Common variants at the promoter region of the APOM confer a risk of rheumatoid arthritis

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 × 10-7). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 × 10-5). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 × 10-10). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.     

In vivo high‐resolution synchrotron radiation imaging of collagen‐induced arthritis in a rodent model

In vivo microstructures of the affected feet of collagen‐induced arthritic (CIA) mice were examined using a high‐resolution synchrotron radiation (SR) X‐ray refraction technique with a polychromatic beam issued from a bending magnet. The CIA models were obtained from six‐week‐old DBA/1J mice that were immunized with bovine type II collagen and grouped as grades 0–3 according to a clinical scoring for the severity of arthritis. An X‐ray shadow of a specimen was converted into a visual image on the surface of a CdWO₄ scintillator that was magnified using a microscopic objective lens before being captured with a digital charge‐coupled‐device camera. Various changes in the joint microstructure, including cartilage destruction, periosteal born formation, articular bone thinning and erosion, marrow invasion by pannus progression, and widening joint space, were clearly identified at each level of arthritis severity with an equivalent pixel size of 2.7 µm. These high‐resolution features of destruction in the CIA models have not previously been available from any other conventional imaging modalities except histological light microscopy. However, thickening of the synovial membrane was not resolved in composite images by the SR refraction imaging method. In conclusion, in vivo SR X‐ray microscopic imaging may have potential as a diagnostic tool in small animals that does not require a histochemical preparation stage in examining microstructural changes in joints affected with arthritis. The findings from the SR images are comparable with standard histopathology findings.     

Incorporation of Baccharis dracunculifolia DC (Asteraceae) leaf extract into phosphatidylcholine-cholesterol liposomes improves its anti-inflammatory effect in vivo

The aerial parts of Baccharis dracunculifolia (BdE) is used in the Brazilian folk medicine to treat inflammatory conditions. Here we examined the ability of free and liposomal BdE to modulate reactive oxygen species generation in human neutrophils in vitro and zymosan-induced acute joint inflammation in Wistar rats. We prepared biocompatible liposomes of soya phosphatidylcholine and cholesterol with low diameter, homogeneous size distribution, and neutral surface charge. Free BdE decreased joint swelling, total leucocyte and neutrophil infiltration, and the synovial levels of tumour necrosis factor-α and interleukins 6 and 1β. Incorporation of BdE into liposomes preserved its capacity to inhibit the neutrophil superoxide anion and total reactive oxygen species generation, and improved its anti-inflammatory effect in vivo by decreasing the effective BdE dose by nearly sixfold. The same liposome type lowered the effective dose of caffeic acid by nearly sixteenfold. Therefore, incorporation of BdE into phosphatidylcholine-cholesterol liposomes improves its anti-inflammatory effect.     

Metabolite assignment of ultrafiltered synovial fluid extracted from knee joints of reactive arthritis patients using high resolution NMR spectroscopy

Currently, there are no reliable biomarkers available that can aid early differential diagnosis of reactive arthritis (ReA) from other inflammatory joint diseases. Metabolic profiling of synovial fluid (SF)—obtained from joints affected in ReA—holds great promise in this regard and will further aid monitoring treatment and improving our understanding about disease mechanism. As a first step in this direction, we report here the metabolite specific assignment of ¹H and ¹³C resonances detected in the NMR spectra of SF samples extracted from human patients with established ReA. The metabolite characterization has been carried out on both normal and ultrafiltered (deproteinized) SF samples of eight ReA patients (n = 8) using high-resolution (800 MHz) ¹H and ¹H─¹³C NMR spectroscopy methods such as one-dimensional ¹H CPMG and two-dimensional J-resolved¹H NMR and homonuclear ¹H─¹H TOCSY and heteronuclear¹H─¹³C HSQC correlation spectra. Compared with normal SF samples, several distinctive ¹H NMR signals were identified and assigned to metabolites in the ¹H NMR spectra of ultrafiltered SF samples. Overall, we assigned 53 metabolites in normal filtered SF and 64 metabolites in filtered pooled SF sample compared with nonfiltered SF samples for which only 48 metabolites (including lipid/membrane metabolites as well) have been identified. The established NMR characterization of SF metabolites will serve to guide future metabolomics studies aiming to identify/evaluate the SF-based metabolic biomarkers of diagnostic/prognostic potential or seeking biochemical insights into disease mechanisms in a clinical perspective.     

Comparative pharmacokinetic study of two boswellic acids in normal and arthritic rat plasma after oral administration of Boswellia serrata extract or Huo Luo Xiao Ling Dan by LC‐MS

Huo Luo Xiao Ling Dan (HLXLD), a Chinese herbal formula composed of 11 different herbs, has been used traditionally for the treatment of arthritis and other chronic inflammatory diseases. However, the pharmacokinetic profile of its anti‐inflammatory bioactive compounds has not been elucidated. Boswellic acids are the bioactive compounds with potent anti‐inflammatory activity isolated from Boswellia serrate which is one of the 11 herbs of HLXLD. The objective of the study was to compare the pharmacokinetics of the two bioactive bowsellic acids: 11‐keto‐β‐boswellic acid and 3‐O‐acetyl‐11‐keto‐β‐boswellic following oral administration of HLXLD or Boswellia serrata extract alone in normal and arthritic rats. An LC‐MS method was developed and validated for the determination of 11‐keto‐β‐boswellic acid and 3‐O‐acetyl‐11‐keto‐β‐boswellic in the comparative pharmacokinetic study. The results showed that there were significant differences in pharmacokinetic parameters between normal and arthritic groups. Interestingly, the absorptions of two boswellic acids were significantly higher in HLXLD than Boswellia serrata extract alone, indicating the synergistic effect of other herbal ingredients in HLXLD. This comparative pharmacokinetic study provided direct evidence supporting the notion that the efficacy of a complex mixture such as HLXLD is better than that of single components in treating human diseases. Copyright © 2014 John Wiley & Sons, Ltd.     

IL-17 induces the production of IL-16 in rheumatoid arthritis

The purpose of this study was to investigate the expression of IL-16 in the rheumatoid synovium and the role of inflammatory cytokines and Toll-like receptor (TLR) ligands in IL-16 production by fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA) patients. Immunohistochemical staining was performed with a monoclonal antibody to IL-16 in synovial tissues from patients with RA and likewise in patients with osteoarthritis (OA). FLS were isolated from RA synovial tissues and stimulated with IL-15, IL-1beta, IFN-gamma, and IL-17. The IL-16 mRNA level was assessed by semiquantitative RT-PCR and real time (RT) PCR and a comparison was made between IL-16 mRNA levels produced by RA-FLS and OA-FLS. Production of IL-16 was identified by a western blot assay, and IL-16 production after stimulation by specific ligands of TLR2 and TLR4 was assessed by RT-PCR. While immunohistochemical staining demonstrated strong expression of IL-16 mRNA in synovial tissues from patients with RA, similar findings were not present in the OA group. Moreover, mRNA expression of IL-16 by RA-FLS increased after treatment with IL-17 but not with IL-15, IL-1beta, and IFN-gamma. Specifically, IL-17 increased IL-16 mRNA level by RA-FLS and peripheral blood mononuclear cells in a dose-dependent manner. However, IL-17 did not stimulate IL-16 production in OA-FLS. Peptidoglycan, a selective TLR2 ligand, also increased production of IL-16 by RA-FLS dose- dependently, whereas LPS, a selective TLR4 ligand, had no such stimulatory effect. The results from our data demonstrate that IL-17 and TLR2 ligands stimulate the production of IL-16 by RA-FLS.     

Molecular design and structural optimization of potent peptide hydroxamate inhibitors to selectively target human ADAM metallopeptidase domain 17

Human ADAMs (a disintegrin and metalloproteinases) have been established as an attractive therapeutic target of inflammatory disorders such as inflammatory bowel disease (IBD). The ADAM metallopeptidase domain 17 (ADAM17 or TACE) and its close relative ADAM10 are two of the most important ADAM members that share high conservation in sequence, structure and function, but exhibit subtle difference in regulation of downstream cell signaling events. Here, we described a systematic protocol that combined computational modeling and experimental assay to discover novel peptide hydroxamate derivatives as potent and selective inhibitors for ADAM17 over ADAM10. In the procedure, a virtual combinatorial library of peptide hydroxamate compounds was generated by exploiting intermolecular interactions involved in crystal and modeled structures. The library was examined in detail to identify few promising candidates with both high affinity to ADAM17 and low affinity to ADAM10, which were then tested in vitro with enzyme inhibition assay. Consequently, two peptide hydroxamates Hxm-Phe-Ser-Asn and Hxm-Phe-Arg-Gln were found to exhibit potent inhibition against ADAM17 (K_i = 92 and 47 nM, respectively) and strong selectivity for ADAM17 over ADAM10 (∼7-fold and ∼5-fold, S = 0.86 and 0.71, respectively). The structural basis and energetic property of ADAM17 and ADAM10 interactions with the designed inhibitors were also investigated systematically. It is found that the exquisite network of nonbonded interactions involving the side chains of peptide hydroxamates is primarily responsible for inhibitor selectivity, while the coordination interactions and hydrogen bonds formed by the hydroxamate moiety and backbone of peptide hydroxamates confer high affinity to inhibitor binding.     

Common Factors of Alzheimer’s Disease and Rheumatoid Arthritis—Pathomechanism and Treatment

The accumulation of amyloid plaques, or misfolded fragments of proteins, leads to the development of a condition known as amyloidosis, which is clinically recognized as a systemic disease. Amyloidosis plays a special role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease, and rheumatoid arthritis (RA). The occurrence of amyloidosis correlates with the aging process of the organism, and since nowadays, old age is determined by the comfort of functioning and the elimination of unpleasant disease symptoms in the elderly, exposure to this subject is justified. In Alzheimer’s disease, amyloid plaques negatively affect glutaminergic and cholinergic transmission and loss of sympathetic protein, while in RA, amyloids stimulated by the activity of the immune system affect the degradation of the osteoarticular bond. The following monograph draws attention to the over-reactivity of the immune system in AD and RA, describes the functionality of the blood–brain barrier as an intermediary medium between RA and AD, and indicates the direction of research to date, focusing on determining the relationship and the cause–effect link between these disorders. The paper presents possible directions for the treatment of amyloidosis, with particular emphasis on innovative therapies.     

Syntheses,Characterization and Anti-inflammatory Activity of a Benzamide Derivative and its Metal Chelates

N-Phenylbenzamide-2,2′-dicarboxylic acid (PBDA) and its copper(II), nickel(II), cobalt (II), zinc(II) and manganese(II) chelates have been synthesized and characterized by their physical measurements, infrared and electronic spectra and magnetic moment data. In an acute anti-inflammatory test, the cobalt chelate was most active (31.1% inhibition) followed by the zinc and copper chelates, whereas the copper chelate (22.3% inhibition) was most active in an adjuvant arthritis test. Again the cobalt chelate was most active in the cotton-wad granuloma test. Gastric irritancy was markedly reduced after chelation by copper, followed in order by zinc, cobalt, manganese and nickel chelates. © 1997 John Wiley & Sons, Ltd.