Impact of IL-21 on Natural Killer cell proliferation and function - a mathematical and functional assessment.

Natural killer (NK) cells are currently in use as immunotherapeutic agents for cancer. Many different cytokines are used to generate NK cells including IL-2, IL-12, IL-15 and IL-18 in solution and membrane bound IL-21. These cytokines drive NK cell activation through the integration of STAT and NF-κB pathways, which overlap and synergize, making it challenging to predict optimal cytokine combinations. We integrated functional assays for NK cells cultured in a variety of cytokine combinations with feature selection and mechanistic regression models. Our regression model successfully predicts NK cell proliferation for different cytokine combinations and indicates synergy between STAT3 and NF-κB transcription factors. Use of IL-21 in solution in the priming, but not post-priming phase of NK cell culture resulted in optimal NK cell proliferation, without compromising cytotoxicity or IFN-γ secretion against hepatocellular carcinoma cell lines. Our work provides a mathematical framework for interrogating NK cell activation for cancer immunotherapy.

BioNetGMMFit: estimating parameters of a BioNetGen model from time-stamped snapshots of single cells.

Mechanistic models are commonly employed to describe signaling and gene regulatory kinetics in single cells and cell populations. Recent advances in single-cell technologies have produced multidimensional datasets where snapshots of copy numbers (or abundances) of a large number of proteins and mRNA are measured across time in single cells. The availability of such datasets presents an attractive scenario where mechanistic models are validated against experiments, and estimated model parameters enable quantitative predictions of signaling or gene regulatory kinetics. To empower the systems biology community to easily estimate parameters accurately from multidimensional single-cell data, we have merged a widely used rule-based modeling software package BioNetGen, which provides a user-friendly way to code for mechanistic models describing biochemical reactions, and the recently introduced CyGMM, that uses cell-to-cell differences to improve parameter estimation for such networks, into a single software package: BioNetGMMFit. BioNetGMMFit provides parameter estimates of the model, supplied by the user in the BioNetGen markup language (BNGL), which yield the best fit for the observed single-cell, time-stamped data of cellular components. Furthermore, for more precise estimates, our software generates confidence intervals around each model parameter. BioNetGMMFit is capable of fitting datasets of increasing cell population sizes for any mechanistic model specified in the BioNetGen markup language. By streamlining the process of developing mechanistic models for large single-cell datasets, BioNetGMMFit provides an easily-accessible modeling framework designed for scale and the broader biochemical signaling community.

The radiation response measurement of a single and multiple cell ionization of neuroblastoma cells by infrared laser trap.

Neuroblastoma (NB) is a common type of cancer found mostly in infants and arising from the immature neural crest cells of the sympathetic nervous system. Using laser trapping (LT) technique, the present work contributes to advancing radiotherapy (RT), a leading treatment method for cancer. A single, 2-cells, 3-cells, 4-cells, and 5-cells were trapped using the high-intensity gradient infrared laser at 1064 nm and allowed to become ionized. In this work, a systematic study of Threshold Ionization Energy (TIE) and Threshold Radiation Dose (TRD) versus mass for both single and multi-cell ionization using laser trapping (LT) techniques on NB is presented. The results show that TIE increased as the mass of cells increased, meanwhile TRD decreased with the increase of cell mass. We observed an inverse correlation between TRD and cell mass. We demonstrate how to compute the maximum radiation dosage for cell death using the LT technique. Results show a possible blueprint for computing the TRD in vivo. The use of multiple cell ionization to determine radiation dosage along with better data accuracy concerning the tumor size and density will have profound implications for radiation dosimetry. The diminution in TRD becomes more significant in multiple cell ionization as we see in TRD vs the number of cells entering the trap. This is due to the chain effect generated by radiation and the absorption by water molecules at 1064 nm. This result provides us with better insight into the optimization of the therapeutic ratio.

Revisiting the Wald Test in Small Case-Control Studies With a Skewed Covariate.

The Wald test is routinely used in case-control studies to test for association between a covariate and disease. However, when the evidence for association is high, the Wald test tends to inflate small P values as a result of the Hauck-Donner effect (HDE). Here, we investigate the HDE in the context of genetic burden, both with and without additional covariates. First, we examine the burden-based P values in the absence of association using whole-exome sequence data from 1000 Genomes Project reference samples (n = 54) and selected preterm infants with neonatal complications (n = 74). Our careful analysis of the burden-based P values shows that the HDE is present and that the cause of the HDE in this setting is likely a natural extension of the well-known cause of the HDE in 2 × 2 contingency tables. Second, in a reanalysis of real data, we find that the permutation test provides increased power over the Wald, Firth, and likelihood ratio tests, which agrees with our intuition since the permutation test is valid for any sample size and since it does not suffer from the HDE. Therefore, we propose a powerful and computationally efficient permutation-based approach for the analysis and reanalysis of small case-control association studies.

FREQMAX provides an alternative approach for determining high-resolution allele frequency thresholds in carrier screening.

As whole-genome data become available for increasing numbers of individuals across diverse populations, the list of genomic variants of unknown significance (VOUS) continues to grow. One powerful tool in VOUS interpretation is determining whether an allele is too common to be considered pathogenic. As genetic and epidemiological parameters vary across disease models, so too does the pathogenic allele frequency threshold for each disease gene. One threshold-setting approach is the maximum credible allele frequency (MCAF) method. However, estimating some of the input values MCAF requires, especially those involving heterogeneity, can present nontrivial statistical challenges. Here, we introduce FREQMAX, our alternative approach for determining allele frequency thresholds in carrier screening. FREQMAX makes efficient use of the data available for well-studied traits and exhibits flexibility for traits where information may be less complete. For cystic fibrosis, more alleles are excluded as benign by FREQMAX than by MCAF. For less-comprehensively characterized traits like ciliary dyskinesia and Smith-Lemli-Opitz syndrome, FREQMAX is able to set the allele frequency threshold without requiring a priori estimates of maximum genetic and allelic contributions. Furthermore, though we describe FREQMAX in the context of carrier screening, its classical population genetics framework also provides context for adaptation to other trait models.

Brd2 haploinsufficiency extends lifespan and healthspan in C57B6/J mice.

Aging in mammals is the gradual decline of an organism's physical, mental, and physiological capacity. Aging leads to increased risk for disease and eventually to death. Here, we show that Brd2 haploinsufficiency (Brd2+/-) extends lifespan and increases healthspan in C57B6/J mice. In Brd2+/- mice, longevity is increased by 23% (p<0.0001), and, relative to wildtype animals (Brd2+/+), cancer incidence is reduced by 43% (p<0.001). In addition, relative to age-matched wildtype mice, Brd2 heterozygotes show healthier aging including: improved grooming, extended period of fertility, and lack of age-related decline in kidney function and morphology. Our data support a role for haploinsufficiency of Brd2 in promoting healthy aging. We hypothesize that Brd2 affects aging by protecting against the accumulation of molecular and cellular damage. Given the recent advances in the development of BET inhibitors, our research provides impetus to test drugs that target BRD2 as a way to understand and treat/prevent age-related diseases.

A new linear regression-like residual for survival analysis, with application to genome wide association studies of time-to-event data.

In linear regression, a residual measures how far a subject's observation is from expectation; in survival analysis, a subject's Martingale or deviance residual is sometimes interpreted similarly. Here we consider ways in which a linear regression-like interpretation is not appropriate for Martingale and deviance residuals, and we develop a novel time-to-event residual which does have a linear regression-like interpretation. We illustrate the utility of this new residual via simulation of a time-to-event genome-wide association study, motivated by a real study seeking genetic modifiers of Duchenne Muscular Dystrophy. By virtue of its linear regression-like characteristics, our new residual may prove useful in other contexts as well.

Prediction of short-term neonatal complications in preterm infants using exome-wide genetic variation and gestational age: a pilot study.

BACKGROUND: Preterm birth is the leading cause of mortality and morbidity in young children, with over a million deaths per year worldwide arising from neonatal complications (NCs). NCs are moderately heritable although the genetic causes are largely unknown. Therefore, we investigated the impact of accumulated genetic variation (burden) on NCs in non-Hispanic White (NHW) and non-Hispanic Black (NHB) preterm infants. METHODS: We sequenced 182 exomes from infants with gestational ages from 26 to 31 weeks. These infants were cared for in the same time period and hospital environment. Eighty-one preterm infants did not develop NCs, whereas 101 developed at least one severe complication. We measured the effect of burden at the single-gene and exome-wide levels and derived a polygenic risk score (PRS) from the top 10 genes to predict NCs. RESULTS: Burden across the exome was associated with NCs in NHW (p = 0.05) preterm infants suggesting that multiple genes influence susceptibility. In a post hoc analysis, we find that PRS alone predicts NCs (AUC = 0.67) and that PRS is uncorrelated with GA ([Formula: see text] = 0.05; p = 0.53). When PRS and GA at birth are combined, the AUC is 0.87. CONCLUSIONS: Our results support the hypothesis that genetic burden influences NCs in NHW preterm infants.

Mathematical modelling identifies the role of adaptive immunity as a key controller of respiratory syncytial virus in cotton rats.

Respiratory syncytial virus (RSV) is a common virus that can have varying effects ranging from mild cold-like symptoms to mortality depending on the age and immune status of the individual. We combined mathematical modelling using ordinary differential equations (ODEs) with measurement of RSV infection kinetics in primary well-differentiated human bronchial epithelial cultures in vitro and in immunocompetent and immunosuppressed cotton rats to glean mechanistic details that underlie RSV infection kinetics in the lung. Quantitative analysis of viral titre kinetics in our mathematical model showed that the elimination of infected cells by the adaptive immune response generates unique RSV titre kinetic features including a faster timescale of viral titre clearance than viral production, and a monotonic decrease in the peak RSV titre with decreasing inoculum dose. Parameter estimation in the ODE model using a nonlinear mixed effects approach revealed a very low rate (average single-cell lifetime > 10 days) of cell lysis by RSV before the adaptive immune response is initiated. Our model predicted negligible changes in the RSV titre kinetics at early times post-infection (less than 5 dpi) but a slower decay in RSV titre in immunosuppressed cotton rats compared to that in non-suppressed cotton rats at later times (greater than 5 dpi) in silico. These predictions were in excellent agreement with the experimental results. Our combined approach quantified the importance of the adaptive immune response in suppressing RSV infection in cotton rats, which could be useful in testing RSV vaccine candidates.

Replication, reanalysis, and gene expression: ME2 and genetic generalized epilepsy.

OBJECTIVE: Genetic generalized epilepsy (GGE) consists of epileptic syndromes with overlapping symptoms and is considered to be largely genetic. Previous cosegregation and association studies have pointed to malic enzyme 2 (ME2) as a candidate susceptibility gene for adolescent-onset GGE. In this article, we present new evidence supporting ME2's involvement in GGE. METHODS: To definitively test ME2's influence on GGE, we used 3 different approaches. First, we compared a newly recruited GGE cohort with an ethnically matched reference sample from 1000 Genomes Project, using an efficient test of association (POPFAM+). Second, we used POPFAM+ to reanalyze a previously collected data set, wherein the original controls were replaced with ethnically matched reference samples to minimize the confounding effect of population stratification. Third, in a post hoc analysis of expression data from healthy human prefrontal cortex, we identified single nucleotide polymorphisms (SNPs) influencing ME2 messenger RNA (mRNA) expression; and then we tested those same SNPs for association with GGE in a large case-control cohort. RESULTS: First, in the analysis of our newly recruited GGE Cohort, we found a strong association between an ME2 SNP and GGE (P = 0.0006 at rs608781). Second, in the reanalysis of previously collected data, we confirmed the Greenberg et al (2005) finding of a GGE-associated ME2 risk haplotype. Third, in the post hoc ME2 expression analysis, we found evidence for a possible link between GGE and ME2 gene expression in human brain. SIGNIFICANCE: Overall, our research, and the research of others, provides compelling evidence that ME2 influences susceptibility to adolescent-onset GGE.

Lung Allocation Score Thresholds Prioritize Survival After Lung Transplantation.

BACKGROUND: The lung allocation score (LAS) prioritizes lung transplant (LTx) candidates with poor transplant-free survival and expected survival benefit from LTx. Although patients with the highest LAS have the shortest waiting time, mortality benefit is unclear in this group, raising criticism that the LAS inappropriately prioritizes critically ill candidates. We aim to identify a threshold above which increasing LAS values do not predict increasing survival benefit. METHODS: The United Network for Organ Sharing Registry was queried for first-time adult LTx candidates with LAS ≥ 30 between May 2005 and December 2016. Survival was tracked from the time of listing through the posttransplant period and compared with survival while remaining on the waitlist, using proportional hazards regression. The survival benefit of LTx was modeled as a piecewise-constant time-dependent covariate, moderated by candidate LAS. RESULTS: Of the overall cohort (N = 21,157), LTx was particularly protective for 365 patients with an initial LAS of 70 to 79 (hazard ratio of death after undergoing LTx relative to remaining on the waitlist, 0.2; 95% CI, 0.1-0.3). However, the survival benefit of LTx did not meaningfully increase for 1,042 patients listed with even higher LAS. Among patients with cystic fibrosis, the survival benefit of LTx was constant above an LAS of approximately 50. CONCLUSIONS: Consistent survival benefit of LTx was observed among patients with an initial LAS of 70 and greater. This result supports equalizing priority for donor lung allocation for patients with LAS ≥ 70. A lower LAS threshold for maximum priority is indicated in patients with cystic fibrosis.

Mutations in bacterial genes induce unanticipated changes in the relationship between bacterial pathogens in experimental otitis media.

Otitis media (OM) is a common polymicrobial infection of the middle ear in children under the age of 15 years. A widely used experimental strategy to analyse roles of specific phenotypes of bacterial pathogens of OM is to study changes in co-infection kinetics of bacterial populations in animal models when a wild-type bacterial strain is replaced by a specific isogenic mutant strain in the co-inoculating mixtures. As relationships between the OM bacterial pathogens within the host are regulated by many interlinked processes, connecting the changes in the co-infection kinetics to a bacterial phenotype can be challenging. We investigated middle ear co-infections in adult chinchillas (Chinchilla lanigera) by two major OM pathogens: non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat), as well as isogenic mutant strains in each bacterial species. We analysed the infection kinetic data using Lotka-Volterra population dynamics, maximum entropy inference and Akaike information criteria-(AIC)-based model selection. We found that changes in relationships between the bacterial pathogens that were not anticipated in the design of the co-infection experiments involving mutant strains are common and were strong regulators of the co-infecting bacterial populations. The framework developed here allows for a systematic analysis of host-host variations of bacterial populations and small sizes of animal cohorts in co-infection experiments to quantify the role of specific mutant strains in changing the infection kinetics. Our combined approach can be used to analyse the functional footprint of mutant strains in regulating co-infection kinetics in models of experimental OM and other polymicrobial diseases.

Predictive Utility of Lung Allocation Score for Retransplantation Outcomes.

BACKGROUND: Treatment of primary graft failure after lung transplantation (LTx) may include retransplantation (rLTx). The number of rLTx cases has doubled since implementation of the Lung Allocation Score in 2005. The Lung Allocation Score was intended to predict LTx outcomes, but its predictive utility has not been assessed in rLTx. We investigated whether 1-year outcomes of LTx and rLTX were equally well predicted by the Lung Allocation Score. METHODS: Recipients of LTx and rLTx aged 18 years or more were identified in 2005 to 2015 United Network for Organ Sharing data. The Lung Allocation Score was entered in multivariable logistic regression models of 1-year retransplant-free survival. Areas under the receiver-operating characteristics curve summarized model predictive value. We examined whether the Lung Allocation Score and its components were differentially associated with outcomes of LTx and rLTx. RESULTS: There were 16,837 LTx and 765 rLTx cases meeting inclusion criteria. Crude 1-year retransplant-free survival rates were 86% after LTx compared with 74% after rLTx. On univariate analysis, both LTx and rLTx cohorts showed poor predictive utility of the Lung Allocation Score (area under the curve 0.55 and 0.57, respectively; difference by transplant type, p = 0.307). Neither the Lung Allocation Score nor its components was differentially associated with LTx compared with rLTx outcomes. CONCLUSIONS: The Lung Allocation Score achieved comparable, but poor, predictive utility for 1-year outcomes of primary LTx and rLTx. We found no evidence that Lung Allocation Score components should be weighted differently for rLTx candidates.

Logistic Bayesian LASSO for detecting association combining family and case-control data.

Because of the limited information from the GAW20 samples when only case-control or trio data are considered, we propose eLBL, an extension of the Logistic Bayesian LASSO (least absolute shrinkage and selection operator) methodology so that both types of data can be analyzed jointly in the hope of obtaining an increased statistical power, especially for detecting association between rare haplotypes and complex diseases. The methodology is further extended to account for familial correlation among the case-control individuals and the trios. A 2-step analysis strategy was taken to first perform a genome-wise single single-nucleotide polymorphism (SNP) search using the Monte Carlo pedigree disequilibrium test (MCPDT) to determine interesting regions for the Adult Treatment Panel (ATP) binary trait. Then eLBL was applied to haplotype blocks covering the flagged SNPs in Step 1. Several significantly associated haplotypes were identified; most are in blocks contained in protein coding genes that appear to be relevant for metabolic syndrome. The results are further substantiated with a Type I error study and by an additional analysis using the triglyceride measurements directly as a quantitative trait.

Ophthalmic community perception of new medication needs.

AIM: To survey ophthalmologists (who have participated previously in clinical research) and ophthalmic industry professionals (who have been involved in ocular research and development) to indicate perceived needs for new pharmaceuticals in various ophthalmic subspecialties. METHODS: A prospective, industry-based survey was sent to ophthalmologists and ophthalmic industry professionals about the perceived needs for new pharmaceutical products. RESULTS: This survey was sent to 559 ophthalmic pharma professionals and ophthalmologists. We received 82 (15%) responses. The results showed that the most commonly perceived need for new pharmaceuticals were dry and wet age-related macular degeneration, glaucoma, diabetic macular edema and dry eye. There was a statistical difference found between response groups (P<0.0001). Respondents indicated they would express their commitment to a new product they perceived as needed by recommending to colleagues (63%), prescribing (60%), participating as principle investigator in a related clinical trial (52%), advising the company (52%), lecturing on behalf of the product (43%), investing in the product (38%), taking no action (7%) or obtain a position in the company (1%). CONCLUSION: Ophthalmic pharma professionals and ophthalmologists perceive the greatest need for new medicines in ophthalmology to be in dry and wet age-related macular degeneration, glaucoma, diabetic macular edema and dry eye.

DNA methylation of the BRD2 promoter is associated with juvenile myoclonic epilepsy in Caucasians.

OBJECTIVE: Juvenile myoclonic epilepsy (JME) is a common adolescent-onset genetic generalized epilepsy (GGE) syndrome. Multiple linkage and association studies have found that BRD2 influences the expression of JME. The BRD2-JME connection is further corroborated by our murine model; Brd2 haploinsufficiency produces characteristics that typify the clinical hallmarks of JME. Neither we, nor several large-scale studies of JME, found JME-related BRD2 coding mutations. Therefore, we investigated noncoding BRD2 regions, seeking the origin of BRD2's JME influence. BRD2's promoter harbors a JME-associated single nucleotide polymorphism (rs3918149) and a CpG (C-phosphate-G dinucleotides) island (CpG76), making it a potential "hotspot" for JME-associated epigenetic variants. Methylating promoter CpG sites causes gene silencing, often resulting in reduced gene expression. We tested for differences in DNA methylation at CpG76 in 3 different subgroups: (1) JME patients versus their unaffected family members, (2) JME versus patients with other forms of GGE, and (3) Caucasian versus non-Caucasian JME patients. METHODS: We used DNA pyrosequencing to analyze the methylation status of 10 BRD2 promoter CpG sites in lymphoblastoid cells from JME patients of Caucasian and non-Caucasian origin, unaffected family members, and also non-JME GGE patients. We also measured global methylation levels and DNA methyl transferase 1 (DNMT1) transcript expression in JME families by standard methods. RESULTS: CpG76 is highly methylated in JME patients compared to unaffected family members. In families with non-JME GGE, we found no relationship between promoter methylation and epilepsy. In non-Caucasian JME families, promoter methylation was mostly not associated with epilepsy. This makes the BRD2 promoter a JME-specific, ethnicity-specific, differentially methylated region. Global methylation was constant across groups. SIGNIFICANCE: BRD2 promoter methylation in JME, and the lack of methylation in unaffected relatives, in non-JME GGE patients, and in non-Caucasian JME, demonstrate that methylation specificity is a possible seizure susceptibility motif in JME risk and suggests JME therapeutics targeting BRD2.

Ophthalmic Start-Up Chief Executive Officers' Perceptions of Development Hurdles.

PURPOSE: To identify current challenges facing ophthalmic pharmaceutical start-ups in developing new products. METHODS: Surveys were distributed to the chief executive officer (CEO) or president of ophthalmic start-ups. RESULTS: The survey attracted 24 responses from 78 surveys distributed (31%). The CEOs stated that a lack of financial capital (n = 18, 75%), FDA regulations (n = 6, 25%), and failure to meet clinical endpoints (n = 6, 25%) were their greatest development hurdles. Risk aversion to medicines in early development (n = 18, 75%), mergers and acquisitions reducing corporate choice for licensing agreements (n = 7, 29%), the emergence of large pharmaceutical-based venture capital funding groups (n = 12, 50%), and the failure of many large pharmaceutical companies to develop their own medicines (n = 10, 42%) were noted as recent prominent trends affecting fundraising. CONCLUSION: The study suggests that development funding, regulatory burden, and meeting clinical endpoints are the greatest development challenges faced by ophthalmic start-up CEOs.

Comparison of the Lund and Browder table to computed tomography scan three-dimensional surface area measurement for a pediatric cohort.

BACKGROUND: Treating burns effectively requires accurately assessing the percentage of the total body surface area (%TBSA) affected by burns. Current methods for estimating %TBSA, such as Lund and Browder (L&B) tables, rely on historic body statistics. An increasingly obese population has been blamed for increasing errors in %TBSA estimates. However, this assumption has not been experimentally validated. We hypothesized that errors in %TBSA estimates using L&B were due to differences in the physical proportions of today's children compared with children in the early 1940s when the chart was developed and that these differences would appear as body mass index (BMI)-associated systematic errors in the L&B values versus actual body surface areas. MATERIALS AND METHODS: We measured the TBSA of human pediatric cadavers using computed tomography scans. Subjects ranged from 9 mo to 15 y in age. We chose outliers of the BMI distribution (from the 31st percentile at the low through the 99th percentile at the high). We examined surface area proportions corresponding to L&B regions. RESULTS: Measured regional proportions based on computed tomography scans were in reasonable agreement with L&B, even with subjects in the tails of the BMI range. The largest deviation was 3.4%, significantly less than the error seen in real-world %TBSA estimates. CONCLUSIONS: While today's population is more obese than those studied by L&B, their body region proportions scale surprisingly well. The primary error in %TBSA estimation is not due to changing physical proportions of today's children and may instead lie in the application of the L&B table.