Mobilan: a recombinant adenovirus carrying Toll-like receptor 5 self-activating cassette for cancer immunotherapy.

Toll-like receptor 5 (TLR5) is considered an attractive target for anticancer immunotherapy. TLR5 agonists, bacterial flagellin and engineered flagellin derivatives, have been shown to have potent antitumor and metastasis-suppressive effects in multiple animal models and to be safe in both animals and humans. Anticancer efficacy of TLR5 agonists stems from TLR5-dependent activation of nuclear factor-κB (NF-κB) that mediates innate and adaptive antitumor immune responses. To extend application of TLR5-targeted anticancer immunotherapy to tumors that do not naturally express TLR5, we created an adenovirus-based vector for intratumor delivery, named Mobilan that drives expression of self-activating TLR5 signaling cassette comprising of human TLR5 and a secreted derivative of Salmonella flagellin structurally analogous to a clinical stage TLR5 agonist, entolimod. Co-expression of TLR5 receptor and agonist in Mobilan-infected cells established an autocrine/paracrine TLR5 signaling loop resulting in constitutive activation of NF-κB both in vitro and in vivo. Injection of Mobilan into primary tumors of the prostate cancer-prone transgenic adenocarcinoma of the mouse prostate (TRAMP) mice resulted in a strong induction of multiple genes involved in inflammatory responses and mobilization of innate immune cells into the tumors including neutrophils and NK cells and suppressed tumor progression. Intratumoral injection of Mobilan into subcutaneously growing syngeneic prostate tumors in immunocompetent hosts improved animal survival after surgical resection of the tumors, by suppression of tumor metastasis. In addition, vaccination of mice with irradiated Mobilan-transduced prostate tumor cells protected mice against subsequent tumor challenge. These results provide proof-of-concept for Mobilan as a tool for antitumor vaccination that directs TLR5-mediated immune response toward cancer cells and does not require identification of tumor antigens.

Rb inactivation accelerates neoplastic growth and substitutes for recurrent amplification of cIAP1, cIAP2 and Yap1 in sporadic mammary carcinoma associated with p53 deficiency.

Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis. We used newly generated MMTV-Cre105Ayn mice to inactivate p53 and/or Rb strictly in the mammary epithelium, and to determine recurrent genomic changes associated with deficiencies of these genes. p53 inactivation led to formation of estrogen receptor-positive raloxifene-responsive mammary carcinomas with features of luminal subtype B. Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation. Genome-wide analysis of mammary carcinomas identified a recurrent amplification at chromosome band 9A1, a locus orthologous to human 11q22, which contains protooncogenes cIAP1 (Birc2), cIAP2 (Birc3) and Yap1. It is interesting that this amplicon was preferentially detected in carcinomas carrying wild-type Rb. However, all three genes were overexpressed in carcinomas with p53 and Rb inactivation, likely due to E2F-mediated transactivation, and cooperated in carcinogenesis according to gene knockdown experiments. These findings establish a model of luminal subtype B mammary carcinoma, identify critical role of cIAP1, cIAP2 and Yap1 co-expression in mammary carcinogenesis and provide an explanation for the lack of recurrent amplifications of cIAP1, cIAP2 and Yap1 in some tumors with frequent Rb deficiency, such as mammary carcinoma.

Antiviral activity of clevudine [L-FMAU, (1-(2-fluoro-5-methyl-beta, L-arabinofuranosyl) uracil)] against woodchuck hepatitis virus replication and gene expression in chronically infected woodchucks (Marmota monax).

L: -FMAU [1-(2-fluoro-5-methyl-beta,L-arabinofuranosyl) uracil] has been shown to be an effective inhibitor of hepatitis B virus (HBV) and duck hepatitis B virus replication in cell culture and duck hepatitis B virus replication in acutely infected Peking ducks. The woodchuck hepatitis virus (WHV) and its natural host, the Eastern woodchuck (Marmota monax), have been established as a predictive model for the evaluation of antiviral therapies against chronic HBV infection. In this report, the antiviral activity of l-FMAU against WHV replication in chronically infected woodchucks is described. Four weeks of once-daily oral administration of L-FMAU significantly reduced viremia, antigenemia, intrahepatic WHV replication, and intrahepatic expression of woodchuck hepatitis virus core antigen (WHcAg) in a dose-dependent manner. At the highest dose administered (10 mg/kg/d), significant reductions of intrahepatic WHV RNA and covalently closed circular (ccc)WHV-DNA levels also were observed. The reduction in viremia was remarkably rapid at the higher doses of L-FMAU, with greater than 1,000-fold reductions in WHV-DNA serum levels observed after as little as 2 to 3 days of therapy. Following the withdrawal of therapy, a dose-related delay in viremia rebound was observed. At the highest doses used, viremia remained significantly suppressed in at least one half of the treated animals for 10 to 12 weeks' posttreatment. No evidence of drug-related toxicity was observed in the treated animals. L-FMAU is an exceptionally potent antihepadnaviral agent in vitro and in vivo, and is a suitable candidate for antiviral therapy of chronic HBV infection.

Hepatic expression of the woodchuck hepatitis virus X-antigen during acute and chronic infection and detection of a woodchuck hepatitis virus X-antigen antibody response.

The expression and localization of the woodchuck hepatitis virus X-antigen (WHxAg) was examined and compared with other markers of a woodchuck hepatitis virus (WHV) infection using rabbit antisera generated against recombinant WHxAg produced in bacteria. Cellular fractionation studies showed that WHxAg was localized to the soluble and cytoskeletal fractions of the cell when assayed by immunoprecipitation of [35S]-met-cys labeled extracts derived from primary cultures of acute WHV-infected hepatocytes. Immunohistochemical examination of liver from chronic WHV-infected animals showed WHV core antigen (WHcAg) and WHxAg expression in non-neoplastic tissue. The WHxAg was found localized to the cytoplasm of infected cells, similar to WHcAg. WHxAg expression was diminished in the foci of altered hepatocytes and in hepatocellular adenomas but was found in only 1 of 11 hepatocellular carcinomas (HCC). Hepatic biopsies from woodchucks experimentally inoculated with WHV were examined during the acute phase of infection and during convalescence for WHcAg and WHxAg expression by immunohistochemistry. Concurrent expression of WHcAg and WHxAg was observed during the viremic phase of infection. The two antigens exhibited similar localization to the cell cytoplasm, similar distribution within the liver lobule, and similar patterns of clearance during convalescence. An immune response to WHxAg was documented in some woodchucks following acute WHV infection. These studies further define the woodchuck model of HBV infection and should allow for the investigation of the role of hepadnaviral X-antigen expression in the pathogenesis of chronic hepatitis and HCC.

A novel adhesion factor produced by hamster pancreatic cancer cell line is effective on normal and carcinoma cell lines of different species.

An adhesion factor, produced by the hamster pancreatic cancer cell line PC-1.0, was tested for its efficiency in promoting the in vitro adhesion of normal and tumor cells (pancreas, lung, kidney, colon, breast, skin, prostate, neuroblast, melanocyte) derived from human, monkey, bovine, hamster, and rat sources. Using a modification of the dimethylthyazol diphenyl tetrazolium (MTT) assay, the factor was found to induce adhesion in all cell lines in a dose-dependent manner. Although the effect was variously expressed, there was a statistically significant difference between the MTT absorbance of cells incubated in the presence or absence of the factor. Conditioned medium of each cell line tested showed significantly less adhesion effect than that produced by PC-1.0 cells. Because our previous study indicated that the adhesion factor produced by PC-1.0 cells differed from known growth factors and adhesion molecules including fibronectin, vibronectin, laminin, and collagen, it appears that PC-1.0 cells produce a novel adhesion factor that enhances adherence of normal and malignant cells of different species.

Dietary energy restriction-induced modulation of protein kinase C zeta isozyme in the hamster pancreas.

Dietary restriction in experimental animals enhances life span, delays disease, inhibits immunological perturbations, and ameliorates cancer. Protein kinase C (PKC) isozymes mediate signals generated by hormones, growth factors, and neurotransmitters for cell proliferation and differentiation. The results of our study showed that a C-terminally directed anti-PKC zeta antibody detected an 81-kDa band in the pancreases of control and energy-restricted hamsters. Syrian golden hamsters were fed energy-restricted diets formulated such that the hamsters received 90% (10% energy restriction (ER)), 80% (20% ER), or 60% (40% ER) of the total energy consumed by control hamsters, with the energy reduced proportionally from fat and carbohydrate. ER decreased PKC zeta isozyme levels by 40-75% in hamsters fed 10, 20, and 40% ER diets for 8 wk. PKC zeta isozyme expression was decreased by 75-80% in hamsters fed ER diets for 15 wk. Although ER caused significant decreases in PKC zeta isozyme levels compared with those of control hamsters at both time points, the relative differences in PKC zeta levels between the dietary ER groups (10, 20, and 40%) were small and not significant. A significant decrease in the body weights of ER animals compared with those of controls was observed at both time points. No differences in tomato lectin and phytohemagglutinin reactivity were observed between control animals and animals fed 10, 20, and 40% ER diets. Furthermore, the cellular expression of PKC zeta in the hamster pancreas did not differ among hamsters fed the various ER diets. These observations may be important for understanding not only the role of dietary ER in pancreatic cancers but also PKC zeta signal transduction mechanisms in normal pancreatic physiology.

Pathogenic effect of osteopetrosis virus strain MAV-2(0) on guinea fowl pancreas.

Osteopetrosis virus strain MAV-2(0) in guinea fowl pancreas induces early unspecific changes in separate acini and the development of neoplasia among apparently unaffected acinar tissue. The tumors were classified as serous and mucinous adenomas and adenocarcinomas, as well as poorly differentiated carcinomas. Foci of phenotypically altered acinar cells were observed in single experimental birds.

Virus-induced duodenal adenomas in guinea fowl.

Polypous growths of the duodenal mucosa were induced in guinea fowls after iv inoculation o virus strain Pts-56. The growths developed in about 60% of the experimental birds after a latent period of 4-6 months. The chronologic study of the alterations in the duodenal mucosa revealed consecutive occurrence of focal lymphoid cell infiltrations of the propria, restricted crypt hyperplasia, and enlargement of the villi followed by the formation of tubulovillous and tubulous adenomas. The virus-induced neoplasia of the guinea fowl duodenal mucosa morphologically closely resembled small intestinal adenomas in humans. Their formal genesis was similar to that of experimental intestinal tumors induced by chemical carcinogens.

Virus-induced pancreatic cancer in guinea fowl: a morphologic study.

The morphologic lesions in the pancreas of 350 guinea fowls infected with avian osteopetrosis virus strain Pts-56 were studied. Focal unspecific changes in the acinar tissue, including reduction of zymogen content, diminution of cytoplasm, and dilatation of acinar lumens accompanied with substitution of the affected acini by tubular structures lined with centroacinar-like cells, developed 2 months from post infection (p.i.) in about 30% of the birds. Adenomatous growths of ductules with mucin-producing or mucin-nonproducing epithelium were observed independently or parallelly to these changes after 3 months p.i. Pancreatic carcinomas arose as early as 4 months p.i. The number and size of the neoplastic foci increased with time, and after the 6th month neoplastic foci were established in almost all infected guinea fowls. Neoplastic proliferations of type A and D endocrine cells were identified in the carcinoma foci of separate cases. Other associated neoplasms were bone tumors and papillomatous growths of the duodenal mucosa.