Granulocyte Protein Research Articles

Fecal and Serum Granulocyte Protein Levels in Inflammatory Bowel Disease and Irritable Bowel Syndrome and Their Relation to Disease Activity.

Neutrophilic calprotectin (CP) and myeloperoxidase (MPO), neutrophil gelatinase-associated lipocalin (NGAL), and eosinophil-derived neurotoxin (EDN) are suggested proxy markers for gut inflammation. However, there are insufficient supporting data for MPO, NGAL, and EDN. In a cross-sectional investigation including adult patients, we studied the ability of CP, MPO, NGAL, and EDN, measured in fecal and serum samples, to differentiate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), and to predict disease activity. Fifty-nine patients had ulcerative colitis (UC), 38 had Crohn's disease, and 100 patients had IBS. The protein concentrations were higher in patients with IBD in the fecal samples ( P < 0.001) and the serum samples ( P < 0.01), and they correlated weakly (r s ≤0.38) between the sample sources. Fecal EDN was higher in patients with Crohn's disease compared with UC (1.79 vs 0.50 mg/kg, P = 0.016). The neutrophilic proteins were superior to EDN in the fecal samples for differentiating between patients with IBD and IBS. Fecal MPO (cutoff: 0.86 mg/kg) had the highest sensitivity (74.7%) and specificity (84.6%). Combining fecal CP and MPO increased the sensitivity to 82.3% (specificity: 73.6%). NGAL (cutoff: 196.9 μg/L) showed the best discriminating performance in serum (sensitivity: 62.9%; specificity: 68.0%). Serum NGAL (cutoff: 272.4 μg/L) predicted active disease in UC (Partial Mayo Score ≥2) with a sensitivity and specificity of 57.1% and 83.3%, respectively. Fecal MPO and serum NGAL are promising novel biomarkers, in addition to fecal CP, for differentiating between IBD and IBS. Serum NGAL may also predict disease activity in patients with UC.

Exploring CD26-/lo subpopulations of lymphocytes in asthma phenotype and severity: A novel CD4+ T cell subset expressing archetypical granulocyte proteins.

Asthma pathology may induce changes in naïve/memory lymphocyte proportions assessable through the evaluation of surface CD26 (dipeptidyl peptidase 4/DPP4) levels. Our aim was to investigate the association of asthma phenotype/severity with the relative frequency of CD26-/lo, CD26int and CD26hi subsets within different lymphocyte populations. The proportion of CD26-/lo, CD26int and CD26hi subsets within CD4+ effector T cells (Teff), total CD4- lymphocytes, γδ-T cells, NK cells and NKT cells was measured in peripheral blood samples from healthy (N = 30) and asthma (N = 119) donors with different phenotypes/severities by flow cytometry. We performed K-means clustering analysis and further characterised the CD4+CD26-/lo Teff cell subset by LC-MS/MS and immunofluorescence. Cluster analysis including clinical and flow cytometry data resulted in four groups, two of them with opposite inflammatory profiles (neutrophilic vs. eosinophilic). Neutrophilic asthma presented reduced CD4-CD26hi cells, which negatively correlated with systemic inflammation. Eosinophilic asthma displayed a general expansion of CD26-/lo subsets. Specifically, CD4+CD26-/lo Teff expansion was confirmed in asthma, especially in atopic patients. Proteomic characterisation of this subset with a TEM/TEMRA phenotype revealed upregulated levels of innate (e.g. MPO and RNASE2) and cytoskeleton/extracellular matrix (e.g. MMP9 and ACTN1) proteins. Immunofluorescence assays confirmed the presence of atypical proteins for CD4+ T cells, and an enrichment in 'flower-like' nuclei and MMP9/RNASE2 levels in CD4+CD26-/lo Teff compared to CD4+ T lymphocytes. There is an association between CD26 levels in different lymphocyte subsets and asthma phenotype/severity. CD4+CD26-/loTEMRA cells expressing innate proteins specific to eosinophils/neutrophils could be determinant in sustaining long-term inflammation in adult allergic asthma.

Serum calprotectin correlates more strongly with inflammation and disease activity in ACPA positive than ACPA negative rheumatoid arthritis.

The aim of the present study was to evaluate performance of serum and synovial fluid levels of the granulocyte protein calprotectin as inflammatory biomarker in rheumatoid arthritis (RA) patients with knee synovitis. 76 RA patients with ongoing knee synovitis were included. Data on disease activity score with 28 joints and their subcomponents and radiological destruction of the affected knee were collected. White blood cell count, C-reactive protein, anti-citrullinated peptide antibodies (ACPA) against cyclic citrullinated peptide version 2 (anti-CCP2), IgM rheumatoid factor (RF) and calprotectin were analysed in parallel in circulation and in synovial fluid (SF). Counts of polynuclear and mononuclear cells were measured in SF. Serum (S) calprotectin correlated stronger than SF-calprotectin with inflammatory markers and disease activity. Instead, SF-calprotectin showed a strong correlation to SF counts of white blood cells, and especially to polymorphonuclear cell counts (Spearman's rho = 0.72, p< 0.001). S-calprotectin showed markedly stronger correlation with inflammatory markers and disease activity in ACPA positive as compared with ACPA negative RA patients; a similar difference was observed for patients with and without IgM RF. The particularly strong association between circulating calprotectin and inflammation in ACPA positive RA is a new argument for a specific role for polymorphonuclear granulocytes/neutrophils in this RA subset. Measurement of calprotectin in SF does not convey any additional benefit compared with measurement in the circulation in RA patients with knee synovitis.

Evaluation of Intestinal Microbiota in Children With Sickle Cell Disease.

Sickle cell disease (SCD) is a chronic hemolytic anemia that may be life-threatening due to multisystemic effects. Identification of the factors which affect the pathophysiology of the disease is important in reducing mortality and morbidity. This study aimed to determine gut microbial diversity in children and adolescents with SCA compared with healthy volunteers and to evaluate the clinical impact of microbiota. The study included 34 children and young adolescents with SCD and 41 healthy volunteer participants. The microbiome was assessed by 16S rRNA sequencing in stool samples. Laboratory parameters of all participants, such as complete blood count and C-reactive protein values and clinical characteristics of SCD patients, were determined and compared, as well as clinical conditions of the patients, such as vascular occlusive crisis and/or acute chest syndrome, frequency of transfusions, intake of penicillin, hydroxyurea, and chelation therapy were recorded. White blood cell count, hemoglobin, immature granulocyte and C-reactive protein levels were significantly higher in the patient group ( P <0.05). Microbiota analysis revealed 3 different clusters among subjects; controls and 2 clusters in the SCD patients (patient G1 and G2 groups). Bacteroides spp. were more prevalent, while Dialester spp. and Prevotella spp. were less prevalent in SCD compared with controls ( t =2.142, P <0.05). Patient G2 (n=9) had a higher prevalence of Bacteroides and a lower prevalence of Prevotella than patient G1 (n=25). In our study, there was a difference between SCD patients and the control group, while 2 different microbiota profiles were encountered in SCD patients. This difference between the microbiota of the patients was not found to affect the clinical picture (such as vascular occlusive crisis, acute chest syndrome).

A new uses of calprotectin as an inflammation biomarker

Calprotectin is a cytosol protein of granulocytes. It is presently used in diagnostics andmonitoring of gastrointestinal diseases, mainly in inflammatory bowel diseases. This paperpresents methods of calprotectin testing and its current role in clinical laboratory. In addition,the paper includes the review of current research and potential new applications of calprotectin.

Applied autecology on the model of cationic proteins of neutrophil granulocytes of carnivores by adverse environmental effects

The article presents studies related to the evaluation of the reaction of cationic proteins of neutrophil granulocytes as markers of environmental impact on the animal body on the example of carnivores (dogs). As is known, neutrophils are one of the first lines of defense of the macroorganism against many impacts, such as environmental pollution by various products of anthropogenic activity, bacterial and viral infections, etc. Therefore, taking into account the reaction of neutrophil cationic proteins to adverse environmental effects may be of interest for the early detection of immunological changes. We found that the level of neutrophil cationic proteins in stray dogs was significantly higher than in domestic dogs. This fact may indicate a longer adaptation process of the body of homeless animals in response to changes in the environmental situation and requires further more detailed study.

AQP5-1364A/C Polymorphism Affects AQP5 Promoter Methylation.

The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin (AQP5)-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele—leading to lower AQP5 expression—has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that AQP5 promoter methylation differs between genotypes in specific types of immune cells.: AQP5 promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of AQP5-1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils (p = 0.0055) and a four-fold increase in monocytes (p = 0.0005) and lymphocytes (p = 0.0184) in septic patients and healthy controls as well. In addition, a decreased AQP5 promoter methylation was accompanied by an increased AQP5 expression in HL-60 (p = 0.0102) and REH cells (p = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the AQP5 promoter. Hence, AQP5 promoter methylation could depict a key mechanism in genotype-dependent expression.

Peroxisome proliferator-activated receptors alpha and beta mediate the anti-inflammatory effects of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 in fish granulocytes

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1β (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparβ, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparβ was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparβ in the resolution of inflammation mediated by 15dPGJ2.

CgATP synthase β subunit involved in the regulation of haemocytes proliferation as a CgAstakine receptor in Crassostrea gigas

Astakine is considered as an endogenous cytokine-like factor of prokineticin homologue in invertebrate. Recently, an astakine homologue (CgAstakine) has been identified and characterized in oyster Crassostrea gigas. In the present study, a CgATP synthase β subunit was identified as the receptor of CgAstakine in C. gigas. There was an ATP-synt_ab_N domain and an AAA domain in the CgATP synthase β subunit protein. The mRNA transcripts of CgATP synthase β subunit were detected in all tested tissues, with the highest expression level in hepatopancreas and gills, which was 109.11-fold (p < 0.01) and 97.21-fold (p < 0.01) of that in labial palps, respectively. After rCgAstakine stimulation, the mRNA transcripts of CgATP synthase β subunit in agranulocytes and semi-granulocytes were significantly increased at 24 h (2.44-fold, and 9.01-fold of that in control group, p < 0.01), and those in granulocytes were significantly increased at 6 h (1.83-fold, p < 0.01), 12 h (1.92-fold, p < 0.01) and 24 h (3.47-fold, p < 0.01). The expression level of CgATP synthase β subunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation, which was 1.64-fold (p < 0.05) and 1.85-fold (p < 0.05) of that in control group, respectively, while there were no significant changes in semi-granulocytes. The immunofluorescence assay showed that CgATP synthase β subunit positive signals were mainly located on the membrane of haemocytes. The number of haemocytes with EdU positive signals was significantly increased after rCgAstakine stimulation (2.04-fold of seawater group, p < 0.01), while significantly decreased after the RNA interference (RNAi) of CgATP synthase β subunit, which was 0.28-fold of that in NC group (p < 0.01). Bio-layer interferometry (BLI) assay confirmed in vitro interaction between rCgAstakine and rCgATP synthase β subunit. There results suggested that CgATP synthase β subunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C. gigas.

Evaluation of Eosinophilic Cationic Protein as a Marker of Alveolar and Cystic Echinococcosis.

Echinococcosis is a neglected zoonotic disease and a worldwide public health problem caused by infection with the larval stages of taeniid cestodes of the genus Echinococcus. In vitro studies have demonstrated a protoscolecidal effect of eosinophilic cationic protein (ECP), a granule protein of eosinophilic granulocytes, against E. granulosus. Therefore, the main objective of this study was to evaluate ECP as a biomarker in the treatment of alveolar echinococcosis (AE) and cystic echinococcosis (CE). Data were collected retrospectively from the Vienna Echinococcosis Cohort over 7 years until December 2020. Altogether, 32 patients (16 AE and 16 CE) were included. In the selected patients, serum ECP values were compared before and after the beginning of an operative and/or benzimidazole (BMZ) therapy. Mean ECP serum levels before intervention were significantly (p < 0.05) elevated at 34.0 ± 22.9 μg/L in AE patients and at 38.6 ± 19.9 μg/L in CE patients compared to the control group. After the intervention, mean ECP levels decreased significantly (p < 0.05) to 20.4 ± 14.6 μg/L in AE patients and to 22.4 ± 8.3 μg/L in CE patients. Furthermore, ECP showed a significant (p < 0.05) correlation of k = 0.56 with PET–CTI. Based on the significant decrease after operative and/or BMZ treatment and the correlation with clinical markers such as PET–CTI, it is recommended to investigate ECP more intensively as a marker of AE and CE in prospective studies with larger cohorts.

Early kinetics of calprotectin in plasma following inguinal hernia surgery.

Calprotectin is one of the most abundant proteins of neutrophil granulocytes. It is released upon neutrophil activation and is considered a sensitive and clinically useful marker for neutrophil-mediated inflammation, including bacterial infections. However, early kinetics of calprotectin activation following inflammatory activation has hitherto been unknown. The aim of the present study was to determine the early phase of the kinetics of calprotectin, in comparison with the inflammatory markers CRP, IL-6, TNF-α, and procalcitonin, in plasma following a standardized temporary mild inflammatory response, using uncomplicated inguinal hernia surgery as a model. The study cohort consisted of 17 adult patients (15 male and 2 female) undergoing elective surgery for hernia. Values of calprotectin increased significantly at 2 h following surgery, and continued to increase to reach the highest level at 24–36 h after surgery, values still not exceeding upper normal reference level. This contrasts to IL-6 and CRP, for which an elevation was found first later, 4 h and 24–36 h post-surgery, respectively, for IL-6, and CRP. No significant increase was seen for TNF-α, or procalcitonin. The data demonstrate a very rapid and significant but modest increase in calprotectin following induction of mild inflammation, supporting that calprotectin can be useful for early detection of inflammatory response.

Assessment of Fecal Calprotectin level in Inflammatory, Infectious and Malignant Gastrointestinal Diseases

Background: Calprotectin is a marker of inflammation as it is a cytosolic protein in the neutrophilic granulocytes. Objectives: We aimedto assess fecal calprotectin (FC) in the inflammatory, infectious and malignant gastrointestinal (G.I.T) diseases. Patients and Methods: 169 patients presented with G.I.T symptoms and proved with inflammatory, infectious or malignant condition by histopathological examination of the G.I.T endoscopic specimens were recruited. Symptomatic subjects with normal findings were considered as a non-organic; comparable group. Complete blood count, E.S.R, C.R.P, liver function tests, kidney function tests and stool analysis followed by endoscopic examination and multiple biopsies were taken for histopathological examination. FC was measured for all subjects. Results: Out of 169 patients; 79 patients (53.7%) showed inflammatory/infectious lesions (34 of them were inflammatory bowel disease (I.B.D)), 68 (46.3%) were malignant lesions and, 22 of them showed non-organic lesions (13%). FC levels (median, IQR; interquartile range) were significantly higher in inflammatory, infection group and malignant group than in the non-organic group (145 (53-2467) and 136 (45-212) versus 17 (10-57) respectively, P value =0.000). Patients with I.B.D showed significantly higher values for FC than in those with non I.B.D, P value= 0.000. Colorectal cancer patients showed higher FC values than gastric or esophageal cancer, P value= 0.000. Conclusions: FC is a useful marker in the diagnosis of G.I.T inflammatory, infectious and malignant conditions especially in I.B.D and colorectal cancer.

Deviant proteome profile of equine granulocytes associates to latent activation status in organ specific autoimmune disease

Equine recurrent uveitis (ERU) is a spontaneous, remitting-relapsing autoimmune disease driven by the adaptive immune system. Although T cells are described as the main effector cells in pathogenesis, granulocytes have also emerged as possible disease mediators. To explore the role of these innate immune cells, we investigated the whole cell proteome of granulocytes from equine recurrent uveitis cases and healthy controls. Among the 2362 proteins identified by mass spectrometry, we found 96 proteins with significantly changed abundance between groups (p < 0.05, fold change >1.2), representing 4.1% of total granulocyte proteome. Within these differential identifications, calgranulin B, a protein associated with pathogenesis in other autoimmune diseases, showed highest abundance in equine recurrent uveitis (18 fold).For a better interpretation of the results from our hypothesis-generating approach, we added a threshold for biological significance (ratio ERU/controls >2: 36 proteins) to the proteins with increased abundance in equine recurrent uveitis and analyzed their allocation to the subsets within the Immune System superpathway. The 36 differentially abundant proteins predominantly associated to RAF/MAP kinase cascade, MHC-I-mediated antigen presentation and neutrophil degranulation, suggesting a latently activated phenotype of these innate immune cells in disease. Raw data are available via ProteomeXchange with identifier PXD013648. SignificanceOur study provides new insights into the protein repertoire of primary equine granulocytes and identifies protein abundance changes associated to equine recurrent uveitis (ERU), an organ specific, spontaneously occurring autoimmune disease. We show that granulocyte proteins with increased abundance in ERU strongly associate to RAF/MAP kinase signaling, MHC-I antigen presentation and neutrophil degranulation, pointing to a more activated state of these cells in ERU cases. Since cells were obtained in quiescent stage of disease, latent activation of granulocytes underlines the role of these innate immune cells in ERU.These findings are highly relevant for veterinary medicine, further establishing the importance of granulocytes in this T cell-driven autoimmune disease. Moreover, they have translational quality for autoimmune uveitis in man, due to strong similarity in disease occurrence, progression and pathogenesis.

Heparin-Binding Protein: A Novel Biomarker Linking Four Different Cardiovascular Diseases.

Cardiovascular diseases are an important group of diseases that seriously affect quality of life. Thus, their treatment warrants further study. Heparin-binding protein (HBP) is a granulocyte protein derived from neutrophils. When an infection occurs, neutrophils release HBP, which can lead to elevated HBP levels in the blood. Therefore, HBP family members are said to be important indicators of infection. However, basic evidence is still lacking to confirm the possible relationship between HBP and cardiovascular diseases. Using bioinformatics methods, we investigated the role of the HBP network in normal hearts and hearts from patients with cardiovascular disease. First, we used the Open Targets database to obtain a list of HBP-encoding mRNAs related to atherosclerosis, myocarditis, myocardial infarction, and myocardial ischemia. Then, we constructed an HBP gene interaction network map using STRING. Clustering coefficients were calculated using Cytoscape, and MCODE was used for subnet analysis. Finally, the proposed interstitial network of HBPs was established and analyzed by Metascape enrichment analysis of the relevant signaling pathways. The aggregation coefficient of the HBP interaction network was higher among hearts with the four cardiovascular diseases, atherosclerosis (0.496), myocarditis (0.631), myocardial infarction (0.532), and myocardial ischemia (0.551), than in normal hearts. Metascape analysis showed that “NABA_MATRISOME_ASSOCIATED” was a typical pathway with the highest p value associated with epithelialization in all four diseases. Moreover, a large number of important HBPs were identified that may be significant for the treatment of these diseases. Therefore, HBPs do have a highly atopic connectivity network in cardiovascular diseases, and specific HBPs or signaling pathways may be used as targets for the development of new treatments for cardiovascular diseases.

THU0129 SLEEP DISTURBANCE AND LOW INFLAMMATION PREDICT A PATTERN OF CHRONIC FATIGUE IN ACTIVELY TREATED PATIENTS WITH ESTABLISHED RA.

Background:Fatigue is common among patients with rheumatoid arthritis (RA) and has major impact on the burden of disease. There is little knowledge regarding the factors predicting the longitudinal development of chronic fatigue.Objectives:To identify baseline predictors for the development of chronic fatigue in patients with RA who initiate biological DMARD (bDMARD) treatment, and to compare disease courses across categories of fatigue for 12 months follow-up.Methods:Different trajectories of fatigue were calculated from a cohort of 209 established RA patients initiating bDMARDs. Fatigue was assessed by use of the fatigue Numeric Rating Scale (0-10) from the Rheumatoid Arthritis Impact of Disease (RAID) questionnaire. The patients were assessed at 0, 1, 2, 3, 6 and 12 months. We defined three groups: no fatigue (≤3 at all visits), improved fatigue (&gt;3 at baseline but ≤3 at follow-up) and chronic fatigue (≥ 4 at all visits). All patients had clinical/subjective assessments (28 tender/swollen joint count, assessor’s/patient’s global VAS, RAID score, widespread pain, pain catastrophizing, the Hospital Anxiety and Depression Scale and inflammatory markers (ESR, CRP and calprotectin (a major granulocyte protein sensitive for inflammation in RA patients)). All patients were assessed by ultrasound (grey scale (GS) and power Doppler (PD)) of 36 joints and 4 tendons with semi-quantitative scoring (0-3). Differences between groups at baseline was assessed by bivariate analyses, and logistic regression models adjusted for age and gender were used to explore baseline predictors of chronic vs improved fatigue. Trajectories of different groups were plotted as estimated marginal means in figures, and differences between groups assessed by mixed models with maximum likelihood random effects, adjusted for age and sex.Results:Table 1 describes demographics and clinical factors of the three groups with significant differences shown in bold. Logistic regression with multivariate assessments found anti-CCP and low inflammation (calprotectin) to be predictors of chronic versus improved fatigue. Sleep disturbance was highly predictive of chronic fatigue. Figure 1 illustrates the trajectories for the three groups at all visits, showing the chronic fatigue group to have significantly higher DAS28, level of widespread pain, depression and sleep disturbance in contrast to no higher level of inflammation assessed by CRP and ultrasound PD.Table 1.No fatigueImproved fatigueChronicfatigueNo fatigue vs Improved fatigueImproved fatigue vs chronic fatigueNo fatigue vs. chronic fatigue482943pppAge, mean (SD) years51 (2)48 (2)54 (2)0.280.090.28Female gender (%)35 (73)24 (83)38 (88)0.400.500.09Higher Education (%)31 (65)23 (79)20 (47)0.170.010.17Anti-CCP positive (%)29 (60)20 (69)36 (84)0.720.010.002RF positive (%)27 (56)17 (59)30 (70)0.760.110.15Disease duration, mean (SD) years7 (1)8 (1)11 (1)0.810.110.03RA disease activityDAS28CRP3.2 (0.1)3.9 (0.2)4.7 (0.2)0.0030.004&lt;0.001Swollen joints (28)5.7 (0.7)5.6 (1.0)6.2 (0.7)0.900.600.63CRP mg/L mean (SD)9.4 (2.4)15.6 (4.1)11.0 (2.6)0.020.020.58Calprotectin mg/L mean (SD)1.6 (0.3)2.0 (0.4)1.5 (0.2)0.440.200.92Sum score PD mean (SD)14.3 (1.8)13.8 (2.5)12.3 (1.9)0.850.620.43Sum score GS mean (SD)31.6 (2.8)29.3 (3.4)28.2 (2.7)0.610.810.39Psychosocial factorsRAID sleep (VAS 0-10)1.2 (0.3)4.3 (0.6)6.7(0.4)&lt;0.001&lt;0.001&lt;0.001RAID fatigue (VAS 0-10)1.4 (0.2)5.6 (0.3)7.1 (0.3)&lt;0.0010.003&lt;0.001Widespread pain (0-25)4.3 (0.4)7.0 (0.8)8.6 (0.7)0.0010.16&lt;0.001HADS anxiety1.5 (0.3)1.4 (0.6)3.4 (0.7)0.260.580.10HADS depression0.8 (0.2)0.9 (0.4)3.0 (0.8)0.980.360.05Pain Catastrophizing (0-6)1.0 (0.2)2.5 (0.3)2.9 (0.3)&lt;0.0010.31&lt;0.001Conclusion:Sleep disturbance is a modifiable factor presently found to predict chronic versus improved fatigue. Thus, attention should be given to RA patients with sleep problems to seek to avoid development of chronic fatigue. This issue should be explored in further studies.Disclosure of Interests:Hilde Berner Hammer Consultant of: Has received fees as consultant from Roche, AbbVie and Novartis., Speakers bureau: Has received fees for speaking from AbbVie, BMS, Pfizer, UCB, Roche, MSD and Novartis, Brigitte Michelsen Grant/research support from: Research support from Novartis, Consultant of: Consulting fees Novartis, Joe Sexton: None declared, Till Uhlig Consultant of: Lilly, Pfizer, Speakers bureau: Grünenthal, Novartis, Sella Aarrestad Provan Consultant of: Novartis

Differences in proteomic profile between two haemocyte types, granulocytes and hyalinocytes, of the flat oyster Ostrea edulis

Haemocytes play a dominant role in shellfish immunity, being considered the main defence effector cells in molluscs. These cells are known to be responsible for many functions, including chemotaxis, cellular recognition, attachment, aggregation, shell repair and nutrient transport and digestion. There are two basic cell types of bivalve haemocytes morphologically distinguishable, hyalinocytes and granulocytes; however, functional differences and specific abilities are poorly understood: granulocytes are believed to be more efficient in killing microorganisms, while hyalinocytes are thought to be more specialised in clotting and wound healing. A proteomic approach was implemented to find qualitative differences in the protein profile between granulocytes and hyalinocytes of the European flat oyster, Ostrea edulis, as a way to evaluate functional differences. Oyster haemolymph cells were differentially separated by Percoll® density gradient centrifugation. Granulocyte and hyalinocyte proteins were separated by 2D-PAGE and their protein profiles were analysed and compared with PD Quest software; the protein spots exclusive for each haemocyte type were excised from gels and analysed by MALDI-TOF/TOF with a combination of mass spectrometry (MS) and MS/MS for sequencing and protein identification. A total of 34 proteins were identified, 20 unique to granulocytes and 14 to hyalinocytes. The results suggested differences between the haemocyte types in signal transduction, apoptosis, oxidation reduction processes, cytoskeleton, phagocytosis and pathogen recognition. These results contribute to identify differential roles of each haemocyte type and to better understand the oyster immunity mechanisms, which should help to fight oyster diseases.

A Novel Mutation in PIGA Associated with Multiple Congenital Anomalies-Hypotonia-Seizure Syndrome 2 (MCAHS2) in a Boy with a Combination of Severe Epilepsy and Gingival Hyperplasia

Multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) is a rare disease caused by mutations in the X chromosomal PIGA gene. Clinically it is characterized by early-onset epilepsy, hypotonia, dysmorphic features, and variable congenital anomalies. PIGA codes for the phosphatidylinositol glycan-class A protein, which forms a subunit of an enzymatic complex involved in glycophosphatidylinositol (GPI) biosynthesis. We present a new case of MCAHS2 and perform a comprehensive review of the available literature to delineate the phenotypical traits associated with germline PIGA mutations. Furthermore, we provide functional evidence of pathogenicity of the novel missense mutation, c.154C>T; (p.His52Tyr), in the PIGA gene causative of MCAHS2 in our patient. By flow cytometry, we observed reduced expression of GPI-anchored surface proteins in patient granulocytes compared to control samples, proving GPI-biogenesis impairment. The patient's severe epilepsy with several daily attacks was refractory to treatment, but the frequency of seizures reduced temporarily under triple therapy with perampanel, rufinamide and vigabatrin. Our study delineates the known MCAHS2 phenotype and discusses challenges of diagnosis and clinical management in this complex, rare disease. Furthermore, we present a novel mutation with functional evidence of pathogenicity.

Serum Calprotectin Is a Potential Marker in Patients with Asthma.

BackgroundCalprotectin is the major cytosolic protein in neutrophil granulocytes. Although asthma is known to cause eosinophilic inflammation, some patients with asthma have non-eosinophilic inflammation, which is characterized by local neutrophilic inflammation. The aim of this study was to assess calprotectin expression levels in a mouse model of asthma, and to observe the relationship of serum calprotectin level and clinical variables in patients with asthma.MethodsMice were sensitized and challenged with 10 μg and 20 μg of Aspergillus fumigatus, respectively; mice treated with saline were used as a control. The levels of calprotectin were determined using enzyme-linked immunosorbent assay, immunoblotting, and immunohistochemical analysis. The serum levels of calprotectin were also assessed in patients with asthma. The relationship between calprotectin and clinicopathological characteristics was determined.ResultsCalprotectin, S100A8, and S100A9 expression was elevated in the mouse lungs, calprotectin levels were higher in the serum of patients with asthma (n = 33) compared with those of healthy individuals (n = 28). Calprotectin levels correlated with forced expiratory volume in one second/forced vital capacity (r = −0.215, P = 0.043), smoke amount (r = 0.413, P = 0.017), body mass index (r = −0.445, P = 0.000), and blood neutrophil percentage (r = 0.300, P = 0.004) in patients with asthma.ConclusionOur data suggest that calprotectin could potentially be used as a biomarker for asthma.

Galectin-9 Is a Possible Promoter of Immunopathology in Rheumatoid Arthritis by Activation of Peptidyl Arginine Deiminase 4 (PAD-4) in Granulocytes.

The aetiology of rheumatoid arthritis (RA) is unknown, but citrullination of proteins is thought to be an initiating event. In addition, it is increasingly evident that the lung can be a potential site for the generation of autoimmune triggers before the development of joint disease. Here, we identified that serum levels of galectin-9 (Gal-9), a pleiotropic immunomodulatory protein, are elevated in RA patients, and are even further increased in patients with comorbid bronchiectasis, a lung disease caused by chronic inflammation. The serum concentrations of Gal-9 correlate with C-reactive protein levels and DAS-28 score. Gal-9 activated polymorphonuclear leukocytes (granulocytes) in vitro, which was characterized by increased cytokine secretion, migration, and survival. Further, granulocytes treated with Gal-9 upregulated expression of peptidyl arginine deiminase 4 (PAD-4), a key enzyme required for RA-associated citrullination of proteins. Correspondingly, treatment with Gal-9 triggered citrullination of intracellular granulocyte proteins that are known contributors to RA pathogenesis (i.e., myeloperoxidase, alpha-enolase, MMP-9, lactoferrin). In conclusion, this study identifies for the first time an immunomodulatory protein, Gal-9, that triggers activation of granulocytes leading to increased PAD-4 expression and generation of citrullinated autoantigens. This pathway may represent a potentially important mechanism for development of RA.

DNA released from neutrophil extracellular traps (NETs) activates pancreatic stellate cells and enhances pancreatic tumor growth

ABSTRACTNeutrophil extracellular trap (NET) formation results in the expulsion of granulocyte proteins and DNA into the extracellular space. This process is mediated by the enzyme peptidyl arginine deiminase 4 (PADI4) and translocation of elastase to the nucleus. NET formation, marked by increased levels of extracellular DNA, promotes pancreatic cancer proliferation and metastasis. Mice deficient in Padi4 demonstrate decreased pancreatic tumor growth, associated with a reduction in circulating extracellular DNA levels, diminished pancreatic stromal activation and improved survival in murine orthotopic pancreatic adenocarcinoma. Transplantation of Padi4−/− bone marrow into genetically engineered mice with Kras driven pancreatic adenocarcinoma (Pdx1-Cre:KrasG12D/+, KC mice) limits the frequency of invasive cancers when compared with syngeneic controls. DNA from neutrophils activates pancreatic stellate cells that form dense, fibrous stroma which can promote and enable tumor proliferation. DNase treatment diminishes murine tumor growth and stromal activation to reverse the effect of NETs within the tumor microenvironment. Furthermore, deletion of the receptor for advanced glycation end products (RAGE) in pancreatic stellate cells abrogates the effects of DNA in promoting stellate cell proliferation and decreases tumor growth. Circulating neutrophil-derived DNA correlates with the stage in patients with pancreatic ductal adenocarcinoma, confirming the role of NETs in human pancreatic cancer. These findings support further investigation into targeting of NETs, PADI4 and extracellular DNA as a potential treatment strategy in patients with pancreatic cancer.Trial Registration: This study reports correlative data from a clinical trial registered with clinicaltrials.gov, NCT01978184 (November 7, 2013).