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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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 ³⁷⁴ARGSVI-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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Investigation of the direct effects of salmon calcitonin on human osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>Calcitonin has been demonstrated to have chondroprotective effects under pre-clinical settings. It is debated whether this effect is mediated through subchondral-bone, directly on cartilage or both in combination. We investigated possible direct effects of salmon calcitonin on proteoglycans and collagen-type-II synthesis in osteoarthritic (OA) cartilage.</p> <p>Methods</p> <p>Human OA cartilage explants were cultured with salmon calcitonin [100 pM-100 nM]. Direct effects of calcitonin on articular cartilage were evaluated by 1) measurement of proteoglycan synthesis by incorporation of radioactive labeled ³⁵SO₄[5 μCi] 2) quantification of collagen-type-II formation by pro-peptides of collagen type II (PIINP) ELISA, 3) QPCR expression of the calcitonin receptor in OA chondrocytes using four individual primer pairs, 4) activation of the cAMP signaling pathway by EIA and, 5) investigations of metabolic activity by AlamarBlue.</p> <p>Results</p> <p>QPCR analysis and subsequent sequencing confirmed expression of the calcitonin receptor in human chondrocytes. All doses of salmon calcitonin significantly elevated cAMP levels (P < 0.01 and P < 0.001). Calcitonin significantly and concentration-dependently [100 pM-100 nM] induced proteoglycan synthesis measured by radioactive ³⁵SO₄incorporation, with a 96% maximal induction at 10 nM (P < 0.001) corresponding to an 80% induction of 100 ng/ml IGF, (P < 0.05). In alignment with calcitonin treatments [100 pM-100 nM] resulted in 35% (P < 0.01) increased PIINP levels.</p> <p>Conclusion</p> <p>Calcitonin treatment increased proteoglycan and collagen synthesis in human OA cartilage. In addition to its well-established effect on subchondral bone, calcitonin may prove beneficial to the management of joint diseases through direct effects on chondrocytes.</p

Insulin growth factor (IGF) stimulates local replenishment of cartilage

Articular cartilage explants were cultured with either oncostatin M plus tumour necrosis factor (OSM + TNF) or vehicle for 7, 11, and 17 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. Aggrecan is completely depleted from the tissue at 7, 11, and 17 days. Other cultures were treated with either OSM + TNF or vehicle for 7, 11, and 17 days followed by stimulation with either IGF or vehicle control for 14 days. Subsequently, cartilage explants were paraffin-embedded and stained for aggrecan content as described in Materials and methods. As a control experiment, articular cartilage explants were cultured for 21 days with vehicle, OSM + TNF, IGF, or metabolically inactive (MI) control for 21 days as controls (lower panel). W/O, without stimulation.<p><b>Copyright information:</b></p><p>Taken from "Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity"</p><p>http://arthritis-research.com/content/10/3/R63</p><p>Arthritis Research & Therapy 2008;10(3):R63-R63.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2483454.</p><p></p

Quantification of aggrecan and collagen degradation products at days 7, 11, and 17

Articular cartilage explants were cultured in the presence or absence of oncostatin M plus tumour necrosis factor (OSM + TNF). Conditioned medium was collected at days 7, 11, and 17. Aggrecanase-mediated aggrecan degradation was measured by the ARGSV-G2 enzyme-linked immunosorbent assay (ELISA), matrix metalloproteinase (MMP)-mediated aggrecan degradation was quantified by the FFGVG-G2 ELISA, and MMP-mediated collagen type II degradation was quantified in the CTX-II ELISA. **< 0.01, ***< 0.001. CTX-II, crosslinked C-terminal neo-epitopes of type II collagen.<p><b>Copyright information:</b></p><p>Taken from "Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity"</p><p>http://arthritis-research.com/content/10/3/R63</p><p>Arthritis Research & Therapy 2008;10(3):R63-R63.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2483454.</p><p></p

Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity-0

II synthesis in cartilage explants was measured by the concentration of N-terminal pro-peptides of type II collagen in the conditioned medium using the PIINP enzyme-linked immunosorbent assay (ELISA). The curves represent the release found at the specific day, where the conditioned medium was fully replaced, and the values were accumulated over the entire period. Vehicle control, metabolically inactive (MI), O + T, oncostatin M plus tumour necrosis factor. Quantification of collagen type II formation 2 weeks after the catabolic induction. The collagen type II synthesis in the identical 14-day periods with or without IGF stimulation following the three different periods of catabolic stimulation was measured by the PIINP ELISA. The conditioned medium was fully replaced three times a week. The results show the accumulated release of collagen type II pro-peptide during the two weeks with anabolic stimulation (insulin growth factor, IGF) and without stimulation (vehicle). IGF-I significantly induced collagen type II formation at low and intermediate catabolic insult, but not at maximal insult. *< 0.05. PIINP, N-terminal pro-peptide of pro-collagen type II.<p><b>Copyright information:</b></p><p>Taken from "Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity"</p><p>http://arthritis-research.com/content/10/3/R63</p><p>Arthritis Research & Therapy 2008;10(3):R63-R63.</p><p>Published online 30 May 2008</p><p>PMCID:PMC2483454.</p><p></p

Treatment with anti-C5aR mAb leads to early-onset clinical and mechanistic effects in the murine delayed-type hypersensitivity arthritis model

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