rTNF alpha facilitates human polymorphonuclear leukocyte adherence to fibrinogen matrices with mobilization of specific and tertiary but not azurophilic granule markers.

rTNF alpha facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen-coated surfaces in a concentration- and time-dependent manner. The adhesion was maximal with 1.0 nM rTNF alpha within 40-50 min at 37 degrees C. A monoclonal antibody (1B4) directed toward the beta 2-chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNF alpha induced adhesion. TNF alpha caused a time-dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of A alpha(1-21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNF alpha could be further stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and A alpha(1-21) production over time. Thus the rTNF alpha-facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.     

Burkholderia pseudomallei triggers altered inflammatory profiles in a whole-blood model of type 2 diabetes-melioidosis comorbidity

Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei. Type 2 diabetes (T2D) is the most common comorbidity associated with melioidosis. B. pseudomallei isolates from melioidosis patients with T2D are less virulent in animal models than those from patients with melioidosis and no identifiable risk factors. We developed an ex vivo whole-blood assay as a tool for comparison of early inflammatory profiles generated by T2D and nondiabetic (ND) individuals in response to a B. pseudomallei strain of low virulence. Peripheral blood from individuals with T2D, with either poorly controlled glycemia (PC-T2D [n = 6]) or well-controlled glycemia (WC-T2D [n = 8]), and healthy ND (n = 13) individuals was stimulated with B. pseudomallei. Oxidative burst, myeloperoxidase (MPO) release, expression of pathogen recognition receptors (TLR2, TLR4, and CD14), and activation markers (CD11b and HLA-DR) were measured on polymorphonuclear (PMN) leukocytes and monocytes. Concentrations of plasma inflammatory cytokine (interleukin-6 [IL-6], IL-12p70, tumor necrosis factor alpha [TNF-α], monocyte chemoattractant protein 1 [MCP-1], IL-8, IL-1β, and IL-10) were also determined. Following stimulation, oxidative burst and MPO levels were significantly elevated in blood from PC-T2D subjects compared to controls. Differences were also observed in expression of Toll-like receptor 2 (TLR2), CD14, and CD11b on phagocytes from T2D and ND individuals. Levels of IL-12p70, MCP-1, and IL-8 were significantly elevated in blood from PC-T2D subjects compared to ND individuals. Notably, differential inflammatory responses of PC-T2D, WC-T2D, and ND individuals to B. pseudomallei occur independently of bacterial load and confirm the efficacy of this model of T2D-melioidosis comorbidity as a tool for investigation of dysregulated PMN and monocyte responses to B. pseudomallei underlying susceptibility of T2D individuals to melioidosis.     

Polymorphism in the proximal promoter region of the perforin gene and its impact on the course of HIV infection

Cytotoxic T lymphocytes (CTLs) play an essential role in the control of viral replication during human immunodeficiency virus (HIV) infection. However, the efficacy of the CTL response varies between individuals. We tested the hypothesis that genetic polymorphisms in the lytic effector molecule perforin could influence the progression of HIV infection. The perforin gene was screened for single nucleotide polymorphisms (SNPs) by denaturing high-performance liquid chromatography (dHPLC). Correlations were sought between perforin genotype, perforin expression and lytic function of CD8+ T lymphocytes from HIV-positive patients. Association of perforin genotype with disease progression was investigated in 426 seroconverters enrolled in the French SEROCO cohort. AIDS-free survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test. Three SNPs were found in the proximal promoter region of the perforin gene: 63G (allelic frequency 0.029), 112G (allelic frequency 0.071) and 1012T (allelic frequency 0.070). The presence of the 1012T genotype correlated with fewer perforin+ cells among circulating CD8+ CTL. However, CTL lines from HIV(-positive) individuals heterozygous for the perforin 1012T SNP displayed normal lysis of target cells, and within the SEROCO cohort, patients heterozygous for the 1012T SNP showed normal disease progression. However, 1012T/T homozygotes showed a tendency towards slower disease progression (P = 0.08). In conclusion, polymorphism in the perforin gene is limited, and although the 1012T genotype appears to influence perforin expression, it was not conclusively associated with disease progression in HIV infection.     

The association of CD18 alleles with anti-myeloperoxidase subtypes of ANCA-associated systemic vasculitides

Wegener granulomatosis (WG), microscopic polyangiitis (MP), and Churg Strauss syndrome (CSS) are rare systemic autoimmune disorders. Common features are anti-neutrophil cytoplasmic antibodies (ANCA) in patient sera. Whereas WG patients show mainly anti-proteinase 3 ANCA, MP and CSS patients typically present anti-myeloperoxidase (MPO) ANCA. ANCA play an important role in the pathogenesis in the vessel wall by activating polymorphonuclear cells (PMN) and increased adhesivity between PMN and endothelial cells via adhesion molecules. Here we investigated major adhesion molecules as predisposition factors via common polymorphisms in or in the vicinity of the candidate genes ICAM-1, e-selectin, PLAUR, CD11b, and CD18. A restriction fragment-length polymorphism in exon 11 of the CD18 gene was associated with MPO-ANCA(+) systemic vasculitis. Our data indicate that a common variant of the CD18 gene confers increased risk for CSS and MP, supporting that genetic factors are involved in the etiology and pathogenesis of ANCA-associated systemic vasculitides.     

The bactericidal/permeability-increasing protein (BPI) is membrane-associated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation

Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability-increasing protein (BPI). BPI binds to lipopolysaccharides (LPS), and exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy. BPI was found to colocate with myeloperoxidase (MPO), a marker for azurophil granules, and it also showed the same pattern of distribution as CD63, a transmembrane-anchored protein. This suggests that BPI is membrane-associated in the azurophil granules in neutrophils. Its presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow. Induction of selective release of azurophilic granules by the Na-ionophore monensin resulted in fusion of endosomes with azurophil granules, leading to the formation of large vacuoles containing MPO, CD63, and BPI. After phagocytosis of serum-treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments. The results show that BPI is present in azurophil granules, is probably primarily membrane-associated, and is relocated after activation, following the same route as MPO and CD63.     

IgE-dependent release of myeloperoxidase by neutrophils from allergic patients

BACKGROUND: The three forms of IgE receptor: the heterotrimeric high-affinity receptor for IgE (FcepsilonRI), the low-affinity receptor for IgE (FcepsilonRII/CD23) and the Mac-2/IgE-binding protein (epsilonBP), have previously been found on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present in the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes that might potentiate inflammation, such as myeloperoxidase (MPO). It is not known whether specific allergens are able to elicit MPO release by neutrophils from allergic patients. METHODS: Neutrophils were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. Also, the cells were challenged with allergens that the patients were not sensitive to. Neutrophils from normal subjects were also challenged with allergens. RESULTS: The in vitro challenge of neutrophils with allergens that the patients were sensitive to elicited a release of MPO by these cells. The in vitro activation of neutrophils was highly allergen-specific, in such a way that allergens other than those accounting for clinical symptoms did not evoke MPO release, and allergens were ineffective on neutrophils from healthy donors. CONCLUSION: An IgE-dependent mechanism might promote MPO release by neutrophils at allergic sites.     

Functional polymorphisms in CD86 gene are associated with susceptibility to pneumonia‐induced sepsis

Sepsis is an illness in which the body has a severe response to bacteria or other germs. A bacterial infection in the body such as lungs may set off the response that leads to the disease. CD86 (B7‐2) is expressed on various immune cells and plays critical roles in immune responses. Genetic polymorphisms in CD86 gene may affect the development of several diseases. Here, we evaluated the association between two CD86 polymorphisms (rs1915087C/T and rs2332096T/G) and susceptibility to pneumonia‐induced sepsis. CD86 rs1915087C/T and rs2332096T/G were identified in 186 pneumonia‐induced septic patients and 196 healthy controls in the Chinese population. Results revealed that subjects with rs1915087CT and TT genotypes had significantly lower risk of pneumonia‐induced sepsis than those with CC genotype [odds ratio (OR) = 0.58, 95% confidence interval (CI), 0.37–0.91, p = 0.017, and OR = 0.40, 95%CI, 0.21–0.76, p = 0.005]. However, prevalence of rs2332096GG genotype and G allele were significantly increased in patients than in healthy controls (OR = 2.75, 95%CI, 1.46–5.16, p = 0.001, and OR = 1.65, 95%CI, 1.21–2.24, p = 0.001]. We further investigated functions of these two polymorphisms by assessing gene expression in peripheral blood mononuclear cells and in monocytes. Data showed subjects carrying rs2332096GG genotype had significantly decreased level of CD86 in monocytes than those carrying rs2332096TT genotype. These results indicate that CD86 polymorphisms are associated with susceptibility to pneumonia‐induced sepsis and may affect gene expression in monocytes.     

Genetic polymorphisms in molecules of innate immunity and susceptibility to infection with Wuchereria bancrofti in South India

A pilot study was conducted to determine if host genetic factors influence susceptibility and outcomes in human filariasis. Using the candidate gene approach, a well-characterized population in South India was studied using common polymorphisms in six genes (CHIT1, MPO, NRAMP, CYBA, NCF2, and MBL2). A total of 216 individuals from South India were genotyped; 67 normal (N), 63 asymptomatic microfilaria positive (MF+), 50 with chronic lymphatic dysfunction/elephantiasis (CP), and 36 tropical pulmonary eosinophilia (TPE). An association was observed between the HH variant CHIT1 genotype, which correlates with decreased activity and levels of chitotriosidase and susceptibility to filarial infection (MF+ and CP; P = 0.013). The heterozygosity of CHIT1 gene was over-represented in the normal individuals (P = 0.034). The XX genotype of the promoter region in MBL2 was associated with susceptibility to filariasis (P = 0.0093). Since analysis for MBL-sufficient vs insufficient haplotypes was not informative, it is possible the MBL2 promoter association results from linkage disequilibrium with neighboring loci. We have identified two polymorphisms, CHIT1 and MBL2 that are associated with susceptibility to human filarial infection, findings that merit further follow-up in a larger study.     

Complement receptor 1 gene polymorphisms in sarcoidosis

Sarcoidosis is likely to result from exposure of genetically susceptible hosts to environmental agents. Erythrocyte (E) complement receptor 1 (CR1) is a membrane protein mediating the transport of immune complexes (ICs) to phagocytes, and at least three polymorphisms on the CR1 gene are related to erythrocyte surface density of CR1 molecules, in turn related to the rate of IC clearance from circulation. We hypothesized that sarcoidosis could be associated with increased frequency of the CR1 gene alleles coding for reduced CR1/E ratio. We studied 91 sarcoid patients and two control groups: 94 healthy volunteers and 71 patients with chronic obstructive pulmonary disease (COPD). Three polymorphic sites of CR1 gene, His1208Arg, intron 27 HindIII/RFLP, and Pro1827Arg, were analyzed. The three polymorphisms were in linkage disequilibrium. The GG genotype for the Pro1827Arg (C(5507)G) polymorphism was significantly associated with sarcoidosis in comparison to both control groups (odds ratio [OR] = 3.13; 95% confidence interval [CI] 1.49-6.69 versus healthy control subjects, and OR= 2.82, 95% CI 1.27-6.39 versus COPD control subjects). The same genotype was particularly associated to disease in females (OR = 7.05; 95% CI 3.10-16.61 versus healthy control subjects). These findings agree with speculations on the role of CR1 gene as a possible susceptibility factor.     

Studies on the association of Fc gamma receptor IIA,IIB, IIIA and IIIB polymorphisms with rheumatoid arthritis in the Japanese: evidence for a genetic interaction between HLA-DRB1 and FCGR3A

We recently detected a new single nucleotide polymorphism of FcgammaRIIB gene, which alters an amino acid within the transmembrane domain from Ile to Thr (I232T), and its association with SLE in the Japanese. This study was performed to examine whether FCGR2B-I232T was associated with susceptibility to rheumatoid arthritis in the Japanese. At the same time, FCGR2A, 3A and 3B polymorphisms were also examined. Genotyping of FCGR2B-I232T, FCGR2A-H131R, FCGR3A-F176V and FCGR3B-NA1/2 polymorphisms were performed using genomic DNA. Association with RA was analyzed in 382 Japanese patients with RA and 303 healthy individuals using a case-control approach. In addition, the same groups of patients and controls were genotyped for HLA-DRB1 to examine possible interaction with FCGR genes. Significantly different distribution of genotype, allele carrier and allele frequencies was not observed between patients with RA and healthy individuals in any of the four polymorphisms. When the subjects were stratified according to the carriage of HLA-DRB1 shared epitope (SE), significant increase of FCGR3A-176F/F genotype was observed in SE positive patients compared with SE positive healthy individuals (P=0.009, P(corr)=0.07). In conclusion, FCGR3A-176F/F genotype was considered to confer risk through genetic interaction with HLA-DRB1 SE.     

Natural and disease associated anti-myeloperoxidase (MPO) autoantibodies

Myeloperoxidase (MPO) is a cationic protein present in primary azurophilic granules of neutrophils and monocytes. MPO produces a highly deleterious reactive oxygen species, the hypochlorous acid (HOCl), using hydrogen peroxide (H(2)O(2)) and chloride ions as substrate. Anti-MPO antibodies (Abs) are present in 70% of the cases in patients with microscopic polyangiitis (MPA), a small-sized vessel vasculitis. Anti-MPO Abs from patients with MPA can trigger the release of MPO by neutrophils and monocytes. Anti-MPO Abs can activate MPO to generate an oxidative stress deleterious for the endothelium. Thus, we recently demonstrated that MPA sera with anti-MPO Abs activated MPO in vitro, and generated hypochlorous acid, whereas sera from MPA patients with no anti-MPO Abs or healthy individuals did not. Both hypochlorous acid production and endothelial lysis were abrogated by N-acetylcysteine (NAC), an antioxidant molecule. Thus, anti-MPO Abs could play a pathogenic role in vivo by triggering an oxidative burst leading to severe endothelial damages.     

血管紧张素转换酶基因多态性与慢性阻塞性肺病易感性的关系

探讨ACE基因插入 (insertion ,I)与缺失 (deletion ,D)多态性对COPD易感性、气道重构和肺功能损害程度的影响。从外周血白细胞中提取人类基因组DNA ,应用血管紧张素转换酶 (ACE)基因第 16内含子多态区两侧序列作为引物 ,采用PCR方法测定 12 2例COPD患者、15 9例健康人的ACE基因型。用多元线性逐步回归分析纠正性别、年龄等影响因素后确定独立危险因子。COPD组中DD基因型的分布频率显著高于正常对照组 (分别为 0 4 7、0 16 ,P<0 0 5 ) ;D等位基因频率也高 (分别为 0 6 2、0 4 3,P <0 0 5 ) ;DD型COPD患者肺通气功能损害较II型者为重 (P <0 0 5 )。ACE基因D型纯合子可能与COPD的遗传易感性相关 ,DD型基因可能是COPD发病新的独立危险因素。     

Polymorphism of glutathione S-transferase M1 and T1 gene LOCI in COPD

Chronic obstructive pulmonary disease (COPD) is a complex combination of signs and symptoms in patients with chronic bronchitis and emphysema, diseases that largely result from cigarette smoking. A little information is available for the underlying molecular mechanisms that are responsible for its occurrence. Polymorphisms in genes of xenobiotics metabolizing enzymes are expected to modulate individual responses to genotoxic carcinogens. Present study was a case–control study of COPD patients and healthy controls. Genetic polymorphisms of GSTM1 and GSTT1 genes in 50 COPD patients and 50 healthy controls were investigated using multiplex polymerase chain reaction–restriction fragment length polymorphism techniques to determine whether polymorphisms of these genes are linked to genetic susceptibility to COPD. All subjects were males and smokers. The frequency of GSTM1 homozygous null genotype was 28.0% in COPD cases when compared with controls (32.0%). The difference was not significant showing that risk of COPD was not associated with the GSTM1 null genotypes. The frequencies of homozygous null genotypes of GSTT1 were significantly higher in COPD cases as compared with controls (40% versus 14.0%) suggesting that the theta-glutathione S-transferases null genotype may be associated with the susceptibility to COPD. No significant differences were observed when comparisons were performed according to severity of disease and smoking for GSTM1 and GSTT1. It was also observed that COPD developed in the early age and with a shorter pack-year history in Indian population.     

IGF-I gene variability is associated with an increased risk for AD

Insulin-like growth factor I (IGF-I), a neuroprotective factor with a wide spectrum of actions in the adult brain, is involved in the pathogenesis of Alzheimer's disease (AD). Circulating levels of IGF-I change in AD patients and are implicated in the clearance of brain amyloid beta (Aβ) complexes. To investigate this hypothesis, we screened the IGF-I gene for various well known single nucleotide polymorphisms (SNPs) covering % of the gene variability in a population of 2352 individuals. Genetic analysis indicated different distribution of genotypes of 1 single nucleotide polymorphism, and 1 extended haplotype in the AD population compared with healthy control subjects. In particular, the frequency of rs972936 GG genotype was significantly greater in AD patients than in control subjects (63% vs. 55%). The rs972936 GG genotype was associated with an increased risk for disease, independently of apolipoprotein E genotype, and with enhanced circulating levels of IGF-I. These findings suggest that polymorphisms within the IGF-I gene could infer greater risk for AD through their effect on IGF-I levels, and confirm the physiological role IGF-I in the pathogenesis of AD.     

Promoter region polymorphism of the CD14 gene (C–159T) is not associated with psoriasis vulgaris

Keratinocytes of psoriatic skin show aberrant expression of membrane-bound CD14 (mCD14). In addition, soluble CD14 (sCD14) is elevated in the sera of psoriatic patients. The mechanisms leading to increased CD14 expression and secretion in psoriasis are poorly understood. A bi-allelic polymorphism in the promoter region of the CD14 gene controls CD14 expression on monocytes and sCD14 levels in the sera of healthy subjects. In this context, we explored the CD14 promoter region genotypes of 63 Finnish patients with psoriasis and 126 non-psoriatic controls using a new ARMS-PCR method. No differences in the CD14 genotype frequencies were found between the groups. Thus, our results suggest that the enhanced CD14 expression in psoriasis is not attributable to functional variants of CD14 (–159C/T).     

Association of single nucleotide polymorphisms in the IL-18 gene with sarcoidosis in a Japanese population

Interleukin-18 (IL-18) and interleukin-12 (IL-12) synergistically stimulate interferon-gamma (IFN-gamma) production from Th1 cells. The levels of serum IL-18 and IFN-gamma and bronchoalveolar lavage fluid IL-18 were elevated in patients with sarcoidosis. The polymorphisms of the IL-18 gene may play a possible role in expression regulation of the gene. We investigated the roles of the polymorphisms in the development of sarcoidosis. We examined two single nucleotide polymorphisms of the IL-18 gene in 119 patients with sarcoidosis and 130 healthy control subjects. Our results showed that the frequency of sarcoidosis patients with the CA genotype at position -607 was significantly higher than that with the AA genotype (OR = 2.200) and a significantly higher proportion of patients had the C allele at -607 compared with that of the controls (OR = 2.123). No significant differences were seen in the distribution of the genotypes or phenotype frequencies at position -137. There was no specific organ involvement associated with a certain genotype or phenotype. In IL-18 gene polymorphisms, the C allele at position -607 might be a genetic risk factor for sarcoidosis in this Japanese population.     

Promoter region polymorphism of the CD14 gene (C-159T) is not associated with psoriasis vulgaris.

Keratinocytes of psoriatic skin show aberrant expression of membrane-bound CD14 (mCD14). In addition, soluble CD14 (sCD14) is elevated in the sera of psoriatic patients. The mechanisms leading to increased CD14 expression and secretion in psoriasis are poorly understood. A bi-allelic polymorphism in the promoter region of the CD14 gene controls CD14 expression on monocytes and sCD14 levels in the sera of healthy subjects. In this context, we explored the CD14 promoter region genotypes of 63 Finnish patients with psoriasis and 126 non-psoriatic controls using a new ARMS-PCR method. No differences in the CD14 genotype frequencies were found between the groups. Thus, our results suggest that the enhanced CD14 expression in psoriasis is not attributable to functional variants of CD14 (-159C/T).     

The polymorphism ‐2548 G/A in leptin and severity of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by airway obstruction that is not fully reversible, and there is evidence of a hereditary component in COPD. We aimed to determine whether the polymorphisms ‐2548G/A of leptin (LEP) gene were associated with COPD and its severity in Chinese. A total of 456 subjects with COPD and 422 healthy controls from West China Hospital were enrolled in this study. COPD patients had been undergone a spirometry and a physical examination to refer the GOLD I–IV stages. The polymorphisms in the leptin promoter region at position ‐2548 G/A were detected by Polymerase chain reaction‐restriction fragment length polymorphism analysis. The genotypes and alleles were scored, and the frequencies of the alleles and genotypes in patients and controls were compared. A significantly higher risk for COPD was observed for carriers of the LEP ‐2548 AA genotype [odds ratio (OR) = 7.87, 95% confidence interval (CI) 4.19–14.77, P < 0.001] and carriers of the LEP ‐2548 GA genotype (OR = 2.98, 95% CI 1.57–5.66, P = 0.001). The LEP ‐2548 A allele: frequency was significantly higher in the patient group compared with the control group (OR = 2.75, 95% CI: 2.20–3.44, P < 0.001). We also found a significant relationship between leptin gene polymorphism and the severity of COPD. In the present case–control study, we found an association between the ‐2548 G/A variant of the leptin gene and pathogenesis, severity of COPD in the Chinese population. It suggests that leptin ‐2548 G/A should be used as a genetic marker of COPD severity.