Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.     

Antimutagenic and anticarcinogenic potentials of some Thai vegetables

HPLC assays were developed and validated for the quantitation of the novel orally active nonsteroidal antiestrogen EM-800 ?(S)-(+)-4-[7-(2,2-dimethyl-l-oxopropoxy)-4-methyl-2-[4-[2-(1-pipe ridinyl)- ethoxy]phenyl]-2H-l-benzopyran-3-yl]-phenyl 2,2-dimethylpropanoate?. The assay involves reversed-phase C18 or C4 columns using different mobile phases with ammonium acetate buffers and UV detection at lambda = 240 nm. The standard curve was linear over the concentration range of 10-1100 micrograms/ml. The precision (% relative standard deviation) values of these methods were in the range of 0.38-0.52 and 1.89-3.45% with C4 and C18 reversed phases, respectively. The limit of detection was found to be 1 microgram/ml. Enantiomeric separation was also obtained using a chiral method (ChiralPak AD column) using a mixture of hexane-reagent alcohol-diethylamine (94.9:5.0:0.1) as mobile phase. These methods were applied to stability studies, evaluation of pharmaceutical dosage forms and in the framework of toxicological studies. Details of some of these applications will be presented.     

Complications resulting from saphenous vein patch graft after carotid endarterectomy

OBJECTIVE: Reducing surgical risks to the minimum in carotid endarterectomy has become crucial, especially with the results of recent clinical trials extending indications to asymptomatic patients. The use of the saphenous vein patch graft (SVPG) has been suggested to reduce early postoperative thrombosis and cerebral infarct as well as late recurrent stenosis. However, the exact risks and complications involved in this technique are not known. METHODS: During a 23-year period (1972-1994), 2888 carotid endarterectomies with SVPG for primary carotid stenosis were performed by the Neurosurgical Cerebrovascular Service at the Mayo Clinic. The data from all patients were retrospectively analyzed, emphasizing postoperative complications related to SVPG. RESULTS: There were five postoperative vein ruptures (0.17%), four cases of aneurysm formation, and three cases of deep infection necessitating surgical intervention. The vein patch ruptured in one male patient and four female patients (mean age, 69 yr). All ruptures occurred within 4 days of the primary operation, including two during the first 24 hours. All patients with rupture underwent emergency surgery and were found to have intact suture lines and tears in the middle of the grafts. Two patients recovered without deficits, one suffered major disability, and the other two died. Aneurysm of the patch developed in two male patients and two female patients (mean age, 71 yr). All of the patients developed painless pulsatile neck masses 1 to 9 years after the initial surgery; two also had recurrent ischemic symptoms. All of the patients with aneurysms underwent surgical correction without consequences. CONCLUSION: Although the benefit of routine use of SVPG in carotid endarterectomy is still the focus of debate, this analysis showed that its use adds a small but definite risk of serious complications related to inherent weakness of the venous tissue. If a surgeon chooses to use a patch graft, our recommendation is for use of a synthetic material rather than vein.     

Effects of removal of weight-bearing function on contractility and myosin isoform composition in single human skeletal muscle cells

The purpose of this study was to investigate the effects of a 6-week period without weight bearing, achieved by bed rest, on the contractile behaviour, myosin isoform expression and myofibrillar protein content of single human muscle fibres. Percutaneous biopsied specimens of the quadriceps muscle were taken from three healthy male volunteers before and at the end of the experimental period. Maximum force normalised to cross-sectional area (specific tension), maximum velocity of unloaded shortening (V0), and myosin heavy chain (MyHC) and light chain (MyLC) isoform composition were measured in single membrane-permeabilised muscle cells obtained from these specimens. At the end of the experimental period, specific tension was reduced (P < 0.001) by 40% and there was a parallel decline in myofibrillar protein content per muscle cell volume. V0 did not change significantly in response to bed rest when data from all muscle cells were pooled. In two of the subjects, however, V0 decreased (P < 0.01-0.001) in muscle cells expressing the beta/slow (type I) MyHC isoform, but there was no change in fibres expressing type IIA or a combination of type IIA and IIB MyHCs. The slowing in type I MyHC fibres was associated with a change in the isoform composition of the regulatory MyLC.     

Physical limitations on Salmonella typhi entry into cultured human intestinal epithelial cells

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >/=40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of approximately 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta1 integrin superfamily.     

Candida albicans endocarditis associated with a contaminated aortic valve allograft: implications for regulation of allograft processing

A patient developed Candida albicans endocarditis and fungemia after undergoing aortic valve replacement with an allograft. The allograft had been found during tissue bank processing to be contaminated with C. albicans, but it was culture-negative for C. albicans after routine disinfection with an antifungal-containing antimicrobial solution. Comparison of the preimplantation and postimplantation C. albicans isolates revealed remarkable genetic similarity, but antifungal susceptibility testing showed that the postimplantation isolate was more resistant to fluconazole and amphotericin B than the preimplantation isolate, suggesting emergence of resistance after disinfection. Implantation of a contaminated heart valve allograft can occur despite disinfection during processing and can result in endocarditis in the recipient. Antimicrobial disinfection protocols that include antifungal drugs may be ineffective. Current U.S. Food and Drug Administration regulations do not require companies to specify details concerning allograft processing. Additional measures may be required to prevent tissue bank release of allografts contaminated with C. albicans or other pathogens.