Inflammation-inducible anti-TNF gene expression mediated by intra-articular injection of serotype 5 adeno-associated virus reduces arthritis

BACKGROUND: The tumor necrosis factor (TNF)-alpha plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF-alpha have a major impact on RA treatment. The long-term safety concerns associated with the repetitive TNF blockade prompt optimization of therapeutic anti-TNF approaches. Since we recently demonstrated that intra-articular gene transfer using a recombinant adeno-associated virus serotype 5 (rAAV5) efficiently transduces arthritic joints, we evaluate its effect on collagen-induced arthritis (CIA) when encoding TNF antagonists. METHODS: Recombinant AAV5 vectors encoding the human TNFRp55 extracellular domain fused to the Fc region of mice IgG1 (TR1) or a small molecular weight dimeric human TNFRp75 extracellular domain (TR2), under two different promoters, the CMV or a chimeric NF-kappaB-based promoter inducible by inflammation, were injected into mouse CIA joints. RESULTS: Best protection against arthritis was obtained with the rAAV5 encoding the TR1, as reflected by delayed disease onset, decreased incidence and severity of joint damage. This effect was associated with a transient expression of the anti-TNF agent when expressed under a NF-kappaB-responsive promoter, only detectable during disease flare, while the antagonist expression was rapidly increased and stable when expressed from a CMV promoter. Importantly, using the intra-articular administration of the rAAV5-NF-kappaB-TR1 vector, we observed a striking correlation between local TR1 expression and inflammation. CONCLUSIONS: These findings strongly support the feasibility of improving the safety of anti-TNF approaches for the treatment of arthritis by local rAAV5-mediated gene expression under an inflammation-responsive promoter, able to provide a limited, transient and therapeutically relevant expression of anti-TNF compounds.     

Involvement of interleukin-1β mediated nuclear factor κB signalling pathways to down-regulate prostate-specific antigen and cell proliferation in LNCaP prostate cancer cells

Involvement of NF-κB (nuclear factor κB) mediated by IL-1β (interleukin-1β) on cell proliferation and PSA (prostate-specific antigen) production of LNCaP prostate cell lines and the possible cross-talk with Akt (also known as protein kinase B) signalling pathway has been investigated. NF-κB and Akt were analysed by Western blotting from LNCaP cells treated by IL-1β before proliferation and PSA production were measured. IL-1β inhibited proliferation and decreased PSA production. The Akt pathway was not sensitive, whereas NF-κB phosphorylation occurred as a result of treatment. PSA production and proliferation of LNCaP cells were down-regulated by NF-κB mediated by IL-1β promoting anti-apoptotic signalling and co-suppressor factors of PSA expression. IL-1β through NF-κB activation provides a rationale for therapeutic approaches in the anticancer treatment of prostate.     

TLR3 signaling in a hepatoma cell line is skewed towards apoptosis

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double-stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain-containing adaptor-inducing IFNbeta) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF-kappaB and IRF3 (interferon regulatory factor 3) and induced IFNbeta (interferon beta) promoter expression, which are known to lead to pro-inflammatory cytokine secretion. In Huh7 cells, there was only a short-term IRF3 activation, and a very low level of IFNbeta expression. In HepG2 cells on the other hand, while no induction of pro-inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.     

Ghrelin attenuates plasminogen activator inhibitor-1 production induced by tumor necrosis factor-alpha in HepG2 cells via NF-kappaB pathway

Plasminogen activator inhibitor type 1 (PAI-1), produced partly from liver is a risk factor for macrovascular and microvascular complications of diabetes. Ghrelin, a recently described orexigenic peptide hormone, attenuates PAI-1 induced by TNF-alpha in the human hepatoma cell line (HepG2). Exposure to TNF-alpha (1 ng/ml) for 24h caused a significant increase in PAI-1 mRNA expression and protein secretion, as evaluated by RT-PCR and ELISA, but pretreatment with ghrelin (1-100 ng/ml) inhibited both basal and TNF-alpha-induced PAI-1 release in a dose and time-dependent manner in HepG2. PDTC, selective NF-kappaB inhibitor, had no additive inhibitory effects with ghrelin. The results indicate that ghrelin inhibits both basal and TNF-alpha-induced PAI-1 production via NF-kappaB pathway in HepG2 cells, and suggest that the peptide plays a therapeutic role in atherosclerosis, especially in obese patients with insulin resistance, in whom ghrelin levels were reduced.     

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.