Aberrant expression of regulatory cytokine IL-35 in patients with systemic lupus erythematosus.

OBJECTIVE: This study characterizes an IL-35-mediated regulatory role in patients with systemic lupus erythematosus (SLE). METHODS: Plasma of SLE patients and healthy controls (HCs) was analyzed for the concentrations of IL-35 and soluble gp130 by using ELISA. mRNA expression of IL-35 subunit (p35 and EBI3) and its receptor (gp130 and IL-12Rß2) in peripheral blood mononuclear cells (PBMCs) was assessed by RT-qPCR. Flow cytometry was performed to evaluate the number of CD4(+)CD25(high)CD127(-)Treg cells and the expression of IL-35 receptor on the CD4+ helper (Th) cells and CD19+ B cells. Plasma collected from SLE patients and HCs was assayed for cytokine and chemokine expression by Luminex multiplex assay. RESULTS: Plasma IL-35 and soluble gp130 levels positively correlated with each other and were significantly higher in patients with severe SLE compared with HCs. Significantly higher levels of inflammatory cytokines/chemokines CCL2, CXCL8, IL-6, interferon (IFN)-γ, IL-10 and IL-17A were observed in plasma of SLE patients than HCs. mRNA levels of IL-35 and its receptor were significantly positively correlated in PBMCs from SLE patients and their levels were higher in SLE than HCs. The increase significantly correlated with changes in SLE Disease Activity Index (SLEDAI) (all p < 0.05). In addition, the number of Treg cells in severe and moderate SLE patients were both significantly lower than HCs, where the ratio of CD4(+)CD25(-)effector T cell %/CD4(+)CD25(high)CD127(-)Treg % was found to be significantly higher in severe SLE patients. Furthermore, the expression of gp130 on CD4+ Th cells and percentage of Tregs were positively correlated with each other, and both were negatively correlated with SLEDAI. CONCLUSION: Our findings indicate that high level of plasma IL-35 in active SLE patients expressed with low level of IL-35 receptor (gp130) on CD4+ Th cells. These data raise the possibility that the level of IL-35 expression in SLE patients is not sufficient to induce the production of CD4(+)CD25(high)CD127(-)Tregs, and subsequently suppress the release of inflammatory cytokines and chemokines upon inflammation.

Remission of systemic lupus erythematosus disease activity with regulatory cytokine interleukin (IL)-35 in Murphy Roths Large (MRL)/lpr mice.

The immunological mechanisms mediated by regulatory cytokine interleukin (IL)-35 are unclear in systemic lupus erythematosus (SLE). We investigated the frequency of CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) regulatory T (Treg ) and IL-10(+) regulatory B (Breg ) cells and related immunoregulatory mechanisms in a female Murphy Roths Large (MRL)/lpr mouse model of spontaneous lupus-like disease, with or without IL-35 treatment. A remission of histopathology characteristics of lupus flare and nephritis was observed in the MRL/lpr mice upon IL-35 treatment. Accordingly, IL-35 and IL-35 receptor subunits (gp130 and IL-12Rß2) and cytokines of MRL/lpr and BALB/c mice (normal controls) were measured. The increased anti-inflammatory cytokines and decreased proinflammatory cytokines were possibly associated with the restoration of Treg and Breg frequency in MRL/lpr mice with IL-35 treatment, compared to phosphate-buffered saline (PBS) treatment. mRNA expressions of Treg -related FoxP3, IL-35 subunit (p35 and EBI3) and soluble IL-35 receptor subunit (gp130 and IL12Rß2) in splenic cells were up-regulated significantly in IL-35-treated mice. Compared with the PBS treatment group, IL-35-treated MRL/lpr mice showed an up-regulation of Treg -related genes and the activation of IL-35-related intracellular Janus kinase/signal transducer and activator of transcription signal pathways, thereby indicating the immunoregulatory role of IL-35 in SLE. These in vivo findings may provide a biochemical basis for further investigation of the regulatory mechanisms of IL-35 for the treatment of autoimmune-mediated inflammation.

Amelioration of allergic airway inflammation in mice by regulatory IL-35 through dampening inflammatory dendritic cells.

BACKGROUND: IL-35, a new member of the IL-12 family, is an inhibitory cytokine produced by regulatory T and B lymphocytes that play a suppressive role in the inflammatory diseases. This study focuses on the cellular mechanism regulating the anti-inflammatory activity of IL-35 in asthmatic mice. METHODS: Ovalbumin-induced asthmatic and humanized asthmatic mice were adopted to evaluate the in vivo anti-inflammatory activities of IL-35. For monitoring the airway, Penh value (% baseline) was measured using a whole-body plethysmograph. RESULTS: In this study using ovalbumin-induced asthmatic mice, we observed that intraperitoneal injection of IL-35 during the allergen sensitization stage was more efficient than administration in the challenge stage for the amelioration of airway hyper-responsiveness. This was reflected by the significantly reduced concentration of asthma-related Th2 cytokines IL-5 and IL-13, as well as eosinophil counts in bronchoalveolar lavage fluid (all P < 0.05). IL-35 also significantly attenuated the accumulation of migratory CD11b+CD103(-) dendritic cells (DC) in the mediastinal lymph node (mLN) and lung of mice (all P < 0.05). IL-35 markedly inhibited the ovalbumin-induced conversion of recruited monocytes into inflammatory DC, which were then substantially reduced in mLN to cause less T-cell proliferation (all P < 0.05). Further study using the humanized asthmatic murine model also indicated human IL-35 exhibited a regulatory impact on allergic asthma. CONCLUSION: Our findings suggest that IL-35 can act as a crucial regulatory cytokine to inhibit the development of allergic airway inflammation via suppressing the formation of inflammatory DC at the inflammatory site and their accumulation in the draining lymph nodes.

Muramyl dipeptide mediated activation of human bronchial epithelial cells interacting with basophils: a novel mechanism of airway inflammation.

Respiratory tract bacterial infection can amplify and sustain airway inflammation. Intracytosolic nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is one member of the nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) family, which senses the conserved structural peptidoglycan component muramyl dipeptide (MDP) in almost all bacteria. In the present study, activation of the NOD2 ligand MDP on primary human bronchial epithelial cells (HBE) co-cultured with human basophils was investigated. Cytokines, NOD2, adhesion molecules and intracellular signalling molecules were assayed by enzyme-linked immunosorbent assay or flow cytometry. The protein expression of NOD2 was confirmed in basophils/KU812 cells and HBE/human bronchial epithelial cell line (BEAS-2B) cells. MDP was found to up-regulate significantly the cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 on basophils and HBE in the co-culture system with or without basophil priming by interleukin (IL)-33 (all P < 0·05). MDP could further enhance the release of inflammatory cytokine IL-6 and chemokine CXCL8, and epithelium-derived anti-microbial peptide ß-defensin 2 in the co-culture. HBE cells were the major source for the release of IL-6, CXCL8 and ß-defensin2 upon stimulation by MDP in the co-culture system. The expression of ICAM-1 and VCAM-1 and release of IL-6 and CXCL8 were suppressed by various signalling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be regulated differentially by the mitogen-activated protein kinase pathways and nuclear transcription factors. The results therefore provide a new insight into the functional role of basophils in innate immunity, and the link between respiratory bacteria-mediated innate immunity and subsequent amplification of allergic inflammation in the airway.

Study of heavy metal poisoning in frequent users of Chinese medicines in Hong Kong and Macau.

This study is a direct assessment of blood heavy metal concentrations of frequent users of Chinese medicines (CM), who had been taking prescribed CM at least 6 days per week for not less than 3 months, to determine whether their intake of CM could cause an increased load of toxic heavy metals in the body. From November 2009 to June 2010, 85 subjects were recruited with informed consent, and their blood samples were collected for measurement of arsenic, cadmium, lead and mercury concentrations. Results showed that blood concentrations of four heavy metals of nearly all 85 subjects were within reference ranges. Only one subject who had consumed plentiful seafood was found to have transiently increased blood arsenic concentration (29% higher than the upper limit of the reference range). However, after refraining from eating seafood for 1 month, his blood arsenic concentration returned to normal. Eighty commonly prescribed CM in both raw medicine and powder concentrate supplied by local distributors were also tested for the four heavy metals. Twelve out of the 80 raw medicines were found to contain one or more of the heavy metals that exceeded the respective maximum permitted content. Cadmium was most frequently found in the contaminated samples. None of the powder concentrates had heavy metal content exceeding their respective maximum permitted level.

Spirometric reference standards for preschool children in Hong Kong.

1. Prediction equations and normograms are established using incentive spirometry in a community cohort of 770 Hong Kong Chinese children aged 2 to 6 years. 2. All spirometric parameters depend mainly on standing height. Boys have higher values than girls. 3. Forced expiratory volumes depend on birth weight, place of birth, history of wheezing, and environmental tobacco smoke (ETS) exposure. 4. High urinary cotinine level as a biomarker of ETS exposure is noted in about one tenth of the children. 5. Urinary cotinine level is inversely associated with all spirometric parameters. This supports implementation of the smoking cessation programme.

Serum small-dense LDL abnormalities in chronic renal disease patients.

Cardiovascular disease (CVD) is the principal cause of mortality in chronic kidney disease (CKD) patients. Dyslipoproteinaemia is a common metabolic derangement in CKD and a traditional risk factor for CVD. This study investigates serum lipoprotein, especially small-dense low-density lipoprotein (sd-LDL), abnormalities in CKD patients. A total of 131 CKD patients (age: 59 +/- 12 years, male = 64) diagnosed according to Kidney Disease: Improving Global Outcomes, 2004 (KDIGO) and 121 age- and gender-matched control subjects (age: 58 +/- 6 years, male = 62) were recruited from Hong Kong and Macau. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C) and direct LDL-C were assayed enzymatically. In addition, sd-LDL, together with very low density and intermediate-density lipoproteins (VLDL and IDL) were measured by US Food and Drug Administration (FDA)-approved polyacrylamide gradient gel electrophoresis. Compared to controls, CKD patients showed significantly decreased TC, LDL-C, normal-size LDL and HDL-C with increased TG, VLDL, IDL and sd-LDL (all P < 0.01). The increased sd-LDL and decreased normal-size LDL fractions resulted in a significantly elevated sd-LDL:LDL ratio in CKD (P < 0.005). In contrast to the low TC and LDL-C, sd-LDL and sd-LDL:LDL ratio were significantly elevated in CKD. Thus, sd-LDL will be used increasingly for CVD risk assessment in CKD and other diseases that show lipoprotein derangement.

Human papillomavirus infection in Macau women.

OBJECTIVE: To investigate the prevalence and genotypes of human papillomavirus (HPV) infection in Macau women. STUDY DESIGN: Female patients presenting for a medical consultation or medical check-up were recruited with informed consent. METHODS: Cytology and HPV-DNA genotyping were performed on 402 cervical specimens that were collected from Macau women. RESULTS: Of the specimens, 29.9% were found to be HPV-DNA positive; 26.4% were infected with one HPV genotype, while 3.0% and 0.5% were infected with two and three HPV genotypes, respectively. The most prevalent HPV genotype was type 52 (11.1%), followed by type 16 (9.7%). Both types 51 and 62 ranked third (9.0%). CONCLUSIONS: The HPV infection rate in Macau appears to be higher than that in the neighbouring city of Hong Kong. The most prevalent genotypes were similar to those in South-west and Southern China.

Sonographic measurement of mesenteric fat predicts presence of fatty liver among subjects with polycystic ovary syndrome.

OBJECTIVE: Visceral fat is believed to be important in the pathogenesis of metabolic syndrome and fatty liver. In this study, we examined the relationship between mesenteric fat thickness and other sonographic indices of adiposity and the presence of fatty liver among subjects with polycystic ovary syndrome (PCOS). SUBJECTS AND METHODS: A total of 117 Chinese subjects with PCOS were evaluated (mean age, 28.6 ± 6.5 yr; mean body mass index, 24.3 ± 5.3 kg/m(2)). Anthropometric measurements and metabolic risk profile, including a standard oral glucose tolerance test, were assessed in all subjects. All subjects underwent an ultrasound examination for measurement of thickness of mesenteric, preperitoneal, and sc fat as well as evaluation for fatty liver. RESULTS: Forty-six (39.3%) of the subjects had fatty liver. PCOS subjects with fatty liver had higher body mass index, waist circumference, waist-hip ratio, and systolic blood pressure; a more unfavorable lipid profile with higher triglyceride; lower high-density lipoprotein cholesterol; higher fasting glucose and insulin; higher 2-h glucose during oral glucose tolerance test; lower SHBG; and higher alanine aminotransferase. Subjects with fatty liver had increased thickness of preperitoneal, mesenteric, and sc fat, as well as increased carotid intima-media thickness. Abdominal fat thickness showed moderate correlation to alanine aminotransferase as well as fasting insulin. On multivariate logistic regression, fasting insulin and mesenteric fat thickness were identified as independent predictors of fatty liver among subjects with PCOS. CONCLUSION: Fatty liver is present in a significant proportion of Chinese patients with PCOS. Sonographic measurement of mesenteric fat is an independent determinant of fatty liver among subjects with PCOS and identifies subjects at increased cardiovascular risk.

Chemical pattern-aided classification to simplify the intricacy of morphological taxonomy of Epimedium species using chromatographic fingerprinting.

Epimedium herb (Yinyanghuo), one of the popular Chinese materia medica, is a multiple species colony of Epimedium genus belonging to Berberidaceae. There are five species of Epimedium that have been officially adopted in Chinese Pharmacopoeia under the same crude drug name 'Yinyanghuo' comprising Epimedium brevicornu, E. koreanum, E. sagittatum, E. pubescens, and E. wushanense. In addition, non-official species like E. acuminatum, E. miryanthum and E. leptorrhizum are also mix-used. Frequently, the morphological taxonomical identification is very difficult during on-site inspection for species authentication in the market. Researchers are often bewildered by the multiple species ambiguity when putting this crude drug in use. Referring to the bioactive constituents that are vital for therapeutic efficacy, the key to clarifying the multiple species confusion should rely on analysis of the bioactive composition. It is well known that medicinal Epimedium herbs contain special C-8 prenylated flavonol glycosides which contribute to various bioactivities and the major four, epimedin A (A), epimedin B (B), epimedin C (C) and icariin (I), are unanimously used as bioactive markers for quality control. In this study, HPLC-DAD fingerprinting was performed for investigating the molecular spectrum of various Epimedium species. It was found that the four major flavonoids constitute the middle part of the chromatographic profiles to form a specific region (named as 'ABCI fingerprint region') being dominant in the HPLC profiles of all medicinal Epimedium species, and the five official species express five different 'ABCI' patterns (different peak: peak ratios). Our study found that the convergent tendency of the 'ABCI region' among multiple species of Epimedium could facilitate differentiation of complex commercial samples based on similar bioactive composition should confer similar bioactivities. Merging the different species that possess the same 'ABCI region' pattern into the same group can create a simpler bioactive-fraction-aided classification array by clustering the commercial samples into three bioactive ingredients-based fingerprint patterns - 'E.b. pattern', 'E.k. pattern' and 'extensive E.w. pattern'. This approach offers the feasibility of characterizing and quality-controlling complex samples in the same genus designated under a single herbal drug entity on the premise of possessing the same bioactive ingredients pattern and supported by long-term traditional usage.

Metabolic syndrome, endothelial injury, and subclinical atherosclerosis in patients with systemic lupus erythematosus.

OBJECTIVES: To study the link between metabolic syndrome (MetS), endothelial injury, and atherosclerosis in patients with systemic lupus erythematosus (SLE). METHODS: Consecutive SLE patients without a history of arterial thrombosis were screened for atherosclerosis at the carotid and coronary arteries by B-mode ultrasound [intima-media thickness (IMT)] and multidetector computed tomography (MDCT) scan (Agatston calcium scores), respectively. Plasma levels of homocysteine, high-sensitivity C-reactive protein (hsCRP), soluble vascular cell adhesion molecule (sVCAM)-1, P-selectin, and soluble thrombomodulin (sTM) were assayed. Patients were stratified according to the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) criteria for MetS, using the Asian criteria for abdominal obesity. Risk factors for atherosclerosis were studied. RESULTS: Of the 123 SLE patients (93% women; age 47.9+/-11 years; SLE duration 10.9+/-7.0 years) studied, 20 (16.3%) had MetS. The prevalence of MetS in the SLE patients was significantly higher than in 492 age- and sex-matched healthy controls (9.6%; p=0.03). Coronary calcification and abnormal carotid IMT were detected in 38 (31%) and 72 (59%) of SLE patients, respectively. Patients with MetS had a significantly higher Agatston score (69.5+/-95 vs. 16.4+/-57; p=0.03) and a numerically higher carotid IMT (p=0.43) than those without. In a logistic regression model, the MetS [odds ratio (OR) 3.11, 95% confidence interval (CI) 1.01-9.59, p=0.049] was associated with coronary atherosclerosis after adjustment for age and other risk factors. In addition, patients with MetS had significantly higher levels of hsCRP (p=0.002), homocysteine (p=0.03), and sTM (p=0.01). CONCLUSIONS: The MetS is more prevalent in SLE patients than the general population and is associated with endothelial injury and coronary atherosclerosis. More aggressive control of risk factors is justified in these patients.

Effects of 25-hydroxyvitamin D level and its change on parathyroid hormone in premenopausal Chinese women.

UNLABELLED: Optimal levels of 25-hydroxyvitamin D [25(OH)D] were investigated in premenopausal Chinese women. Parathyroid hormone (PTH) change at 3 months was associated with change in 25(OH)D but not with baseline levels, and PTH fell even when starting levels of 25(OH)D were >40 nmol/L, consistent with optimal values for 25(OH)D of ≥40 nmol/l. INTRODUCTION: The upper level of 25-hydroxyvitamin D [25(OH)D] which constitutes a long-term bone health risk by causing elevated PTH levels is uncertain. Although many studies have addressed this question using cross-sectional data, the present study is one of few employing a prospective approach to determine 25(OH)D levels required to minimize PTH. METHODS: Relationships among baseline values and 3-month changes (Δ) in PTH and 25(OH)D were assessed in 221 Chinese women, aged 28.0±4.4 years (mean±SD), taking part in a placebo-controlled dairy product intervention delivering 200 IU vitamin D(3)/day. RESULTS: Baseline 25(OH)D was 34±11 nmol/L and was inversely related to baseline PTH (r=-0.18, P=0.007), with a plateau in PTH levels when 25(OH)D was >40 nmol/L. After 3 months intervention, PTH fell 11% and neither Δ25(OH)D nor ΔPTH differed between treatment and control groups. ΔPTH was inversely related to Δ25(OH)D (P<0.001) but not to baseline 25(OH)D. Similarly, ΔPTH differed between quartiles of Δ25(OH)D (P<0.001), but not between quartiles of baseline 25(OH)D and no interaction was observed between quartiles of baseline 25(OH)D and Δ25(OH)D. Even in the highest quartile of baseline 25(OH)D (>40 nmol/L), PTH fell 0.4±0.1 pmol/L (mean±SEM; P=0.008). CONCLUSIONS: We conclude that vitamin D deficiency is common in young women in Hong Kong. The cross-sectional analysis indicates that optimal 25(OH)D is >40 nmol/L, and the longitudinal data is consistent with a higher optimal value which is not defined in this study's results.

Interleukin-17A activation on bronchial epithelium and basophils: a novel inflammatory mechanism.

Basophils are the accessory cell type for T-helper (Th)2 induction and initiators in immunoglobulin E-mediated chronic allergic inflammation. Basophils and Th17 cells accumulate at the inflammatory sites, such as the airways of allergic asthmatic patients. We investigated the activation of interleukin (IL)-17A on the primary human basophils/KU812 basophilic cells and primary human bronchial epithelial cells/BEAS-2B bronchial epithelial cells. Cytokines, chemokines, adhesion molecules and intracellular signalling molecules were assayed by ELISA or flow cytometry. Co-culture of bronchial epithelial cells and basophils could significantly induce the release of IL-6, an epithelial inflammatory cytokine, and CCL2, a chemokine for basophils, esosinophils and monocytes. Such induction was synergistically enhanced by IL-17A, and direct interaction between these two cells was necessary for IL-17A-induced IL-6 and CCL2 release. Surface expression of intercellular adhesion molecule-1 on bronchial epithelial cells was also upregulated upon their interaction. The interaction of basophils and bronchial epithelial cells under IL-17A stimulation was differentially regulated by extracellular signal-regulated kinase, c-Jun N-terminal protein kinase, p38 mitogen-activated protein kinase and nuclear factor-kappaB pathways. These findings suggest a novel immunopathological role of Th17 cells and basophils in allergic asthma through the activation of granulocyte-mediated inflammation initiated by the direct interaction between basophils and bronchial epithelial cells.

Activation profile of Toll-like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll-like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR-1-9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR-3, -8, -9) and extracellular TLRs (TLR-1, -2, -4, -5, -6) were elevated in monocytes, CD4(+) T lymphocytes, CD8(+) T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0.001). Moreover, cell surface expression of TLR-4 on CD4(+) T lymphocytes and CD8(+) T lymphocytes, and TLR-6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR-4 on CD4(+) T lymphocytes and CD8(+) T lymphocytes: r = 0.536, P = 0.04; r = 0.713, P = 0.003; TLR-6 in B lymphocytes: r = 0.572, P = 0.026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)-1beta, IL-6, IL-10 and IL-12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR-3 ligand), lipopolysaccharide (TLR-4 ligand), peptidoglycan (TLR-2 ligand), flagellin (TLR-5 ligand), R837 (TLR-7 ligand) and CpG DNA (TLR-9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self-originated DNA plays immunopathological roles via TLR activation in SLE.

Decreased expression of T lymphocyte co-stimulatory molecule CD26 on invariant natural killer T cells in systemic lupus erythematosus.

CD26, a T cell co-stimulatory molecule and dipeptidyl peptidase IV for the degradation of interferon-gamma-induced chemokine, participates in multiple immunopathological roles in leukocyte homing and inflammation. Decreased circulating concentration of soluble (s)CD26 in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis and murine model of arthritis and encephalomyelitis have been reported. In the present study, the plasma concentration of sCD26 and chemokines, and cell surface expression of CD26 on monocytes, CD4+T lymphocytes, CD8+T lymphocytes, CD19+B lymphocytes and invariant natural killer T (iNKT) lymphocytes were analyzed using ELISA and flow cytometry, respectively, in 23 SLE patients and 14 sex- and age-matched control subjects. Although there was no significant difference between plasma concentrations of soluble CD26 in SLE patients with controls (p > 0.05), there was significant elevated Th1 chemokines CXCL10 and CXCL9 but not Th2 chemokine CCL2, and down-regulation in iNKT lymphocytes number and cell surface expression of CD26 on CD4+T and iNKT lymphocytes of SLE patients compared with controls (all p < 0.05). Decreased circulating number of iNKT cells and CD26 on iNKT cells can be important for the immunopathogenesis by exacerbating Th1-related inflammation in SLE.