Preface

The human hepatitis B virus (HBV) is a small-enveloped DNA virus causing acute and chronic hepatitis. Despite the existence of an effective prophylactic vaccine and the strong capacity of approved antiviral drugs to suppress viral replication, chronic HBV infection (CHB) continues to be a major health burden worldwide. Both the inability of the immune system to resolve CHB and the unique replication strategy employed by HBV, which forms a stable viral covalently closed circular DNA (cccDNA) minichromosome in the hepatocyte nucleus, enable infection persistence. Knowledge of the complex network of interactions that HBV engages with its host is still limited but accumulating evidence indicates that epigenetic modifications occurring both on the cccDNA and on the host genome in the course of infection are essential to modulate viral activity and likely contribute to pathogenesis and cancer development. Thus, a deeper understanding of epigenetic regulatory processes may open new venues to control and eventually cure CHB. This review summarizes major findings in HBV epigenetic research, focusing on the epigenetic mechanisms regulating cccDNA activity and the modifications determined in infected host cells and tumor liver tissues

From preschool wheezing to asthma: Immunological determinants

Asthma represents a chronic respiratory disease affecting millions of children worldwide. The transition from preschool wheezing to school-age asthma involves a multifaceted interplay of various factors, including immunological aspects in early childhood. These factors include complex cellular interactions among different immune cell subsets, induction of pro-inflammatory mediators and the molecular impact of environmental factors like allergens or viral infections on the developing immune system. Furthermore, the activation of specific genes and signalling pathways during this early phase plays a pivotal role in the manifestation of symptoms and subsequent development of asthma. Early identification of the propensity or risk for asthma development, for example by allergen sensitisation and viral infections during this critical period, is crucial for understanding the transition from wheeze to asthma. Favourable immune regulation during a critical ‘window of opportunity’ in early childhood can induce persistent changes in immune cell behaviour. In this context, trained immunity, including memory function of innate immune cells, has significant implications for understanding immune responses, potentially shaping long-term immunological outcomes based on early-life environmental exposures. Exploration of these underlying immune mechanisms that drive disease progression will provide valuable insights to understand childhood asthma development. This will be instrumental to develop preventive strategies at different stages of disease development for (i) inhibiting progression from wheeze to asthma or (ii) reducing disease severity and (iii) uncovering novel therapeutic strategies and contributing to more tailored and effective treatments for childhood asthma. In the long term, this shall empower healthcare professionals to develop evidence-based interventions that reduce the burden of asthma for children, families and society overall

Novel statistical approaches for non-normal censored immunological data: analysis of cytokine and gene expression data

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects. Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding. Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping. Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression. Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets

Third Follow-Up of the Study on Occupational Allergy Risks (SOLAR III) in Germany: Design, Methods, and Initial Data Analysis

Introduction: Asthma and allergies are complex diseases affected by genetic and environmental factors, such as occupational and psychosocial factors, as well as interactions between them. Although childhood is a critical phase in the development of asthma and allergies, few cohort studies on occupational outcomes followed up participants from childhood onwards. We present design, methods, and initial data analysis for the third follow-up of SOLAR (Study on Occupational Allergy Risks), a prospective and population-based German asthma and allergy cohort. Methods: The SOLAR cohort was initially recruited in 1995–1996 for Phase II of the German branch of the International Study of Asthma and Allergies in Childhood (ISAAC II) and followed up three times since, in 2002–2003, 2007–2009, and 2017–2018. During the third follow-up (SOLAR III), participants were between 29 and 34 years old. Since SOLAR focuses on occupational exposures, follow-ups were conducted at important points in time of the development of participants' career. To evaluate the potential of selection bias, responders and non-responders were compared based on variables from earlier study phases. In responders, frequency and pattern of missing values were examined and compared within the subsets of paper and online versions of the used questionnaires. Results: In total, 1,359 participants completed the questionnaire of the third follow-up (47.3% of eligible participants). Initially, the cohort started with 6,399 participants from the ISAAC II questionnaire study. A selection process led to a study population that is more female, higher educated, smokes less and has a higher proportion of certain asthma and allergy symptoms (also in their parents) than the initial cohort. Pattern and frequency of missing values were different for paper and online questionnaires. Discussion: The third follow-up of the SOLAR cohort offers the opportunity to analyze the course of asthma and allergies and their associations to environmental, occupational and psychosocial risk factors over more than 20 years from childhood to adulthood. Selection processes within the cohort might lead to bias that needs to be considered in future analyses

A Computed Tomographic Study of the Premolar Teeth of Babyrousa spp.

A photographic and computed tomography (CT) scanning study was carried out on the premolar teeth of 18 adult male Babyrousa babyrussa skulls, 10 skulls of Babyrousa celebensis, including 6 adult males, 1 adult female, 1 subadult male, 1 subadult female, and 1 juvenile male. The occlusal morphology of the permanent maxillary premolar teeth of B. babyrussa was very similar to that of B. celebensis. Almost all the maxillary third premolar teeth (107/207) had 2 roots, whereas maxillary fourth premolar teeth (108/208) had 3 or 4 roots. All of the mesial tooth roots of 107/207 and 108/208 were tapering rod-like structures; each contained a single pulp canal. Almost all distal roots of 107/207 were “C” shaped and contained 2 pulp canals. The 108/208 palatal roots were “C” shaped and contained 2 pulp canals. The mesial and distal roots of the mandibular third premolar teeth (307/407) teeth were uniformly rod-like, as were the mesial roots of the mandibular fourth premolar teeth (308/408) teeth. The distal roots of the 308/408 teeth were “C” shaped. All B. babyrussa 307/407 teeth have a single pulp canal located in each of the mesial and distal roots. The 308/408 mesial tooth root contained 1 pulp canal. In all but 3 of the 36 distal 308/408 roots of B. babyrussa teeth and in 7 of the 14 distal roots of B. celebensis teeth there was a single pulp canal; in the other 7 teeth there were 2 pulp canals. Each of the 3 medial roots contained 1 pulp canal

Integrating farm and air pollution studies in search for immunoregulatory mechanisms operating in protective and high-risk environments

Background Studies conducted in farm environments suggest that diverse microbial exposure promotes children's lung health. The underlying mechanisms are unclear, and the development of asthma-preventive strategies has been delayed. More comprehensive investigation of the environment-induced immunoregulation is required for better understanding of asthma pathogenesis and prevention. Exposure to air pollution, including particulate matter (PM), is a risk factor for asthma, thus providing an excellent counterpoint for the farm-effect research. Lack of comparable data, however, complicates interpretation of the existing information. We aimed to explore the immunoregulatory effects of cattle farm dust (protective, Finland) and urban air PM (high-risk, China) for the first time using identical research methods. Methods We stimulated PBMCs of 4-year-old children (N = 18) with farm dust and size-segregated PM and assessed the expression of immune receptors CD80 and ILT4 on dendritic cells and monocytes as well as cytokine production of PBMCs. Environmental samples were analysed for their composition. Results Farm dust increased the percentage of cells expressing CD80 and the cytokine production of children's immune cells, whereas PM inhibited the expression of important receptors and the production of soluble mediators. Although PM samples induced parallel immune reactions, the size-fraction determined the strength of the effects. Conclusions Our study demonstrates the significance of using the same research framework when disentangling shared and distinctive immune pathways operating in different environments. Observed stimulatory effects of farm dust and inhibitory effects of PM could shape responses towards respiratory pathogens and allergens, and partly explain differences in asthma prevalence between studied environments.Peer reviewe

T Regulatory Cells in Cord Blood—FOXP3 Demethylation as Reliable Quantitative Marker

Regulatory T-cells (Tregs), characterized as CD4+CD25(hi) T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4+CD25(hi), FOXP3 mRNA, protein expression, and suppressive Treg function

Increased breath naphthalene in children with asthma and wheeze of the All Age Asthma Cohort (ALLIANCE).

Background
Exhaled breath contains numerous volatile organic compounds (VOCs) known to be related to lung disease like asthma. Its collection is non-invasive, simple to perform and therefore an attractive method for the use even in young children. We analysed breath in children of the multicenter All Age Asthma Cohort (ALLIANCE) to evaluate if "breathomics" have the potential to phenotype patients with asthma and wheeze, and to identify extrinsic risk factors for underlying disease mechanisms.
Methods
A breath sample was collected from 142 children (asthma: 51, pre-school wheezers: 55, healthy controls: 36) and analysed using gas chromatography-mass spectrometry (GC/MS). Children were diagnosed according to GINA guidelines and comprehensively examined each year over up to seven years. Forty children repeated the breath collection after 24 or 48 months. 
Results
Most breath VOCs differing between groups reflect the exposome of the children. We observed lower levels of lifestyle-related VOCs and higher levels of the environmental pollutants, especially naphthalene, in children with asthma or wheeze. Naphthalene was also higher in symptomatic patients and in wheezers with recent inhaled corticosteroid use. No relationships with lung function or TH2 inflammation were detected.
Conclusion
Increased levels of naphthalene in asthmatics and wheezers and the relationship to disease severity could indicate a role of environmental or indoor air pollution for the development or progress of asthma. Breath VOCs might help to elucidate the role of the exposome for the development of asthma.
The study was registered at ClinicalTrials.gov (NCT02496468).
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Excessive Unbalanced Meat Consumption in the First Year of Life Increases Asthma Risk in the PASTURE and LUKAS2 Birth Cohorts.

A higher diversity of food items introduced in the first year of life has been inversely related to subsequent development of asthma. In the current analysis, we applied latent class analysis (LCA) to systematically assess feeding patterns and to relate them to asthma risk at school age. PASTURE (N=1133) and LUKAS2 (N=228) are prospective birth cohort studies designed to evaluate protective and risk factors for atopic diseases, including dietary patterns. Feeding practices were reported by parents in monthly diaries between the 4th and 12th month of life. For 17 common food items parents indicated frequency of feeding during the last 4 weeks in 4 categories. The resulting 153 ordinal variables were entered in a LCA. The intestinal microbiome was assessed at the age of 12 months by 16S rRNA sequencing. Data on feeding practice with at least one reported time point was available in 1042 of the 1133 recruited children. Best LCA model fit was achieved by the 4-class solution. One class showed an elevated risk of asthma at age 6 as compared to the other classes (adjusted odds ratio (aOR): 8.47, 95% CI 2.52-28.56, p = 0.001) and was characterized by daily meat consumption and rare consumption of milk and yoghurt. A refined LCA restricted to meat, milk, and yoghurt confirmed the asthma risk effect of a particular class in PASTURE and independently in LUKAS2, which we thus termed unbalanced meat consumption (UMC). The effect of UMC was particularly strong for non-atopic asthma and asthma irrespectively of early bronchitis (aOR: 17.0, 95% CI 5.2-56.1, p < 0.001). UMC fostered growth of iron scavenging bacteria such as Acinetobacter (aOR: 1.28, 95% CI 1.00-1.63, p = 0.048), which was also related to asthma (aOR: 1.55, 95% CI 1.18-2.03, p = 0.001). When reconstructing bacterial metabolic pathways from 16S rRNA sequencing data, biosynthesis of siderophore group nonribosomal peptides emerged as top hit (aOR: 1.58, 95% CI 1.13-2.19, p = 0.007). By a data-driven approach we found a pattern of overly meat consumption at the expense of other protein sources to confer risk of asthma. Microbiome analysis of fecal samples pointed towards overgrowth of iron-dependent bacteria and bacterial iron metabolism as a potential explanation

Continuous Rather Than Solely Early Farm Exposure Protects From Hay Fever Development

BACKGROUND: An important window of opportunity for early-life exposures has been proposed for the development of atopic eczema and asthma.OBJECTIVE: However, it is unknown whether hay fever with a peak incidence around late school age to adolescence is similarly determined very early in life.METHODS: In the Protection against Allergy-Study in Rural Environments (PASTURE) birth cohort potentially relevant exposures such as farm milk consumption and exposure to animal sheds were assessed at multiple time points from infancy to age 10.5 years and classified by repeated measure latent class analyses (n [ 769). Fecal samples at ages 2 and 12 months were sequenced by 16S rRNA. Hay fever was defined by parent -reported symptoms and/or physician's diagnosis of hay fever in the last 12 months using questionnaires at 10.5 years.RESULTS: Farm children had half the risk of hay fever at 10.5 years (adjusted odds ratio [aOR] 0.50; 95% CI 0.31-0.79) than that of nonfarm children. Whereas early life events such as gut microbiome richness at 12 months (aOR 0.66; 95% CI 0.46-0.96) and exposure to animal sheds in the first 3 years of life (aOR 0.26; 95% CI 0.06-1.15) were determinants of hay fever, the continuous consumption of farm milk from infancy up to school age was necessary to exert the protective effect (aOR 0.35; 95% CI 0.17-0.72).CONCLUSIONS: While early life events determine the risk of subsequent hay fever, continuous exposure is necessary to achieve protection. These findings argue against the notion that only early life exposures set long-lasting trajectories. (c) 2022 The Authors. Published by Elsevier IncPeer reviewe