Developments in the field of allergy mechanisms in 2015 through the eyes of Clinical & Experimental Allergy

In the first of two papers we described the development in the field of allergy mechanisms as described by Clinical and Experimental Allergy in 2015. Experimental models of allergic disease, basic mechanisms, clinical mechanisms and allergens are all covered. A second paper will cover clinical aspects.     

Genomic context sensitivity of insulator function

The specificity of interactions between genomic regulatory elements and potential target genes is influenced by the binding of insulator proteins such as CTCF, which can act as potent enhancer blockers when interposed between an enhancer and a promoter in a reporter assay. But not all CTCF sites genome-wide function as insulator elements, depending on cellular and genomic context. To dissect the influence of genomic context on enhancer blocker activity, we integrated reporter constructs with promoter-only, promoter and enhancer, and enhancer blocker configurations at hundreds of thousands of genomic sites using the Sleeping Beauty transposase. Deconvolution of reporter activity by genomic position reveals distinct expression patterns subject to genomic context, including a compartment of enhancer blocker reporter integrations with robust expression. The high density of integration sites permits quantitative delineation of characteristic genomic context sensitivity profiles and their decomposition into sensitivity to both local and distant DNase I hypersensitive sites. Furthermore, using a single-cell expression approach to test the effect of integrated reporters for differential expression of nearby endogenous genes reveals that CTCF insulator elements do not completely abrogate reporter effects on endogenous gene expression. Collectively, our results lend new insight into genomic regulatory compartmentalization and its influence on the determinants of promoter–enhancer specificity.

A boundary model offers an attractive paradigm for understanding regulatory specificity in mammalian genomes through the delineation of independent regulatory domains. Insulators are a class of genomic regulatory elements that block interaction of enhancers with their cognate promoters (Phillips and Corces 2009). Enhancer blocker activity is canonically defined by a reporter assay that interposes a candidate insulator element between a weak promoter and an enhancer (Chung et al. 1993), while barrier insulators protect transgenes from silencing owing to spreading of heterochromatin (West et al. 2002). Insulators have also been used to counter genotoxicity from transgene enhancer activation of endogenous oncogenes (Li et al. 2009; Liu et al. 2015). Known insulators such as the chicken beta-globin hypersensitive site 4 element or the Igf2/H19 imprinting control region (Bell and Felsenfeld 2000) are composite elements with enhancer blocker, barrier, and other activities (Dickson et al. 2010), and often have secondary functions, such as silencers (Qi et al. 2015).The architectural protein CTCF is the only known vertebrate insulator protein, and its binding can confer a potent enhancer blocking effect (Phillips and Corces 2009). Additionally, binding sites for CTCF colocalize with genomic features such as topologically associated domain boundaries (Dixon et al. 2012), but direct functional analysis of these sites is impeded by the difficulty of genome engineering at the relevant scales. Although binding affinity, DNA methylation, and recognition sequence orientation appear to confer some specificity for CTCF sites involved in domain organization (de Wit et al. 2015; Guo et al. 2015; Sanborn et al. 2015), these factors alone remain inadequate to distinguish true insulator elements impacting expression of nearby genes from the ∼100,000 CTCF sites genome-wide (Maurano et al. 2015; Tycko et al. 2019). Stably integrated reporter assays have shed light on the mechanics of insulator function, but such methods do not assess interaction with the surrounding endogenous genomic elements (Walters et al. 1999). In contrast, integrated barcoded reporter assays (Akhtar et al. 2013; Maricque et al. 2019; Moudgil et al. 2020) offer the potential to directly assess the interaction between novel CTCF sites and the endogenous genomic landscape.Here, we aim to functionally characterize endogenous genomic regulatory elements through their effect on integrated reporters. We describe a high-throughput randomly integrated barcoded reporter platform based on a previously described enhancer blocker construct interposing a potent CTCF insulator element (Liu et al. 2015) between a weak promoter and a potent enhancer. We integrate these reporters, both with and without insulator elements, randomly throughout the genome of K562 erythroleukemia cells using the Sleeping Beauty transposase system. We use the unique reporter barcodes to map individual insertion locations and enable position-specific readout of genomic context effects. Finally, we apply single-cell RNA-seq (scRNA-seq) to detect specific reporter integrations that perturb endogenous gene expression.     

Children's color trails.

Color Trails for Children was developed in response to the need for instruments which minimize cultural bias in neuropsychological testing. The test, similar in format to Trail Making, was designed to provide an evaluation of speeded visuomotor tracking while minimizing the influence of language. The present research involves two exploratory studies which examine the relationship between Color Trails for Children and Trail Making, factors that may affect performance times, and discriminant validity. Results indicate that the tests appear to measure the same neuropsychological domains, and administration of Trail Making did not significantly alter performance times on Color Trails. Increasing age and IQ were related to quicker completion time for both tests. Females were found to complete Color Trails 2 and Trail Making Part B more quickly than males in this sample. Comparison between children diagnosed with learning disabilities, attention deficits, or mild neurological conditions and a preliminary standardization sample supported the discriminant validity of Color Traits to distinguish between normal controls and children with altered neuropsychological functioning. Comparison between clinical conditions indicated that Color Trails 2 was particularly sensitive in discriminating among the groups. Although further research is needed, results suggest that Color Trails has the potential to be an effective research and clinical tool in child neuropsychological assessment.     

Challenges to recovery following early psychosis: Nursing implications of recovery rate and timing

Early intervention in first episode psychosis is based on an indicated prevention approach that has early illness identification and timely recovery as primary goals. Nurses are instrumental in helping individuals and families achieve both aims. To better understand recovery following a first episode, a prospective cohort of 260 individuals participating in a three-year early intervention program was monitored for achievement of recovery outcomes. Two outcome measures were used to examine the recovery rate and timing of the cohort: (1) partial recovery was comprised of two criteria: (a) symptom control (psychosis and mania), and (b) daily functioning, and 2) comprehensive recovery was measured by three criteria: (a) symptom control; (b) daily functioning; and, (c) quality of life. Survival analysis, including the Kaplan-Meier statistic, and Cox hazard regression were used to examine the cohort's rate and timing for both measures. One hundred and seventy-four individuals attained partial recovery with half (51.1%) reaching the target within nine months. Comprehensive recovery was achieved by 59 individuals (22.7%), primarily in year two and three of treatment. Issues impacting quality of life delayed recovery for the majority of program participants. The gap between psychosis remission and satisfaction/fulfillment with one's everyday life is troubling, but could be improved with stronger nursing support and influence. Sharing the recovery experience with individuals and families that supports their life goals and the discovery of meaning, hope and purpose in the face of illness is the work of nurses. Suggestions for strengthening nursing's impact are considered.     

An essential role for an inositol polyphosphate multikinase, Ipk2, in mouse embryogenesis and second messenger production

Phospholipase C and several inositol polyphosphate kinase (IPK) activities generate a branched ensemble of inositol polyphosphate second messengers that regulate cellular signaling pathways in the nucleus and cytoplasm. Here, we report that mice deficient for Ipk2 (also known as inositol polyphosphate multikinase), an inositol trisphosphate and tetrakisphosphate 6/5/3-kinase active at several places in the inositol metabolic pathways, die around embryonic day 9.5 with multiple morphological defects, including abnormal folding of the neural tube. Metabolic analysis of Ipk2-deficient cells demonstrates that synthesis of the majority of inositol pentakisphosphate, hexakisphosphate and pyrophosphate species are disrupted, although the presence of 10% residual inositol hexakisphosphate indicates the existence of a minor alternative pathway. Agonist induced inositol tris- and bis-phosphate production and calcium release responses are present in homozygous mutant cells, indicating that the observed mouse phenotypes are a result of failure to produce higher inositol polyphosphates. Our data demonstrate that Ipk2 plays a major role in the synthesis of inositol polyphosphate messengers derived from inositol 1,4,5-trisphosphate and uncovers a role for their production in embryogenesis and normal development.     

Prevalence of allergic sensitization to indoor fungi in West Virginia

Exposure to indoor fungi is of growing concern in residential and occupational environments in the United States. The purpose of this study was to determine the prevalence of sensitization to common indoor fungal species in an atopic population. We evaluated 102 patients (73 female and 29 male patients) for immunoglobulin E (IgE) reactivity to a panel of skin-prick test (SPT) reagents used for routine allergy testing. Patients also were tested for six additional fungi that are common indoor contaminants. All patients had symptoms consistent with allergic rhinitis or asthma. The presence of specific IgE against the fungal species was determined using immunoblotting. Of the 102 eligible patients, 68% had at least one positive skin test. The most prevalent positive SPTs were to dust mites, cats, vernal grass, and short ragweed. Overall, 21/102 (21%) patients with asthma or allergic rhinitis were skin test positive to at least one fungal extract. Of the patients with a positive SPT to fungi, 12/21 (58%) showed sensitivity to one or more of the newly tested species; most notably Trichoderma viride (8%), Chaetomium globosum (7%), Paecilomyces variotii (7%), and Acremonium strictum (6%). Immunoblotting revealed specific IgE against a number of protein bands belonging to these fungal species. The prevalence of fungal sensitization was common, particularly for indoor fungal contaminants that are not routinely included in SPT panels. Cross-reactivity with other fungi may partially explain our results; however, skin testing for these indoor fungi may provide useful diagnostic information.     

Predicting who will develop dementia in a cohort of Canadian seniors

OBJECTIVES: We examined whether easily attainable variables were useful in predicting who became demented over a five year period and determined the rates of incident dementia for different categories of mild cognitive impairment. METHODS: This was a cohort study of subjects recruited nationally in a population-based survey of Canadians 65 years and older (the Canadian Study of Health and Aging). After standardized clinical assessments, a subset of subjects (n = 1782) was categorized as not demented at time one. Identical study methods allowed a reassessment of the cognitive status of surviving subjects (n = 892) five years later. RESULTS: Three baseline variables (Modified Mini Mental State (3MS) score, subject's age, and an informant's report of the presence of memory problems) were statistically significant predictors of the development of a dementia. An equation incorporating these three variables had a sensitivity of 79% and a specificity of 56% for predicting dementia among survivors at time two. An equation substituting the MMSE for the 3MS showed similar results. The various categories of mild cognitive impairment examined showed significantly different likelihoods for the subsequent development of a dementia. Some categories with a higher dementia risk were characterized by inclusion criteria requiring neuropsychological test scores that were greater than one standard deviation (SD) below the mean of age based normative data. CONCLUSION: In the absence of extensive laboratory, radiologic or neuropsychological tests, simple variables that can be easily determined in the course of a single clinical encounter were useful in predicting subjects with a higher risk of developing dementia. Attempts to use neuropsychological results to predict the development of dementia should look for significant impairments on age-standardized tests.