We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.

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Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.Hepatitis C virus (HCV), a hepatotropic, positive-stranded RNA virus of the Flaviviridae family, is estimated to infect at least 400 million people worldwide and is a major etiologic agent of liver failure and hepatocellular carcinoma (11,66,70). HCV comprises a 9.6-kb genome with a conserved 5Ј untranslated region that functions as an internal ribosome entry site (33, 69). The untranslated region precedes a long open reading frame that encodes a 3,010-amino-acid (aa) polypeptide that is subsequently cleaved into 10 protein products (33). The amino-terminal region of the viral polypeptide is posttranslationally processed by host cell proteases to generate three structural protein products, Core (nucleocapsid) and envelope glycoproteins E1 and E2 (31, 43). Nonstructural proteins that facilitate virus replication (NS2, -3, -4a, -4b, -5a, and -5b) reside in the carboxy region of the polypeptide and are cleaved by virus-encoded proteases comprising 68).Standard therapeutic intervention for HCV infection consists of the administration of interferon (IFN) in combination with ribavirin. However, less than 50% of infected patients respond to this regimen and few alternative therapies exist (13, 14, 26, 52). There is presently no tissue culture system to efficiently cultivate HCV, which not only hampers research efforts aimed at elucidating the molecular mechanisms of virus replication but also impedes attempts at producing candidate vaccines and immunotherapies that target HCV-related disease. Consequently, a number of recombinant subunit-based HCV vaccine strategies, involving genetic immunization and purified proteins, have been attempted, as well as virus vectors expressing the HCV envelope glycoproteins (3,8,25,30,56,63,65). Determining an effective vaccination strategy has proven di...

Reactivation of latent HSV is exceptionally frequent in cancer patients. The results of this study suggest that virus reactivation occurs independently of cancer chemotherapy. The potential role of HSV reactivation in oral mucosa damage remains unclear.

FlhD is a positive regulator of cadA. A mutant with a transposon-mediated lacZ fusion to cadA exhibited a cell division phenotype similar to that of the flhD mutant and had FlhD-dependent ␤-galactosidase activity. Under different growth conditions, the cell division rate correlated with the level of expression of cadA.In a previous study (13), we demonstrated that Escherichia coli flhD is involved in a process other than flagellar expression, namely, reducing the cell division rate as cells enter stationary phase. It was demonstrated that flhD mutant cells grown in tryptone broth (TB) (1% tryptone, 1% NaCl) continued to divide at a rate that is typical for mid-exponential growth at a time when wild-type cells started to reduce their cell division rate. flhD mutant cells were smaller than wild-type cells as they entered stationary phase, and it was demonstrated that this characteristic correlated with their inability to sense the depletion of serine from the medium, which signals wild-type cells to reduce their cell division rate. It was discussed that the signal cascade probably included phosphorylation of OmpR (15) by acetyl phosphate (14). It was unclear how the expression of flhD led to the reduction of the cell division rate.We now demonstrate that the effect of FlhD upon the cell division rate is mediated through induction of cadA. The cad operon consists of cadB and cadA, encoding the lysine/cadaverine antiporter CadB and the enzyme lysine decarboxylase CadA. Expression of this operon is dependent on the regulator CadC, whose gene is located upstream of cadA. The expression from the p cad promoter is induced by low pH, low oxygen, rich medium, and excess lysine. All of these signals activate cadBA expression via a cadC-dependent pathway (10)(11)(12)18).Construction of the cadA transposon mutant. All strains, plasmids, and phage used in this study are listed in Table 1. Plasmid pPB10 was constructed by cloning the flhD gene, obtained from plasmid pPM61 (1) by PCR, into the arabinoseinducible plasmid pKB130 (17), kindly provided by L. Katz (Abbott Laboratories, Chicago, Ill.). pcnB1 zad-981::mini-Kn from strain RP7947 (8), kindly provided by J. S. Parkinson (University of Utah, Salt Lake City), was used to reduce the plasmid copy number and the background expression. The resulting strain BP78 exhibited complementation of the flhD mutation in the presence of arabinose. The cadA::Tn10-lacZ fusion was obtained by transposon mutagenesis. Cells of strain BP78 were infected with phage lysate TnphoAЈ-2 (19), kindly provided by B. Wanner (Purdue University, West Lafayette, Ind.). Cells were plated on tetracycline to identify chromosomal insertions. One tetracycline-resistant colony was blue in the presence of arabinose and white in its absence. This strain was designated BP87 and is characterized in this report. cadA::Tn10-lacZ was transduced by P1 transduction into the wild-type strain YK410 and the flhD mutant strain YK4131. In Luria-Bertani medium (LB) (TB plus 0.5% yeast extract) and late-exponential phase, the ...

The aim of this study was to determine surface roughness and topography of polished dental resin-based nanocomposites. Four representative dental resin-based nanocomposites were tested in the study: two nanohybrids (Filtek Z550 and Tetric EvoCeram) and two nanofilled (Filtek Ultimate Body and Filtek Ultimate Translucent); and two reference materials: one microfilled (Gradia Direct) and one microhybrid (Filtek Z250). Polymerized cylindrical specimens (4 mm x 2 mm) were polished with multi-step polishing system- Super Snap. Immediately after the polishing, topography of each specimen was examined by Veeco di CP-II Atomic Force Microscope. Specimen's surface has been scanned in 6 points in contact mode with CONT20A-CP tips. 1 Hz scan rate and 256 × 256 resolution were used to obtain topography on a 90 µm × 90 µm scanning area. Measured topography data were processed by Image Processing and Data Analysis v2.1.15 software. Following parameters were compared among specimens: average roughness and maximum peak-to-valley distance. All of the tested materials had similar average surface roughness after finishing and polishing procedure. The lowest values occurred in the material Filtek Ultimate Body, and the highest in the Filtek Z550. When interpreting maximum peak-to-valley distance the larger differences in values (up to 100%) occurred in Filtek Z550, Filtek Z250 and Filtek Ultimate Body, which is a result of the deep polishing channels and tracks. Type, size, distribution of fillers and filler loading in tested materials, didn't influence average roughness values, but had an impact on maximum peak-to-valley distance values.

The objective of this work was to measure and correlate the degree of conversion (DC), mechanical properties and monomer elution from self-, dual- and light-cured core composites. Five samples of each of the following materials were prepared for each test: Clearfil (Core, Photo Core, Automix), Bisco (Core-Flo, Light-Core and Bis-Core). DC was determined using FTIR, compressive and flexural strength and modulus of elasticity using a universal testing machine and microhardness using Vickers hardness. Elution was measured using HPLC. One-way ANOVA with Tukey’s post-test and Pearson’s correlation were used to statistically analyze the data. DC of Clearfil-Dual (70.1%) and Clerafil-Photo (66.8%) were higher than Clearfil-Self (55.4%) and all Bisco materials (51.4–55.3%). Flexural strength of Clearfilwas higher than that of Bisco composites. The Microhardness of Clearfil-Dual (119.8VHN) and Clearfil-Photo (118.0VHN) were higher compared to other materials. The greatest elution was detected from self-cured materials. DC positively correlated to microhardness and compressive/flexural strength and negatively to BisGMA elution. Clearfil-Photo and Automix showed higher conversion, lower monomer elution and, generally, better mechanical properties. Self-cured composites should not be recommended for routine clinical use as their performance was inferior to dual- and light-cured composites. Microhardness may be used as an indicator of elution.

Low nanosilica addition proved more effective in improving mechanical properties compared to higher additions. Furthermore, handling properties are unaffected by nanosilica addition.