Bailey, Kent R.

Abstract Background: Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays. Methods: We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias. Results: Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose–response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms. Conclusions: Multiplex measurements present difficult challenges that require further analytical and statistical developments.

The objective of this study was to determine the impact of lupus nephritis disease activity on maternal and foetal outcomes in pregnant patients with systemic lupus erythematosus (SLE). Medical records of all pregnant patients with SLE treated at our institution between 1976 and 2007 were reviewed. All patients met American College of Rheumatology classification criteria for SLE. Demographic data, history of lupus nephritis, nephritis disease activity and maternal and foetal outcomes of pregnancy were abstracted. Active lupus nephritis was defined as the presence of proteinuria >0.5 g/day and/or active urinary sediment with or without an elevation in serum creatinine (Cr). Quiescent lupus nephritis was confirmed in the presence of proteinuria <0.5 mg/day and inactive urinary sediment. We identified 58 patients with 90 pregnancies. Compared with pregnancies in SLE patients without renal involvement ( n = 47), pregnancies in patients with active lupus nephritis ( n = 23) were associated with a higher incidence of maternal complications (57% vs 11%, P < 0.001), whereas those with quiescent lupus nephritis ( n = 20) were not (35% vs 11%, P = 0.10). Women with active lupus nephritis were more likely to deliver preterm than women without lupus nephritis, median of 34 weeks vs 40 gestational weeks, respectively ( P = 0.002) and were more likely to suffer foetal loss (35% vs 9%, P = 0.031). Active, but not quiescent, lupus nephritis during pregnancy is associated with a higher incidence of maternal and foetal complications compared with pregnancies in SLE patients without renal involvement.

Background— We previously reported the feasibility of an acute, orally delivered, newly developed, conjugated form of human B-type natriuretic peptide (hBNP) in normal animals. The objective of the present study was to extend our findings and to define the chronic actions of an advanced oral conjugated hBNP (hBNP-054) administered for 6 days on sodium excretion and blood pressure. We also sought to establish the ability of this new conjugate to acutely activate cGMP and to reduce blood pressure in an experimental model of angiotensin II (ANG II) –mediated hypertension. Methods and Results— First, we developed additional novel conjugated forms of oral hBNP that were superior to our previously reported hBNP-021 in reducing blood pressure in 6 normal dogs. We then tested the new conjugate, hBNP-054, chronically in 2 normal dogs to assess its biological actions as a blood pressure–lowering agent and as a natriuretic factor. Second, we investigated the effects of acute oral hBNP-054 or vehicle in 6 dogs that received continuous infusion of ANG II to induce hypertension. After baseline determination of mean blood pressure (MAP) and blood collection for plasma hBNP and cGMP, all dogs received continuous ANG II infusion (20 ng · kg −1 · min −1 , 1 mL/min) for 4 hours. After 30 minutes of ANG II, dogs received oral hBNP-054 (400 μg/kg) or vehicle in a random crossover fashion with a 1-week interval between dosing. Blood sampling and MAP measurements were repeated 30 minutes after ANG II administration and 10, 30, 60, 120, 180, and 240 minutes after oral administration of hBNP-054 or vehicle. In the chronic study in normal dogs, oral hBNP-054 effectively reduced MAP for 6 days and induced a significant increase in 24-hour sodium excretion. hBNP was not present in the plasma at baseline in any dogs, and it was not detected at any time in the vehicle group. However, hBNP was detected throughout the duration of the study after oral hBNP-054, with a peak concentration at 30 minutes of 1060±818 pg/mL. In the acute study, after ANG II administration, plasma cGMP was not activated after vehicle, whereas it was significantly increased after oral hBNP-054 ( P =0.01 between the 2 groups). Importantly, MAP was significantly increased after ANG II throughout the acute study protocol. However, although no changes occurred in MAP after vehicle administration, oral hBNP-054 reduced MAP for >2 hours (from 138±1 mm Hg after ANG II to 124±2 mm Hg at 30 minutes, 124±2 mm Hg at 1 hour, and 130±5 mm Hg at 2 hours after oral hBNP-054; P <0.001). Conclusions— This study reports for the first time that a novel conjugated oral hBNP possesses blood pressure–lowering and natriuretic actions over a 6-day period in normal dogs. Furthermore, hBNP-054 activates cGMP and reduces MAP in a model of acute hypertension. These findings advance the concept that orally administered chronic BNP is a potential therapeutic strategy for cardiovascular diseases such as hypertension.

Endothelin plays an important role in the pathogenesis of atherosclerosis. The aim of the study was to evaluate the safety and hemodynamic and metabolic responses to 6 months treatment with atrasentan, the selective endothelin-A receptor antagonist. Seventy-two patients with multiple cardiovascular risk factors and nonobstructive coronary artery disease on coronary angiogram were randomly assigned in a double-blind manner to atrasentan or placebo. Mean aortic blood pressure decreased from 92±10 to 80±10 mm Hg ( P <0.001) in the atrasentan group and did not change in the placebo group (93±10 and 92±11 mm Hg; P =0.84). The difference between the groups was significant ( P <0.001). No effect on heart rate was observed. In a subgroup of patients not treated with angiotensin-converting enzyme inhibitor, creatinine level decreased in the atrasentan versus the placebo group ( P =0.011). Fasting glucose ( P =0.026), glycosylated hemoglobin level ( P =0.041), triglyceride l ( P =0.013), lipoprotein-A ( P =0.046), and uric acid levels ( P =0.048) decreased significantly in the atrasentan group compared with the placebo group. No progression of angiographic coronary disease was observed. The most common adverse effects with atrasentan were nasal stuffiness, headache, and edema. In conclusion, 6 months of treatment with atrasentan results in a reduction of blood pressure and improvement in glucose and lipid metabolism. These findings suggest the beneficial role of atrasentan in the treatment of hypertension and metabolic syndrome.

We conducted a genome-wide association study to identify novel genes influencing diastolic blood pressure (BP) response to hydrochlorothiazide, a commonly prescribed thiazide diuretic preferred for the treatment of high BP. Affymetrix GeneChip Human Mapping 100K Arrays were used to measure single nucleotide polymorphisms across the 22 autosomes in 194 non-Hispanic black subjects and 195 non-Hispanic white subjects with essential hypertension selected from opposite tertiles of the race- and sex-specific distributions of age-adjusted diastolic BP response to hydrochlorothiazide (25 mg daily, PO, for 4 weeks). The black sample consisted of 97 “good” responders (diastolic BP response [mean±SD]=−18.3±4.2 mm Hg; age=47.1±6.1 years; 51.5% women) and 97 “poor” responders (diastolic BP response=−0.18±4.3; age=47.4±6.5 years; 51.5% women). Haplotype trend regression identified a region of chromosome 12q15 in which haplotypes constructed from 3 successive single nucleotide polymorphisms (rs317689, rs315135, and rs7297610) in proximity to lysozyme ( LYZ ), YEATS domain containing 4 ( YEATS4 ), and fibroblast growth receptor substrate 2 ( FRS2 ) were significantly associated with diastolic BP response (nominal P =2.39×10 −7 ; Bonferroni corrected P =0.024; simulated experiment-wise P =0.040). Genotyping of 35 additional single nucleotide polymorphisms selected to “tag” linkage disequilibrium blocks in these genes provided corroboration that variation in LYZ and YEATS4 was associated with diastolic BP response in a statistically independent data set of 291 black subjects and in the sample of 294 white subjects. These results support the use of genome-wide association analyses to identify novel genes influencing antihypertensive drug responses.

Despite 40 years of research demonstrating the efficacy of antihypertensive medications for lowering blood pressure and decreasing cardiovascular disease, hypertension control rates worldwide remain low. We explored here how both medication efficacy rates and patient/physician decision-making (discontinuation rates) affect overall hypertension control rates. To do this we introduced the use of Kaplan–Meier methodology to predict hypertension control rates separately within age strata. An important aspect of our analysis is the use of a “treatment intensity score,” including both the number of agent(s) and the percentage of maximal dose of agent(s). We investigated how blood pressure control rates vary with age, gender, and treatment intensity. Our analysis found that both efficacy and discontinuation rates at different treatment intensities vary with age and gender, so that for each increase in age by 1 decade, the log hazard of achieving controlled blood pressure at any given intensity decreases by 0.10 in men and 0.20 in women. Overall hypertension control rates ranged from 80.8% for persons age 15 to 39 years to only 42.1% for persons age ≥80 years. Our analysis more accurately represents achievable hypertension control rates, with differences by gender, than previous work, because we investigated the contributions of efficacy and treatment aggressiveness in the control of hypertension.

The objective of this study was to assess the usefulness of a comparison of clinical failure and restenosis rates of endovascular procedures at 1 year in patients with peripheral arterial disease. The resulting comparison is presented as “clinical failure/restenosis coordinate.” The authors screened 171 papers describing the outcome of lower extremity angioplasty or stent placement. In 20 of them, authors reported detailed outcomes of interest, including baseline demographic measurements, location of arterial occlusive lesions, a measure of restenosis (measured by ankle-brachial indices, ultrasonography, or angiography), and clinical outcomes (mortality, repeat percutaneous transluminal angioplasty, or amputation). An overview of these 20 angioplasty papers was performed. Besides the usual meta-analyses of each end point separately, data were also plotted as coordinates of clinical failure versus restenosis. The clinical failure-to-restenosis coordinate was calculated and reported for percutaneous transluminal angioplasty of the aortoiliac and femoropopliteal distributions. Clinically reported outcomes in the literature were used to calculate the clinical failure/restenosis coordinate. This value was significantly different for various locations of the angioplasty and various baseline angiographic characteristics. A numeric coordinate pair of clinical failure and restenosis is identifiable in patients undergoing endovascular treatment of peripheral arterial disease. The varying coordinates may be important in elucidating the incidence and mechanisms of clinical failure after endovascular treatment. The coordinate reported in this article is hypothesis-generating about mechanisms of endovascular treatment failure. This coordinate is important in determining the role of restenosis in the clinical failure of endovascular therapy of peripheral arterial disease.

Cerebral white matter hyperintensities on brain MRI (leukoaraiosis) are associated with increased risk of stroke and dementia. To assess the relationships of blood pressure level and circadian pattern with leukoaraiosis, we obtained 24-hour ambulatory blood pressure recordings and brain magnetic resonance images in 343 white and 267 black adults who were members of sibships that had ≥2 siblings with essential hypertension. In multiple linear regression models, factors associated with greater leukoaraiosis in both racial groups included age ( P ≤0.002), homocysteine levels ( P ≤0.006), and brain volume ( P ≤0.008). In blacks, ambulatory blood pressure measures associated with greater leukoaraiosis were higher awake, asleep, and 24-hour systolic and diastolic levels ( P ≤0.009 for each). In addition, there was a trend for smaller nocturnal declines in systolic and diastolic levels (ie, nondipping patterns) to be associated with greater leukoaraiosis, and all of these associations, except nondipping of diastolic level, remained or became significant after controlling for office blood pressure ( P <0.05 for each). In whites, among ambulatory blood pressure measures, only higher asleep diastolic levels trended toward association with greater leukoaraiosis. However, similar to findings in blacks, nondipping of systolic and diastolic ambulatory blood pressure levels were each associated with greater leukoaraiosis ( P ≤0.008), and all of these associations remained or became significant after controlling for office blood pressure ( P ≤0.009 for each). Higher ambulatory blood pressure levels and a nondipping circadian pattern contribute to greater leukoaraiosis volume after controlling for office blood pressure.

OBJECTIVE—Birth weight is a risk factor for both diabetes and mortality. Diabetes is a risk factor for mortality. Whether the excess mortality observed for diabetes varies with birth weight is unclear. RESEARCH DESIGN AND METHODS—Among all 2,508 Rochester, Minnesota, residents who first met research criteria for adult-onset diabetes in 1960–1995, 171 were born locally in-hospital after 1922 (i.e., birth weights available) as singleton, term infants. Each case subject and two age- and sex-matched nondiabetic control subjects (born locally, residing locally when the case subject met the criteria for diabetes) were followed through 31 December 2000 for vital status. RESULTS—Of the diabetic case subjects, 16% (27 of 171) died vs. 7% (25 of 342) of control subjects (P = 0.004). The difference was less for normal-birth-weight (NBW) (2,948–&lt;3,856 g) individuals (12% [12 of 102] vs. 8% [20 of 246], P = 0.31) than for abnormal-birth-weight individuals (low birth weight [LBW] 20% [8 of 39] vs. 2% [1 of 46], P = 0.01; high birth weight [HBW] 23% [7 of 30] vs. 8% [4 of 50], P = 0.16), as confirmed with age- and sex-adjusted Cox proportional hazards (diabetes-associated hazard ratio 1.4 [95% CI 0.69–2.90] for NBW vs. 4.8 [1.7–13.3] for abnormal birth weight, test for interaction P = 0.056). The observed diabetes deaths were greater than expected, based on mortality for the general population (27 vs. 13.3, P &lt; 0.001), with 70% of excess deaths occurring among LBW (8 vs. 2.2, P &lt; 0.001) and HBW (7 vs. 3.1, P = 0.03) individuals. CONCLUSIONS—The excess mortality observed for diabetes appears disproportionately concentrated among abnormal-birth-weight individuals, thus identifying a subset of at-risk diabetic individuals and reinforcing the importance of NBW deliveries.