Chlamydia pneumoniae heat shock protein 60 enhances expression of ERK, TLR-4 and IL-8 in atheromatous plaques of coronary artery disease patients

Chlamydia pneumoniae heat shock protein (cHSP) 60 is produced during chronic chlamydial infection and activate innate immune and inflammatory responses thereby contributing to atherogenesis. However, to date there is no apparent signaling cascade delineated in human atherosclerotic plaques in C. pneumoniae positive coronary artery disease (CAD) patients. Atherosclerotic plaques were obtained from 40 CAD patients (28 men, 12 women) attending Department of Cardio Thoracic and Vascular Surgery Safdarjung Hospital, New Delhi. Atherosclerotic plaques were used for gene expression studies at RNA level by real-time PCR and to study expression of ERK1/2, JNK1/2, NF-kB, IkkB and MCP-1 at protein level by immunoblotting. Significantly higher (p < 0.001) RNA expression was found for IL-8, TLR-2/4, TGF-β, ICAM1, VCAM1 and MAPKinase genes, whereas significantly lower (p < 0.001) RNA expression for SMAD4, IkkB, BRCA1 and IL-10 was detected in cHSP60-positive atheromatous plaque of CAD patients. Moreover, at proteins level pERK1/2 (p = 0.05), NF-kB (p = 0.017), MCP-1 (p = 0.011) was higher and IkkB expression was lower (p = 0.038) in cHSP60-positive atheromatous plaque of CAD patients. This study by using human atheromatous plaques at RNA and protein levels demonstrated higher expression of TLR-2/4, IL-8, ICAM1, VCAM1, ERK1/2 and NF-kB in cHSP60-positive CAD patients.     

Toll-like receptor 2 and 4 induced interleukin-19 dampens immune reactions and associates inversely with spondyloarthritis disease activity

Spondyloarthritis (SpA) is a group of immune mediated inflammatory diseases affecting joints, gut, skin and entheses. The inflammatory process involves activation of Toll-like receptor (TLR)-2 and TLR-4 and production of cytokines and chemokines such as monocyte chemoattractant protein 1 (CCL2/MCP-1). This proinflammatory chemokine recruits monocytes to sites of inflammation and is central in the development of several immune-mediated inflammatory diseases. Interleukin (IL)-19 is a member of the IL-10 family of cytokines. IL-19-deficient mice are more susceptible to innate-mediated colitis and develop more severe inflammation in response to injury. In this work, we studied inducers of IL-19 production and effect of IL-19 on the production of CCL2/MCP-1 and proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and in PBMCs and synovial fluid mononuclear cells (SFMCs) from SpA patients. Further, we measured IL-19 in plasma from HCs and in plasma and synovial fluid from SpA patients. Constitutive IL-19 expression was present in both PBMCs and SFMCs and the secretion of IL-19 was increased by TLR-2 and TLR-4 ligands. Neutralizing IL-19 in HC PBMCs and SpA SFMCs resulted in increased production of CCL-2/MCP-1. IL-19 concentrations were decreased in synovial fluid compared with plasma and associated inversely with disease activity in SpA. SpA SFMCs produced less IL-19 in response to LPS compared with HC PBMCs. These findings indicate that IL-19 production is diminished in SpA. Taken together, impaired IL-19 control of the innate immune system might be involved in the pathogenesis of SpA.     

Levels of soluble adhesion molecules among normal and hypertensive type 2 diabetic patients

BACKGROUND: Hypertension and type-2 diabetes affect endothelial function, which in turn increases the expression of soluble adhesion molecules and lead to the development of vascular damage. The aim of this study was to assess soluble adhesion molecule levels among normotensive and hypertensive diabetic patients. MATERIAL AND METHODS: Serum levels of soluble VCAM1, ICAM1 and e-selectin were measured in 80 type-2 diabetic patients, (40 normotensive and 40 hypertensive), and in 40 normotensive non-diabetic subjects by ELISA (RyDSystems Minneapolis). Statistical analysis was performed with ANOVA. RESULTS: Among diabetic patients, levels of all three soluble adhesion molecules were significantly increased when compared with non-diabetic patients (p < 0.001 for all three molecules), In diabetic hypertensive patients, higher levels of ICAM1 were detected in comparison to normotensive diabetic patients (316 vs. 295 ng/ml p < 0.01), VCAM1 and e-selectin levels were not different between diabetic patients with and without hypertension. CONCLUSIONS: Diabetes is associated with increased levels of soluble adhesion molecules, suggesting a role of these molecules may play in endothelial damage. ICAM1 is further increased when hypertension and diabetes are present. The latter may explain why diabetic-hypertensive patients displayed more complications than normotensive patients.     

Production of tumor necrosis factor and interleukin-1 by macrophages from human atheromatous plaques.

The production of cytokines by atheromatous plaque macrophages from human endarterectomy tissue was assessed in vitro by short-term cell culture and in situ by immunohistology. Macrophages were isolated from plaques of 14 patients undergoing carotid endarterectomy and 7 patients undergoing reconstructive procedures on atheromatous distal aortic and femoral arteries. Tumor necrosis factor (TNF) and interleukin 1 (IL-1) production by plaque macrophages and blood monocytes isolated concurrently from these patients was assessed. TNF release by macrophages from carotid plaques (0.39 +/- 0.12 ng/10(6) cells/24 hours) was significantly augmented compared to the release by corresponding blood monocytes (0.014 +/- 0.011 ng/10(6) cells/24 hours, P = 0.03), and by macrophages from noncarotid lesions (0.038 +/- 0.036 ng/10(6) cells/24 hours, P < 0.04). Cellular TNF expression by macrophages within carotid plaques was also more prominent than in noncarotid lesions. By contrast, IL-1 production by plaque macrophages from both carotid and noncarotid plaques was not augmented compared to blood monocytes, and only infrequent and low-intensity labeling for IL-1 was present on macrophages within plaques from either group. These results provide functional and immunohistological evidence for increased production of TNF but not IL-1 by activated macrophages, indicating local and selective augmentation of cytokine production within carotid plaques. This suggests that macrophages play an active role in the inflammatory response within atheromatous carotid plaques.     

Chlamydial Heat Shock Protein 60 Induces Acute Pulmonary Inflammation in Mice via the Toll-Like Receptor 4- and MyD88-Dependent Pathway

Heat shock protein 60 derived from Chlamydia pneumoniae (cHSP60) activates Toll-like receptor 4 (TLR4) signaling through the MyD88 pathway in vitro, but it is not known how cHSP60 contributes to C. pneumoniae-induced lung inflammation. We treated wild-type (WT), TLR2^−/−, TLR4^−/−, or MyD88^−/− mice intratracheally (i.t.) with recombinant cHSP60 (50 μg), UV-killed C. pneumoniae (UVCP; 5 × 10⁶ inclusion-forming units/mouse), lipopolysaccharide (2 μg), or phosphate-buffered saline (PBS) and sacrificed mice 24 h later. Bronchoalveolar lavage (BAL) was obtained to measure cell counts and cytokine levels, lungs were analyzed for histopathology, and lung homogenate chemokine concentrations were determined. Bone marrow-derived dendritic cells (BMDDCs) were generated and stimulated with live C. pneumoniae (multiplicity of infection [MOI], 5), UVCP (MOI, 5), or cHSP60 for 24 h, and the expression of costimulatory molecules (CD80 and CD86) was measured by fluorescence-activated cell sorting. cHSP60 induced acute lung inflammation with the same intensity as that of UVCP-induced inflammation in WT mice but not in TLR4^−/− or MyD88^−/− mice. cHSP60- and UVCP-induced lung inflammation was associated with increased numbers of cells in BAL, increased neutrophil recruitment, and elevated BAL interleukin-6 (IL-6) levels. Both cHSP60 and UVCP induced IL-6 release and CD80 and CD86 expression in WT cells but not in MyD88^−/− BMDDCs. cHSP60 stimulated DC activation in a TLR4- and MyD88-dependent manner with an intensity similar to that induced by UVCP. These data suggest that cHSP60 promotes lung inflammation and DC activation via TLR4 and MyD88 and therefore may play a significant role in the pathogenesis of C. pneumoniae-induced chronic inflammatory lung diseases.Chlamydia pneumoniae is an obligate intracellular gram-negative bacterium that causes upper and lower respiratory tract infections throughout the world; it is responsible for 10% of community-acquired pneumonia (17). The estimated number of cases of C. pneumoniae-induced pneumonia is 300,000 cases per year. Approximately 50% of young adults and 75% of the elderly population have serological evidence of previous infection (8, 17). In addition to acute infection, there is increasing evidence that implicates C. pneumoniae and heat shock protein 60 derived from C. pneumoniae (cHSP60) in the pathogenesis of atherosclerosis and chronic inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and bronchitis (7, 9, 10, 29).The three major effector antigens of Chlamydia are lipopolysaccharide (LPS), the major outer membrane protein (MOMP), and cHSP60 (16). Chlamydia pneumoniae has a unique biphasic life cycle. The metabolically inactive infectious elementary bodies (EBs) attach and enter the host cell, where they differentiate into the metabolically active reticulate bodies. The reticulate bodies replicate within the expanding endosome, resulting in the development of characteristic cytoplasmic inclusions. These persistent intracellular inclusions contain increased quantities of cHSP60, a highly immunogenic protein that has been implicated in the stimulation of the innate immune system and the pathogenesis of chronic inflammatory lung diseases (9). Chlamydia can achieve a state of chronic intracellular infection in which they remain viable but quiescent and do not replicate. During such persistent infections, cHSP60 is abundantly produced and might stimulate the innate immune and inflammatory responses, thus contributing to chronic inflammatory lung diseases (9, 14, 16).The involvement of the cHSP60 of C. trachomatis in the immunopathogenesis of trachoma, pelvic inflammatory disease, and tubal infertility is well established, but the role of cHSP60 in the pathogenesis of C. pneumoniae-induced infections and chronic lung disease has not been discerned (25, 26, 32). Evidence that cHSP60 indeed plays a role comes from studies showing the presence of anti-cHSP60 antibodies (Abs) in adult patients with asthma who developed symptoms after an acute respiratory illness (11). However, the cHSP60 responses might simply represent a marker for exposure to chlamydial infection. Conversely, Huittinen et al. showed an independent association between cHSP60 immunoglobulin A (IgA) Abs and the severity of allergic asthma, even after controlling for the effects of migration inhibition factor IgA Abs to C. pneumoniae (12). Importantly, these authors demonstrated that only Abs against cHSP60, and not those against C. pneumoniae EBs, were associated with chronic airway disease (31). These results clearly suggest the possibility that the persistent presence of cHSP60 after C. pneumoniae infection in the lungs participates in the immunopathology of chronic airway disease. Furthermore, several studies have linked cHSP60 with asthma and decreased pulmonary functions (11, 12, 34).Previous reports indicate that C. pneumoniae infection and cHSP60 alone can activate the innate immune system through Toll-like receptors (TLRs), one of the sensors of innate immunity (5, 24, 28, 30, 33). Live Chlamydia pneumoniae infection can activate the innate immune response by both TLR2 and TLR4, and we have demonstrated a critical role of MyD88 in host defense against C. pneumoniae (22, 28). Some studies with dendritic cells (DCs) indicate that the immune recognition of live Chlamydia pneumoniae was dependent largely on TLR2 and only to a lesser extent on TLR4 (24, 27). However, we reported that cHSP60 is a potent inducer of vascular endothelial cells (EC) and macrophage inflammatory responses, and these inflammatory effects are mediated through the innate immune receptor complex TLR4-MD2 (4). These responses proceed via the MyD88-dependent signaling pathway.In this study, we show that the i.t. administration of cHSP60 induces acute lung inflammation in a TLR4- and MyD88-dependent manner with severity comparable to that seen after the inoculation of UVCP in mice. We also demonstrate that cHSP60 induces lung inflammation and stimulates DC activation and maturation in a MyD88-dependent manner and with intensity similar to that induced by UVCP. These data suggest that cHSP60 is a key inflammatory component of Chlamydia pneumoniae that induces lung inflammation and DC activation, and therefore it may play a significant role in the pathogenesis of C. pneumoniae-induced acute and chronic inflammatory lung diseases.     

细胞因子白细胞介素-1α、S100β 在阿尔茨海默病不同类型老年斑中的表达

目的 探讨小胶质细胞、星形胶质细胞及其所产生的炎性介质白细胞介素(IL)-1α、S100β在阿尔茨海默病（Alzheimer's disease,AD）不同类型的老年斑形成和进展中的意义。方法 从北京医院病理科1982至2008年尸检资料中选出AD 34例,所有脑标本的海马部切片均进行IL-1α/β-淀粉蛋白、S100β/β-淀粉蛋白免疫组织化学双标记染色。结果 IL-1α/β-淀粉蛋白及S100 β/ β-淀粉蛋白免疫组织化学双标记染色均显示老年斑有4种类型,即弥漫性非神经突斑、弥漫性神经突斑、有致密核心的神经突斑、有致密核心的非神经突斑。在4种类型的老年斑中,与弥漫性神经突斑相关的小胶质细胞和星形胶质细胞数量最多,分别为(7.29±3.04)和(6.49±2.20)个/ mm2,与弥漫性非神经突斑、有致密核心的神经突斑及有致密核心的非神经突斑的小胶质细胞和星形胶质细胞的数量,分别为(3.24±1.53)和(4.14±1.77)个/mm2,(2.09±1.37)和(2.25±0.83)个/ mm2,(1.38±0.90)和(0.58±0.36)个/mm2,与弥漫性神经突斑相关的小胶质细胞和星形胶质细胞数量均高于其他类型的老年斑(P＜0.05)。结论 小胶质细胞、星形胶质细胞及其所产生的炎性介质IL-1α、S100β可能在AD弥漫性非神经突斑转化为弥漫性神经突斑的过程中具有一定意义。     

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses.

BACKGROUND: Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES: To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS: Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS: Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS: Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.     

Role of chemokines in promoting instability of coronary atherosclerotic plaques and the underlying molecular mechanism

Our aim was to investigate the role of chemokines in promoting instability of coronary atherosclerotic plaques and the underlying molecular mechanism. Coronary angiography and intravascular ultrasound (IVUS) were performed in 60 stable angina pectoris (SAP) patients and 60 unstable angina pectoris (UAP) patients. The chemotactic activity of monocytes in the 2 groups of patients was examined in Transwell chambers. High-sensitivity C-reactive protein (hs-CRP), monocyte chemoattractant protein-1 (MCP-1), regulated on activation in normal T-cell expressed and secreted (RANTES), and fractalkine in serum were examined with ELISA kits, and expression of MCP-1, RANTES, and fractalkine mRNA was examined with real-time PCR. In the SAP group, 92 plaques were detected with IVUS. In the UAP group, 96 plaques were detected with IVUS. The plaques in the UAP group were mainly lipid 51.04% (49/96) and the plaques in the SAP group were mainly fibrous 52.17% (48/92). Compared with the SAP group, the plaque burden and vascular remodeling index in the UAP group were significantly greater than in the SAP group (P<0.01). Chemotactic activity and the number of mobile monocytes in the UAP group were significantly greater than in the SAP group (P<0.01). Concentrations of hs-CRP, MCP-1, RANTES, and fractalkine in the serum of the UAP group were significantly higher than in the serum of the SAP group (P<0.05 or P<0.01), and expression of MCP-1, RANTES, and fractalkine mRNA was significantly higher than in the SAP group (P<0.05). MCP-1, RANTES, and fractalkine probably promote instability of coronary atherosclerotic plaque.     

Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients

Jha HC, Srivastava P, Divya A, Prasad J, Mittal A. Prevalence of Chlamydophila pneumoniae is higher in aorta and coronary artery than in carotid artery of coronary artery disease patients. APMIS 2009; 117: 905–11. Coronary artery disease (CAD) is a public health problem accounting for an estimated one‐third of deaths overall. A potential link between infectious agents and atherosclerosis has been suggested. Data obtained from several seroepidemiological studies have suggested that infection with Chlamydiophila pneumoniae, Helicobacter pylori, cytomegalovirus and herpes simplex virus‐1 can initiate or maintain the atherosclerotic process. However, there is no single study in which multiple infectious agents have been detected together in different vascular locations in the same population. This would help in determining if there is any leading pathogen in atheromatous plaques of CAD patients. Hence, we screened for C. pneumoniae, H. pylori, CMV and HSV‐1 in different vascular locations of CAD patients using quantitative real‐time (RT) PCR. We performed multiplex RT‐PCR for detecting pathogens, viz. C. pneumoniae, H. pylori, CMV and HSV‐1 in different vascular locations of CAD patients. Percent positivity scores for C. pneumoniae, H. pylori, CMV and HSV‐1 in different vascular locations were as follows: aorta (64.7, 35.3, 11.7 and 11.7 respectively); carotid (27.2, 27.2, 9 and 0 respectively); coronary artery (58.3, 33.3, 16.6 and 8.3 respectively). Combined positivity for C. pneumoniae (C. pneumoniae IgA and RT‐PCR for C. pneumoniae) was the highest compared with all other groups. Aorta and coronary artery were more susceptible to these pathogens as compared with carotid artery. Moreover, CAD patients’ characteristics were associated with C. pneumoniae positivity (C. pneumoniae IgA and RT‐PCR), suggesting thereby that C. pneumoniae may have caused chronic persistent infection in CAD.     

Understanding the expression of Toll-like receptors in Asian Indians predisposed to coronary artery disease

Introduction

Toll-like receptors (TLRs) are an important link between innate and adaptive immunity.

Material and methods

Expression of TLR-2, TLR-4, and TLR-9 genes was assessed in 60 coronary artery disease (CAD) patients and 79 controls by SYBR Green 1 based real time PCR assay.

Results

Expression of the TLR-2 gene was found to be significantly elevated in cases (1.295 ±0.09) compared to the controls (1.033 ±0.08) (p = 0.015) whereas expression of the TLR-9 gene was significantly lower in cases (1.522 ±0.18) than in the controls (2.165 ±0.16) (p = 0.032). There was no difference in TLR-4 expression levels (p = 0.174). A significant correlation of TLR-2 was observed with TLR-4 (r = 0.803, p<0.0001) and TLR-9 (r = 0.264, p = 0.003) as well as between TLR-4 and TLR-9 (r = 0.303, p = 0.001). A significant association was seen between TLR 2 (OR 3.94, 95% CI 1.73-8.99, p = 0.001) and TLR-9 (OR 0.297, 95% CI 0.131-0.672, p = 0.004) with CAD after adjustment for age and gender. Statins did not affect TLR gene expression.

Conclusions

The TLR-2, TLR-4 and TLR-9 genes exhibit a differential pattern of expression between CAD patients and controls in this Asian Indian cohort. This observation warrants further investigation, keeping in mind the infectious and inflammatory elements in perspective, in order to understand the true implications of TLR in the aetiopathology of CAD and consequent therapeutic implications.     

Expression of ICAM1 and VCAM1 Serum Levels in Rheumatoid Arthritis Clinical Activity. Association with Genetic Polymorphisms

To investigate the association of sICAM-1 and sVCAM-1 with ICAM1 721G>A and VCAM1 1238G>C polymorphisms and rheumatoid arthritis (RA) clinical activity, sixty RA patients and 60 healthy non-related subjects (HS) matched for age and sex were recruited. Soluble adhesion molecules were determined by ELISA technique. Rheumatoid factor (RF), C reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) were measured by routine methods. Disability and clinical activity was measured with Spanish-HAQ-DI and DAS28 scores, respectively. The ICAM1 and VCAM1 polymorphism were identified using the PCR-RFLP procedure. Inter-group comparison showed increased levels of sICAM-1 and sVCAM-1 in RA patients (284 and 481 ng/mL) versus HS (132 and 280 ng/mL); in the RA group, significant correlations between sVCAM-1 and RF (r = 0.402), ESR (r = 0.426), Spanish-HAQ-DI (r = 0.276), and DAS28 (r = 0.342) were found, whereas sICAM-1 only correlated with RF (r = 0.445). In RA patients, a significant association with the 721A allele of ICAM1 polymorphism (p = 0.04), was found. In addition, the allele impact (G/A + A/A) of this polymorphism was confirmed, (p = 0.038, OR = 2.3, C.I. 1.1–5.0). sVCAM-1 and sICAM-1 serum levels reflected the clinical status in RA, independently of the ICAM1 and VCAM1 polymorphism. However, the ICAM1 721A allele could be a genetic marker to RA susceptibility.     

辛伐他汀对小鼠巨细胞病毒肺炎Toll样受体2表达的影响

目的:观察辛伐他汀对小鼠巨细胞病毒(MCMV)肺炎小鼠肺组织Toll样受体2(Toll-like receptor2,TLR-2)、干扰素γ(IFN-γ)和单核细胞趋化蛋白1(MCP-1)表达的影响,并探讨辛伐他汀干预MCMV肺炎的可能机制。方法:将40只6~8周龄BALB/c小鼠随机分成5组:正常对照(NC)组、MCMV感染组和辛伐他汀干预(SMV1、SMV2和SMV3)组。SMV1、SMV2和SMV3组分别在腹腔注射MCMV前7 d、与MCMV同时和MCMV感染后3 d给予辛伐他汀(50 mg·kg~(-1)·d~(-1),均连续给药7 d)灌胃,NC组及MCMV组分别予以同体积的生理盐水灌胃。HE染色观察小鼠肺组织的病理改变,real-time PCR检测MCMV DNA的变化,免疫组化和Western blot检测肺组织中TLR-2的表达,ELISA检测肺组织中IFN-γ和MCP-1的变化。结果:与NC组相比,MCMV组肺组织病理染色显示肺泡间质水肿,肺泡壁增宽,内可见大量炎性细胞浸润;肺组织中TLR-2的表达增加,MCMV DNA含量升高,肺组织中炎症因子IFN-γ和MCP-1明显增多(P0.05)。辛伐他汀干预后,与MCMV组相比,肺组织病理改变较前减轻,TLR-2的表达下降(P0.05),MCMV DNA含量降低(P0.05),炎性因子IFN-γ和MCP-1明显下降(P0.05),且SMV1组TLR-2的表达量及MCMV DNA含量下降较SMV2和SMV3组更明显(P0.05),而SMV2和SMV3两组相比差异无统计学显著性。结论:辛伐他汀干预通过下调TLR-2信号通路而降低TLR-2的表达,减少IFN-γ和MCP-1的分泌,并抑制MCMV DNA的复制,从而使肺组织的病理损害得以减轻;且提前给予辛伐他汀干预对预防MCMV的感染及减轻炎症反应具有重要作用。     

Seroprevalence of antibodies to conserved regions of Chlamydia trachomatis heat shock proteins 60 and 10 in women in India

Persistent, untreated chlamydial infection causes chronic stimulation of the host immune system against immunogenic antigens such as chlamydial heat shock proteins (cHSP) 60 and 10. In order to find the seroprevalence of antibodies to cHSPs, enzyme-linked immunosorbent assay (ELISA) is performed using specific peptide sequences to measure antibody response against major outer membrane protein (MOMP), cHSP60 and cHSP10 in patient sera. In this study, 255 patients attending the gynaecology out-patient department (March 2004 to August 2005) of Safdarjung Hospital were enrolled. Of these patients, 107 were diagnosed with cervicitis while 52 had pelvic inflammatory disease (PID)/infertility. Chlamydia trachomatis infection in endocervical specimens is diagnosed by a direct fluorescence assay (DFA) and the polymerase chain reaction (PCR). In 75 (29.4%) of the C. trachomatis-positive women, 50 (66.7%) were ELISA positive for MOMP 48 (64.0%) were positive for cHSP60 and 46 (61.3%) were positive for cHSP10. The anti-MOMP index correlated positively with anti-cHSP60 (R = 0.522, P < 0.01) and anti-cHSP10 (R = 0.286, P < 0.05). Antibody titre for MOMP was significantly higher than that for cHSP60 (1:5; P < 0.01 and 1:25; P < 0.05). Moreover, patients with PID/infertility showed significantly higher antibody titres for cHSP60 and cHSP10 when compared to patients with cervicitis at dilutions of 1 in 50, 1 in 250, 1 in 1250 (P < 0.001) and at 1 in 6250 (P < 0.01).     

Impaired TLR-2 expression and TLR-2-mediated cytokine secretion in macrophages from patients with atopic dermatitis

Background:  In many patients with atopic dermatitis (AD), the disease is complicated by their enhanced susceptibility to bacterial skin infections, especially with Staphylococcus aureus . The pattern recognition receptor toll-like receptor (TLR)-2 recognizes components of S. aureus , for example, lipoteichoic acid (LTA) and peptidoglycan (PGN) and, therefore, might be crucial in the pathogenesis and flare-ups of AD.
Objective:  To investigate TLR-2 expression and cytokine secretion in macrophages from patients with AD compared to healthy controls upon TLR-2 stimulation with PGN, LTA and Pam3Cys.
Methods:  Macrophages were cultivated from highly purified peripheral blood monocytes of AD patients and nonatopic healthy controls and stimulated with PGN, LTA and Pam3Cys in a time and dose–dependent manner. Afterwards, TLR-2 expression and cytokine secretion were measured on protein and mRNA level. TLR-1 and TLR-6 expression were investigated on the mRNA level. Immunohistochemical stainings from punch biopsies were performed to investigate TLR-2 expression in skin macrophages .
Results:  We could clearly show that macrophages from patients with AD expressed significantly less TLR-2, whereas the expression pattern of TLR-1 and TLR-6 were not altered. Macrophages had a reduced capacity to produce pro-inflammatory cytokines such as IL-6, IL-8 and IL-1β after stimulation with TLR-2 ligands.
Conclusion:  Our findings clearly show an impaired TLR-2 expression and functional differences of TLR-2-mediated effects on macrophages of AD patients compared to healthy controls which might contribute to the enhanced susceptibility to skin infections with S. aureus in AD.     

Fluticasone propionate downregulates nasal fibroblast functions involved in airway inflammation and remodeling

BACKGROUND: Besides being highly effective in the treatment of allergic and nonallergic rhinitis with eosinophilia, intranasal corticosteroids appear to be useful in reducing nasal polypoid lesions and the likelihood of polyp recurrence after surgery. We evaluated the ability of fluticasone propionate to downregulate fibroblast functions related to nasal inflammation and remodeling. METHODS: Primary nasal polyp tissue-derived fibroblasts were stimulated with tumor necrosis factor (TNF)-alpha or interleukin (IL)-4 or basic fibroblast growth factor (bFGF) in the presence of fluticasone propionate (0.1-100 nM). Fibroblast proliferation, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression and eotaxin release were then evaluated. RESULTS: As compared with unstimulated cultures, a significant increase in fibroblast proliferation was observed when the cells were stimulated with bFGF (p < 0.05), but not with TNF-alpha or IL-4 (p > 0.05). TNF-alpha induced an upregulation of ICAM-1 expression (p < 0.05), which was not seen in fibroblasts cultured in the presence of IL-4 or bFGF. No changes in VCAM-1 expression were induced by TNF-alpha, IL-4 or bFGF, whereas both TNF-alpha and IL-4 increased eotaxin release (p < 0.05). Both bFGF-induced fibroblast proliferation and TNF-alpha-induced ICAM-1 expression were significantly reduced by fluticasone, starting at the dose of 1 and 10 nM, respectively (p < 0.05). Fluticasone at concentrations of 1-100 nM effectively inhibited eotaxin release by TNF-alpha- or IL-4-stimulated fibroblasts (p < 0.05). CONCLUSIONS: The pharmacologic activity of fluticasone in patients with chronic upper airway inflammatory disease may include inhibition of resident fibroblast functions involved in airway inflammation and remodeling.     

Diagnostic and prognostic roles of peripheral blood Toll-like receptor-4 and stanniocalcin-1 genes expression in acute lung injury

Acute lung injury (ALI) is an acute inflammatory disorder. Toll-like receptor-4 (TLR-4) and Stanniocalcin -1 (STC-1) had roles in lung endothelial protection. This study aims to assess TLR-4 and SCT-1 genes expressions in peripheral blood of ALI patients. Total RNA was extracted from peripheral blood of 48 subjects (20 healthy controls, 28 ALI patients) and expressions of genes were assessed by real-Time qRT-PCR. The expression levels of TLR-4 and SCT-1 genes were significantly lower in ALI patients compared to controls (P < 0.0001). After 10 days, the expression levels of TLR-4 and SCT-1 were increased compared to their baseline levels (p = 0.012 and 0.024, respectively). SCT-1 has 92.9% sensitivity and 100% specificity in ALI detection. SCT-1 gene expression was negatively correlated with severity score (r= −0.54, p = 0.003). The mortality pattern was higher in ALI patients with lower TLR-4 gene expression (p = 0.014). In conclusion, the peripheral blood expressions of TLR-4 and STC-1 genes were decreased in ALI patients. Both genes expressions were increased with patients' recovery. SCT-1 had higher sensitivity and specificity in ALI diagnosis. The peripheral blood expressions of SCT-1 and TLR-4 genes seem to be diagnostic and prognostic markers in ALI.     

OX-LDL诱导内皮细胞条件培养液对内皮细胞VCAM-1和ICAM-1的影响

目的探讨受损内皮细胞的自分泌和旁分泌对内皮细胞自身的影响。方法利用正常内皮细胞条件培养液和用氧化型低密度脂蛋白(OX-LDL)诱导内皮细胞的条件培养液分别作用于正常内皮细胞和受损内皮细胞,用酶联免疫细胞化学法检测血管内皮细胞黏附分子-1(VCAM-1)和细胞间黏附分子-1(ICAM-1)表达的变化。结果正常内皮细胞的条件培养液和OX-LDL诱导的内皮细胞条件培养液对正常内皮细胞VCAM-1和ICAM-1的表达作用不明显(P>0.05),而对受损内皮细胞VCAM-1和ICAM-1的表达具有明显的下调作用(P<0.01)。结论正常和受到氧化损伤的内皮细胞的自分泌和旁分泌作用对正常内皮细胞黏附分子没有影响,而对受损内皮细胞黏附分子有下调作用,说明内皮细胞可通过下调黏附分子的表达来实现自身的抗损伤作用。     

Distinct indirect pathways govern human NK-cell activation by TLR-7 and TLR-8 agonists

NK cells limit the emergence of cancers and viral infections by surveillance of 'missing-self' and 'induced-self' ligands, and by direct recognition of pathogen-associated molecules. We examined individual roles for Toll-like receptors (TLRs)-7 and -8 in human NK-cell activation using synthetic, small molecule agonists of either TLR-7 (imiquimod and 3M-001), TLR-8 (3M-002) or both TLR-7/8 (3M-003 and R-848) for comparison with known ligands of TLR-2 to -9. Tracking cytokine production in PBMC initially revealed that a subset of TLR agonists including polyinosinic-polycytidylic acid (poly I:C), 3M-002, 3M-003, R-848 and single-stranded RNA trigger relatively high levels of IFN-gamma expression by NK cells. Isolated NK cells did not express TLR-7 or TLR-8. Unlike MALP-2 and poly I:C, 3M-001-3 did not induce expression of either CD69 or IFN-gamma by purified NK cells suggesting indirect activation. IL-18 and IL-12p70 were primarily required for induction of IFN-gamma by both synthetic and natural TLR-8 ligands, while type I IFN was required for induction of CD69 on NK cells by the TLR-7 agonist 3M-001. In addition to expression of IFN-gamma and CD69, relative induction of NK-cell cytotoxicity by TLR-7 and TLR-8 agonists was compared. Immune response modifiers (IRMs) with a TLR-8 agonist component (3M-002 and 3M-003) stimulated greater levels of K562 cytolysis than achieved with 3M-001 or IL-2 (1000 units ml(-1)). In vivo NK-cell cytotoxicity was also enhanced by R-848, but not in type I IFNR-deficient mice. We conclude that IRMs can modulate NK-cell function both in vitro and in vivo and that distinct indirect pathways control human NK-cell activation by TLR-7 and TLR-8 agonists.