Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection

Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.     

Serum Levels of Coenzyme Q10 in Patients with Multiple System Atrophy

The COQ2 gene encodes an essential enzyme for biogenesis, coenzyme Q10 (CoQ10). Recessive mutations in this gene have recently been identified in families with multiple system atrophy (MSA). Moreover, specific heterozygous variants in the COQ2 gene have also been reported to confer susceptibility to sporadic MSA in Japanese cohorts. These findings have suggested the potential usefulness of CoQ10 as a blood-based biomarker for diagnosing MSA. This study measured serum levels of CoQ10 in 18 patients with MSA, 20 patients with Parkinson’s disease and 18 control participants. Although differences in total CoQ10 (i.e., total levels of serum CoQ10 and its reduced form) among the three groups were not significant, total CoQ10 level corrected by serum cholesterol was significantly lower in the MSA group than in the Control group. Our findings suggest that serum CoQ10 can be used as a biomarker in the diagnosis of MSA and to provide supportive evidence for the hypothesis that decreased levels of CoQ10 in brain tissue lead to an increased risk of MSA.     

Elevated Levels of Procoagulant Plasma Microvesicles in Dialysis Patients

Cardiovascular (CV) death remains the largest cause of mortality in dialysis patients, unexplained by traditional risk factors. Endothelial microvesicles (EMVs) are elevated in patients with traditional CV risk factors and acute coronary syndromes while platelet MVs (PMVs) are associated with atherosclerotic disease states. This study compared relative concentrations of circulating MVs from endothelial cells and platelets in two groups of dialysis patients and matched controls and investigated their relative thromboembolic risk. MVs were isolated from the blood of 20 haemodialysis (HD), 17 peritoneal dialysis (PD) patients and 20 matched controls. Relative concentrations of EMVs (CD144^{+ ve}) and PMVs (CD42b^{+ ve}) were measured by Western blotting and total MV concentrations were measured using nanoparticle-tracking analysis. The ability to support thrombin generation was measured by reconstituting the MVs in normal plasma, using the Continuous Automated Thrombogram assay triggered with 1µM tissue factor. The total concentration of MVs as well as the measured sub-types was higher in both patient groups compared to controls (p<0.05). MVs from HD and PD patients were able to generate more thrombin than the controls, with higher peak thrombin, and endogenous thrombin potential levels (p<0.02). However there were no differences in either the relative quantity or activity of MVs between the two patient groups (p>0.3). Dialysis patients have higher levels of circulating procoagulant MVs than healthy controls. This may represent a novel and potentially modifiable mediator or predictor of occlusive cardiovascular events in these patients.     

Coenzyme Q10, Copper,Zinc, and Lipid Peroxidation Levels in Serum of Patients with Chronic Obstructive Pulmonary Disease

Severity of chronic obstructive pulmonary disease (COPD) exacerbation is associated with increased level of copper (Cu), zinc (Zn), and lipid peroxidation (malodialdehyde, MDA). The aim of this study was to investigate the levels of lipid peroxidation, Coenzyme Q10 (CoQ10), Zn, and Cu in the COPD exacerbations. Forty-five patients with COPD acute exacerbation and 45 healthy smokers as control group were used in the study. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were lower in exacerbation group than in control. C- reactive protein levels, white blood cell count, and sedimentation rate were significantly (p < 0.001) higher in patients than in control. CoQ10 level and Cu/Zn ratio was significantly (p < 0.05) lower in patients than in control, although MDA, Cu, and Zn levels were significantly (p < 0.05) higher in patients than in control. Negative correlations were found among MDA, Cu, Zn, FEV1, and FVC values in exacerbation and control subjects (p < 0.05). In conclusion, we observed that oxidative stress in the exacerbation period of COPD patients was increased. The decrease in CoQ10 level and Cu/Zn ratio and elevation in Cu and Zn levels observed in the patients probably result from the defense response of organism and are mediated by inflammatory-like substances.     

Serum Copper,Zinc, and Magnesium Levels in Patients with Chronic Fluorosis

Although there are many studies on effect of fluoride on trace elements in experimental animals, few studies exist on serum trace elements levels in patients with endemic fluorosis. We aimed to determine the serum levels of trace elements including serum copper (Cu), zinc (Zn), and serum levels of minerals including calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K) in patients with endemic fluorosis. The study group consisted of 30 patients with endemic fluorosis (17 females, 13 males, mean age 33.53 ± 9.85 years). An age, gender, and body mass index matched 30 healthy volunteers comprised control group (21 females, ten males with a mean age 33.93 ± 7.39 years). Urine fluoride levels of chronic fluorosis patients were significantly higher than that of control subjects as expected (1.92 ± 0.10 mg/l vs. 0.41 ± 0.09 mg/l, respectively; P < 0.001). Serum Cu levels (89.14 ± 16.77 μg/dL vs. 102.69 ± 25.04 μg/dL, respectively, P = 0.017), serum Zn levels (77.98 ± 20.58 μg/dL vs. 94.57 ± 35.87μg/dL, respectively, P = 0.032), and serum Mg levels (1.92 ± 0.18 mg/dL vs. 2.07 ± 0.31 mg/dL, respectively, p = 0.022) was significantly lower in chronic fluorosis patients than in controls. There were no statistically significant differences between the fluorosis group and control group with respect to serum levels of Na, K, Ca, and P. We concluded that chronic fluorosis is associated with reduced serum levels of Cu, Zn, and Mg.     

Elevated Serum Levels of Mannose-Binding Lectin and Diabetic Nephropathy in Type 2 Diabetes

Objective

Inflammation and complement activation initiated by mannose-binding lectin (MBL) may be implicated in the pathogenesis of diabetic vascular complications. We investigated serum MBL levels in type 2 diabetes with diabetic nephropathy (DN) and with persistent normoalbuminuria.

Method

Serum MBL levels were determined in 242 type 2 diabetes with overt nephropathy and 242 type 2 diabetes with persistent normoalbuminuria matched for age, sex, and duration of diabetes, as well as in 100 healthy control subjects. The prediction value of MBL was compared with HbA1c, Hs-CRP and with other known predictors. Multivariate analyses were performed using logistic regression models.

Results

The serum MBL levels were significantly higher in diabetes with DN as compared to with persistent normoalbuminuria (P<0.0001). Multivariate logistic regression analysis adjusted for common factors showed that serum MBL levels≥2950ug/L was an independent indictor of DN (OR=7.55; 95%CI: 3.44–19.04). Based on the ROC curve, the optimal cutoff value of serum MBL levels as an indicator for diagnosis of DN was projected to be 2950ug/L, which yielded a sensitivity of 77.2 % and a specificity of 80.8%, with the area under the curve at 0.809 (95%CI, 0.769—0.848).

Conclusion

Our findings suggested that MBL may be involved in the pathogenesis of DN in type 2 diabetes, and that determination of MBL status might be used to identify patients at increased risk of developing nephropathy complications.     

Elevated Serum Levels of Macrophage Migration Inhibitory Factor Are Associated with Progressive Chronic Cardiomyopathy in Patients with Chagas Disease

Clinical symptoms of chronic Chagas disease occur in around 30% of the individuals infected with Trypanosoma cruzi and are characterized by heart inflammation and dysfunction. The pathogenesis of chronic chagasic cardiomyopathy (CCC) is not completely understood yet, partially because disease evolution depends on complex host-parasite interactions. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that promotes numerous pathophysiological processes. In the current study, we investigated the link between MIF and CCC progression.Immunohistochemical analysis demonstrated MIF overexpression in the hearts from chronically T. cruzi-infected mice, particularly those showing intense inflammatory infiltration. We also found that MIF exogenously added to parasite-infected murine macrophage cultures is capable of enhancing the production of TNF-α and reactive oxygen species, both with pathogenic roles in CCC. Thus, the integrated action of MIF and other cytokines and chemokines may account for leukocyte influx to the infected myocardium, accompanied by enhanced local production of multiple inflammatory mediators. We further examined by ELISA the level of MIF in the sera from chronic indeterminate and cardiomyopathic chagasic patients, and healthy subjects. CCC patients displayed significantly higher MIF concentrations than those recorded in asymptomatic T. cruzi-infected and uninfected individuals. Interestingly, increased MIF levels were associated with severe progressive Chagas heart disease, in correlation with elevated serum concentration of high sensitivity C-reactive protein and also with several echocardiographic indicators of left ventricular dysfunction, one of the hallmarks of CCC. Our present findings represent the first evidence that enhanced MIF production is associated with progressive cardiac impairment in chronic human infection with T. cruzi, strengthening the relationship between inflammatory response and parasite-driven pathology. These observations contribute to unravel the elements involved in the pathogenesis of CCC and may also be helpful for the design of novel therapies aimed to control long-term morbidity in chagasic patients.     

IL-10、IL-18和内毒素在肝硬化患者自发性细菌性腹膜炎中的作用

目的:探讨由肝硬化合并自发性细菌性腹膜炎(spontaneous bacterial peritonitis,SBP)患者血清中IL-10、IL-18及内毒素的水平及其临床意义.方法:采用ELISA方法检测各组血清IL-10、IL-18水平,采用偶氮显色基质法检测内毒素水平.结果:SBP患者血清中IL-10、IL-18及内毒素的水平明显高于正常对照组,P＜0.05,SBP患者治疗后血清中IL-10、IL-18及内毒素的水平都明显降低,P＜0.05.结论:检测血清中IL-10、IL-18及内毒素在肝硬化患者自发性细菌性腹膜炎中的诊断及预后中具有一定指导意义.     

Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations.     

Serum Copper and Zinc Levels in Patients with Endometrial Cancer

Biological Trace Element Research - The aim of the present study was to evaluate serum concentrations of copper (Cu) and zinc (Zn), in relation with metabolic profile and clinicopathologic features...     

Elevated Serum Triglyceride and Retinol-Binding Protein 4 Levels Associated with Fructose-Sweetened Beverages in Adolescents

Background

The metabolic effect of fructose in sugar-sweetened beverages (SSB) has been linked to de novo lipogenesis and uric acid (UA) production.

Objectives

This study investigated the biological effects of SSB consumption on serum lipid profiles and retinol-binding protein 4 (RBP4) among Taiwanese adolescents.

Methods

We evaluated the anthropometric parameters and biochemical outcomes of 200 representative adolescents (98 boys and 102 girls) who were randomly selected from a large-scale cross-sectional study. Data were analyzed using multiple regression models adjusted for covariates.

Results

Increased SSB consumption was associated with increased waist and hip circumferences, body mass index (BMI) values and serum UA, triglyceride (TG) and RBP4 levels. Adolescents who consumed >500 ml/day of beverages half-to-heavily sweetened with high-fructose corn syrup (HFCS) exhibited TG and RBP4 levels 22.7 mg/dl and 13.92 ng/ml higher than non-drinkers, respectively. HFCS drinkers with hyperuricemia had higher TG levels than HFCS drinkers with normal UA levels (98.6 vs. 81.6 mg/dl). The intake of HFCS-rich SSBs and high value of BMI (≥24) interactively reinforced RBP4 levels among overweight/obese adolescents. Circulating RBP4 levels were significantly correlated with weight-related outcomes and TG and UA concentration among HFCS drinkers (r = 0.253 to 0.404), but not among non-drinkers.

Conclusions

High-quantity HFCS-rich beverage consumption is associated with higher TG and RBP4 levels. Hyperuricemia is likely to intensify the influence of HFCS-rich SSB intake on elevated TG levels, and in overweight and obese adolescents, high BMI may modify the action of fructose on higher circulating levels of RBP4.     

Circulating Heat Shock Protein 60 Levels Are Elevated in HIV Patients and Are Reduced by Anti-Retroviral Therapy

Circulating heat shock protein 60 (Hsp60) and heat shock protein 10 (Hsp10) have been associated with pro- and anti-inflammatory activity, respectively. To determine whether these heat shock proteins might be associated with the immune activation seen in HIV-infected patients, the plasma levels of Hsp60 and Hsp10 were determined in a cohort of 20 HIV-infected patients before and after effective combination anti-retroviral therapy (cART). We show for the first time that circulating Hsp60 levels are elevated in HIV-infected patients, with levels significantly reduced after cART, but still higher than those in HIV-negative individuals. Hsp60 levels correlated significantly with viral load, CD4 counts, and circulating soluble CD14 and lipopolysaccharide levels. No differences or correlations were seen for Hsp10 levels. Elevated circulating Hsp60 may contribute to the immune dysfunction and non-AIDS clinical events seen in HIV-infected patients.     

Angiopoietin-2 and Biliary Diseases: Elevated Serum,but Not Bile Levels Are Associated with Cholangiocarcinoma

Background

The diagnosis of cholangiocarcinoma (CC) is challenging especially in patients with primary sclerosing cholangitis (PSC) and often delayed due to the lack of reliable markers. Angiopoietin-2 (Angpt-2) has been employed as a biomarker of angiogenesis and might be involved in tumor neoangiogenesis.

Aim

To evaluate the diagnostic potential of Angpt-2 as a biomarker to detect patients with CC.

Methods

Bile and serum Angpt-2 levels were measured in patients with CC (n = 45), PSC (n = 74), CC complicating PSC (CC/PSC) (n = 11) and patients with bile duct stones (n = 37) in a cross sectional study. Diagnostic accuracy of Angpt-2 was compared to carbohydrate antigen 19-9 (CA19-9). Fluorescent immunohistochemistry from human CC liver tissue samples was performed to localize the origin of Angpt-2.

Results

Serum Angpt-2 concentration was significantly elevated in patients with CC compared to control patients (p<0.05). Diagnostic accuracy of Angpt-2 as determined by receiver operating characteristic (ROC) analysis resulted in a higher area under the curve (AUC) value compared to CA19-9 (AUC: 0.85 versus 0.77; 95% confidence interval (CI): 0.74–0.93 versus 0.65–0.87, respectively). Angpt-2 was also detectable in bile, but was not associated with the presence of CC. Immunohistochemistry revealed a strong induction of Angpt-2 expression in the tumor vasculature.

Conclusions

Circulating Angpt-2 in serum might be a promising protein candidate locally derived from the tumor vasculature in patients with CC. Measurement of Angpt-2 in serum may be useful for diagnosis and further clinical management of patients with CC.     

Evaluation of Oxidative Stress, Antioxidant Status and Serum Vitamin C Levels in Cancer Patients

Taking into account the importance role of lipid peroxidation and antioxidants in the prevention and incidence of cancer, the present study was carried out to determine oxidative stress, serum total antioxidant (TAS), and vitamin C levels in cancer patients. Malondialdehyde(MDA), total antioxidant status, and vitamin C levels of 57cancer patients aged 19–80 years and 22 healthy subjects (control group) aged 22–76 years were evaluated. Serum concentrations of MDA as thiobarbitaric acid complexes were measured by fluorometry method, the serum TAS by using commercial test kits from Randox Laboratories, and vitamin C by using spectrocolorimetric method. The mean serum MDA concentrations of all cancer groups except lung cancer were significantly higher than control group (P < 0.004). The mean total antioxidant status was insignificantly higher than control group. The mean serum vitamin C level was significantly lower in patients as compared to the healthy subjects (PV < 0.0001). In conclusion, an alteration in the lipid peroxidation with concomitant changes in antioxidant defense system in cancer patients may be due to excessive oxidative stress. Serum low levels of vitamin C in the different type of cancer patients in spite of adequate daily intake may be due to increased utilization to scavenge lipid peroxides as well as their sequestration by tumor cells.