KRAS mutations in the parental tumour accelerate in vitro growth of tumoroids established from colorectal adenocarcinoma.

The aim of the present study was to characterize a patient-derived in vitro 3D model (ie tumoroid) established from colorectal adenocarcinoma. This study investigated the growth rate of tumoroids and whether the Kirsten rat sarcoma (KRAS) mutations in the parental tumour accelerate this rate. The tumoroids were established from surgical resections of primary and metastatic colorectal adenocarcinoma from 26 patients. The in vitro growth rate of these tumoroids was monitored by automated imaging and recorded as relative growth rate. The KRAS hotspot mutations were investigated on the parental tumours by Ion Torrent™ next-generation sequencing. The KRAS mutations were detected in 58% of the parental tumours, and a significantly higher growth rate was observed for tumoroids established from the KRAS-mutated tumours compared to wild-type tumours (P < 0.0001). The average relative growth rate (log10) on day 10 was 0.360 ± 0.180 (mean ± SD) for the KRAS-mutated group and 0.098 ± 0.135 (mean ± SD) for the KRAS wild-type group. These results showed that the presence of KRAS mutations in parental tumours is associated with an acceleration of the growth rate of tumoroids. The future perspective for such a model could be the implementation of chemoassays for personalized medicine.

Season of birth shapes neonatal immune function.

BACKGROUND: Birth season has been reported to be a risk factor for several immune-mediated diseases. We hypothesized that this association is mediated by differential changes in neonatal immune phenotype and function with birth season. OBJECTIVE: We sought to investigate the influence of season of birth on cord blood immune cell subsets and inflammatory mediators in neonatal airways. METHODS: Cord blood was phenotyped for 26 different immune cell subsets, and at 1 month of age, 20 cytokines and chemokines were quantified in airway mucosal lining fluid. Multivariate partial least squares discriminant analyses were applied to determine whether certain immune profiles dominate by birth season, and correlations between individual cord blood immune cells and early airway immune mediators were defined. RESULTS: We found a birth season-related fluctuation in neonatal immune cell subsets and in early-life airway mucosal immune function. The seasonal airway immune pattern was associated with the number of activated and regulatory T cells in cord blood whereas it was independent of concomitant presence of pathogenic airway microbes. Specifically, summer newborns presented with the lowest levels of all cell types and mediators; fall newborns displayed high levels of activated T cells and mucosal IL-12p70, TNF-α, IL-13, IL-10, and IL-2; and winter newborns had the highest levels of innate immune cells, IL-5, type 17-related immune mediators, and activated T cells. CONCLUSION: Birth season fluctuations seem to affect neonatal immune development and result in differential potentiation of cord blood immune cells and early airway mucosal immune function.

Distinct inflammatory and cytopathic characteristics of Escherichia coli isolates from inflammatory bowel disease patients.

Escherichia coli (E. coli) may be implicated in the pathogenesis of inflammatory bowel disease (IBD), as implied from a higher prevalence of mucosa-associated E. coli in the gut of IBD-affected individuals. However, it is unclear whether different non-diarrheagenic E. coli spp. segregate from each other in their ability to promote intestinal inflammation. Herein we compared the inflammation-inducing properties of non-diarrheagenic LF82, 691-04A, E. coli Nissle 1917 (ECN) and eleven new intestinal isolates from different locations in five IBD patients and one healthy control. Viable E. coli were cultured with human monocyte-derived dendritic cells (moDCs) and monolayers of intestinal epithelial cells (IECs), followed by analysis of secreted cytokines, intracellular levels of reactive oxygen species and cellular death. The IBD-associated E. coli LF82 induced the same dose-dependent inflammatory cytokine profile as ECN and ten of the new E. coli isolates displayed as high level IL-12p70, IL-1ß, IL-23 and TNF-α from moDCs irrespective of their site of isolation (ileum/colon/faeces), disease origin (diseased/non-diseased) or known virulence factors. Contrarily, 691-04A and one new IBD E. coli isolate induced a different cellular phenotype with enhanced killing of moDCs and IECs, coupled to elevated IL-18. The cytopathic nature of 691-04A and one other IBD E. coli isolate suggests that colonization with specific non-diarrheagenic E. coli could promote intestinal barrier leakage and profound intestinal inflammation, while LF82, ECN and the remaining non-diarrheagenic E. coli isolates hold notorious pro-inflammatory characteristics that can progress inflammation in case of intestinal barrier leakage.

Children with asthma by school age display aberrant immune responses to pathogenic airway bacteria as infants.

BACKGROUND: Asthma is a highly prevalent chronic lung disease that commonly originates in early childhood. Colonization of neonatal airways with the pathogenic bacterial strains Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae is associated with increased risk of later childhood asthma. We hypothesized that children with asthma have an abnormal immune response to pathogenic bacteria in infancy. OBJECTIVE: We aimed to assess the bacterial immune response in asymptomatic infants and the association with later development of asthma by age 7 years. METHODS: The Copenhagen Prospective Studies on Asthma in Childhood birth cohort was followed prospectively, and asthma was diagnosed at age 7 years. The immune response to H influenzae, M catarrhalis, and S pneumoniae was analyzed in 292 infants using PBMCs isolated and stored since the age of 6 months. The immune response was assessed based on the pattern of cytokines produced and T-cell activation. RESULTS: The immune response to pathogenic bacteria was different in infants with asthma by 7 years of age (P = .0007). In particular, prospective asthmatic subjects had aberrant production of IL-5 (P = .008), IL-13 (P = .057), IL-17 (P = .001), and IL-10 (P = .028), whereas there were no differences in T-cell activation or peripheral T-cell composition. CONCLUSIONS: Children with asthma by school age exhibited an aberrant immune response to pathogenic bacteria in infancy. We propose that an abnormal immune response to pathogenic bacteria colonizing the airways in early life might lead to chronic airway inflammation and childhood asthma.

The glycosylation and characterization of the candidate Gc macrophage activating factor.

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.

Distinct immune phenotypes in infants developing asthma during childhood.

Early exposure to environmental triggers may elicit trajectories to chronic inflammatory disease through deregulated immune responses. To address relations between early immune competence and development of childhood asthma, we performed functional immune profiling of 186 parameters in blood of 541 18-month-old infants and examined links between their response phenotype and development of transient or persistent disease at 6 years of age. An abnormal neutrophil-linked antiviral response was associated with increased risk of transient asthma. Children who exhibited persistent asthma at year 6 showed enhanced interleukin-5 (IL-5) and IL-13 production in stimulated T cells at 18 months of age, which was associated with early life bacterial colonization of the airways. These findings highlight the early appearance of distinct immune characteristics in infants developing different asthma endotypes during childhood.

Susceptibility to Lower Respiratory Infections in Childhood is Associated with Perturbation of the Cytokine Response to Pathogenic Airway Bacteria.

BACKGROUND: Neonatal colonization of the airways with respiratory pathogens is associated with increased risk of lower respiratory infections (LRI) in early childhood. Therefore, we hypothesized that children developing LRI have an aberrant immune response to pathogenic bacteria in infancy. The objective was to characterize in vitro the early life systemic immune response to pathogenic bacteria and study the possible association with incidence of LRI during the first 3 years of life. METHODS: The Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) is a clinical birth cohort study of 411 children born of mothers with asthma. LRI incidence was prospectively captured from 6-monthly planned visits and visits at acute respiratory episodes. The in vitro systemic immune response to Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae was characterized by the production of TNF-α, IFN-γ, IL-2, IL-5, IL-10, IL-13 and IL-17 in peripheral blood mononuclear cells isolated at age 6 months from 291 infants. Data were analyzed by Poisson regression against incidence of LRI in infancy. RESULTS: A multivariable model including all cytokine responses from the 3 different bacterial stimulations significantly identified children at risk of LRI (P = 0.006). The immune response pattern associated with LRI was characterized by perturbed production of several cytokines rather than production of one specific cytokine, and was independent of concurrent asthma. TNF-α and IL-5 were key drivers but did not explain the entire variation in LRI susceptibility. CONCLUSIONS: Children at risk of future LRI present a perturbed systemic immune response upon exposure to common airway pathogens in early life.