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Sixteen patients with active rheumatoid arthritis, despite conventional synthetic disease-modifying antirheumatic drugs, started additional tofacitinib treatment. Blood samples were drawn at baseline and at 3-month follow-up visits, and plasma levels of 92 inflammation-related proteins were measured with proximity extension assay. Levels of 21 proteins changed substantially (>20%) and statistically significantly upon tofacitinib treatment, and the baseline levels of interleukin-6, C-C motif chemokine 11 and Delta and Notch-like epidermal growth factor-related receptor associated with the treatment response. The data on the effects of tofacitinib on soluble proteins in patients with rheumatoid arthritis (RA) is currently very limited. We analyzed how tofacitinib treatment and thus inhibition of the Janus kinase-signal transducer and activation of transcription pathway affects the in vivo levels of inflammation-related plasma proteins in RA patients. In this study, 16 patients with active RA [28-joint disease activity score (DAS28) >3.2] despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) started tofacitinib treatment 5 mg twice daily. Levels of 92 inflammation-related plasma proteins were determined by proximity extension assay at baseline and at 3 months. Tofacitinib treatment for 3 months, in csDMARD background, decreased the mean DAS28 from 4.4 to 2.6 (P < 0.001). Marked (>20%) and statistically significant (P < 0.05) changes were found in the levels of 21 proteins, 18 of which decreased and 3 increased. Of these proteins, 17 are directly involved in inflammatory responses or in the cellular response to cytokines. The highest (>50%) decrease was observed for interleukin-6 (IL-6), C-X-C motif chemokine ligand 1, matrix metalloproteinase-1, and AXIN1. Higher baseline levels of IL-6 and lower levels of C-C motif chemokine 11 and Delta and Notch-like epidermal growth factor-related receptors were associated with DAS28 improvement. Our results indicate that tofacitinib downregulates several proinflammatory plasma proteins that may contribute to the clinical efficacy of tofacitinib. In addition, soluble biomarkers may predict the treatment response to tofacitinib.

Objectives: Severe acute pancreatitis (SAP) has high morbidity and mortality but there are no widely accepted predictive biomarkers in clinical use. Matrix metalloproteinases (MMPs) are active in tissue destruction and inflammatory responses. We studied whether serum levels of activated MMP-8 (aMMP8), MMP-9 and their regulators tissue inhibitor of matrix metalloproteinases (TIMP)-1, myeloperoxidase (MPO) and human neutrophil elastase (HNE) could predict the development of SAP. Methods: The study comprised 214 AP patients (revised Atlanta classification: 142 mild, MAP; 54 moderately severe, MSAP; 18 SAP) referred to Helsinki University Hospital. A venous blood sample was taken within 72 h from the onset of symptoms. Serum levels of aMMP-8 were determined using immunofluorometric assay, and those of MMP-9, TIMP-1, MPO and HNE using enzyme-linked immunosorbent assay. AP groups were compared using Jonckheere-Terpstra test and predictive value for SAP was analyzed using receiver operating characteristics (ROC) analysis. Results: Serum aMMP-8 levels were higher in SAP (median 657 ng/ml, interquartile range 542-738 ng/ ml) compared to MSAP (358 ng/ml, 175-564 ng/ml; p < 0.001) and MAP (231 ng/ml, 128-507 ng/ml; p < 0.001). Similar trend was seen with TIMP-1 and MPO. In ROC analysis aMMP-8, MPO and TIMP-1 emerged as potential markers for the development of SAP (areas under ROC curves 0.83, 0.71 and 0.69, respectively). Conclusions: Serum aMMP-8 measured early in the course of AP (within 72 h of symptom onset) predicted the development of SAP. (c) 2021 The Authors. Published by Elsevier B.V. on behalf of IAP and EPC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Objectives Clinical practice lacks biomarkers to predict the severity of acute pancreatitis (AP). We studied if intracellular signaling of circulating leukocytes could predict persistent organ dysfunction (OD) and secondary infections in AP. Methods A venous blood sample was taken from 174 patients with AP 72 hours or less from onset of symptoms and 31 healthy controls. Phosphorylation levels (p) of appropriately stimulated signal transducer and activator of transcription 1 (STAT1), STAT6, nuclear factor-kappa B (NF-kappa B), Akt, and nonstimulated STAT3 in monocytes, neutrophils, and lymphocytes was measured using phosphospecific flow cytometry. Results The patients showed higher pSTAT3 and lower pSTAT1, pSTAT6, pNF-kappa B, and pAkt than healthy controls. pSTAT3 in all leukocyte subtypes studied increased, and pSTAT1 in monocytes and T cells decreased in an AP severity-wise manner. In patients without OD at sampling, high pSTAT3 in monocytes and T lymphocytes were associated with development of persistent OD. In patients with OD, low interleukin-4-stimulated pSTAT6 in monocytes and neutrophils and Escherichia coli-stimulated pNF-kappa B in neutrophils predicted OD persistence. High pSTAT3 in monocytes, CD8(+) T cells, and neutrophils; low pSTAT1 in monocytes and T cells; and low pNF-kappa B in lymphocytes predicted secondary infections. Conclusions Leukocyte STAT3, STAT1, STAT6, and NF-kappa Beta phosphorylations are potential predictors of AP severity.

Background/objectives: Inflammation is related to the development and progression of pancreatic cancer (PC). Locally, anti-inflammatory macrophages (M2), and systemically, high levels of certain inflammation-modulating cytokines associate with poor prognosis in PC. The detailed effects of systemic inflammation on circulating monocytes and macrophage polarisation remain unknown. We aimed to find out how intracellular signalling of peripheral blood monocytes is affected by the systemic inflammatory state in PC patients and how it affects their differentiation into macrophages. Methods: Monocytes were isolated from 50 consenting PC patients and 20 healthy controls (HC). The phosphorylation status of the signalling molecules was assessed by flow cytometry both from unstimulated and appropriately stimulated monocytes. Monocytes derived from HC and PC patients were cocultured with cancer cells (MIA PaCa-2 and HPAF-II) in media supplemented with autologous serum, and the CD marker expression of the obtained macrophages was assessed by flow cytometry. Results: Phosphorylation levels of unstimulated STAT2, STAT3 and STAT6 were higher (p

Background: There is a wide variety of disease severity in patients with complicated intraabdominal infection (cIAI). The prognostic role of intraabdominal view (IAV) was recently studied, and an IAV score was introduced. The aim of this study was to analyze the associations between the preoperative levels of eight relevant circulating cytokines and IAV components, the IAV score, as well as outcome. Materials and methods: This was a single-center prospective study. The study cohort consisted of operatively managed adult patients with a cIAI. Preoperative plasma levels of eight cytokines were determined. The operating surgeon filled a form describing IAV. Outcomes analyzed were 30-day mortality and the development of organ dysfunctions requiring intensive care unit admission. Results: A total of 131 patients with cIAI were analyzed, 30-day mortality was 9.9% (n = 13), and 28 (21.4%) patients had postoperative organ dysfunctions. All components of IAV, the IAV score, and outcomes were associated with various cytokine levels. Interleukin-8 was the most competent marker associating with all the variables assessed in this study: diffuse peritonitis (P

Intracellular signalling in blood leucocytes shows multiple aberrations in acute pancreatitis (AP) complicated by organ dysfunction (OD). We studied whether the aberrations associate with severity of AP and occur in sepsis complicated by OD. The study comprises 14 sepsis patients (11 with shock), 18 AP patients (nine mild; six moderately severe; three severe) and 28 healthy volunteers. Within 48 h after admission to hospital, phosphorylation of nuclear factor-ĸB (NF-ĸB), signal transducers and activators of transcription (STATs) 1,3, and extracellular signal-regulated kinases 1/2 were measured from stimulated or non-stimulated leucocytes using phosphospecific whole blood flow cytometry. In sepsis, as compared with healthy subjects, phosphorylated NF-ĸB levels of monocytes promoted by bacterial lipopolysaccharides, tumour necrosis factor or Escherichia coli cells were lower (P < 0.001 for all), pSTAT1 levels of monocytes promoted by IL-6 were lower (P < 0.05 for all), and STAT3 was constitutively phosphorylated in monocytes, neutrophils and lymphocytes (P < 0.001 for all). In AP, severity was associated with proportions of pSTAT1-positive monocytes and lymphocytes promoted by IL-6 (P < 0.01 for both), constitutive STAT3 phosphorylation in neutrophils (P < 0.05), but not with any of the pNF-ĸB levels. Monocyte pSTAT3 fluorescence intensity, promoted by IL-6, was lower in sepsis and AP patients with OD than in AP patients without OD (P < 0.001). Collectively, signalling aberrations in sepsis with OD mimic those described previously in AP with OD. Possibility that aberrations in STAT1 and STAT3 pathways provide novel markers predicting evolution of OD warrants studies including patients presenting without OD but developing it during follow-up.

Background:Cytokines are important mediators of inflammation and tissue destruction in rheumatoid arthritis (RA) 1. Several cytokines involved in RA pathogenesis act through Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway 2. The effects of JAK-inhibitor tofacitinib on cytokine signaling in vitro are well established, while in vivo evidence in patients remains scarce.Objectives:To investigate in vivo in rheumatoid arthritis patients i) which JAK-STAT pathways are inhibited by tofacitinib and ii) if baseline signaling profile is associated with the treatment response.Methods:Sixteen patients with active RA, despite treatment with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), received tofacitinib 5 mg twice daily for three months. Levels of basal and cytokine-induced phosphorylated STATs and total STAT1 and STAT3 in peripheral blood monocytes, T cells and B cells were measured by flow cytometry. mRNA expression of JAKs, STATs and suppressors of cytokine signaling (SOCS) were measured from peripheral blood mononuclear cells (PBMCs) by quantitative PCR. Association of baseline signaling profile with treatment response (the change from baseline in disease activity score (DAS28)) was studied by calculating correlation coefficients.Results:Treatment with tofacitinib and csDMARDs decreased median DAS28 from 4.4 to 2.6 (p < 0.001). Tofacitinib significantly decreased cytokine-induced phosphorylation of all JAK-STAT pathways studied. Basal STAT1, STAT3, STAT4 and STAT5 phosphorylation in monocytes and/or T cells was downregulated by tofacitinib. No changes were observed in STAT1 and STAT3 protein levels, while gene expression of STAT3, STAT4, STAT5A, JAK1, JAK3 and all studied SOCSs was significantly suppressed. Baseline STAT phosphorylation levels in T cells and monocytes and SOCS3 expression in PBMCs correlated with treatment response.Conclusion:Tofacitinib suppresses multiple JAK-STAT pathways in RA patients in vivo. Baseline JAK-STAT signaling profile may be applicable as a prognostic marker for treatment response to tofacitinib.References:[1]McInnes, I. B., Buckley, C. D. & Isaacs, J. D. Cytokines in rheumatoid arthritis-shaping the immunological landscape. Nature Reviews Rheumatology vol. 12 63–68 (2016).[2]Schwartz, D. M., Bonelli, M., Gadina, M. & O’Shea, J. J. Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases. Nat. Rev. Rheumatol.12, 25–36 (2016).Acknowledgements:This study was supported by Pfizer Inc.Disclosure of Interests:Maaria Palmroth Consultant of: Pfizer and from 1/21 a part-time employee of MedEngine and consultant for Pfizer, Krista Kuuliala Grant/research support from: Pfizer, Ritva Peltomaa Speakers bureau: Boehringer Ingelmheim, Pfizer, Sanofi, Paid instructor for: Boehringer Ingelheim, Eli Lilly and Company, Janssen, Abbvie, UCB Pharma, Anniina Virtanen: None declared, Antti Kuuliala: None declared, Antti Kurttila: None declared, Anna Kinnunen: None declared, Marjatta Leirisalo-Repo: None declared, Olli Silvennoinen Speakers bureau: Pfizer, AbbVie, Pia Isomäki Speakers bureau: Abbvie, Eli Lilly and Company, Pfizer, Roche, Paid instructor for: Abbvie, Eli Lilly and Company, Pfizer, Roche, Grant/research support from: Pfizer

Background: We found recently that baseline signal transducer and activator of transcription 3 phosphorylation in peripheral blood CD4(+) T cells of patients with early rheumatoid arthritis (RA) is associated with treatment response to synthetic disease-modifying antirheumatic drugs (DMARDs). This prompted us to study the baseline phosphorylation profiles of Janus kinases (JAKs) in blood leukocytes with respect to treatment response in early RA. Methods: Thirty-five DMARD-naive patients with early RA provided blood samples for whole blood flow cytometric determination of phosphorylation of JAKs in CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes. Treatment response was determined after 1 year of treatment with synthetic DMARDs, with remission defined as absence of tender and swollen joints and normal erythrocyte sedimentation rate. Exact logistic regression was used to investigate the association of baseline variables with treatment response. Ninety-five percent CIs of means were estimated by bias-corrected bootstrapping. Results: High JAK3 phosphorylation in CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes and low JAK2 phosphorylation in CD14(+) monocytes were significantly associated with remission following treatment with synthetic DMARDs. Conclusions: Baseline JAK phosphorylation profile in peripheral blood leukocytes may provide a means to predict treatment response achieved by synthetic DMARDs among patients with early RA.

Objectives: We aimed to study if interleukin (IL) 8 and hepatocyte growth factor (HGF) predict development of severe acute pancreatitis (SAP) among patients without organ dysfunction (OD) at presentation, and if they discriminate transient OD from persistent OD among patients presenting with OD. Methods: From prospectively collected cohort of 176 AP patients and 32 healthy controls, plasma levels of IL-8 and HGF were determined within 5 days after symptom onset using an enzyme-linked immunosorbent assay. Results: AP was severe in 23 patients, of whom 10 did not have clinical signs of OD at presentation. IL-8 and HGF levels increased along with the severity of AP (P

Objective To find novel predictors of treatment response to disease-modifying antirheumatic drugs (DMARDs), we studied activation of STAT (signal transducers and activators of transcription) 6 and 1 in circulating leukocytes of patients with rheumatoid arthritis (RA). Methods 19 patients with untreated recent-onset RA, 16 patients with chronic RA irresponsive to synthetic DMARDs and 37 healthy volunteers provided blood samples for whole blood flow cytometric determination of intracellular STAT6 and STAT1 phosphorylation, expressed as relative fluorescence units, in response to IL-4 and IFN-γ, respectively. Phosphorylation was restudied and treatment response (according to European League Against Rheumatism) determined after 1-year treatment with synthetic DMARDs in recent-onset RA and with biological DMARD in synthetic DMARD-irresponsive RA. Estimation-based exact logistic regression was used to investigate relation of baseline variables to treatment response. 95% confidence intervals of means were estimated by bias-corrected bootstrapping and the significance between baseline and follow-up values was calculated by permutation test. Results At baseline, levels of phosphorylated STAT6 (pSTAT6) induced by IL-4 in monocytes were higher in those who achieved good treatment response to synthetic DMARDs than in those who did not among patients with untreated RA (OR 2.74, 95% CI 1.05 to 9.47), and IFN-γ -stimulated lymphocyte pSTAT1 levels were higher in those who achieved good treatment response to a biological drug than in those who did not among patients with chronic RA (OR 3.91, 95% CI 1.12 to 20.68). During follow-up, in recent-onset RA patients with good treatment response to synthetic DMARDS, the lymphocyte pSTAT6 levels decreased (p = 0.011), and, consequently, the ratio of pSTAT1/pSTAT6 in lymphocytes increased (p = 0.042). Conclusion Cytokine-stimulated STAT6 and STAT1 phosphorylation in circulating leukocytes was associated with treatment response to DMARDs in this pilot study. The result, if confirmed in larger studies, may aid in developing personalized medicine in RA.

It has been proposed that the Akt kinase pathway provides a regulatory mechanism to limit the inflammatory response. We examined the activation of Akt upon LPS challenge in monocytes of patients with rheumatoid arthritis (RA), and correlated it with disease activity. Twelve subjects with recent-onset, DMARD-naive RA, thirteen patients with chronic, DMARD therapy -nonresponding RA and 27 healthy volunteers provided whole blood samples for phosphospecific flow cytometric measurement of unstimulated and LPS-stimulated Akt phosphorylation at serine 473 in monocytes, determined in relative fluorescence units (RFU). Activation capability, i.e. responsiveness of monocytes, was determined as the difference between stimulated and unstimulated samples, and compared between groups using Mann-Whitney test. CRP and ESR, swollen and tender joint counts, patients’ global assessment of disease activity, DAS28 score, and plasma IL-6 determined by ELISA were correlated with Akt activation using Spearman method. Median (interquartile range) Akt activation capability was significantly lower in DMARD-naive (379 RFU [285, 432],], p=0.016) and even lower in DMARD-nonresponding RA (258 RFU [213, 338], p

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.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.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.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.

The aim of this study was to determine whether the +896 A→G substitution of the Toll-like receptor 4 ( TLR4 ) gene, causing the Asp299→Gly change in the extracellular domain of TLR4, influences treatment response in recent-onset rheumatoid arthritis. 169 patients with rheumatoid arthritis were genotyped from the Finnish Rheumatoid Arthritis Combination Therapy trial, in which they were treated either with only one disease-modifying antirheumatic drug (DMARD) with or without prednisolone (single group), or with three DMARDs and prednisolone (combination group). Patients homozygotic for the wild-type +896A allele were compared with those having the polymorphic G allele in terms of early clinical response (at 6 months) by the 28-joint Disease Activity Score (DAS28). 1 of 20 (5%; (95% (confidence interval (CI) 1 to 5)) patients of the single group with TLR4 +896AG or GG and 29 of 67 (43%; (95% CI 31 to 56)) patients with AA were in remission (p = 0.001). DAS28 of the single group with TLR4 +896AG or GG was higher than with AA (p = 0.019). In the combination group, remission rates and DAS28 values were comparable between the genotypes. The polymorphic TLR4 +896G allele may impair treatment response to single DMARD treatment in recent-onset rheumatoid arthritis.

Peripheral blood mononuclear cells of Crohn's disease (CD) patients with the common 1007fs mutation of the caspase recruitment domain-containing 15/nucleotide-binding oligomerization domain-containing 2 (CARD15/NOD2) gene show impaired nuclear factor kappa B (NF-κB) activation in response to muramyl dipeptide (MDP), as determined by Western blotting. We applied phospho-specific flow cytometry to examine NF-κB and p38 activation in whole blood monocytes of 16 CD patients with or without the 1007fs and previously described rare mutations of the CARD15 gene, and healthy reference subjects. Aliquots of whole blood were supplemented with MDP (0-1000 ng/mL), incubated for 10-40 min and processed for flow cytometry. Bacterial lipopolysaccharide (LPS) was used as a positive control agonist. We found that NF-κB and p38 phosphorylation induced by MDP was not detectable in monocytes of patients homozygous for the CARD15 1007fs mutation, while those induced by LPS were normal. We also determined MDP-induced NF-κB phosphorylation levels in nuclear extracts of mononuclear cells separated from blood using enzyme-linked immunosorbent assay (ELISA), and observed that the levels decreased in a 1007fs mutation-dose dependent manner. We conclude that phospho-specific whole blood flow cytometry provides a means to study phosphorylation of NF-κB and p38 in clinical samples and can be applied to screening of CD patients homozygous for the CARD15 1007fs mutation.

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.

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

Background and objectives Clonally expanded CD8 + lymphocytes are known to exist in patients with rheumatoid arthritis (RA). Interestingly, in large granular lymphocyte (LGL) leukaemia (a haematological malignancy with indolent disease course), CD8 + lymphocytes expand and the expanded clones harbour somatic mutations. LGL leukaemia patients with STAT3 mutated CD8 + lymphocytes have significantly increased risk of developing autoimmune manifestations, RA being a common autoimmune co-morbidity. As somatic mutations in CD8 + lymphocytes associate with autoimmunity in LGL leukaemia, we wanted to examine if similar phenomenon exists in newly diagnosed RA patients without known LGL lymphoproliferation. Materials and methods Lymphocyte clonality was analysed by flow cytometry using vbβ -staining kit (Beckman Coulter) and the major clones were confirmed by TCR deep sequencing. Deep sequencing of 986 selected immune-related genes was performed from CD8 + T cells using CD4 + lymphocytes as germline control. The coverage was 200–6000x enabling reliable calling of low frequency mutations. Candidate mutations were validated using targeted deep amplicon sequencing. Results 43% (n = 31) of the newly diagnosed RA patients in our patient cohort (n = 68) had CD8 + lymphocyte expansions exceeding 10% of the total CD8 + T cells. The largest clones were subjected to targeted deep sequencing. We identified somatic gene variants in five (n = 5) patients and in one healthy control. Most of these variants were non-synonymous coding single nucleotide changes in genes important for immune functions ( C5 , PADI4 , IRF1 , SLAMF6 , BST1 and CLEC10A ), or proliferation ( PTPRO , PADI4, PROM1 , CDK12 and CLIP2 ). Sequencing of FACS sorted CD8 + lymphocytes confirmed that the expanded clone harboured the identified mutations. Conclusions In RA patients, somatic mutations accumulate in the CD8 + T cells during clonal expansion. Related findings have recently been reported in the myeloid lineage, where somatic variants are found to accumulate even in healthy subjects in a process called clonal haematopoiesis. Importantly, we focused on CD8 + lymphocytes that have undergone thymic education and thus we report acquired somatic variants in mature cytotoxic lymphocytes in a non-malignant set-up. Although more research is needed to address the possible pathogenicity of the identified mutations, these findings provide an interesting link between autoimmunity and cancer.

Background/objectives Circulating polymorphonuclear leukocytes (PMNLs) may contribute to development of organ dysfunction in acute pancreatitis (AP). We outlined aberrations in PMNL signaling profiles in patients with AP complicated by organ dysfunction and immune suppression. Methods Study comprised 13 patients treated at intensive care unit due to severe AP complicated by vital organ dysfunction. Mean proportion (SEM) of HLA-DR-positive monocytes was 55.0% (4.1%). 13 healthy volunteers served as reference subjects. Phosphorylation of PMNL NFκB, p38, ERK1/2 and STAT3, -5 and -6 was determined using whole blood flow cytometry. Transmigration of PMNLs was studied using endothelial EA-HY cell monolayer. Results Proportions of NFκB phosphorylation-positive PMNLs were lower in the patients' than in reference subjects' blood samples supplemented with tumor necrosis factor. p38 phosphorylation was normal while ERK1/2 phosphorylation was decreased. STAT3 was constitutively activated in five patients. Proportion of patients' pSTAT6-positive cells was normal while fluorescence intensity was decreased. STAT5 phosphorylation was normal. Transmigration of patients' PMNLs was increased. Conclusions In patients with AP complicated by organ dysfunction proportion of pNFκB-positive PMNLs is decreased. This impairs patients' defense mechanisms against infection. Despite immune suppression, PMNL transmigration was increased and p38 phosphorylation capacity was not depressed, which may contribute to end organ inflammation and dysfunction.

OBJECTIVES: To study innate immune responsiveness of HLA-B27 positive subjects recovered from Yersinia-triggered reactive arthritis (B27 + ReA+). METHODS: Whole blood samples from 15 B27 + ReA+, 15 B27 + ReA- and 15 B27 - ReA- subjects were heparinized, aliquoted and (i) kept at 0 degree C to preserve constitutive cell surface marker status, or (ii) cultured with or without bacterial lipopolysaccharide (LPS) supplement, in adherent and non-adherent conditions at 37 degrees C for 4 h. Neutrophil surface expression of CD11b, CD14 and CD16 was quantified flow cytometrically, and compared between the subject groups using Jonckheere-Terpstra test. RESULTS: The B27 + ReA+ group showed significantly higher CD11b levels than the B27 - ReA- group on non-adherent neutrophils cultured with LPS as 100 pg/ml (P = 0.027), 10 ng/ml (P = 0.048) or 1 microg/ml (P = 0.024), or on adherent neutrophils without LPS supplement (P = 0.040). CD14 and CD16 expression on cultured neutrophils and constitutive expression of all three markers were comparable between the groups. CONCLUSIONS: Enhanced neutrophil reactivity observed may exacerbate innate immune inflammation in HLA-B27 positive ReA patients.

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. Public Library of Science open access

Introduction Severe acute pancreatitis is associated with systemic inflammation, compensatory immune suppression, secondary infections, vital organ dysfunction, and death. Our study purpose was to delineate signaling profiles of circulating lymphocytes in acute pancreatitis complicated by organ dysfunction. Methods Sixteen patients with acute pancreatitis, dysfunction of vital organ(s), and immune suppression (proportion of HLA-DR Human Leukocyte Antigen - DR - positive monocytes < 80%) participated. Healthy volunteers served as reference subjects. Using phospho-specific whole blood flow cytometry we studied lymphocyte phosphorylation of nuclear factor-κB (NFκB), mitogen-activated protein kinases p38 and extracellular signal-regulated kinases (ERK)1/2, and signal transducers and activators of transcription (STATs) 1, 3, and 6. Statistical comparisons were performed with the Wilcoxon-Mann-Whitney test. Results In blood samples supplemented with tumor necrosis factor, E. coli or S. aureus, phosphorylation levels of NFκB were lower and levels of p38 were higher in patients with acute pancreatitis than healthy subjects. Low NFκB activation involved CD3+CD4+ and CD3+CD8+ lymphocytes. ERK1/2 phosphorylation induced by co-stimulation with phorbol 12-myristate 13-acetate and calcium ionophore A23187 was depressed in patients. STAT3 was constitutively activated in patients' CD3+CD4+ and CD3+CD8+ lymphocytes. Also, IL-6-induced STAT1 phosphorylation was impaired while IL-4-induced STAT6 phosphorylation was enhanced. Conclusions Lymphocytes of patients with acute pancreatitis, organ dysfunction and immune suppression show impaired NFκB activation, which increases infection risk and enhanced p38 activation, which sustains inflammation. Secondly, they indicate constitutive STAT3 activation, which may favor Th17 lineage of CD4+ lymphocyte differentiation. Thirdly, they reveal impaired STAT1 activation and enhanced STAT6 activation, denoting a shift from Th1 towards Th2 differentiation.

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., Accumulation of somatic mutations in lymphocytes is a feature of some cancers. Here the authors show that patients with recent onset of rheumatoid arthritis also accumulate mutations in their expanded CD8+ effector memory T cell pool independent of cancer association.