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Background: Adverse reactions to wheat-containing skin care products have been linked to food allergy development.Objectives: To determine the role of skin barrier dysfunction and inflammation in sensitization to gluten-derived hydrolysates via the skin in Brown Norway rats with and without oral tolerance to wheat.Methods: Skin barrier defect was induced by mechanical disruption, and skin inflammation was induced by topical application of SLS or MC903. Unmodified, enzyme hydrolyzed, or acid hydrolyzed gluten products were applied to the skin 3 times per week for 5 weeks. Subsequently, rats were orally gavaged with unmodified gluten.Results: Wheat-naïve rats were readily sensitized to gluten hydrolysates via the skin. Skin barrier defect and skin inflammation had little effect on the skin sensitization and hydrolysate-specific IgE levels. Oral administration of unmodified gluten promoted the production of unmodified gluten-specific IgE in rats sensitized via the skin. Sensitization through intact skin, disrupted skin barrier, or inflamed skin was unable to break tolerance to unmodified gluten in rats on a wheat-containing diet.Conclusions: Mechanical skin barrier disruption and skin inflammation play a limited role in experimental skin sensitization to gluten-derived hydrolysates.

Allergen-specific immunotherapy (IT) is emerging as a viable avenue for the treatment of food allergies. Clinical trials currently investigate raw or slightly processed foods as therapeutic agents, as trials using food-grade agents can be performed without the strict regulations to which conventional drugs are subjected. However, this limits the ability of standardization and may affect clinical trial outcomes and reproducibility. Herein, we provide an overview of methods used in the production of immunotherapeutic agents for the treatment of food allergies, including processed foods, allergen extracts, recombinant allergens, and synthetic peptides, as well as the physical and chemical processes for the reduction of protein allergenicity. Commercial interests currently favor producing standardized drug-grade allergen extracts for therapeutic use, and clinical trials are ongoing. In the near future, recombinant production could replace purification strategies since it allows the manufacturing of pure, native allergens or sequence-modified allergens with reduced allergenicity. A recurring issue within this field is the inadequate reporting of production procedures, quality control, product physicochemical characteristics, allergenicity, and immunological properties. This information is of vital importance in assessing therapeutic standardization and clinical safety profile, which are central parameters for the development of future therapeutic agents.

The use of antibiotics as well as changes in the gut microbiota have been linked to development of food allergy in childhood. It remains unknown whether administration of a single clinically relevant antibiotic directly promotes food allergy development when administrated during the sensitisation phase in an experimental animal model. We investigated whether the antibiotic amoxicillin affected gut microbiota composition, development of cow's milk allergy (CMA) and frequencies of allergic effector cells and regulatory T cells in the intestine. Brown Norway rats were given daily oral gavages of amoxicillin for six weeks and whey protein concentrate (WPC) with or without cholera toxin three times per week for the last five weeks. Microbiota composition in faeces and small intestine was analysed by 16S rRNA sequencing. The development of CMA was assessed by WPC-specific IgE in serum, ear swelling response to WPC and body hypothermia following oral gavage of WPC. Allergic effector cells were analysed by histology, and frequencies of regulatory and activated T cells were analysed by flow cytometry. Amoxicillin administration reduced faecal microbiota diversity, reduced the relative abundance of Firmicutes and increased the abundance of Bacteroidetes and Proteobacteria. Despite these effects, amoxicillin did not affect the development of CMA, nor the frequencies of allergic effector cells or regulatory T cells. Thus, amoxicillin does not carry a direct risk for food allergy development when administrated in an experimental model of allergic sensitisation to WPC via the gut. This finding suggests that confounding factors may better explain the epidemiological link between antibiotic use and food allergy.

Background: Autoimmunity and allergy have been associated with decreased number and function of regulatory T-cells (Tregs) and low interleukin-2 (IL-2) levels. We aimed to investigate if the release of IL-2 from peripheral blood mononuclear cells (PBMCs) stimulated with pathogenic airway bacteria was associated with development of allergy-outcomes in early childhood. Methods: PBMCs were isolated at age 6 months in 331 infants from the Copenhagen Prospective Studies on Asthma in Childhood 2000 (COPSAC2000) mother-child cohort, and subsequently stimulated with H. influenzae, M. catarrhalis and S. pneumoniae in in vitro cultures. Levels of cytokines (IL-2, IL-10, IFN-γ, TNF-α, IL-5, IL-13 and IL-17A) were determined in the supernatant by electrochemiluminescence immunoassays. The immune profiles were analyzed for association with development of total-IgE, allergic sensitization and rhinitis during the first 7 years of life using regression models and principal component analysis (PCA). Findings: An attenuated IL-2 response to stimulation with H. influenzae (p = 0∙011) and M. catarrhalis (p = 0∙027) was associated with elevated total-IgE at age 7, which was confirmed in a multivariate PCA model including all cytokine measurements (PC2, p = 0∙032). An immune profile with both reduced IL-2 and elevated IL-5 was associated with increased risk of allergic rhinitis (PC3, p = 0∙038). We found no associations with development of allergic sensitization. Interpretation: A reduced IL-2 response from PBMCs exposed to common pathogenic airway bacteria at age 6 months was associated with elevated total-IgE and allergic rhinitis during the first 7 years of life. These findings suggest that suppressed Treg activity in early life may herald onset of allergy in early childhood, which could be a target for low-dose IL-2 trials in the future. Fund: COPSAC is funded by private and public research funds all listed on www.copsac.com. Keywords: IL-2, Airway bacteria, Birth cohort, IgE, Sensitization

BackgroundIt remains largely unknown how physicochemical properties of hydrolysed infant formulas influence their allergy preventive capacity, and results from clinical and animal studies comparing the preventive capacity of hydrolysed infant formula with conventional infant formula are inconclusive. Thus, the use of hydrolysed infant formula for allergy prevention in atopy-prone infants is highly debated. Furthermore, knowledge on how gut microbiota influences allergy prevention remains scarce.ObjectiveTo gain knowledge on (1) how physicochemical properties of hydrolysed whey products influence the allergy preventive capacity, (2) whether host microbiota disturbance influences allergy prevention, and (3) to what extent hydrolysed whey products influence gut microbiota composition.MethodsThe preventive capacity of four different ad libitum administered whey products was investigated in Brown Norway rats with either a conventional or an amoxicillin-disturbed gut microbiota. The preventive capacity of products was evaluated as the capacity to reduce whey-specific sensitisation and allergic reactions to intact whey after intraperitoneal post-immunisations with intact whey. Additionally, the direct effect of the whey products on the growth of gut bacteria derived from healthy human infant donors was evaluated by in vitro incubation.ResultsTwo partially hydrolysed whey products with different physicochemical characteristics were found to be superior in preventing whey-specific sensitisation compared to intact and extensively hydrolysed whey products. Daily oral amoxicillin administration, initiated one week prior to intervention with whey products, disturbed the gut microbiota but did not impair the prevention of whey-specific sensitisation. The in vitro incubation of infant faecal samples with whey products indicated that partially hydrolysed whey products might confer a selective advantage to enterococci.ConclusionsOur results support the use of partially hydrolysed whey products for prevention of cow’s milk allergy in atopy-predisposed infants regardless of their microbiota status. However, possible direct effects of partially hydrolysed whey products on gut microbiota composition warrants further investigation.

Scope Within the last decade, quinoa seeds have gained much popularity as a new food and have recently been proposed as an appropriate food for early introduction in infants. Quinoa contains high levels of saponins, which are known for their adjuvant activity and effect on the intestinal barrier function. The aim of this study is to investigate the impact of quinoa on intestinal permeability and inflammation in comparison with the positive controls; cholera toxin (CT), and capsaicin. Methods and results The effect of quinoa on intestinal barrier function and inflammation is investigated in vitro using a Caco-2 cell line and in vivo using a Brown Norway rat model. Effects in vivo are analyzed by protein uptake, histology, gene expression, antibody levels, and flow cytometry. Quinoa and the positive controls all increased the intestinal permeability, but distinct patterns of absorbed protein are observed in the epithelium, Peyer's patches, lamina propria, and serum. The quinoa-mediated effect on intestinal barrier function is found to be distinct from the effect of the two positive controls. Conclusion The findings demonstrate the ability of quinoa to increase intestinal permeability and to promote compartment-specific protein uptake via mechanisms that may differ from CT and capsaicin.

Background: Several childhood asthma risk loci that relate to immune function have been identified by genome-wide association studies (GWAS), but the underlying mechanisms remain unknown. Objective: Here, we examined whether perturbed innate immune responses mediate the association between known genetic risk variants and development of childhood asthma. Methods: Peripheral blood mononuclear cells from 336 six-month-old infants from the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC2000) cohort were stimulated in vitro with six different innate ligands (LPS, CpG, poly(I:C), R848, HDMAPP and aluminium hydroxide together with low levels of LPS) followed by quantification of 18 released cytokines and chemokines 40 h after the stimulations. The innate immune response profiles were decomposed by principal component (PC) analysis, and PC1-5 were used in mediation analyses of the effect of 25 known genetic risk variants on childhood asthma until age 7. Results: The effects of two variants from the 17q21 locus (rs7216389, rs2305480) on asthma and exacerbation risk were significantly mediated by immune parameters induced in response to ligands mimicking intracellular colonization; bacterial DNA (CpG) and double-stranded viral RNA (poly(I:C)). The Th17 and innate lymphoid cell type 3-amplifying cytokine IL-23 was the most prominent cytokine involved. Conclusion: The 17q21 effect on childhood asthma and exacerbations was partly mediated by deregulation of IL-23 in response to intracellular microbial ligands, which may suggest ineffective clearance of intracellular pathogens in the lungs.

Cow’s milk allergy is a health problem of growing concern. However, it is possible to modify the inherent allergenic properties of milk proteins by proteolysis typically for use in hypoallergenic infant formulas (HA IFs). HA IFs are widely used in the management cow’s milk allergy (CMA) and may be applicable in the prevention and treatment of CMA. Here we provide an overview of milk protein allergens and the factors involved in the in vivo digestibility of these, including intestinal tract proteases, pH, surfactant, matrix, processing, and glycation. The industrial-scale production of HA IF involves the use of common commercially available proteases followed by drying and additions of other dietary nutrients. To some extent, the degree of hydrolysis determines the overall allergenicity of the HA IF, which is influenced by parameters such as the specific proteases used, hydrolysis duration, enzyme to protein ratio, matrix, and processing. Several molecular assays can be used to determine the degree of hydrolysis and evaluate product allergenicity in vitro, however animal models may represent an important tool for evaluation of sensitising and primary preventing capacities of HA IFs.

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.

Food allergy is an adverse reaction to otherwise harmless proteins in food. The disease is a major health problem of growing concern, affecting approximately 5%-8% of young children and 2%-4% of adults. No accepted strategy exists for prevention and treatment of food allergy, and strict avoidance of the offending food is presently the only viable management option. Living with food avoidance may have a huge impact on the quality of life of food allergic patients, with daily fear of serious or even fatal reactions. The urgent need for safe and efficient food allergy treatment options has led to massive research efforts to develop and improve strategies for food allergy immunotherapeutic approaches. A first step in developing new and improved strategies of immunotherapy often involves the use of animal models. In present review, we provide an overview of animal studies of allergen-specific immunotherapy highlighting opportunities and challenges for each approach. The presented models, almost exclusively performed in mice, assess therapeutic efficacy and immunological outcomes following oral, intraperitoneal, subcutaneous, epicutaneous, and sublingual administration of native allergens, or preparations of hydrolyzed allergen, T-cell-directed peptides, or allergen with immunomodulatory adjuvants. Recently, approaches using immune cell therapy have demonstrated efficacy. Current models mainly assess anaphylaxis as the primary clinical outcome. With the increased appreciation that food allergy is a heterogeneous disease presenting different phenotypes, there is a continued need to develop new disease-relevant therapeutic models of food allergy.

Background Deviation in blood collection procedures is a central source of preanalytical variation affecting overall analytical and diagnostic precision. The order of draw of venous sampling is suspected to affect analytical results, in particular for coagulation analysis. Here we compare the procedures in venous blood sampling among clinical biochemistry departments to assess the uniformity of order of blood draw and adherence to international guidelines in the Danish health care system. Methods We collected venous order of draw procedures from 49 clinical biochemistry departments at 22 public hospitals in Denmark. Procedures were compared to the international guidelines fromthe Clinical Laboratory Standards Institute (CLSI) and World Health Organization (WHO), and assessed in relation to department ISO 15189:2012 accreditation. Results We observed seven different order of draw procedures related to citrate , serum, heparin, and EDTA tubes, and the use of discard tubes in relation to coagulation assays. 31 departments (63.3%) were found to adhere to CLSI and WHO guidelines. A majority of departments instructs the use of discard tubes before collection for coagulation assays in citrate tubes (44 departments; 89.8%). The citrate tube was the first sample tube to be drawn for most departments (35 departments; 75.5%); and the preferred order of non-citrate tubes was serum-heparin-EDTA (36 departments; 73.5%). Adherence to the CLSI and WHO guidelines was not associated with department ISO 15189:2012 accreditation ( p = .57). Conclusions Venous order of draw procedures is diverse at Danish clinical biochemistry departments and show moderate adherence to international guidelines.

ScopePersonal care products containing hydrolysed gluten have been linked to spontaneous sensitisation through the skin, however the impact of the hydrolysate characteristics on the sensitising capacity is generally unknown. Methods and ResultsThe physicochemical properties of five different wheat-derived gluten products (1 unmodified, 1 enzyme hydrolysed, and 3 acid hydrolysed) were investigated, and the skin sensitising capacity was determined in allergy-prone Brown Norway rats. Acid hydrolysed gluten products exhibited the strongest intrinsic sensitising capacity via the skin. All hydrolysed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolysed products was associated with product-specific sensitisation in wheat tolerant rats. Sensitisation to acid hydrolysed gluten products was associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitisation induced by unmodified gluten or enzyme hydrolysed gluten. ConclusionAcid hydrolysis enhances the skin sensitising capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolysed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

The microbiota plays a central role in human health and disease by shaping immune development, immune responses and metabolism, and by protecting from invading pathogens. Technical advances that allow comprehensive characterization of microbial communities by genetic sequencing have sparked the hunt for disease‐modulating bacteria. Emerging studies in humans have linked the increased abundance of Prevotella species at mucosal sites to localized and systemic disease, including periodontitis, bacterial vaginosis, rheumatoid arthritis, metabolic disorders and low‐grade systemic inflammation. Intriguingly, Prevotella abundance is reduced within the lung microbiota of patients with asthma and chronic obstructive pulmonary disease. Increased Prevotella abundance is associated with augmented T helper type 17 (Th17) ‐mediated mucosal inflammation, which is in line with the marked capacity of Prevotella in driving Th17 immune responses in vitro. Studies indicate that Prevotella predominantly activate Toll‐like receptor 2, leading to production of Th17‐polarizing cytokines by antigen‐presenting cells, including interleukin‐23 (IL‐23) and IL‐1. Furthermore, Prevotella stimulate epithelial cells to produce IL‐8, IL‐6 and CCL20, which can promote mucosal Th17 immune responses and neutrophil recruitment. Prevotella‐mediated mucosal inflammation leads to systemic dissemination of inflammatory mediators, bacteria and bacterial products, which in turn may affect systemic disease outcomes. Studies in mice support a causal role of Prevotella as colonization experiments promote clinical and inflammatory features of human disease. When compared with strict commensal bacteria, Prevotella exhibit increased inflammatory properties, as demonstrated by augmented release of inflammatory mediators from immune cells and various stromal cells. These findings indicate that some Prevotella strains may be clinically important pathobionts that can participate in human disease by promoting chronic inflammation.

The intestinal gut microbiota is essential for maintaining host health. Concerns have been raised about the possible connection between antibiotic use, causing microbiota disturbances, and the increase in allergic and autoimmune diseases observed during the last decades. To elucidate the putative connection between antibiotic use and immune regulation, we have assessed the effects of the antibiotic amoxicillin on immune regulation, protein uptake, and bacterial community structure in a Brown Norway rat model. Daily intra-gastric administration of amoxicillin resulted in an immediate and dramatic shift in fecal microbiota, characterized by a reduction of within sample (α) diversity, reduced variation between animals (β diversity), increased relative abundance of Bacteroidetes and Gammaproteobacteria, with concurrent reduction of Firmicutes, compared to a water control group. In the small intestine, amoxicillin also affected microbiota composition significantly, but in a different way than observed in feces. The small intestine of control animals was vastly dominated by Lactobacillus, but this genus was much less abundant in the amoxicillin group. Instead, multiple different genera expanded after amoxicillin administration, with high variation between individual animals, thus the small intestinal α and β diversity were higher in the amoxicillin group compared to controls. After 1 week of daily amoxicillin administration, total fecal IgA level, relative abundance of small intestinal regulatory T cells and goblet cell numbers were higher in the amoxicillin group compared to controls. Several bacterial genera, including Escherichia/Shigella, Klebsiella (Gammaproteobacteria), and Bifidobacterium, for which the relative abundance was higher in the small intestine in the amoxicillin group than in controls, were positively correlated with the fraction of small intestinal regulatory T cells. Despite of epidemiologic studies showing an association between early life antibiotic consumption and later prevalence of inflammatory bowel diseases and food allergies, our findings surprisingly indicated that amoxicillin-induced perturbation of the gut microbiota promotes acute immune regulation. We speculate that the observed increase in relative abundance of small intestinal regulatory T cells is partly mediated by immunomodulatory lipopolysaccharides derived from outgrowth of Gammaproteobacteria.

Cyclooxygenases are known as important regulators of metabolism and immune processes via conversion of C20 fatty acids into various regulatory lipid mediators, and cyclooxygenase activity has been implicated in browning of white adipose tissues. We generated transgenic (TG) C57BL/6 mice expressing the Ptgs2 gene encoding cyclooxygenase-2 (COX-2) in mature adipocytes. TG mice fed a high-fat diet displayed marginally lower weight gain with less hepatic steatosis and a slight improvement in insulin sensitivity, but no difference in glucose tolerance. Compared to littermate wildtype mice, TG mice selectively reduced inguinal white adipose tissue (iWAT) mass and fat cell size, whereas the epididymal (eWAT) fat depot remained unchanged. The changes in iWAT were accompanied by increased levels of specific COX-derived lipid mediators and increased mRNA levels of interleukin-33, interleukin-4 and arginase-1, but not increased expression of uncoupling protein 1 or increased energy expenditure. Epididymal WAT (eWAT) in TG mice exhibited few changes except from increased infiltration with eosinophils. Our findings suggest a role for COX-2-derived lipid mediators from adipocytes in mediating type 2 immunity cues in subcutaneous WAT associated with decreased hepatic steatosis, but with no accompanying induction of browning and increased energy expenditure.

Background: Infant formulas (IFs) based on hydrolysed cow’s milk proteins are central in the management of cow’s milk allergy (CMA) in infants and small children. New IF compositions with improved prevention and treatment properties are needed, along with appropriate preclinical animal models, to evaluate these properties before introduction into humans. Objectives: We aimed to develop preclinical models for the assessment of the primary preventive and desensitising capacity of cow’s milk IF in allergy-prone, high-IgE responder Brown Norway rats. Method: Preventive capacity was assessed in cow’s milk-naïve rats given a 2- or 4-week regimen of whey-based extensively hydrolysed IF (eHF), partially hydrolysed IF (pHF), or intact β-lactoglobulin (BLG) ad libitum in drinking bottles, followed by intraperitoneal (i.p.) immunisation with BLG. Desensitising capacity was assessed in orally BLG-sensitised rats after a 3- or 6-week regimen of eHF, pHF, or intact BLG administration in drinking bottles, followed by i.p. challenge with BLG. Primary preventive and desensitising capacity were analysed by serum BLG-specific IgG1 and IgE. Results: The preventive regimens did not induce detectable BLG-specific IgG1 or IgE in cow’s milk-naïve rats. A preventive regimen consisting of pHF or BLG, but not eHF, induced complete tolerance to BLG, as demonstrated by the absence of BLG-specific IgE following i.p. immunisation. Desensitising regimens had a limited effect on BLG-specific IgG1 or IgE when comparing sensitised rats before and after treatment. Challenge with BLG (i.p.) increased BLG-specific IgE in all treatment regimens except for in the BLG group, suggesting a limited desensitising capacity of IF based on hydrolysates and a need for the presence of intact allergen for desensitisation. Conclusions: The presented models highlight that different mechanisms are at play in the induction of de novo tolerance to cow’s milk proteins and the desensitisation of CMA. Different IF products may be needed for the primary prevention and treatment of CMA.

Breast milk long-chain PUFA (LCPUFA) have been associated with changes in early life immune responses and may modulate T-cell function in infancy. We studied the effect of maternal fatty acid desaturase (FADS) genotype and breast milk LCPUFA levels on infants’ blood T-cell profiles and ex vivo-produced cytokines after anti-CD3/CD28 stimulation of peripheral blood mononuclear cells in 6-month-old infants from the Copenhagen Prospective Study of Asthma in Childhood birth cohort. LCPUFA concentrations of breast milk were assessed at 4 weeks of age, and FADS SNP were determined in both mothers and infants (n 109). In general, breast milk arachidonic acid (AA) levels were inversely correlated with the production of IL-10 (r −0·25; P=0·004), IL-17 (r −0·24; P=0·005), IL-5 (r −0·21; P=0·014) and IL-13 (r −0·17; P=0·047), whereas EPA was positively correlated with the counts of blood regulatory T-cells and cytotoxic T-cells and decreased T-helper cell counts. The minor FADS alleles were associated with lower breast milk AA and EPA, and infants of mothers carrying the minor allele of FADS SNP rs174556 had higher production of IL-10 (r −0·23; P=0·018), IL-17 (r −0·25; P=0·009) and IL-5 (r −0·21; P=0·038) from ex vivo-activated immune cells. We observed no association between T-cell distribution and maternal or infant FADS gene variants. We conclude that increased maternal LCPUFA synthesis and breast milk AA are associated with decreased levels of IL-5, IL-13 (type-2 related), IL-17 (type-17 related) and IL-10 (regulatory immune responses), but not with interferon-γ and TNF-α, which could be due to an effect of the maternal FADS variants on the offspring immune response transferred via breast milk LCPUFA.

Deregulated immune responses to environmental triggers may propagate disease by eliciting chronic inflammatory disease trajectories. To address relations between early immune competence and development of common childhood diseases in humans, we performed a large-scale immunological profiling in 18-month-old infants and linked ex vivo functional immune response phenotypes to development of disease until school-age. Several distinct and non-overlapping response phenotypes were identified to selectively associate with development of asthma, allergic rhinoconjunctivitis, or atopic dermatitis. These findings pinpoint the diversity of immune responses between infants in early life and its importance in relation to the development of common childhood diseases.

Recent studies of healthy human airways have revealed colonization by a distinct commensal bacterial microbiota containing Gram-negative Prevotella spp. However, the immunological properties of these bacteria in the respiratory system remain unknown. Here we compare the innate respiratory immune response to three Gram-negative commensal Prevotella strains (Prevotella melaninogenica, Prevotella nanceiensis and Prevotella salivae) and three Gram-negative pathogenic Proteobacteria known to colonize lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma (Haemophilus influenzae B, non-typeable Haemophilus influenzae and Moraxella catarrhalis). The commensal Prevotella spp. and pathogenic Proteobacteria were found to exhibit intrinsic differences in innate inflammatory capacities on murine lung cells in vitro. In vivo in mice, non-typeable H. influenzae induced severe Toll-like receptor 2 (TLR2)-independent COPD-like inflammation characterized by predominant airway neutrophilia, expression of a neutrophilic cytokine/chemokine profile in lung tissue, and lung immunopathology. In comparison, P. nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram-negative bacteria P. nanceiensis was completely TLR2-dependent. These findings demonstrate weak inflammatory properties of Gram-negative airway commensal Prevotella spp. that may make colonization by these bacteria tolerable by the respiratory immune system.

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.

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.

Background p-Phenylenediamine (PPD) and related substances are ingredients of more than two-thirds of oxidative (permanent) hair dyes currently used. Although PPD is a potent skin sensitizer in predictive assays, the extent to which permanent hair dyes sensitize humans has been questioned due to the in-use conditions, e.g. the presence of couplers in the hair dye gel and rapid oxidation using a developer. Objectives To study the skin sensitizing potential of permanent hair dyes in mice. Methods Two different permanent hair dye products containing PPD were studied in CBA mice using a modified version of the local lymph node assay. The colour gel and developer (oxidant) were tested separately and in combination. Response was measured by ear swelling and cytokine production in ear tissue and serum by enzyme-linked immunosorbent assay. The immune cellular response in the draining lymph nodes was analysed by flow cytometry. Results Application of the colour gel both alone and mixed with the developer induced skin production of interleukin (IL)-1beta, tumour necrosis factor-alpha and IL-6 as well as systemic IL-6 release. Both treatments induced B- and T-cell infiltration as well as T-cell proliferation within the draining lymph nodes. Treatment with the mixture induced at least 20% more skin inflammation, cytokine production and CD4+ T-cell activation compared with the colour gel alone. Conclusions Consumer available PPD-containing permanent hair dyes can be potent and rapid immune activators. Mixing the colour gel and developer (oxidant) increased the induction of skin inflammation compared with application of the colour gel alone.

The CD3γ di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3γ di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8+ T cells is impaired in mice with a mutated CD3γ di-leucine-based motif. The CD3γ mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8+ T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3γ-mediated TCR down-regulation in virus-specific CD8+ T cell responses.

Background: The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody-induced depletion of specific subtypes of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations. Objectives: To develop a method for studying the collective cellular dynamics of immune cells in the draining lymph nodes during CHS in intact animals. Patients/Methods: Mice were sensitized and/or challenged with 2,4-dinitrofluorobenzene or oxazolone. Using multi-parameter flow cytometry we determined the proliferation, activation state, and absolute number of helper T cells, cytotoxic T cells, B cells, and natural killer cells in the draining lymph nodes. Results: The presented method can be applied to evaluate the effect of different contact allergens on various cell populations of the immune system. Conclusions: Our study support recent findings that several cell types seem to be involved in CHS.

Summary Mycobacterium bovis bacillus Calmette–Guerin (BCG) vaccine has been associated with beneficial effects on overall childhood mortality in low-income countries; this cannot be explained merely by the prevention of tuberculosis (TB) deaths. The beneficial effects of BCG vaccine could be the result of either strengthening of pro-inflammatory mechanisms, helping neonates to fight infections, or the induction of an immune-regulatory network restricting overt inflammation and intense pathology. We aimed to study the effect of live BCG on the ability of dendritic cells (DCs) to polarize T-cell responses. Monocyte-derived DCs were matured in the presence or absence of BCG. The DC phenotype was assessed by CD83 expression, interleukin-12 (IL-12) and IL-10 production, as well as for the ability to polarize T-cell responses. Following stimulation with CD40 ligand, DCs matured in the presence of BCG showed enhanced IL-10 and diminished IL-12 production. These DCs primed naive T cells to develop into IL-10-producing T cells, with no T helper 1 or T helper 2 bias. These results suggest that BCG vaccination might result in the development of IL-10-producing DCs as well as IL-10-producing T cells that could contribute to restricting overt inflammation in infants exposed to pathogens and thus lead to lower infant mortality.

Background: First-born children are at higher risk for development of a range of immune-mediated diseases. The underlying mechanism of ‘birth-order-effects’ on disease risk is largely unknown, but in utero programming of the child's immune system may play a role. Objective: We studied the association between birth-order and the functional response of stimulated cord blood T cells. Method: Purified cord blood T cells were polyclonally activated with anti-CD3/CD28-coated beads in a subgroup of 28 children enrolled in the COPSAC2010 birth cohort. Expression levels of seven activation markers on helper and cytotoxic T cells as well as the percentage of CD4+CD25+ T cells were assessed by flow cytometry. Production of IFN-γ, TNF-α, IL-17, IL-4, IL-5, IL-13 and IL-10 was measured in supernatants. Results: IL-10 secretion (P = 0.007) and CD25 expression on CD4+ helper T cells (P = 0.0003) in activated cord blood T cells were selectively reduced in first-born children, while the percentage of CD4+CD25+ cord blood T cells was independent of birth-order. Conclusion: First-born infants display a reduced anti-inflammatory profile in T cells at birth. This possible in utero ‘birth-order’ T cell programing may contribute to later development of immune-mediated diseases by increasing overall immune reactivity in first-born children as compared to younger siblings.

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.

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.

TCR and cytokine receptor signaling play key roles in the complex homeostatic mechanisms that maintain a relative stable number of T cells throughout life. Despite the homeostatic mechanisms, a slow decline in naive T cells is typically observed with age. The CD3γ di-leucine-based motif controls TCR down-regulation and plays a central role in fine-tuning TCR expression and signaling in T cells. In this study, we show that the age-associated decline of naive T cells is strongly accelerated in CD3γLLAA knock-in mice homozygous for a double leucine to alanine mutation in the CD3γ di-leucine-based motif, whereas the number of memory T cells is unaffected by the mutation. This results in premature T cell population senescence with a severe dominance of memory T cells and very few naive T cells in middle-aged to old CD3γ mutant mice. The reduced number of naive T cells in CD3γ mutant mice was caused by the combination of reduced thymic output, decreased T cell apoptosis, and increased transition of naive T cells to memory T cells. Experiments with bone marrow chimeric mice confirmed that the CD3γLLAA mutation exerted a T cell intrinsic effect on T cell homeostasis that resulted in an increased transition of CD3γLLAA naive T cells to memory T cells and a survival advantage of CD3γLLAA T cells compared with wild-type T cells. The experimental observations were further supported by mathematical modeling of T cell homeostasis. Our study thus identifies an important role of CD3γ-mediated TCR down-regulation in T cell homeostasis.

Background IL-17–producing T H (T H 17) cells are key mediators of chronic inflammation in mice. Recent studies have implicated T H 17-mediated inflammation in the pathogenesis of human autoimmune diseases; however, the involvement of T H 17 cells in allergic disorders remains largely elusive. Objective To investigate T H 17-mediated inflammation in human beings with allergic contact dermatitis; in particular, the innate response of keratinocytes to contact allergen, the induction of allergen-specific T H 17 cells, and the presence of T H 17-related effector cells in inflamed skin. Methods Human keratinocytes were stimulated with nickel in vitro followed by measurements of IL-23 and IL-12 production by quantitative PCR and ELISA. Allergen-specific memory T cells from the blood of individuals with nickel allergy and healthy controls were identified and characterized by using a short-term ex vivo assay. Nickel patch test lesions and normal skin were analyzed for the expression of T H 17-related cells and molecules by using immunohistochemistry. Results Keratinocytes were found to produce IL-23, but no detectable IL-12, in a response to nickel stimulation. Memory T cells isolated from peripheral blood of individuals with nickel allergy, but not healthy controls, contained T H 17 and T H 1 cells proliferating in response to nickel-pulsed DCs. Inflamed skin of nickel-challenged allergic individuals contained infiltrating neutrophils and cells expressing IL-17, IL-22, CCR6, and IL-22R. Conclusion Our results demonstrate the involvement of T H 17-mediated immunopathology in human allergic contact dermatitis, including both innate and adaptive immune responses to contact allergens.

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCα and PKCθ were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser126 and the di-leucine-based receptor-sorting motif of the CD3γ chain. Finally, we found that PKCθ was mainly implicated in down-regulation of directly engaged TCR, whereas PKCα was involved in down-regulation of nonengaged TCR.