Treatment of ischemic wounds using cultured dermal fibroblasts transduced retrovirally with PDGF-B and VEGF121 genes.

The healing of ischemic wounds is a particularly difficult clinical challenge. In this study, rabbit dermal fibroblasts transduced retrovirally with human platelet-derived growth factor B (PDGF-B) and human vascular endothelial growth factor 121 (VEGF121) genes were used to treat wounds in a rabbit ischemic ear model. The PDGF-B and VEGF121 genes were obtained from human umbilical vein endothelial cells (HUVECs) by reverse transcription-polymerase chain reaction, cloned into retroviral vectors under control of the beta-actin promoter, and introduced into primary rabbit dermal fibroblast cells. In vitro results demonstrated that rabbit dermal fibroblasts are transduced and selected readily using retroviral vectors, and are engineered to secrete PDGF-B and VEGF121 at steady-state levels of 150 ng per 10(6) cells per 24 hours and 230 ng per 10(6) cells per 24 hours respectively. These cells were then seeded onto polyglycolic acid (PGA) scaffold matrices and used to treat ischemic rabbit ear wounds. Immunohistochemistry showed intense staining for PDGF-B and VEGF121 in the wounds treated with these transduced cells compared with the control treatment groups. For the relatively more ischemic distal ear wounds, granulation tissue deposition was increased significantly in the wounds treated with PDGF-B- and VEGF121-transduced cells compared with wounds treated with PGA alone. These results demonstrate that gene augmentation of rabbit dermal fibroblasts with the PDGF-B and VEGF121 genes introduced into this ischemic wound model via PGA matrices modulates wound healing, and may have clinical potential in the treatment of ischemic wounds.

Evaluation of two current generation enzyme immunoassays and an improved isolation-based assay for the rapid detection and isolation of rotavirus from stool.

BACKGROUND: Rapid and accurate rotavirus testing is important in decisions involving patient care and management. Quality assurance testing needs to be periodically performed, especially among widely used assays having a direct impact on patient care. OBJECTIVES: To evaluate the current generation Kallestad Pathfinder Direct antigen Detection system (PTH), and the widely used Rotaclone(R) Rotavirus EIA Diagnostic Kit (RTC), in comparison with an improved cell culture amplification-antigen detection (CCA-Ag) isolation-based assay. STUDY DESIGN: Two hundred stool specimens (specimen stored at > or =-75 degrees C), which had been previously tested by PTH, were tested by RTC and CCA-Ag. Discordant specimens were retested by PTH, blocking assay, polyacrylamide gel electrophoresis (PAGE), and/or electron microscopy (EM). RESULTS: Among 200 stool specimens, 197 were in accord by PTH, RTC and CCA-Ag. The sensitivity, specificity, positive and negative predictive values for RTC, PTH and CCA-Ag were, 100, 99, 99, 100, 100, 99, 99, 100; and 98, 100, 100, 98%, respectively. Among five initially discordant specimens, two required a period of 10 days to affect isolation. A non-cultivatable (CCA-Ag negative) but true positive specimen, was identified as rotavirus group A serotype G2 by RT-PCR. Four true positive but discordant specimens were blocking assay negative using one or both EIA kits. CONCLUSIONS: PTH and RTC are excellent rotavirus detection system. However, PTH is more expensive (ca. $3.50 vs. $2.00 per test), mandates a slightly longer turn-around time (ca. 1 vs. 1.5 h), and necessitates slightly more hands-on manipulative/preparative steps. Blocking assay was not a reliable confirmatory test for the resolution of specimen discordancy. A combination of CCA-Ag, PAGE, EM, and/or perhaps RT-PCR, is recommended as an appropriate test panel for the resolution of discordant results during assay evaluation. The newly modified and simplified 48-h rotavirus isolation-based assay may serve as a base line methodology in laboratory evalaution studies, as a laboratory support methodology during drug/vaccine efficacy trials, or for the testing of sources (e.g., biopsy/autopsy tissues) not approved for assay by commercial rotavirus kits.

Cartilage and bone regeneration using gene-enhanced tissue engineering.

Joint cartilage injury remains a major problem in orthopaedics with more than 500,000 cartilage repair procedures performed yearly in the United States at a cost of hundreds of millions of dollars. No consistently reliable means to regenerate joint cartilage currently exists. The technologies of gene therapy and tissue engineering were combined using a retroviral vector to stably introduce the human bone morphogenic protein-7 complementary deoxyribonucleic acid into periosteal-derived rabbit mesenchymal stem cells. Bone morphogenic protein-7 secreting gene modified cells subsequently were expanded in monolayer culture, seeded onto polyglycolic acid grafts, implanted into a rabbit knee osteochondral defect model, and evaluated for bone and cartilage repair after 4, 8, and 12 weeks. The grafts containing bone morphogenic protein-7 gene modified cells consistently showed complete or near complete bone and articular cartilage regeneration at 8 and 12 weeks whereas the grafts from the control groups had poor repair as judged by macroscopic, histologic, and immunohistologic criteria. This is the first report of articular cartilage regeneration using a combined gene therapy and tissue engineering approach.

Rab6c, a new member of the rab gene family, is involved in drug resistance in MCF7/AdrR cells.

A new Rab6 homolog cDNA, Rab6c, was discovered by a hypermethylated DNA fragment probe that was isolated from a human multidrug resistant (MDR) breast cancer cell line, MCF7/AdrR, by the methylation sensitive-representational difference analysis (MS-RDA) technique. Rab6c was found to be under-expressed in MCF7/AdrR and MES-SA/Dx5 (a human MDR uterine sarcoma cell line) compared with their non-MDR parental cell lines. MCF7/AdrR cells expressing the exogenous Rab6c exhibited less resistance to several anti-cancer drugs, such as doxorubicin (DOX), taxol, vinblastine, and vincristine, than the control cells containing the empty vector. Flow cytometry experiments confirmed that the transfectants' diminished resistance to DOX was caused by increased drug accumulation induced by the exogenous Rab6c. These results indicate that Rab6c is involved in drug resistance in MCF7/AdrR cells.

Hepatocyte growth factor decreases sensitivity to chemotherapeutic agents and stimulates cell adhesion, invasion, and migration.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), plays an important role in cell:cell adhesion, cell proliferation, motility, and invasiveness of epithelial cells and tumor cells. In this study, we examined the effects of HGF on these types of biological activities and chemosensitivity in Chinese hamster ovary (CHO) cells by stable transfection of the HGF gene. HGF-transfected clones produced very high titers of HGF protein, whereas control vector-transfected clones did not produce detectable HGF protein. HGF-transfected clones showed modestly increased proliferation rates and became more resistant to cell death and apoptosis caused by two anticancer drugs, adriamycin (ADR) and camptothecin (CPT), compared to controlvector-transfected clones. Furthermore, HGF-transfected clones also exhibited increased activities of cell adhesion, migration, and invasion. The current study is the first demonstration that overexpression of the HGF gene affects chemosensitivity and cell metastasis behaviors, suggesting that HGF signaling pathway is a promising new target of therapeutic intervention of tumors.

Human serum-resistant retroviral vector particles from galactosyl (alpha1-3) galactosyl containing nonprimate cell lines.

Retroviral vector particles (RVP) which are resistant to inactivation by human serum will be needed for many in vivo gene therapy applications. Murine-based producer cell lines generate RVP which are inactivated by human serum, reportedly due to the presence of the galactosyl (alpha1-3) galactosyl carbohydrate moiety (alphaGal) on these and other nonprimate producer cells and RVP. Consequently, human cells (which lack the alphaGal moiety) have been developed as producer cell lines for generation of human serum-resistant RVP. In this study, we report that contrary to earlier reports, the presence of the alphaGal moiety on producer cells and RVP does not necessarily correlate with cell killing or RVP inactivation by human serum. We show that the alphaGal-positive ferret brain cell line, Mpf, is an excellent basal cell line for generation of RVP which have titers and serum resistance levels equal to or greater than RVP produced in human cell lines such as HT1080. Therefore, packaging cell lines need not be limited to those of human or primate origin for production of human serum-resistant RVP.

The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.