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Biomedical subjects

J R Wands

Publications and source records attributed to J R Wands.

At least 55 records · Page 3Linked to original sources
PubMed1999

Targeted gene transfer to hepatocellular carcinoma cells in vitro using a novel monoclonal antibody-based gene delivery system.

Gene therapy approaches for the treatment of malignant tumors will require high-level expression of therapeutic genes in tumors compared with normal tissues. This may be achieved either by targeted gene delivery to tumor cells or by the use of tumor-specific promoters. Here, we describe the use of a novel conjugate consisting of a tumor-reactive monoclonal antibody (mAb), designated AF-20, coupled to a DNA-binding cationic amphiphile, cholesteryl-spermine, for gene delivery to hepatocellular carcinoma (HCC) cells. The high-affinity mAb, AF-20, recognizes a rapidly internalized 180-kd cell-surface glycoprotein that is abundantly expressed on HCC and other human tumors. The AF-20 mAb and an isotype-matched control antibody (C7-57) were covalently coupled to cholesteryl-spermine. Binding and internalization of AF-20-cholesteryl-spermine was confirmed by fluorescence microscopy using fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG antibody. Following transfection of FITC-labeled oligonucleotides and ethidium monoazide-labeled plasmid DNA, cellular uptake and intracellular localization of nucleic acids were examined by laser scanning confocal microscopy. Transfection of luciferase or beta-galactosidase reporter genes complexed to AF-20-cholesteryl-spermine resulted in high levels of gene expression in AF-20 antigen-positive tumor cells. Very low levels of gene expression were observed using the control compound (C7-57-cholesteryl-spermine), which does not recognize the AF-20 tumor antigen or when AF-20 antigen-negative NIH 3T3 cells were transfected with AF-20-cholesteryl-spermine. Thus, covalent coupling of the AF-20 mAb to cholesteryl-spermine generated a highly specific and efficient nonviral vector system for targeted gene delivery to AF-20 antigen-positive HCC cells.

Animals↗
PubMed1999

Specific inhibition of hepatitis B virus replication by sense RNA.

We describe effects of sense RNA molecules on hepatitis B virus (HBV) replication and antigen synthesis in transiently transfected cells. When certain subgenomic fragments of HBV were expressed as sense RNA together with a replication-competent genome of HBV, they inhibited HBV replication by up to 75% and HBsAg secretion by up to 60%. The corresponding antisense sequences had a 50% inhibitory effect in one case and no effect in another case. The sense RNA species did not inhibit duck hepatitis B virus (DHBV) replication, suggesting specific inhibitory effects. HBV transcript levels were unaltered in the presence of sense RNA species, consistent with an inhibitory effect mediated at the posttranscriptional level. The inhibition of HBV replication by overexpression of sense RNA derived from the viral genome represents an example of sense cosuppression of an animal virus.

Cell Line↗
PubMed1999

Differential effects of ethanol on insulin-signaling through the insulin receptor substrate-1.

Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1deltaGrb2), Syp (IRS-1deltaSyp), or Grb2 and Syp (IRS-1deltaGrb2deltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1deltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1deltaSyp and IRS-1deltaGrb2deltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1deltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.

Cell Division↗
PubMed1999

Carboxypeptidase D is an avian hepatitis B virus receptor.

The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4 degrees C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.

Animals↗
PubMed1999

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein.

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.

Animals↗
PubMed1999

Intracellular retention of hepatitis B virus surface proteins reduces interleukin-2 augmentation after genetic immunizations.

We have previously shown that hepatitis B virus (HBV) surface antigens (HBsAgs) are highly immunogenic after genetic immunization. Compared to the secreted middle HBV surface proteins (MHBs) or small HBV surface proteins (SHBs), the nonsecreted large HBV surface protein (LHBs), however, induced significantly weaker humoral and cellular immune responses that could not be augmented by genetic coimmunizations with cytokine expression plasmids. In order to understand the mechanisms underlying this phenomenon, we examined the effect of coimmunizations with an interleukin-2 (IL-2) DNA expression plasmid on the immunogenicity at the B- and T-cell level of nonsecreted wild-type LHBs, a secreted mutant LHBs, wild-type SHBs, and a nonsecreted mutant SHBs. Coimmunizations of mice with plasmids encoding wild-type SHBs or the secreted mutant LHBs and IL-2 increased anti-HBs responses, helper T-cell proliferative activity and cytotoxic T-lymphocyte killing. By contrast, coimmunizations of plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells.

Animals↗
PubMed1999

Combinatorial screening and intracellular antiviral activity of hairpin ribozymes directed against hepatitis B virus.

A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.

Antiviral Agents↗
PubMed1999

DNA vaccines.

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) virus are worldwide problems often leading to the development of chronic liver disease and hepatocellular carcinoma. Genetic immunizations with DNA encoding for structural and nonstructural proteins of HCV and HBV in experimental mice generate a broad base CD4+ and CD8+ cellular immune response which may be required for viral clearance from the host. DNA based immunization is a promising antiviral approach for the development of therapeutic and prophylactic vaccine against HBV and HCV.

Animals↗
PubMed1999

Biologic significance of angiopoietin-2 expression in human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is generally a highly vascular tumor, but the mechanisms of neovascularization that permit rapid growth have not been defined. Angiopoietins (Ang) recently have been identified as ligands for vascular endothelial-specific Tie2 receptor tyrosine kinase and may be important growth factors in the generation of new blood vessels. We investigated Ang expression in 23 samples of HCC and paired adjacent uninvolved liver samples to determine if these genes have a potential role in the growth and spread of this disease. The full coding sequence of a variant angiopoietin-2 (Ang2) cDNA was obtained from HCC specimens, and the biologic consequences of overexpression on tumor formation and hemorrhage were determined in an animal model system. Angiopoietin-1 (Ang1) was equally expressed in HCC and adjacent noncarcinomatous liver tissue. Surprisingly, Ang2 was found to be highly expressed only in tumor tissue. In addition, Ang2 was expressed in 10 of 12 hypervascular HCC, but only in 2 of 11 hypovascular HCC. Ectopic expression of Ang2 in nonexpressing HCC cells promotes the rapid development of human hepatomas and produces hemorrhage within tumors in nude mice. These results suggest a role for Ang2 in the neovascularization of HCC. This enhanced gene expression may contribute to the clinical hypervascular phenotype, as well as tumor formation and progression.

Amino Acid Sequence↗
PubMed1999

Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression.

We developed approaches using antisense RNA to inhibit hepatitis C virus (HCV) RNA translation and HCV core protein expression. An HCV genotype 1b cDNA comprising nt 1-1321 or a fusion construct consisting of HCV (nt 1-584) and luciferase cDNAs were inserted downstream of T7 and CMV promoter sequences and used to generate HCV RNA target molecules. Such constructs will produce HCV core or HCV coreluciferase fusion proteins in vitro or within transfected cells. Seven different antisense RNA constructs were designed to target the highly conserved 5' region of HCV RNA at nt positions 1-402. For in vitro experiments, synthesized HCV RNA target sequences and antisense RNAs were mixed at various molar ratios and subsequently translated in a rabbit reticulocyte lysate system. In cell culture studies, the HCV core-luciferase fusion cDNA was co-transfected with antisense RNA-producing constructs into human hepatocellular carcinoma (HCC) cells. Luciferase activity in cell lysates was measured to determine quantitatively antiviral effects within the cell. It was found that translation of HCV RNAs was efficiently inhibited by antisense RNA in vitro. The specificity of this inhibition was confirmed using control target RNA sequences or nonrelevant antisense RNA constructs. Co-transfection studies demonstrated that antisense RNA inhibited HCV core-luciferase fusion protein expression by 41-57% in HuH-7 HCC cells. These studies indicate that antisense RNA will find viral target RNA sequences in HuH-7 cells and inhibit HCV RNA translation. More important, these studies have defined critical viral RNA target sequences susceptible to antisense inhibitory effects within the cell.

Animals↗
PubMed1998

Genetic immunization generates cellular and humoral immune responses against the nonstructural proteins of the hepatitis C virus in a murine model.

Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.

Animals↗
PubMed1998

Cloning, bacterial synthesis, and characterization of immunoglobulin variable regions of a monoclonal antibody specific for the hepatitis B virus X protein.

The nucleotide (nt) sequences encoding the variable regions of the heavy (H) and light (L) chains were determined for a murine monoclonal antibody, 12/231/93, which is specific for a linear epitope located between amino acids 90 and 102 of the hepatitis B virus (HBV) X protein (HBx). The variable (V) regions of the H and L chains were shown to belong to the mouse H chain subgroup II (C) and kappa L chain group III, respectively. The cloned variable region sequences were used for the production of a Fab fragment in Escherichia coli, which had binding activity for membrane immobilized recombinant HBx. These gene sequences may be useful for the study of HBx function in cells that will support HBV replication.

Amino Acid Sequence↗
PubMed1998

A novel frizzled gene identified in human esophageal carcinoma mediates APC/beta-catenin signals.

A novel member of the human frizzled (Fz) gene family was cloned and found to be specifically expressed in 3 of 13 well differentiated (23%), 13 of 20 moderately differentiated (62%), and 12 of 14 poorly differentiated (86%) squamous cell esophageal carcinomas compared with the adjacent uninvolved normal mucosa. The FzE3 cDNA encodes a protein of 574 amino acids and shares high sequence homology with the human FzD2 gene particularly in the putative ligand binding region of the cysteine-rich extracellular domain. Functional analysis revealed that transfection and expression of the FzE3 cDNA in esophageal carcinoma cells stimulates complex formation between adenomatous polyposis coli (APC) and beta-catenin followed by nuclear translocation of beta-catenin. Furthermore, cotransfection of a mutant construct encoding a FzE3 protein with a C-terminal truncation completely inhibited the interaction of APC with beta-catenin in cells. Finally, coexpression of FzE3 with Lef-1 transcription factor enhanced beta-catenin translocation to the nucleus. These observations suggest that FzE3 gene expression may down-regulate APC function and enhance beta-catenin mediated signals in poorly differentiated human esophageal carcinomas.

Adenomatous Polyposis Coli Protein↗
PubMed1998

A novel variant of human Grb7 is associated with invasive esophageal carcinoma.

The cDNAs of a putative growth factor-bound (Grb) 7 signal transduction molecule and Grb7V novel splice variant were isolated from an invasive human esophageal carcinoma. Although both Grb7 isoforms share homology with the Mig-10 cell migration gene, the Grb7V isoform lacks 88 base pairs in the C terminus; the resultant frame shift leads to substitution of an SH2 domain with a short hydrophobic sequence. The wild-type Grb7 protein, but not the Grb7V isoform, is rapidly tyrosyl phosphorylated in response to EGF stimulation in esophageal carcinoma cells. Analysis of human esophageal tumor tissues and regional lymph nodes with metastases revealed that Grb7V was expressed in 40% of Grb7-positive esophageal carcinomas. More importantly, Grb7V expression was enhanced after metastatic spread to lymph nodes as compared to the original tumor tissues. Finally, transfection of an antisense Grb7 RNA expression construct lowered endogenous Grb7 protein levels and suppressed the invasive phenotype exhibited by esophageal carcinoma cells. These findings suggest that Grb7 isoforms are involved in cell invasion and metastatic progression of human esophageal carcinomas.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Tightly regulated expression of the entire hepatitis C virus structural region in continuous human cell lines.

Investigation of the hepatitis C virus (HCV) life cycle is limited by the lack of an efficient cell culture system. Employing a tetracycline-regulated gene expression system we generated a panel of continuous human cell lines allowing the inducible expression and faithful processing of HCV structural proteins as well as of a functional NS2-3 autoprotease. HCV proteins were found in the cytoplasm in a pattern characteristic for the endoplasmic reticulum. High-level expression of HCV proteins was found to be cytotoxic. These cell lines represent a unique in vitro system in which to further investigate the structural proteins of HCV and to evaluate novel antiviral strategies against hepatitis C in a well-defined and reproducible cellular context.

Cell Compartmentation↗
PubMed1998

Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective.

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC). HBV mutants result from isoleucine (I) or valine (V) substitutions in the methionine (M) of the YMDD motif in the viral reverse-transcriptase catalytic domain. In addition, other mutations in the reverse-transcriptase "B domain" involving either a phenylalanine (F)-to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well. To determine the biologic consequences of these mutations on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 293 human embryo kidney-derived cell lines. In transiently transfected HCC cells, the viruses bearing the YI/VDD or F501L mutations had greatly impaired replication as compared to wild-type virus, whereas the virus carrying the L515M substitution showed the least defect. Double mutants with the L515M substitution showed intermediate defect between the YI/VDD or F501L and the L515M single-mutant strains. In contrast, when transfected into HEK 293 cells, the viruses bearing the YI/VDD or L515M mutation replicated as wild-type. However, under conditions of deoxynucleotide depletion produced by hydroxyurea treatment of HEK 293 cells, all mutants but not the wild-type virus exhibited a reduced replication phenotype similar to that observed in HCC cells. In both HCC and HEK 293 cells, the mutant viruses carrying the F501L substitution showed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level. These findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired viral replication within the cell.

2-Aminopurine↗
PubMed1998

Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice.

Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.

Animals↗