Comprehensive Evaluation and Optimization of Amplicon Library Preparation Methods for High-Throughput Antibody Sequencing

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.     

The Role of Estrogen Signaling in a Mouse Model of Inflammatory Bowel Disease: A Helicobacter Hepaticus Model

The pathogenesis of inflammatory bowel diseases (IBD), Crohn''s disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with Helicobacter hepaticus triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and β knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERβ on a non-CD4⁺ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.     

Gastroesophageal Reflux in Relation to Adenocarcinomas of the Esophagus: A Pooled Analysis from the Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON)

Background

Previous studies have evidenced an association between gastroesophageal reflux and esophageal adenocarcinoma (EA). It is unknown to what extent these associations vary by population, age, sex, body mass index, and cigarette smoking, or whether duration and frequency of symptoms interact in predicting risk. The Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON) allowed an in-depth assessment of these issues.

Methods

Detailed information on heartburn and regurgitation symptoms and covariates were available from five BEACON case-control studies of EA and esophagogastric junction adenocarcinoma (EGJA). We conducted single-study multivariable logistic regressions followed by random-effects meta-analysis. Stratified analyses, meta-regressions, and sensitivity analyses were also conducted.

Results

Five studies provided 1,128 EA cases, 1,229 EGJA cases, and 4,057 controls for analysis. All summary estimates indicated positive, significant associations between heartburn/regurgitation symptoms and EA. Increasing heartburn duration was associated with increasing EA risk; odds ratios were 2.80, 3.85, and 6.24 for symptom durations of <10 years, 10 to <20 years, and ≥20 years. Associations with EGJA were slighter weaker, but still statistically significant for those with the highest exposure. Both frequency and duration of heartburn/regurgitation symptoms were independently associated with higher risk. We observed similar strengths of associations when stratified by age, sex, cigarette smoking, and body mass index.

Conclusions

This analysis indicates that the association between heartburn/regurgitation symptoms and EA is strong, increases with increased duration and/or frequency, and is consistent across major risk factors. Weaker associations for EGJA suggest that this cancer site has a dissimilar pathogenesis or represents a mixed population of patients.     

Is Peripheral Immunity Regulated by Blood-Brain Barrier Permeability Changes?

S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses.     

Population Connectivity Shifts at High Frequency within an Open-Coast Marine Protected Area Network

A complete understanding of population connectivity via larval dispersal is of great value to the effective design and management of marine protected areas (MPA). However empirical estimates of larval dispersal distance, self-recruitment, and within season variability of population connectivity patterns and their influence on metapopulation structure remain rare. We used high-resolution otolith microchemistry data from the temperate reef fish Hypsypops rubicundus to explore biweekly, seasonal, and annual connectivity patterns in an open-coast MPA network. The three MPAs, spanning 46 km along the southern California coastline were connected by larval dispersal, but the magnitude and direction of connections reversed between 2008 and 2009. Self-recruitment, i.e. spawning, dispersal, and settlement to the same location, was observed at two locations, one of which is a MPA. Self-recruitment to this MPA ranged from 50–84%; within the entire 60 km study region, self-recruitment accounted for 45% of all individuals settling to study reefs. On biweekly time scales we observed directional variability in alongshore current data and larval dispersal trajectories; if viewed in isolation these data suggest the system behaves as a source-sink metapopulation. However aggregate biweekly data over two years reveal a reef network in which H. rubicundus behaves more like a well-mixed metapopulation. As one of the few empirical studies of population connectivity within a temperate open coast reef network, this work can inform the MPA design process, implementation of ecosystem based management plans, and facilitate conservation decisions.     

The Efficacy of the Ribonucleotide Reductase Inhibitor Didox in Preclinical Models of AML

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC₅₀ values in the low micromolar range (mean IC₅₀ 37 µM [range 25.89–52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML.     

Nesting Habitat Creation Enhances Recruitment in a Predator‐Free Environment: Malaclemys Nesting at the Paul S. Sarbanes Ecosystem Restoration Project

Aquatic turtles worldwide are plagued with habitat loss due to development and shoreline alteration that destroys the terrestrial–aquatic linkage which they must cross to reproduce successfully. Furthermore, nesting habitat loss can concentrate nesting, increasing nest predator efficiency. We describe how the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island created nesting habitat for Malaclemys terrapin (Diamondback Terrapin), and document nesting success in response to construction progress and the absence of raccoons and foxes, the primary nest predators. We monitored terrapin nests throughout the nesting seasons from 2002 to 2011 to determine overall and within‐nest survivorship. Female terrapins began nesting on the restoration project within 1 year but planned construction during the study eliminated some nesting areas and opened previously inaccessible areas. Overall, nest survivorship was considerably higher than mainland nesting areas due to the absence of raccoons and foxes on the island and within‐nest survivorship was similar. Egg size, hatchling size, and the frequency of shell scute anomalies were similar to other terrapin populations, suggesting normal developmental conditions on the island. We documented annual variation in hatchling size that correlated negatively with mean air temperature during the incubation season. Our results indicate that restored or created isolated island habitat can be located rapidly by terrapins and can become an important source of recruitment in regions where nesting habitat is limited and predation is high. Poplar Island illustrates how habitat loss and restoration can affect turtle populations by revealing the changes in nesting patterns and success in newly created, predator‐free habitat.