Early-life gut microbiome and egg allergy.

BACKGROUND: Gut microbiota may play a role in egg allergy. We sought to examine the association between early-life gut microbiota and egg allergy. METHODS: We studied 141 children with egg allergy and controls from the multicenter Consortium of Food Allergy Research study. At enrollment (age 3 to 16 months), fecal samples were collected, and clinical evaluation, egg-specific IgE measurement, and egg skin prick test were performed. Gut microbiome was profiled by 16S rRNA sequencing. Analyses for the primary outcome of egg allergy at enrollment, and the secondary outcomes of egg sensitization at enrollment and resolution of egg allergy by age 8 years, were performed using Quantitative Insights into Microbial Ecology, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, and Statistical Analysis of Metagenomic Profiles. RESULTS: Compared to controls, increased alpha diversity and distinct taxa (PERMANOVA P = 5.0 × 10-4 ) characterized the early-life gut microbiome of children with egg allergy. Genera from the Lachnospiraceae, Streptococcaceae, and Leuconostocaceae families were differentially abundant in children with egg allergy. Predicted metagenome functional analyses showed differential purine metabolism by the gut microbiota of egg-allergic subjects (Kruskal-Wallis Padj  = 0.021). Greater gut microbiome diversity and genera from Lachnospiraceae and Ruminococcaceae were associated with egg sensitization (PERMANOVA P = 5.0 × 10-4 ). Among those with egg allergy, there was no association between early-life gut microbiota and egg allergy resolution by age 8 years. CONCLUSION: The distinct early-life gut microbiota in egg-allergic and egg-sensitized children identified by our study may point to targets for preventive or therapeutic intervention.

Utility of component analyses in subjects undergoing sublingual immunotherapy for peanut allergy.

BACKGROUND: Sublingual immunotherapy (SLIT) with peanut changes clinical and immune responses in most peanut-allergic individuals, but the response is highly variable. OBJECTIVE: We sought to examine the component-specific effects of peanut SLIT and determine whether peanut component testing could predict the outcome of a double-blind, placebo-controlled food challenge (DBPCFC) after 12 months of peanut SLIT. METHODS: We included 33 subjects who underwent peanut SLIT with a DBPCFC of 2500 mg of peanut protein performed after 12 months of therapy. Plasma samples from baseline and after 12 months of peanut SLIT were assayed using ImmunoCAP for IgE and IgG4 against whole peanut, Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9. RESULTS: Following 12 months of SLIT, 10 subjects (30%) passed the DBPCFC without symptoms and were considered desensitized. Subjects that failed the DBPCFC tolerated a median of 460 mg peanut protein (range: 10-1710 mg). The desensitized group had significantly lower baseline levels of IgE against peanut (median 40.8 vs. 231 kUA /L, P = 0.0082), Ara h 2 (median 17 vs. 113 kUA /L, P = 0.0082), and Ara h 3 (median 0.3 vs. 8.5 kUA /L, P = 0.0396). ROC curves indicated that baseline IgE against peanut and Ara h 2 were equally effective at discriminating between the two groups (AUC = 0.7957, P = 0.007752 for both). CONCLUSION AND CLINICAL RELEVANCE: In this cohort of subjects undergoing SLIT for peanut allergy, lower baseline levels of IgE against Ara h 2, Ara h 3, and peanut were associated with successful desensitization.

Component-resolved analysis of IgA, IgE, and IgG4 during egg OIT identifies markers associated with sustained unresponsiveness.

BACKGROUND: In a previously reported CoFAR study, 55 subjects with egg allergy underwent randomized, placebo-controlled egg oral immunotherapy (eOIT). Active treatment induced desensitization in most and sustained unresponsiveness (SU) in a smaller subset. We hypothesized that component-resolved analysis of IgE, IgG4, IgA, IgA1, and IgA2 may identify potential biomarkers of SU in OIT subjects. METHODS: Longitudinal samples for 51 egg-allergic subjects (37 active and 14 placebo) were available. Egg white (EW)-, ovalbumin (OVA)-, and ovomucoid (OVM)-specific levels of IgA, IgA1, and IgA2 were quantified by ELISA. IgE and IgG4 to these antigens were quantified using ImmunoCAP® . Clinical responders achieved SU to egg; all others were considered nonresponders. Between-group comparisons were made among active and placebo, as well as responders and nonresponders. RESULTS: No placebo subjects achieved responder status. Through month 48, among the 37 active subjects, baseline IgE-OVM was lower in responders (median 3.97 kU/l, n = 19) than in nonresponders (10.9 kU/l, n = 18, P = 0.010). Logistic regression analysis revealed that lower baseline IgE-EW (P = 0.038), IgE-OVM (P = 0.032), and a higher IgG4/IgE-OVM ratio (P = 0.013) were associated with clinical response. Relative increases in IgG4-EW, IgA-EW, and IgA2-EW were observed in responders (P = 0.024, 0.024, and 0.029, respectively). IgG4/IgE, IgA/IgE, and IgA2/IgE ratios for EW and IgA/IgE ratio for OVA were found to be significantly elevated among responders (P = 0.004, 0.009, 0.028, and 0.008, respectively). CONCLUSIONS: Increased IgG4-EW, IgA-EW, and IgA2-EW during eOIT are associated with clinical response to eOIT. Lower pretreatment IgE-EW and IgE-OVM are also associated with SU. Future studies are needed to evaluate and validate these potential biomarkers.

Effects of a pre-existing food allergy on the oral introduction of food proteins: findings from a murine model.

Cashew-allergic mice develop elevated walnut-specific IgE upon oral feeding of walnut proteins. Ingestion of tree nuts in the presence of a known nut allergy could lead to additional sensitizations and anaphylaxis following subsequent exposure.

A phase 1 study of heat/phenol-killed, E. coli-encapsulated, recombinant modified peanut proteins Ara h 1, Ara h 2, and Ara h 3 (EMP-123) for the treatment of peanut allergy.

BACKGROUND: Immunotherapy for peanut allergy may be limited by the risk of adverse reactions. OBJECTIVE: To investigate the safety and immunologic effects of a vaccine containing modified peanut proteins. METHODS: This was a phase 1 trial of EMP-123, a rectally administered suspension of recombinant Ara h 1, Ara h 2, and Ara h 3, modified by amino acid substitutions at major IgE-binding epitopes, encapsulated in heat/phenol-killed E. coli. Five healthy adults were treated with 4 weekly escalating doses after which 10 peanut-allergic adults received weekly dose escalations over 10 weeks from 10 mcg to 3063 mcg, followed by three biweekly doses of 3063 mcg. RESULTS: There were no significant adverse effects in the healthy volunteers. Of the 10 peanut-allergic subjects [4 with intermittent asthma, median peanut IgE 33.3 kUA /l (7.2-120.2), and median peanut skin prick test wheal 11.3 mm (6.5-18)]; four experienced no symptoms; one had mild rectal symptoms; and the remaining five experienced adverse reactions preventing completion of dosing. Two were categorized as mild, but the remaining three were more severe, including one moderate reaction and two anaphylactic reactions. Baseline peanut IgE was significantly higher in the five reactive subjects (median 82.4 vs 17.2 kUA /l, P = 0.032), as was baseline anti-Ara h 2 IgE (43.3 versus 8.3, P = 0.036). Peanut skin test titration and basophil activation (at a single dilution) were significantly reduced after treatment, but no significant changes were detected for total IgE, peanut IgE, or peanut IgG4. CONCLUSIONS: Rectal administration of EMP-123 resulted in frequent adverse reactions, including severe allergic reactions in 20%.

Immunology in the Clinic Review Series; focus on allergies: immunotherapy for food allergy.

There is no approved therapy for food allergy. The current standard of care is elimination of the triggering food from the diet and accessibility to epinephrine. Immunotherapy is a promising treatment approach. While desensitization to most foods seems feasible, it remains unclear if a permanent state of tolerance is achievable. The research team at Duke is pioneering immunotherapy for food allergies. Work here has evolved over time from small open-label pilot studies to larger randomized designs. Our data show that immunological changes associated with immunotherapy include reduction in mast cell reactivity, decreased basophil responses, decreased specific-immunoglobulin (Ig)E, increased IgG4 and induction of regulatory T cells. Immunotherapy has generated much excitement in the food allergy community; however, further studies are needed before it is ready for clinical use.

How do we know when peanut and tree nut allergy have resolved, and how do we keep it resolved?

Over the last two decades, the prevalence of peanut and tree nut allergy has increased throughout the western world. Adverse reactions to these foods account for over 50% of all deaths resulting from food-related anaphylaxis. Until recently, evidence suggested that all peanut and tree nut allergy were permanent. It is now known that about 20% and 10%, respectively, of young patients outgrow peanut and tree nut allergies. Achieving tolerance is associated with increasing circulating T regulatory cells and reduced production of allergen-specific IgE. Reliable predictors of resolution are not yet available. A direct correlation between skin test weal size and allergen-specific IgE, at the time of diagnosis and likelihood of resolution, has been reported. Resolution of peanut or tree nut allergy cannot be determined conclusively by either allergen-specific IgE analysis or by skin prick testing. Oral food challenge is the gold standard for determining resolution of food allergy. Food challenges should only be undertaken in a clinical setting fully equipped to deal with a potential severe adverse reaction. Approximately 8% of patients who outgrow peanut allergy may suffer a recurrence, but recurrent tree nut allergy has not been reported to date. Infrequent ingestion of peanut may be related to the re-emergence of allergy. Induction of tolerance through oral immunotherapy or sublingual immunotherapy is now being actively studied, but remains experimental. Studies have reported short-term desensitization to peanut, but ongoing follow-up will determine whether tolerance is achieved long term.

Food allergy QoL questionnaire for children aged 0-12 years: content, construct, and cross-cultural validity.

BACKGROUND: To date, there is no food allergy-specific questionnaire that allows parents to report children's health-related QoL (HRQL) from the child's perspective. OBJECTIVE: The aim of this study was to develop a sensitive, multi-dimensional measure to assess parental perception of HRQL in children aged 0-12 years with food allergy. METHODS: The Food Allergy QoL - Parent Form (FAQLQ-PF) was developed and validated in four stages: (1) item generation using focus groups, expert opinion, and literature review; (2) item reduction, using clinical impact and factor analysis; (3) internal and test-retest reliability and construct validity were evaluated using relevant scales of the Child Health Questionnaire (CHQ)-28 and the disease-specific food allergy independent measure (FAIM); and (4) cross-cultural and content validity was examined by administering the questionnaire in a US sample. RESULTS: Stage 1: Saturation was reached at 110 items. Stage 2: The reduced instrument has 14 items for children <4 years and 26 and 30 items for children aged 4-6 years and 7-12 years, respectively. Factor analysis revealed three subscales: emotional impact, food anxiety, and social and dietary limitations, accounting for 68% of the variance. Stage 3: Cronbach's alpha >0.7 for subscales and total score. Construct validity was demonstrated by significant correlations between relevant scales of the CHQ-28 and FAQLQ-PF subscales (r=0.69-0.77, P<0.01), and between FAQLQ-PF subscales and the FAIM. Sensitivity was shown by significant within-group differences in a sample of 124 food-allergic children. Stage 4: The FAQLQ-PF was validated in a sample of US children, Cronbach's alpha >0.7 for subscales and total score. Construct validity was demonstrated by significant correlations between FAQLQ-PF and the FAIM (parent report) and between the FAQLQ-PF and the FAIM (child report). No differences were observed between the US and Irish scores. CONCLUSION: The FAQLQ-PF is psychometrically robust, with excellent reliability and validity.

Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity.

Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly (A)+ RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in E. coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general

Molecular cloning and epitope analysis of the peanut allergen Ara h 3.

Peanut allergy is a significant IgE-mediated health problem because of the increased prevalence, potential severity, and chronicity of the reaction. Following our characterization of the two peanut allergens Ara h 1 and Ara h 2, we have isolated a cDNA clone encoding a third peanut allergen, Ara h 3. The deduced amino acid sequence of Ara h 3 shows homology to 11S seed-storage proteins. The recombinant form of this protein was expressed in a bacterial system and was recognized by serum IgE from approximately 45% of our peanut-allergic patient population. Serum IgE from these patients and overlapping, synthetic peptides were used to map the linear, IgE-binding epitopes of Ara h 3. Four epitopes, between 10 and 15 amino acids in length, were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope is recognized by all Ara h 3-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding. By determining which amino acids are critical for IgE binding, it might be possible to alter the Ara h 3 cDNA to encode a protein with a reduced IgE-binding capacity. These results will enable the design of improved diagnostic and therapeutic approaches for food-hypersensitivity reactions.

Mechanisms of food allergy.

The prevalence of food allergy continues to rise, particularly in 'westernized' societies; it has been linked to the 'hygiene hypothesis' and the increased diversity of food consumption worldwide. The pathogenic mechanisms and Th1/Th2 paradigm are being closely examined with respect to the occurrence of inflammatory and injury/repair responses at different mucosal sites. Genetically modified plants as potential food sources and allergenicity are current topics of controversy.

Lack of immunogenicity of ice structuring protein type III HPLC12 preparation administered by the oral route to human volunteers.

Before a novel protein can be used in foods, its potential allergenicity must be assessed. In this study, healthy volunteers consumed ice structuring protein (ISP) Type III preparation or a control material 5 days a week for a total of 8 weeks. General measures of health were recorded during the study, and the immunogenicity of the protein was assessed by monitoring the levels of IgG and IgE antibodies specific for ISP Type III. The participants remained in good health throughout the study and during the 4 week follow-up period. No IgG or IgE antibodies specific for ISP Type III were detected in the blood of the participants. Investigations of immunogenicity in man have not been previously applied in the context of safety evaluation and they do not form part of the regimens proposed for the evaluation of protein allergenicity. Consequently no standardised protocols exist for such studies, nor any background against which to interpret the results. Nevertheless, the absence of an immune response using a protocol which could have been expected to result in a response with a strongly immunogenic protein, confirms the conclusions of earlier published work, and attests to the lack of allergenicity of ISP Type III preparation.

Integrative transcriptomic analysis reveals key drivers of acute peanut allergic reactions.

Mechanisms driving acute food allergic reactions have not been fully characterized. We profile the dynamic transcriptome of acute peanut allergic reactions using serial peripheral blood samples obtained from 19 children before, during, and after randomized, double-blind, placebo-controlled oral challenges to peanut. We identify genes with changes in expression triggered by peanut, but not placebo, during acute peanut allergic reactions. Network analysis reveals that these genes comprise coexpression networks for acute-phase response and pro-inflammatory processes. Key driver analysis identifies six genes (LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3) predicted to causally modulate the state of coregulated networks in response to peanut. Leukocyte deconvolution analysis identifies changes in neutrophil, naive CD4+ T cell, and macrophage populations during peanut challenge. Analyses in 21 additional peanut allergic subjects replicate major findings. These results highlight key genes, biological processes, and cell types that can be targeted for mechanistic study and therapeutic targeting of peanut allergy.

In vitro translation of RNA from the German cockroach Blattella germanica.

The translation of mRNA within total RNA of German (Blattella germanica) cockroaches was performed using a rabbit reticulocyte lysate system. Analysis of the translation products by SDS-PAGE and combined autoradiography revealed several synthesized proteins with apparent molecular weights ranging from 20 kD to 110 kD. SDS-PAGE/Western blotting of non-radiolabeled translation products and incubation with human serum with IgE to cockroach allergens showed the presence of a 36 kD and 50 kD allergen. The confirmation of the translation of the cockroach allergens from total RNA is an important first step in the cloning of cockroach allergens.

Egg hypersensitivity and measles-mumps-rubella vaccine administration.

Because reports have described egg-sensitive individuals in whom anaphylaxis developed after measles vaccination, current recommendations include delaying administration of egg-derived vaccines until skin testing can be performed. Specifically, the 1988 Red Book recommends skin testing via scratch, prick, or puncture with 1:10 dilution of the vaccine and, if the result is negative, intradermal testing is suggested. The purpose of this study was to evaluate the likelihood of reaction to measles-mumps-rubella (MMR) vaccine in patients with documented egg sensitivity and to delineate the efficacy of skin-prick testing (SPT) to MMR as a predictor of hypersensitivity to the vaccine. Egg sensitivity was documented by initial SPT to egg and then, if possible, double-blind placebo-controlled food challenge (DBPCFC). Patients with a positive DBPCFC to egg or a history of anaphylactic egg sensitivity had a SPT with the MMR vaccine and then were given the MMR vaccine. Additionally, children with atopic dermatitis who had been previously proven egg sensitive via DBPCFCs were evaluated retrospectively for sensitivity to the MMR vaccine. Sixteen children with a history of egg sensitivity underwent SPT to egg, with a positive result 3 mm greater than the negative control found in 12 patients. Eight of these children had a positive DBPCFC to egg. The SPT to MMR vaccine was negative in all 16 children; vaccine administration followed with no resultant systemic problems. Three children had a local reaction at the site of injection. Twelve additional children with atopic dermatitis and egg sensitivity were reviewed. Each child had a positive SPT and DBPCFC to egg.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune function in patients treated with phenytoin.

Multiple immunologic side effects have been ascribed to phenytoin. Numerous reports in the literature discuss the possible cellular and humoral abnormalities that appear to be present in patients given phenytoin. The most consistent finding is a reduction in serum IgA found in up to 20% of patients. To resolve some of the conflicting studies on cellular immune status, 191 patients taking phenytoin were evaluated initially with an serum IgA determination, and then further immune studies were done on the 11% with IgA values lower than two standard deviations below the mean. Data collected included total lymphocyte counts, lymphocyte population studies, and responses to in vitro mitogen stimulation. Only 2 of 191 patients had serum IgA values less than 5 mg/dL, which is an incidence not significantly different than that in the population at large. The patients with decreased serum IgA values did not have an increased incidence of autoimmune phenomena, allergic disorders, gastrointestinal manifestations, or recurrent upper respiratory tract infections. Their cellular immune status showed no significant variations from control values. Thus it appears that routine monitoring of patients on phenytoin with serum IgA determinations is of limited value, and the immunologic side effects of phenytoin are not expressed as a cellular abnormality.

Impact of dietary yogurt on immune function.

Studies of the effects of yogurt on immunity and atopic diseases have suggested improvements in cytokine (interleukin-2 and interferon-gamma) responses and clinical scores in patients with allergic rhinitis. This study compares prospectively immune parameters of participants who received 16 oz of yogurt versus 16 oz of milk/day in a randomized cross-over design. Yogurt that contained live, active Lactobacillus bulgaricus and Streptococcus thermophilus or 2% milk was consumed for one month each. Twenty otherwise healthy adults with atopic histories documented by skin testing were enrolled. Immune studies were performed at the beginning and end of the two 1-month study phases, separated by a 2-week washout period. These studies included measurements of cellular, humoral, and phagocytic function. No adverse events were noted in either group. No significant improvements in any immune parameter were noted. The consumption of yogurt that contained the live active bacteria L bulgaricus and S thermophilus does not appear to enhance immune function in atopic individuals at the dosage and duration used in this study.

Flow cytometric analysis of lymphocytes from rats following chronic ethanol treatment.

The current investigation focused on lymphoid cell populations of adult male Sprague-Dawley rats fed a total enteral nutrition diet in which ethanol provided 38% of the total calories. Rats received the National Research Council (NRC) recommended daily intake of nutrients 35 days. An evaluation of lymphocyte populations from peripheral blood demonstrated a decrease in the absolute number of B cells (p < or = 0.007) and absolute numbers of CD4 T cells (p < or = 0.06) in the ethanol-treated animals. Spleen and thymus weights were significantly reduced (p = 0.0001) in the ethanol-treated rats and the CD4/CD8 ratio of splenic lymphocytes decreased in the ethanol group (p < or = 0.03). Thymus T-cell recovery from the ethanol-treated group was significantly reduced with no apparent redistribution in subset numbers with the exception of a minor, yet significant, decrease (p < or = 0.05) in the CD4/CD8 ratio. These data are the first to demonstrate that chronic alcohol intake alters lymphoid cell populations in the peripheral blood and primary organs of the immune systems in the presence of adequate nutrition.

Effect of soy protein ingestion on total and specific immunoglobulin G concentrations in neonatal porcine serum measured by enzyme-linked immunosorbent assay.

Crossbred neonatal pigs from spring and summer farrowings were used to evaluate the systemic humoral immune response in porcine serum after ingestion of soy protein. At 10 to 12 d of age (average BW 3.8 kg), pigs were randomly allotted to three treatment groups according to litter, weight, and sex. Treatments were intermittent gavage feedings two or three times daily for six consecutive days with nonfat dry milk (NFDM) or textured vegetable protein (TVP) and a nongavaged control group. Pigs were weaned at 20 to 22 d of age (average BW 5.7 kg) and fed a corn-soybean meal-based starter diet. Total serum immunoglobulin G (IgG) and IgG concentrations specific for soy protein were measured by ELISA. Blood samples were taken at 1 d of age after colostrum intake and at 4-d intervals from the beginning of treatment to 31 d of age. When averaged from d 1 to 31, spring-farrowed pigs had greater (P less than .005) total IgG and soy-protein-specific IgG concentrations than did summer-farrowed pigs (8.70 vs 6.51 mg/mL and 1.59 vs .55 micrograms/mL, respectively). Total serum IgG concentrations changed with time (P = .005); they initially decreased, then recovered after weaning. These changes were independent of treatment, sex, or farrowing season. Soy-protein-specific IgG concentrations also changed with time (P = .003); however, this trend was dependent on season (P = .014). Summer-farrowed pigs exhibited a more rapid and severe decrease in serum IgG concentrations specific for soy protein than did spring-farrowed pigs.(ABSTRACT TRUNCATED AT 250 WORDS)