Regulatory T cells (CD4CD25FoxP3) expansion in systemic sclerosis correlates with disease activity and severity

Background

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc).

Methods

Ten patients with SSc donated 20 ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4⁺ cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-β and IL-10 by activated CD4⁺ cells was measured by ELISA in culture supernatants.

Results

The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r = 0.71, p = 0.034 and r = 0.67, p = 0.044, respectively). ELISA-measured production of TGF-β and IL-10 by CD4⁺ cells was similar in patients and controls.

Conclusions

Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-β or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.     

Polymorphism of stromal cell-derived factor-1 selectively upregulates gene expression and is associated with increased susceptibility to coronary artery disease

Stromal cell-derived factor-1 (SDF-1) plays critical roles in vascular development and hematopoiesis. Here, we investigated the function of SDF-1 rs1801157G/A polymorphism in various immune cells and examined its association with susceptibility to coronary artery disease (CAD). Protein and mRNA levels of SDF-1 were tested in peripheral CD4+ T cell, CD8+ T cells, monocytes, and natural killer (NK) T cells from healthy donors with different genotypes of rs1801157G/A polymorphism. Prevalence of the polymorphism was compared between CAD patients and healthy controls. Data revealed that SDF-1 mRNA and protein were detectable in CD4+ T cells, CD8+ T cells, monocytes and NK T cells. Interestingly, both protein level and mRNA level of SDF-1 were significantly increased in the monocytes with rs1801157AA genotype, whereas the same phenomenon was not observed in the other three cell types. Blockage of CD14 completely inhibited the upregulation of SDF-1 in the monocytes with rs1801157AA genotype. Association analysis showed that frequencies of the rs1801157AA genotype and A allele were significantly higher in CAD cases than in controls (odds ratio [OR] = 2.28, 95% confidence interval [CI], 1.50–3.29, p < 0.0001, and OR = 1.46, 95% CI, 1.21–3.73, p < 0.0001, respectively). Also, prevalence of rs1801157AA genotype was further increased in cases with ST-elevation myocardial infarction (OR = 1.65, 95% CI, 1.04–2.56, p = 0.028). Our data suggest a novel pathway for regulating SDF-1 and a new risk factor for CAD.     

Association study of CD36 single nucleotide polymorphisms with essential hypertension in the Northeastern Han Chinese

We found that cluster determinant 36 (CD36) gene is up-regulated in essential hypertension (EH) patients in our former research, but the association between CD36 gene variations and EH has not yet been clearly demonstrated. The relationship between CD36 gene single nucleotide polymorphisms (SNPs) and EH in the northeastern Han Chinese was examined in the present study through direct sequencing and genotype-detection. A total of 589 unrelated northeastern Han Chinese including 276 with EH and 313 controls were studied. SNPs in exon 7, exon 13 and intron 4 were detected using PCR-sequencing. The genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). + 216T/C, + 273A/G, + 132C/T, + 217T/C, + 212T/G and + 233T/C polymorphisms were identified. Distributions of genotypes AA, GA and GG of + 273A/G polymorphism were significantly different between EH group and the control group (χ²: 9.056, p = 0.011) and G allelic frequency was higher in EH (p = 0.006, OR = 1.629, 95% CI [1.224–2.168]). Logistic regression analysis showed that + 273A/G polymorphism was closely associated with blood pressure (BP) after adjusting for ages. When subclassified by sex, the genotype distribution of + 273A/G (p = 0.011) and allelic frequency of G allele (p = 0.006) were significantly different between EH participants and controls in males, but not in females. Subgroup analysis performed by body mass index (BMI) suggested that the genotype distribution of + 273A/G and allelic frequency were significantly different in non-obese group and non-obese men, but the associations were not significant (non-obese group: p = 0.016, OR = 1.664, 95% CI [1.459–2.409]; non-obese men: p = 0.073, OR = 1.898, 95% CI [1.033–3.487]). + 273A/G polymorphism in CD36 gene was associated with EH, and + 273G could be an independent predictor.     

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-β and FoxP3 mRNA. Ιn active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.     

Allergen-specific B cell subset responses in cow’s milk allergy of late eczematous reactions in atopic dermatitis

B cells have regulatory functions in immune responses. Antigen-specific responses of B cell subsets by allergen stimulation ex vivo were examined in milk allergy of late eczematous reactions. Eight milk allergy subjects and 13 milk tolerant subjects were selected by DBPCFC. PBMCs were stimulated by casein ex vivo and stained for B cell subsets using monoclonal antibodies. CD19+ B cells unchanged from 8.7 ± 3.8% to 8.0 ± 5.1% (p = 0.504, n = 8) in the milk allergy group and decreased in the milk tolerant group from 8.5 ± 3.2% to 5.0 ± 1.6% (p = 0.001, n = 13). The fraction of apoptotic B cells in B cells significantly decreased 4.4 ± 3.1% to 1.3 ± 0.4% (p = 0.027, n = 4) in the allergy group and insignificantly increased from 2.8 ± 0.6% to 5.4 ± 2.6% (p = 0.059, n = 6) in the milk tolerant group. CD5+ regulatory B1 cell% in B cells decreased in milk allergy subjects from 36.2 ± 5.0% to 31.0 ± 5.7% (p = 0.010) and unchanged in milk tolerant subjects from 41.6 ± 10.2% to 43.8 ± 10.0% (p = 0.413). IL-10 producing CD19+CD5+ regulatory B cell% in CD19+CD5+ regulatory B cells significantly decreased from 24.9 ± 6.5% to 13.8 ± 5.6% (p = 0.002, n = 5) by casein stimulation in milk allergy group and unchanged from 44.8 ± 11.3% to 43.9 ± 10.0% (p = 0.297, n = 5) in the milk tolerant group. B cell subset responses to IL-4 and IL-5 were also similar in both groups. B cell subset changes seemed to have diagnostic value. Exact immunologic roles of regulatory CD5+ B1 cells need further investigation.     

Analysis of CD95 and CCR7 expression on circulating CD4+ lymphocytes revealed disparate immunoregulatory potentials in systemic lupus erythematosus

Emerging data have implicated a critical role for CD4 in the pathogenesis of systemic lupus erythematosus (SLE). This study was designed to delineate the contribution of CD4⁺ T cells in the pathogenesis of SLE disease. Forty-four patients (3 male: 41 female) and 20 healthy volunteers (4 male: 16 female) were included in the study. CD4⁺ lymphocytes analysis was done using three-color flow cytometry with antibodies against human-CD95, a prototype cell death receptor, and the chemokine receptor-7 (CCR7) after gating for lymphocytes based on the forward and side scatter. Serum levels of IL-6, IL-12, IL-17, TNF-α and IL-10 cytokines were assayed using ELISA. Disease activity was assessed using the SLE disease activity index (SLEDAI). Based on the expression of CCR7 and CD95, CD4⁺ lymphocytes were subdivided into three particular subsets; CD4⁺CD95⁺CCR7⁺ cells, CD4⁺CD95⁻CCR7⁺ cells and CD4⁺CD95⁺CCR7⁻ cells. Percentage of CD4⁺CD95⁺CCR7⁺ cell subset was significantly higher in patients with SLE with active disease (SLEDAI > 6) and inactive (SLEDAI < 6) as compared with controls (P = 0.005), and it showed a significant positive correlation with ANA titer (P = 0.01), and a negative correlation with WBCs count (P = 0.001). CD4⁺CD95⁺CCR7⁻ cell subset was significantly higher in active SLE patients in comparison to patients with inactive disease and controls (P = 0.05, P = 0.005 respectively), and it correlates positively with SLEDAI, IL-6 and IL-17 levels (P = 0.001, 0.05, 0.01 respectively), and negatively with blood WBCs counts (P = 0.001). The third CD4⁺CD95⁻CCR7⁺cell subset was found significantly lower in SLE patients compared with controls, and it was found negatively correlated with IL-10, IL-6, and IL-17. The results show that CD4⁺CD95⁺subset lacking expression of CCR7 is associated with cell mediated inflammatory response as manifested by its correlation with signs of inflammation, inflammatory cytokines and disease activity index. Whereas, CD4⁺CD95⁺CCR7⁺ correlate more with antibody immune responses as manifested by association with serum ANA. These data suggest disparate roles of these cell subsets in the pathophysiology of SLE. A better understanding of the characteristics of CD4 cell subsets may shed light on the pathogenesis of autoimmune diseases, particularly SLE.     

Changes of frequency and expression level of CD161 in CD8+ T cells and natural killer T cells in peripheral blood of patients with systemic lupus erythematosus

Immunologic abnormalities of natural killer (NK) cells and T cells play a role in the pathogenesis of systemic lupus erythematosus (SLE). CD161 is expressed on most of the NK cells and on some T cells. The quantities of CD161-expressing cells and expression levels of CD161 were analyzed in T cells and NK cells from patients with SLE compared with normal controls. The expression of CD161 on NK cells, NKT cells, CD4⁺ T cells, and CD8⁺ T cells in peripheral blood from patients with inactive SLE and active SLE, and from the normal controls group were determined using flow cytometry. The frequency and expression level of CD161 in the lymphocyte subsets and its relationship with the quantity of regulatory T cells, anti-double stranded DNA antibody, and the titer of antinuclear antibody were evaluated. Both the percentages of the CD161⁺ subpopulation and the mean fluorescence intensities (MFIs) of CD161 in CD8⁺ T cells and NKT cells decreased significantly in SLE patients compared with normal controls (P < .001). The CD161 expression in CD8⁺ T cells and NKT cells also decreased in the anti-dsDNA (+) group (P < .05). The counts of Treg cells were lower in SLE patients and were weakly correlated with the percentage of the CD161 subpopulation (r = 0.229, P = .016) and the MFIs of CD161 expression in CD8⁺ T cells (r = .232, P = .014). The frequencies and levels of CD161 expression on CD8⁺ T cells and NKT cells were reduced in SLE patients, suggesting that an abnormality of these cells was related to the pathogenesis of SLE.     

microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients

Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs’ function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25^hi+ and CD127− by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p = 0.05) and decreased expression of both miRNA-342 (p < 0.0001) and miRNA-191 (p = 0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.     

Alterations of cholesterol precursor levels in Alzheimer's disease

Cerebral and extracerebral cholesterol metabolism are altered in Alzheimer's disease (AD) as indicated by reduced plasma levels of the cholesterol elimination products 24S-hydroxycholesterol, which is of cerebral origin, and of 27-hydroxycholesterol, which is formed extracerebrally. However, it has to be evaluated, if changes of cholesterol metabolism in the whole body or in the CNS are exclusively due to the altered elimination of cholesterol or are also due to altered de novo synthesis in AD. We investigated CSF and plasma levels of cholesterol and of its precursors lanosterol, lathosterol and desmosterol in AD patients and non-demented controls. We found CSF levels of cholesterol (p = 0.011), absolute levels of all investigated cholesterol precursors (each p < 0.001) and ratios of cholesterol precursors/cholesterol (each < 0.01) to be lower in AD patients as compared to controls. In plasma, the absolute levels of lanosterol (p = 0.026) and lathosterol (p < 0.001) and the ratio of lathosterol/cholesterol (p = 0.002) but none of the other investigated parameters were reduced in AD patients (p > 0.1). Furthermore, ratios of desmosterol/lathosterol in CSF (p = 0.023) and plasma (p = 0.009) were higher in AD patients as compared to controls. Our data support the hypothesis that cholesterol metabolism is altered in AD and further suggest that especially cholesterol de novo synthesis within the CNS of AD patients might be reduced. These findings raise doubt on a beneficial effect of cholesterol lowering treatment in manifest AD.     

Numerical defects in CD8CD28 T-suppressor lymphocyte population in patients with type 1 diabetes mellitus and multiple sclerosis

Type 1 diabetes mellitus (T1D) and multiple sclerosis (MS) are organ-specific autoimmune diseases leading to an attack of auto-aggressive lymphocytes against the pancreatic β-cells and central nervous system, respectively. Using four-colour flow cytometry, T-lymphocyte populations having an important function in autoimmune processes were analyzed. T-regulatory cells (Treg) CD4⁺CD25⁺CD127^low, T-suppressor cells (Ts) CD8⁺CD28⁻, activated helper CD4⁺CD25⁺CD127⁺ and cytotoxic CD8⁺CD25⁺ T-cells and also naive CD4⁺CD45RA⁺ and memory T-cells CD4⁺CD45RO⁺ were compared in the group of patients with T1D (n = 30), MS (n = 31) and in the group of healthy controls (n = 29). Significant differences in Ts cells, activated helper and cytotoxic cells and also memory T-cells were recognized in the group of T1D patients compared to healthy controls. Ts population was significantly lowered in MS patients as well. However, no significant differences were noticed in Treg population. The observed data demonstrate significant differences among patients with T1D and MS in comparison to healthy individuals.     

The skewed frequency of B-cell subpopulation CD19+CD24 hiCD38 hi cells in peripheral blood mononuclear cells is correlated with the elevated serum sCD40L in patients with active systemic lupus erythematosus

CD19⁺CD24^hiCD38^hi cells play an essential role in maintaining immune homeostasis. CD40 signaling is involved in regulating the induction and function of CD19⁺CD24^hiCD38^hi cells. Changes in B-cell subpopulations and CD19⁺CD24^hiCD38^hi cells have been observed in systemic lupus erythematosus (SLE) patients. Whether changes in the B-cell subpopulation are related to the aberrant CD40 signaling in SLE patients remains unclear. In this study, we examined changes in the levels of CD19⁺CD24^hiCD38^hi cells and CD19⁺CD24^hiCD38^low cells in peripheral blood mononuclear cells and the serum level of soluble CD40 ligand (sCD40L) in 30 patients with SLE. Through routine biochemical assays and flow cytometry assay, we found that (1) the CD19⁺CD24^hiCD38^hi cell subset was upregulated in SLE patients compared to that in healthy controls (HCs) (P < 0.05); (2) the CD19⁺CD24^hiCD38^low cell subset was downregulated in SLE patients compared with that in HCs; and (3) CD38 expression was positively correlated with SLE manifestations and the serum sCD40L level (P < 0.05). In conclusion, the relative level of Bregs is significantly higher in SLE patients than in HCs and is positively correlated with disease activity and sCD40L level.     

Down-regulation of HLA class II and costimulatory CD86/B7-2 on circulating monocytes from melanoma patients

Antigen-presenting cells are crucial for the induction of an antigen-specific antitumoral immune response. Deteriorations in the expression pattern of cell surface molecules important for the presentation of antigens might therefore be indicative of an impaired immune response status in cancer patients. In the present study we investigated the expression of MHC class I and class II molecules, of the costimulatory molecules CD80/B7-1 and CD86/B7-2, of the adhesion molecule CD11c, and of the marker of activation CD71 on CD14⁺ peripheral blood monocytes (PBMs) from 144 melanoma patients in different stages of disease and 43 healthy controls, by flow cytometric analysis. We found a decreased expression of HLA-DR (p<0.0005), HLA-DQ (p=0.006), HLA-DP (p<0.0005), and CD86/B7-2 (p=0.001) on PBMs from melanoma patients compared with healthy controls, whereas no significant difference could be detected in the expression of HLA class I antigens and CD80/B7-1. This down-regulated expression was associated with disease progression. In contrast, CD71 expression was stage-dependently increased on PBMs from melanoma patients compared with healthy controls (p=0.024). No correlation was found between the PBM surface expression pattern and age, gender, tumor load, and current mode of therapy of the patients. The observed down-regulation of HLA class II and CD86/B7-2 on melanoma patients PBMs might reflect an ineffective antigen-presenting function contributing to an impaired antigen-specific immune response in these patients.Both authors S. Ugurel and D. Uhlig contributed equally to this work     

CD53, a suppressor of inflammatory cytokine production,is associated with population asthma risk via the functional promoter polymorphism − 1560 C>T

Background

In this study, the association of asthma with CD53, a member of the tetraspanin family, was assessed for the first time in a mechanism-based study.

Methods

Genetic polymorphisms of CD53 were analyzed in 591 subjects and confirmed in a replication study of 1001 subjects. CD53 mRNA and protein levels were measured in peripheral blood leukocytes, and the effects of the promoter polymorphisms on nuclear factor binding were examined by electrophoretic mobility shift assay. Cellular functional studies were conducted by siRNA transfections.

Results

Among tagging SNPs of CD53, the − 1560 C>T in the promoter region was significantly associated with asthma risk. Compared with the CC genotype, the CT and TT genotypes were associated with a higher asthma risk, with odd ratios of 1.74 (P = 0.009) and 2.03 (P = 0.004), respectively. These findings were confirmed in the replication study with odd ratios of 1.355 (P = 0.047) and 1.495 (P = 0.039), respectively. The − 1560 C>T promoter SNP had functional effects on nuclear protein binding as well as mRNA and protein expression levels in peripheral blood leukocytes. When CD53 was knocked down by siRNA in THP-1 human monocytic cells stimulated with house dust mite, the production of inflammatory cytokines as well as NFκB activity was significantly over-activated, suggesting that CD53 suppresses over-activation of inflammatory responses.

Conclusions

The − 1560 C>T SNP is a functional promoter polymorphism that is significantly associated with population asthma risk, and is thought to act by directly modulating nuclear protein binding, thereby altering the expression of CD53, a suppressor of inflammatory cytokine production.     

Characterisation of allergen-specific responses of IL-10-producing regulatory B cells (Br1) in Cow Milk Allergy

CD19+CD5+ regulatory B cells regulate immune responses by producing IL-10. IL-10-producing regulatory B cell (Br1) responses by allergen stimulation were investigated in human food allergy. Six milk allergy patients and eight milk-tolerant subjects were selected according to DBPCFC. PBMCs were stimulated by casein in vitro and stained for intracellular IL-10 and apoptosis. In response to allergen stimulation, Br1 decreased from 26.2 ± 18.3 to 15.5 ± 8.9% (p = 0.031, n = 6) in the milk allergy group and increased from 15.4 ± 9.0 to 23.7 ± 11.2% (p = 0.023, n = 8) in the milk-tolerant group. Apoptotic non-IL-10-producing regulatory B cells increased from 21.8 ± 9.3 to 38.0 ± 16.1% (p = 0.031, n = 6) in the milk allergy group and unchanged from 28.8 ± 13.8 to 28.0 ± 15.0% (p = 0.844, n = 8) in the milk-tolerant group. Br1 may be involved in the immune tolerance of food allergies by producing IL-10 and simultaneously undergoing apoptosis in humans. The exact roles for Br1 in immune tolerance needs to be further investigated.     

Decreased regulatory T‐cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4⁺/CD8⁺ ratio and CD4⁺CD25^hiFoxP3⁺ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4⁺/CD8⁺ ratio and Tregs in 82 patients and 50 controls. CD8⁺ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8⁺ T‐cell counts compared with stable GV patients (p = 0.001). The CD4⁺/CD8⁺ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4⁺/CD8⁺ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.     

SLE带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+FoxP3~+调节性T细胞的表达及相关性研究

目的:检测系统性红斑狼疮(systemic lupus erythematosus,SLE)合并带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+Fox P3~+调节性T细胞的表达及相关性,探讨其在SLE合并带状疱疹发病中的临床意义。方法:采用流式细胞术检测30例SLE患者、30例SLE合并带状疱疹患者及30例健康对照者外周血中CD4~+/CD8~+T淋巴细胞亚群表面CD28的表达及CD4~+CD25~+Fox P3~+Treg细胞的表达水平,并分析SLE合并带状疱疹患者外周血CD4~+CD28~+和CD4~+CD25~+Fox P3~+调节性T细胞表达的相关性。结果:SLE合并带状疱疹组患者急性期外周血CD4~+T淋巴细胞比率、绝对计数显著降低,CD4~+、CD8~+T淋巴细胞表面的CD28表达下调,CD4~+CD25~+Fox P3~+Treg细胞水平显著高于SLE组及健康对照组,SLE合并带状疱疹组患者外周血CD4~+CD25~+Fox P3~+Treg水平与CD4~+CD28~+水平成负相关(P均0.05)。结论:SLE合并带状疱疹患者CD4~+、CD8~+T细胞活化异常,CD4~+CD25~+Fox P3~+Treg细胞可能参与抑制了T细胞的活化。     

Expression of CD45 and proliferative response to CD3 as suitable classification markers of patients with gastric adenocarcinoma

25 patients with resectable gastric adenocarcinoma, subdivided according to the absence or presence of residual neoplasic disease (RND− or RND+, respectively), were studied. Cytofluorometric analysis and proliferative responses to mitogens was performed in peripheral blood mononuclear cells of patients. When compared to healthy subjects, the percentage of CD3-expressing cells was significantly reduced in both groups of patients studied (p<0.0001 in all instances). However, when CD45 is considered instead of (CD3, its expression is found to be significantly reduced only in the RND+ patients (72%±11), when compared with the control group (96±1%, p<0.0001). Likewise, cells from these patients significantly less proliferated when stimulated with monoclonal antibodies to CD3 than control cells (18,920±6,019 cpm vs. 42,697±1,798 cpm, p=0.0036); a difference not found if RND− patients (33,619±11,733 cpm) were considered. We propose that the low expression of CD45 and the poor response to CD3 are markers that are able to identify the subgroup of patients in whom the disease will tend to progress more rapidly.We also suggest the use of such markers as additional criteria for the classification of patients with gastric adenocarcinoma or to identify patients who require more aggressive therapeutic strategies.     

Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles (MP) plays an important role in etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells and platelets, exhibits high affinity for PS and has been shown to inhibit several of these PS-mediated pathophysiological processes. We determined plasma annexin A5 levels and MP-associated procoagulant activity, a measure of MP-PS exposure, in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty-five HbAA blood donors served as controls.Both annexin A5 and MP-PS were highest in HbSS patients (5.7 ng/mL, IQR 3.7-7.6 and 37.9 nM, IQR 31.9-69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7-7.6 and 20.9 nM, IQR 10.9-29.6) and healthy controls (2.5 ng/mL, IQR 1.4-4.4 and 13.1 nM, IQR 9.5-18.5) (p = 0.01 and p < 0.001, respectively). At presentation with a painful crisis, annexin A5 and MP-PS had increased in 16 of 17 patients (p = 0.001 and p < 0.001, respectively). Most interestingly, in 7 HbSS patients the proportional increase in MP-PS exposure was higher than the proportional increase in plasma annexin A5 concentration, leading to lower annexin A5/MP-PS ratio of HbSS patients during crisis than HbAA controls (0.0027 (0.0017-0.0049) vs 0.0048 (0.0027-0.0085), p = 0.05). In conclusion, patients with SCD have elevated plasma levels of annexin A5- and PS-exposing MP. During crisis both levels increase, but in most HbSS patients MP-PS exposure increases more than annexin A5. Future studies must address a potential role of annexin A5 in modulating PS-related pathophysiological processes in SCD.     

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus

Introduction

Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.

Methods

The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10⁻D16^low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.

Results

SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10⁻CD16^low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.

Conclusions

Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.