Antti Kuuliala

Low circulating soluble interleukin 2 receptor level predicts rapid response in patients with refractory rheumatoid arthritis treated with infliximab.

Background: Treatment with infliximab induces a rapid therapeutic response in most patients with active rheumatoid arthritis. Factors predicting good response are not well known. Objective: To study the predictive value of baseline level of soluble interleukin 2 receptor (sIL2R), a marker of lymphocyte activation, on the treatment response. Methods: 24 patients with active rheumatoid arthritis received intravenous infusions of infliximab at study entry, at two weeks, at six weeks, and at eight week intervals thereafter. Outcome was evaluated at six weeks and 22 weeks. Clinical assessment and standard laboratory tests were made and the DAS28 disease activity score was calculated. Serum sIL2R level at entry was measured by automated immunoassay analyser (Immulite®). The mean change in DAS28 score from entry to six weeks and 22 weeks was calculated and related to sIL2R level using baseline adjusted robust regression analysis. Results: Baseline level of serum sIL2R (mean (SD), 621 (325) U/ml) did not correlate with baseline DAS28 score (r = 0.24 (95% confidence interval, −0.18 to 0.58)). At six weeks DAS28 scores improved, with a mean change of −2.53 (−3.08 to −1.98) (p<0.001). This change was predicted by low baseline sIL2R level (regression coefficient per 100 U/ml: 0.205 (0.003 to 0.407) (p = 0.047)). At 22 weeks the DAS28 scores improved, with a mean change of −2.26 (−2.75 to −1.77) (p<0.001). The change was not predicted by baseline sIL2R level. Conclusions: Low baseline sIL2R level may predict a rapid clinical response in patients with refractory rheumatoid arthritis treated with infliximab.

Cord blood monocytes, neutrophils and lymphocytes from preterm and full-term neonates show multiple aberrations in signalling profiles measured using phospho-specific whole-blood flow cytometry.

Immaturity of the immune system renders newborns susceptible to infections. We searched for aberrations in leucocyte signalling profiles, using phospho‐specific whole‐blood flow cytometry, in cord blood of nine preterm (two born before 32nd gestational week) and nine full‐term infants, born by caesarean section. Thirteen adults served as reference subjects. Monocyte NF‐κB phosphorylation following tumour necrosis factor (TNF) or bacterial stimulation was higher in preterm neonates than in full‐term neonates or adults, p38 phosphorylation following bacterial stimulation was higher in both preterm and full‐term neonates than in adults, while STAT1 phosphorylation by IFN‐γ or IL‐6, STAT3 phosphorylation by IL‐6 and STAT5 phosphorylation by GM‐CSF were lower in both full‐term and preterm neonates than in adults. Neutrophil STAT1 and STAT3 phosphorylation following IFN‐γ stimulation and STAT5 phosphorylation following GM‐CSF stimulation were lower in newborn neonates than in adults. In both CD3+CD4+ and CD3+CD8+ lymphocytes, NF‐κB phosphorylation by TNF was higher and STAT5 phosphorylation by IL‐2 was lower in preterm and full‐term newborns than in adults. STAT6 phosphorylation by IL‐4 was comparable in monocytes and lymphocytes of newborns and adults. The results suggest that innate immune signalling pathways responding to inflammatory stimuli are strongly functional in leucocytes of preterm neonates, which may render these neonates susceptible to inappropriate tissue injury. In leucocytes of both preterm and full‐term newborns, responses needed against intracellular pathogens, and regulatory functions show immaturities, possibly contributing to worse control of infections.

Constitutive STAT3 Phosphorylation in Circulating CD4(+) T Lymphocytes Associates with Disease Activity and Treatment Response in Recent-Onset Rheumatoid Arthritis

The aim of the present study was to examine constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation in circulating leukocytes as a candidate biomarker in rheumatoid arthritis (RA). 25 patients with recent-onset, untreated RA provided samples for whole blood flow cytometric determination of intracellular STAT3 phosphorylation, expressed as relative fluorescence units. The occurrence of constitutive STAT3 phosphorylation was evaluated by determining proportion of STAT3-phosphorylated cells among different leukocyte subtypes. Plasma levels of interleukin (IL)-6, IL-17 and IL-21 were measured by immunoassay, radiographs of hands and feet were examined and disease activity score (DAS28) was determined. Biomarkers were restudied and treatment response (according to European League Against Rheumatism) was determined after 12 months of treatment with disease-modifying antirheumatic drugs. At baseline, constitutive phosphorylation of STAT3 occurred in CD4+ T cells of 14 (56%) patients, CD8+ T cells of 13 (52%) patients, in CD19+ B cells of 7 (28%) patients, and in CD14+ monocytes of 12 (48%) patients. STAT3 phosphorylation levels of CD4+ T cells associated with DAS28, and those of all leukocyte subtypes studied associated with erosive disease. The presence of constitutive STAT3 phosphorylation in CD4+ T lymphocytes, pSTAT3 fluorescence intensity of CD4+ and CD8+ T cells and C-reactive protein (CRP) levels at baseline associated with good treatment response. In conclusion, constitutive STAT3 phosphorylation in circulating CD4+ T cells is common in recent-onset untreated RA and associates with good treatment response in patients characterized by high disease activity and the presence of systemic inflammation.

Periodontitis in early and chronic rheumatoid arthritis: a prospective follow-up study in Finnish population

Objectives To investigate the association between rheumatoid arthritis (RA) and periodontitis with special emphasis on the role of antirheumatic drugs in periodontal health. Design Prospective follow-up study. Patients with early untreated RA and chronic active RA were examined at baseline and 16 months later. Controls were examined once. Settings and participants The study was conducted in Finland from September 2005 to May 2014 at the Helsinki University Hospital. Overall, 124 participants were recruited for dental and medical examinations: 53 were patients with early disease-modifying antirheumatic drug (DMARD) naїve RA (ERA), 28 were patients with chronic RA (CRA) with insufficient response to conventional DMARDs. After baseline examination, patients with ERA started treatment with synthetic DMARDs and patients with CRA with biological DMARDs. Controls were 43 age-matched, gender-matched and community-matched participants. Outcome measures Degree of periodontitis (defined according to the Center for Disease Control and Prevention and the American Academy of Periodontology). Prevalence of periodontal bacteria (analysed from plaque samples), clinical rheumatological status by Disease Activity Score, 28-joint count (DAS28), function by Health Assessment Questionnaire (HAQ) and treatment response by European League Against Rheumatism (EULAR) criteria. Results Moderate periodontitis was present in 67.3% of patients with ERA, 64.3% of patients with CRA and 39.5% of control participants (p=0.001). Further, patients with RA had significantly more periodontal findings compared with controls, recorded with common periodontal indexes. In the re-examination, patients with RA still showed poor periodontal health in spite of treatment with DMARDs after baseline examination. The prevalence of Porphyromonas gingivalis was higher in patients with ERA with periodontal probing depth ≥4 mm compared with patients with CRA and controls. Antirheumatic medication did not seem to affect the results. Conclusions Moderate periodontitis was more frequent in patients with RA than in controls. Patients with ERA and CRA exhibited poorer periodontal health parameters when compared with controls. There was no association between antirheumatic treatment and periodontal parameters.

Cellular and humoral markers of systemic inflammation in acute reactive arthritis and early rheumatoid arthritis

Objective: To compare systemic inflammation in reactive arthritis (ReA), rheumatoid arthritis (RA), and sepsis using novel markers of systemic inflammation, and to study whether they are helpful in distinguishing between ReA and RA. Methods: In 28 patients with acute ReA, 16 patients with early untreated RA, and 25 patients with blood culture‐positive sepsis, phagocyte CD11b expression was measured by flow cytometry, serum procalcitonin (PCT) levels by immunoluminometric assay, and soluble E‐selectin (sE‐selectin) levels by enzyme‐linked immunosorbent assay (ELISA). Results: Neutrophil and monocyte CD11b expression and serum levels of PCT and sE‐selectin were higher in patients with sepsis than patients with ReA or RA, or in healthy subjects (all p<0.01). They were comparable in healthy subjects, ReA, and RA. Conclusion: Patients with acute ReA and early RA have normal CD11b expression levels on phagocytes and normal PCT and sE‐selectin levels in serum. Elevated levels suggest possible sepsis.

Somatic mutations in clonally expanded cytotoxic T lymphocytes in patients with newly diagnosed rheumatoid arthritis

Somatic mutations contribute to tumorigenesis. Although these mutations occur in all proliferating cells, their accumulation under non-malignant conditions, such as in autoimmune disorders, has not been investigated. Here, we show that patients with newly diagnosed rheumatoid arthritis have expanded CD8+ T-cell clones; in 20% (5/25) of patients CD8+ T cells, but not CD4+ T cells, harbour somatic mutations. In healthy controls (n=20), only one mutation is identified in the CD8+ T-cell pool. Mutations exist exclusively in the expanded CD8+ effector-memory subset, persist during follow-up, and are predicted to change protein functions. Some of the mutated genes (SLAMF6, IRF1) have previously been associated with autoimmunity. RNA sequencing of mutation-harbouring cells shows signatures corresponding to cell proliferation. Our data provide evidence of accumulation of somatic mutations in expanded CD8+ T cells, which may have pathogenic significance for RA and other autoimmune diseases.

Signalling profiles of circulating leucocytes in patients recovered from reactive arthritis

Objectives: Reactive arthritis (ReA) is a sterile joint inflammation triggered by a remote infection and associated with human leucocyte antigen (HLA)-B27. Its pathogenesis is unknown, but abnormal response to microbial structures or endogenous inflammatory mediators may be involved. We studied responses in leucocyte signalling profiles in patients with previous ReA after a full recovery. Method: The study comprised 10 HLA-B27-positive healthy subjects with a history of Yersinia enterocolitica-triggered ReA (B27+ReA+) and 20 healthy reference subjects, of whom 10 carried HLA-B27 (B27+ReA–) and 10 did not (B27–ReA–). Phosphospecific fluorescent monoclonal antibodies and flow cytometry were used to determine activation of nuclear factor kappa B (NF-κB), signal transducers and activators of transcription (STATs) 1, 3, 5, and 6, and two mitogen-activated protein (MAP) kinases, p38 and extracellular signal-regulated kinase (ERK)1/2, in monocytes, lymphocytes, lymphocyte subsets, and neutrophils. B27+ReA+ and B27–ReA– whole-blood samples were incubated with Yersinia with or without infliximab to study the role of tumour necrosis factor (TNF) in lymphocyte subset activation. Samples of the three subject groups were studied using soluble bacterial or endogenous stimuli. Fluorescence levels were determined as relative fluorescence units (RFU) and the proportion of positively fluorescing cells. Results: The intracellular activation of circulating leucocytes in response to soluble stimuli was consistently comparable in B27+ReA+, B27+ReA–, and B27–ReA– subjects. Infliximab inhibited Yersinia-induced lymphocyte NF-κB phosphorylation similarly in B27+ReA+ and B27–ReA– groups. Conclusions: ReA susceptibility is not reflected in leucocyte signalling profiles elicited by phlogistic stimuli. However, the possibility remains that aberrations occur in response to combinations of stimuli, such as those associated with leucocyte adhesion.

Circulating soluble interleukin‐2 receptor level predicts remission in very early reactive arthritis

Objectives: To assess the predictive value of serum soluble interleukin‐2 receptor (sIL‐2R) levels in patients with acute reactive arthritis (ReA). Methods: The study includes 26 patients with acute ReA who had participated in a prospective population‐based cohort study of very early arthritis. The patients had had arthritis of at least one joint with a maximum duration of 3 months. They were assessed by a rheumatologist on presentation and 6 months later. Serum sIL‐2R levels on presentation were measured by the Immulite automated immunoassay analyser. Remission at 6 months, defined by the absence of swollen and tender joints, was related to the baseline sIL‐2R level using a permutation test with general scores. Bootstrap estimation was used to derive the 95% confidence interval (CI). Results: A total of 17 patients (65%) were in remission at 6 months and nine patients (35%) still had joint symptoms. In patients reaching remission within 6 months, the mean baseline sIL‐2R level, 891 U/mL (95% CI: 658 to 1123), was higher than in patients not reaching remission, 501 U/mL (95% CI: 436 to 566), p = 0.022. Conclusions: A high serum sIL‐2R level at baseline is a predictor of remission in patients with acute ReA.

Association of Matrix Metalloproteinases-7,-8 and-9 and TIMP-1 with Disease Severity in Acute Pancreatitis. A Cohort Study

Objectives Several biomarkers for early detection of severe acute pancreatitis (SAP) have been presented. Matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) are released early in inflammation. We aimed to assess levels of MMP-7, -8, -9 and TIMP-1 in acute pancreatitis (AP) and explore their ability to detect disease severity. Our second aim was to find an association between MMPs, TIMP and creatinine. Methods We collected plasma samples for MMP-7, -8, -9 and TIMP-1 analyses from 176 patients presenting within 96 h from onset of acute pancreatitis (AP) symptoms. We used samples from 32 control subjects as comparison. The revised Atlanta Classification was utilised to assess severity of disease. Receiver operating characteristic curve analysis and Spearman´s Rho-test were utilised for statistical calculations. Results Compared with controls, patients showed higher levels of all studied markers. MMP-8 was higher in moderately severe AP than in mild AP (p = 0.005) and MMP-8, -9 and TIMP-1 were higher in severe than in mild AP (p<0.001, p = 0.005 and p = 0.019). MMP-8 detected SAP with an AUC of 0.939 [95% CI 0.894–0.984], LR+ 9.03 [5.30–15.39]. MMP-8, -9 and TIMP-1 failed to discern moderately severe AP from SAP. MMP-7 was not different between patient groups. MMP-7 and TIMP-1 correlated weakly with creatinine (Rho = 0.221 and 0.243). MMP-8 might be a useful biomarker in early detection of SAP.

Inflammatory biomarkers in saliva and serum of patients with rheumatoid arthritis with respect to periodontal status

Abstract Objective: To study prospectively the association of salivary and serum matrix metalloproteinase (MMP)-8, tissue inhibitor of MMPs (TIMP)-1 and interleukin (IL)-6 with periodontal and systemic inflammation in rheumatoid arthritis (RA). We hypothesized that biomarker concentrations reflect inflammation. Methods: Fifty three early untreated RA (ERA) and 28 chronic RA (CRA) patients, underwent rheumatological and dental examinations at baseline and one year later after starting first conventional or biological disease modifying antirheumatic drug. We included 43 control subjects. Saliva and serum samples were analyzed for MMP-8, TIMP-1 and IL-6. Periodontal health was assessed by bleeding on probing (BOP), pocket depth (PD) and periodontal inflammatory burden index (PIBI); RA disease activity was assessed by disease activity score DAS28. Joint destruction was analyzed by the modified Sharp-van der Heijde (SHS) method. Results: Serum MMP-8 (p < .001; p < .001) and IL-6 (p < .001; p = .002) were significantly higher in CRA vs. other study groups during the study. Salivary MMP-8 (p = .010) and IL-6 (p = .010) were significantly higher in ERA vs. other study groups at baseline. Salivary MMP-8 was associated with periodontal parameters. Conclusion: Elevated serum concentrations of MMP-8 and IL-6 in CRA patients reflected chronic RA, while elevated salivary concentrations of MMP-8 levels in ERA patients reflected increased periodontal inflammation. Key messages Concentrations of inflammatory biomarkers in serum and saliva were different between patients with RA and healthy controls. Concentrations of MMP-8 and of IL-6 in serum were elevated in patients with chronic RA reflecting joint inflammation and the burden of established RA. Concentrations of MMP-8 in saliva was elevated already at the early stage of RA and the level of salivary MMP-8 was associated with poor periodontal health both in patients with early and in those with chronic RA.