Interpretation of In Vitro Concentration-Response Data for Risk Assessment and Regulatory Decision-making: Report from the 2022 IWGT Quantitative Analysis Expert Working Group Meeting.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations. This article is protected by copyright. All rights reserved

The evolution of the terrestrial-terminating Irish Sea glacier during the last glaciation

Here we reconstruct the last advance to maximum limits and retreat of the Irish Sea Glacier (ISG), the only land‐terminating ice lobe of the western British Irish Ice Sheet. A series of reverse bedrock slopes rendered proglacial lakes endemic, forming time‐transgressive moraine‐ and bedrock‐dammed basins that evolved with ice marginal retreat. Combining, for the first time on glacial sediments, optically stimulated luminescence (OSL) bleaching profiles for cobbles with single grain and small aliquot OSL measurements on sands, has produced a coherent chronology from these heterogeneously bleached samples. This chronology constrains what is globally an early build‐up of ice during late Marine Isotope Stage 3 and Greenland Stadial (GS) 5, with ice margins reaching south Lancashire by 30 ± 1.2 ka, followed by a 120‐km advance at 28.3 ± 1.4 ka reaching its 26.5 ± 1.1 ka maximum extent during GS‐3. Early retreat during GS‐3 reflects piracy of ice sources shared with the Irish‐Sea Ice Stream (ISIS), starving the ISG. With ISG retreat, an opportunistic readvance of Welsh ice during GS‐2 rode over the ISG moraines occupying the space vacated, with ice margins oscillating within a substantial glacial over‐deepening. Our geomorphological chronosequence shows a glacial system forced by climate but mediated by piracy of ice sources shared with the ISIS, changing flow regimes and fronting environments

Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome

BACKGROUND:In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV. METHODOLOGY:Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected. CONCLUSION:XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not

IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?

BACKGROUND: The role of Th2 cells (producing interleukin (IL-)4, IL-5 and IL-13) in allergic asthma is well-defined. A distinct proinflammatory T cell lineage has recently been identified, called Th(17 )cells, producing IL-17A, a cytokine that induces CXCL8 (IL-8) and recruits neutrophils. Neutrophilic infiltration in the airways is prominent in severe asthma exacerbations and may contribute to airway gland hypersecretion, bronchial hyper-reactivity and airway wall remodelling in asthma. AIM: to study the production of IL-17 in asthmatic airways at the mRNA level, and to correlate this with IL-8 mRNA, neutrophilic inflammation and asthma severity. METHODS: We obtained airway cells by sputum induction from healthy individuals (n = 15) and from asthmatic patients (n = 39). Neutrophils were counted on cytospins and IL-17A and IL-8 mRNA expression was quantified by real-time RT-PCR (n = 11 controls and 33 asthmatics). RESULTS: Sputum IL-17A and IL-8 mRNA levels are significantly elevated in asthma patients compared to healthy controls. IL-17 mRNA levels are significantly correlated with CD3γ mRNA levels in asthmatic patients and mRNA levels of IL-17A and IL-8 correlated with each other and with sputum neutrophil counts. High sputum IL-8 and IL-17A mRNA levels were also found in moderate-to-severe (persistent) asthmatics on inhaled steroid treatment. CONCLUSION: The data suggest that Th(17 )cell infiltration in asthmatic airways links T cell activity with neutrophilic inflammation in asthma

Life course trajectories of alcohol consumption in the United Kingdom using longitudinal data from nine cohort studies.

Background Alcohol consumption patterns change across life and this is not fully captured in cross-sectional series data. Analysis of longitudinal data, with repeat alcohol measures, is necessary to reveal changes within the same individuals as they age. Such data are scarce and few studies are able to capture multiple decades of the life course. Therefore, we examined alcohol consumption trajectories, reporting both average weekly volume and frequency, using data from cohorts with repeated measures that cover different and overlapping periods of life. Methods Data were from nine UK-based prospective cohorts with at least three repeated alcohol consumption measures on individuals (combined sample size of 59,397 with 174,666 alcohol observations), with data spanning from adolescence to very old age (90 years plus). Information on volume and frequency of drinking were harmonised across the cohorts. Predicted volume of alcohol by age was estimated using random effect multilevel models fitted to each cohort. Quadratic and cubic polynomial terms were used to describe non-linear age trajectories. Changes in drinking frequency by age were calculated from observed data within each cohort and then smoothed using locally weighted scatterplot smoothing. Models were fitted for men and women separately. Results We found that, for men, mean consumption rose sharply during adolescence, peaked at around 25 years at 20 units per week, and then declined and plateaued during mid-life, before declining from around 60 years. A similar trajectory was seen for women, but with lower overall consumption (peak of around 7 to 8 units per week). Frequent drinking (daily or most days of the week) became more common during mid to older age, most notably among men, reaching above 50% of men. Conclusions This is the first attempt to synthesise longitudinal data on alcohol consumption from several overlapping cohorts to represent the entire life course and illustrates the importance of recognising that this behaviour is dynamic. The aetiological findings from epidemiological studies using just one exposure measure of alcohol, as is typically done, should be treated with caution. Having a better understanding of how drinking changes with age may help design intervention strategies

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock

Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics

© 2018 The Author(s). Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDM S ) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDM S -wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics

Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice

Family structure, parent-child conversation time and substance use among Chinese adolescents

<p>Abstract</p> <p>Background</p> <p>The family plays a vital role in shaping adolescent behaviours. The present study investigated the associations between family structure and substance use among Hong Kong Chinese adolescents.</p> <p>Methods</p> <p>A total of 32,961 Form 1 to 5 (grade 7-12 in the US) Hong Kong students participated in the Youth Smoking Survey in 2003-4. An anonymous questionnaire was used to obtain information about family structure, daily duration of parent-child conversation, smoking, alcohol drinking and drug use. Logistic regression was used to calculate the adjusted odds ratios (OR) for each substance use by family structure.</p> <p>Results</p> <p>Adjusting for sex, age, type of housing, parental smoking and school, adolescents from non-intact families were significantly more likely to be current smokers (OR = 1.62), weekly drinkers (OR = 1.72) and ever drug users (OR = 1.72), with significant linear increases in ORs from maternal, paternal to no-parent families compared with intact families. Furthermore, current smoking (OR = 1.41) and weekly drinking (OR = 1.46) were significantly more common among adolescents from paternal than maternal families. After adjusting for parent-child conversation time, the ORs for non-intact families remained significant compared with intact families, but the paternal-maternal differences were no longer significant.</p> <p>Conclusions</p> <p>Non-intact families were associated with substance use among Hong Kong Chinese adolescents. The apparently stronger associations with substance use in paternal than maternal families were probably mediated by the poorer communication with the father.</p

Coexpression Network Analysis in Abdominal and Gluteal Adipose Tissue Reveals Regulatory Genetic Loci for Metabolic Syndrome and Related Phenotypes

Metabolic Syndrome (MetS) is highly prevalent and has considerable public health impact, but its underlying genetic factors remain elusive. To identify gene networks involved in MetS, we conducted whole-genome expression and genotype profiling on abdominal (ABD) and gluteal (GLU) adipose tissue, and whole blood (WB), from 29 MetS cases and 44 controls. Co-expression network analysis for each tissue independently identified nine, six, and zero MetS–associated modules of coexpressed genes in ABD, GLU, and WB, respectively. Of 8,992 probesets expressed in ABD or GLU, 685 (7.6%) were expressed in ABD and 51 (0.6%) in GLU only. Differential eigengene network analysis of 8,256 shared probesets detected 22 shared modules with high preservation across adipose depots (DABD-GLU = 0.89), seven of which were associated with MetS (FDR P<0.01). The strongest associated module, significantly enriched for immune response–related processes, contained 94/620 (15%) genes with inter-depot differences. In an independent cohort of 145/141 twins with ABD and WB longitudinal expression data, median variability in ABD due to familiality was greater for MetS–associated versus un-associated modules (ABD: 0.48 versus 0.18, P = 0.08; GLU: 0.54 versus 0.20, P = 7.8×10−4). Cis-eQTL analysis of probesets associated with MetS (FDR P<0.01) and/or inter-depot differences (FDR P<0.01) provided evidence for 32 eQTLs. Corresponding eSNPs were tested for association with MetS–related phenotypes in two GWAS of >100,000 individuals; rs10282458, affecting expression of RARRES2 (encoding chemerin), was associated with body mass index (BMI) (P = 6.0×10−4); and rs2395185, affecting inter-depot differences of HLA-DRB1 expression, was associated with high-density lipoprotein (P = 8.7×10−4) and BMI–adjusted waist-to-hip ratio (P = 2.4×10−4). Since many genes and their interactions influence complex traits such as MetS, integrated analysis of genotypes and coexpression networks across multiple tissues relevant to clinical traits is an efficient strategy to identify novel associations