Pediatric intestinal failure associated liver disease (IFALD) is associated with a high morbidity and mortality. Studies have suggested that when intravenous (IV) soybean oil (SO) is replaced with fish oil (FO), direct hyperbilirubinemia is more likely to resolve. The necessary duration of FO has not been established. This study seeks to determine if 24 weeks of FO is an effective and safe therapy for IFALD.

1) Proteomic Data Pre-processing: Quantification and Normalization of Luminex Assay System

High through-put genomic and proteomic technologies allow rapid analysis of molecular targets of thousands of genes at a time, either at the DNA, RNA or protein level. In these type of experiments variations in expression measurements can occur from a variety of sources. Our goal was to examine measurement and normalization techniques to reduce the experimental variation in data derived from a bead-based multiplex Luminex assay system which allows simultaneous measurements of proteins. Normalization for the Luminex assay system requires a fundamentally different approach than the case of traditional microarrays. In the Luminex assay system, each experimental unit is a plate and each plate has results for multiple subjects and analytes. We quantified performance among different measurement systems (fluorescent intensity, background in fluorescent intensity, and observed concentration) in both high and standard scanning systems. Various normalization techniques (scale normalization, quantile normalization, lowess curve normalization) were adapted to the Luminex data scenario and their performance was compared in two datasets.

We used the coefficient of variation across plates to compare the performance of normalization methods. Median and Lowess normalizations appeared to result in reducing plate- to-plate variation the most. Quantile normalization does not appear to work well for these datasets. Our results suggest that simple normalizations such as scale and lowess curve normalizations perform better than complex methods such as quantile normalization. Complex methods may add noise and bias to the normalized adjustment when the assumptions are not met.

2) Integration of microRNA and mRNA by Weighted Gene Co-expression Network Analysis

We focus on the step-by-step network construction and module detection of mRNAs by weighted gene co-expression network analysis (WGCNA), followed by identifying the strong correlation between miRNA and module eigengenes. We then evaluate whether the predicted mRNA targets are differentially present between a given module and other modules by using the Fisher's exact test. We retained miRNAs who are significant in the fisher exact test, and are strongly correlated with eigengenes in a module.

Next we relate modules to disease status by using eigengene network methodology, we found that 11 out of 13 modules are significantly related with disease status. Enrichment analyses by DAVID software are implemented for the 11 modules.

We also run step-by-step network construction and module detection of miRNAs and found 6 modules. We used LASSO regression to explore the relationship between miRNA and mRNAs. The predictors are module eigengene of miRNA and the outcome is the eigengene from each mRNA module.

We found that 1 miRNA "hsa_miR_25" is significantly anti-correlated with mRNA Magenta module. "hsa_miR_25" belongs to the miRNA module "blue" that is also predictive to Magenta mRNA module through LASSO regression. Its putative mRNA targets are found and integrated from the renal dataset.

In conclusion, the weighted co-expression network analysis provides a novel integrative view of miRNA and their putative genes. It also greatly alleviates the multiple testing problems that plague standard gene-centric methods.

Background: Despite extensive research to identify biomarkers of response in patients with non-muscle invasive bladder cancer (NMIBC), there is no biomarker to date that can serve this purpose. Herein, we sought to determine if a large panel of urinary cytokines measured at different time points can predict response to treatment in patients with NMIBC.

Methods and Material: Serial urine samples were collected from 50 patients with intermediate or high-risk NMIBC enrolled in a prospective, phase II randomized study evaluating intravesical BCG +/- intradermal HS-410 therapy. Urines were collected at baseline, week-7, week-13, week-28 and at end of treatment. Using a multiplex immunoassay, 105 cytokines were analyzed. To predict outcome of time to event (either recurrence or progression), univariate and multivariable Cox analyses were performed.

Results: 15 patients (30%) developed recurrence and 4 patients (8%) progressed during the follow-up. Among clinicopathologic variables, only smoking status, ever-smoker vs. non-smoker status, was associated with an improved response rate (HR 0.38, 95% CI 0.14 - 0.99, p=0.0483). In the most relevant multivariable model, the percent change (for 100 units) of IL-18 binding protein-a (HR 1.995, 95% CI 1.157-3.438, p=0.01), IL-23 (HR 1.116, 95% CI 1.012-1.23, p=0.03), IL-8 (HR 0.273, 95% CI 0.069-1.082, p=0.06), and interferon gamma-induced protein-10 (HR 0.955, 0.914-0.997, p=0.04) at week-13 from baseline best predicted time to event.

Conclusion: In a time-dependent manner, urinary cytokines provided additional value to clinicopathologic features to predict response to immune modulating agents in patients with intermediate and high risk NMIBC. Further studies are needed to validate these findings.

Background:

Fibroblast growth factor 23 (FGF23) levels are elevated in cardiopulmonary bypass (CPB)-associated acute kidney injury (AKI); however, it is unknown how much of the circulating FGF23 is intact and bioactive. Hypoxia may induce FGF23 production, yet its impact in humans is unknown. Pediatric cardiac surgery patients have both a high incidence of CPB-associated AKI and a high prevalence of chronic hypoxemia.

Methods:

We assessed the effects of hypoxemia and CPB-associated AKI on C-terminal FGF23 (cFGF23) and intact FGF23 (iFGF23) levels in 32 pediatric cardiac surgery patients with normal eGFR. Plasma cFGF23 and iFGF23 were measured pre-operatively and serially post-operatively.

Results:

Despite normal renal and ventricular function, pre-operative cFGF23 levels were high and elevated out of proportion to iFGF23 levels. Pre-operative oxygen saturation correlated inversely with FGF23. Pre-operative cFGF23 and oxygen saturation both predicted post-operative AKI. Post-operatively, cFGF23 and iFGF23 increased early post-reperfusion; although cFGF23 levels remained elevated, iFGF23 levels soon returned to baseline.

Conclusions:

Pre-operative cFGF23 may predict CPB-associated kidney dysfunction. Changes over time in cFGF23 and iFGF23 levels post-CPB differ. Chronic hypoxemia may affect FGF23 production in humans.

Background: Hepatitis C (HCV) may increase cardiovascular disease (CVD) risk in HIV-infected persons. We hypothesized that HCV virologic clearance reduces CVD risk, as manifested by reduction in non-hepatic CVD biomarkers.

Methods: Of 54 HIV/HCV coinfected subjects who received 72 weeks of pegylated interferon/ribavirin, 27 with and 27 without sustained virologic response (SVR) matched by race/ethnicity and sex, stored serum/plasma before treatment and 24 weeks after end of treatment (EOT) were tested for non-hepatic (sICAM-1, sP-selectin, IL-6, D-dimer, and lipoprotein-associated phospholipase A2 [Lp-PLA2]) and hepatic markers of CVD (cholesterol and hsCRP). Baseline characteristics and biomarkers were compared between SVRs and non-SVRs by Wilcoxon rank sum test. Changes in each biomarker were examined within SVRs/non-SVRs and between groups by t-tests and regression models.

Results: The cohort included 54 subjects, 30 white, 24 black, and 44 male. Baseline levels of non-hepatic markers were not significantly different between groups, including sICAM-1 (overall median [Q1, Q3]=439.2 [365.6, 592.8] ng/mL), sP-selectin (146.7 [94.1, 209.9] ng/mL) and IL-6 (2.32 [1.61, 3.49] pg/mL). Of 52 subjects with baseline Lp-PLA2, 37 (71%) had Lp-PLA2>235 ng/mL. SVRs had a significant decrease in log10 sICAM-1, but not non-SVRs (mean [sd] = -0.09 [0.13] vs -0.01 [0.14], p=0.047 for between group comparison). Adjusting for baseline AST and ALT, SVR was significantly associated with decrease in sICAM-1 (p=0.033), but not after adjusting for change in ALT (p=0.105). At 24 weeks after EOT, 17 (63%) SVRs had Lp-PLA2>235 ng/mL compared to 25 (93%) non-SVRs (p=0.021).

Conclusions: High baseline levels of non-hepatic CVD markers suggest high CVD risk in HIV/HCV coinfection. SVR was associated with decrease in sICAM-1 and lower CVD risk by Lp-PLA2 level. HCV virologic clearance may lower CVD risk through reduction in hepatic, and subsequently systemic, inflammation.

Background and objectives: Studies have demonstrated higher parathyroid hormone (PTH) and lower 25-hydroxy Vitamin D in African-American children. There is minimal information on these differences in pediatric patients on dialysis.

Design, setting, participants and measurements: Differences in the markers of mineral and bone metabolism (MBM) were assessed in 661 pediatric patients. Differences by race and ethnicity over the first year of dialysis treatment were assessed using linear mixed models.

Results: African-American race predicted 23% higher serum PTH when compared to Caucasian patients and Hispanic ethnicity trended toward 14.8% higher PTH. Upon gender stratification, the differences in PTH were magnified in African-American and Hispanic females who had 38% and 28.8% higher PTH compared to Caucasian females, respectively.

Conclusions: The markers of mineral and bone metabolism differ by race and ethnicity within the pediatric dialysis population. Therefore, consideration of these differences may impact the goal-targeted treatment of MBM disorders.

Background: Prior studies have identified risk factors for survival in patients with end-stage heart failure (HF) requiring left ventricular assist device (LVAD) support. However, patients with biventricular HF may represent a unique cohort.

Methods: We retrospectively evaluated a consecutive cohort of 113 adult, end-stage HF patients at UCLA Medical Center who required BIVAD support between 2000 and 2009.

Results: Survival to transplant was 66.4%, with 1-year actuarial survival of 62.8%. All patients were INTERMACS level 1 or 2 and received Thoratec paracorporeal BIVAD as bridge-to-transplant. We generated a scoring system for survival to transplant. Our final model with age, gender, dialysis, cholesterol, ventilator, and albumin gave a c-statistic of 0.870. A simplified system preserved a c-statistic of 0.844. Patients were divided into normal or high risk groups (median survival of 367 and 17 days, respectively), with strong discrimination between groups for mortality. In this study, we generate a scoring system that offers high prognostic ability for patients requiring BIVAD support, in hopes that it may assist in clinical decision making. Further studies are needed to prospectively validate our scoring system.

Background: Candidates may be active or temporarily inactive (status 7) on the kidney transplant waiting list. One reason candidates may be inactive is for a “weight currently inappropriate for transplantation”. We hypothesized that many of these candidates would not achieve active status.

Methods: Using OPTN/UNOS data from 2006-2012, we used competing risks methods to determine the cumulative incidence of conversion to active status (activation), death, and delisting before conversion among 1679 obese adult kidney candidates designated as status 7 due to a weight inappropriate for transplantation. Fine and Gray competing risks regression was performed to characterize factors associated with conversion to active status in the overall study population and of eventual transplantation among a subgroup of activated candidates.

Results: At six years, the cumulative incidence of activation was 49%, of death before conversion was 15%, and of delisting was 21%. Higher body mass index (BMI) was strongly associated with a decreased subhazard of activation (BMI ≥45 vs. 30-34.9, sHR: 0.22; 95% CI: 0.16-0.33). Female gender, diabetic end-stage renal disease, history of a previous transplant, panel reactive antibodies <80%, dialysis-dependence at listing, and UNOS region 5 were negatively associated with activation. Among activated candidates, the cumulative incidence of transplantation at six years after initial waitlisting was 61%.

Conclusions: Our findings indicate that half of obese status 7 candidates with a weight inappropriate for transplantation will not achieve active waitlist status. BMI at listing had a strong association with conversion to active status; however, co-morbid factors and regional variation also impact activation.

Rationale/Objective: Over the past 30 years, the five-year survival rate for lung cancer has increased by only 5%. A better understanding of genomic alterations and the tumor microenvironment along the spectrum of early disease could lead to identification of targetable neoantigens that may form the basis of future interceptive therapies.

Methods: From 41 lobectomy specimens for early stage lung adenocarcinoma, laser capture microdissection was utilized to obtain multiple areas of atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), normal epithelium and the associated ADC for WES. Quantitative IHC and immunofluorescent staining for Granzyme B, PD-1, PDL-1, CD4+, CD8+ and FOXP3 were assessed. Putative neoantigens were identified from ns somatic mutations by prediction of avid binding to patients’ HLA utilizing multiple algorithms in accord with the Immune Epitope Database recommendations.

Results: Non-synonymous (ns) somatic mutations were grouped into the following categories: a) premalignant mutations only observed in premalignant lesions, b) progression-associated mutations (PAMs) located in both premalignant lesions and ADC, and c) malignant-specific mutations only identified in ADC. Neoepitopes derived from PAMs, referred to as progression-associated neoepitopes (PAN), are associated with the highest levels of CD8+ T cell infiltration and PD-L1 expression. Frequent immune-effector cell infiltration and accompanying adaptive immune suppression suggest specific immune antigen recognition of PAN.

Semen HIV-1 level is an important determinant of the risk of HIV-1 sexual transmission. In this prospective observational study, we investigated potential associations between seminal cytokine levels and HIV-1 concentration in the seminal plasma of HIV-infected men, using paired blood and semen samples from 18 HIV-1 chronically-infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood were measured and analyzed using simple linear regressions to screen for an effect of cytokines on seminal plasma HIV-1. Forwards stepwise regression was performed to construct the final multivariate model. The median HIV-1 concentration was 4.42 log10 RNA copies/ml (IQR 2.98, 4.70) and 2.96 log10 RNA copies/ml (IQR 2, 4.18) in blood and semen plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 level (p<0.00005), seminal plasma IFN-gamma level (p=0.03), and seminal plasma IL-17 level (p=0.03) were positively associated with seminal plasma HIV-1 level; seminal plasma IL-5 was negatively associated with seminal HIV-1 level (p=0.0007). These data indicate that, in addition to plasma levels of HIV-1, cytokine profiles in the male genital tract are an important determinant of HIV-1 levels in semen. Th1 and Th17 cell profiles are associated with increased levels of virus while the Th2 cellular profile is associated with decreased levels of virus. These results support the importance of genital tract immunomodulation in HIV-1 transmission.