Antigen presenting cells expressing Fas ligand down-modulate chronic inflammatory disease in Fas ligand-deficient mice

We assessed the effect of modified antigen presenting cells (APCs) expressing high levels of Fas ligand (APC-FasL) on post-viral chronic inflammatory disease. FasL-deficient B6-gld/gld mice infected with murine cytomegalovirus (MCMV) cleared the virus from their lungs, kidneys, and livers within 2 weeks of infection. However, inflammation persisted in these organs for more than 8 weeks, with a chronically increased T-cell response to MCMV-infected APCs and production of autoantibodies. Administration of APC-AdFasL at 4 weeks suppressed this inflammation and diminished the T-cell response and autoantibody production. APC-AdFasL that had been transfected with ultraviolet-irradiated MCMV were more effective than uninfected APC-AdFasL in ameliorating the chronic inflammation. APC-AdFasL migrated preferentially to the spleen, where they triggered apoptosis of lymphocytes in the marginal zone of the spleen. These results confirm that Fas-mediated apoptosis is not required for clearance of virus, but is required for down-modulation of the virally induced chronic inflammatory response. This organwide effect of APC-AdFasL appears to be mediated by elimination of activated T lymphocytes in the spleen before their emigration to the target organs.     

Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification

A simple, rapid, nonradioactive method has been developed to facilitate the direct detection of point mutations that cause genetic disease. The method operates on the basis of the specific amplification of a target allele by the polymerase chain reaction with extension primers designed such that their 3' end is placed at the mutation site. When this base is complementary to that of the specific allele, the DNA segment is amplified; when it is not complementary, the polymerase chain reaction cannot proceed. When alpha 1-antitrypsin (alpha 1AT) deficiency was used as a model, the technique of allele-specific amplification was capable of selective detection of five different mutations that cause the alpha 1AT deficiency state, including three different naturally occurring single-base substitution mutations (alleles Z, S, and Nullbellingham), an insertion mutation (Nullmattawa), and a deletion mutation (Nullgranite falls). Double-blind evaluation of 47 samples of genomic DNA demonstrated 100% accuracy of the method. The technique of allele-specific amplification is rapid, simple, and does not require the existence of a convenient restriction endonuclease site or the use of radioactive materials, and thus should have broad applicability for the detection of known genetic diseases in a highly sensitive and specific fashion.     

Melanoma differentiation associated gene-7 (mda-7)/IL-24: a 'magic bullet' for cancer therapy?

An ideal cancer gene therapy would selectively kill cancer cells without harming normal cells and induce multipronged 'bystander' antitumor effects, facilitating eradication of both primary and metastatic tumors. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 (IL-24) exhibits all of these attributes and more. It induces cancer-selective apoptosis, inhibits angiogenesis, stimulates an antitumor immune response, sensitizes cancer cells to radiation and other modalities of conventional therapies, and exhibits profound 'bystander' activity eliminating both primary and distant tumors in animal models. Moreover, a replication-incompetent adenovirus expressing mda-7/IL-24, Ad.mda-7 (INGN-241), has now undergone evaluation in a Phase I clinical trial for multiple solid tumors, including melanomas, and has demonstrated safety and significant objective clinical activity. Considering these exciting observations, mda-7/IL-24 is being hailed as a 'magic bullet' for cancer gene therapy. This review elaborates on the pleiotropic properties of mda-7/IL-24 and unravels novel aspects of the molecule mandating future studies and expanded clinical applications.     

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells

Oncolytic adenoviruses constitute a new and promising tool for cancer treatment that has been rapidly translated into clinical trials. However, minimal or absent expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells represents a major limitation for Ad5-based oncolysis. Here, we report on the resistance of CAR-negative primary melanoma cells to cell killing by wild-type Ad5 (Ad5wt) even after high titer infection, thus underlining the need for tropism-modification of oncolytic adenoviruses. We engineered a new generation of oncolytic adenoviruses that exhibit both efficient target cell infection by swapping Ad5 fiber domains with those of Ad serotype 3, which binds to a receptor distinct from CAR, and targeted virus replication. Fiber chimerism resulted in efficient cytopathicity to primary melanoma cells, which was at least 10(4)-fold increased relative to Ad5wt. Since viral infectivity mediated by such modified viral capsids was not cell type-specific, it was pivotal to carefully restrict adenoviral replication to target cells. Towards this end, we replaced both E1A and E4 promoters of fiber chimeric viruses by tyrosinase enhancer/promoter constructs. The resulting viruses showed melanoma-specific expression of E1A and E4 and combined efficient virus replication and cell killing in melanoma cell lines and primary melanoma cells with a remarkable specificity profile that implements strong attenuation in nonmelanoma cells, including normal fibroblasts and keratinocytes.     

A highly sensitive and specific chemiluminescent enzyme-linked immunosorbent assay for diagnosis of active Trypanosoma cruzi infection

BACKGROUND: Chagas' disease is transmitted to man either by the bite of insects harboring Trypanosoma cruzi or by the transfusion of blood from infected donors. The conventional serologic testing as presently used in blood banks in South America is unsatisfactory, because of a high number of inconclusive and false-positive results. Other methods such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens have been proposed, but inherent difficulties have so far precluded their adoption in the large-scale screening required by blood banks. STUDY DESIGN AND METHODS: A highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote glycoconjugate antigen and a complex epimastigote antigen was devised for the diagnosis of active T. cruzi infection. RESULTS: Chemiluminescent ELISA was 100-percent sensitive in the diagnosis of 100 cases of confirmed Chagas' disease. Inconclusive results and false-positive reactions were eliminated in a panel of 115 sera.The specificity of the chemiluminescent ELISA was 100 percent with a purified trypomastigote glycoconjugate antigen and 99.7 percent with a complex epimastigote antigen when applied to 1000 normal human sera and 288 heterologous sera from patients with other infections, including leishmaniasis, and vaccinated individuals. CONCLUSION: The chemiluminescent ELISAs provide a test that is highly sensitive (purified trypomastigote glycoconjugate and complex epimastigote antigens) and specific (purified trypomastigote glycoconjugate antigen) for Chagas' disease diagnosis. It can be used in blood bank screening and to monitor the treatment of patients undergoing chemotherapy.     

Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of scFv in situ. To circumvent these limitations, we have developed an approach to express scFv in vivo. An anti-erbB2 scFv was engineered for secretion by eukaryotic cells. The secreted scFv could bind to its target and specifically suppress cell growth of erbB2-positive cells in vitro. Adenoviral vectors expressing the cDNA for the secretory scFv likewise could induce target cells to produce an anti-tumor anti-erbB2 scFv. In vivo gene transfer via the anti-erbB2 scFv encoding adenovirus also showed anti-tumor effects. Thus, by virtue of engineering a secreted version of the anti-tumor anti-erbB-2 scFv, and in vivo expression via adenoviral vector, effective concentrations of scFv were achieved. In vivo gene transfer clearly represents a powerful means to realize effective scFv-based approaches. This method will likely have applicability for a range of disorders amenable to targeted therapeutic approaches.     

Primary adenovirus-specific cytotoxic T lymphocyte response occurs after viral clearance and liver enzyme elevation

The virus-specific cytotoxic T lymphocyte (CTL) response is a major obstacle to effective delivery of adenovirus gene therapy. However, its relative role in viral clearance, transgene elimination and hepatotoxicity remains unclear. In this paper, we present an analysis of viral clearance and liver toxicity in relation to the induction of the virus-specific CD8 T-cell response revealed by an MHC class I tetramer. A surprisingly high number of tetramer+ CD8 T cells were found in the liver and lung and reached peak values at days 8 and 10, respectively, post-infection. Nearly 100% of these tetramer+ CD8 T cells expressed high levels of granzyme B and IFNgamma. Remarkably, liver viral load and liver enzyme elevation peaked early, at days 2 and 4, respectively, post-infection, before the specific CTL response was detectable. After generation of CTLs, there was only minimal liver damage or further decrease in virus titer. These results indicated that the primary peak response of tetramer+ CTLs does not correlate with the elimination of adenovirus or liver cytotoxic response.