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1.  GWAS and admixture mapping identify different asthma-associated loci in Latinos: The GALA II Study 
Background
Asthma is a complex disease with both genetic and environmental causes. Genome-wide association studies of asthma have mostly involved European populations and replication of positive associations has been inconsistent.
Objective
To identify asthma-associated genes in a large Latino population with genome-wide association analysis and admixture mapping.
Methods
Latino children with asthma (n = 1,893) and healthy controls (n = 1,881) were recruited from five sites in the United States: Puerto Rico, New York, Chicago, Houston, and the San Francisco Bay Area. Subjects were genotyped on an Affymetrix World Array IV chip. We performed genome-wide association and admixture mapping to identify asthma-associated loci.
Results
We identified a significant association between ancestry and asthma at 6p21 (lowest p-value: rs2523924, p < 5 × 10−6). This association replicates in a meta-analysis of the EVE Asthma Consortium (p = 0.01). Fine mapping of the region in this study and the EVE Asthma Consortium suggests an association between PSORS1C1 and asthma. We confirmed the strong allelic association between the 17q21 asthma in Latinos (IKZF3, lowest p-value: rs90792, OR: 0.67, 95% CI 0.61 – 0.75, p = 6 × 10−13) and replicated associations in several genes that had previously been associated with asthma in genome-wide association studies.
Conclusions
Admixture mapping and genome-wide association are complementary techniques that provide evidence for multiple asthma-associated loci in Latinos. Admixture mapping identifies a novel locus on 6p21 that replicates in a meta-analysis of several Latino populations, while genome-wide association confirms the previously identified locus on 17q21.
doi:10.1016/j.jaci.2013.08.055
PMCID: PMC4085159  PMID: 24406073
Asthma; Latinos; Admixture Mapping; Genome-wide Association Study; Local Ancestry; 17q21; 6p21
2.  Winter birth in inner city asthmatic children is associated with increased food allergen sensitization risk 
doi:10.1016/j.jaci.2014.05.002
PMCID: PMC4119828  PMID: 24954275
asthma; vitamin D; food allergy; peanut; milk; egg; wheat; soy; inner city
3.  Prevalence of allergic sensitization in the U.S.: Results from the National Health and Nutrition Examination Survey (NHANES) 2005–2006 
Background
Allergic sensitization is an important risk factor for the development of atopic disease. The National Health and Nutrition Examination Survey (NHANES) 2005–2006 provides the most comprehensive information on IgE-mediated sensitization in the general US population.
Objective
We investigated clustering, sociodemographic and regional patterns of allergic sensitization and examined risk factors associated with IgE-mediated sensitization.
Methods
Data for this cross-sectional analysis were obtained from NHANES 2005–2006. Participants aged ≥1 year (N=9440) were tested for sIgEs to inhalant and food allergens; participants ≥6 years were tested for 19 sIgEs, and children aged 1–5 years for 9 sIgEs. Serum samples were analyzed using the ImmunoCAP System. Information on demographics and participant characteristics was collected by questionnaire.
Results
Of the study population aged 6 and older, 44.6% had detectable sIgEs, while 36.2% of children aged 1–5 years were sensitized to ≥1 allergen. Allergen-specific IgEs clustered into 7 groups that might have largely reflected biological cross-reactivity. Although sensitization to individual allergens and allergen types showed regional variation, the overall prevalence of sensitization did not differ across census regions, except in early childhood. In multivariate modeling, young age, male gender, non-Hispanic black race/ethnicity, geographic location (census region), and reported pet avoidance measures were most consistently associated with IgE-mediated sensitization.
Conclusions
The overall prevalence of allergic sensitization does not vary across US census regions, except in early life, although allergen-specific sensitization differs by sociodemographic and regional factors. Biological cross-reactivity may be an important, but not a sole, contributor to the clustering of allergen-specific IgEs.
Clinical implications
IgE-mediated sensitization shows clustering patterns and differs by sociodemographic and regional factors, but the overall prevalence of sensitization may not vary across US census regions.
doi:10.1016/j.jaci.2013.12.1071
PMCID: PMC4119838  PMID: 24522093
allergen; allergy; allergic sensitization; serum IgE
4.  Effects of rhinovirus species on viral replication and cytokine production 
Background
Epidemiological studies provide evidence of differential virulence of rhinovirus (RV) species. We recently reported that RV-A and RV-C induced more severe illnesses than RV-B, suggesting that the biology of RV-B might be different from RV-A or RV-C.
Objective
To test the hypothesis that RV-B has lower replication and induces lesser cytokine responses than RV-A or RV-C.
Methods
We cloned full-length cDNA of RV-A16, A36, B52, B72, C2, C15 and C41 from clinical samples, and grew clinical isolates of RV-A7 and B6 in cultured cells. Sinus epithelial cells were differentiated at air-liquid interface. We tested for differences in viral replication in epithelial cells after infection with purified viruses (108 RNA copies) and measured virus load by quantitative RT-PCR. We measured lactate dehydrogenase (LDH) concentration as a marker of cellular cytotoxicity, and cytokine/chemokine secretion by multiplex ELISA.
Results
At 24 hours post infection, virus load of RV-B (RV-B52, B72, or B6) in adherent cells was lower than that of RV-A or RV-C. The growth kinetics of infection indicated that RV-B types replicate more slowly. Furthermore, RV-B released less LDH than RV-A or RV-C, and induced lower levels of cytokines and chemokines such as CXCL10, even after correction for viral replication. RV-B replicates to lower levels also in primary bronchial epithelial cells.
Conclusions
Our results indicate that RV-B types have lower and slower replication, and lower cellular cytotoxicity and cytokine/chemokine production compared to RV-A or RV-C. These characteristics may contribute to reduced severity of illnesses that has been observed with RV-B infections.
Clinical implications
RV-B types replicate at a lower rate and produce less cytokine/chemokine compared to RV-A or RV-C, which may contribute to the clinical observation that RV-B causes less severe illnesses.
Capsule summary
RV-B types replicate more slowly and to lower levels, and less cytokine/chemokine production compared to RV-A or RV-C. These characteristics may contribute to reduced severity of illnesses that has been observed with RV-B infections.
doi:10.1016/j.jaci.2014.01.029
PMCID: PMC4119842  PMID: 24636084
Asthma; cellular cytotoxicity; chemokine; cytokine; rhinovirus
5.  Loss of Regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation 
BACKGROUND
Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for AHR, suggesting that underlying abnormalities in structural cells such as airway smooth muscle (ASM) contribute to the asthmatic diathesis. Dysregulation of procontractile, G protein-coupled receptor (GPCR) signaling in ASM could mediate enhanced contractility.
OBJECTIVE
We explored the role of a regulator of procontractile GPCR signaling, RGS5, in unprovoked and allergen-induced AHR.
METHODS
We evaluated GPCR-evoked Ca2+ signaling, precision cut lung slice (PCLS) contraction, and lung inflammation in naïve and Aspergillus fumigatus-challenged WT and Rgs5−/− mice. We analyzed lung resistance and dynamic compliance in live, anesthetized mice by invasive plethysmography.
RESULTS
Loss of RGS5 promoted constitutive AHR due to enhanced GPCR-induced Ca2+ mobilization in ASM. PCLS from naïve Rgs5−/− mice contracted maximally at baseline, independent of allergen challenge. RGS5 deficiency had little effect on parameters of allergic inflammation including cell counts in bronchoalveolar lavage fluid (BALF), mucin production, ASM mass, and subepithelial collagen deposition. Unexpectedly, induced IL-13 and IL-33 were much lower in challenged lungs from Rgs5−/− mice relative to WT.
CONCLUSION
Loss of RGS5 confers spontaneous AHR in mice in the absence of allergic inflammation. Because it is selectively expressed in ASM within the lung and does not promote inflammation, RGS5 may be a therapeutic target for asthma.
doi:10.1016/j.jaci.2014.01.019
PMCID: PMC4119844  PMID: 24666695
Asthma; airway hyperresponsivness; G proteins; Regulator of G protein signaling proteins; Aspergillus fumigatus
6.  Neonatal rhinovirus induces mucous metaplasia and airways hyperresponsiveness via IL-25 and ILC2s 
Background
Early-life human rhinovirus (RV) infection has been linked to asthma development in high risk infants and children. Nevertheless, the role of RV infection in the initiation of asthma remains unclear.
Objective
We hypothesized that, in contrast to infection of mature BALB/c mice, neonatal infection with RV promotes an IL-25-driven type 2 response which causes persistent mucous metaplasia and airway hyperresponsiveness.
Methods
Six day-old and eight week-old BALB/c mice were inoculated with sham HeLa cell lysate or RV. Airway responses from 1 to 28 days after infection were assessed by qPCR, ELISA, histology, immunofluorescence microscopy, flow cytometry and methacholine responsiveness. Selected mice were treated with a neutralizing antibody to IL-25.
Results
Compared to mature mice, RV infection in neonatal mice increased lung IL-13 and IL-25 production whereas IFN-γ, IL-12p40 and TNF-α expression were suppressed. In addition, the population of IL-13-secreting type 2 innate lymphoid cells (ILC2s) was expanded with RV infection in neonatal but not in mature mice. ILC2 cells were the major cell type secreting IL-13 in neonates. Finally, anti-IL-25 neutralizing antibody attenuated ILC2 expansion, mucous hypersecretion and airways responsiveness.
Conclusions
These findings suggest that early-life viral infection could contribute to asthma development by provoking age-dependent, IL-25-driven type 2 immune responses.
doi:10.1016/j.jaci.2014.04.020
PMCID: PMC4119851  PMID: 24910174
Asthma; IL-25; mouse; neonatal; rhinovirus; type 2 innate lymphoid cells
7.  Airway Surface Mycosis in Chronic Th2-Associated Airway Disease 
Background
Environmental fungi have been linked to T helper type 2 (Th2) cell-related airway inflammation and the Th2-associated chronic airway diseases asthma, chronic rhinosinusitis with nasal polyps (CRSwNP) and allergic fungal rhinosinusitis (AFRS), but whether these organisms participate directly or indirectly in disease pathology remains unknown.
Objective
To determine the frequency of fungus isolation and fungus-specific immunity in Th2-associated and non-associated airway disease patients.
Methods
Sinus lavage fluid and blood were collected from sinus surgery patients (n=118) including CRS patients with and without nasal polyps and AFRS and non-CRS/non-asthmatic control patients. Asthma status was deteremined from medical history. Sinus lavage fluids were cultured and directly examined for evidence of viable fungi. Peripheral blood mononuclear cells were restimulated with fungal antigens in an enzyme linked immunocell spot (ELISpot) assay to determine total memory fungus-specific IL-4-secreting cells. These data were compared to fungus-specific IgE levels measured from plasma by ELISA.
Results
Filamentous fungi were significantly more commonly cultured from Th2-associated airway disease subjects (asthma, CRSwNP, or AFRS: n=68) compared to non-Th2-associated control patients (n=31); 74% vs 16% respectively, p<0.001. Both fungus-specific IL-4 ELISpot (n=48) and specific IgE (n=70) data correlated with Th2-associated diseases (sensitivity 73% and specificity 100% vs. 50% and 77%, respectively).
Conclusions
The frequent isolation of fungi growing directly within the airways accompanied by specific immunity to these organisms only in patients with Th2-associated chronic airway diseases suggests that fungi participate directly in the pathogenesis of these conditions. Efforts to eradicate airway fungi from the airways should be considered in selected patients.
Clinical Implications
Airway fungi may contribute to the expression of sinusitis with nasal polyps and asthma, suggesting that efforts to eradicate fungi from the airways and environments of these patients should be considered.
doi:10.1016/j.jaci.2014.04.028
PMCID: PMC4119856  PMID: 24928648
Allergic; Airway; Mycosis; Fungal; Asthma; Chronic Rhinosinusitis; Th2-associated airway disease
9.  Gene hunting in the genomic era: Approaches to diagnostic dilemmas in patients with primary immunodeficiencies 
There are more than 180 different genetic causes of primary immunodeficiencies identified to date. Approaches for identifying causative mutations can be broadly classified into 3 strategies: (1) educated guesses based on known signaling pathways essential for immune cell development and function, (2) similarity of clinical phenotypes to mouse models, and (3) unbiased genetic approaches. Next-generation DNA sequencing permits efficient sequencing of whole genomes or exomes but also requires strategies for filtering vast amounts of data. Recent studies have identified ways to solve difficult cases, such as diseases with autosomal dominant inheritance, incomplete penetrance, or mutations in noncoding regions. This review focuses on recently identified primary immunodeficiencies to illustrate the strategies, technologies, and potential pitfalls in finding novel causes of these diseases.
doi:10.1016/j.jaci.2013.08.021
PMCID: PMC3976463  PMID: 24100122
Primary immunodeficiencies; whole-genome sequencing; whole-exome sequencing; linkage analysis; homozygosity mapping
10.  Risks for Infection in Patients With Asthma (or Other Atopic Conditions): Is Asthma More Than a Chronic Airway Disease? 
Most of the research effort regarding asthma has been devoted to its causes, therapy, and prognosis. There is also evidence that the presence of asthma can influence patients’ susceptibility to infections, yet research in this aspect of asthma has been limited. There is additional debate in this field, with current literature tending to view the increased risk of infection among atopic patients as due to opportunistic infections secondary to airway inflammation, especially in severe atopic diseases. Other evidence, however, suggests that such risk and its underlying immune dysfunction may be a phenotypic or clinical feature of atopic conditions. This review argues that 1) improved understanding of the effects of asthma or other atopic conditions on the risk of microbial infections will bring important and new perspectives to clinical practice, research, and public health concerning atopic conditions and that 2) research efforts into the causes and effects of asthma must be juxtaposed because they are likely to guide each other.
doi:10.1016/j.jaci.2014.04.024
PMCID: PMC4122981  PMID: 25087224
adaptive immunity; allergic rhinitis; asthma; atopic dermatitis; epidemiology; immune dysfunction; immune incompetence; infection; innate immunity; phenotype; risk; susceptibility
11.  The CARD11-BCL10-MALT1 (CBM) signalosome complex: Stepping into the limelight of human primary immunodeficiency 
Next-generation DNA sequencing has accelerated the genetic characterization of many human primary immunodeficiency diseases (PIDs). These discoveries can be lifesaving for the affected patients, and also provide the unique opportunity to study the impact of specific genes on human immune function. In the past 18 months, a number of independent groups have begun to define novel PIDs caused by defects in the CARD11–BCL10–MALT1 (CBM) signalasome complex. The CBM complex forms an essential molecular link between the triggering of cell surface antigen receptors and NF-κB activation. Germline mutations affecting the CBM complex are now recognized as the cause of novel combined immunodeficiency phenotypes which all share abnormal NF-κB activation and dysregulated B cell development as defining features. For this Current Perspectives we have engaged experts in both basic biology and clinical immunology to capture the worldwide experience in recognizing and managing patients with PIDs caused by CBM complex mutations.
doi:10.1016/j.jaci.2014.06.015
PMCID: PMC4167767  PMID: 25087226
CARD11–BCL10–MALT1 (CBM) signalosome complex; Primary immunodeficiency diseases; Combined immunodeficiency; Congenital B cell lymphocytosis; Paracaspase; Next generation sequencing; NF-κB; CARMA1
12.  Therapy of autoinflammatory syndromes 
The therapy of autoinflammatory syndromes is an excellent example of the power of translational research. Recent advances in our understanding of the molecular and immunologic basis of this newly identified classification of disease have allowed for the application of novel, effective, targeted treatments with life-changing effects on patients. Although colchicine and TNF-α inhibitors are important therapies for specific autoinflammatory syndromes, the novel IL-1–targeted drugs are particularly effective for many of these diseases. Recently, the pharmaceutical industry has adopted a strategy of confirming the efficacy of new targeted drugs in often-ignored patients with orphan diseases, and US Food and Drug Administration policies have allowed for accelerated approval of these drugs, creating a win-win situation for patients and industry. This article reviews the general approach to the therapy of autoinflammatory diseases, focusing on current approved therapies and novel approaches that might be used in the future.
doi:10.1016/j.jaci.2009.11.001
PMCID: PMC4508191  PMID: 20004774
Autoinflammatory; IL-1; inflammasome
13.  Hematopoietic Stem Cell Transplantation for CD3δ deficiency 
Background
CD3δ deficiency is a fatal form of severe combined immunodeficiency which can be cured by hematopoietic stem cell transplantation (HSCT). The presence of a thymus loaded with T cell progenitors in these patients may require special considerations in choosing the regimen of conditioning and the type of HSCT.
Objectives
To study the outcome of CD3δ deficiency using various modalities of stem cell transplantation.
Methods
We analyzed data on 13 patients with CD3δ deficiency who underwent HSCT in 7 centers. HSCT was performed using different sources of donor stem cells as well as various conditioning regimens.
Results
Two patients who received stem cells from matched related donors and survived, both needed substantial conditioning in order to engraft. Only one of six other patients who received a related mismatched donor (MMRD) transplant survived, two of them had no conditioning while the others received various combinations of conditioning regimens.
Three other patients received stem cells from a matched unrelated donor (MUD), survived and enjoyed full immune reconstitution.
Two other patients received unrelated cord blood without conditioning. One of them has had a partial but stable engraftment, while the other engrafted well but is only 12 months after HSCT. We also report here for the first time that patients with CD3δ deficiency can present with typical features of Omenn syndrome.
Conclusions
HSCT is a successful treatment for patients with CD3δ deficiency. The small number of patients in this report prevent definitive statements on the importance of survival factors, but several are suggested: 1) HLA matched donor transplants are associated with superior reconstitution and survival than mismatched donor transplants; 2) substantial conditioning appears necessary; 3) early diagnosis and absence of opportunistic infections.
doi:10.1016/j.jaci.2011.05.031
PMCID: PMC4490832  PMID: 21757226
CD3delta; severe combined immunodeficiency; bone marrow transplant; stem cell transplant; myeloablative conditioning; engraftment
14.  B-cell development and functions and therapeutic options in adenosine deaminase–deficient patients 
Background
Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor.
Objective
We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function.
Methods
Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation.
Results
Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT.
Conclusions
ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.
doi:10.1016/j.jaci.2013.12.1043
PMCID: PMC4489526  PMID: 24506932
Gene therapy; adenosine deaminase–deficient severe combined immunodeficiency; B-cell development; antibodies
15.  A Mouse Model Links Asthma Susceptibility to Prenatal Exposure to Diesel Exhaust 
Background
Most asthma begins in the first years of life. This early onset cannot be merely attributed to genetic factors, because the prevalence of asthma is increasing. Epidemiological studies have indicated roles for prenatal and early childhood exposures, including exposure to diesel exhaust. However, little is known about the mechanisms. This is largely due to paucity of animal models.
Objective
We aimed to develop a mouse model of asthma susceptibility through prenatal exposure to diesel exhaust.
Methods
Pregnant C57BL/6 female mice were given repeated intranasal applications of diesel exhaust particles (DEP) or phosphate-buffered saline (PBS). Offspring underwent suboptimal immunization and challenge with ovalbumin (OVA) or received PBS. Pups were examined for features of asthma; lung and liver tissues were analyzed for transcription of DEP-regulated genes.
Results
Offspring of mice exposed to DEP were hypersensitive to OVA, indicated by airway inflammation and hyperresponsiveness, increased serum levels of OVA-specific IgE, and increased levels of pulmonary and systemic T-helper (Th)2 and Th17 cytokines. These cytokines were primarily produced by natural killer (NK) cells. Antibody-mediated depletion of NK cells prevented airway inflammation. Asthma susceptibility was associated with increased transcription of genes known to be specifically regulated by the aryl hydrocarbon receptor (AhR) and oxidative stress. Features of asthma were either marginal or absent in OVA-treated pups of PBS-exposed mice.
Conclusion
We created a mouse model that linked maternal exposure to DEP with asthma susceptibility in offspring. Development of asthma was dependent on NK cells and associated with increased transcription from AhR- and oxidative stress-regulated genes.
doi:10.1016/j.jaci.2013.10.047
PMCID: PMC4065237  PMID: 24365139
prenatal exposure; diesel exhaust particles; asthma; mouse model; natural killer cells; aryl hydrocarbon receptor; interleukin 5; interleukin 13; interleukin 17
16.  Mastocytosis associated with a rare germline KIT K509I mutation displays a well-differentiated mast cell phenotype 
Background
Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We report the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation.
Objectives
To investigate the impact of the germline KIT K509I mutation on human mast cell development and function.
Methods
Primary human mast cells derived from CD34+ peripheral blood progenitors were examined for growth, development, survival and IgE-mediated activation. In addition, a mast cell transduction system which stably expressed the KIT K509I mutation was established.
Results
KIT K509I biopsied mast cells were round, CD25(−) and well differentiated. KIT K509I progenitors, cultured in SCF, demonstrated a ten-fold expansion compared to progenitors from healthy subjects and developed into mature, hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived, however lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed the SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction.
Conclusion
Germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype, distinct to that of somatic KIT D816V disease, whose oncogenic potential may be influenced by SCF and selective KIT splicing.
Clinical Implications
Mastocytosis associated with reported germline KIT activating mutations, in this case KIT K509I, display a mature, well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease.
doi:10.1016/j.jaci.2013.12.1090
PMCID: PMC4125511  PMID: 24582309
KIT; K509I; mastocytosis; germline; mast cells; well-differentiated
18.  GAIN OF FUNCTION STAT1 MUTATION-RELATED PRIMARY IMMUNODEFICIENCY IS ASSOCIATED WITH DISSEMINATED MUCORMYCOSIS 
SUMMARY
We identified a novel gain of function mutation in STAT1 in a patient with disseminated Apophysomyces trapeziformis infection who had never had mucocutaneous candidiasis or autoimmunity. To our knowledge this is the first report of a genetic predisposition associated with mucormycosis.
doi:10.1016/j.jaci.2014.02.037
PMCID: PMC4125455  PMID: 24709374
STAT1; Immunodeficiency; Mucormycosis; Apophysomyces trapeziformis; Interferon gamma
19.  Delayed clinical and ex vivo response to mammalian meat in patients with IgE to galactose-alpha-1,3-galactose 
Background
In 2009, we reported a novel form of delayed anaphylaxis to red meat related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Although patients were remarkably consistent in their description of a 3- to 6-hour delay between eating mammalian meat and the appearance of symptoms, this delay has not been demonstrated under observed studies.
Objectives
We sought to formally document the time course of clinical symptoms after the ingestion of mammalian meat in subjects with IgE to alpha-gal and to monitor ex vivo for the appearance of markers of an allergic reaction.
Methods
Open food challenges were performed with mammalian meat in 12 subjects with a history of severe urticarial reactions 3 to 6 hours after eating beef, pork, or lamb, as well as in 13 control subjects. Blood samples were taken hourly during each challenge.
Results
Ten of 12 subjects with IgE to alpha-gal had clinical evidence of a reaction during the food challenge (vs none of the control subjects, P < .001). The reactions occurred 3 to 7 hours after the initial ingestion of mammalian meat and ranged from urticaria to anaphylaxis. Tryptase levels were positive in 3 challenges. Basophil activation, as measured by increased expression of CD63, correlated with the appearance of clinical symptoms.
Conclusion
The results presented provide clear evidence of an IgE-mediated food allergy that occurs several hours after ingestion of the inciting allergen. Moreover, here we report that in vivo basophil activation during a food challenge occurs in the same time frame as clinical symptoms and likely reflects the appearance of the antigen in the bloodstream. (J Allergy Clin Immunol 2014;)
doi:10.1016/j.jaci.2014.01.024
PMCID: PMC4125475  PMID: 24656556
Anaphylaxis; alpha-gal; basophil; mammalian meat; food allergy
20.  APOε 4 is associated with enhanced in vivo innate immune responses in humans 
Background
The genetic determinants of the human innate immune response are poorly understood. Apolipoprotein (apo)E, a lipid-trafficking protein that impacts inflammation, has well-described ‘wild type’ (ε3) and disease-associated (ε2, ε4) alleles, but its connection to human innate immunity is undefined.
Objective
To define the relationship of APOε4 to the human innate immune response.
Methods
We evaluated APOε4 in several functional models of the human innate immune response including intravenous lipopolysaccharide challenge in human subjects, and assessed APOε4 association to organ injury in human severe sepsis, a disease driven by dysregulated innate immunity.
Results
Whole blood from healthy APOε3/APOε4 volunteers induced higher cytokines upon ex vivo stimulation with Toll like Receptor (TLR)2, TLR4, or TLR5 ligands than blood from APOε3/APOε3 subjects, whereas TLR7/8 responses were similar. This was associated with increased lipid rafts in APOε3/APOε4 monocytes. By contrast, APOε3/APOε3 and APOε3/APOε4 serum neutralized lipopolysaccharide equivalently and supported similar lipopolysaccharide responses in Apoe-deficient macrophages, arguing against a differential role for secretory APOE4 protein. After intravenous lipopolysaccharide, APOε3/APOε4 human subjects had higher hyperthermia and plasma TNFα and earlier plasma IL-6 than APOε3/APOε3 subjects. APOE4-targeted replacement mice displayed enhanced hypothermia, plasma cytokines, and hepatic injury, and altered splenic lymphocyte apoptosis after systemic lipopolysaccharide compared with APOE3 counterparts. In a cohort of 828 severe sepsis patients, APOε4 was associated with increased coagulation system failure among European American subjects.
Conclusions
APOε4 is a determinant of the human innate immune response to multiple TLR ligands, and associates with altered patterns of organ injury in human sepsis.
doi:10.1016/j.jaci.2014.01.032
PMCID: PMC4125509  PMID: 24655576
Apolipoprotein; Toll like Receptor; Lipopolysaccharide
21.  MicroRNA-150 regulates the cytotoxicity of natural killers by targeting perforin-1 
Background
Perforin-1 is the predominant cytolytic protein secreted by natural killer cells. For a rapid immune response, resting NK cells contain high Prf1 mRNA concentrations while exhibiting minimal cytotoxicity due to a blockage of Prf1 protein synthesis, implying that an unknown post-transcriptional regulatory mechanism exists.
Objective
We sought to determine that microRNA-150 (miR-150) post-transcriptionally regulates Prf1 translation in both mouse and human NK cells at rest and at various time points after activation.
Methods
To investigate the role of miR-150 in NK cells, mouse NK cells with a targeted deletion of miR-150 (miR-150−/− NK cells), primary human NK cells, and NK92 MI cells were utilized. NK cell cytotoxicity assays and western blotting proved that activated miR-150−/− NK cells expressed upregulated Prf1, augmenting NK cell cytotoxicity. When immunodeficient mice were injected with miR-150−/− NK cells, there was a significant reduction in tumor growth and metastasis of B16F10 melanoma.
Results
We report that miR-150 binds to 3′ untranslated regions of mouse and human Prf1, post-transcriptionally downregulating its expression. Mouse wild-type NK cells displayed downregulated miR-150 expression in response to interleukin-15, which led to corresponding repression and induction of Prf1 during rest and after IL-15 activation, respectively.
Conclusion
Our results indicated that miR-150 is a common post-transcriptional regulator for Prf1 in mouse and human NK cells that represses NK cell lytic activity. Thus the therapeutic control of miR-150 in NK cells could enhance NK cell based immunotherapy against cancer, providing a better clinical outcome.
doi:10.1016/j.jaci.2014.02.018
PMCID: PMC4125537  PMID: 24698324
miR-150; NK cells; Prf1; NK cell cytotoxicity; post-transcriptional regulation; immunotherapy; tumor growth and metastasis
22.  Progress in HIV-1 Vaccine Development 
The past two years have seen a number of basic and translational science advances in the quest for development of an effective HIV-1 vaccine. These advances include discovery of new envelope (Env) targets of potentially protective antibodies, demonstration that CD8+ T cells can control HIV-1 infection, development of immunogens to overcome HIV-1 T cell epitope diversity, identification of correlates of transmission risk in an HIV-1 efficacy trial, and mapping the co-evolution of HIV-1 founder Env mutants in infected individuals who develop bnAbs, thereby defining broad neutralizing antibody (bnAb) developmental pathways. Despite these advances, a promising HIV-1 vaccine efficacy trial published in 2013 failed to prevent infection, and the HIV-1 vaccine field is still years away from deployment of an effective vaccine. This review summarizes what some of the scientific advances have been, what roadblocks still remain, and what the most promising approaches are for progress in design of successful HIV-1 vaccine candidates.
doi:10.1016/j.jaci.2014.04.025
PMCID: PMC4133697  PMID: 25117798
HIV-1; vaccine; T cells; B cells; broadly neutralizing antibodies
23.  The transcription factor Etv5 controls TH17 cell development and allergic airway inflammation 
Background
The differentiation of TH17 cells, which promote pulmonary inflammation, requires the cooperation of a network of transcription factors.
Objectives
We sought to define the role of Etv5, an Ets-family transcription factor, in TH17 cell development and function.
Methods
TH17 development was examined in primary mouse T cells wherein Etv5 expression was altered by retroviral transduction, small interfering RNA targeting a specific gene, and mice with a conditional deletion of Etv5 in T cells. The direct function of Etv5 on the Il17 locus was tested with chromatin immunoprecipitation and reporter assays. The house dust mite–induced allergic inflammation model was used to test the requirement for Etv5-dependent TH17 functions in vivo.
Results
We identify Etv5 as a signal transducer and activator of transcription 3–induced positive regulator of TH17 development. Etv5 controls TH17 differentiation by directly promoting 0a and Il17f expression. Etv5 recruits histone-modifying enzymes to the Il17a–Il17f locus, resulting in increased active histone marks and decreased repressive histone marks. In a model of allergic airway inflammation, mice with Etv5-deficient T cells have reduced airway inflammation and IL-17A/F production in the lung and bronchoalveolar lavage fluid compared with wild-type mice, without changes in TH2 cytokine production.
Conclusions
These data define signal transducer and activator of transcription 3–dependent feed-forward control of TH17 cytokine production and a novel role for Etv5 in promoting T cell–dependent airway inflammation.
doi:10.1016/j.jaci.2013.12.021
PMCID: PMC4209254  PMID: 24486067
TH17 cells; transcription factor; Etv5; epigenetic modifications; allergic inflammation
24.  Fractional exhaled nitric oxide in childhood is associated with 17q11.2-q12 and 17q12-q21 variants 
Background
The fractional concentration of nitric oxide in exhaled air (FeNO) is a biomarker of eosinophilic airway inflammation and associated with childhood asthma. Identification of common genetic variants associated with childhood FeNO may help to define biological mechanisms related to specific asthma phenotypes.
Objective
To identify genetic variants associated with childhood FeNO, and their relation with asthma.
Methods
FeNO was measured in children aged 5 to 15 years. In 14 genome-wide association (GWA) studies (N = 8,858), we examined the associations of ~2.5 million single nucleotide polymorphisms (SNPs) with FeNO. Subsequently, we assessed whether significant SNPs were expression quantitative trait loci (eQTLs) in genome-wide expression datasets of lymphoblastoid cell lines (N = 1,830), and were related with asthma in a previously published GWA dataset (cases: n=10,365; controls: n=16,110).
Results
We identified 3 SNPs associated with FeNO: rs3751972 in LYR motif containing 9 (LYRM9) (P = 1.97×10−10) and rs944722 in inducible nitric oxide synthase 2 (NOS2) (P = 1.28×10−9) both located at 17q11.2-q12, and rs8069176 near gasdermin B (GSDMB) (P = 1.88×10−8) at 17q12-q21. We found a cis eQTL for the transcript soluble galactoside-binding lectin 9 (LGALS9) that is in linkage disequilibrium with rs944722. Rs8069176 was associated with GSDMB and ORM1-like 3 (ORMDL3) expression. Rs8069176 at 17q12-q21, and not rs3751972 and rs944722 at 17q11.2-q12, were associated with physician-diagnosed asthma.
Conclusion
This study identified 3 variants associated with FeNO, explaining 0.95% of the variance. Identification of functional SNPs and haplotypes in these regions might provide novel insight in the regulation of FeNO. This study highlights that both shared and distinct genetic factors affect FeNO and childhood asthma.
doi:10.1016/j.jaci.2013.08.053
PMCID: PMC4334587  PMID: 24315451
airway inflammation; asthma phenotypes; biomarker; genetics; genome-wide association study
25.  PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator–dependent autoimmunity 
Background
PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance.
Objective
We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients.
Methods
Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency.
Results
We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti–calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells.
Conclusion
Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.
doi:10.1016/j.jaci.2015.01.040
PMCID: PMC4487867  PMID: 25842288
Autoimmune regulator; tolerance; DNA-dependent protein kinase catalytic subunit; PRKDC; autoimmunity; VDJ recombination; severe combined immunodeficiency; recombination-activating gene

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