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We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351-364 nm) flow cytometry was used to confirm ABCG2-specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT-PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342-DNA fluorescence in live cells. A live cell 'kinetic SP gate' tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye-DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic-critical for functional assay design and the interpretation of heterogeneity. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Background: Genetic factors play a role in chronic obstructive pulmonary disease (COPD) but are poorly understood. A number of candidate genes have been proposed on the basis of the pathogenesis of COPD. These include the matrix metalloproteinase (MMP) genes which play a role in tissue remodelling and fit in with the protease - antiprotease imbalance theory for the cause of COPD. Previous genetic studies of MMPs in COPD have had inadequate coverage of the genes, and have reported conflicting associations of both single nucleotide polymorphisms (SNPs) and SNP haplotypes, plausibly due to under-powered studies. Methods: To address these issues we genotyped 26 SNPs, providing comprehensive coverage of reported SNP variation, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations. We used logistic regression to adjust for age, gender, centre and smoking history. Results: Haplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), showed an association with severe/very severe disease, corresponding to GOLD Stages III and IV. Conclusions: Those with the common A-A haplotype for these two SNPs were at greater risk of developing severe/very severe disease (p = 0.0039) while possession of the minor G variants at either SNP locus had a protective effect (adjusted odds ratio of 0.76; 95% CI 0.61 - 0.94). The A-A haplotype was also associated with significantly lower predicted FEV1 (42.62% versus 44.79%; p = 0.0129). This implicates haplotypes of MMP-12 as modifiers of disease severity. [ABSTRACT FROM AUTHOR]

Objectives: In order to determine how gene expression is altered in disease it is of fundamental importance that the global distribution of gene expression levels across the disease-free brain are understood and how differences between tissue types might inform tissue choice for investigation of altered expression in disease state. The aim of this pilot project was to use RNA-sequencing to investigate gene expression differences between five general areas of post-mortem human brain (frontal, temporal, occipital, parietal and cerebellum), and in particular changes in gene expression in the cerebellum compared to cortex regions for genes relevant to Alzheimer’s disease, as the cerebellum is largely preserved from disease pathology and could be an area of interest for neuroprotective pathways. Results: General gene expression profiles were found to be similar between cortical regions of the brain, however the cerebellum presented a distinct expression profile. Focused exploration of gene expression for genes associated with Alzheimer’s disease suggest that those involved in the immunity pathway show little expression in the brain. Furthermore some Alzheimer’s disease associated genes display significantly different expression in the cerebellum compared with other brain regions, which might indicate potential neuroprotective measures. [ABSTRACT FROM AUTHOR]

Mature basophils play critical inflammatory roles during helminthic, autoimmune, and allergic diseases through their secretion of histamine and the type 2 cytokines interleukin 4 (IL-4) and IL-13. Basophils are activated typically by allergen-mediated IgE crosslinking but also by endogenous "innate" factors. The aim of this study was to identify the innate stimuli (cytokines, chemokines, growth factors, hormones, neuropeptides, metabolites, and bacterial products) and signaling pathways inducing primary basophil activation. Basophils from naïve mice or helminth-infected mice were cultured with up to 96 distinct stimuli and their influence on basophil survival, activation, degranulation, and IL-4 or IL-13 expression were investigated. Activated basophils show a heterogeneous phenotype and segregate into distinct subsets expressing IL-4, IL-13, activation, or degranulation markers. We find that several innate stimuli including epithelial derived inflammatory cytokines (IL-33, IL-18, TSLP, and GM-CSF), growth factors (IL-3, IL-7, TGFβ, and VEGF), eicosanoids, metabolites, TLR ligands, and type I IFN exert significant direct effects on basophils. Basophil activation mediated by distinct upstream signaling pathways is always sensitive to Syk and IκB kinases-specific inhibitors but not necessarily to NFAT, STAT5, adenylate cyclase, or c-fos/AP-1 inhibitors. Thus, basophils are activated by very diverse mediators, but their activation seem controlled by a core checkpoint involving Syk and IκB kinases. [ABSTRACT FROM AUTHOR]

In this letter to the editor the author responds to criticism of her article "A Reconsideration of the Equitable Building in New York" published in the March, 1990 issue of this journal.

Abstract: We employed gene array technology to investigate the effects of α1-antichymotrypsin (ACT), soluble or fibrillar Alzheimer''s peptide (Aβ1–42) alone and the combination of ACT/Aβ1–42 on human astrocytes. Using a 1.2-fold change as significance threshold, 398 astrocyte genes showed altered expression in response to these treatments compared to controls. Of the 276 genes affected by the ACT/soluble Aβ1–42 combination, 195 (70.6%) were suppressed. The ACT/fibrillar Aβ1–42 combination affected expression of 64 genes of which 58 (90.5%) were up-regulated. The most prominent gene expression changes in response to the ACT/soluble Aβ1–42, were the down-regulation of at least 60 genes involved in transcription, signal transduction, apoptosis and neurogenesis. The ACT/fibril Aβ1–42 increased the expression of genes involved in transcription regulation and signal transduction. Surprisingly, gene expression of astrocytes exposed to soluble or fibrillar Aβ1–42 alone was largely unaffected. Thus, the molecular forms generated by the combination of ACT/Aβ1–42 alter expression of astrocyte genes more profoundly in breadth and magnitude than soluble or fibrillar Aβ1–42 alone, suggesting that pathogenic effects of Aβ1–42 may occur as a consequence of its association with other proteins. [Copyright &y& Elsevier]

Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ 2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11b lo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11b hi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4 + GATA3 + helper T cells (T H ), whereas antifungal IL-17 + RORγt + T H cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: The type 2 cytokines IL-4 and IL-13 promote not only atopic dermatitis (AD) but also the resolution of inflammation. How type 2 cytokines participate in the resolution of AD is poorly known., Objective: Our aim was to determine the mechanisms and cell types governing skin inflammation, barrier dysfunction, and resolution of inflammation in a model of AD., Methods: Mice that exhibit expression of IL-4, IL-13, and MCPT8 or that could be depleted of basophils or eosinophils, be deficient in IL-4 or MHC class II molecules, or have basophils lacking macrophage colony-stimulating factor (M-CSF) were treated with calcipotriol (MC903) as an acute model of AD. Kinetics of the disease; keratinocyte differentiation; and leukocyte accumulation, phenotype, function, and cytokine production were measured by transepidermal water loss, histopathology, molecular biology, or unbiased analysis of spectral flow cytometry., Results: In this model of AD, basophils were activated systemically and were the initial and main source of IL-4 in the skin. Basophils and IL-4 promoted epidermal hyperplasia and skin barrier dysfunction by acting on keratinocyte differentiation during inflammation. Basophils, IL-4, and basophil-derived M-CSF inhibited the accumulation of proinflammatory cells in the skin while promoting the expansion and function of proresolution M2-like macrophages and the expression of probarrier genes. Basophils kept their proresolution properties during AD resolution., Conclusion: Basophils can display both beneficial and detrimental type 2 functions simultaneously during atopic inflammation., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

In this article, we report the number of cyclin B1 proteins tagged with enhanced green fluorescent protein (eGFP) in fixed U-2 OS cells across the cell cycle. We use a quantitative analysis of epifluorescence to determine the number of eGFP molecules in a nondestructive way, and integrated over the cell we find 10 4 to 10 5 molecules. Based on the measured number of eGFP tagged cyclin B1 proteins, knowledge of cyclin B1 dynamics through the cell cycle, and the cell morphology, we identify the stages of cells in the cell cycle. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry., (© 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.)

Preeclampsia, which affects approximately 5% of pregnancies, is a leading cause of maternal and perinatal death. The causes of preeclampsia remain unclear, but there is evidence for inherited susceptibility. Genome-wide association studies (GWAS) have not identified maternal sequence variants of genome-wide significance that replicate in independent data sets. We report the first GWAS of offspring from preeclamptic pregnancies and discovery of the first genome-wide significant susceptibility locus (rs4769613; P = 5.4 × 10 -11 ) in 4,380 cases and 310,238 controls. This locus is near the FLT1 gene encoding Fms-like tyrosine kinase 1, providing biological support, as a placental isoform of this protein (sFlt-1) is implicated in the pathology of preeclampsia. The association was strongest in offspring from pregnancies in which preeclampsia developed during late gestation and offspring birth weights exceeded the tenth centile. An additional nearby variant, rs12050029, associated with preeclampsia independently of rs4769613. The newly discovered locus may enhance understanding of the pathophysiology of preeclampsia and its subtypes.

Background: Several regions of the genome have shown to be associated with COPD in genome-wide association studies of common variants., Objective: To determine rare and potentially functional single nucleotide polymorphisms (SNPs) associated with the risk of COPD and severity of airflow limitation., Methods: 3226 current or former smokers of European ancestry with lung function measures indicative of Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2 COPD or worse were genotyped using an exome array. An analysis of risk of COPD was carried out using ever smoking controls (n=4784). Associations with %predicted FEV1 were tested in cases. We followed-up signals of interest (p<10(-5)) in independent samples from a subset of the UK Biobank population and also undertook a more powerful discovery study by meta-analysing the exome array data and UK Biobank data for variants represented on both arrays., Results: Among the associated variants were two in regions previously unreported for COPD; a low frequency non-synonymous SNP in MOCS3 (rs7269297, pdiscovery=3.08×10(-6), preplication=0.019) and a rare SNP in IFIT3, which emerged in the meta-analysis (rs140549288, pmeta=8.56×10(-6)). In the meta-analysis of % predicted FEV1 in cases, the strongest association was shown for a splice variant in a previously unreported region, SERPINA12 (rs140198372, pmeta=5.72×10(-6)). We also confirmed previously reported associations with COPD risk at MMP12, HHIP, GPR126 and CHRNA5. No associations in novel regions reached a stringent exome-wide significance threshold (p<3.7×10(-7))., Conclusions: This study identified several associations with the risk of COPD and severity of airflow limitation, including novel regions MOCS3, IFIT3 and SERPINA12, which warrant further study., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Alpha-1-antitrypsin (AAT), also called serine proteinase inhibitor A1 (Serpin A1), is the most abundant serpin in human plasma. A major physiological role of AAT is to protect the lung from the destructive effects of excess uninhibited neutrophil elastase. During inflammation, circulating levels of AAT may increase twofold-to-threefold as part of the acute-phase response. The liver is the main contributor to this increase. However, local synthesis may provide an important mechanism for controlling neutrophil elastase activity at sites of inflammation, and previous studies have shown a marked increase in production after cytokine stimulation. In the current study we report a distinct transcription initiation site for AAT expression in the lung alveolar epithelial cell line A549, which is located nine bases upstream of the previously mapped full-length monocyte transcription start-site, and show using site-directed mutagenesis that two Sp1 sites and a putative TATA box are functional. EMSA experiments provide evidence for Sp1 and Sp3 binding to these two Sp1 sites. We have also mapped the minimal promoter region and a cell-specific element essential for expression in A549 cells, both of which reside in an 865bp fragment upstream of the transcription start-site. Understanding the mechanisms of AAT gene regulation in a lung-derived cell line has important implications for understanding the control of localised lung tissue damage which occurs as a result of excess proteolytic activity.

Unlabelled: Alpha(1)-antitrypsin deficiency (AATD) due to homozygosity of the protease inhibitor (Pi) Z variant predisposes to childhood liver disease and pulmonary emphysema. About 10% of all neonates with AATD develop liver disease, and about 3% overall progress to severe disease. AATD is a principal genetic indication for liver transplantation in children. The liver pathology is associated with accumulation of abnormally folded protein in hepatocytes, the principal producers of circulating alpha(1)-antitrypsin (AAT). It is currently unknown why only a small proportion of Pi ZZ individuals progress to clinically significant cirrhosis. The AAT gene shows significant variation, and we hypothesized that cryptic genetic variants within the AAT gene may contribute to susceptibility to liver disease. In a case-control study consisting of 42 patients with established moderate-to-severe liver disease and 335 homozygous Pi ZZ patients who mostly presented with chronic obstructive pulmonary disease (n = 322: 242 index cases and 80 unaffected sibs) or were asymptomatic (n = 13) with no evidence of liver disease, we identified a single nucleotide polymorphism (SNP) that conferred a significant risk for liver disease (P = 0.007). The frequency of the SNP was no different in 242 Pi ZZ cases with chronic obstructive pulmonary disease compared with 80 nonindex cases. The SNP therefore appears to confer susceptibility to liver disease, although reporter gene assays failed to show any functional differences between alleles., Conclusion: This is the first description of a genetic modifier of liver disease in homozygous ZZ children and has potential implications for screening and possible therapies that are currently being developed.