Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

BACKGROUND: Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. RESULTS: The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). CONCLUSIONS: The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget

Irreversible renal damage after transient renin-angiotensin system stimulation:involvement of an AT1-receptor mediated immune response

Transient activation of the renin-angiotensin system (RAS) induces irreversible renal damage causing sustained elevation in blood pressure (BP) in Cyp1a1-Ren2 transgenic rats. In our current study we hypothesized that activation of the AT1-receptor (AT1R) leads to a T-cell response causing irreversible impairment of renal function and hypertension. Cyp1a1-Ren2 rats harbor a construct for activation of the RAS by indole-3-carbinol (I3C). Rats were fed a I3C diet between 4-8 weeks of age to induce hypertension. Next, I3C was withdrawn and rats were followed-up for another 12 weeks. Additional groups received losartan (20 mg/kg/day) or hydralazine (100 mg/kg/day) treatment between 4-8 weeks. Rats were placed for 24h in metabolic cages before determining BP at week 8, 12 and 20. At these ages, subsets of animals were sacrificed and the presence of kidney T-cell subpopulations was investigated by immunohistochemistry and molecular marker analysis. The development of sustained hypertension was completely prevented by losartan, whereas hydralazine only caused a partial decrease in BP. Markers of renal damage: KIM-1 and osteopontin were highly expressed in urine and kidney samples of I3C-treated rats, even until 20 weeks of age. Additionally, renal expression of regulatory-T cells (Tregs) was highly increased in I3C-treated rats, whereas the expression of T-helper 1 (Th1) cells demonstrated a strong decrease. Losartan prevented these effects completely, whereas hydralazine was unable to affect these changes. In young Cyp1a1-Ren2 rats AT1R activation leads to induction of an immune response, causing a shift from Th1-cells to Tregs, contributing to the development of irreversible renal damage and hypertension

Arterial structure and function and environmental exposure to cadmium

Objectives: Few studies have addressed the effect of cadmium toxicity on arterial properties. Methods: We investigated the possible association of 24 h urinary cadmium excretion (an index of lifetime exposure) with measures of arterial function in a randomly selected population sample (n= 557) from two rural areas with low and high environmental exposure to cadmium. Results: 24 h urinary cadmium excretion was significantly higher in the high compared with the low exposure group (p < 0.001). Even though systolic (p = 0.42), diastolic (p = 0.14) and mean arterial pressure (p = 0.68) did not differ between the high and low exposure groups, aortic pulse wave velocity (p = 0.008), brachial pulse pressure (p = 0.026) and femoral pulse pressure (p = 0.008) were significantly lower in the high exposure group. Additionally, femoral distensibility (p, 0.001) and compliance (p = 0.001) were significantly higher with high exposure. Across quartiles of 24 h urinary cadmium excretion (adjusted for sex and age), brachial (p for trend = 0.015) and femoral (p for trend= 0.018) pulse pressure significantly decreased and femoral distensibility (p for trend= 0.008) and compliance (p for trend= 0.007) significantly increased with higher cadmium excretion. After full adjustment, the partial regression coefficients confirmed these associations. Pulse wave velocity (beta = -0.79 +/- 0.27; p = 0.004) and carotid (beta = -4.20 +/- 1.51; p = 0.006), brachial (beta = -5.43 +/- 1.41; p = 0.001) and femoral (beta = -4.72 +/- 1.74; p = 0.007) pulse pressures correlated negatively, whereas femoral compliance (beta = 0.11 +/- 0.05; p = 0.016) and distensibility (beta = 1.70 +/- 0.70; p = 0.014) correlated positively with cadmium excretion. Conclusion: Increased cadmium body burden is associated with lower aortic pulse wave velocity, lower pulse pressure throughout the arterial system, and higher femoral distensibility

Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

Background: Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. Results: The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). Conclusions: The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget

Telomere lenght and its associations with oxidized-LDL, carotid artery distensibility and smoking

Oxidative stress is a key factor driving the aging of cells and arteries. Studies suggest that white blood cell (WBC) telomere length is an index of systemic aging. We, therefore, investigated the association between WBC telomere length and oxidized-LDL, and vascular aging, expressed by the distensibility of the carotid artery. We studied a random population sample of 216 non-smokers and 89, smokers. In all subjects, age and gender- adjusted telomere length was inversely correlated with plasma oxidized-LDL (regression coefficient = -0.656 kb/mg/dL; p=0.0006). Independent of gender, age and mean blood pressure, carotid distensibility increased with telomere length (2.33+/- 1.18 10-3/kPa/kb; p=0.05) but decreased with higher plasma levels of oxidized LDL (-10.7+/- 3.91 10-3/kPa/ mg/dL; p=0.006). Adjusted for gender and age, smokers' telomere length was shorter (6.72 vs 6.91 kb; p=0.014) and plasma oxidized-LDL level higher (0.52 vs 0.46 mg/dL; p=0.03) than in non-smokers. Higher level of oxidized-LDL, is associated with shorter WBC telomeres and increased stiffness of the carotid artery. Smoking is marked by increased oxidative stress in concert with shortened WBC telomere length

Telomere length and its associations with oxidized-LDL, carotid artery distensibility and smoking

Oxidative stress is a key factor driving the aging of cells and arteries. Studies suggest that white blood cell (WBC) telomere length is an index of systemic aging. We, therefore, investigated the association between WBC telomere length and oxidized-LDL, and vascular aging, expressed by the distensibility of the carotid artery. We studied a random population sample of 216 non-smokers and 89, smokers. In all subjects, age and gender- adjusted telomere length was inversely correlated with plasma oxidized-LDL (regression coefficient = -0.656 kb/mg/dL; p=0.0006). Independent of gender, age and mean blood pressure, carotid distensibility increased with telomere length (2.33+/- 1.18 10-3/kPa/kb; p=0.05) but decreased with higher plasma levels of oxidized LDL (-10.7+/- 3.91 10-3/kPa/ mg/dL; p=0.006). Adjusted for gender and age, smokers' telomere length was shorter (6.72 vs 6.91 kb; p=0.014) and plasma oxidized-LDL level higher (0.52 vs 0.46 mg/dL; p=0.03) than in non-smokers. Higher level of oxidized-LDL, is associated with shorter WBC telomeres and increased stiffness of the carotid artery. Smoking is marked by increased oxidative stress in concert with shortened WBC telomere length

CXCL10_{10} Is a Circulating Inflammatory Marker in Patients with Advanced Heart Failure: a Pilot Study

Chemokines are involved in the remodeling of the heart; however, their significance as biomarkers in heart failure is unknown. We observed that circulating CXCR3 receptor chemokines CXCL9 and CXCL10 in a rat model of heart failure were increased 1 week after myocardial infarction. CXCL10 was also increased in both remote and infarcted regions of the heart and remained elevated at 16 weeks; CXCL9 was elevated in the remote area at 1 week. In humans, hierarchical clustering and principal component analysis revealed that circulating CXCL10, MIP-1α, and CD40 ligand were the best indicators for differentiating healthy and heart failure subjects. Serum CXCL10 levels were increased in patients with symptomatic heart failure as indexed by NYHA classification II through IV. The presence of CXCL10, MIP-1α, and CD40 ligand appears to be dominant in patients with advanced heart failure. These findings identify a distinct profile of inflammatory mediators in heart failure patient