Effectiveness of the soluble form of the interleukin-1 receptor accessory protein as an inhibitor of interleukin-1 in collagen-induced arthritis.

OBJECTIVE: To investigate whether the soluble form of interleukin-1 (IL-1) receptor accessory protein (sIL-1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL-1 signaling in vitro, and to evaluate its applicability in collagen-induced arthritis (CIA). METHODS: Soluble IL-1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL-1RAcP or a stable-transfected NIH3T3 fibroblast cell line. The ability of affinity-purified sIL-1RAcP to inhibit IL-1 signaling was tested on NF-kappaB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL-1RAcP was both locally (knee joint) and systemically overexpressed in collagen-immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL-6 and anti-bovine type II collagen (BCII) antibody levels. RESULTS: Incubation of the NF-kappaB reporter fibroblast with purified sIL-1RAcP protein showed a marked reduction of IL-1-induced, but not tumor necrosis factor-induced, NF-kappaB activation. This showed a novel role for sIL-1RAcP as a specific inhibitor of IL-1 signaling. Local transplantation of sIL-1RAcP-producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL-1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL-1RAcP had no profound effect on serum levels of IL-6 and anti-BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL-1RAcP markedly ameliorated CIA in all joints. CONCLUSION: In this study we demonstrated that sIL-1RAcP is a biologically active and innovative inhibitor of IL-1, and treatment of mice with sIL-1RAcP had a profound prophylactic effect on collagen-induced arthritis.

Combined transcriptional and transductional targeting improves the specificity and efficacy of adenoviral gene delivery to ovarian carcinoma.

Adenoviruses are efficient gene delivery vehicles but have broad native tropism. To this end, finding ways to target this virus specifically to carcinomas has become an important focus of cancer gene therapy. Transductional and transcriptional forms of targeting have been used with promising results in ovarian carcinoma. Therefore, we combined both forms of targeting to investigate the effect on the specificity and efficiency of transgene expression in this disease. We used the tissue-specific SLPI promoter and the ovarian cancer associated targeting adaptor protein, sCARfC6.5. This bispecific protein contains the coxsackie-adenovirus receptor ectodomain and a single-chain antibody specific for c-erbB-2. Viruses containing the SLPI or the ubiquitously expressed CMV promoter, with or without sCARfC6.5, were used for infection of ovarian cancer cell lines, primary ovarian tumor cells, and in an orthotopic model of disseminated ovarian carcinoma. This dual-targeting strategy increased the efficiency and specificity of transgene expression in vitro in reporter and cell-killing assays, and in vivo. By using both the SLPI promoter and sCARfC6.5, transgene expression was increased in ovarian tumors and decreased in normal tissues, including the liver. Thus, we show that combining transcriptional and transductional targeting can increase the efficacy and specificity of adenoviral gene therapy for ovarian carcinoma.

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.

A tropism-modified adenoviral vector increased the effectiveness of gene therapy for arthritis.

Adenoviral vectors (AdV) are used for anti-inflammatory cytokine therapy in experimental arthritis. Cell entry of AdV is dependent on the initial recognition of the coxsackie-adenovirus receptor (CAR) on cells. Recently, an Arg-Gly-Asp (RGD) motif was introduced in the HI loop of the fiber knob, this enables the adenovirus to bypass CAR and mediate cell entry via RGD binding integrins. In this study, we explored the transduction efficiency of the RGD-modified adenovirus in synovium and compared the RGD-modified with the conventional adenoviral vector for their effectiveness to modulate the murine collagen-induced arthritis (CIA) model when used to overexpress mIL-1Ra in the knee joint. Twenty-four hours after intra-articular injection of 10(7) fluorescent forming units (ffu) virus, luciferase (luc) activity in Ad5LucRGD-injected joints was up to 38 times higher than in AdCMVLuc-injected joints, and in arthritic joints the transduction efficiency was up to 69 times higher for the Ad5LucRGD viruses. Transduction of the synovial lining by the RGD-modified adenovirus containing the mIL-1Ra transgene, markedly improved the inhibition of CIA compared with the conventional virus in both a prophylactic and therapeutic treatment protocol. These results show that targeting integrins with the RGD-modified AdV improved the outcome of gene therapy for arthritis.

[Studying the possibility of respiratory immunization against tick-borne encephalitis]. / Izuchenie vozmozhnosti respiratornoi immunizatsii protiv kleshchevogo éntsefalita.

There are known 3 likely mechanisms of virus conveyance into the central nervous system (CNS). These include hematogenic penetration, spread along the peripheral nerves, and the olfactory pathway which begins from the infected olfactory neuroepithelial cells. The possibility of viral spread into CNS via the olfactory pathway was shown for the representatives of togaviruses, herpesviruses, coronaviruses, rhabdoviruses, and for some others. This study suggests that the olfactory pathway of viral conveyance into CNS may be blocked by specific mucosal antibodies in the nasal mucosa. The recombinant TK- variant of WR vaccinia strain with inserted genes coding structural and nonstructural proteins of TBE virus is accumulated in the branches of the respiratory tract only while the parenteral vaccinia strain is detected in the brain regions, spleen, respiratory tract, and in blood. The protective activity of recombinant strain and inactivated TBE vaccine after mice immunization by escarification or intranasally, or subcutaneously was comparatively studied. The findings indicate that intranasal immunization by recombinant strain is the most protective against intraperitoneal challenge by TBE virus. The mucosal and humoral immune response that was induced by intranasal immunization seems to provide the highest levels of protection, which was experimentally observed.

[Immunobiological properties of vp24 protein of Ebola virus expressed by recombinant vaccinia virus]. / Izuchenie immunobiologicheskikh svoistv belka vp24 virusa Ebola v sostave rekombinantnogo virusa ospovaktsiny.

Immunological and biochemical parameters were studied in guinea pigs immunized with recombinant vaccinia virus containing full-sized gene of Ebola virus vp24 protein and then infected with virulent strain of Ebola virus. The majority of the studied parameters changed similarly in guinea pigs immunized with recombinant vaccinia virus and control guinea pigs inoculated with vaccinia virus both before and after challenge with Ebola virus. However, in animals immunized with recombinant vaccinia virus producing vp24 some biochemical parameters, the mean life span after challenge with Ebola virus, the level of antibodies to the virus, and the phagocytic activity of neutrophils indicated the development of immunological processes other than in controls, namely, the development of immune response to vp24. Although these processes did not eventually lead to the survival of animals, they prolonged the mean life span and resulted in the production of anti-Ebola antibodies, though the level thereof was low. These data demonstrate that recombinant vaccines against Ebola fever are a promising trend of research.

Immunization with recombinant vaccinia viruses expressing structural and part of the nonstructural region of tick-borne encephalitis virus cDNA protect mice against lethal encephalitis.

Three recombinant vaccinia viruses containing different fragments of tick-borne encephalitis virus (TBEV) cDNA representing the 5'-noncoding region (5'NCR), all structural and part of the nonstructural regions were constructed. Western blot analysis showed that E and NS1 proteins were expressed and processed correctly in cells infected with recombinant viruses vC-NS1 (coding for C-prM-E-NS1 region) and vC-NS3 (coding for C-prM-E-NS1-NS2A-NS2B-NS3 region). In contrast, in cells infected with recombinant virus v5'C-NS2A (coding for 5'NCR and C-prM-E-NS1-NS2A regions) expression of NS1 protein was greatly reduced and no E protein was detected. Immunization of mice with vC-NS3 induced high levels of TBEV-specific antibodies and protected them against intraperitoneal challenge with 10(7) LD50 of TBEV. The level of protection was very similar to the level of protection achieved by immunization with commercially available inactivated TBEV vaccine. Although the immunization of mice with recombinants vC-NS1 and v5'C-NS2A induced much lower levels of TBEV-specific antibodies, they were still protected against intraperitoneal challenge with 10(4) and 10(3.6) LD50 of TBEV, respectively. The high level of protection against TBEV infection achieved by the immunization of mice with the recombinant vaccinia virus vC-NS3 makes this virus a very attractive candidate for development of a live recombinant vaccine against TBEV.

[Composition and degree of sulfation of glycosaminoglycans from tissues of different animal species: heterogeneity and tissue specificity of heparan sulfates]. / Sostav i stepen' sul'fatirovaniia glikozaminoglikanov iz tkanei zhivotnykh raznykh vidov: geterogennost' i tkanevaia spetsifichnost' geparansul'fatov.

The composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans from various animal tissues were studied. It was shown that sulfated GAG contain chondroitin sulfates AC and B as well as heparan sulfates. The bulk of GAG in the majority of tissues under study is represented by 2-3 types of heparan sulfate molecules differing in the degree of sulfation. According to the degree of sulfation heparan sulfates from all tissues studied can be classified into three groups. Homologous tissues of various animal species are characterized by a similar composition and the degree of sulfation The data obtained are discussed in terms of the feasible role of proteoheparan sulfates in specific cell-to-cell interactions.

[Comparative characteristics of the composition and degree of sulfation of glycosaminoglycans in resting and actively proliferating tissues]. / Sravnitel'naia kharakteristika sostava i stepeni sul'fatirovaniia glikozaminoglikanov pokoiashchikhsia a aktivno proliferiruiushchikh tkanei.

A comparative study of the composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans isolated from normal animal tissues and from actively proliferating embryonic, tumour and regenerating tissues was carried out. Significant differences in the ratios of various types of sulfated GAG were revealed. The relative content of chondroitin sulfate AC in actively proliferating tissues was considerably increased. It was found that all types of sulfated GAG in actively proliferating tissues, with the exception of regenerating tissue, are characterized by a lower degree of sulfation as compared to GAG from resting tissues. The experimental results are discussed in terms of the role of proteoglycans in the organization of extracellular matrix and in control of cell proliferation.