Autophagy-associated immune dysregulation and hyperplasia in a patient with compound heterozygous mutations in ATG9A.

ABBREVIATIONS: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

Cellular assays to evaluate B-cell function.

Inborn errors of immunity (IEI) that present with recurrent infections are largely due to antibody (Ab) deficiencies. Therefore, assessment of the B-cell and Ab compartment is a major part of immunologic evaluation. Here we provide an overview about cellular assays used to study B-cell function and focus on lymphocyte proliferation assay (LPA), opsonophagocytic assay (OPA), and the Enzyme-linked Immunosorbent Spot Assay (ELISPOT) including clinical applications and limitations of these techniques. LPAs assess ex-vivo cell proliferation in response to various stimuli. Clinically available LPAs utilize peripheral blood mononuclear cells and mostly assess T-cell proliferation with pokeweed mitogen considered the most B-cell specific stimulus. In the research setting, isolating B cells or using more B-cell specific stimuli such as CD40L with IL-4/IL-21 or the TLR9 ligand CpG can more specifically capture the proliferative ability of B cells. OPAs are functional in-vitro killing assays used to evaluate the ability of IgG Ab to induce phagocytosis applied when assessing the potency of vaccine candidates or along with avidity assays to evaluate the quality of secreted IgG. The B-cell ELISPOT assesses Ab production at a cellular level and can characterize the Ab response of particular B-cell subtypes. It can be used in patients on IgG therapy by capturing specific Abs produced by individual B cells, which is not affected by exogenous IgG from plasma donors, and when assessing the vaccine response in patients on immunomodulatory drugs that can affect memory B-cell function. Emerging approaches that are only available in research settings are also briefly introduced.

Molecular networks in atopic mothers impact the risk of infant atopy.

BACKGROUND: The prevalence of atopic diseases has increased with atopic dermatitis (AD) as the earliest manifestation. We assessed if molecular risk factors in atopic mothers influence their infants' susceptibility to an atopic disease. METHODS: Pregnant women and their infants with (n = 174, high-risk) or without (n = 126, low-risk) parental atopy were enrolled in a prospective birth cohort. Global differentially methylated regions (DMRs) were determined in atopic (n = 92) and non-atopic (n = 82) mothers. Principal component analysis was used to predict atopy risk in children dependent on maternal atopy. Genome-wide transcriptomic analyses were performed in paired atopic (n = 20) and non-atopic (n = 15) mothers and cord blood. Integrative genomic analyses were conducted to define methylation-gene expression relationships. RESULTS: Atopic dermatitis was more prevalent in high-risk compared to low-risk children by age 2. Differential methylation analyses identified 165 DMRs distinguishing atopic from non-atopic mothers. Inclusion of DMRs in addition to maternal atopy significantly increased the odds ratio to develop AD in children from 2.56 to 4.26. In atopic compared to non-atopic mothers, 139 differentially expressed genes (DEGs) were identified significantly enriched of genes within the interferon signaling pathway. Expression quantitative trait methylation analyses dependent on maternal atopy identified 29 DEGs controlled by 136 trans-acting methylation marks, some located near transcription factors. Differential expression for the same nine genes, including MX1 and IFI6 within the interferon pathway, was identified in atopic and non-atopic mothers and high-risk and low-risk children. CONCLUSION: These data suggest that in utero epigenetic and transcriptomic mechanisms predominantly involving the interferon pathway may impact and predict the development of infant atopy.

An enzyme-linked immunospot assay to evaluate Pneumovax response when on intravenous immunoglobulin.

BACKGROUND: Testing for common variable immunodeficiency (CVID) requires evaluation of specific antibody responses to vaccines. Current practice of evaluating pneumococcal serotype-specific immunoglobulin (Ig)G levels after Pneumovax (P23) has several limitations and is not accurate for patients already on immunoglobulin replacement. In contrast, the enzyme-linked immunospot (ELISPOT) assay can be interpreted in patients on immunoglobulin replacement as ex vivo measurement of specific antibody-secreting cells (ASCs). OBJECTIVE: To optimize and test an ELISPOT assay to evaluate vaccination response to P23 and compare with P23 serotype-specific IgG for patients on intravenous immunoglobulin (IVIG). METHODS: We prospectively enrolled a total of 15 adults: normal controls (n = 8), patients with CVID on IVIG replacement (n = 2), patients on IVIG replacement for recurrent infections who did not meet diagnostic criteria for CVID, considered IgG deficiency (n = 2), and patients without immunodeficiency on high-dose IVIG for other diagnosis (n = 3). We measured P23 serotype-specific IgG before and 4 weeks after P23 and ELISPOT ASCs before and 1 week after P23 (with B-cell subpopulation analysis by flow cytometry in patients on IVIG). RESULTS: Normal controls had a vaccination response by P23 serotype-specific IgG, whereas patients on IVIG did not. Except for true patients with CVID on IVIG, a P23 ELISPOT ASC response was found in normal controls (highest) and most patients on IVIG for recurrent infections or other diagnosis. CONCLUSION: Our pilot study suggests that an optimized ELISPOT protocol has utility to evaluate the P23-specific antibody response after vaccination. Our ELISPOT assay seemed reliable for patients on IVIG and may help differentiate true patients with CVID from those with a less stringent diagnosis while on IVIG.

Impact of atopic dermatitis treatment on child and parent sleep, daytime functioning, and quality of life.

BACKGROUND: Atopic dermatitis (AD) is a common childhood disorder that is associated with a variety of negative health outcomes in children and parents, including poor sleep and daytime functioning. Despite this, few studies have examined the impact of treatment for AD on sleep, and even fewer have included validated sleep questionnaires, child report of sleep disturbance, or objective measures of sleep. OBJECTIVE: To address limitations in the literature by examining objective and subjective reports of sleep, as well as measures of daytime functioning before and after admission to an intensive treatment program for AD. METHODS: Twenty-nine parent-child dyads who presented to an intensive day treatment program participated in this study. Sleep was objectively measured with 1 week of actigraphy both 1 week before admission and 1 month after discharge. Subjective questionnaires of sleep, daytime functioning, and quality of life were completed by children and parents at admission, discharge, 1 month after discharge, and 3 months after discharge. RESULTS: Study results highlight the benefit of the treatment program on reducing AD severity, as well as improvements in objectively measured sleep duration and efficiency, self-reported measures of sleep, daytime functioning, and quality of life in children and parents up to 3 months after discharge. CONCLUSION: This study highlights the importance of treatment for child AD on both child and parent health outcomes.

Corrigendum: Germline hypomorphic CARD11 mutations in severe atopic disease.

This corrects the article DOI: 10.1038/ng.3898.

Germline hypomorphic CARD11 mutations in severe atopic disease.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Decreased serum vitamin D levels in children with asthma are associated with increased corticosteroid use.

BACKGROUND: There is little knowledge about clinical variables associated with vitamin D (VitD) insufficiency in asthmatic children. OBJECTIVE: We sought to investigate disease variables associated with VitD insufficiency in patients with childhood asthma and interaction of VitD with corticosteroid-mediated anti-inflammatory responses. METHODS: We analyzed 25-hydroxyvitamin D serum levels in 100 asthmatic children to investigate relationships between 25-hydroxyvitamin D levels and patients' characteristics. We determined VitD's effects on dexamethasone (DEX) induction of mitogen-activated protein kinase phosphatase 1 and IL-10 in PBMCs. RESULTS: The median 25-hydroxyvitamin D serum level was 31 ng/mL. Forty-seven percent of subjects had VitD levels in the insufficient range (<30 ng/mL), whereas 17% were VitD deficient (<20 ng/mL). Log(10) IgE (P = .01, rho = -0.25) and the number of positive aeroallergen skin prick test responses (P = .02, rho = -0.23) showed a significant inverse correlation with VitD levels, whereas FEV(1) percent predicted (P = .004, rho = 0.34) and FEV(1)/forced vital capacity ratio (P = .01, rho = 0.30) showed a significant positive correlation with VitD levels. The use of inhaled steroids (P = .0475), use of oral steroids (P = .02), and total steroid dose (P = .001) all showed significant inverse correlations with VitD levels. The amount of mitogen-activated protein kinase phosphatase 1 and IL10 mRNA induced by VitD plus DEX was significantly greater than that induced by DEX alone (P < .01). In an experimental model of steroid resistance in which DEX alone did not inhibit T-cell proliferation, addition of VitD to DEX resulted in significant dose-dependent suppression of cell proliferation. CONCLUSIONS: Corticosteroid use and worsening airflow limitation are associated with lower VitD serum levels in asthmatic patients. VitD enhances glucocorticoid action in PBMCs from asthmatic patients and enhances the immunosuppressive function of DEX in vitro.

Corticosteroid-resistant asthma is associated with classical antimicrobial activation of airway macrophages.

BACKGROUND: The cause of corticosteroid-resistant (CR) asthma is unknown. OBJECTIVE: We sought to perform gene microarray analyses by using bronchoalveolar lavage (BAL) cells from well-characterized subjects with CR asthma and subject with corticosteroid-sensitive (CS) asthma to elucidate the differential expression of genes that contribute to the development of corticosteroid resistance. METHODS: The patients were characterized as having CR or CS asthma based on FEV(1) percent predicted improvement after a 1-week course of oral prednisone. Expression of selected gene targets was verified by means of real-time PCR and ELISA. RESULTS: Microarray analyses demonstrated significantly higher levels (>3-fold increase, P < .05) of transcripts for TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, CXCL1, CXCL2, CXCL3, CXCL8 (IL-8), CCL3, CCL4, and CCL20 in BAL cells of subjects with CR asthma. These findings, confirmed by means of RT-PCR in additional BAL samples, were consistent with classical macrophage activation by bacterial products. In contrast, markers of alternatively activated macrophages, arginase I and CCL24, were decreased. Genes associated with activation of the LPS signaling pathway (early growth response 1, dual-specificity phosphatase 2, molecule possessing ankyrin repeats induced by LPS, and TNF-alpha-induced protein 3) were significantly increased in BAL samples from subjects with CR asthma (P < .05). These patients had significantly higher amounts (1444.0 +/- 457.3 pg/mg total protein) of LPS in BAL fluid than seen in subjects with CS asthma (270.5 +/- 216.0 pg, P < .05), as detected by using the LAL assay and confirmed by means of gas chromatographic/mass spectrometric analysis. Prolonged exposure to LPS induced functional steroid resistance to dexamethasone in normal human monocytes, as demonstrated by persistently increased IL-6 levels in the presence of dexamethasone. CONCLUSIONS: Classical macrophage activation and induction of LPS signaling pathways along with high endotoxin levels detected in BAL fluid from subjects with CR asthma suggest that LPS exposure might contribute to CR asthma.

Neutrophilic airway inflammation and association with bacterial lipopolysaccharide in children with asthma and wheezing.

Asthma is a leading chronic childhood illness in the US. To gain further insight into the pathophysiology of childhood asthma, we studied markers of airway inflammation and possible triggers such as bacterial lipopolysaccharide (LPS) in 18 children with chronic asthma and persistent wheezing who underwent clinically indicated bronchoscopy and bronchoalveolar lavage (BAL). We predominantly found neutrophilic airway inflammation associated with increased levels of IL-8, metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP-1) and MMP-9/TIMP-1 ratio. A significant correlation was found between levels of LPS in BAL and airway neutrophils in BAL from a subgroup of children who had a tendency of increased levels of MMP-9 and TIMP-1, suggesting that increased LPS levels in BAL may contribute to chronic airway inflammation and early remodeling. Our data highlight the importance of defining chronic triggers of early airway inflammation in children and characterizing their inflammation, considering the use of bronchoscopy and BAL. Increased knowledge of airway inflammation in children may help prevent a more severe asthma phenotype and lead to environmental control measures and new treatment strategies to intervene against the establishment of irreversible inflammation.

The role of food allergy in atopic dermatitis.

Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disease affecting more than 10% of all children. Sensitization to foods triggers isolated skin symptoms in about 30% of children. These symptoms include immediate reactions within minutes after ingesting food without exacerbation of AD and early and late exacerbations of AD. It is important to identify clinically relevant sensitizations to foods using skin prick tests, a specific IgE blood test (ImmunoCAP; Phadia, Portage, MI, USA), and double-blind, placebo-controlled food challenges to initiate appropriate dietary interventions and avoid unnecessary dietary restrictions. Children with AD triggered by food allergens demonstrate a distinct immune response upon stimulation of their peripheral blood mononuclear cells with food allergen. A defective skin barrier and increased intestinal permeability appear to facilitate allergen sensitization. Appropriate skin care to maintain skin barrier function and dietary avoidance of highly allergenic foods during infancy may help to prevent allergen sensitization, thereby reducing the severity of AD and food allergies.

Airway remodeling and lack of bronchodilator response in steroid-resistant asthma.

BACKGROUND: Steroid-resistant (SR) asthma is characterized by airway inflammation that fails to resolve despite treatment with corticosteroids, raising concerns that resistance to steroid therapy in asthma could lead to airway remodeling. OBJECTIVE: We sought to determine whether SR asthma is accompanied by decreased airflow reversibility and could lead to airway remodeling. METHODS: Spirometric results were evaluated for 40 asthmatic patients defined as having SR or steroid-sensitive (SS) asthma on the basis of a 1-week course of oral prednisone. Twenty-three asthmatic patients underwent bronchoscopy with collection of bronchoalveolar lavage (BAL) fluid to analyze markers of airway remodeling in BAL fluid and cells. RESULTS: Prednisone significantly improved FEV(1) percent predicted in SS asthma (62.0% +/- 10.9% [mean +/- SD] to 79.4% +/- 11.3%, P < .001) but not in SR asthma (66.9% +/- 10.0% to 65.9% +/- 12.1%). The bronchodilator response was significantly greater in the SS than in the SR group (Delta FEV(1) percent predicted, 33.5% +/- 22.5% vs 15.2% +/- 7.9%; P = .001), regardless of inhaled corticosteroid use. No difference in amounts of matrix metalloproteinase (MMP) 9, PMN elastase, or vascular endothelial growth factor was found in BAL fluid from both groups. Tissue inhibitor of metalloproteinases (TIMP) 1 levels were, however, significantly less in BAL fluid of patients with SR asthma compared with those in patients with SS asthma (921.9 +/- 313.4 vs 2267.0 +/- 456.8 pg/mL, P < .05), resulting in significantly higher MMP-9/TIMP-1 ratios in the BAL fluid of patients with SR asthma (0.24 +/- 0.04 vs 0.11 +/- 0.03, P < .01). Finally, dexamethasone treatment induced TIMP-1 mRNA in BAL fluid cells from patients with SS asthma (P < .01) but not in cells from patients with SR asthma. CONCLUSION: Bronchodilator reversibility is impaired in SR asthma and is associated with a shift in MMP-9/TIMP-1 ratio caused by inability of steroids to enhance TIMP-1 production, potentially promoting proteolytic activity in airways of patients with SR asthma and contributing to chronic airway remodeling. CLINICAL IMPLICATIONS: SR asthma might lead to irreversible airways disease.

Increased glucocorticoid receptor Beta expression converts mouse hybridoma cells to a corticosteroid-insensitive phenotype.

Glucocorticoid (GC) insensitivity is a challenging clinical problem associated with many chronic inflammatory disorders and life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRbeta, which does not bind GC but antagonizes the transactivating activity of the classic GCR, termed GCRalpha. GC-insensitive conditions have been associated with increased GCRbeta expression. Whether or not increased GCRbeta expression can contribute to GC insensitivity, however, remains controversial. To more precisely demonstrate the effect of GCRbeta on steroid responsiveness, we virally transduced GCRbeta cDNA into mouse DO-11.10 hybridoma cells, as mice are known to be deficient in the GCRbeta gene. We demonstrate that viral transduction of GCRbeta cDNA into mouse hybridoma cells to induce stable expression of GCRbeta results in GC insensitivity of these cells. Furthermore, in such cells GCRalpha is complexed with GCRbeta. Such heterodimer formation may account for the reduced effectiveness of GC action in cells overexpressing GCRbeta.

Bee-venom allergy in children: long-term predictive value of standardized challenge tests.

Venom immunotherapy (VIT) is able to protect insect venom-allergic patients against life-threatening sting reactions. Standardized sting challenges can be used as a diagnostic tool to check whether VIT is required. No data are available on the long-term predictive value of sting challenge tests. The purpose of this study was to investigate the long-term predictive value of sequential bee-sting challenges with respect to the ability to predict future sting reactions in bee-venom (BV) allergic children. Between 1988 and 1992, 92 BV-allergic children had been challenged with sequential bee stings at intervals of 2-6 weeks to determine the necessity of VIT. In 1996, all 92 families were followed-up using standardized telephone interviews. Until the follow-up, 61 children (66.3%) had experienced at least one natural bee sting. Based on the results of the initial challenge tests, 13 of the 61 patients had been started on VIT. Two of these 13 (15.4%) developed systemic reactions 1 year after VIT of 5 years, of which one was mild and one was severe. Among the 48 re-stung patients who were not treated with VIT, three children (6.3%) experienced mild systemic reactions, whereas 45 children reported no more than a local reaction. The long-term predictive value of sequential bee-sting challenge tests for systemic reactions in children not treated with VIT remained at a level of 93.8% (95% confidence interval: 82.8-98.7%) even over a period of more than 6 years. Based on this data, we conclude that sequential bee-sting challenges are a powerful tool to determine the necessity for VIT in BV-allergic children.