Reference genes for the developing mouse lung under consideration of biological, technical and experimental confounders.

For gene expression analysis, the raw data obtained from RT-qPCR are preferably normalized to reference genes, which should be constantly expressed regardless of experimental conditions. Selection of reference genes is particularly challenging for the developing lung because of the complex transcriptional and epigenetic regulation of genes during organ maturation and injury repair. To date, there are only limited experimental data addressing reliable reference genes for this biological circumstance. In this study, we evaluated reference genes for the lung in neonatal C57BL/6 mice under consideration of biological, technical and experimental conditions. For that, we thoroughly selected candidates from commonly used reference genes side-by-side with novel ones by analyzing publicly available microarray datasets. We performed RT-qPCR of the selected candidate genes and analyzed their expression variability using GeNorm and Normfinder. Cell-specific expression of the candidate genes was analyzed using our own single-cell RNA-sequencing data from the developing mouse lung. Depending on the investigated conditions, i.e., developmental stages, sex, RNA quality, experimental condition (hyperoxia) and cell types, distinct candidate genes demonstrated stable expression confirming their eligibility as reliable reference genes. Our results provide valuable information for the selection of proper reference genes in studies investigating the neonatal mouse lung.

Erratum: Epigenome-wide association of father's smoking with offspring DNA methylation: a hypothesis-generating study.

[This corrects the article DOI: 10.1093/eep/dvz023.][This corrects the article DOI: 10.1093/eep/dvz023.].

Epigenome-wide association of father's smoking with offspring DNA methylation: a hypothesis-generating study.

Epidemiological studies suggest that father's smoking might influence their future children's health, but few studies have addressed whether paternal line effects might be related to altered DNA methylation patterns in the offspring. To investigate a potential association between fathers' smoking exposures and offspring DNA methylation using epigenome-wide association studies. We used data from 195 males and females (11-54 years) participating in two population-based cohorts. DNA methylation was quantified in whole blood using Illumina Infinium MethylationEPIC Beadchip. Comb-p was used to analyse differentially methylated regions (DMRs). Robust multivariate linear models, adjusted for personal/maternal smoking and cell-type proportion, were used to analyse offspring differentially associated probes (DMPs) related to paternal smoking. In sensitivity analyses, we adjusted for socio-economic position and clustering by family. Adjustment for inflation was based on estimation of the empirical null distribution in BACON. Enrichment and pathway analyses were performed on genes annotated to cytosine-phosphate-guanine (CpG) sites using the gometh function in missMethyl. We identified six significant DMRs (Sidak-corrected P values: 0.0006-0.0173), associated with paternal smoking, annotated to genes involved in innate and adaptive immunity, fatty acid synthesis, development and function of neuronal systems and cellular processes. DMP analysis identified 33 CpGs [false discovery rate (FDR) < 0.05]. Following adjustment for genomic control (λ = 1.462), no DMPs remained epigenome-wide significant (FDR < 0.05). This hypothesis-generating study found that fathers' smoking was associated with differential methylation in their adolescent and adult offspring. Future studies are needed to explore the intriguing hypothesis that fathers' exposures might persistently modify their future offspring's epigenome.

Influences of environmental bacteria and their metabolites on allergies, asthma, and host microbiota.

The prevalence of allergic diseases and asthma has dramatically increased over the last decades, resulting in a high burden for patients and healthcare systems. Thus, there is an unmet need to develop preventative strategies for these diseases. Epidemiological studies show that reduced exposure to environmental bacteria in early life (eg, birth by cesarean section, being formula-fed, growing up in an urban environment or with less contact to various persons) is associated with an increased risk to develop allergies and asthma later in life. Conversely, a reduced risk for asthma is consistently found in children growing up on traditional farms, thereby being exposed to a wide spectrum of microbes. However, clinical studies with bacteria to prevent allergic diseases are still rare and to some extent contradicting. A detailed mechanistic understanding of how environmental microbes influence the development of the human microbiome and the immune system is important to enable the development of novel preventative approaches that are based on the early modulation of the host microbiota and immunity. In this mini-review, we summarize current knowledge and experimental evidence for the potential of bacteria and their metabolites to be used for the prevention of asthma and allergic diseases.

Identification of a plasma miRNA biomarker signature for allergic asthma: A translational approach.

BACKGROUND: Asthma is a heterogeneous chronic disease with different phenotypes and treatment responses. Thus, there is a high clinical need for molecular disease biomarkers to aid in differentiating these distinct phenotypes. As MicroRNAs (miRNAs), that regulate gene expression at the post-transcriptional level, are altered in experimental and human asthma, circulating miRNAs are attractive candidates for the identification of novel biomarkers. This study aimed to identify plasmatic miRNA-based biomarkers of asthma, through a translational approach. METHODS: We prescreened miRNAs in plasma samples from two different murine models of experimental asthma (ovalbumin and house dust mite); miRNAs deregulated in both models were further tested in a human training cohort of 20 asthma patients and 9 healthy controls. Candidate miRNAs were then validated in a second, independent group of 26 asthma patients and 12 healthy controls. RESULTS: Ten miRNA ratios consisting of 13 miRNAs were differentially regulated in both murine models. Measuring these miRNAs in the training cohort identified a biomarker signature consisting of five miRNA ratios (7 miRNAs). This signature showed a good sensitivity and specificity in the test cohort with an area under the receiver operating characteristic curve (AUC) of 0.92. Correlation of miRNA ratios with clinical characteristics further revealed associations with FVC % predicted, and oral corticosteroid or antileukotriene use. CONCLUSION: Distinct plasma miRNAs are differentially regulated both in murine and in human allergic asthma and were associated with clinical characteristics of patients. Thus, we suggest that miRNA levels in plasma might have future potential to subphenotype patients with asthma.

Prenatal exposure to tobacco smoke sex dependently influences methylation and mRNA levels of the Igf axis in lungs of mouse offspring.

Prenatal smoke exposure is a risk factor for abnormal lung development and increased sex-dependent susceptibility for asthma and chronic obstructive pulmonary disease (COPD). Birth cohort studies show genome-wide DNA methylation changes in children from smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. The insulin-like growth factor (IGF) system plays an important role in lung development. We hypothesized that prenatal exposure to smoke induces lasting changes in promoter methylation patterns of Igf1 and Igf1r, thus influencing transcriptional activity and contributing to abnormal lung development. We measured and compared mRNA levels along with promoter methylation of Igf1 and Igf1r and their protein concentrations in lung tissue of 30-day-old mice that had been prenatally exposed to cigarette smoke (PSE) or filtered air (control). Body weight at 30 days after birth was measured as global indicator of normal development. Female PSE mice showed lower mRNA levels of Igf1 and its receptor (Igf1: P = 0.05; Igf1r: P = 0.03). Furthermore, CpG-site-specific methylation changes were detected in Igf1r in a sex-dependent manner and the body weight of female offspring was reduced after prenatal exposure to smoke, while protein concentrations were unaffected. Prenatal exposure to smoke induces a CpG-site-specific loss of Igf1r promoter methylation, which can be associated with body weight. These findings highlight the sex-dependent and potentially detrimental effects of in utero smoke exposure on DNA methylation and Igf1 and Igf1r mRNA levels. The observations support a role for Igf1 and Igf1r in abnormal development.

[Asthma Update 2015--What Cell Biology in Basic Pulmonary Research Can Offer to the Pneumologist]. / Asthma Update 2015--Was die zellbiologisch-pneumologische Grundlagenforschung dem Lungenarzt anbieten kann.

Bronchial asthma is one of the most common chronic inflammatory diseases world-wide causing an enormous socio-economic burden especially in industrialized countries. Currently, asthma is increasingly considered to be a poly-symptomatic disease comprising a variety of different asthma phenotypes and endotypes. This heterogeneity of asthma explains why the standard treatment with corticosteroids and ß-agonists cannot achieve full symptom control in all cases, especially not during acute exacerbations. Therefore, current asthma research focuses on primary prevention of asthma as well as on novel approaches towards a phenotype- and endotype-specific asthma therapy.

Pregnancy and Infants' Outcome: Nutritional and Metabolic Implications.

Pregnancy is a complex period of human growth, development, and imprinting. Nutrition and metabolism play a crucial role for the health and well-being of both mother and fetus, as well as for the long-term health of the offspring. Nevertheless, several biological and physiological mechanisms related to nutritive requirements together with their transfer and utilization across the placenta are still poorly understood. In February 2009, the Child Health Foundation invited leading experts of this field to a workshop to critically review and discuss current knowledge, with the aim to highlight priorities for future research. This paper summarizes our main conclusions with regards to maternal preconceptional body mass index, gestational weight gain, placental and fetal requirements in relation to adverse pregnancy and long-term outcomes of the fetus (nutritional programming). We conclude that there is an urgent need to develop further human investigations aimed at better understanding of the basis of biochemical mechanisms and pathophysiological events related to maternal-fetal nutrition and offspring health. An improved knowledge would help to optimize nutritional recommendations for pregnancy.

Developmental determinants in non-communicable chronic diseases and ageing.

Prenatal and peri-natal events play a fundamental role in health, development of diseases and ageing (Developmental Origins of Health and Disease (DOHaD)). Research on the determinants of active and healthy ageing is a priority to: (i) inform strategies for reducing societal and individual costs of an ageing population and (ii) develop effective novel prevention strategies. It is important to compare the trajectories of respiratory diseases with those of other chronic diseases.

Peripheral monocytes of obese women display increased chemokine receptor expression and migration capacity.

CONTEXT: The activation of peripheral immune cells and the infiltration of immune cells into adipose tissue in obesity are implicated in the development of type 2 diabetes mellitus. OBJECTIVE: The aim of the study was to compare peripheral immune cells from obese and normal-weight women with regard to composition of immune cell subpopulations, surface expression of the chemokine receptors (CCRs) CCR2, CCR3, CCR5, and CXCR3 (chemokine (C-X-C motif) receptor 3) and cell-intrinsic migration capacity. DESIGN: This was a case-control study. SETTING: The study was conducted at a university clinical study center. PATIENTS: Obese females and normal-weight females were included for fluorescence-activated cell sorting analysis and migration assays. MAIN OUTCOME MEASURES: Peripheral blood mononuclear cells were prepared from fasting blood samples and used for fluorescence-activated cell sorting analysis and migration assays. RESULTS: An increase in the percentages of CD14(+)CD16(+) monocytes was observed in obese subjects compared with controls. The CCR profile of monocytes differed significantly in the obese state; in particular, CCR2 levels were increased. In addition, a higher chemotactic activity of monocytes from obese subjects was observed in a migration assay, which was associated with both insulin resistance and CCR2 expression. CONCLUSION: Our results suggest that the enhanced intrinsic migratory capacity of peripheral monocytes in obese women may be due to the increased CCR expression, further supporting a link between peripheral immune cell dysfunction and obesity.

Inverse association between trans isomeric and long-chain polyunsaturated fatty acids in pregnant women and their newborns: data from three European countries.

BACKGROUND: trans unsaturated fatty acids are thought to interfere with essential fatty acid metabolism. To extend our knowledge of this phenomenon, we investigated the relationship between trans isomeric and long-chain polyunsaturated fatty acids (LCPUFA) in mothers during pregnancy and in their infants at birth. METHODS: Fatty acid composition of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was determined in Spanish (n = 120), German (n = 78) and Hungarian (n = 43) women at the 20th and 30th week of gestation, at delivery and in their newborns. RESULTS: At the 20th week of gestation, the sum of trans fatty acids in PE was significantly (p < 0.01) lower in Hungarian [0.73 (0.51), % wt/wt, median (IQR)] than in Spanish [1.42 (1.36)] and German [1.30 (1.21)] women. Docosahexaenoic acid (DHA) values in PE were significantly (p < 0.01) higher in Hungarian [5.65 (2.09)] than in Spanish [4.37 (2.60)] or German [4.39 (3.3.2)] women. The sum of trans fatty acids significantly inversely correlated to DHA in PCs in Spanish (r = -0.37, p < 0.001), German (n = -0.77, p < 0.001) and Hungarian (r = -0.35, p < 0.05) women, and in PEs in Spanish (r = -0.67, p < 0.001) and German (r = -0.71, p < 0.001), but not in Hungarian (r = -0.02) women. Significant inverse correlations were seen between trans fatty acids and DHA in PEs at the 30th week of gestation (n = 241, r = -0.52, p < 0.001), at delivery (n = 241, r = -0.40, p < 0.001) and in cord lipids (n = 218, r = -0.28, p < 0.001). CONCLUSION: Because humans cannot synthesize trans isomeric fatty acids, the data obtained in the present study support the concept that high maternal trans isomeric fatty acid intake may interfere with the availability of LCPUFA both for the mother and the fetus.

Maternal vitamin D intake during pregnancy increases gene expression of ILT3 and ILT4 in cord blood.

BACKGROUND: Recent studies indicate that prenatal vitamin D intake may protect against the development of atopic diseases in young children. Vitamin D has been shown to induce tolerogenic antigen-presenting cells such as dendritic cells. Whether the allergy-protective potential of prenatal vitamin D is mediated through such mechanisms is, however, unknown. OBJECTIVE: To evaluate the association between prenatal vitamin D supplementation and tolerogenic antigen-presenting cells in cord blood (CB) as determined by mRNA measurement of immunoglobulin-like transcripts (ILT)3 and ILT4. METHODS: A prospective multi-centre birth cohort was established in rural areas of five European countries. Information on maternal exposures including vitamin D intake was collected by questionnaires during pregnancy. The gene expression of ILT3 and ILT4 was analysed by real-time PCR in the CB of 927 children. Maternal vitamin D supplementation was assessed in Finland and France (n=349). RESULTS: Maternal vitamin D supplementation during pregnancy was associated with an increase in the gene expression of ILT3 (P=0.012) and ILT4 (P<0.001). This association remained significant for ILT4 (P=0.020) and showed a positive trend for the gene expression of ILT3 (P=0.059) after multivariate analysis controlling for various confounders. CONCLUSIONS: Vitamin D supplementation during pregnancy may increase the mRNA levels of ILT3 and ILT4 in CB. This finding may point towards an early induction of tolerogenic immune responses by maternal vitamin D intake.

Specific IgE to allergens in cord blood is associated with maternal immunity to Toxoplasma gondii and rubella virus.

BACKGROUND: Various studies have found reduced prevalences of atopic sensitization and atopic diseases in children previously exposed to infections or living conditions with a high microbial burden, such as the farming environment. OBJECTIVE: We sought to determine the relationships of cord blood immunoglobulin E (IgE) with maternal health conditions before and during pregnancy. METHODS: Pregnant women living in rural areas in five European countries were recruited in the third trimester of pregnancy. Information on maternal health during pregnancy was collected from maternity records and by questionnaires (n = 497). Specific IgE for inhalant and food allergens was assessed in cord blood and peripheral blood samples of the mothers. RESULTS: Inverse associations of cord blood IgE to seasonal allergens with positive maternal records for Toxoplasma gondii (adjusted odds ratio = 0.37 [0.17-0.81]) and rubella virus (adjusted odds ratio = 0.35 [0.13-0.96]) were found. The previously described effect of prenatal farm exposure on IgE to seasonal allergens was partly confounded by a positive maternal record for T. gondii. The number of maternal siblings, maternal contact to cats during pregnancy or during her first year of life, predicted a positive maternal record for T. gondii. CONCLUSIONS: Maternal immunity to T. gondii and rubella may impact on atopic sensitization in the fetus. A positive T. gondii record explained the previously identified effect of prenatal farm exposure on IgE to seasonal allergens only to a minor extent.

Inhaled glutathione decreases PGE2 and increases lymphocytes in cystic fibrosis lungs.

Reduced glutathione (GSH), a major antioxidant and modulator of cell proliferation, is decreased in the bronchoalveolar lavage fluid (BALF) of cystic fibrosis (CF) patients. We previously have shown that GSH inhalation in CF patients significantly increased GSH levels in BALF and improved lung function (M. Griese et al., 2004, Am. J. Respir. Crit. Care Med.169, 822-828). GSH depletion in vitro enhances susceptibility to oxidative stress, increases inflammatory cytokine release, and impairs T cell responses. We therefore hypothesized that an increase in GSH in BALF reduces oxidative stress, decreases inflammation, and modulates T cell responses in lungs of CF patients. BALF from 17 CF patients (median FEV1 67% (43-105%) of predicted) was assessed before and after GSH inhalation for total protein, markers of oxidative stress (8-isoprostane, myeloperoxidase, and ascorbic and uric acid), pattern of protein oxidation, prostaglandin E2 (PGE2), and proinflammatory cytokines. BALF cells were differentiated using cytospin slides, and lymphocytes were further analyzed by flow cytometry. Inhalation of GSH decreased BALF levels of PGE2 and increased CD4+ and CD8+ lymphocytes in BALF significantly but had no effect on markers of oxidative stress. BALF lymphocytes correlated positively with lung function, whereas levels of PGE2 showed an inverse correlation. The patients with the greatest improvement in lung function after GSH treatment also had the largest decline in PGE2 levels. We conclude that GSH inhalation in CF patients increases lymphocytes and suppresses PGE2 in the bronchoalveolar space. Thus, GSH primarily affected the pulmonary immune response rather than the oxidative status in CF patients. The effect of GSH inhalation on PGE2 levels and lymphocytes in CF warrants further investigation.

Chemokine receptor 5 expression in gastric mucosa of Helicobacter pylori-infected and noninfected children.

Experimental data from human adults or animal models indicate that the Helicobacter pylori-specific immune response is dominated by inflammatory T cells of the Th1 type. To investigate whether a Th1 immune response is established in early H. pylori infection, gastric biopsy samples from 70 children were subjected to immunohistochemical analysis. To this end, T cells, B cells, monocytes, neutrophils, and chemokine receptor 5 (CCR5)-expressing (CCR5(+)) cells, which are associated with Th1 immune responses, were quantified. Children were classified according to H. pylori status and clinical, laboratory, and macroscopic (during endoscopy) findings, without knowledge of histological findings. Group 1 included 31 H. pylori-infected children, group 2 contained 24 children with other conditions possibly affecting the stomach, and group 3 contained 15 children without verifiable pathological findings in the stomach. Lymphoid follicles were present in 90% of biopsy samples from group 1 and 48% of those from group 2 but absent in group 3 biopsy samples. Intraepithelial T cells and CCR5(+) cells were regularly detected in all groups without significant differences. B cells, monocytes, and neutrophils were not found. In contrast, the numbers of lamina propria T cells (P < 0.003) and CCR5(+) cells (P < 0.001) were increased significantly in H. pylori-infected children. B cells (in 13 of 66 children) were detected in children with active (n = 11) or previously cleared (n = 2) H. pylori infections but were absent in healthy children. The numbers of monocytes (in 10 of 67 children) did not differ among the groups. Calculations indicated that the majority of gastric T cells express CCR5; this finding is in contrast to the low percentage of CCR5(+) T cells in the peripheral circulation. Thus, an increase in the numbers of CCR5(+) cells in H. pylori-infected stomach mucosa suggests that this molecule may play an important role in gastric immune responses.

Influence of age on 13C-urea breath test results in children.

BACKGROUND: The 13C-urea breath test for diagnosis of Helicobacter pylori infection has not been validated in infants and young children. The influence of age on the test results was studied by conventional validation against invasive methods and by mathematical estimation in a large pediatric population. METHODS: The breath test was performed in 1499 children aged 2 months to 18 years. After a fasting period of 4 hours or more, 75 mg 13C-urea was ingested with cold apple juice, breath samples were taken at baseline and at 15 and 30 minutes. The distribution of the natural logarithms of the delta-over baseline (DOB) values were calculated, and the optimal cutoff values between positive and negative test results and gray zones with a risk of misclassification more than 10% were determined for both time points. In a subgroup of 149 children results of the breath test were compared with concordant results of histology and rapid urease test; 53 of them were less than 6 years of age. RESULTS: Logarithmic results of 1499 breath tests revealed two normally distributed subgroups with minimal overlap. The calculated optimal cutoff values were 4.7/1000 at 15 minutes and 5.0/1000 at 30 minutes. At 30 minutes, only 2.6% of all results were in the calculated gray zone (2.6-6.5/1000). Age was negatively correlated to DOB values of both negative (r = -0.223) and positive results (r = -0.291; P < 0.001). Breath test-negative and -positive children 6 or less years of age had significantly higher mean DOB values (P < 0.02) and a larger proportion of results within the gray zone than older children. Compared with biopsy-based results, the least discrepancies occurred at a cutoff of 5.0/1000: 0 of 61 infected (sensitivity 100%) and 6 of 88 noninfected children. Because five of the false-positive results were obtained in children less than 6 years of age, specificity and positive predictive values were lower in this age group than in older patients (88.1% vs. 97.8% and 68.8% vs. 98.0%, respectively). CONCLUSIONS: Under the applied conditions, the 13C-urea breath test shows an excellent separation between positive and negative results. Because of some overlap and a strong age effect, definition of a gray zone appears more meaningful than a threshold value. Because infants and young children have a high risk for false-positive breath test results, the values for cutoff and gray zones may have to be adapted. Further validation studies against invasive methods are warranted in this age group.

Atypical CD3+ CD4(low) cell population in a boy with fatal EBV-infection.

A previously healthy 10-year-old Greek boy born to nonconsanguineous healthy parents developed progressive liver disease after acute infectious mononucleosis. EBV-induced autoimmune hepatitis was suspected and treatment was started with high-dose prednisolone, acyclovir and intravenous immunoglobulins. Despite therapy, his liver function continuously deteriorated and the child died 9 months later in profound immune deficiency from candida septicemia. Flow cytometric analysis of his lymphocytes revealed a major subpopulation of atypical cells (20.3%) which were CD3+, fitted into the lymphocyte gate but showed a very low level of CD4 expression, comparable to that of monocytes. After short-time cell culture, the cells became adherent and developed granules and dendrites. We conclude that these cells may represent strongly activated CD4+ T lymphocytes with downregulated CD4 expression or a subtype of dendritic cells.