Active Disease Patients Research Articles

Efficacy of Pneumococcal Polysaccharide Conjugate Vaccine (13-valent, Adsorbed) in Patients with Systemic Juvenile Idiopathic Arthritis Treated with Genetically Engineered Biologic Drugs (Tocilizumab or Canakinumab): Prospective Cohort Study

Background. Immunological potency of 13-valent pneumococcal vaccine (PCV-13) in children with systemic juvenile idiopathic arthritis (SJIA) is still unstudied. Estimates of the genetically engineered biologic drugs (GEBD) effects on pneumococcal vaccination results also remain controversial.Objective. The aim of the study was to explore the PCV-13 efficacy in patients with SJIA and who is on treatment with monoclonal antibodies against interleukin 6 receptor (tocilizumab) and interleukin 8 receptor beta (canakinumab).Methods. The study included patients under the age of 18 with SJIA in remission or active form of disease vaccinated with PCV-13. The vaccine was administered in single dose of 0.5 ml intramuscularly in patients on treatment with GEBD or 3 weeks before GEBD administration for the first time (for patients with active disease). Vaccination was considered effective at achievement of the minimum protective level of antibodies to capsular polysaccharide of pneumococcus (anti-SPP IgG; ≥ 7 U/ml) or increase of anti-SPP IgG level ≥ 2 times in 4 weeks after vaccination. The anti-SPP IgG levels were measured with enzyme immunoassay.Results. The study included 53 patients (27 girls) in remission of SJIA and 25 (16 girls) in active disease. Median age was 13.3 and 10.8 years respectively. Tocilizumab/canakinumab was administrated in 43/10 and 18/7 patients respectively. Minimum significant anti-SPP IgG level and two-fold increase in anti-SPP IgG level were recorded in 49/53 (92%) and 32/53 (60%) patients with SJIA in remission, as well as in 22/25 (88%) and 18/25 (72%) patients in active disease respectively. PCV-13 immunological potency in patients with SJIA in remission and in active disease (in those who were initially administrated and who did not receive GEBD) did not differ.Conclusion. PCV-13 vaccination allows to achieve protective antibodies level in most of the patients with SJIA in children population regardless of the disease stage and the history of GEBD administration.

Detrimental effects of hypercortisolism on brain structure and related risk factors

Brain structural abnormalities are often observed on magnetic resonance imaging (MRI) scans of Cushing's syndrome patients, but the pathogenesis is not fully understood. To understand the relationship between brain structural abnormalities and potential risk factors in active Cushing's disease (CD) patients, a total of 101 treatment-naïve CD patients and 95 sex-, age- and education matched controls with non-functioning adenomas (NFA) underwent clinical evaluation and MRI investigation, and the relative risk factors were analyzed. 14 patients in sustained remission after transsphenoidal surgery were followed. Compared with the NFA subjects, the patients with CD had more cortical (P < 0.01) and subcortical atrophy (P < 0.01) and a higher prevalence of white matter hyperintensity (WMH) (P < 0.01). WMH severity in CD patients positively correlated with age (r = 0.532, P = 0.000), disease course (r = 0.257, P = 0.009), postprandial glucose (r = 0.278, P = 0.005), frequency of left ventricular hypertrophy (r = 0.398, P = 0.001) and hypothyroidism (r = 0.246, P = 0.014). The markers of cortical and subcortical atrophy (sylvian fissure ratio, bifrontal ratio, bicaudate ratio and third ventricle width) were positively associated with the progression of WMH in the CD patients. In the follow-up of 14 patients with CD, brain atrophy and WMH was partially reversible after correction of hypercortisolism. In conclusions, brain atrophy and WMH were more likely to appear in CD patients and were possibly partially reversible following correction of hypercortisolism.

The onset of active disease in systemic lupus erythematosus patients is characterised by excessive regulatory CD4+-T-cell differentiation

An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in systemic lupus erythematosus (SLE) patients. Both cell subsets consist of highly proliferating Tregs/Tresps expressing inducible T-cell co-stimulatory molecule (ICOS) and less proliferating ICOS--Tregs/Tresps. Six-colour-flow-cytometric analysis was used to examine the effect of ICOS+- and ICOS--Treg/Tresp cell differentiation on the composition of the total CD4+-T-helper cell pool with ICOS+- and ICOS--Tregs/Tresps. Functionality of Tregs was examined using suppression assays. In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE patients (SLEDAI <7), disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In these patients, the functional activity of ICOS+-Tregs, but not of ICOS--Tregs, was preserved. In 13 markedly active disease patients (SLEDAI >7), the percentage of both ICOS+-Tregs and ICOS+-Tresps, was strongly increased within total CD4+-T-helper cells. However, the increased ratio of ICOS+-Tregs/ICOS+-Tresps was not maintained in these patients, due to terminal differentiation and accumulation of naïve cells within total ICOS+-Tregs. Despite increased differentiation of both ICOS--Tregs and ICOS--Tresps, the percentage of ICOS--Tregs increased within CD4+-T-helper cells, while that of ICOS--Tresps decreased, resulting in a significantly increased ratio of ICOS--Tregs/ICOS--Tresps independent of age. Our data reveal a crucial role of Treg immune senescence for the occurrence of disease flares in SLE patients, with ICOS+-Treg cells being most affected.

P0413THE ICOS+ TREG/ICOS+ TRESP BALANCE HAS A DECISIVE ROLE REGARDING THE PRESERVATION OF THE REMISSION PHASE IN SLE PATIENTS

Abstract Background and Aims An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in Systemic Lupus Erythematosus (SLE) patients. Both cell subsets consist of highly proliferative inducible T-cell co-stimulatory molecule expressing (ICOS+-) and less proliferating ICOS--Treg/Tresp cells. Method In this study we aimed to examine the role of age-dependent differentiation for maintaining the ICOS+-Treg/ICOS+-Tresp or ICOS--Treg/ICOS--Tresp balance in SLE remission patients compared to active disease patients and to evaluate the influence of proliferation inhibitor or glucocorticoid therapy concerning the maintenance of these equilibria. Results In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE remission patients, medication controlled disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In 13 active disease patients this increased ICOS+-Treg/ICOS+-Tresp ratio could not be maintained, due to an age-dependent earlier terminal differentiation of ICOS+-Tregs than of ICOS+-Tresps. The increased ratio of ICOS--Treg/ICOS--Tresp was maintained in active disease patients, but it decreased with age. This was attributable to an age-independent aberrant differentiation of ICOS--Tresps and a resulting loss of age-dependent differentiation of ICOS--Tregs. Especially the administration of proliferation inhibitors (azathioprine or mycophenolic acid) ensured prolonged differentiation and maintenance of functional ICOS+-Tregs with age. There was a similar but weaker effect of this medication on ICOS+-Tresps, causing a significant decrease of these cells with age. The proliferation capacity was neither affected in ICOS+-Tregs/Tresps nor in ICOS--Tregs/Tresps. As a result, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age and kept the patients in remission. In contrast, glucocorticoids increased the differentiation of ICOS--Tregs especially in younger individuals, but had an adverse effect on their proliferation capacity. Conclusion Our data reveal a crucial role of the ICOS+-Treg/ICOS+-Tresp balance for the maintenance of self-tolerance and avoidance of disease flares in SLE. The used immunosuppressive therapy, especially the administration of proliferation inhibitors, has a decisive effect on the preservation of regular differentiation of all Treg/Tresp subsets with age, and most importantly avoids the terminal differentiation and immune senescence of ICOS+-Tregs.

Prothrombotic fibrin network characteristics in patients with acromegaly: a novel mechanism for vascular complications.

There remains increased cardiovascular mortality in patients with acromegaly. This study aims to evaluate whether GH/IGF-1 excess increases vascular disease by adversely affecting fibrin network characteristics. Cross-sectional study in 40 patients with acromegaly (21 males, age 53 ± 13 years) and 40 age/gender-matched controls. Clot structure was analysed using a validated turbidimetric assay and fibrin networks were visualised by laser scanning confocal microscopy (LSCM). Metabolic profile parameters, body composition, plasma fibrinogen and PAI-1 were also assessed. Twenty-two patients had active acromegaly and 18 were in remission. There was no difference in qualitative patient characteristics between the two groups. Both groups had less favourable body composition and cardiovascular risk profile compared with controls. Despite no difference in clot formation and lysis parameters between the two patient groups, active disease patients had higher fibrinogen and clot maximum absorbance compared with controls, after adjusting for BMI (3.8 ± 0.2 vs 2.6 ± 0.2 mg/mL, P < 0.001; and 0.39 ± 0.02 vs 0.33 ± 0.01 arbitrary units, P = 0.03, respectively). Patients in remission had higher fibrinogen compared with controls following adjustment for BMI (3.3 ± 0.2 vs 2.6 ± 0.2 mg/mL, P = 0.02) but not clot maximum absorbance (0.35 ± 0.03 vs 0.33 ± 0.02 arbitrary units, P = 0.6). LSCM showed increased fibrin network density only in active disease patients, consistent with turbidimetric analysis. In addition to active disease, BMI, fat mass and skinfold thickness were associated with higher clot density and longer lysis time. Patients with active acromegaly have more compact clots, thus conferring increased thrombosis risk. Prothrombotic fibrin networks may represent one mechanism for enhanced vascular risk in active acromegaly.

Impaired brain network architecture in Cushing's disease based on graph theoretical analysis.

To investigate the whole functional brain networks of active Cushing disease (CD) patients about topological parameters (small world and rich club et al.) and compared with healthy control (NC). Nineteen active CD patients and twenty-two healthy control subjects, matched in age, gender, and education, underwent resting-state fMRI. Graph theoretical analysis was used to calculate the functional brain network organizations for all participants, and those for active CD patients were compared for and NCs. Active CD patients revealed higher global efficiency, shortest path length and reduced cluster efficiency compared with healthy control. Additionally, small world organization was present in active CD patients but higher than healthy control. Moreover, rich club connections, feeder connections and local connections were significantly decreased in active CD patients. Functional network properties appeared to be disrupted in active CD patients compared with healthy control. Analyzing the changes that lead to abnormal network metrics will improve our understanding of the pathophysiological mechanisms underlying CD.

Increased sMer, but not sAxl, sTyro3, and Gas6 relate with active disease in juvenile systemic lupus erythematosus.

Tyro3, Axl, and Mer (TAM) receptors and ligands mediate apoptotic bodies engulfment which alteration has been related with juvenile systemic lupus erythematosus (JSLE) pathogenesis. Thus, the aim was to determine their soluble levels. Serum sTyro3, sAxl, sMer, and Gas6 levels were measured using ELISA in 67 JSLE patients, 12 juvenile idiopathic arthritis (JIA) inflammatory and 20 healthy controls and related with SLEDAI-2K score, anti-dsDNA antibody, ESR, CRP, C3, C4 levels, and nephritis. JSLE patients with active disease (SLEDAI-2K> 4) had significantly increased sMer levels compared with healthy controls (median 8.4 vs. 6.0 ng/mL, p = 0.009) and inactive disease patients (5.2 ng/mL, p = 0.0003). sMer levels correlated with SLEDAI-2K (r = 0.44; p = 0.0004) and ESR (r = 0.24; p = 0.04), while sAxl correlated with SLEDAI-2K (r = 0.33; p = 0.008) and C4 levels (r = - 0.24; p = 0.04). JSLE patients taking glucocorticoid had increased sAxl and sMer levels. Moreover, sAxl correlated with sMer and sTyro3 levels. Patients with nephritis and those with focal or diffuse proliferative glomerulonephritis had these protein levels similar to healthy controls and patients without renal involvement. sTyro3 levels of JSLE patients taking glucocorticoid were decreased, and correlated with Gas6 and sAxl, while Gas6 levels correlated with age upon enrollment. JIA controls had protein levels similar to healthy controls and JSLE patients. This study reinforces that sMer is increased in active JSLE patients, yet sMer and sAxl correlates with disease activity parameters, and their alterations are disease-specific. However, further studies are needed to determine exact roles of sTyro3 and Gas6 in disease pathogenesis. Key Points • sMer and sAxl serum levels are related with active disease in JSLE patients • sMer correlated with SLEDAI-2K score in JSLE • sTyro3, sAxl, sMer and Gas6 levels did not related with nephritis in JSLE patients • sTyro3 and Gas6 exact roles in JSLE are not established and further studies are needed.

351 Intrinsic Sex-Specific Differences in Inflammatory and Fibrotic Gene Expression and Remodeling Contribute to Male Disease Predisposition in Eosinophilic Esophagitis

INTRODUCTION: Eosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal inflammation and fibrosis with a higher disease prevalence in males than females (3:1). The underlying mechanisms behind this sexual dimorphism remain unclear. METHODS: Primary human fibroblasts from males and females with active EoE were isolated from esophageal biopsies. Normal fibroblasts were isolated from donor esophageal explants. Fibroblasts were treated with recombinant IL-4, IL-13, or TGFβ1 and analyzed for inflammatory and fibrotic gene expression by qPCR. Esophageal biopsy specimens from active disease patients were scored using a standardized histology score. RESULTS: IL-4 or IL-13 induced significantly more eotaxin-3/ccl26 mRNA expression in esophageal fibroblasts from males as compared with females (P &lt; 0.001). This trend was observed in fibroblasts derived from adults or children, consistent with increased EoE onset in males of all ages. TGFβ1 induced significantly higher levels of collagen-1, α-smooth muscle actin, fibronectin, and serpinE1 (P &lt; 0.03) mRNA in male (n = 5) EoE esophageal fibroblasts as compared with females (n = 3). Hematoxylin and eosin staining of EoE esophageal biopsies (n = 153, 125 males and 28 females) revealed that while overall eosinophil count correlated with epithelial remodeling score (r = 0.3550, P &lt; 0.0001) within the distal esophagus, this correlation was distinctly stronger in the male patients (r = 0.4062, P &lt; 0.0001) than within the female patients (r = 0.1546, P = 0.4323). The same pattern was found in the middle (male r = 0.6080, P &lt; 0.0001; female r = 0.4723, P = 0.013) and proximal esophageal levels (male r = 0.8504, P &lt; 0.0001; female r = 0.5235, P = 0.006). Immunohistochemical staining of EoE esophageal biopsies showed significantly more TGFβ1-positive cells in female than male patients (P &lt; 0.001) but no differences in Smad2/3 phosphorylation. CONCLUSION: Male esophageal fibroblasts have a more robust response to Th2 interleukins and TGFβ1 as compared with female fibroblasts in adults and children and during the normal and active disease states, suggesting an intrinsic sexual dimorphism in esophageal cells. Epithelial histologic disease severity in males also correlates more strongly with elevated eosinophil counts than in females, suggesting that the male esophagus may have more profound responses to cytokines or antigens as compared to females. These data support a potential mechanism for the sex biased disease expression of EoE.

The Clinical Importance of 21-Day Combined Parenteral and Enteral Nutrition in Active Inflammatory Bowel Disease Patients.

The aim of the study was to show the clinical magnitude of short-term feeding: enteral nutrition (EN) combined with parenteral nutrition (PN) in active Crohn’s disease and ulcerative colitis patients. Among 122 eligible inflammatory bowel disease (IBD) patients, 65 met the inclusion criteria. Combined EN and PN was administered for 21 days, wherein over the first 3–5 days of treatment, trophic enteral nutrition (300 kcal/day) was used with an energy increase of up to 1500 kcal. An EN was administered using a nasogastric tube or, in case of intolerance, using a naso-jejunal tube. For PN, the “All in One” system was used according to individually prepared admixtures (ESPEN Guidelines). In addition to routine blood measurement (i.e., ALAT, ASPAT, GGTP, creatinine, lipid profile), the following parameters were assessed: adiponectin, leptin, (hs)TNF-α, hsIL-6 and hsIL-10, TSH, NT-proBNP, serum vitamin B12 concentration, and tHcy. The results showed a considerable improvement in all clinically significant parameters (p < 0.05), showing the benefits and importance of short-term well-balanced EN combined with PN for nutritional and clinical status in IBD patients with active disease. The daily work at hospitals with active IBD patients demonstrates the potential of continued administration of home-based nutrition by patients.

Effects of tapering tumor necrosis factor inhibitor on the achievement of inactive disease in patients with axial spondyloarthritis: a nationwide cohort study

ObjectivesTo investigate the association between the extent of tapering tumor necrosis factor inhibitor (TNFi) and the likelihood of achieving inactive disease in patients with axial spondyloarthritis (axSpA)MethodsWe analyzed 1575 1-year follow-up interval data of 776 axSpA patients treated with TNFi for more than 1 year in a nationwide observational cohort. The decision on tapering TNFi was made by patients and their physicians. We quantified TNFi used during interval as a dose quotient (DQ). The intervals were classified into the heavy-tapering (DQ < 50), mild-tapering (DQ 50–99), and control groups (DQ = 100). Outcome variables included achieving Ankylosing Spondylitis Disease Activity Score-inactive disease (ASDAS-ID) and major clinical response of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI50) in the follow-up visit. The effects of TNFi tapering on the outcome were analyzed using the generalized estimating equation.ResultsAt the baseline visit, 91.1% of the patients showed a high disease activity (ASDAS-CRP ≥ 2.1). DQ of each interval was significantly influenced by the ASDAS-CRP measure in the prior follow-up (P < 0.001). ASDAS-ID was observed in 42.3% of the intervals. A multivariable analysis showed that the likelihood of outcome achievement was comparable between the control and mild-tapering groups, but significantly decreased in the heavy-tapering group (vs. the control group, adjusted OR = 0.28, [95% CI, 0.08–0.94]). In contrast, the likelihood to achieve BASDAI50 response was not different among the groups. In the subgroup of patients who reached ASDAS-ID 1 year after TNFi treatment (n = 327), ASDAS-ID was observed in 66.1% of the subsequent intervals, and only the mild-tapering group showed a likelihood of target maintenance comparable with that of the control group (adjusted OR = 1.25 [0.41–3.80]). This likelihood decreased with an increase in ASDAS-CRP.ConclusionMild tapering of TNFi has efficacy comparable with that of the standard-dose treatment for ASDAS-ID achievement in patients with axSpA.

P080 Association between intestinal mucosa and plasmatic levels of infliximab and TNFa in Chilean patients with inflammatory bowel disease

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation. Tumor Necrosis Factor a (TNFa) levels have been found to be elevated in the intestinal mucosa of patients with Crohn's Disease (CD) and Ulcerative Colitis (UC), which turned the anti-TNFa agents as one of the mainstays of the treatment. Nevertheless, some patients fail to respond or loose response, despite adequate plasmatic levels of anti-TNFa agents. The aim of this study was to determine intestinal mucosa levels of infliximab (IFX) and TNFa and its possible association with their plasmatic levels and the endoscopic activity in IBD patients. METHODS: Cross–sectional study of patients with IBD enrolled at the IBD program of Clínica Las Condes, Chile. The enrollment was done between May 2016 and January 2017. Tissue and plasmatic IFX and TNFa samples were taken the same day just before the next IFX infusion. The endoscopic activity was determined by colonoscopy using Mayo Score for UC and SES-CD/Rutgeerts for CD. Samples of mucosa were taken from colon in patients in remission and from inflamed and non-inflamed colon tissue in active patients. Mucosal levels of IFX and TNFa were normalized by total protein levels. The statistical analysis was performed using paired t-test analysis for comparison between inflamed and non-inflamed sites and an alpha value of &lt;0.05 was considered statistically significant. RESULTS: Fourteen patients were included (9 UC and 5 CD) of which 6 were in remission and 8 with active disease (2 mild, 5 moderate and 1 severe). Plasmatic levels of IFX were higher in patients in remission compared to patients with active disease (4.488 AU/mL vs 2.378 AU/mL; P = 0.1355). In this last group, higher levels were observed in patients with mild than moderate endoscopic activity (4.785 AU/mL vs 1.552 AU/mL; P = 0.0264). Plasmatic TNFa levels were higher in patients with active disease compared to patients in remission (111.938 pg/mL vs 23.467 pg/mL; P = 0.0011), as opposed to IFX levels. Mucosal IFX levels were higher in patients with active disease vs remission, not statistically significant (6.136 ng/mL vs 3.917 ng/mL; P = 0.1186). On the contrary, mucosal TNFa levels were significantly higher in patients in remission vs active disease (65.062 pg/mL vs 36.479 pg/mL; P = 0.0330). In patients with active disease (n = 8), there was no statistical difference between the IFX level in the inflamed compared to the non-inflamed mucosa (6.136 ng/mL vs 5.000 ng/mL; P = 0.3534). TNFa in the non-inflamed mucosa was significantly higher compared to inflamed mucosa (64.846 pg/mL vs 36.479 pg/mL; P = 0.0066). In both, no difference was observed according to endoscopic activity level. The proportion of IFX/TNFα in mucosa was higher in patients with mild and moderate activity (0.182 and 0.179, respectively) than patients with severe activity (0.074). CONCLUSION(S): The discordance between plasmatic and intestinal mucosa IFX and TNFα, in patients with active disease, indicate that the inflammatory cascade might be determined by others specific inflammatory pathways. Further studies need to be performed in a larger sample of patients to better elucidate the mechanistic explanation for persistent inflammation in certain IBD patients.

Overexpression of Long Noncoding RNAs (lncRNA) NF-κβ-Interacting Long Noncoding RNA (NKILA) in Ankylosing Spondylitis is Correlated with Transforming Growth Factor β1 (TGF-β1), Active Disease and Predicts Length of Treatment.

BackgroundLong noncoding RNAs (lncRNA) NF-κβ-interacting long noncoding RNA (NKILA) is downregulated in various types of cancers, while its involvement in other diseases is unknown. In the present study we found that plasma lncRNA NKILA was expressed at higher levels in active ankylosing spondylitis patients than in healthy controls.Material/MethodsAccording to Youden’s index, active disease patients were divided into high and low lncRNA NKILA groups.ResultsPatients in the high lncRNA NKILA level group had significantly longer length of treatment and higher re-hospitalization rate at 3 years after discharge. Plasma levels of TGF-β1 were also higher in active ankylosing spondylitis patients than in healthy controls. Levels of plasma lncRNA NKILA and TGF-β1 were significantly and positively correlated in ankylosing spondylitis patients but not in healthy controls.ConclusionsOverexpression of lncRNA NKILA in ankylosing spondylitis is correlated with active disease and predicts length of treatment. LncRNA NKILA may participate in ankylosing spondylitis through the interaction with TGF-β1, which is a key player in this disease.

Decreased expression of FcγRII in active Graves' disease patients.

BackgroundGraves' disease (GD) is a common autoimmune disease characterized by genetic and environmental factors. Fcγ receptors (FcγRs) are involved in several autoimmune disorders through recognizing immunoglobulin (Ig) G antibodies and mediating immune response. The study on the expression of FcγRs in GD patients is scarce. The purpose of this study was to evaluate the expression of three different types of FcγRs in patients with active and remissive GD.MethodsBlood samples of patients and healthy subjects were collected to analyze the percentage of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) on peripheral blood mononuclear cells (PBMCs) and monocytes by flow cytometry and Western blotting. CD32 isotypes were also examined in cases and controls by real‐time PCR.ResultsThe cell percentages expressed CD32 and protein expressions of CD32 on PBMCs, and monocytes from patients with active GD were significantly reduced compared to controls and patients with remissive GD. In particular, the expression of CD32B on PBMC was also decreased in active GD patients. However, the cell percentages expressed CD16 and CD64 from PBMCs and monocytes were comparable between three groups. Besides, the percentages of CD14+CD32+ cells were negatively correlated with TRAb titers in active GD patients (r = −0.5825, P ＜ 0.001).ConclusionThese results suggested that CD32 may act as a novel marker for active GDs. The expression of monocytic CD32, in particular CD32B, in GD patients might play a crucial role in maintaining FcγRs function and be a therapeutic target in GD patients.

Elevated levels of IL-24 in systemic lupus erythematosus patients.

This study aimed to assess IL-24 levels and their association with clinical manifestations in patients with systemic lupus erythematosus (SLE). There were 75 patients with SLE and 58 healthy controls recruited in this study. Serum levels of IL-24 were measured by enzyme-linked immunosorbent assays, and mRNA levels of IL-24 were tested by quantitative real-time polymerase chain reaction . The area under the curve of the receiver operating characteristic (ROC) curve was used for diagnostic ability of the inflammatory cytokine. Serum IL-24 levels were significantly higher in SLE patients than that in healthy controls. SLE patients with nephritis had higher IL-24 levels than those without nephritis. Active SLE patients showed higher expression of IL-24 as compared to less active disease patients. The mRNA levels of IL-24 were much higher in SLE patients. Correlation analysis showed significant correlation between serum IL-24 levels and SLE disease activity index. In addition, ROC analysis may suggest good ability of serum IL-24 in differentiating SLE. The inflammatory cytokine correlated with SLE disease activity, and may be involved in this disease pathogenesis.

Food avoidance in outpatients with Inflammatory Bowel Disease – Who, what and why

Food avoidance is common with Inflammatory Bowel Disease (IBD) and adherence to dietary guidelines is poor, contributing to under and over nutrition. Reasons for food avoidance have not been previously explored in detail. This study of IBD outpatients aimed to describe food avoidance patterns and rationale behind this, and describe source and confidence with dietary advice. A prospective cross-sectional study using structured interview, nutritional assessment and medical record review was conducted in patients with confirmed diagnosis of IBD (n=117) attending outpatient clinics over a six-month period. Participants were interviewed on foods avoided, rationale for food avoidance and previous dietary advice (source and confidence). Means±SD or medians (IQR), percentages and counts were used to describe participant characteristics, food avoidance, and source and confidence in dietary advice. Bivariate analysis was used to explore relationships between food avoidance and disease factors (IBD subtype; disease activity: active disease vs remission), and between confidence in dietary advice and disease activity. Almost all participants reported food avoidance (90%), with more foods avoided during active disease (5.2±3.6 foods/food categories, versus remission 2.9±2.5, p<0.001). Lactose-containing foods were avoided by 40% of patients in active disease and 33% in remission. Pain/cramping, increased bowel motions and diarrhea were the most common reasons for avoiding foods/food categories during both active disease and remission. Participants were most confident in advice received from the internet (3.3±1.2; dietitian: 2.8±1.5) in active disease; in remission participants had greatest confidence in advice received from gastroenterologists (4.1±0.8; dietitian: 3.5±1.2). High prevalence of avoidance of nutritious foods and low confidence in dietetic advice amongst people with IBD is of concern. Further work is needed to build trust and ensure patients are provided with evidence-based nutrition recommendations to manage their symptoms whilst optimizing nutritional quality of their diet.

Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients – A pilot study

The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.

Identification of LncRNA Linc00513 Containing Lupus-Associated Genetic Variants as a Novel Regulator of Interferon Signaling Pathway.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by augmented type I interferon signaling. High-throughput technologies have identified plenty of SLE susceptibility single-nucleotide polymorphisms (SNPs) yet the exact roles of most of them are still unknown. Functional studies are principally focused on SNPs in the coding regions, with limited attention paid to the SNPs in non-coding regions. Long non-coding RNAs (lncRNAs) are important players in shaping the immune response and show relationship to autoimmune diseases. In order to reveal the role of SNPs located near SLE related lncRNAs, we performed a transcriptome profiling of SLE patients and identified linc00513 as a significantly over expressed lncRNA containing functional SLE susceptibility loci in the promoter region. The risk-associated G allele of rs205764 and A allele of rs547311 enhanced linc00513 promoter activity and related to increased expression of linc00513 in SLE. We also identified linc00513 to be a novel positive regulator of type I interferon pathway by promoting the phosphorylation of STAT1 and STAT2. Elevated linc00513 expression positively correlated with IFN score in SLE patients. Linc00513 expression was higher in active disease patients than those inactive ones. In conclusion, our data identify two functional promoter variants of linc00513 that contribute to increased level of linc00513 and confer susceptibility on SLE. The study provides new insights into the genetics of SLE and extends the role of lncRNAs in the pathogenesis of SLE.

Decreased Suppression and Increased Phosphorylated STAT3 in Regulatory T Cells are Associated with Benefit from Adjuvant PD-1 Blockade in Resected Metastatic Melanoma.

PD-1 blockade induces durable responses in patients with metastatic melanoma and prolongs relapse-free survival in patients with resected melanoma; however, current biomarkers do not consistently associate with patient responses. In this study, we investigated the impact of nivolumab therapy on peripheral blood regulatory T cells (Treg) and its relation to patient outcomes. Peripheral blood Tregs and conventional CD4+ T cells from patients with resected high-risk melanoma treated with adjuvant nivolumab were assessed for gene expression changes by RNA-seq. Percentages of circulating Tregs and phosphorylated-STAT3 (pSTAT3) expression levels were assessed by flow cytometry and validated in an independent cohort of active disease patients. Suppressive function of Tregs was assessed in allogeneic mixed lymphocyte reactions. Tregs from non-relapse patients had increased expression of proliferation associated genes. An increase in the proportion of circulating Tregs and pSTAT3 expression and a reduction in Treg-suppressive capacity were observed in non-relapsing, but not relapsing patient samples 13 weeks after starting treatment. In vitro blockade of PD-1 increased Treg percentages and pSTAT3 expression, and reduced Treg-suppressive function. PD-1 blockade also led to IL10 production by T cells, resulting in higher Treg proliferation. The addition of a STAT3 inhibitor ameliorated the increase in Tregs, enhanced suppressive function, and decreased T-cell IL10 production in vitro. These results demonstrate that induction of pSTAT3, reduced suppressive function, and a paradoxical increase in Treg proliferation are novel correlates of patient benefit from PD-1 blockade.

Active Immune Thrombocytopenia (ITP) Disease Is Characterised By a Reduced Treg:CD8 Effector T Cell Ratio Which Is Modulated By Thrombopoietin-Receptor Agonists (TPO-RA)

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated low platelet count and a skewed proinflammatory Th1/Th17 profile. However, little is known about the involvement of CD8+ cytotoxic T cells in ITP pathophysiology and whether they are regulated by regulatory T cells (Treg). Immunosuppressive therapy has been the mainstay treatment in ITP. More recently, Thrombopoietin receptor agonists (TPO-RA); Romiplostim (Romi) and Eltrombopag (EPAG), have been increasingly used to stimulate megakaryocytopoiesis to produce more platelets. TPO-RAs are reported to induce complete remission in up to 30% of cases, with limited understanding of their impact on the immune system. Here we describe changes in T cell subsets in patients with ITP: how these changes are affected by disease activity and how TPO-RA may induce remission through modulating the immune system.Methods: Multi-color flow cytometric panels were designed to characterize peripheral blood T cell subsets, including CD8+ T cell and Treg subsets, phenotypically as well as functionally through intracellular cytokine expression. To determine whether CD8+ cells were platelet specific, an IFNγ ELISpot assay was performed using platelets from a healthy donor and PBMC from both HC and patients. Forty patients with ITP were included: 13 were on Romi, 11 on EPAG and 16 on no treatment at the time of analysis. Of these 40 patients, 15 patients had active disease (AD) (platelet-count less than 30 x 109/L) and 25 had stable disease (SD) (> 30 x 109/L). These patients were compared with 26 age and gender-matched healthy controls (HC). Data were presented as median values; Mann Whitney U and Kruskal Wallis tests were used with Dunn's multiple comparisons correction; a P value of < 0.05 was considered significant.Results: CD4/CD8 T cell ratio was significantly lower in patients compared to HC [1.77 vs. 3.97; P value < 0.001]. CD45RA+CD62L- Terminally-differentiated CD8+ T cells were significantly higher in patients compared to HC [66.3% vs. 8.56%; P value < 0.001]. This finding was more prominent in AD patients than those with SD [66% vs. 44.4%; P value < 0.05]. This effector population is polyfunctional, expressing high levels of proinflammatory cytokines including TNFα, IFNγ and Granzyme B when compared to HC [P value < 0.05]. Additionally, this population lacks the exhaustion markers PD-1 and Tim-3. Furthermore, these cells were reactive to platelets showing higher IFNγ-producing cells when co-cultured with platelets.CD3+CD4+CD25hiCD127lo Treg frequency did not differ between patients and HC [P value>0. 05]. Treg functionality was preserved in these patients; no important changes were observed in their capacity to express interlukin2 intracellularly, nor in the cell surface receptor (CD25) [P value > 0. 05]. However, Tregs were significantly lower in AD patients with compared to SD patients [2.32% vs. 4.46%; P value < 0.05]. Treg function is interactive with other T cell subsets and depends on its relative abundance in relation to other subsets; The Treg/effector CD8+ T cell ratio was significantly lower in patients compared to HC [0.06 vs. 0.16; P value < 0.01] and was also significantly lower in AD compared to SD patients [0.03 vs. 0.09 ; P value < 0.05].EPAG-treated patients had a significantly lower effector CD8+ T cells compared to Romi-treated patients [42.4% vs 76.8%; P value <0.01]. Although EPAG did not have a direct effect on the frequency of Treg, the Treg/effector CD8+ T cell ratio was significantly lower than that in patients on Romi [0.04 vs. 0.11; P value < 0.05] because of the CD8+.T cell difference. The Treg/effector CD8+ T cell ratio in EPAG-treated patients was comparable to the ratio of HC [0.11 vs. 0.16; P value > 0.05].Conclusion: While Th1/Th2 cell ratio is often considered as driving ITP, these results demonstrate the involvement of cytokine secreting effector CD8+ T cells in the disease pathogenesis. The imbalance in immune tolerance is also highlighted in the form of a significant reduction in the Treg/effector CD8 T cell ratio. The differences seen in T cell subsets between EPAG- and Romi-treated patients suggest the potential for an additional, differential immunomodulatory effect between the two agents which is currently being explored.Acknowledgment: The authors wish to thank Prof James B Bussel for his insightful comments and support of this work. DisclosuresCooper:Amgen, Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

CLL Intraclonal Fractions Defined By Time Since Cell Birth/Division Promote a Leukemia-Supportive, Immune-Tolerant Microenvironment By Distinct Mechanisms

Chronic lymphocytic leukemia (CLL) cells actively participate in the formation of the tumor microenvironment (TME). The interplay of CLL cells and leukemia-supporting cells such as Th2 cells and regulatory T cells (Tregs) promotes a leukemia-supportive, immune-tolerant TME. In these supportive/tolerogenic niches of lymph nodes (LN) and bone marrow (BM), CLL cells slowly proliferate, and the rate of proliferation correlates with disease progression. However, how these dividing/recently-divided cells and other intraclonal CLL fractions interact with non-neoplastic cells to shape the TME to support growth and accelerate disease is unclear.To address these questions, matched LN and PB samples collected at day 13 after 2H2O ingestion from treatment-naïve patients (7 with stable disease and 7 with active disease) were sorted to determine in vivo growth rates of CLL cells within the proliferative fraction (PF, CXCR4DimCD5Bright), resting fraction (RF, CXCR4BrightCD5Dim) and intermediate fraction (IF, CXCR4IntCD5Int). In LN, PF cells had the highest 2H-DNA levels, and only the growth rate of PF but not IF or RF cells correlated with disease aggressiveness. In the PB, PF cells also had the highest growth rate; however, all 3 fractions (PF, IF, RF) had 2H-DNA levels that correlated with disease aggressiveness. Thus, PF cells from patients with aggressive clinical course undergo quicker transitions to the IF and then to the RF which might promote faster tissue homing and disease progression. Indeed, more PF cells from active than stable disease patients were found in the spleen (SP) and BM and less remained in PB, 18 hours post-injection into NSG mice.Gene expression profiling (GEP) was then performed on RF, IF, and PF from 7 paired PB and LN samples. GEP signatures of the PF from PB and LN were similar, consistent with 2H2O data that the PF are recent emigrants from TME. These GEP also inferred enhanced cell proliferation (CCND2, CDK2AP1), adhesion and motility (FERMT3, CD49d, CD11a, CD21), antigen presentation (CD1C), and promotion of T-cell trafficking (CCL3, CCL4) in LN CLL cells within the PF but not the IF or RF. Thus, CLL cells within the three intraclonal fractions might promote distinct biologic functions.To test this, we studied T cell responses stimulated by the CXCR4/CD5 fractions in vitro and in vivo. In an antigen-driven allo-MLR, the whole clone of CLL cells triggered the division of normal T cells, but PF induced the highest level of T-cell division that is ≥ 3 times more than any other fraction. Similarly, in an autologous polyclonal CD4 T cell response stimulated by anti-CD3/28 Dynabeads and IL-2, T-cell division was suppressed by unseparated CLL cells and each fraction. However, the least suppression was seen in T cells co-cultured with PF cells compared to those cultured alone or with IF or RF. In both settings, PF cells induced significantly more IL-4+ T cells, and RF cells triggered more Tregs. Similar numbers of Th1 cells were seen in all cultures. The RF and IF, but not the PF, produced the immunosuppressive cytokine IL-10.Finally, when dividing cases based on disease aggressiveness, significantly more T-cell division was triggered by PF and RF from active patients than stable patients; the percentages of Th1 and Th2 cells however were similar. These results were confirmed in vivo; in NSG mice injected with autologous T cells together with the PF, IF or RF sorted from 2 sets of active versus stable disease patients, PF from all 4 cases induced the highest levels of CLL B and T cell expansion in SP and BM, and RF from active but not stable disease patients triggered the growth of CLL T and B cells.In summary, CLL disease progression correlates with the rate of CLL cell division, and the rapidity that CLL cells home to the TME. The intraclonal fractions of CLL clones exhibit distinct biologic properties that can be further differentiated based on disease aggressiveness. This appears especially relevant for the development of a leukemia-supportive, immune-tolerant TME contributed by all 3 CXCR4/CD5 fractions, albeit by different mechanisms. The PF creates this by superior antigen presentation capacity and skewing T cell function to an immunosuppressive Th2 phenotype. For the IF and RF, this is done by inducing IL-10 secretion and amplifying Tregs. Together, these findings suggest the possibility of targeting specific subpopulations in CLL clones with distinct immunoregulatory modalities as a novel form of therapy. DisclosuresChen:Beigene: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.