Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V77-E92 of αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

Beneficial effects associated to a healthy lifestyle in systemic lupus erythematosus: A cross-sectional study.

OBJECTIVE: To assess whether a healthy lifestyle is associated to beneficial effects on various systemic lupus erythematosus (SLE) health domains. METHODS: In a cross-sectional study, Mediterranean Diet Adherence Score (MEDAS), physical activity energy expenditure (PAEE), and smoking status were assessed by questionnaires, along with clinical parameters and various health domains including Systemic Lupus Disease Activity Score (SLEDAI), Depression Scale (CES-D), Fatigue Severity (FSS), functional status (FFbH), physical and mental quality of life (PCS, MCS). Lifestyle choices were assessed with respect to health domains by linear regression modeling. Additionally, SLE patients with a healthy lifestyle (MEDAS ≥ 4, ≥ 1 h sport per week, no smoking) were compared to those without by Wilcoxon's signed-rank test. RESULTS: 49 of 145 SLE patients (44.3 ± 31.7 years, 87.6% female) followed a healthy lifestyle and showed a higher physical quality of life (ß = 4.5 (95%-CI 1.5-7.9) p = 0.01), lower depression (ß = -5.0 (-8.2 to -0.2) p = 0.02) and lower fatigue (ß = -0.8 (-1.5 to -0.2) p = 0.01) independently of SLE disease activity. Furthermore, dsDNA-antibodies were lower (146 ± 540 vs 266 ± 146 U/mL, p = 0.049). In a more detailed analysis, physical activity had the highest impact on the various health domains when compared to smoking or diet adherence, which was consistent even after adjusting for multiple potential confounders. Each 1,000 kcal of weekly PAEE was associated to a 1.8 (0.9-2.6) point increase in the PCS (p = 0.0001), a 0.2 (0.03-0.4) point decrease in the CES-D (p = 0.01) and a 2.8 (1.2-4.4) point increase in the FFbH (p = 0.0006). CONCLUSION: A healthy lifestyle, especially physical activity is associated with beneficial effects including quality of life, depression and fatigue in SLE.

[Adult-onset Still's disease : Diagnosis and treatment according to the new S2e guidelines of the German Society of Rheumatology (DGRh)]. / Adultes Still-Syndrom : Diagnostik und Therapie unter Berücksichtigung der neuen DGRh(Deutsche Gesellschaft für Rheumatologie)-S2e-Leitlinie.

Adult-onset Still's disease (AOSD) is a rare autoinflammatory disease characterized by intermittent fever and a combination of symptoms, such as an evanescent rash synchronous with fever, arthralgia/arthritis, lymphadenopathy and hepatosplenomegaly. The diagnosis is based on a characteristic constellation of symptoms and the exclusion of infections, hemato-oncological diseases, infectious diseases and alternative rheumatological causes. The systemic inflammatory reaction is reflected by high levels of ferritin and C­reactive protein (CRP). The pharmacological treatment concept includes glucocorticoids often in combination with methotrexate (MTX) and ciclosporine (CSA) for reduction of steroids. The interleukin 1 (IL-1) receptor antagonist anakinra, the IL-1beta antibody canakinumab or an IL­6 receptor blockage with tocilizumab (off label for AOSD) are used where there is no response to MTX or CSA. Anakinra or canakinumab can be used as a primary option in AOSD in cases of moderate and severe disease activity.

Associations of site-specific CD4+-T-cell hypomethylation within CD40-ligand promotor and enhancer regions with disease activity of women with systemic lupus erythematosus.

OBJECTIVE: To comprehensively assess associations of site-specific CD4+-T-cell hypomethylation of the CD40-Ligand gene (CD40L) with disease activity of women with systemic lupus erythematosus (SLE). METHODS: CpG-sites within the DNA of the promotor and two enhancer regions (n = 22) of CD40L were identified and numbered consecutively. The rate of methylated DNA in isolated CD4+-T-cells of women with SLE were quantified for each methylation site by MALDI-TOF. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Associations of site-specific methylation rates with the SLEDAI scores were assessed by linear regression modelling. P values were adjusted according to Bonferroni-Holm as indicated. RESULTS: 60 female SLE patients participated in the study (age 45.7 ± 11.1 years, disease duration 17.0 ± 8.3 years). Significant associations to the SLEDAI were noted for CpG22 hypomethylation of the promotor (ß = -40.1, p = 0.017, adjusted p = 0.027), trends were noted for CpG17 hypomethylation of the promotor (ß = -30.5, p = 0.032, adjusted p = 0.6), and for CpG11 hypermethylation of the second enhancer (ß = 15.0, p = 0.046, adjusted p = 0.8). CONCLUSION: Site-specific hypomethylation of the CD40L promotor in CD4+-T-cells show associations with disease activity in female SLE patients.

Methyl donor micronutrients, CD40-ligand methylation and disease activity in systemic lupus erythematosus: A cross-sectional association study.

OBJECTIVE: Hypomethylation of CD40-ligand (CD40L) in T-cells is associated with increased disease activity in systemic lupus erythematosus (SLE). We therefore investigated possible associations of dietary methyl donors and products with CD40L methylation status in SLE. METHODS: Food frequency questionnaires were employed to calculate methyl donor micronutrients in 61 female SLE patients (age 45.7 ± 12.0 years, disease duration 16.2 ± 8.4 years) and compared to methylation levels of previously identified key DNA methylation sites (CpG17 and CpG22) within CD40L promotor of T-cells using quantitative DNA methylation analysis on the EpiTYPER mass spectrometry platform. Disease activity was assessed by SLE Disease Activity Index (SLEDAI). Linear regression modelling was used. P values were adjusted according to Benjamini & Hochberg. RESULTS: Amongst the micronutrients assessed (g per day), methionine and cysteine were associated with methylation of CpG17 (ß = 5.0 (95%CI: 0.6-9.4), p = 0.04; and ß = 2.4 (0.6-4.1), p = 0.02, respectively). Methionine, choline, and cysteine were additionally associated with the mean methylation of the entire CD40L (ß = 9.5 (1.0-18.0), p = 0.04; ß = 1.6 (0.4-3.0), p = 0.04; and ß = 4.3 (0.9-7.7), p = 0.02, respectively). Associations of the SLEDAI with hypomethylation were confirmed for CpG17 (ß=-32.6 (-60.6 to -4.6), p = 0.04) and CpG22 (ß=-38.3 (-61.2 to -15.4), p = 0.004), but not the mean methylation of CD40L. Dietary products with the highest impact on methylation included meat, ice cream, white bread, and cooked potatoes. CONCLUSIONS: Dietary methyl donors may influence DNA methylation levels and thereby disease activity in SLE.

High diagnostic accuracy of histone H4-IgG autoantibodies in systemic lupus erythematosus.

Objective: Diagnosis of SLE relies on the detection of autoantibodies. We aimed to assess the diagnostic potential of histone H4 and H2A variant antibodies in SLE. Methods: IgG-autoantibodies to histones H4 (HIST1H4A), H2A type 2-A (HIST2H2AA3) and H2A type 2-C (HIST2H2AC) were measured along with a standard antibody (SA) set including SSA, SSB, Sm, U1-RNP and RPLP2 in a multiplex magnetic microsphere-based assay in 153 SLE patients [85% female, 41 (13.5) years] and 81 healthy controls [77% female, 43.3 (12.4) years]. Receiver operating characteristic analysis was performed to assess diagnostic performance of individual markers. Logistic regression analysis was performed on a random split of samples to determine the additional value of histone antibodies in comparison with SA by likelihood ratio test and determination of diagnostic accuracy in the remaining validation samples. Results: Microsphere-based assay showed good interclass correlation (mean 0.85, range 0.73-0.99) and diagnostic performance in receiver operating characteristic analysis (area under the curve (AUC) range 84.8-93.2) compared with routine assay for SA parameters. HIST1H4A-IgG was the marker with the best individual diagnostic performance for SLE vs healthy (AUC 0.97, sensitivity 95% at 90% specificity). HIST1H4A-IgG was an independent significant predictor for the diagnosis of SLE in multivariate modelling (P < 0.0001), and significantly improved prediction of SLE over SA parameters alone (residual deviance 45.9 vs 97.1, P = 4.3 × 10-11). Diagnostic accuracy in the training and validation samples was 89 and 86% for SA, and 95 and 89% with the addition of HIST1H4A-IgG. Conclusion: HIST1H4A-IgG antibodies improve diagnostic accuracy for SLE vs healthy.

Update on imaging in rheumatic diseases: cartilage.

In recent years, the role of articular cartilage for understanding pathogenesis as well as for clinical research has become increasingly important. Whereas previously cartilage could only be assessed invasively, various imaging procedures are available for its evaluation now. Although still widely used, conventional radiography bears significant limitations since it assesses cartilage indirectly by joint space width. Today, the cartilage thickness and structure can be reliably evaluated using ultrasound, although the molecular structure cannot be determined, yet. Besides ultrasound, MRI offers the possibility to image morphological changes with a very high resolution. In addition, the quality and composition of joint cartilage can already be measured due to a constant technical improvement and new MRI sequences such as dGEMRIC even in small joints (e.g. MCP or MTP joints). Despite the advantages of contrast agents for the detection of inflammation, its use is reevaluated today. Regarding that contrast agent-free imaging techniques for the assessment of joint cartilage are developed with great effort to depict its quality and changes over time. These novel MRI methods such as T2/T2*- and T1ρ-mapping, gagCEST, and sodium imaging provide promising quantitative imaging biomarkers that can detect early cartilage changes before morphological alterations occur. Hence, US and MRI will likely be of paramount importance in future clinical trials and clinical assessment of inflammatory and degenerative joint diseases not only for understanding pathogenesis but also for using its possible value in daily practice.

Validation and evaluation of the German version of the Systemic Lupus Activity Questionnaire (SLAQ).

OBJECTIVES: Disease activity accounts for damage, overall mortality and co-morbidities in SLE and should frequently be assessed to adapt therapeutic patient management. The Systemic Lupus Activity Questionnaire (SLAQ) is a patient-reported instrument for the assessment of disease activity derived from the Systemic Lupus Erythematosus Activity Measure (SLAM) and was originally developed in English. Our aim was to validate the SLAQ in German and evaluate its use in a large cohort. METHODS: We translated and adapted the SLAQ questionnaire in German. It was applied to SLE outpatients at a tertiary centre (n=328) and compared to the SLAMnolab and other SLE outcome parameters. Internal consistency, criterion validity, inter-rater and test-retest reliability as well as construct validity were examined. Correlation, Cronbach's alpha, Mann-Whitney U-test or the Kruskal-Wallis one-way analysis of variance test were ascertained where appropriate. Levels of statistical significance were defined at 5% (p<0.05). All reported p-values are two-tailed. RESULTS: The German SLAQ showed a comparable strong correlation with the SLAMnolab (r=0.632, p<0.0001) as the original version of the SLAQ and presented a good to excellent internal consistency reliability (Cronbach's alpha=0.89). Accrued damage as well as low disease activity are factors possibly influencing the score. Amongst others, scores were higher in patients with more reported flares, lower self-reported overall health, lower functional status and higher daily doses of prednisolone. CONCLUSIONS: Our German version of the SLAQ shows a comparable validity as the original SLAQ and is a promising instrument to survey disease activity in clinical routine as well as in clinical and epidemiological studies. Possible interacting factors need to be considered when applying.

Inflammation and vascularisation markers of arthroscopically-guided finger joint synovial biospies reflect global disease activity in rheumatoid arthritis.

OBJECTIVES: To analyse whether synovial markers of the clinically dominant metacarpophalangeal (MCP) joint reflect global disease activity measures in rheumatoid arthritis (RA). METHODS: Arthroscopically-guided synovial biopsies from the dominant metacarpophalangeal (MCP) joint of 10 patients with RA (DAS28 >3.2) were stained for determination of the synovitis score, CD68, vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α). MRI and ultrasound were used to calculate the RAMRIS and US7 score respectively. Arthroscopy of the same joint was repeated in 6 patients after 6 months. RESULTS: The synovitis score significantly correlated to DAS28 (Spearman r=0.74), CRP (r=0.69), and US7 (r=0.66); sublining CD68 macrophages to CRP (r=0.6); HIF-1α to DAS28 (r=0.77), CRP (r=0.73); and VEGF to DAS28 (r=0.753) and RAMRIS (r=0.663). All patients showed a reduction of the DAS28 after 6 months (mean±SD: 5.2±1.5 vs. 2.75±1.1; p<0.05). There were three patients with a good EULAR response, and only these showed declining sublining CD68 macrophages in the control biopsy (χ2 test: LR 8.3, p=0.05). Two of the remaining patients with increasing CD68 sublining macrophages showed a deterioration of the RAMRIS. CONCLUSIONS: Some histological findings in arthroscopically-guided biopsies of the dominantly affected MCP joint reflect global disease activity measures and their changes in RA patients. Moreover, repeated MCP synovial biopsy may distinguish true responders from individuals with residual disease activity, who are not readily recognized by clinical means.

Advantages of a combined rheumatoid arthritis magnetic resonance imaging score (RAMRIS) for hand and feet: does the RAMRIS of the hand alone underestimate disease activity and progression?

BACKGROUND: To evaluate a combined rheumatoid arthritis magnetic resonance imaging score (RAMRIS) for hand and foot (HaF-score) in rheumatoid arthritis (RA). METHODS: Magnetic resonance imaging (MRI, 0.2 Tesla) of the dominant hand and foot of 26 ACPA positive RA patients before and 6 months after initiation of methotrexate was obtained. RAMRIS of the hand was complemented by corresponding scoring of the foot (MTP I-V; HaF-score). Disease Activity Score 28 (DAS28) and a tender and swollen joint count (JC) of the joints scored in MRI were recorded. Changes in these scores (Δ) were assessed. RESULTS: ΔHaF-score correlated significantly with ΔDAS28 (r = 0.820, 95%-CI 0.633-0.916). Correlations to ΔDAS28 were best for changes in the synovitis subscore (0.648) and bone marrow edema (0.703). Correlations to ΔDAS28 were significantly better for of the ΔHaF-score than ΔRAMRIS (0.499, 0.139-0.743, p = 0.0368).All patients with at least moderate response (EULAR criteria, n = 11) had continuing disease activity on MRI, including five cases with new erosions, three of them at the feet. Improvements of the hand JC or foot JC were seen in 16 and 15 cases, respectively. However, MRI of the hand or feet improved in only 10 and 9 cases, respectively. No patient fulfilled SDAI remission criteria. CONCLUSIONS: The HaF-score identifies patients with continuing disease activity despite clinical response that would have been missed by consideration of the traditional RAMRIS or the DAS28 alone. Response as opposed to remission may be an insufficient goal in RA as all patients showed continuing disease activity, especially at the feet.

Effect of Lifestyle Counselling via a Mobile Application on Disease Activity Control in Inflammatory Arthritis: A Single-Blinded, Randomized Controlled Study.

BACKGROUND: Mobile applications (apps) are a resource for information on lifestyle and nutrition which are associated to improved outcomes in inflammatory arthritis. OBJECTIVE: The aim of this study was to explore whether targeted lifestyle counselling via an app improves disease activity in arthritis patients. METHODS: Patients with rheumatoid arthritis (RA), spondyloarthritis (SpA), psoriatic arthritis (PsA) were randomized to 12 weeks of lifestyle counselling via an app (Mida, Midaia GmbH, Germany) pertaining to a healthy Mediterranean Diet, physical activity, and mental health. Disease activity was measured with specific instruments by a blinded physician and categorized (remission, low, moderate, high). Dietary adherence was assessed by the Mediterranean Diet Adherence Screener (MEDAS). Mixed effects logistic regression adjusted to baseline disease activity, age, and sex were calculated. RESULTS: Of 158 patients included (73% female, 53.3 ± 11.7 years), 74 were in the active counselling group (ACG). All showed improvement in low disease activity or remission. ACG patients had an odds ratio (OR) of 2.8 (95%-CI 1.1-7.2, p = 0.035), while OR in the control group was not significant OR = 2.1 (0.9-5.0, p = 0.097). The control group was less likely to reach a MEDAS >= 4 (OR = 0.16 (0.03-0.77), p = 0.02), while this was not seen in the ACG (OR = 0.54 (0.06-4.63), p = 0.6). Patients in the ACG showed a tendency towards improved adhesion to a Mediterranean Diet (MEDAS) (ß = 0.35 (-0.05-0.74), p = 0.086). This tendency was not observed in the control group (ß = 0.09 (-0.29-0.46), p = 0.64). CONCLUSIONS: Individualized lifestyle and dietary counselling via app may help to improve disease control in inflammatory arthritis patients.

Human casein alpha s1 (CSN1S1) skews in vitro differentiation of monocytes towards macrophages.

BACKGROUND: The milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. In the present study, we investigated the effect of CSN1S1 on the differentiation of monocytes. METHODS: Primary human monocytes were stimulated with recombinant CSN1S1 and compared to cells stimulated with GM-CSF/IL-4 or M-CSF/IFNγ. Morphological changes were assessed by microscopy and quantification of surface markers of differentiation by FACS analysis. Phagocytic activity of CSN1S1 stimulated cells was measured by quantification of zymosan labeled particle uptake. The role of mitogen activated protein kinases for CSN1S1-induced differentiation of monocytes and proinflammatory cytokine expression was assessed by supplementation of specific inhibitors. RESULTS: CSN1S1 at a concentration of 10 µg/ml resulted in morphological changes (irregular shape, pseudopodia) and aggregation of cells, comparable to changes observed in M-CSF/IFNγ differentiated macrophages. Surface marker expression was altered after 24 h with an upregulation of CD14 (mean 2.5 fold) and CD64 (1.9 fold) in CSN1S1 stimulated cells. CSN1S1 treated cells showed a characteristic surface marker pattern for macrophages after 120 h of incubation (CD14high, CD64high, CD83low, CD1alow) comparable to changes observed in M-CSF/IFNγ treated monocytes. Furthermore, phagocytic activity was increased 1.4 and 1.9 fold following stimulation with 10 µg/ml CSN1S1 after 24 and 48 h, respectively. Early GM-CSF, but not GM-CSF/IL-4 induced differentiation of monocytes towards dendritic cells (DC) was inhibited by addition of CSN1S1. Finally, CSN1S1 induced upregulation of CD14 was impeded by inhibition of ERK1/2, while inhibition of the mitogen activated protein kinases JNK and p38 did not influence cellular differentiation. However, JNK and p38 inhibitors impeded CSN1S1 induced secretion of the proinflammatory cytokines IL-1b or IL-6. CONCLUSIONS: CSN1S1 skews in vitro differentiation of monocytes towards a macrophage-like phenotype. Data is accumulating that functions of CSN1S1 are beyond nutritional properties and include immunomodulatory effects.