Unique Antibody Responses to Malondialdehyde-Acetaldehyde (MAA)-Protein Adducts Predict Coronary Artery Disease

Malondialdehyde-acetaldehyde adducts (MAA) have been implicated in atherosclerosis. The purpose of this study was to investigate the role of MAA in atherosclerotic disease. Serum samples from controls (n = 82) and patients with; non-obstructive coronary artery disease (CAD), (n = 40), acute myocardial infarction (AMI) (n = 42), or coronary artery bypass graft (CABG) surgery due to obstructive multi-vessel CAD (n = 72), were collected and tested for antibody isotypes to MAA-modifed human serum albumin (MAA-HSA). CAD patients had elevated relative levels of IgG and IgA anti-MAA, compared to control patients (p<0.001). AMI patients had a significantly increased relative levels of circulating IgG anti-MAA-HSA antibodies as compared to stable angina (p<0.03) or CABG patients (p<0.003). CABG patients had significantly increased relative levels of circulating IgA anti-MAA-HSA antibodies as compared to non-obstructive CAD (p<0.001) and AMI patients (p<0.001). Additionally, MAA-modified proteins were detected in the tissue of human AMI lesions. In conclusion, the IgM, IgG and IgA anti-MAA-HSA antibody isotypes are differentially and significantly associated with non-obstructive CAD, AMI, or obstructive multi-vessel CAD and may serve as biomarkers of atherosclerotic disease.     

Extracellular Matrix Biomarker,Fibulin-1, Is Closely Related to NT-proBNP and Soluble Urokinase Plasminogen Activator Receptor in Patients with Aortic Valve Stenosis (The SEAS Study)

Background

Fibulin-1, a circulating extracellular matrix glycoprotein, has been associated with arterial disease and elevated N-terminal prohormone B-type natriuretic peptide (NT-proBNP) in diabetes. Soluble urokinase plasminogen activator receptor (suPAR), a marker of inflammation, has been associated with subclinical atherosclerosis. Therefore, we aimed to explore the interplay between these biomarkers and mild to moderate aortic valve stenosis (AS).

Methods

In 374 patients with mild to moderate AS, we investigated the relationship of fibulin-1 with NT-proBNP, levels of suPAR and the degree of AS at baseline and after one and four years of treatment with Simvastatin 40 mg and Ezetimibe 10 mg or placebo.

Results

During treatment, fibulin-1 became more closely associated with NT-proBNP (β_year0 = 0.10, p = 0.08, β_year1 = 0.16, p = 0.005, β_year4 = 0.22, p<0.001) and suPAR (β_year0 = 0.05, p = 0.34, β_year1 = 0.16, p = 0.006, β_year4 = 0.13, p = 0.03) at the expense of the association to aortic valve area index (AVAI) (β_year0 = −0.14, p = 0.005, β_year1 = −0.08, p = 0.11, β_year4 = −0.06, p = 0.22) independently of age, gender, creatinine, and serum aspartate aminotransferase (Adj.R_year0 ² = 0.19, Adj.R_year1 ² = 0.22, Adj.R_year4 ² = 0.27). Fibulin-1 was unrelated to aortic regurgitation, left ventricular mass, and ejection fraction. In patients with baseline AVAI<0.58 cm²/m² (median value), fibulin-1 was more closely associated to NT-proBNP (β_year0 = 0.25, β_year1 = 0.21, β_year4 = 0.22, all p<0.01), and suPAR (β_year0 = 0.09, p = 0.26, β_year1 = 0.23, β_year4 = 0.21, both p<0.01) independently of age, gender, AST and treatment allocation.

Conclusions

Increased levels of fibulin-1 were independently associated with higher levels of suPAR and NT-proBNP especially in patients with lower AVAI, suggesting that fibulin-1 may be an early marker of AS as well as cardiac fibrosis secondarily to elevated left ventricular hemodynamic load.     

Serum Dipeptidyl Peptidase-4 Activity in Insulin Resistant Patients with Non-Alcoholic Fatty Liver Disease: A Novel Liver Disease Biomarker

Background

In a cross-sectional study we studied the fasting serum DPP-4 enzymatic activity (sDPP-4) and the insulin resistance index (HOMA2-IR) in gliptin naïve patients with type 2 diabetes and in non-alcoholic fatty liver disease (NAFLD) and in healthy controls (CNTRL).

Methods and Findings

sDPP-4 was measured by kinetic assay in 39 NAFLD (F/M:19/20, mean age: 47.42 yrs) and 82 type 2 diabetes (F/M:48/34, 62.8 yrs) patients and 26 (F/M:14/12, 35.3 yrs) controls. Definition of T2D group as patients with type 2 diabetes but without clinically obvious liver disease created non-overlapping study groups. Diagnosis of NAFLD was based on ultrasonography and the exclusion of other etiololgy. Patients in T2D and NAFLD groups were similarly obese. 75 g CH OGTT in 39 NAFLD patients: 24-NGT, 4-IGT or IFG (“prediabetes”), 11-type 2 diabetes. HOMA2-IR: CNTRL: 1.44; T2D-group: 2.62 (p = 0.046 vs CNTRL, parametric tests); NAFLD(NGTonly): 3.23 (p = 0.0013 vs CNTRL); NAFLD(IFG/IGT/type 2 diabetes): 3.82 (p<0.001 vs CNTRL, p = 0.049 vs 2TD group). sDPP-4 activity was higher in NAFLD both with NGT (mean:33.08U/L) and abnormal glucose metabolism (30.38U/L) than in CNTRL (25.89U/L, p<0.001 and p = 0.013) or in T2D groups (23.97U/L, p<0.001 and p = 0.004). Correlations in NAFLD among sDPP-4 and ALT: r = 0.4637,p = 0.0038 and γGT: r = 0.4991,p = 0.0017 and HOMA2-IR: r = 0.5295,p = 0.0026 and among HOMA2-IR and ALT: r = 0.4340,p = 0.0147 and γGT: r = 0.4128,p = 0.0210.

Conclusions

The fasting serum DPP-4 activity was not increased in T2D provided that patients with liver disease were intentionally excluded. The high serum DPP-4 activities in NAFLD were correlated with liver tests but not with the fasting plasma glucose or HbA1C supporting that the excess is of hepatic origin and it might contribute to the speedup of metabolic deterioration. The correlation among γGT, ALT and serum DPP-4 activity and also between serum DPP-4 activity and HOMA2-IR in NAFLD strongly suggests that serum DPP-4 activity should be considered as a novel liver disease biomarker.     

Serum Thyroid-Stimulating Hormone and Anti-Thyroglobulin Antibody Are Independently Associated with Lesions in Spinal Cord in Central Nervous System Demyelinating Diseases

Transverse myelitis (TM) is associated with neuromyelitis optica (NMO) and multiple sclerosis (MS). Early recognition of useful parameters may be helpful to distinguish their difference. This retrospective study analyzed thyroid parameters from 243 serum samples (relapse = 128; remission = 115) of 178 patients with demyelinating diseases (NMO, n = 25; TM, n = 48; MS, n = 105). The relationship between thyroid and clinical parameters was analyzed. Patients with NMO and TM had a higher frequency of abnormal thyroid-stimulating hormone (TSH), anti-thyroglobulin antibodies (TG-Ab), and antithyroid peroxidase antibody (TPO-Ab) than MS patients (p<0.05). The level of TSH and TG-Ab returned to normal levels after administration of high-dose intravenous methylprednisolone (p<0.05). In 96 patients (NMO, n = 19; TM, n = 25; MS, n = 52) without treatment, serum levels of TSH, TG-Ab and TPO-Ab were significantly different between patients with and without myelitis (p<0.01). Patients positive for aquaporin-4 (AQP4) antibodies showed higher abnormalities of TSH (p = 0.001), TG-Ab (p = 0.004) and TPO-Ab (p<0.0001) levels than AQP4 antibodies negative patients. Logistic regression analyses revealed independent relationships between TSH (odds ratio [OR]  = 33.994; p<0.0001), TG-Ab (OR = 7.703; p = 0.017) and myelitis occurrence in 96 patients at the active stage. In 52 MS patients experiencing their first attack, MS patients with myelitis were associated with TSH abnormalities (OR = 42.778; p<0.0001). This study showed increased abnormalities of thyroid parameters in patients with NMO and TM than in MS patients. MS patients with myelitis also had greater TSH abnormality than in MS patients without myelitis. Abnormal TSH and TG-Ab were independently associated with myelitis occurrence in central nervous system demyelinating disorders.     

Red Blood Cell Distribution Width during the First Week Is Associated with Severity and Mortality in Septic Patients

Objective

Higher values of red blood cell distribution width (RDW) have been found in non-surviving than in surviving septic patients. However, it is unknown whether RDW during the first week of sepsis evolution is associated with sepsis severity and early mortality, oxidative stress and inflammation states, and these were the aims of the study.

Methods

We performed a prospective, observational, multicenter study in six Spanish Intensive Care Units with 297 severe septic patients. We measured RDW, serum levels of malondialdehyde (MDA) to assess oxidative stress, and tumour necrosis factor (TNF)-α to assess inflammation at days 1, 4, and 8. The end-point was 30-day mortality.

Results

We found higher RDW in non-surviving (n = 104) than in surviving (n = 193) septic patients at day 1 (p = 0.001), day 4 (p = 0.001), and day 8 (p = 0.002) of ICU admission. Cox regression analyses showed that RDW at day 1 (p<0.001), 4 (p = 0.005) and 8 (p = 0.03) were associated with 30-day mortality. Receiver operating characteristic curves showed that RDW at day 1 (p<0.001), 4 (p<0.001), and 8 (p<0.001) could be used to predict 30-day mortality. RDW showed a positive correlation with serum MDA levels at day 1 and day 4, with serum TNF-α levels at days 4 and 8, and with SOFA score at days 1, 4 and 8.

Conclusions

The major findings of our study were that non-surviving septic patients showed persistently higher RDW during the first week of ICU stay than survivors, that RDW during the first week was associated with sepsis severity and mortality, that RDW during the first week could be used as biomarker of outcome in septic patients, and that there was an association between RDW, serum MDA levels, and serum TNF-α levels during the first week.     

Increased Serum C-Reactive Protein Level Is Associated with Increased Storage Lower Urinary Tract Symptoms in Men with Benign Prostatic Hyperplasia

Objective

Chronic inflammation is considered as one of the contributing mechanisms of lower urinary tract symptoms (LUTS). Serum C-reactive protein (CRP) level is the widely used biomarker of inflammatory status. This study investigated the association between serum CRP level in men with benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) before and after medical treatment.

Methods

A total of 853 men with BPH and LUTS were enrolled. All patients completed the International Prostate Symptoms Score (IPSS) questionnaire and urological examinations. The parameters of uroflowmetry (maximum flow rate, Qmax; voided volume, VV), post-void residual (PVR), total prostate volume (TPV) and transition zone index (TZI), serum prostate specific antigen (PSA), and serum CRP levels were obtained. All patients were treated with alpha-blocker or antimuscarinic agent based on the IPSS voiding to storage subscore ratio (IPSS-V/S). Correlation analyses were performed between serum CRP levels with age, IPSS, TPV, TZI, Qmax, PVR, VV, PSA and between baseline and post treatment.

Results

The mean age was 66.9±11.6 years old and the mean serum CRP levels were 0.31±0.43 mg/dL. Univariate analyses revealed serum CRP levels were significantly associated with age (p<0.001), PSA levels (p = 0.005) and VV (p = 0.017), but not significantly associated with TPV (p = 0.854) or PVR (p = 0.068). CRP levels were positively associated with urgency (p<0.001) and nocturia (p<0.001) subscore of IPSS, total IPSS (p = 0.008) and storage IPSS (p<0.001) and negatively associated with IPSS- V/S ratio (p = 0.014). Multivariate analyses revealed that serum CRP levels were significantly associated with age (p = 0.004) and storage IPSS subscore p<0.001). Patients with IPSS-V/S<1 and treated with tolterodine for 3 months had significant decrease of CRP levels after treatment.

Conclusion

Serum CRP levels are associated with storage LUTS and sensory bladder disorders, suggesting chronic inflammation might play a role in the patients with storage predominant LUTS.     

Platelet Turnover in Stable Coronary Artery Disease – Influence of Thrombopoietin and Low-Grade Inflammation

Background

Newly formed platelets are associated with increased aggregation and adverse outcomes in patients with coronary artery disease (CAD). The mechanisms involved in the regulation of platelet turnover in patients with CAD are largely unknown.

Aim

To investigate associations between platelet turnover parameters, thrombopoietin and markers of low-grade inflammation in patients with stable CAD. Furthermore, to explore the relationship between platelet turnover parameters and type 2 diabetes, prior myocardial infarction, smoking, age, gender and renal insufficiency.

Methods

We studied 581 stable CAD patients. Platelet turnover parameters (immature platelet fraction, immature platelet count, mean platelet volume, platelet distribution width and platelet large cell-ratio) were determined using automated flow cytometry (Sysmex XE-2100). Furthermore, we measured thrombopoietin and evaluated low-grade inflammation by measurement of high-sensitive CRP and interleukin-6.

Results

We found strong associations between the immature platelet fraction, immature platelet count, mean platelet volume, platelet distribution width and platelet large cell ratio (r = 0.61–0.99, p<0.0001). Thrombopoietin levels were inversely related to all of the platelet turnover parameters (r = −0.17–−0.25, p<0.0001). Moreover, thrombopoietin levels were significantly increased in patients with diabetes (p = 0.03) and in smokers (p = 0.003). Low-grade inflammation evaluated by high-sensitive CRP correlated significantly, yet weakly, with immature platelet count (r = 0.10, p = 0.03) and thrombopoietin (r = 0.16, p<0.001). Also interleukin-6 correlated with thrombopoietin (r = 0.10, p = 0.02).

Conclusion

In stable CAD patients, thrombopoietin was inversely associated with platelet turnover parameters. Furthermore, thrombopoietin levels were increased in patients with diabetes and in smokers. However, low-grade inflammation did not seem to have a substantial impact on platelet turnover parameters.     

Liver microRNA hsa-miR-125a-5p in HBV Chronic Infection: Correlation with HBV Replication and Disease Progression

To study in HBsAg chronic carriers the expression of liver hsa-miR-125a-5p and its correlation with liver HBV-DNA values and clinical presentation, 27 consecutive Caucasian, HBsAg/anti-HBe/HBV-DNA-positive patients who were naive to nucleos(t)ide analogues and interferon therapy and had no marker of HCV, HDV or HIV infection and no history of alcohol intake were enrolled. For each patient, liver HBV DNA and liver hsa-miR-125a-5p were quantified by real-time PCR in relation to β-globin DNA or RNU6B, respectively. Liver fibrosis and necroinflammation were graded by applying Ishak''s scoring system. Liver hsa-miR-125a-5p was detected in all patients enrolled and a correlation between its concentration and liver HBV DNA was demonstrated (p<0.0001). Higher liver hsa-miR-125a-5p concentrations were observed in patients with HBV-DNA plasma level >10³ IU/ml (p<0.02), in those with HAI >6 (p = 0.02) and those with fibrosis score >2 (p<0.02) than in patients with lower scores. Higher HBV-DNA liver concentrations were found in patients with abnormal AST (p = 0.005) and ALT serum levels (p = 0.05), in those with serum HBV DNA higher than 10E3 IU/mL (p = 0.001) and those with fibrosis score >2 (p = 0.02) than in patients with a lower load. By multivariate logistic regression analysis, liver hsa-miR-125a-5p was identified as an independent predictor of disease progression: O.R. = 4.21, C.I. 95%  = 1.08–16.43, p<0.05, for HAI >6; O.R. = 3.12, C.I. 95%  = 1.17–8.27, p<0.05, for fibrosis score >2. In conclusion, in HBsAg/anti-HBe-positive patients, the liver hsa-miR-125a-5p level correlated with liver and plasma HBV-DNA values and was associated to a more severe disease progression.     

Diagnostic and Prognostic Impact of Circulating YKL-40, IL-6, and CA 19.9 in Patients with Pancreatic Cancer

Purpose

We tested the hypothesis that high plasma YKL-40 and IL-6 associate with pancreatic cancer and short overall survival.

Patients and Methods

In all, 559 patients with pancreatic cancer from prospective biomarker studies from Denmark (n = 448) and Germany (n = 111) were studied. Plasma YKL-40 and IL-6 were determined by ELISAs and serum CA 19.9 by chemiluminescent immunometric assay.

Results

Odds ratios (ORs) for prediction of pancreatic cancer were significant for all biomarkers, with CA 19.9 having the highest AUC (CA 19.9: OR = 2.28, 95% CI 1.97 to 2.68, p<0.0001, AUC = 0.94; YKL-40: OR = 4.50, 3.99 to 5.08, p<0.0001, AUC = 0.87; IL-6: OR = 3.68, 3.08 to 4.44, p<0.0001, AUC = 0.87). Multivariate Cox analysis (YKL-40, IL-6, CA 19.9, age, stage, gender) in patients operated on showed that high preoperative IL-6 and CA 19.9 (dichotomized according to normal values) were independently associated with short overall survival (CA 19.9: HR = 2.51, 1.22–5.15, p = 0.013; IL-6: HR = 2.03, 1.11 to 3.70, p = 0.021). Multivariate Cox analysis of non-operable patients (Stage IIB-IV) showed that high pre-treatment levels of each biomarker were independently associated with short overall survival (YKL-40: HR = 1.30, 1.03 to 1.64, p = 0.029; IL-6: HR = 1.71, 1.33 to 2.20, p<0.0001; CA 19.9: HR = 1.54, 1.06 to 2.24, p = 0.022). Patients with preoperative elevation of both IL-6 and CA 19.9 had shorter overall survival (p<0.005) compared to patients with normal levels of both biomarkers (45% vs. 92% alive after 12 months).

Conclusions

Plasma YKL-40 and IL-6 had less diagnostic impact than CA 19.9. Combination of pretreatment YKL-40, IL-6, and CA 19.9 may have clinical value to identify pancreatic cancer patients with the poorest prognosis.     

Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity

Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005) in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG), CXCL10 (IP-10) and CCL19 (MIP3B) were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively). There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.     

Soluble Markers of the Toll-Like Receptor 4 Pathway Differentiate between Active and Latent Tuberculosis and Are Associated with Treatment Responses

Background

Biomarkers to differentiate between active tuberculosis (TB) and latent TB infection (LTBI) and to monitor treatment responses are requested to complement TB diagnostics and control, particularly in patients with multi-drug resistant TB. We have studied soluble markers of the Toll-like-receptor 4 (TLR-4) pathway in various stages of TB disease and during anti-TB treatment.

Methods

Plasma samples from patients with culture confirmed drug-sensitive TB (n = 19) were collected before and after 2, 8 and 24 weeks of efficient anti-TB treatment and in a LTBI group (n = 6). Soluble (s) CD14 and myeloid differentiation-2 (MD-2) were analyzed by the Enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was analyzed by the Limulus Amebocyte Lysate colorimetric assay. Nonparametric statistics were applied.

Results

Plasma levels of sCD14 (p<0.001), MD-2 (p = 0.036) and LPS (p = 0.069) were elevated at baseline in patients with untreated active TB compared to the LTBI group. MD-2 concentrations decreased after 2 weeks of treatment (p = 0.011), while LPS levels decreased after 8 weeks (p = 0.005). In contrast, sCD14 levels increased after 2 weeks (p = 0.047) with a subsequent modest decrease throughout the treatment period. There was no significant difference in concentrations of any of these markers between patients with pulmonary and extrapulmonary TB or between patients with or without symptoms.

Conclusion

Our data suggest that plasma levels of LPS, MD-2 and sCD14 can discriminate between active TB and LTBI. A decline in LPS and MD-2 concentrations was associated with response to anti-TB treatment. The clinical potential of these soluble TLR-4 pathway proteins needs to be further explored.     

Study on Risk Factors for Severe Hand,Foot and Mouth Disease in China

Background

Epidemics of HFMD are elevated every year globally, especially in mainland China. The disease now presents as an increasing threat to public health worldwide.

Methods

Five hundred and seventy-one EV71-infected HFMD patients in Beijing You''an Hospital were grouped by disease severity: Mild (no severe complication) (n = 221), and Severe group (complicated with brainstem encephalitis (BE), and/or pulmonary edema (PE) (n = 350)). Clinical and laboratory findings and levels of 7 serum cytokines were analyzed.

Results

Univariate analysis showed that (RR)>26/min (p<0.001), age<4 yo (p = 0.001), GLU>8.3 mmol/L (p = 0.008), CL<98 mmol/L (p = 0.026), and WBC>1.2×10⁹/L (p = 0.040) were associated with severe cases. Results of multivariate analysis indicated five independent risk factors (RR>26/min (p<0.001), Age<4 yo (p<0.001), GLU>8.3 mmol/L (p = 0.011), LYM>40% (p = 0.010), and ALT>40 U/L (p = 0.045)). In addition to single-factor analysis, we further analyzed the use of different combinations of risk factors. “GLU>8.3 and CL<98 and RR>26” (confidence ration (CR) = 100%) is the top indicator, followed by “ALT>40 and LYM>40% and RR>26 and Age<4 yo” (CR = 92.9%).Serum levels of IL-2, IL-4, IL-10, IFNγ, GM-CSF, and TNFα were higher in severe cases than in mild cases. A new evaluation scoring system by scoring each risk factor 1 and independent risk factor 2 was developed for early identification of severe HFMD cases.

Conclusions

Five independent risk factors, along with indicative combinations of risk factors, for severe cases were identified, and a scoring system was created to facilitate the use of indicators for early medical intervention.     

A Higher Frequency of Circulating IL-22+CD4+ T Cells in Chinese Patients with Newly Diagnosed Hashimoto’s Thyroiditis

Background

IL-22 and IL-17A are implicated in the pathogenesis of autoimmune diseases. However, the role of IL-22⁺ and IL-17A⁺ CD4⁺ T cells in the pathogenesis of Hashimoto’s thyroiditis (HT) is not fully understood. This study investigates serum IL-22 and IL-17A levels and determines the frequency of circulating IL-22⁺ CD4⁺ T cells in HT patients to understand their roles in the pathogenesis of HT.

Methods

The levels of serum IL-22, IL-17A and IFN-γ and the frequency of circulating IL-22⁺CD4⁺ and IL-17A⁺CD4⁺ T cells in 17 HT patients and 17 healthy controls (HC) were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The levels of serum free triiodothyronine (FT4), free thyroxine (FT3), thyroid stimulating hormone (TSH), anti-thyroid peroxidase (TPO) and anti-thyroglobulin antibodies (TgAb) by chemiluminescent enzyme immunoassay and radioimmunoassay.

Results

The percentages of circulating IL-22⁺CD4⁺ and IL-17⁺CD4⁺ T cells (p<0.0001, p<0.0001) and the levels of serum IL-22, IL-17A and IFN-γ (p<0.0001, p<0.0001, p = 0.0210) in the HT patients were significantly higher than that in the HC. The percentages of IL-22⁺CD4⁺ T cells were positively correlated with Th17 cells (r = 0.8815, p<0.0001) and IL-17A⁺IL-22⁺CD4⁺ T cells (r = 0.8914, p<0.0001), but were negatively correlated with Th1 cells (r = −0.6110, p<0.0092) in the HT patients. The percentages of Th22 cells, Th17 cells and IL-17A⁺IL-22⁺CD4⁺ T cells were negatively correlated with the levels of serum TSH in the HT patients (r = −0.8402, p<0.0001; r = −0.8589, p<0.0001; r = −0.8289 p<0.0001, respectively).

Conclusions

A higher frequency of circulating IL-22⁺CD4⁺ and IL-17A⁺CD4⁺ T cells may be associated with the development of HT in Chinese patients.     

Fibrin Clot Structure and Platelet Aggregation in Patients with Aspirin Treatment Failure

Background

Aspirin is a cornerstone in prevention of cardiovascular events and modulates both platelet aggregation and fibrin clot formation. Some patients experience cardiovascular events whilst on aspirin, often termed aspirin treatment failure (ATF). This study evaluated both platelet aggregation and fibrin clot structure in patients with ATF.

Methods

We included 177 stable coronary artery disease patients on aspirin monotherapy. Among these, 116 (66%) had ATF defined as myocardial infarction (MI) whilst on aspirin. Platelet aggregation was assessed by Multiplate® aggregometry and VerifyNow®, whereas turbidimetric assays and scanning electron microscopy were employed to study fibrin clot characteristics.

Results

Enhanced platelet aggregation was observed in patients with ATF compared with non-MI patients following stimulation with arachidonic acid 1.0 mM (median 161 (IQR 95; 222) vs. 97 (60; 1776) AU*min, p = 0.005) and collagen 1.0 µg/mL (293 (198; 427) vs. 220 (165; 370) AU*min, p = 0.03). Similarly, clot maximum absorbance, a measure of fibrin network density, was increased in patients with ATF (0.48 (0.41; 0.52) vs. 0.42 (0.38; 0.50), p = 0.02), and this was associated with thinner fibres (mean ± SD: 119.7±27.5 vs. 127.8±31.1 nm, p = 0.003) and prolonged lysis time (552 (498; 756) vs. 519 (468; 633) seconds; p = 0.02). Patients with ATF also had increased levels of C-reactive protein (CRP) (1.34 (0.48; 2.94) and 0.88 (0.32; 1.77) mg/L, p = 0.01) compared with the non-MI group. Clot maximum absorbance correlated with platelet aggregation (r = 0.31–0.35, p-values<0.001) and CRP levels (r = 0.60, p<0.001).

Conclusions

Patients with aspirin treatment failure showed increased platelet aggregation and altered clot structure with impaired fibrinolysis compared with stable CAD patients without previous MI. These findings suggest that an increased risk of aspirin treatment failure may be identified by measuring both platelet function and fibrin clot structure.     

High Levels of Asymmetric Dimethylarginine Are Strongly Associated with Low HDL in Patients with Acute Myocardial Infarction

Objectives

Low levels of high-density lipoprotein (HDL) cholesterol are associated with an increased risk of acute myocardial infarction possibly through impaired endothelial atheroprotection and decreased nitric oxide (NO) bioavailability. Asymmetric dimethylarginine (ADMA) mediates endothelial function by inhibiting nitric oxide synthase activity. In patients with acute myocardial infarction, we investigated the relationship between serum levels of HDL and ADMA.

Approach and Results

Blood samples from 612 consecutive patients hospitalized for acute MI <24 hours after symptom onset were taken on admission. Serum levels of ADMA, its stereoisomer, symmetric dimethylarginine (SDMA) and L-arginine were determined using high-performance liquid chromatography. Patients with low HDL (<40 mg/dL for men and <50 mg/dL for women) were compared with patients with higher HDL. Most patients (59%) had low HDL levels. Median ADMA levels were markedly higher in the low HDL group (0.69 vs. 0.50 µmole/L, p<0.001). In contrast, SDMA and L-arginine levels were similar for the two groups (p = 0.120 and p = 0.064). Notably, ADMA, but not SDMA or L-arginine, was inversely correlated with HDL (r = −0.311, p<0.001). In stratified analysis, this relationship was only found for low HDL levels (r = −0.265, p<0.001), but not when HDL levels were higher (r = −0.077, p = 0.225). By multivariate logistic regression analysis, ADMA level was strongly associated with low HDL levels (OR(95%CI):6.06(3.48–10.53), p<0.001), beyond traditional confounding factors.

Conclusions

Our large population-based study showed for the first time a strong inverse relationship between HDL and ADMA in myocardial infarction patients, suggesting a functional interaction between HDL and endothelium, beyond metabolic conditions associated with low HDL levels.     

Complement Alternative Pathway Activation in Human Nonalcoholic Steatohepatitis

The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (r_s = 0.69; p<0.05) and C3c/C3 activation ratio (r_s = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway activation in driving hepatic inflammation in NASH. Therefore, alternative pathway factors may be considered attractive targets for treating NASH by inhibiting complement activation.     

Osteoprotegerin,Pericytes and Bone-Like Vascular Calcification Are Associated with Carotid Plaque Stability

Background and Purpose

Vascular calcification, recapitulating bone formation, has a profound impact on plaque stability. The aim of the present study was to determine the influence of bone-like vascular calcification (named osteoid metaplasia = OM) and of osteoprotegerin on plaque stability.

Methods

Tissue from carotid endarterectomies were analysed for the presence of calcification and signs of vulnerability according to AHA grading system. Osteoprotegerin (OPG), pericytes and endothelial cells were sought using immuno-histochemistry. Symptoms and preoperative imaging findings (CT-scan, MRI and Doppler-scan) were analyzed. Human pericytes were cultured to evaluate their ability to secrete OPG and to influence mineralization in the plaque.

Results

Seventy-three carotid plaques (49 asymptomatic and 24 symptomatic) were harvested. A significantly higher presence of OM (18.4% vs 0%, p<0.01), OPG (10.2% of ROI vs 3.4% of ROI, p<0.05) and pericytes (19% of ROI vs 3.8% of ROI, p<0.05) were noted in asymptomatic compared to symptomatic plaques. Consistently, circulating OPG levels were higher in the plasma of asymptomatic patients (3.2 ng/mL vs 2.5 ng/mL, p = 0.05). In vitro, human vascular pericytes secreted considerable amounts of OPG and underwent osteoblastic differentiation. Pericytes also inhibited the osteoclastic differentiation of CD14+ cells through their secretion of OPG.

Conclusions

OPG (intraplaque an plasmatic) and OM are associated with carotid plaque stability. Pericytes may be involved in the secretion of intraplaque OPG and in the formation of OM.     

Low Serum 25-Hydroxyvitamin D Concentrations Are Associated with Increased Risk for Melanoma and Unfavourable Prognosis

Background

Low vitamin D status (serum 25(OH)D concentration) is associated with increased incidence and unfavourable outcome of various types of cancer. However, there are limited data on influence of serum 25(OH)D on risk and prognosis of malignant melanoma.

Methods

Basal serum 25(OH)D concentrations were retrospectively analyzed in a cohort of melanoma patients (n = 324) and healthy controls (n = 141). We tested the hypothesis that serum 25(OH)D concentrations are predictive of melanoma risk, thickness of primary melanomas, and overall survival (OS).

Results

Median serum 25(OH)D concentrations were significantly lower (p = 0.004) in melanoma patients (median = 13.6 ng/ml) as compared to controls (median = 15.6 ng/ml). Primary tumors of patients with low serum 25(OH)D concentrations (<10 ng/ml) had significantly (p = 0.006) greater Breslow thickness (median: 1.9 mm) as compared to patients with higher levels (>20 ng/ml; median: 1.00 mm). Patients with 25(OH)D serum concentrations in the lowest quartile had inferior overall survival (median: 80 months) comparing with the highest quartile (median: 195 months; p = 0.049).

Conclusions

Our data support the concept that serum 25(OH)D concentrations are associated with risk and prognosis of melanoma. Whether normalizing serum 25(OH)D concentrations in these patients improves outcomes will require testing in future clinical trials.     

Overexpression of CCN3 Inhibits Inflammation and Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice

Background

Cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed (CCN) 3 has been recently reported to play a role in regulating inflammation of vascular endothelial cells. However, the role of CCN3 in atherosclerosis, which is characterized by vascular inflammation, remains unclear.

Hypothesis and Objectives

Overexpression of CCN3 may relieve the inflammation response in and inhibit the progress of atherosclerosis. We aimed to explore the potential roles of CCN3 in inflammation in atherosclerosis.

Strategy and Main Results

In in vitro studies using cultured human aortic endothelial cells and human umbilical vein endothelial cells, CCN3 mRNA and protein expression significantly decreased in response to tumor necrosis factor-α and interleukin-1β treatments (p<0.05), when analyzed by quantitative real-time polymerase chain reaction and Western blot. Using a mouse model of atherosclerosis, the mRNA and protein levels of CCN3 decreased by 72.2% (p = 0.041) and 86.4% (p = 0.036), respectively, compared with levels in wild-type control mice, respectively. Overexpression of CCN3 by adenovirus-mediated gene overexpression decreased low-density lipoprotein cholesterol by 48.9% (p = 0.017), total cholesterol by 58.9% (p = 0.031), and triglycerides by 56.8% (p = 0.022), and it increased high-density lipoprotein cholesterol level by 2.16-fold (p = 0.039), compared with control groups. Additionally, a reduced plaque area and increased fibrous cap were observed (p<0.05). Furthermore, CCN3 overexpression decreased cell adhesion molecule-1 mRNA expression by 84.7% (p = 0.007) and intercellular adhesion molecule-1 mRNA expression by 61.2% (p = 0.044). Inflammatory factors, including matrix metalloproteinases, cyclooxygenase 2, and tissue factor also significantly (p<0.05) decreased with CCN3 overexpression in the atherosclerotic mouse model. Additionally, CCN1 and CCN2, which have been reported to be highly expressed in aortic atherosclerotic plaques, were significantly downregulated (p<0.05) by CCN3 overexpression.

Conclusion

CCN3 overexpression is associated with control of inflammatory processes and reversion of dyslipidemia in the process of atherosclerosis, which implies that CCN3 may be a promising target in the treatment of atherosclerosis.