Andrew A Hill

Wnt/β-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone

A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/β-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/β-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/β-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3β inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3β inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/β-catenin signaling is a normal physiological response to load and that activation of the Wnt/β-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.

Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome.

Temporal profiles of transcript abundance during embryonic development were obtained by whole-genome expression analysis from precisely staged C. elegans embryos. The result is a highly resolved time course that commences with the zygote and extends into mid-gastrulation, spanning the transition from maternal to embryonic control of development and including the presumptive specification of most major cell fates. Transcripts for nearly half (8890) of the predicted open reading frames are detected and expression levels for the majority of them (>70%) change over time. The transcriptome is stable up to the four-cell stage where it begins rapidly changing until the rate of change plateaus before gastrulation. At gastrulation temporal patterns of maternal degradation and embryonic expression intersect indicating a mid-blastula transition from maternal to embryonic control of development. In addition, we find that embryonic genes tend to be expressed transiently on a time scale consistent with developmental decisions being made with each cell cycle. Furthermore, overall rates of synthesis and degradation are matched such that the transcriptome maintains a steady-state frequency distribution. Finally, a versatile analytical platform based on cluster analysis and developmental classification of genes is provided.

Pathways Activated during Human Asthma Exacerbation as Revealed by Gene Expression Patterns in Blood

Background Asthma exacerbations remain a major unmet clinical need. The difficulty in obtaining airway tissue and bronchoalveolar lavage samples during exacerbations has greatly hampered study of naturally occurring exacerbations. This study was conducted to determine if mRNA profiling of peripheral blood mononuclear cells (PBMCs) could provide information on the systemic molecular pathways involved during asthma exacerbations. Methodology/Principal Findings Over the course of one year, gene expression levels during stable asthma, exacerbation, and two weeks after an exacerbation were compared using oligonucleotide arrays. For each of 118 subjects who experienced at least one asthma exacerbation, the gene expression patterns in a sample of peripheral blood mononuclear cells collected during an exacerbation episode were compared to patterns observed in multiple samples from the same subject collected during quiescent asthma. Analysis of covariance identified genes whose levels of expression changed during exacerbations and returned to quiescent levels by two weeks. Heterogeneity among visits in expression profiles was examined using K-means clustering. Three distinct exacerbation-associated gene expression signatures were identified. One signature indicated that, even among patients without symptoms of respiratory infection, genes of innate immunity were activated. Antigen-independent T cell activation mediated by IL15 was also indicated by this signature. A second signature revealed strong evidence of lymphocyte activation through antigen receptors and subsequent downstream events of adaptive immunity. The number of genes identified in the third signature was too few to draw conclusions on the mechanisms driving those exacerbations. Conclusions/Significance This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs.

The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo

Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multipotent C blastomere lineage in the C. elegans embryo. To identify PAL-1 regulatory target genes, we used microarrays to compare transcript abundance in wild-type embryos with mutant embryos lacking a C blastomere and to mutant embryos with extra C blastomeres. pal-1-dependent C-lineage expression was verified for select candidate target genes by reporter gene analysis, though many of the target genes are expressed in additional lineages as well. The set of validated target genes includes 12 transcription factors, an uncharacterized wingless ligand and five uncharacterized genes. Phenotypic analysis demonstrates that the identified PAL-1 target genes affect specification, differentiation and morphogenesis of C-lineage cells. In particular, we show that cell fate-specific genes (or tissue identity genes) and a posterior HOX gene are activated in lineage-specific fashion. Transcription of targets is initiated in four temporal phases, which together with their spatial expression patterns leads to a model of the regulatory network specified by PAL-1.

Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression

Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time series, whole‐genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage‐specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one‐hybrid and promoter sequence analyses. Based on these results, we define and characterize two modules composed of muscle‐ and epidermal‐specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross‐inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches.

The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls

BackgroundThe R620W variant in protein tyrosine phosphatase non-receptor 22 (PTPN22) is associated with rheumatoid arthritis (RA). The PTPN22 gene has alternatively spliced transcripts and at least two of the splice forms have been confirmed to encode different PTPN22 (LYP) proteins, but detailed information regarding expression of these is lacking, especially with regard to autoimmune diseases.MethodsWe have investigated the mRNA expression of known PTPN22 splice forms with TaqMan real-time PCR in relation to ZNF592 as an endogenous reference in peripheral blood cells from three independent cohorts with RA patients (n = 139) and controls (n = 111) of Caucasian origin. Polymorphisms in the PTPN22 locus (25 SNPs) and phenotypic data (gender, disease activity, ACPA and RF status) were used for analysis. Additionally, we addressed possible effects of methotrexate treatment on PTPN22 expression.ResultsWe found consistent differences in the expression of the PTPN22 splice forms in unstimulated peripheral blood mononuclear cells between RA patients and normal controls. This difference was more pronounced when comparing the ratio of splice forms and was not affected by methotrexate treatment.ConclusionsOur data show that RA patients and healthy controls have a shift in balance of expression of splice forms derived from the PTPN22 gene. This balance seems not to be caused by treatment and may be of importance during immune response due to great structural differences in the encoded PTPN22 proteins.

Development and application of a biomarker assay for determining the pharmacodynamic activity of an antagonist candidate biotherapeutic antibody to IL21R in whole blood.

BackgroundIn preparation for potential clinical development of Ab-01, an antagonistic antibody directed against the IL21R, studies were undertaken to address translational medicine needs that fall into four categories: 1) development of a pharmacodynamic biomarker assay suitable for use in the clinic, 2) demonstration that Ab-01 has the desired biological activity in vitro and in vivo in cynomolgus monkeys, the preferred safety study species, 3) pre-clinical in vivo proof-of-concept that the assay can be used to detect Ab-01 pharmacodynamic (PD) activity in treated subjects, and 4) comprehensive assessment of the agonistic potential of Ab-01 when cross-linked. This report and a recently published companion report address the first three of these needs. The fourth has been addressed in a separate study.MethodsGenes that change RNA expression upon ex vivo rhIL21 stimulation of whole blood were identified in human and cynomolgus monkey. The inhibitory effects of exogenously added Ab-01 were measured ex vivo in human and monkey, and the in vivo inhibitory effects of Ab-01 treatment were measured in monkey.ResultsStimulation of whole human blood for 2 hours with rhIL21 induced robust increases in RNA expression of 6 genes. This response was blocked by Ab-01, indicating that the assay is suitable for measuring Ab-01 activity in blood. rhIL21 induced expression of a similar set of genes in cynomolgus monkey blood. This response was blocked with Ab-01, thus demonstrating that Ab-01 has the desired activity in the species, and that safety studies done in cynomolgus monkeys are relevant. Proof -of-concept for using this assay system to detect PD activity in vivo was generated by measuring the response in monkey blood to ex vivo rhIL21 stimulation before and 5 minutes following in vivo Ab-01 administration.ConclusionsA robust PD biomarker assay suitable for clinical use has been developed in human whole blood. The successful adaptation of the assay to cynomolgus monkeys has enabled the demonstration of Ab-01 activity both in vitro and in vivo in monkey, thus validating the use of this species in safety studies and establishing proof-of-concept for using this PD assay system to aid in dose selection in clinical studies.

The Biomarkers of Lupus Disease Study: A Bold Approach May Mitigate Interference of Background Immunosuppressants in Clinical Trials

Molecular medicine raised expectations for strategically targeted biologic agents in systemic lupus erythematosus (SLE), but clinical trial results have been disappointing and difficult to interpret. Most studies add investigational agents to various, often effective, standard therapy immunosuppressants used at baseline, with unknown treatment interactions. Eliminating polypharmacy in trials of active lupus remains controversial. We undertook the Biomarkers of Lupus Disease study to test withdrawal of immunosuppressants as a novel approach to rendering SLE trials interpretable.

Assessing agonistic potential of a candidate therapeutic anti-IL21R antibody

BackgroundSelective neutralization of the IL21/IL21R signaling pathway is a promising approach for the treatment of a variety of autoimmune diseases. Ab-01 is a human neutralizing anti-IL21R antibody. In order to ensure that the activities of Ab-01 are restricted to neutralization even under in vitro cross-linking and in vivo conditions, a comprehensive assessment of agonistic potential of Ab-01 was undertaken.MethodsIn vitro antibody cross-linking and cell culture protocols reported for studies with a human agonistic antibody, TGN1412, were followed for Ab-01. rhIL21, the agonist ligand of the targeted receptor, and cross-linked anti-CD28 were used as positive controls for signal transduction. In vivo agonistic potential of Ab-01 was assessed by measuring expression levels of cytokine storm-associated and IL21 pathway genes in blood of cynomolgus monkeys before and after IV administration of Ab-01.ResultsUsing a comprehensive set of assays that detected multiple activation signals in the presence of the positive control agonists, in vitro Ab-01-dependent activation was not detected in either PBMCs or the rhIL21-responsive cell line Daudi. Furthermore, no difference in gene expression levels was detected in blood before and after in vivo Ab-01 dosing of cynomolgus monkeys.ConclusionsDespite efforts to intentionally force an agonistic signal from Ab-01, none could be detected.

Stepwise Distributed Open Innovation Contests for Software Development: Acceleration of Genome-Wide Association Analysis

Abstract Background: The association of differing genotypes with disease-related phenotypic traits offers great potential to both help identify new therapeutic targets and support stratification of patients who would gain the greatest benefit from specific drug classes. Development of low-cost genotyping and sequencing has made collecting large-scale genotyping data routine in population and therapeutic intervention studies. In addition, a range of new technologies is being used to capture numerous new and complex phenotypic descriptors. As a result, genotype and phenotype datasets have grown exponentially. Genome-wide association studies associate genotypes and phenotypes using methods such as logistic regression. As existing tools for association analysis limit the efficiency by which value can be extracted from increasing volumes of data, there is a pressing need for new software tools that can accelerate association analyses on large genotype-phenotype datasets. Results: Using open innovation (OI) and contest-based crowdsourcing, the logistic regression analysis in a leading, community-standard genetics software package (PLINK 1.07) was substantially accelerated. OI allowed us to do this in <6 months by providing rapid access to highly skilled programmers with specialized, difficult-to-find skill sets. Through a crowd-based contest a combination of computational, numeric, and algorithmic approaches was identified that accelerated the logistic regression in PLINK 1.07 by 18- to 45-fold. Combining contest-derived logistic regression code with coarse-grained parallelization, multithreading, and associated changes to data initialization code further developed through distributed innovation, we achieved an end-to-end speedup of 591-fold for a data set size of 6678 subjects by 645 863 variants, compared to PLINK 1.07s logistic regression. This represents a reduction in run time from 4.8 hours to 29 seconds. Accelerated logistic regression code developed in this project has been incorporated into the PLINK2 project. Conclusions: Using iterative competition-based OI, we have developed a new, faster implementation of logistic regression for genome-wide association studies analysis. We present lessons learned and recommendations on running a successful OI process for bioinformatics.