Effect of PKCδ on iNOS mRNA expression and stability.

<p>(A) L929 fibroblasts were stimulated with a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with PKCδ inhibitor rottlerin for 4 or 10 h. At indicated time points incubations were terminated and extracted total RNA was subjected to RT-PCR. (B) J774 macrophages were transiently transfected with PKCδ siRNA using DharmaFECT 4 transfection reagent. Treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 3 or 9 h before incubations were terminated and extracted total RNA was subjected to RT-PCR. (C) L929 fibroblasts were activated by a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with rottlerin. After 6 h, actinomycin D (2 µg/ml) was added into the cell culture to stop transcription. Incubations were terminated at indicated time points after actinomycin D and extracted total RNA was subjected to RT-PCR. iNOS mRNA levels were normalized against GAPDH mRNA. Values are mean+SEM, n = 3. *p<0.05.</p

Effect of PKCδ on NF-κB activity.

<p>(A) J774 cells were stimulated with LPS and treated with rottlerin for 30 min before the preparation of nuclear extracts. NF-κB DNA binding activity was analyzed by EMSA. Gels shown are representatives of three others with similar results. (B) L929 cells were transfected with NF-κB reporter to form L929 pNF-κB cell line. The transfected cells were stimulated with a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with rottlerin for 1 h before the total mRNA was extracted and subjected to RT-PCR. Luciferase mRNA levels were normalized against GAPDH mRNA. IκB kinase inhibitor BMS3445541 was used as a control agent to inhibit NF-κB mediated transcription. Values are mean+SEM, n = 4. **p<0.01.</p

Effects of PKCδ inhibitor rottlerin on the degradation of iNOS protein.

<p>J774 macrophages were treated with LPS and rottlerin for 8 h before the addition of proteasome inhibitor lactacystin. After 6 h, incubations were terminated and immunoblots were run using iNOS specific antibody. Actin was determined as a loading control. Chemiluminescent signal was quantified as described under the Methods section. Values are mean+SEM, n = 3.</p

Effect of rottlerin on carrageenan induced paw inflammation model in mice.

<p>L-NIL and rottlerin were administered i.p. 2 h prior to carrageenan (1.5%) was injected into a hind paw. Paw edema was measured before, 3 h and 6 h after carrageenan injection. Edema is expressed as the difference between the volume changes of the carrageenan treated paw and the control paw (injected with saline). Values are mean+SEM, n = 5–6, **p<0.01.</p

Effect of PKCδ on NO production.

<p>(A) Effects of rottlerin on LPS-induced NO production in J774 macrophages was measured after 24 h incubation as nitrite accumulated in the culture medium by the method of Griess. Values are mean+SEM, n = 4. (B) Effects of rottlerin on cytokine-induced (IL-1β, IFNγ, and TNFα) NO production in L929 fibroblasts after 24 h incubation. Values are mean+SEM, n = 4. (C) J774 macrophages were transiently transfected with PKCδ siRNA using DharmaFECT 4 transfection reagent. Treatment with non-targeting siRNA was used as control. Macrophages were stimulated with LPS for 24 h before the NO production was measured. Values are mean+SEM, n = 3. (D) L929 fibroblasts were transiently transfected with PKCδ siRNA using DharmaFECT 1 transfection reagent and treatment with non-targeting siRNA was used as control. L929 fibroblasts were stimulated with a combination of cytokines (IL-1β, IFNγ, and TNFα) and treated with PKCδ inhibitor rottlerin for 24 h before NO production was measured. Values are mean+SEM, n = 7,** p<0.01.</p

Additional file 1: of Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes

Supplementary data. Supplementary information to the “Methods”. (DOCX 23 kb

YKL-40 was associated with disease activity and inflammation during intensive treatment with DMARDs.

<p>The figure shows quadratic relationships of YKL-40 with the disease activity measured as DAS28 (A) and with the inflammatory cytokine IL-6 (B) during the 26 weeks of treatment. The patients were treated for the first four weeks with a combination of sulphasalazine, methotrexate, hydroxychloroquine and low dose prednisolone, and thereafter randomized to receive either placebo infusions or infliximab infusions added to the treatment for another 22 weeks. Measurements were carried out at weeks 0, 4, 10, 18, and 26 during the treatment and area under the curve analysis over time (AUC_{0-26 weeks}) adjusted for age, gender and RF positivity was used. Gray-shaded areas represent 95% confidence intervals around the mean. YKL-40 AUC_{0-26 weeks} showed a statistically significant correlation to DAS28 AUC_{0-26 weeks} and IL-6 AUC_{0-26 weeks}.</p

CONSORT flow diagram for the NEO-RACo study.

<p>CONSORT flow diagram for the NEO-RACo study.</p

YKL-40 levels decreased during the intensive anti-rheumatic treatment with a combination of csDMARDs.

<p>The figure shows the mean change in plasma YKL-40 levels in 88 patients in the NEO-RACo -study treated with a combination of sulphasalazine, methotrexate, hydroxychloroquine and low dose prednisolone for the first four weeks, and thereafter randomized to receive either placebo infusions (Fin-RACo + Pla) or infliximab infusions (Fin-RACo + INFL) added on the csDMARD combination for another 22 weeks. YKL-40 levels decreased significantly (p<0.001) during the first four weeks of treatment with csDMARDs; when infliximab (or placebo) was added to the treatment there was a minor further decrease (groups combined, p = 0.031) but no difference between placebo and infliximab treatment groups was found. The change is presented as ng/ml, mean ± 95% CI, n = 88.</p

Generalized estimating equations models for the effect of YKL40, time and interaction in measures of disease activity from baseline to 26 weeks.

<p>Generalized estimating equations models for the effect of YKL40, time and interaction in measures of disease activity from baseline to 26 weeks.</p