A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis

BACKGROUND: Electric pulse mediated gene transfer has been applied successfully in vivo for increasing naked DNA administration in various tissues. To achieve non-viral gene transfer into arthritic joint tissue, we investigated the use of electrotransfer (ET). Because anti-inflammatory cytokine strategies have proven efficient in experimental models of arthritis, we compared the therapeutic efficiency of local versus systemic delivery of the interleukin-10 (IL-10) using in vivo ET. METHODS: A plasmid vector expressing IL-10 was transferred into DBA/1 mouse knee joints by ET with 12 pulses of variable duration and voltage. The kinetics of transgene expression were analyzed by specific enzyme-linked immunosorbent assay (ELISA) in sera and knees. Optimal conditions were then used to deliver increasing amounts of IL-10 plasmid intra-articularly (i.a.) in the collagen-induced arthritis (CIA) mouse model. The therapeutic efficiency was compared with the potency of intra-muscular (i.m.) ET. RESULTS: Following i.a. ET, local IL-10 secretion peaked on day 7 and dropped 2 weeks after. A second ET produced the same kinetics without enhancing gene transfer efficiency, while transgene was still detected in injected muscles 4 weeks after ET. Only the i.m. ET of 25 microg of IL-10 significantly inhibited all the clinical and biological features of arthritis. The i.a. ET only showed mild improvement of arthritis when 100 microg of IL-10 plasmid were electrotransfered weekly from day 18 following arthritis induction. CONCLUSIONS: The present results suggest that gene transfer into arthritic joints by ET is an effective means to deliver anti-inflammatory cytokines. However, short duration of transgene expression impedes a significant effect for the treatment of arthritis, making i.m. ET more potent than i.a. ET for clinical benefit in CIA.     

Polymorphism in the tumour necrosis factor receptor II gene is associated with circulating levels of soluble tumour necrosis factor receptors in rheumatoid arthritis

Levels of soluble tumour necrosis factor receptors (sTNFRs) are elevated in the circulation of patients with rheumatoid arthritis (RA). Although these receptors can act as natural inhibitors of tumour necrosis factor-α, levels of sTNFRs in RA appear to be insufficient to prevent tumour necrosis factor-α induced inflammation. The factors that regulate circulating levels of sTNFRs are unclear, but polymorphisms in the tumour necrosis factor receptor genes may play a role. We investigated the relationship between polymorphisms in the tumour necrosis factor receptor I (TNF-RI) and II (TNF-RII) genes and levels of sTNFRs in two groups of Caucasian RA patients: one with early (disease duration ≤2 years; n = 103) and one with established disease (disease duration ≥5 years; n = 151). PCR restriction fragment length polymorphism analysis was used to genotype patients for the A36G polymorphism in the TNF-RI gene and the T676G polymorphism in TNF-RII. Levels of sTNFRs were measured using ELISA. We also isolated T cells from peripheral blood of 58 patients with established RA with known TNF-R genotypes, and release of sTNFRs into the culture medium was measured in cells incubated with or without phytohaemagglutinin. Serum levels of the two sTNFRs (sTNF-RI and sTNF-RII) were positively correlated in both populations, and the level of each sTNFR was significantly higher in the patients with established disease (P < 0.0001). Multiple regression analyses corrected for age, sex and disease duration revealed a significant trend toward decreasing sTNF-RI and sTNF-RII levels across the TNF-RII genotypes (TT > TG > GG) of patients with established disease (P for trend = 0.01 and P for trend = 0.03, respectively). A similar nonsignificant trend was seen for early disease. No relationship with the TNF-RI A36G polymorphism was observed. sTNFRs released by isolated T cells exhibited a similar trend toward decreasing levels according to TNF-RII genotype, although only the association with levels of sTNF-RII was significant. Strong correlations were found between levels of circulating sTNFRs and levels released by T cells in vitro. Our data indicate that the T676G polymorphism in TNF-RII is associated with levels of sTNFRs released from peripheral blood T cells, and with circulating levels of sTNFR in patients with RA.     

Mutation of the extracellular domain of tumour necrosis factor receptor 1 causes reduced NF-kappaB activation due to decreased surface expression

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) results from point mutations in the extracellular domain of TNF receptor 1 (TNFRSF1A), but the effects of the mutations are controversial. This study shows that reduced NF-kappaB signalling is a feature of four TRAPS mutations. Reduced signalling correlates with reduced surface expression, measured by flow cytometry and microscopy. This suggests that correct formation of the extracellular domain of TNFRSF1A is important for localisation and receptor function. Importantly, our data provides a mechanism for the reduced TNFRSF1 signalling observed in a patient cell line.     

Effect of H2 treatment in a mouse model of rheumatoid arthritis‐associated interstitial lung disease

Rheumatoid arthritis (RA)‐associated interstitial lung disease (ILD), a primary cause of mortality in patients with RA, has limited treatment options. A previously established RA model in D1CC transgenic mice aberrantly expressed major histocompatibility complex class II genes in joints, developing collagen II‐induced polyarthritis and anti‐cyclic citrullinated peptide antibodies and interstitial pneumonitis, similar to those in humans. Molecular hydrogen (H₂) is an efficient antioxidant that permeates cell membranes and alleviates the reactive oxygen species‐induced injury implicated in RA pathogenesis. We used D1CC mice to analyse chronic lung fibrosis development and evaluate H₂ treatment effects. We injected D1CC mice with type II collagen and supplied them with H₂‐rich or control water until analysis. Increased serum surfactant protein D values and lung densities images were observed 10 months after injection. Inflammation was patchy within the perilymphatic stromal area, with increased 8‐hydroxy‐2?‐deoxyguanosine‐positive cell numbers and tumour necrosis factor‐α, BAX, transforming growth factor‐β, interleukin‐6 and soluble collagen levels in the lungs. Inflammatory and fibrotic changes developed diffusely within the perilymphatic stromal area, as observed in humans. H₂ treatment decreased these effects in the lungs. Thus, this model is valuable for studying the effects of H₂ treatment and chronic interstitial pneumonia pathophysiology in humans. H₂ appears to protect against RA‐ILD by alleviating oxidative stress.     

Dialysable leucocyte extract (DLE) reduces lipopolysaccharide- induced tumour necrosis factor secretion in human leucocytes

Dialysable leucocyte extract (DLE), obtained from lysed leucocytes, provide clinical effectiveness in a broad spectrum of diseases. Tumour necrosis factor (TNF) is raised in AIDS patients leading to an increase in human immunodeficiency virus (HIV) replication in vitro [1,2], whereas progression to AIDS in asymptomatic HIV infected individuals is retarded under treatment with DLE. In the present study we tested the DLE effect in vitro on both TNF biological activity (cytotoxicity) in L929 cells and its induction by lipopolysaccharide (LPS) in human monocytes as well as in whole blood from healthy donors. When monocytic cells were simultaneously exposed to LPS and DLE during a period of 5 1/2 hours, the induction of TNF was strongly diminished. The same inhibitory effect of DLE on TNF induction was observed when LPS was added to the culture medium prior to DLE. No significant effect of DLE on TNF-mediated cytotoxicity, even in the presence of the highest concentrations of DLE tested, was detected. DLE treatment of whole human blood regulates responses to LPS: simultaneous in vitro exposure to endotoxin provokes a remarkable decrease (4- and 1.6-fold) of TNF release. In pre-incubation experiments, TNF production was largely reduced or completed abrogated. These results could, in part, explain the in vivo observed effect, when under treatment with this extract, the progression to AIDS of HIV-infected individuals was retarded. The results suggest that ‘natural’ substances like DLE may be important immunomodulators in inflammatory diseases.     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

Association of tumor necrosis factor alpha and its receptor polymorphisms with rheumatoid arthritis in female patients

Rheumatoid arthritis (RA) is a chronic autoimmune disorder associated with altered expression of pro-inflammatory cytokines. We aim to elucidate the association between the −308G/A polymorphism of the TNF-α gene and 196M/R polymorphism in TNFRII gene and susceptibility and severity of RA. One hundred and seventy-two RA patients and one hundred and sixty controls were enrolled in the study. Polymorphisms (SNPs) at position −308 of TNF and −196 of TNFRII genes were determined using restriction fragment length polymorphism–polymerase chain reaction (PCR–RFLP). TNF AA genotype was more prevalent among the patients. GG genotype was significantly more likely to have erosive arthropathy. TNFRII RR genotype was more prevalent among the patients. Our findings suggest that the 308AA genotype of TNF-α and TNFRII 196M/R polymorphism are associated with RA susceptibility. While only the 308GG genotype of TNF-α is associated with RA severity.     

Structure of CrmE, a virus-encoded tumour necrosis factor receptor

Vaccinia virus (VACV), the smallpox vaccine, encodes many proteins that subvert the host immune response. One of these, cytokine response modifier E (CrmE), is secreted by infected cells and protects these cells from apoptotic challenge by tumour necrosis factor alpha (TNFalpha). We have expressed recombinant CrmE from VACV strain Lister in Escherichia coli, shown that the purified protein is monomeric in solution and competent to bind TNFalpha, and solved the structure to 2.0 A resolution. This is the first structure of a virus-encoded tumour necrosis factor receptor (TNFR). CrmE shares significant sequence similarity with mammalian type 2 TNF receptors (TNFSFR1B, p75; TNFR type 2). The structure confirms that CrmE adopts the canonical TNFR fold but only one of the two "ligand-binding" loops of TNFRSF1A is conserved in CrmE, suggesting a mechanism for the higher affinity of poxvirus TNFRs for TNFalpha over lymphotoxin-alpha. The roles of dimerisation and pre-ligand-assembly domains (PLADs) in poxvirus and mammalian TNFR activity are discussed.     

Effect of sclerostin-neutralising antibody on periarticular and systemic bone in a murine model of rheumatoid arthritis: a microCT study

Introduction

Patients with chronic inflammatory diseases have increased bone loss and bone fragility and are at increased risk of fracture. Although anti-resorptive drugs are effective in blocking inflammation-induced bone loss, they are less effective at rebuilding bone. We have previously shown that treatment with sclerostin antibody (Scl-AbI) builds bone and can prevent or restore bone loss in a murine model of inflammatory bowel disease. In this study, we tested the effect of Scl-AbI in a murine model of rheumatoid arthritis (the collagen-induced arthritis model, CIA). We hypothesised that sclerostin blockade can protect and restore bone both locally and systemically without affecting progression of inflammation.

Methods

CIA was induced in male DBA/1 mice, which were treated with either PBS or Scl-AbI (10 mg/kg, weekly) prophylactically for 55 days or therapeutically for 21 days (starting 14 days post onset of arthritis). Systemic inflammation was assessed by measuring the serum concentration of anti-CII IgG1, IgG2a and IgG2b by ELISA. Changes in bone mass and structure, either at sites remote from the joints or at periarticular sites, were measured using DEXA and microCT. Bone focal erosion was assessed in microCT scans of ankle and knee joints.

Results

Circulating anti-CII immunoglobulins were significantly elevated in mice with CIA and there were no significant differences in the levels of anti-CII immunoglobulins in mice treated with PBS or Scl-ABI. Prophylactic Scl-AbI treatment prevented the decrease in whole body bone mineral density (BMD) and in the bone volume fraction at axial (vertebral body) and appendicular (tibial proximal metaphysis trabecular and mid-diaphysis cortical bone) sites seen in PBS-treated CIA mice, but did not prevent the formation of focal bone erosions on the periarticular bone in the knee and ankle joints. In the therapeutic study, Scl-AbI restored BMD and bone volume fraction at all assessed sites but was unable to repair focal erosions.

Conclusions

Sclerostin blockade prevented or reversed the decrease in axial and appendicular bone mass in the murine model of rheumatoid arthritis, but did not affect systemic inflammation and was unable to prevent or repair local focal erosion.     

Angiotensin II type 2 receptor correlates with therapeutic effects of losartan in rats with adjuvant‐induced arthritis

The angiotensin II type 1 receptor (AT1R) blocker losartan ameliorates rheumatoid arthritis (RA) in an experimental model. In RA, AT2R mainly opposes AT1R, but the mechanism by which this occurs still remains obscure. In the present study, we investigated the role of AT2R in the treatment of rats with adjuvant-induced arthritis (AIA) by losartan. Adjuvant-induced arthritis rats were treated with losartan (5, 10 and 15 mg/kg) and methotrexate (MTX; 0.5 mg/kg) in vivo from day 14 to day 28. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumour necrosis factor-α, and VEGF levels were examined by ELISA. The expression of AT1R and AT2R was detected by western blot and immunohistochemistry analysis. After stimulation with interleukin-1β in vitro, the effects of the AT2R agonist {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (10⁻⁸–10⁻⁵ M) on the chemotaxis of monocytes induced by 10% foetal calf serum (FCS) were analysed by using Transwell assay. Subsequently, the therapeutic effects of {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (5, 10 and 20 μg/kg) were evaluated in vivo by intra-articular injection in AIA rats. After treatment with losartan, the down-regulation of AT1R expression and up-regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with reduction in the polyarthritis index. Treatment with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 inhibited the chemotaxis of AIA monocytes in vitro, possibly because of the up-regulation of AT2R expression. Intra-articular injection with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (10 and 20 μg/kg) ameliorated the arthritis index and histological signs of arthritis. In summary, the present study strongly suggests that the up-regulation of AT2R might be an additional mechanism by which losartan exerts its therapeutic effects in AIA rats.     

Assignment of the porcine tumour necrosis factor alpha and beta genes to the chromosome region 7p11-q11 by in situ hybridization

The loci of the porcine tumour necrosis factor genes, alpha (TNFA) and beta (TNFB), have been chromosomally assigned by radioactive in situ hybridization. The genomic probes for TNFA and TNFB yielded signals above 7p11-q11, a region that has been shown earlier to carry the porcine major histocompatibility locus (SLA). These mapping data along with preliminary molecular studies suggest a genomic organization of the SLA that is similar to that of human and murine major histocompatibility complexes.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

Regional Intra‐Subject Variability in Abdominal Adiposity Limits Usefulness of Computed Tomography

Objective: Computed tomography (CT) and magnetic resonance imaging, the most accurate methods of abdominal fat measurement, have been applied using a number of protocols, ranging from single‐slice area determination to multiple‐slice volume calculation. The aim of this study was to assess the validity of single‐slice CT for abdominal fat area measurement by estimating the intra‐subject variability in abdominal fat areas and comparing the ranking of subjects across four contiguous abdominal levels. Research Methods and Procedures: Nineteen premenopausal women (age, 35.3 ± 1.4 years; mean ± SE) were studied. CT was used to measure intra‐abdominal fat (IAF) area, percentage of total intra‐abdominal area (%IAF), subcutaneous abdominal fat (SAF) area, and IAF/SAF at four adjacent cross‐sectional lumbar levels (L2–L4). Intra‐subject variability (percentage) was defined as SD/mean × 100. Total body fat was measured by DXA, which was further analyzed for central abdominal fat. Results: Mean body mass index was 24.9 ± 1.0 kg/m². The average (range) intra‐subject variability was 28% (8% to 61%) for IAF, 46% (19% to 124%) for %IAF, 26% (14% to 38%) for SAF area, and 19% (7% to 71%) for IAF/SAF. The pattern of this variability was not uniform between subjects, because their ranking by IAF area was markedly different at each CT level. Discussion: We demonstrated significant intra‐subject variability in CT‐measured adipose tissue areas across four predetermined sites. This resulted in a difference in the ordering of subjects by IAF at each of the four imaging sites, suggesting that the usefulness of single‐slice CT in the assessment of abdominal adiposity in premenopausal women may be limited, particularly when performed for the purpose of making comparisons between subjects based on abdominal fat area.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.