Faculty Bibliography

Reverse phase protein arrays (RPPA) can assess protein expression and activation states in large numbers of samples (n > 1000) and evidence suggests feasibility in the setting of multi-institution clinical trials. Despite evidence in solid tumors, little is known about protein stability in leukemia. Proteins collected from leukemia cells in blood and bone marrow biopsies must be sufficiently stable for analysis. Using 58 leukemia samples, we initially assessed protein/phospho-protein integrity for the following preanalytical variables: 1) shipping vs local processing, 2) temperature (4 °C vs ambient temperature), 3) collection tube type (heparin vs Cell Save (CS) preservation tubes), 4) treatment effect (pre- vs post-chemotherapy) and 5) transit time. Next, we assessed 1515 samples from the Children's Oncology Group Phase 3 AML clinical trial (AAML1031, NCT01371981) for the effects of transit time and tube type. Protein expression from shipped blood samples was stable if processed in ≤72 h. While protein expression in pre-chemotherapy samples was stable in both heparin and CS tubes, post-chemotherapy samples were stable in only CS tubes. RPPA protein extremes is a successful quality control measure to identify and exclude poor quality samples. These data demonstrate that a majority of shipped proteins can be accurately assessed using RPPA. SIGNIFICANCE: RPPA can assess protein abundance and activation states in large numbers of samples using small amounts of material, making this method ideal for use in multi-institution clinical trials. However, there is little known about the effect of preanalytical handling variables on protein stability and the integrity of protein concentrations after sample collection and shipping. In this study, we used RPPA to assess preanalytical variables that could potentially affect protein concentrations. We found that the preanalytical variables of shipping, transit time, and temperature had minimal effects on RPPA protein concentration distributions in peripheral blood and bone marrow, demonstrating that these preanalytical variables could be successfully managed in a multi-site clinical trial setting.

The goal of this study was to assess biomarkers of exposure to glyphosate and assess potential associations with renal function in children. Glyphosate is used ubiquitously in agriculture worldwide. While previous studies have indicated that glyphosate may have nephrotoxic effects, few have examined potential effects on kidney function in children. We leveraged three cohorts across different phases of child development and measured urinary levels of glyphosate. We evaluated associations of glyphosate with three biomarkers of kidney injury: albuminuria (ACR), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury marker 1 (KIM-1). Multivariable regression analyses examined associations of glyphosate with kidney injury biomarkers controlling for covariates. We identified glyphosate in 11.1% of the total participants. The herbicide was detected more frequently in the neonate population (30%). Multivariable regression models failed to identify significant associations of log-transformed glyphosate with any of the kidney injury biomarkers, controlling for covariates age, sex, and maternal education. While we confirm detectability of glyphosate in children's urine at various ages and stages of life, there is no evidence in this study for renal injury in children exposed to low levels of glyphosate. Further studies of larger sample size are indicated to better understand putative deleterious effects of the herbicide after different levels of exposure.

Objective: We conducted this study to test the hypothesis that plasma zonulin levels are elevated in pediatric patients with nephrotic syndrome compared to healthy controls. Study Design: Plasma zonulin levels were measured by ELISA in 114 children enrolled in the NEPTUNE study. Clinical and laboratory data were retrieved from the NEPTUNE database. Results: The median age of the patients was 10 (IQR = 5 to 14) years, 59 were male, 64 had minimal change disease, 47 focal segmental glomerulosclerosis, median eGFR was 96 (IQR = 80 to 114) ml/min/1.73 m², and median urine protein:creatinine ratio was 0.5 (IQR = 0.1 to 3.4) (g:g). The plasma zonulin level was 14.2 ± 5.0 vs. 10.2 ± 2.5 ng/ml in healthy adults in a report using the same assay kit, P = 0.0025. These findings were confirmed in an independent cohort of children with nephrotic syndrome compared to healthy age-matched controls, P = 0.01. Zonulin concentrations did not differ in children with minimal change disease vs. focal segmental glomerulosclerosis, frequently relapsing vs. steroid-dependent vs. steroid-resistant clinical course, and were not influenced by the immunosuppressive treatment regimen. There was no relationship between plasma zonulin levels and the absolute or percentage change in proteinuria from enrollment until the time of the zonulin assay. Conclusion: Plasma zonulin levels are elevated in childhood nephrotic syndrome regardless of level of proteinuria or specific treatment. The cause of the high plasma zonulin levels and whether zonulin contributes to glomerular injury requires further study.

BACKGROUND:Melamine and cyanuric acid, which are currently used in a variety of common consumer products and present in foods, have been implicated in the development of urolithiasis and acute kidney injury in Chinese children. To determine whether US children have measurable concentrations of these chemicals in their bodies and whether they are at greater risk of acute kidney injury, we measured melamine and cyanuric acid exposure in a cohort of US children and determined their relationship with markers of kidney injury. METHODS:We measured urinary melamine and cyanuric acid in a convenience sample of 109 children (4 months - 8 years) from Seattle, WA and New York City, NY using liquid chromatography with tandem mass spectrometry. We measured several urinary markers of kidney injury: fatty acid binding protein 3 (FABP3), kidney injury molecule 1 (KIM1), neutrophil gelatinase-associated lipocalin (NGAL) using Luminex xMAP methods, and urine urea was measured using standard laboratory methods. We described urinary melamine and cyanuric acid concentrations and assessed predictors of the exposures. We used multivariable linear regression to assess relationships between melamine/cyanuric acid and kidney injury markers in unadjusted and adjusted (creatinine, age, sex) analyses. RESULTS:Melamine and cyanuric acid were above the limit of detection (LOD) in 78% and 95% of all samples, respectively. The mean concentrations (SD) for melamine and cyanuric acid were 27.4 ng/ml (141.9 ng/ml) and 35.3 ng/ml (42.4 ng/ml). In unadjusted analyses, we observed statistically significant increases in the percentages of FABP3 and KIM1 in relation to a one log unit change in melamine and cyanuric acid, respectively. In adjusted analyses, we observed a 55% (95% CI 0, 141) increase in KIM1 in relation to a one log unit increase in cyanuric acid. CONCLUSIONS:US children have detectable concentrations of melamine and cyanuric acid in urine, and these concentrations are higher than those reported in children from other countries. This is a novel finding that improves upon previous exposure estimates using questionnaires only and suggests widespread exposure in the population. Cyanuric acid is associated with increased KIM 1 concentrations, suggesting kidney injury. Given the potential widespread exposure, future analyses should examine melamine and cyanuric acid in relation to chronic kidney disease and markers of kidney injury in a larger cohort that is representative of the general population.

Background: Case reports suggest that NS is responsive to dietary modifications including a gluten-free diet (GFD). In celiac disease, zonulin is released from enterocytes after exposure to gliadin, activates protease activated receptor 2 (PAR2), and perturbs the actin cytoskeleton and cell-cell junctions in the gut. PAR2 is present on podocytes and, therefore, zonulin may increase glomerular permeability in NS. We conducted this study to test the hypothesis that plasma zonulin levels are elevated in pediatric patients with NS.
Method(s): Plasma specimens collected from patients <=18 yr old with minimal change disease or FSGS enrolled in the NEPTUNE study, were tested. Clinical and laboratory data were retrieved coincident with the visit when the zonulin level was measured. Samples were available for testing from the 4 or 8 month visit. Plasma zonulin levels were measured by ELISA. Results (mean+/-SD or median (IQR)) were analyzed by t-test, Wilcoxon, Kruskal-Wallis, or linear regression and considered significant if P<0.05 Results: There were 113 patients, 9.5+/-4.9 yr, 53% male, 42% white, 40% black and 18% other. Disease classification was infrequent relapser in 27%, frequent relapser/steroid dependent 42% and steroid resistant 30%. The mean BP, eGFR, and serum albumin were normal. Urine protein:creatinine (UPC) ratio was 3.9+/-6.9 (g:g). The plasma zonulin level in NS children was 14.2+/-6.0 vs 10+/-2.5 ng/ml in healthy adults (P<0.01) and was >3 standard deviations above the mean in 27%. There was a trend toward lower zonulin levels in children with UPC >=2 vs <2, 12.9(7.4) vs 16.7(8.0) (P=0.051). Plasma zonulin levels did not differ by eGFR, disease classification, or BP. Plasma zonulin and serum albumin concentrations were directly correlated, r=0.24, P=0.04.
Conclusion(s): The plasma zonulin level was significantly elevated in more than a quarter of children with NS and was unrelated to BP or eGFR. We observed a significant relationship between zonulin values and serum albumin but not proteinuria. There was a trend to lower zonulin levels in children with nephrotic-range proteinuria. Further study is needed to determine the relationship between plasma zonulin levels and proteinuria and to test whether the plasma zonulin level can be used to predict response to a GFD in children with NS

BACKGROUND: The health effects of bisphenol A (BPA) and di-(2-ethylhexyl) phthalate (DEHP) have been studied extensively in children. The impact of other chemicals in these two classes has not been investigated as fully. METHODS: We conducted a cross-sectional pilot study of 10-13 year old healthy children. We assessed descriptive, univariable and multivariable associations of urinary metabolites of bisphenols and phthalates with oxidant stress, insulin resistance, body mass, and endothelial dysfunction. Possible associations with brachial artery distensibility, pulse wave velocity (markers of vascular stiffness), and serum endothelial cell-derived microparticle levels were also assessed. RESULTS: We enrolled 41 participants, 12.1 +/- 1.0 years, most of whom were Mexican-Americans (42%) or other Hispanics (34%). Increased BPA levels were associated with increased levels of F2-isoprostane (ng/ml) (P=0.02), with a similar trend for DEHP metabolites. Each log unit increase of high molecular weight (HMW) phthalate metabolites was associated with 0.550 increase in HOMA-IR units (p=0.019) and altered circulating levels of activated endothelial cell-derived microparticles (% per ml) (P=0.026). Bisphenol S (BPS), a replacement for BPA, was associated with increased albumin (mg):creatinine (g) ratio (P=0.04). Metabolites of HMW phthalates were also associated with decreased brachial artery distensibility (P=0.047). CONCLUSIONS: Exposure to bisphenols and phthalates, including a BPA replacement, is associated with increased oxidant stress, insulin resistance, albuminuria, as well as disturbances in vascular function in healthy children.Pediatric Research (2017); doi:10.1038/pr.2017.16.

RATIONALE: The World Trade Center (WTC) collapse generated caustic airborne particulates that caused chronic rhinosinusitis in exposed fire department of New York (FDNY) firefighters. Surgery was performed when symptoms remained uncontrolled despite medical management. OBJECTIVES: To identify predictors of surgical intervention for chronic rhinosinusitis in firefighters exposed to airborne irritants at the WTC collapse site. METHODS: We assessed in 8,227 firefighters with WTC-exposure between 9/11/2001 (9/11) and 9/25/2001, including WTC-site arrival time, months of rescue/recovery work, and eosinophil concentration measured between 9/11 and 3/10/2003. We assessed the association of serum cytokines and immunoglobulins with eosinophil concentration and surgery for rhinosinusitis in 112 surgical cases and 376 controls with serum available from the first 6 months after exposure to the WTC collapse site. MEASUREMENTS AND MAIN RESULTS: Between 9/11 and 3/10/2015, the surgery rate was 0.47 cases per 100 person years. In the first 18 months post 9/11, surgical patients had higher mean blood eosinophil levels than study cohort patients(219+/-155 vs. 191+/-134; P <0.0001). Increased surgery risk was associated with increasing blood eosinophil counts (HR 1.12 per 100 cells/uL; 95% CI 1.07 to1.17; P <0.001); arriving at the WTC site 9/11 or 9/12/2001 (HR 1.43; 95% CI 1.04 to 1.99; P=0.03); and working >/=6 months at the WTC-site (HR 1.48; 95% CI 1.14 to 1.93; P<0.01). Median blood eosinophil levels for surgery patients were above levels for the cohort in all 18-month intervals 3/11/2000 through 3/10/2015 using 51,163 measurements representing 97,733 person-years of observation. Increasing age, increasing IL-17A and low IgA in serum from 2001-2002 predicted blood eosinophil concentration in surgical patients but not in controls (R2=0.26, p<0.0001 vs. R2=0.008, p=0.56). CONCLUSIONS: Increasing blood eosinophil concentration predicts surgical intervention for chronic rhinosinusitis, particularly in those with intense acute and prolonged exposure to airborne irritants. WTC-exposed FDNY firefighters who underwent irritant-associated sinus surgery are immunologically different from the cohort. Surgical patients have a higher blood eosinophil levels that is associated with mediators of mucosal immunity.