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

Implication of proteins containing tetratricopeptide repeats in conditional virulence phenotypes of Legionella pneumophila

Legionella pneumophila, the causative agent of Legionnaires\u27 disease, is a ubiquitous freshwater bacterium whose virulence phenotypes require a type IV secretion system (T4SS). L. pneumophila strain JR32 contains two virulence-associated T4SSs, the Dot/ Icm and Lvh T4SSs. Defective entry and phagosome acidification phenotypes of dot/icm mutants are conditional and reversed by incubating broth-grown stationary-phase cultures in water (WS treatment) prior to infection, as a mimic of the aquatic environment of Legionella. Reversal of dot/icm virulence defects requires the Lvh T4SS and is associated with a\u3e 10-fold induction of LpnE, a tetratricopeptide repeat (TPR)-containing protein. In the current study, we demonstrated that defective entry and phagosome acidification phenotypes of mutants with changes in LpnE and EnhC, another TPR-containing protein, were similarly reversed by WS treatment. In contrast to dot/icm mutants for which the Lvh T4SS was required, reversal for the ΔlpnE or the ΔenhC mutant required that the other TPR-containing protein be present. The single and double ΔlpnE and ΔenhC mutants showed a hypersensitivity to sodium ion, a phenotype associated with dysfunction of the Dot/Icm T4SS. The ΔlpnE single and the ΔlpnE ΔenhC double mutant showed 3- to 9-fold increases in translocation of Dot/Icm T4SS substrates, LegS2/SplY and LepB. Taken together, these data identify TPR-containing proteins in a second mechanism by which the WS mimic of a Legionella environmental niche can reverse virulence defects of broth-grown cultures and implicate LpnE and EnhC directly or indirectly in translocation of Dot/Icm T4SS protein substrates. © 2012, American Society for Microbiology

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-6

, or treated with pro-inflammatory cytokines 10 ng/ml oncostatin M (OSM) in combination with 20 ng/ml tumour necrosis factor alpha (TNFα) (-◆-). The conditioned medium from four independent wells was measured for the presence of G1/G2 molecules at each collected time-point , or accumulated throughout the study-period. As negative control, explants were frozen and thawed four times in liquid nitrogen (--). The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-3

Thritis (RA) (N = 38) were analyzed in the G1/G2 assay. The concentrations are log-transformed data and the values are mean + SEM. The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t-tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-4

From patients with Rheumatoid arthritis (RA) (lane 3) and the membrane was stained with the monoclonal antibody F-78 raised against intact bovine aggrecan, binding to G1 and G2, or with BC-3 raised against the aggrecanase-generated ARGSVI sequence. As control, plasma samples from 22 healthy individuals were used (lane 2). A standard molecular weight marker was also run to determine the size of the detected fragments (lane 1).<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-5

, or treated with pro-inflammatory cytokines 10 ng/ml oncostatin M (OSM) in combination with 20 ng/ml tumour necrosis factor alpha (TNFα) (-◆-). The conditioned medium from four independent wells was measured for the presence of aggrecanase-generated ARGSVI-G2 fragments at each collected time-point , or accumulated throughout the study-period. As negative control, explants were frozen and thawed four times in liquid nitrogen (--). The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile-2

Thritis (RA) (N = 38) were analyzed for their anti-CCP level. The activities are log-transformed data and the values are mean + SEM. The asterisks indicate significant differences (P < 0.05). For the statistical analysis, two-tailed non-parametric t-tests were used.<p><b>Copyright information:</b></p><p>Taken from "Patients with rheumatoid arthritis have an altered circulatory aggrecan profile"</p><p>http://www.biomedcentral.com/1471-2474/9/74</p><p>BMC Musculoskeletal Disorders 2008;9():74-74.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2426686.</p><p></p