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

Heritability of The Retinal Microcirculation in Flemish Families

BACKGROUND Few population studies have described the heritability and intrafamilial concordance of the retinal microvessels, or the genetic or environmental correlations of the phenotypes of these vessels. METHODS We randomly selected 413 participants from 70 families (mean age, 51.5 years; 50.1% women) from a Flemish population. We postprocessed retinal images using IVAN software to generate the central retinal arteriole equivalent (CRAE), central retinal venule equivalent (CRVE), and arteriole-to-venule-ratio (AVR) from these images. We used SAGE version 6.2 and SAS version 9.2 to compute multivariate-adjusted estimates of heritability and intrafamilial correlations of the CRAE, CRVE, and AVR of the retinal microvessels in the images. RESULTS Sex, age, mean arterial pressure, and smoking explained up to 12.7% of the variance of the phenotypes of the retinal microvessels of the study participants. With adjustments applied for these covariates, the heritability estimates of CRAE, CRVE, and AVR were 0.213 (P = 0.044), 0.339 (P = 0.010), and 0.272 (P = 0.004), respectively. The parent-offspring correlations for CRAE, CRVE, and AVR were 0.118 (NS), 0.225 (P < 0.01), and 0.215 (P < 0.05), respectively. The corresponding values were 0.222 (P < 0.05), 0.213 (P < 0.05), and 0.390 (P < 0.001) for sib-sib correlations, respectively. The genetic and environmental correlations between CRAE and CRVE were 0.360 and 0.545 (P < 0.001 for both). CONCLUSION Our study showed moderate heritability for CRAE, CRVE, and AVR, and a significant genetic correlation of CRAE with CRVE in the Flemish population of our study. These findings suggest that genetic factors influence the diameter of the retinal microvessels, and that CRAE and CRVE share some genetic determinant

Retinal and Renal Microvasculature in Relation to Central Hemodynamics in 11‐Year‐Old Children Born Preterm or At Term

Background Prematurity disrupts the perinatal maturation of the microvasculature and macrovasculature and confers high risk of vascular dysfunction later in life. No previous studies have investigated the crosstalk between the microvasculature and macrovasculature in childhood. Methods and Results In a case‐control study, we enrolled 55 children aged 11 years weighing \u3c1000 g at birth and 71 matched controls (October 2014–November 2015). We derived central blood pressure (BP) wave by applanation tonometry and calculated the forward/backward pulse waves by an automated pressure–based wave separation algorithm. We measured the renal resistive index by pulsed wave Doppler and the central retinal arteriolar equivalent by computer‐assisted program software. Compared with controls, patients had higher central systolic BP (101.5 versus 95.2 mm Hg, P\u3c0.001) and backward wave amplitude (15.5 versus 14.2 mm Hg, P=0.029), and smaller central retinal arteriolar equivalent (163.2 versus 175.4 µm, P\u3c0.001). In multivariable analyses, central retinal arteriolar equivalent was smaller with higher values (+1 SD) of central systolic BP (−2.94 µm; 95% CI, −5.18 to −0.70 µm [P=0.011]) and forward (−2.57 µm; CI, −4.81 to −0.32 µm [P=0.026]) and backward (−3.20 µm; CI, −5.47 to −0.94 µm [P=0.006]) wave amplitudes. Greater renal resistive index was associated with higher backward wave amplitude (0.92 mm Hg, P=0.036). Conclusions In childhood, prematurity compared with term birth is associated with higher central systolic BP and forward/backward wave amplitudes. Higher renal resistive index likely moves reflection points closer to the heart, thereby explaining the inverse association of central retinal arteriolar equivalent with central systolic BP and backward wave amplitude. These observations highlight the crosstalk between the microcirculation and macrocirculation in children. Registration URL: http://www.clinicaltrials.gov. Unique Identifier: NCT02147457

Arterial Properties in Relation to Genetic Variations in the Adducin Subunits in a White Population

Background Adducin is a membrane skeleton protein, which consists of either α- and β- or α- and γ-subunits. We investigated whether arterial characteristics might be related to the genes encoding ADD1 (Gly460Trp-rs4961), ADD2 (C1797T-rs4984), and ADD3 (IVS11+386A>G-rs3731566). Methods We randomly recruited 1,126 Flemish subjects (mean age, 43.8 years; 50.3% women). Using a wall-tracking ultrasound system, we measured the properties of the carotid, femoral, and brachial arteries. We studied multivariate-adjusted phenotype-genotype associations, using a population- and family-based approach. Results In single-gene analyses, brachial diameter was 0.15 mm (P = 0.0022) larger, and brachial distensibility and cross-sectional compliance were 1.55 × 10-3/kPa (P = 0.013) and 0.017 mm2/kPa (P = 0.0029) lower in ADD3 AA than ADD3 GG homozygotes with an additive effect of the G allele. In multiple-gene analyses, the association of brachial diameter and distensibility with the ADD3 G allele occurred only in ADD1 GlyGly homozygotes. Otherwise, the associations between the arterial phenotypes in the three vascular beds and the ADD1 or ADD2 polymorphisms were not significant. In family-based analyses, the multivariate-adjusted heritability was 0.52, 0.38, and 0.30 for brachial diameter, distensibility, and cross-sectional compliance, respectively (P < 0.001). There was no evidence for population stratification (0.07 ≤ P ≤ 0.96). Transmission of the mutated ADD3 G allele was associated with smaller brachial diameter in 342 informative offspring (-0.12 ± 0.04 mm; P = 0.0085) and in 209 offspring, who were ADD1 GlyGly homozygotes (-0.14 ± 0.06 mm; P = 0.018). Conclusions In ADD1 GlyGly homozygotes, the properties of the brachial artery are related to the ADD3 (A386G) polymorphism, but the underlying mechanism needs further clarification. American Journal of Hypertension (2009). doi: 10.1038/ajh.2008.26

Heritability and intrafamilial aggregation of arterial characteristics

BACKGROUND: We investigated the heritability and familial aggregation of various indexes of arterial stiffness and wave reflection and we partitioned the phenotypic correlation between these traits into shared genetic and environmental components. METHODS: Using a family-based population sample, we recruited 204 parents (mean age, 51.7 years) and 290 offspring (29.4 years) from the population in Cracow, Poland (62 families), Hechtel-Eksel, Belgium (36), and Pilsen, the Czech Republic (50). We measured peripheral pulse pressure (PPp) sphygmomanometrically at the brachial artery; central pulse pressure (PPc), the peripheral augmentation indexes (PAIxs) and central augmentation indexes (CAIxs) by applanation tonometry at the radial artery; and aortic pulse wave velocity (PWV) by tonometry or ultrasound. In multivariate-adjusted analyses, we used the ASSOC and PROC GENMOD procedures as implemented in SAGE and SAS, respectively. RESULTS: We found significant heritability for PAIx, CAIx, PPc and mean arterial pressure ranging from 0.37 to 0.41; P &lt;/= 0.0001. The method of intrafamilial concordance confirmed these results; intrafamilial correlation coefficients were significant for all arterial indexes (r &gt;/= 0.12; P &lt;/= 0.02) with the exception of PPc (r = -0.007; P = 0.90) in parent-offspring pairs. The sib-sib correlations were also significant for CAIx (r = 0.22; P = 0.001). The genetic correlation between PWV and the other arterial indexes were significant (rhoG &gt;/= 0.29; P &lt; 0.0001). The corresponding environmental correlations were only significantly positive for PPp (rhoE = 0.10, P = 0.03). CONCLUSION: The observation of significant intrafamilial concordance and heritability of various indexes of arterial stiffness as well as the genetic correlations among arterial phenotypes strongly support the search for shared genetic determinants underlying these traits

Conventional and Ambulatory Blood Pressure as Predictors of Retinal Arteriolar Narrowing

At variance with the long established paradigm that retinal arteriolar narrowing trails hypertension, several longitudinal studies, all based on conventional blood pressure (CBP) measurement, proposed that retinal arteriolar narrowing indicates heightened microvascular resistance and precedes hypertension. In 783 randomly recruited Flemish (mean age, 38.2 years; 51.3% women), we investigated to what extent CBP and daytime (10 am to 8 pm) ambulatory blood pressure (ABP) measured at baseline (1989-2008) predicted the central retinal arteriolar equivalent (CRAE) in retinal photographs obtained at follow-up (2008-2015). Systolic/diastolic hypertension thresholds were 140/90 mm Hg for CBP and 135/85 mm Hg for ABP. In multivariable-adjusted models including both baseline CBP and ABP, CRAE after 10.3 years (median) of follow-up was unrelated to CBP (P≥0.14), whereas ABP predicted CRAE narrowing (P≤0.011). Per 1-SD increment in systolic/diastolic blood pressure, the association sizes were -0.95 μm (95% confidence interval, -2.20 to 0.30)/-0.75 μm (-1.93 to 0.42) for CBP and -1.76 μm (-2.95 to -0.58)/-1.48 μm (-2.61 to -0.34) for ABP. Patients with ambulatory hypertension at baseline (17.0%) had smaller CRAE (146.5 versus 152.6 μm; P&lt;0.001) at follow-up. CRAE was not different (P≥0.31) between true normotension (normal CBP and ABP; prevalence, 77.6%) and white-coat hypertension (elevated CBP and normal ABP, 5.4%) and between masked hypertension (normal CBP and elevated ABP, 10.2%) and hypertension (elevated CBP and ABP, 6.8%). In conclusion, the paradigm that retinal arteriolar narrowing precedes hypertension can be explained by the limitations of CBP measurement, including nonidentification of masked and white-coat hypertension.</p

Association between cognition and the retinal microvasculature in 11-year old children born preterm or at term

Background: Retinal microvessels can be visualized non-invasively and mirror the status of the cerebral microvasculature. Aims: To investigate whether in young children born prematurely or at term cognitive performance is related to retinal microvascular traits. Study design, subjects: In 93 prematurely born infants (birth weight < 1000 g) and 87 controls born at term, we measured head circumference (HC) and determined intelligence quotient (IQ) by combining matrix reasoning and spatial span (Wechsler Non-Verbal test, Dutch version) and post-processed retinal photographs using Singapore I Vessel Assessment software (version 3.6). Outcome measures, results: Compared with controls, cases had smaller HC (51.7 vs 53.4 cm; p < 0.001), lower IQ (93.9 vs 109.2; p < 0.001), smaller retinal arteriolar (CRAE; 162.7 vs 174.0 μm; p < 0.001) and venular (CRVE; 234.9 vs 242.8 μm; p = 0.003) diameters and CRAE/CRVE ratio (0.69 vs 0.72; p = 0.001). A 1-SD decrease in CRAE was associated with smaller HC (−0.53 cm; p < 0.001) and lower total IQ (−3.74; p < 0.001), matrix reasoning (−1.77; p = 0.004) and spatial span (−2.03; p = 0.002). These associations persisted after adjustment for sex and age and risk factors for cognitive impairment, including blood pressure, body mass index and parental educational attainment. Conclusions: HC, total IQ, matrix reasoning and spatial span decrease with smaller retinal arteriolar diameter. Our findings suggest that maldevelopment of the cerebral microcirculation, as mirrored by the retinal microvasculature, has lasting effects on the growth of the brain and cognitive performance of prematurely born children

Reproducibility of Retinal Microvascular Traits Decoded by the Singapore i Vessel Assessment Software Across the Human Age Range

Retinal microvascular traits predict adverse health outcomes. The Singapore I Vessel Assessment (SIVA) software improved automated postprocessing of retinal photographs. In addition to microvessel caliber, it generates measure

Cardiovascular end points and mortality are not closer associated with central than peripheral pulsatile blood pressure components

none32Pulsatile blood pressure (BP) confers cardiovascular risk. Whether associations of cardiovascular end points are tighter for central systolic BP (cSBP) than peripheral systolic BP (pSBP) or central pulse pressure (cPP) than peripheral pulse pressure (pPP) is uncertain. Among 5608 participants (54.1% women; mean age, 54.2 years) enrolled in nine studies, median follow-up was 4.1 years. cSBP and cPP, estimated tonometrically from the radial waveform, averaged 123.7 and 42.5 mm Hg, and pSBP and pPP 134.1 and 53.9 mm Hg. The primary composite cardiovascular end point occurred in 255 participants (4.5%). Across fourths of the cPP distribution, rates increased exponentially (4.1, 5.0, 7.3, and 22.0 per 1000 person-years) with comparable estimates for cSBP, pSBP, and pPP. The multivariable-adjusted hazard ratios, expressing the risk per 1-SD increment in BP, were 1.50 (95% CI, 1.33-1.70) for cSBP, 1.36 (95% CI, 1.19-1.54) for cPP, 1.49 (95% CI, 1.33-1.67) for pSBP, and 1.34 (95% CI, 1.19-1.51) for pPP (P<0.001). Further adjustment of cSBP and cPP, respectively, for pSBP and pPP, and vice versa, removed the significance of all hazard ratios. Adding cSBP, cPP, pSBP, pPP to a base model including covariables increased the model fit (P<0.001) with generalized R2 increments ranging from 0.37% to 0.74% but adding a second BP to a model including already one did not. Analyses of the secondary end points, including total mortality (204 deaths), coronary end points (109) and strokes (89), and various sensitivity analyses produced consistent results. In conclusion, associations of the primary and secondary end points with SBP and pulse pressure were not stronger if BP was measured centrally compared with peripherally.noneHuang, Qi-Fang; Aparicio, Lucas S; Thijs, Lutgarde; Wei, Fang-Fei; Melgarejo, Jesus D; Cheng, Yi-Bang; Sheng, Chang-Sheng; Yang, Wen-Yi; Gilis-Malinowska, Natasza; Boggia, José; Niiranen, Teemu J; Wojciechowska, Wiktoria; Stolarz-Skrzypek, Katarzyna; Barochiner, Jessica; Ackermann, Daniel; Tikhonoff, Valérie; Ponte, Belen; Pruijm, Menno; Casiglia, Edoardo; Narkiewicz, Krzysztof; Filipovský, Jan; Czarnecka, Danuta; Kawecka-Jaszcz, Kalina; Jula, Antti M; Bochud, Murielle; Vanassche, Thomas; Verhamme, Peter; Struijker-Boudier, Harry A J; Wang, Ji-Guang; Zhang, Zhen-Yu; Li, Yan; Staessen, Jan AHuang, Qi-Fang; Aparicio, Lucas S; Thijs, Lutgarde; Wei, Fang-Fei; Melgarejo, Jesus D; Cheng, Yi-Bang; Sheng, Chang-Sheng; Yang, Wen-Yi; Gilis-Malinowska, Natasza; Boggia, José; Niiranen, Teemu J; Wojciechowska, Wiktoria; Stolarz-Skrzypek, Katarzyna; Barochiner, Jessica; Ackermann, Daniel; Tikhonoff, Valérie; Ponte, Belen; Pruijm, Menno; Casiglia, Edoardo; Narkiewicz, Krzysztof; Filipovský, Jan; Czarnecka, Danuta; Kawecka-Jaszcz, Kalina; Jula, Antti M; Bochud, Murielle; Vanassche, Thomas; Verhamme, Peter; Struijker-Boudier, Harry A J; Wang, Ji-Guang; Zhang, Zhen-Yu; Li, Yan; Staessen, Jan

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