Immunocytokines: the long awaited therapeutic magic bullet in rheumatoid arthritis?

Modulatory cytokines such as IL-4 and IL-10 looked promising biologicals, but suffered from poor exposure at the inflamed joints when administered via the patient-friendly subcutaneous route. Immunocytokines have now been engineered with tissue targeting potential and are a possible solution to this problem, although challenges still exist. Local inflammatory processes cause destruction of extracellular matrix (ECM) components, leading to neo-eptitopes, and/or elicit the synthesis of new ECM components. This makes ECM elements interesting targets for antibody-mediated recognition and retention, to achieve higher levels of immunocytokines at the site of therapeutic interference. The study presented by Schwager and colleagues shows that targeted delivery of IL-10 is more efficacious in experimental arthritis. Clinical studies are warranted to show whether this strategy works for all rheumatoid arthritis patients or is better for subgroups with a defined ECM phenotype. In principle, the scFv-targeting system is plastic enough to allow for personalized strategies

Trapped in a vicious loop: Toll-like receptors sustain the spontaneous cytokine production by rheumatoid synovium

Synovial tissue of patients with rheumatoid arthritis (RA) spontaneously produces several cytokines, of which a fundamental role in joint inflammation and destruction has been established. However, the factors sustaining this phenomenon remain poorly understood. In a recent report, blockade of Toll-like receptor 2 (TLR2) was found to inhibit the spontaneous release of inflammatory cytokines by intact RA synovial explant cultures. Adding to the recent evidence implicating other TLRs (in particular, TLR4), this observation highlights the potential of TLRs as therapeutic targets to suppress the local production of multiple cytokines and to control the chronic inflammatory loop in RA

Gene therapy in animal models of rheumatoid arthritis: are we ready for the patients?

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints, with progressive destruction of cartilage and bone. Anti-tumour necrosis factor-α therapies (e.g. soluble tumour necrosis factor receptors) ameliorate disease in 60–70% of patients with RA. However, the need for repeated systemic administration of relatively high doses in order to achieve constant therapeutic levels in the joints, and the reported side effects are downsides to this systemic approach. Several gene therapeutic approaches have been developed to ameliorate disease in animal models of arthritis either by restoring the cytokine balance or by genetic synovectomy. In this review we summarize strategies to improve transduction of synovial cells, to achieve stable transgene expression using integrating viruses such as adeno-associated viruses, and to achieve transcriptionally regulated expression so that drug release can meet the variable demands imposed by the intermittent course of RA. Evidence from animal models convincingly supports the application of gene therapy in RA, and the feasibility of gene therapy was recently demonstrated in phase I clinical trials

Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling

Bone morphogenetic protein-2 (BMP-2) has been proposed as a tool for cartilage repair and as a stimulant of chondrogenesis. In healthy cartilage, BMP-2 is hardly present, whereas it is highly expressed during osteoarthritis. To assess its function in cartilage, BMP-2 was overexpressed in healthy murine knee joints and the effects on proteoglycan (PG) synthesis and degradation were evaluated. Moreover, the contribution of BMP in repairing damage induced by interleukin-1 (IL-1) was investigated. Ad-BMP-2 was injected intra-articularly into murine knee joints, which were isolated 3, 7, and 21 days after injection for histology, immunohistochemistry, and autoradiography. In addition, patellar and tibial cartilage was isolated for RNA isolation or measurement of PG synthesis by means of 35SO4 2- incorporation. To investigate the role for BMP-2 in cartilage repair, cartilage damage was induced by intra-articular injection of IL-1. After 2 days, Ad-BMP-2, Ad-BMP-2 + Ad-gremlin, Ad-gremlin, or a control virus was injected. Whole knee joints were isolated for histology at day 4 or patellae were isolated to measure 35SO42- incorporation. BMP-2 stimulated PG synthesis in patellar cartilage on all days and in tibial cartilage on day 21. Aggrecan mRNA expression had increased on all days in patellar cartilage, with the highest increase on day 7. Collagen type II expression showed a similar expression pattern. In tibial cartilage, collagen type II and aggrecan mRNA expression had increased on days 7 and 21. BMP-2 overexpression also induced increased aggrecan degradation in cartilage. VDIPEN staining (indicating matrix metalloproteinase activity) was elevated on day 3 in tibial cartilage and on days 3 and 7 in patellar cartilage, but no longer was by day 21. Increased NITEGE staining (indicating aggrecanase activity) was found on days 7 and 21. In IL-1-damaged patellar cartilage, BMP-2 boosted PG synthesis. Blocking of BMP activity resulted in a decreased PG synthesis compared with IL-1 alone. This decreased PG synthesis was associated with PG depletion in the cartilage. These data show that BMP-2 boosts matrix turnover in intact and IL-damaged cartilage. Moreover, BMP contributes to the intrinsic repair capacity of damaged cartilage. Increased matrix turnover might be functional in replacing matrix molecules in the repair of a damaged cartilage matrix

A crucial role for tumor necrosis factor receptor 1 in synovial lining cells and the reticuloendothelial system in mediating experimental arthritis

Contains fulltext : 89310.pdf (publisher's version ) (Open Access)INTRODUCTION: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that mainly affects synovial joints. Biologics directed against tumor-necrosis-factor (TNF)-alpha are efficacious in the treatment of RA. However, the role of TNF receptor-1 (TNFR1) in mediating the TNFalpha effects in RA has not been elucidated and conflicting data exist in experimental arthritis models. The objective is to investigate the role of TNFR1 in the synovial lining cells (SLC) and the reticuloendothelial system (RES) during experimental arthritis. METHODS: Third generation of adenovirus serotype 5 were either injected locally in the knee joint cavity or systemically by intravenous injection into the retro-orbital venous sinus to specifically target SLC and RES, respectively. Transduction of organs was detected by immunohistochemistry of the eGFP transgene. An adenoviral vector containing a short hairpin (sh) RNA directed against TNFR1 (HpTNFR1) was constructed and functionally evaluated in vitro using a nuclear factor-kappaB (NF-kappaB) reporter assay and in vivo in streptococcal cell wall-induced arthritis (SCW) and collagen-induced arthritis (CIA). Adenoviruses were administered before onset of CIA, and the effect of TNFR1 targeting on the clinical development of arthritis, histology, quantitative polymerase chain reaction (qPCR), cytokine analyses and T-cell assays was evaluated. RESULTS: Systemic delivery of Ad5.CMV-eGFP predominantly transduced the RES in liver and spleen. Local delivery transduced the synovium and not the RES in liver, spleen and draining lymph nodes. In vitro, HpTNFR1 reduced the TNFR1 mRNA expression by three-fold resulting in a 70% reduction of TNFalpha-induced NF-kappaB activation. Local treatment with HpTNFR1 markedly reduced mRNA and protein levels of interleukin (IL)-1beta and IL-6 in SLC during SCW arthritis and ameliorated CIA. Systemic targeting of TNFR1 in RES of liver and spleen by systemic delivery of Ad5 virus encoding for a small hairpin RNA against TNFR1 markedly ameliorated CIA and simultaneously reduced the mRNA expression of IL-1beta, IL-6 and Saa1 (75%), in the liver and that of Th1/2/17-specific transcription factors T-bet, GATA-3 and RORgammaT in the spleen. Flow cytometry confirmed that HpTNFR1 reduced the numbers of interferon (IFN)gamma (Th1)-, IL-4 (Th2)- and IL-17 (Th17)-producing cells in spleen. CONCLUSIONS: TNFR1-mediated signaling in both synovial lining cells and the reticuloendothelial system independently played a major pro-inflammatory and immunoregulatory role in the development of experimental arthritis

A Tail-Anchored Myotonic Dystrophy Protein Kinase Isoform Induces Perinuclear Clustering of Mitochondria, Autophagy, and Apoptosis

Contains fulltext : 79678.pdf (publisher's version ) (Open Access)BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients

NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox(-/- )mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox(-/- )mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox(-/- )mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox(-/- )mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox(-/- )mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction

The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPβ-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPβ, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPβ plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to No. Y)

Rheumatoid Arthritis Patients With Circulating Extracellular Vesicles Positive for IgM Rheumatoid Factor Have Higher Disease Activity

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that mainly affects synovial joints. Validated laboratory parameters for RA diagnosis are higher blood levels of rheumatoid factor IgM (IgM-RF), anti-citrullinated protein autoantibodies (ACPA), C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR). Clinical parameters used are the number of tender (TJC) and swollen joints (SJC) and the global patient visual analog score (VAS). To determine disease remission in patients a disease activity score (DAS28) can be calculated based on SJC, TJC, VAS, and ESR (or alternatively CRP). However, subtle and better predictive changes to follow treatment responses in individual patients cannot be measured by the above mentioned parameters nor by measuring cytokine levels in blood. As extracellular vesicles (EVs) play a role in intercellular communication and carry a multitude of signals we set out to determine their value as a biomarker for disease activity. EVs were isolated from platelet-free plasma of 41 RA patients and 24 healthy controls (HC) by size exclusion chromatography (SEC). We quantified the particle and protein concentration, using NanoSight particle tracking analysis and micro-BCA, respectively, and observed no differences between RA patients and HC. In plasma of 28 out of 41 RA patients IgM-RF was detectable by ELISA, and in 13 out of these 28 seropositive RA patients (RF+RA) IgM-RF was also detected on their isolated pEVs (IgM-RF+). In seronegative RA patients (RF−RA) we did not find any RF present on pEVs. When comparing disease parameters we found no differences between RF+RA and RF−RA patients, except for increased ESR levels in RF+RA patients. However, RF+RA patients with IgM-RF+ pEVs showed significantly higher levels of CRP and ESR and also VAS and DAS28 were significantly increased compared to RA+ patients without IgM-RF+ pEVs. This study shows for the first time the presence of IgM-RF on pEVs in a proportion of RF+RA patients with a higher disease activity

Organização do trabalho de enfermagem na Atenção Básica: uma questão para a saúde do trabalhador

Contains fulltext : 208073.pdf (publisher's version ) (Open Access