Treatment with a Monoclonal Anti-IL-12p40 Antibody Induces Substantial Gut Microbiota Changes in an Experimental Colitis Model.

Background and Aim. Crohn's disease is associated with gut microbiota (GM) dysbiosis. Treatment with the anti-IL-12p40 monoclonal antibody (12p40-mAb) has therapeutic effect in Crohn's disease patients. This study addresses whether a 12p40-mAb treatment influences gut microbiota (GM) composition in mice with adoptive transfer colitis (AdTr-colitis). Methods. AdTr-colitis mice were treated with 12p40-mAb or rat-IgG2a or NaCl from days 21 to 47. Disease was monitored by changes in body weight, stool, endoscopic and histopathology scores, immunohistochemistry, and colonic cytokine/chemokine profiles. GM was characterized through DGGE and 16S rRNA gene-amplicon high-throughput sequencing. Results. Following 12p40-mAb treatment, most clinical and pathological parameters associated with colitis were either reduced or absent. GM was shifted towards a higher Firmicutes-to-Bacteroidetes ratio compared to rat-IgG2a treated mice. Significant correlations between 17 bacterial genera and biological markers were found. The relative abundances of the RF32 order (Alphaproteobacteria) and Akkermansia muciniphila were positively correlated with damaged histopathology and colonic inflammation. Conclusions. Shifts in GM distribution were observed with clinical response to 12p40-mAb treatment, whereas specific GM members correlated with colitis symptoms. Our study implicates that specific changes in GM may be connected with positive clinical outcomes and suggests preventing or correcting GM dysbiosis as a treatment goal in inflammatory bowel disease.

Identification of the calcitonin receptor in osteoarthritic chondrocytes.

BACKGROUND: Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA). However, the presence of the calcitonin receptor (CTR) in articular cartilage chondrocytes is yet to be identified. In this study, we sought to further investigate the expression of the CTR in naïve human OA articular chondrocytes to gain further confirmation of the existents of the CTR in articular cartilage. METHODS: Total RNA was purified from primary chondrocytes from articular cartilage biopsies from four OA patients undergoing total knee replacement. High quality cDNA was produced using a dedicated reverse transcription polymerase chain reaction (RT-PCR) protocol. From this a nested PCR assay amplifying the full coding region of the CTR mRNA was completed. Western blotting and immunohistochemistry were used to characterize CTR protein on protein level in chondrocytes. RESULTS: The full coding transcript of the CTR isoform 2 was identified in all four individuals. DNA sequencing revealed a number of allelic variants of the gene including two potentially novel polymorphisms: a frame shift mutation, +473del, producing a shorter form of the receptor protein, and a single nucleotide polymorphism in the 3' non coding region of the transcript, +1443 C>T. A 53 kDa protein band, consistent with non-glycosylated CTR isoform 2, was detected in chondrocytes with a similar size to that expressed in osteoclasts. Moreover the CTR was identified in the plasma membrane and the chondrocyte lacuna of both primary chondrocytes and OA cartilage section. CONCLUSIONS: Human OA articular cartilage chondrocytes do indeed express the CTR, which makes the articular a pharmacological target of salmon calcitonin. In addition, the results support previous findings suggesting that calcitonin has a direct anabolic effect on articular cartilage.

Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity.

INTRODUCTION: Physiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation. METHODS: Cartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue. RESULTS: Catabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points. CONCLUSION: Cartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic auto-immune disease with extensive articular cartilage destruction. Aggrecan depletion, mediated by aggrecanases is one of the first signs of early cartilage erosion. We investigated, whether measurement of aggrecan and fragments thereof in serum, could be used as biomarkers for joint-disease in RA patients and furthermore characterized the fragments found in the circulation. METHODS: The study consisted of 38 patients, 12 males (62.2 +/- 16.0 years) and 26 females (59.8 +/- 20.7 years) diagnosed with RA: 41.5 +/- 27.5 mm/h erythrocyte sedimentation rate (ESR), 38.4 +/- 34.7 mg/ml C-reactive protein (CRP) and 4.8 +/- 1.7 disease activity score (DAS) and 108 healthy age-matched controls. Aggrecan levels were measured using two immunoassays, i.e. the (374)ARGSVI-G2 sandwich ELISA measuring aggrecanase-mediated aggrecan degradation and the G1/G2 sandwich assay, detecting aggrecan molecules containing G1 and/or G2 (total aggrecan) We further characterized serum samples by western blots, by using monoclonal antibodies F-78, binding to G1 and G2, or by BC-3, detecting the aggrecanase-generated N-terminal 374ARGSVI neo-epitope. RESULTS: Total aggrecan levels in RA patients were significantly decreased from 824.8 +/- 31 ng/ml in healthy controls to 570.5 +/- 30 ng/ml (31% decrease, P < 0.0001), as measured by the G1/G2 ELISA. Western blot analysis with F-78 showed one strong band at 10 kDa, and weaker bands at 25 and 45 kDa in both healthy controls and RA patients. In contrast, staining for aggrecanase-activity revealed only one strong band in RA patients of 45 kDa. CONCLUSION: This is the first study, which characterizes different aggrecan fragments in human serum. The data strongly suggests that total aggrecan levels, i.e. aggrecan molecules containing G1 and/or G2 are lower in RA patients, and that RA patients have at least one specific subpopulation of aggrecan fragments, namely aggrecanse generated 374ARGSVI fragments. Further clinical studies are needed to investigate the potential of G1/G2 as a structure-related biochemical marker in destructive joint-diseases.

Calcitonin affects both bone and cartilage: a dual action treatment for osteoarthritis?

Osteoarthritis (OA) is the most common form of degenerative joint diseases and a major cause of disability and impaired quality of life in the elderly. OA is a complex disease involving both bone and cartilage properties, and may therefore require alternative approaches for treatment. Recent lines of evidence suggest that calcitonin acts on both osteoclasts and chondrocytes. The review summarizes emerging observations from cell biology to preliminary clinical trials, describing possible chondroprotective effects of calcitonin. This review summarizes peer-reviewed articles found using predefined search criteria and published in the PubMed database before June 2007. In addition, abstracts from the OsteoArthritis Research Society International (OARSI) conferences in the time period 2000 to 2006 were included. A range of studies, at the cellular level, in animal models, and in clinical trials, describe positive effects of calcitonin on bone health. Regarding articular cartilage, direct effects of calcitonin on chondrocytes on matrix synthesis, as well as inhibition of cartilage degradation, have been presented. In addition, clinical evidence for a chondroprotective effect of calcitonin is emerging. Several lines of evidence suggest direct anabolic effects of calcitonin on articular chondrocytes, resulting in increased proteoglycan synthesis. The anticatabolic effects of calcitonin may involve induction of cAMP, resulting in attenuation of MMP-mediated cartilage degradation. Presently there is limited availability of chondroprotective agents. Therefore, the current clinical research on calcitonin is highly anticipated, and may prove calcitonin treatment efficacious for the prevention and treatment of OA.

Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: implications of the time of initiation.

OBJECTIVE: To investigate how the time of initiation influences the effects of estrogen therapy on type II collagen (CII) turnover and the structural integrity of articular cartilage in ovariectomized rats and to determine whether estrogen exerts direct effects on the catabolic function of chondrocytes ex vivo. METHODS: A total of 46 Sprague-Dawley rats were distributed into 1 of the following treatment groups: 1) ovariectomy, 2) ovariectomy plus early estrogen therapy, 3) ovariectomy plus delayed estrogen therapy, or 4) sham operation. Cartilage turnover was estimated by measuring the serum levels of C-telopeptide of type II collagen (CTX-II). Cartilage lesions at week 9 were quantified using a published scoring technique. The presence of the CTX-II epitope in articular cartilage was assessed by immunohistochemistry. The effects of estrogen (1-100 nM) on chondrocytes were investigated in bovine cartilage explants subjected to catabolic cytokines (tumor necrosis factor alpha [TNFalpha] and oncostatin M [OSM]). RESULTS: In ovariectomized rats, estrogen therapy evoked significant decreases in serum CTX-II independently of the time of initiation; yet, delayed initiation resulted in diminished efficacy in terms of preventing cartilage lesions. CTX-II fragments were present in articular cartilage, colocalizing with early lesions at the cartilage surface. In untreated animals, the early relative increases in serum CTX-II were proportional to the severity of cartilage lesions at week 9 (r = 0.73, P < 0.01). Estrogen significantly and dose-dependently countered CTX-II release from TNFalpha plus OSM-stimulated cartilage explants ex vivo. CONCLUSION: Our results suggest that estrogen counters the acceleration of CII degradation and related structural alterations, and these benefits can be maximized by early initiation after menopause. The protective effect of estrogen seems to involve direct inhibition of the catabolic function of chondrocytes.