Role of beta-defensin 2 and interleukin-4 receptor as stroke outcome biomarkers.

Acute ischemic stroke is a complex disease with huge interindividual evolution variability that makes challenging the prediction of an adverse outcome. Our aim was to study the association of bloodstream signatures to early neurological outcome after stroke, by combining a subpooling of samples strategy with protein array discovery approach. Plasma samples from 36 acute stroke patients (< 4.5 h from onset) were equally pooled within outcome groups: worsening, stability, and improvement (n = 3 pools of four patients each, for each outcome group). These nine pools were screened using a 177 antibodies library, and 35 proteins were found altered regarding outcome classification (p < 0.1). Processes of inflammation, immune response, coagulation, and apoptosis were regulated by these proteins. Ten representative candidates, mainly cytokines and chemokines, were assayed for replication in individual baseline plasma samples from 80 new stroke patients: ß-defensin2, MIP-3b, plasminogen activator inhibitor 1 active, ß-cell-attracting chemokine 1, Exodus-2, interleukin-4 receptor (IL-4R), IL-12p40, leukemia inhibitor factor, MIP-1b, and tumor necrosis factor-related weak inducer of apoptosis. Multivariate logistic regression analysis showed ß-defensin 2 (ORadj 4.87 [1.13-20.91] p = 0.033) and IL-4R (ORadj 3.52 [1.03-12.08] p = 0.045) as independent predictors of worsening at 24 h after adjustment by clinical variables. Both biomarkers improve the prediction by 19% as compared to clinical information, suggesting a potential role for risk stratification in acute thrombolyzed stroke patients. Early neurological deterioration after stroke is not easily predictable. The use of blood biomarkers might help in decision-making processes regarding this complication. By combining a sub-pooling of samples strategy with protein array discovery approach, we have found two new biomarkers: beta-defensin-2 and interleukin-4 receptor. Both biomarkers improve the prediction of poor-outcome over clinical variables in the acute phase of stroke.

Biomarkers of inflammation and endothelial dysfunction in stroke with and without sleep apnea.

OBJECTIVE: Although sleep apnea (SA) is a risk factor for ischemic stroke and an important prognostic factor in affected patients, the exact pathophysiological link between SA and stroke is unknown. We investigated whether the plasma concentration of biomarkers of inflammation and endothelial dysfunction, including soluble tumor necrosis factor receptor-1 and -2 (sTNF-R1 and sTNF-R2), tumor necrosis factor-ß (TNF-ß), soluble intercellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) are increased in patients with acute stroke and SA compared with stroke patients without SA. DESIGN/METHODS: In total, 76 patients with ischemic stroke admitted to the stroke unit were included in this study. Plasma concentrations of biomarkers were determined after CT scans on admission. All patients received cardiorespiratory polygraphy within the first 72 h after admission. In all patients, demographic data, National Institutes of Health Stroke Scale scores and cerebrovascular risk factors were assessed. RESULTS: An apnea-hypopnea index (AHI) ≥10/h was found in 37 of our patients (48.7%). In these patients with SA, sTNF-R1 and sTNF-R2 levels were significantly higher than in patients with an AHI lower than 10/h. TNF-ß, however, showed no significant difference between both groups, just like the soluble intercellular and vascular cell adhesion molecules sICAM-1 and sVCAM-1. CONCLUSIONS/RELEVANCE: SA is associated with raised levels of sTNF-R1 and sTNF-R2 in patients with acute ischemic stroke. Taking into account the established impact of these two markers on the causation and course of cerebrovascular disease, these proteins may be part of the pathophysiological pathway linking SA to stroke.

B-cell-activating factor receptor expression on naive and memory B cells: relationship with relapse in patients with rheumatoid arthritis following B-cell depletion therapy.

OBJECTIVES: To examine the expression of B-cell-activating factor receptor (BAFF-R) on naive CD27- and memory CD27+ B cells in normal individuals and patients with rheumatoid arthritis (RA) undergoing B-cell depletion therapy with rituximab. PATIENTS AND METHODS: BAFF-R expression on B-cell subsets was determined in normal controls (NC; n = 11), active patients with RA pre-rituximab (pre-RX; n = 15), relapsing patients either concordant for B-cell repopulation (C-R, n = 13) or discordant, with relapse more than 3 months after repopulation (D-R, n = 11) and patients in remission over 3 months postrepopulation (discordant non-relapsing (D-NR), n = 5). Serum BAFF was measured by ELISA and analysed using Mann-Whitney. RESULTS: There was no significant difference between NC, pre-RX and D-NR patients in %BAFF-R-positive B cells or mean fluorescence intensity (MFI) in naive and memory B cells. Relapsing patients had significantly lower MFI and %BAFF-R-positive cells in both naive and memory compartments from NC and pre-RX (C-R and D-R; p < 0.01). BAFF levels in pre-RX patients were within the normal range and did not correlate with BAFF-R expression in any patient group. D-NR patients had relatively lower proportions of pre and postswitch CD27+ B cells than pre-RX patients (D-NR vs pre-RX; p < 0.05 for both) and also lower numbers of postswitch B cells than D-R patients (D-NR vs D-R, p < 0.05). CONCLUSION: BAFF-R expression was significantly reduced on both naive and memory B cells in patients at relapse, regardless of the relationship with B-cell repopulation or serum BAFF levels. Re-establishment of active disease was also associated with an increase in class-switch recombination. Factors responsible for lower levels of BAFF-R may relate to altered thresholds for autoreactive B-cell generation at relapse in patients with RA.

Favorable impact of the interleukin-4 receptor allelic variant I75 on the survival of diffuse large B-cell lymphoma patients demonstrated in a large prospective clinical trial.

BACKGROUND: Recently published data indicate that host germline variations in immune genes can influence the outcome of lymphoma patients. Interleukin (IL)-4 and IL13 are crucial immune factors and may influence the course of the disease. Both cytokines signal through the interleukin-4 receptor (IL4R). Therefore, we investigated whether polymorphisms of IL4, IL13 and IL4R genes could predict the outcome of diffuse large B-cell lymphoma (DLBCL) patients. METHODS: In 228 DLBCL samples of the German High-Grade Non-Hodgkin's Lymphoma Study Group, the polymorphisms of IL4 (-524CT, rs2243250), IL13 (-1069CT, rs1800925) and IL4R (I75V, rs1805010; S503P, rs1805015; Q576R, rs1801275) were analyzed and the soluble interleukin-4 receptor (sIL4R) serum level was measured before the start of chemotherapy. RESULTS: Patients harboring IL4R V75 (IL4R(I75V-AG) and IL4R(I75V-GG)) had shorter overall survival (OS) (P = 0.044) and event-free survival (EFS) (P = 0.056) periods compared with I75 carriers (IL4R(I75V-AA)). Multivariate analysis adjusted to the International Prognostic Index revealed a relative risk of 1.9 for carriers of the IL4R V75 (P = 0.011) in relation to OS. DLBCL patients homozygous for the IL4R I75 and low sIL4R serum levels have the most favorable OS and EFS. CONCLUSIONS: These data support the role for host germline gene variations of immunologically important factors like the IL4R I75V gene variation to predict the survival in DLBCL patients.

Decreased B cell activating factor receptor expression on peripheral lymphocytes associated with increased disease activity in primary Sjögren's syndrome and systemic lupus erythematosus.

OBJECTIVE: To analyse B cell activating factor (BAFF) receptor (BAFF-R) expression on peripheral lymphocytes from patients with primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE). PATIENTS AND METHODS: Peripheral blood mononuclear cells from 20 patients with pSS, 19 patients with SLE and 15 controls were examined by flow cytometry to investigate BAFF-R mean fluorescence intensity (MFI) on lymphocytes. BAFF-R mRNA level from isolated blood B cells of nine patients with pSS and eight controls was assessed by real-time quantitative reverse transcription-PCR. BAFF serum level was determined by ELISA. RESULTS: In all subjects, BAFF-R was expressed on all naïve CD27- and memory CD27+ B-cells and was present on <0.5% of T cells. The expression of BAFF-R on B cells was significantly decreased in patients with pSS as compared with controls (MFI = 7.8 vs 10.6, p = 0.001), and was intermediate in patients with SLE (MFI = 9.5). Serum BAFF level was inversely correlated with BAFF-R MFI (p = 0.007), but not because of competition between endogenous BAFF (at observed concentrations in patients) and the monoclonal antibody (11C1) detecting BAFF-R. BAFF-R mRNA levels did not differ between patients with pSS and controls (p = 0.48). BAFF-R MFI decreased after overnight culture with recombinant human BAFF (from 32.5 to 25.4, p = 0.03). Contrary to the serum BAFF level, BAFF-R expression was correlated with extraglandular involvement in pSS and SLE Disease Activity Index. CONCLUSIONS: BAFF-R expression is reduced on peripheral B cells of patients with pSS and SLE. This down-regulation occurs through a post-transcriptional mechanism and could be the consequence of chronic increase in BAFF. BAFF-R levels on B cells could be a novel activity biomarker in autoimmune diseases.

Cytokine mapping of sera from women with preeclampsia and normal pregnancies.

INTRODUCTION: Preeclampsia is a pregnancy-specific syndrome. The immune system in preeclampsia is changed with an increased innate activity and there is a hypothesis of a shift towards Th1-type immunity. The aim of this study was to determine a spectrum of soluble immunological factors denoting different aspects of immune activation in third trimester sera from women with preeclampsia (N=15) and compare with levels in sera from normal pregnant women (N=15). MATERIAL AND METHODS: IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40, IL-13, IL-15, IL-17, IFN-alpha, IFN-gamma, TNF-alpha, GM-CSF, MIP-lalpha, MIP-1beta, MCP-1, eotaxin and RANTES were measured in serum using multiplex bead arrays. The levels of soluble CD14 and soluble IL-4 receptor were measured by enzyme-linked immunoassay (ELISA). RESULTS: Preeclamptic women had significantly increased levels of circulating IL-6 (p=0.002), IL-8 (p=0.003) and soluble IL-4R (p=0.037), compared to women with normal pregnancies. CONCLUSION: This study supports the hypothesis of increased inflammatory responses in preeclampsia, illustrated by the increased levels of IL-6 and IL-8. The finding of increased levels of soluble IL-4 receptor is an intriguing finding with several interpretations, which may partly support the hypothesis of a Th1 shift in preeclampsia.

Evaluation of selected peripheral blood leukocyte functions in patients with various forms of periodontal disease.

INTRODUCTION: Periodontitis (P) is an infectious disease that develops in the supporting tissues of the tooth. One of the risk factors leading to it may be dysfunction of some immune system cells. Therefore, the object of the study was to assess selected functions of peripheral blood leukocytes in patients with various forms of P. As leukocytes are able to secrete interleukin (IL)-4 and IL-6, concentrations of their soluble receptors and the expression of their membrane receptors were investigated. MATERIAL AND METHODS: Twenty generally healthy subjects with aggressive (AP)and chronic periodontitis (CP)were enrolled in the study. The control group consisted of 8 healthy subjects,with no changes in periodontium. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured. Levels of IL-4,IL-6,and their soluble receptors sIL-4R and sIL-6R were determined in the supernatant by ELISA. The expressions of cell surface IL-4R and IL-6R were assayed on PBMC using flow cytometry. RESULTS: No statistically significant differences were found in the selected parameters between people with periodontal disease and healthy controls. However, in subjects with AP, there was an increasing tendency in IL-6 concentration and IL-4R expression on PBMCs. CONCLUSIONS: Our results show that leukocytes play a significant part in P and their activity is probably lesion-dependent. Estimation of the cytokines secreted by leukocytes may facilitate differentiation and prognosis of the disease progression.

Interleukin-4 and interleukin-4 soluble receptor alpha levels in bronchoalveolar lavage from children with asthma.

BACKGROUND: In asthma there is increased expression of the Th2-type cytokine interleukin-4 (IL-4). IL-4 is important in immunoglobulin isotype switching to immunoglobulin E and adhesion of eosinophils to endothelium. OBJECTIVE: We hypothesized that levels of IL-4 in bronchoalveolar lavage (BAL) fluid would be increased in stable, atopic asthmatic children compared with controls and that levels of its physiologic inhibitor IL-4 soluble receptor alpha (IL-4sR alpha) would be correspondingly decreased. METHODS: One hundred sixteen children attending a children's hospital for elective surgery were recruited. A nonbronchoscopic BAL was performed, and IL-4 and IL-4sR alpha were measured in the BAL supernatants. RESULTS: There was no significant difference in IL-4 concentrations between atopic asthmatic children, atopic normal controls, and nonatopic normal controls [0.13 pg/mL (0.13 to 0.87) vs 0.13 pg/mL (0.13 to 0.41) vs 0.13 pg/mL (0.13 to 0.5), P = 0.65]. IL-4sR alpha levels were significantly increased in asthmatic patients compared with atopic controls [6.4 pg/mL (5.0 to 25.5) vs 5.0 pg/mL (5.0 to 9.9), P = 0.018], but not when compared with the nonatopic controls [5.2 pg/mL (5.0 to 10.6), P = 0.19]. CONCLUSIONS: Contrary to expectation, IL-4sR alpha levels are increased in BAL from stable asthmatic children compared with nonatopic controls, and we speculate that IL-4sR alpha is released by inflammatory cells in the airways to limit the proinflammatory effects of IL-4.

Distinct patterns of cytokine regulation in discrete clinical forms of Plasmodium falciparum malaria.

The pathogenesis of two of the most severe complications of Plasmodium falciparum malaria, cerebral malaria (CM) and severe malarial anaemia (SA) both appear to involve dysregulation of the immune system. We have measured plasma levels of TNF and its two receptors in Ghanaian children with strictly defined cerebral malaria (CM), severe malarial anaemia (SA), or uncomplicated malaria (UM) in two independent studies in an area of seasonal, hyperendemic transmission of P. falciparum. Levels of TNF, soluble TNF receptor 1 (sTNF-R1) and 2 (sTNF-R2) were found to be significantly higher in CM than in the other clinical categories of P. falciparum malaria patients. Levels of both receptors depended on clinical category, whereas only sTNF-R1 levels were significantly dependent on parasitemia. Detailed analysis of the interrelationship between these variables resolved this pattern further, and identified marked differences between the patient categories. While levels of TNF, sTNF-R1 and sTNF-R2 correlated with parasitemia in UM, this was not the case in CM and SA. Rather, there was a tendency towards high levels of TNF and its receptors in CM and low levels in SA without significant correlation to parasitemia in either category. This, and the fact that malaria-induced increases in plasma levels of IL-10 are much lower in SA compared to CM, suggest that distinct forms of dysregulation of the immune response to infection contribute to the pathogenesis of CM and SA.

Cytokines and soluble cytokine receptor induction after IL-12 administration in cancer patients.

This study shows that subcutaneous administration of increasing doses of IL-12, once a week, in 21 cancer patients increased the expression of cytokine genes (interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), IP-10, MIG, IL-10, IL-4) in peripheral blood mononuclear cells even at very low doses (30 ng/kg). Surprisingly, no circulating TNF-alpha or IL-4 could be detected in the plasma of patients treated with IL-12. However, a marked increase of soluble IL-4 receptor was demonstrated in the plasma of five of the six patients studied, which may represent an additional mechanism by which IL-12 inhibits the development of the Th2 response in vivo. A marked decline of IFN-gamma and IP10 induction was recorded after repeated cycles of IL-12. In contrast, in most patients IL-12 increased IL-10 expression with no subsequent decrease during the course of therapy, and even an earlier peak of IL-10 induction at the 6th cycle. In addition, a constant up-regulation of serum soluble IFN-gamma receptor levels was observed after each cycle of IL-12 treatment with a delayed peak compared with the IFN-gamma peak. The constant rise of IL-10 and soluble IFN-gamma receptor during IL-12 therapy may therefore contribute to the inhibition of IFN-gamma activity detected after repeated cycles of IL-12. Lastly, a marked heterogeneity of cytokine induction was observed from one patient to another, which appeared to be independent of the dose of IL-12 administered. These data may lead to a better understanding of the biological activity of IL-12 and the in vivo mechanisms of its regulation.