Effect of Semecarpus anacardium Linn. nut milk extract on rat neutrophil functions in adjuvant arthritis

Neutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA) and various inflammatory conditions, by accumulation and liberation of active proteolytic enzymes. The effect of milk extract of Semecarpus anacardium Linn. nuts (SA) at a dosage of 150 mg kg(-1) body weight day(-1) for 14 days on adjuvant arthritis was studied to gain some insight into this intriguing disease in relation to neutrophil functions. The decreased phagocytic function of neutrophils (phagocytic index and avidity index) found in adjuvant arthritis was significantly increased by the administration of the drug SA. Increased levels of reactive oxygen species (superoxide radical, hydroxyl radical, H2O2 and myeloperoxidase), lysosomal enzymes (acid phosphatase and cathepsin D) and increased accumulation of neutrophils in the joints observed in adjuvant arthritic animals were reverted back to near normal levels by treatment with SA. The results of this study indicate that SA can be considered to be a good therapeutic agent for inflammation and arthritis.     

Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [³H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-dl-penicillamine (SNAP) (500 μM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [³H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.     

Imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fibroblasts

Synovial fibroblast is the key cell type in the growth of the pathological synovial tissue in arthritis. Here, we show that platelet-derived growth factor (PDGF) is a potent mitogen for synovial fibroblasts isolated from patients with rheumatoid arthritis. Inhibition of PDGF-receptor signalling by imatinib mesylate (1muM) completely abrogated the PDGF-stimulated proliferation and inhibited approximately 70% of serum-stimulated proliferation of synovial fibroblasts. Similar extent of inhibition was observed when PDGF was neutralized with anti-PDGF antibodies, suggesting that imatinib mesylate does not inhibit pathways other than those mediated by PDGF-receptors. No signs of apoptosis were detected in synovial fibroblasts cultured in the presence of imatinib. These results suggest that imatinib mesylate specifically inhibits PDGF-stimulated proliferation of synovial fibroblasts, and that inhibition of PDGF-receptors could represent a feasible target for novel antirheumatic therapies.     

Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice

Sphingosine-1-phosophate, generated from the phosphorylation of sphingosine by sphingosine kinase enzymes, is suggested to function as an intracellular second messenger for inflammatory mediators, including formyl peptide, C5a, and Fc. More recently, a role for sphingosine kinases during inflammation has also been proposed. Here we show that sphingosine kinase 1 knockout mice exhibit normal inflammatory cell recruitment during thioglycollate-induced peritonitis and that sphingosine kinase 1-null neutrophils respond normally to formyl peptide. In the collagen-induced arthritis model of rheumatoid arthritis, sphingosine kinase 1 knockout mice developed arthritis with normal incidence and severity. Our findings show that sphingosine kinase 1 is dispensable for inflammatory responses and support the need for more extensive studies of sphingosine kinases in inflammation.     

Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.     

Cross‐reactivity of a human IgG1 anticitrullinated fibrinogen monoclonal antibody to a citrullinated profilaggrin peptide

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70% of RA patients are anticitrullinated protein antibody (ACPA) positive. ACPA responses have been widely studied and are suggested to be heterogeneous, favoring antibody cross‐reactivity to citrullinated proteins. In this study, we examined factors that may influence cross‐reactivity between a commercial human anticitrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated profilaggrin sequence (HQCHQEST‐ Cit‐GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross‐reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA.     

Microrheology of human synovial fluid of arthritis patients studied by diffusing wave spectroscopy

The viscoelastic properties of synovial fluid (SF) are critical to its functions of lubrication and shock‐absorption of joints in human body; a change in the viscoelastic properties, even of only a few percents, is often concomitant with arthritis. In this work, the elastic modulus G ′(f) and the viscous modulus G ′′(f) of SF from patients suffering from three kinds of joint diseases, namely, osteoarthritis (OA), rheumatoid arthritis (RA), and gouty arthritis (GA), were determined as a function of frequency “f ” (in the low frequency range from f ～ 0.1 to 10 Hz) by Diffusing Wave Spectroscopy (DWS) and correlated with the white blood cell (WBC) count. A strong correlation was observed, showing a higher WBC count corresponding to lower elastic and viscous moduli, G ′ and G ′′; further details depend on inflammatory vs. non‐inflammatory, and on the severity of inflammation. Different types of arthritis lead to different degrees of decreasing viscoelasticity. Identical measurements were carried out with a commercial visco‐supplementation (or artificial SF) to serve as reference. In general, the reduction in both G ′ and G ′′ was most severe in the case of GA and least severe in the case of OA. Besides, in all cases, the reduction in G ′ was more prominent than the reduction in G ′′, indicating that in general, the deterioration in the elasticity of SF by inflammation is more severe than that in the viscosity. This simple method for quantitative physical characterization of synovial fluid may serve as a useful complementary metric to the conventional biochemical analysis in clinical diagnosis of arthritis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular basis of recognition of human osteopontin by 23C3, a potential therapeutic antibody for treatment of rheumatoid arthritis

Osteopontin plays an important role in the development and perpetuation of rheumatoid arthritis (RA). Antibodies targeting osteopontin have shown promising therapeutic benefits against this disease. We have previously reported a novel anti-RA monoclonal antibody, namely, 23C3, and shown it capable of alleviating the symptoms of RA in a murine collagen-induced arthritis model, restoring the cytokine production profile in joint tissues, and reducing T-cell recall responses to collagen type II. We describe here the crystal structure of 23C3 in complex with its epitope peptide. Analyses of the complex structure reveal the molecular mechanism of osteopontin recognition by 23C3. The peptide folds into two tandem β-turns, and two key residues of the peptide are identified to be critical for the recognition by 23C3: TrpP43 is deeply embedded into a hydrophobic pocket formed by AlaL34, TyrL36, LeuL46, TyrL49, PheL91, and MetH102 and therefore has extensive hydrophobic interactions with 23C3, while AspP47 has a network of hydrophilic interactions with residues ArgH50, ArgH52, SerH53, and AsnH56 of the antibody. Besides the complementarity-determining region loops, the framework region L2 of 23C3 is also shown to interact with the epitope peptide, which is not common in the antibody-antigen interactions and thus could be exploited in the engineering of 23C3. These results not only provide valuable information for further improvement of 23C3 such as chimerization or humanization for its therapeutic application, but also reveal the features of this specific epitope of osteopontin that may be useful for the development of new antibody drugs against RA.     

Th1、Th2和Th17型细胞在类风湿性关节炎和系统性红斑狼疮中的活化特点

探讨Th1、Th2和Th17型细胞在类风湿性关节炎(RA)和系统性红斑狼疮(SLE)发病机制中的作用。收集37例RA患者、25例SLE患者和34例健康人的抗凝血,应用ELISA检测血清中IFN-γ、IL-10和IL-17的水平。与健康对照组比较,RA和SLE患者血清中IFN-γ的水平均具有统计学意义(P<0.05);SLE患者IL-10水平出现有意义的升高(P<0.05);而RA患者IL-17的升高具有统计学意义(P<0.05)。由此提示Th1、Th2和Th17细胞在自身免疫性疾病中均发挥不同的重要作用。     

关节镜清理术治疗前踝撞击征的疗效

目的:探讨踝关节镜对前踝撞击征的诊疗价值。方法:采用关节镜清理术治疗前踝撞击综合征33例,其中男23例,女10例。年龄平均39岁(18～72岁)。左踝16例,右踝17例。病史6个月至20年,有运动损伤史13例,踝关节扭伤病史17例,踝关节骨折史3例。临床表现为踝关节肿胀、疼痛和下蹲活动受限。X线片显示胫骨前唇和距骨骨质增生,MRI显示距骨软骨损伤,踝关节腔内积液。采用局麻关节镜下骨赘磨削,滑膜切除,踝关节清理术。结果:随访33例,平均时间35个月(4个月-6年)。术后踝关节背伸活动功能正常,按照McGuire踝关节评分系统对术前、术后踝关节功能进行评估,术前平均55分,术后平均80分,术后提高了25分。优13例,良15例,可3例,差2例,优良率84.8%。结论:局麻下踝关节镜骨赘切除、滑膜刨削和软骨修整可有效解除踝关节撞击症状,手术创伤小、功能恢复快,效果满意。