorin, is the erythrocyte receptor for reovirus.

Antibodies, Monoclonal↗
PubMed1987

Reovirus type 3 infection in patients with primary biliary cirrhosis and primary sclerosing cholangitis.

Reovirus type 3 (Reo-3) infection has recently been implicated in the pathogenesis of certain idiopathic, cholestatic liver diseases of newborns. In the present study, antibody titres to Reo-3 virus (anti-Reo-3) were determined in sera from 43 adults with idiopathic cholestatic liver disease, including 34 patients with primary biliary cirrhosis (PBC) and 9 patients with primary sclerosing cholangitis (PSC). Seventy-four adults with various other causes of chronic liver disease and 16 healthy volunteers served as controls. Geometric mean titres of anti-Reo-3 were significantly higher in PBC and PSC sera than chronic liver disease and healthy controls (P less than 0.005). Mean antibody titres for all patient groups, however, were within the 95% confidence limits for normals. Seven of 34 (21%) PBC patients and 3/9 (33%) PSC patients had elevated titres of anti-Reo-3, as compared to only 4/74 (5%) chronic liver disease (P less than 0.05) and 0/16 (0%) healthy control subjects (P less than 0.05) (Fisher's Exact Test). Antibody titres to five other common viruses were normal in patients with high anti-Reo-3 titres when compared to age- and sex-matched controls with liver disease. Immunoperoxidase staining for Reo-3 viral markers and cultures of liver biopsy material for Reo-3 virus were negative in both patients and controls. The results of this study indicate that, although patients with PBC and PSC have higher anti-Reo-3 antibody titres than patients with other forms of chronic liver disease or healthy volunteers, only a minority of these patients have titres that exceed the 95% confidence limits for normals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

The prevalence of antibodies to reovirus type 3 in adults with idiopathic cholestatic liver disease.

Recent evidence suggests that neonatal infection with Reovirus type 3 (Reo-3) may play an important role in the pathogenesis of certain idiopathic cholestatic liver diseases of children. Whether this virus is of pathogenetic importance in similar diseases seen in adults is unclear. In this study, sera from 22 adults with idiopathic cholestatic liver disease (16 primary biliary cirrhosis (PBC) and six primary sclerosing cholangitis (PSC] were tested for antibody to Reo-3 virus by a standard hemagglutination inhibition assay. The results were compared to antibody prevalence in ten adults with various other causes of chronic liver disease and 11 healthy volunteers. Although patients with idiopathic cholestatic liver disease had a high prevalence of anti Reo-3 antibodies (20/22, 91%), similar prevalence rates were found in the two control populations (7/10, 70% in chronic liver disease controls and 11/11, 100% in healthy volunteers). Moreover, the geometric mean titres of antibody to Reo-3 virus were similar in all study groups. While these results do not exclude a pathogenetic role for Reo-3 viral infection, they do imply that some genetic predisposition must exist if Reo-3 infection is truly of pathogenetic importance in idiopathic cholestatic liver disease of adults.

Adult↗
PubMed1984

Studies on reovirus receptors of L cells: virus binding characteristics and comparison with reovirus receptors of erythrocytes.

"Saturation binding experiments" were carried out to characterize the attachment of reovirus to mouse L fibroblasts. Scatchard analysis of data obtained from such experiments suggests that one homogeneous set of noncooperative, high affinity binding sites are involved in reovirus attachment. It is estimated that L cells possess between 3 and 5 X 10(5) reovirus binding sites per cell and that the equilibrium dissociation constant (KD) is approximately 3 X 10(-9) M. Scatchard analysis of data from similar experiments, carried out in the presence of anti-sigma 1 and anti-sigma 3 antibodies, revealed that although both antibodies prevent viral attachment, they exhibit distinct binding inhibition characteristics: anti-sigma 1 effectively abolishes high-affinity, specific binding, whereas anti-sigma 3 apparently blocks low-affinity, nonspecific interactions. The nature of the L-cell receptor was then probed using various enzymes and reagents, and compared with that of the reovirus receptor on human type O erythrocytes. It was found that whereas reovirus hemagglutination (HA) is inhibited by pretreatment of erythrocytes with various proteases or neuraminidase, virus binding to L cells is unaffected by such treatments. Neither HA nor cell binding is inhibited by the various sugars tested, including N-acetyl-D-glucosamine, which was previously reported to inhibit reovirus HA (L. D. Gelb and A. M. Lerner, 1965, Science 147, 404-405). Both L cells and erythrocyte reovirus receptors are nevertheless highly sensitive to periodate treatment, which presumably destroys the high-affinity reovirus binding sites since protein sigma 1, which is capable of attaching to L cells by itself, does not bind to cells pretreated with periodate. It is therefore concluded that sugar residues on the receptor may be involved in this specific interaction. The possibility that gangliosides may serve as reovirus receptors was also probed. It was found that bovine brain gangliosides, but not cerebrosides, readily aggregate reovirus, inhibit HA, and block viral attachment to L cells. However, binding of protein sigma 1 to L cells is unaffected by gangliosides. Inhibition of reovirus HA and L-cell binding by these gangliosides is therefore most likely due to a steric hindrance effect brought about by interactions between the gangliosides and other components of the outer viral capsid.

Animals↗
PubMedDate not supplied

Interleukin-7 gene transfer in non-small-cell lung cancer decreases tumor proliferation, modifies cell surface molecule expression, and enhances antitumor reactivity.

Cytokine gene transfer to tumor cells can augment host antitumor responses and modify tumor phenotype. To evaluate the immunoregulatory and antitumor capacities of lung tumor-derived interleukin-7 (IL-7), we transduced non-small-cell lung cancer (NSCLC) cell lines with the IL-7/HyTK internal ribosomal entry site (IRES) retroviral vector and evaluated modifications in tumor phenotype and cocultured effector activities. In vitro proliferation of IL-7-transduced tumor cells was significantly less than control vector-transduced and parental tumor cells. The decreased proliferation rates of IL-7-transduced cells could be reproduced by adding high concentrations of recombinant IL-7 to the parental cells. Anti-IL-7 monoclonal antibody significantly increased the proliferation of the IL-7-transduced cells (P < .05). Parental NSCLC cells were found to express the IL-7 receptor, and IL-7 gene transduction did not alter expression of the IL-7 receptor. IL-7 transduction significantly altered tumor cell expression of intracellular adhesion molecule 1, major histocompatibility complex 1, lymphocyte function-related antigen 3, very late activation antigen beta 1, and p185neu. Peripheral blood lymphocytes cocultured with either IL-7-transduced tumor cells or tumor supernatants had enhanced cytolytic and proliferative capacities compared with coculture with control vector-transduced or parental cells. Our findings indicate that IL-7 gene transfer in NSCLC significantly augments cocultured effector activities in vitro, inhibits tumor cell proliferation, and modifies tumor cell surface phenotype. These findings suggest that IL-7 gene therapy may be effective in modifying host antitumor responses in NSCLC.

Antibodies, Monoclonal↗
PubMedDate not supplied

Dendritic cells augment granulocyte-macrophage colony-stimulating factor (GM-CSF)/herpes simplex virus thymidine kinase-mediated gene therapy of lung cancer.

Lung cancer, the leading cause of cancer death in the United States, is resistant to most currently available therapies. To evaluate a multicomponent gene therapy approach that replaces tumor-bearing host immune deficits, we genetically modified Line 1 (L1C2), a weakly immunogenic alveolar cell carcinoma cell line. L1C2 was transduced ex vivo with a retroviral construct that contained two components: a cytokine gene (granulocyte-macrophage colony-stimulating factor) and a drug sensitivity gene (herpes simplex virus thymidine kinase). The third component of this therapy, in vitro-activated syngeneic bone marrow-derived dendritic cells, was included to augment antigen presentation. The addition of ganciclovir (GCV) caused the lysis of transduced tumor cells, resulting in the release of potential tumor antigens. Ex vivo-transduced tumor cells regressed in vivo following GCV therapy but were not effective in the treatment of established parental tumors. To treat established tumors, dendritic cells were administered in combination with transduced tumor cells and GCV. A total of 50% of these mice rejected the 5-day-old established tumors and were immune to rechallenge with parental L1C2 cells. Thus, this multicomponent gene therapy system leads to both the regression of established tumors and enhanced immunogenicity in this weakly immunogenic murine lung cancer model.

Adenocarcinoma, Bronchiolo-Alveolar↗