Gustavo Gimenez

Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens.

BACKGROUND Patients with peanut allergy have highly stable pathologic antibody repertoires to the immunodominant B-cell epitopes of the major peanut allergens Ara h 1 to 3. OBJECTIVE We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires. METHODS Measurements of total peanut-specific IgE (psIgE) and peanut-specific IgG(4) (psIgG(4)) were made with CAP-FEIA. We analyzed sera from 22 patients with OIT and 6 control subjects and measured serum specific IgE and IgG(4) binding to epitopes of Ara h 1 to 3 using a high-throughput peptide microarray technique. Antibody affinity was measured by using a competitive peptide microarray, as previously described. RESULTS At baseline, psIgE and psIgG(4) diversity was similar between patients and control subjects, and there was broad variation in epitope recognition. After a median of 41 months of OIT, polyclonal psIgG(4) levels increased from a median of 0.3 μg/mL (interquartile range [25% to 75%], 0.1-0.43 μg/mL) at baseline to 10.5 μg/mL (interquartile range [25% to 75%], 3.95-45.48 μg/mL; P < .0001) and included de novo specificities. psIgE levels were reduced from a median baseline of 85.45 kU(A)/L (23.05-101.0 kU(A)/L) to 7.75 kU(A)/L (2.58-30.55 kU(A)/L, P < .0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated patients, several subjects generated new IgE specificities, even as the total psIgE level decreased. Global epitope-specific shifts from IgE to IgG(4) binding occurred, including at an informative epitope of Ara h 2. CONCLUSION OIT differentially alters Ara h 1 to 3 binding patterns. These changes are variable between patients, are not observed in control subjects, and include a progressive polyclonal increase in IgG(4) levels, with concurrent reduction in IgE amount and diversity.

A bioinformatics approach to identify patients with symptomatic peanut allergy using peptide microarray immunoassay.

BACKGROUND Peanut allergy is relatively common, typically permanent, and often severe. Double-blind, placebo-controlled food challenge is considered the gold standard for the diagnosis of food allergy-related disorders. However, the complexity and potential of double-blind, placebo-controlled food challenge to cause life-threatening allergic reactions affects its clinical application. A laboratory test that could accurately diagnose symptomatic peanut allergy would greatly facilitate clinical practice. OBJECTIVE We sought to develop an allergy diagnostic method that could correctly predict symptomatic peanut allergy by using peptide microarray immunoassays and bioinformatic methods. METHODS Microarray immunoassays were performed by using the sera from 62 patients (31 with symptomatic peanut allergy and 31 who had outgrown their peanut allergy or were sensitized but were clinically tolerant to peanut). Specific IgE and IgG(4) binding to 419 overlapping peptides (15 mers, 3 offset) covering the amino acid sequences of Ara h 1, Ara h 2, and Ara h 3 were measured by using a peptide microarray immunoassay. Bioinformatic methods were applied for data analysis. RESULTS Individuals with peanut allergy showed significantly greater IgE binding and broader epitope diversity than did peanut-tolerant individuals. No significant difference in IgG(4) binding was found between groups. By using machine learning methods, 4 peptide biomarkers were identified and prediction models that can predict the outcome of double-blind, placebo-controlled food challenges with high accuracy were developed by using a combination of the biomarkers. CONCLUSIONS In this study, we developed a novel diagnostic approach that can predict peanut allergy with high accuracy by combining the results of a peptide microarray immunoassay and bioinformatic methods. Further studies are needed to validate the efficacy of this assay in clinical practice.

Use of IgE and IgG4 epitope binding to predict the outcome of oral immunotherapy in cow's milk allergy.

Oral immunotherapy (OIT) with cows milk (CM) has been reported to induce a number of specific antibody responses, but these remain to be fully characterized. Our objective was to explore whether IgE and IgG4 epitope binding profiles could predict the risk of side effects during CM OIT.

In vitro digestibility of bovine β-casein with simulated and human oral and gastrointestinal fluids. Identification and IgE-reactivity of the resultant peptides

Stability during digestion is considered an important feature in determining the allergenicity of food proteins. This study aimed to provide an immunological characterisation of the digestion products of the major cows milk allergen β-casein (β-CN) produced by in vitro orogastrointestinal hydrolysis with simulated and human digestive fluids. β-CN was unaffected by oral digestion, but quickly broke down during the early stages of gastric digestion. The degradation with human fluids was faster than that with commercial enzymes. There were similarities in the peptide patterns of the hydrolysates produced in both models, showing 20 peptides in common after gastric digestion. After gastroduodenal digestion, the human fluids gave less numerous and shorter peptides. The IgE binding of most of the individual sera used to the hydrolysates produced with simulated and human fluids increased at the end of the gastric phase and decreased when the duodenal digestion was completed. Two IgE-binding synthetic peptides: β-CN (57-68) and β-CN (82-93), which matched fragments released by β-CN following in vitro digestion with simulated and human fluids, consisted of the most immunoreactive areas of the protein. The similarities found between the in vitro simulated digestion system and that using human digestive fluids suggest that the former would provide a reasonably good estimation of the potential allergenicity of protein digests.

Dietary antigens, epitope recognition, and immune complex formation in recent onset psychosis and long-term schizophrenia

Peptides derived from dietary antigens such as bovine milk caseins are opioid receptor ligands and contribute to schizophrenia-associated hyperpeptidemia and hyperpeptiduria. The IgG antibody response to bovine caseins is increased in schizophrenia and recent onset psychosis. To identify specific casein peptide sequences that are antigenic in patients vs controls, we measured serum IgG binding to 10-26 amino acid long linear epitopes of casein with immunoassays for the entire group (n=95 recent onset psychosis; n=103 long-term schizophrenia; n=65 control), and with peptide microarray libraries in a casein-sensitive subset (n=14 recent onset; n=10 control). In the entire group, we compared anti-casein peptide IgG vs anti-whole casein IgG and evaluated whether peptide immune complexes contributed to IgG binding results. Anti-whole casein IgG levels correlated with anti-casein peptide IgG in controls only (R2=0.17-0.25, p≤0.002-0.03). In recent onset psychosis, IgG binding to linear peptide sequences was significantly decreased 3.8-5.7-fold compared to controls in immunoassays (OR 0.18-0.26, p≤0.0001-0.001). In peptide microarrays, recent onset patients again showed significantly reduced IgG binding and fewer epitopes than controls (p≤0.00001-0.05). Anti-peptide IgG levels did not differ between patients with long-term schizophrenia and controls. Finally, significantly more recent onset individuals had casein peptide-IgG immune complexes than controls (OR 4.96, p≤0.001). These findings suggest an immunological specificity that differs in early vs later stages of neuropsychiatric diseases and an IgG saturation by casein-derived peptides that may in part explain the reduced IgG binding to small linear epitopes observed in these patients.

Natural tolerance development in cow's milk allergic children: IgE and IgG4 epitope binding

Although most of cows milk (CM) allergic children will outgrow their allergy, the pathomechanism of the natural development of tolerance remains poorly understood. It has been suggested that the balance between milk‐specific IgE and IgG4 plays a major role.

Relationship of IgE to basophil phenotypes in peanut-sensitized adults.

reduced percentage of IFN-g positive cells (see Fig E2, A, in this article’s Online Repository at www.jacionline.org). However, IL17 and IFN-g CD4 lymphocytes did not increase in H2Rdeficient animals, suggesting that CD4 cells are not the cellular source for elevated cytokine secretion. No changes in IL-17 or IFN-g lymphocytes was observed in mesenteric lymph node cells (see Fig E2, B). The percentage of Foxp3 cells within PPs was similar in wild-type and H2R-deficient animals and levels remained unaltered after L saerimneri 30a gavage (results not shown). The biological consequences of biogenic amine secretion in vivo by the resident microbiota are largely unknown. Histamine can have both proinflammatory and anti-inflammatory effects on immunoregulatory processes, depending on which histamine receptor is activated. With the exception of scombroid poisoning, it is currently unknownwhether histamine secretion by the microbiota is altered during, or contributes to, mucosal inflammatory disorders. Interestingly, L saerimneri 30a secretes approximately 100-fold higher levels of histamine, compared with the levels previously described for L rhamnosus. This suggests that the amount of histamine secreted by a microbe may be critical in determining its pathological versus protective effects. Maintenance of mucosal homeostasis is heavily dependent on appropriate sampling and processing of microbial ligands by the innate immune system, in particular dendritic cells. In vitro studies have demonstrated that TLR responses to microbial ligands are significantly influenced by histamine signaling through H2R. In this report, we demonstrate that the H2R is required for IL-6 and IL-17 mucosal responses, suggesting that H2R is a critical immunoregulatory receptor that significantly influences the in vivo immune response to histamine-secreting microbes within the intestine. These observations suggest that histamine from the enteric microbes might exert immunoregulatory effects in vivo; however, it remains to be determined, on a case-by-case basis, whether these effects are protective or pathological. This report also highlights the need to carefully select and screen putative probiotic bacterial strains before human consumption. It is unknown whether the pathological effects seen in mice after the administration of L saerimneri 30a are solely due to histamine secretion because this bacterium also secretes cadaverine, which was previously demonstrated to be toxic in rodents. Regardless of the mechanism, this is a cautionary note, which clearly stresses that not all Lactobacilli strains can be considered safe. Ruth Ferstl, PhD Remo Frei, PhD Elisa Schiavi, PhD Patrycja Konieczna, PhD Weronika Barcik, MSc Mario Ziegler, Dipl-Ing Roger P. Lauener, MD Christophe Chassard, PhD Christophe Lacroix, PhD Cezmi A. Akdis, MD Liam O’Mahony, PhD

Evaluation of flu vaccines with regard to their egg protein content.

Approximately 20% of the US population contracts influenza every year. Flu vaccines have been used as a preventive measure since 1945. However, as a result of the production methods utilized for generating influenza vaccines, significant egg allergy is a contraindication to vaccination. Sensitivity to egg is among the leading causes for food allergy in children, affecting approximately 1.6% of pediatric population in the United States (1). Vaccines that are produced by injecting influenza virus into fertilized chicken eggs may contain small amounts of egg protein allergens, and immunization with these vaccines has been reported to provoke anaphylactic reactions in susceptible children (2). The American Academy of Pediatrics (AAP) recommends that influenza vaccines produced in eggs not be used in patients with severe systemic allergic reactions to egg. The AAP also states that prior to immunization, children with egg allergy and asthma should undergo prick skin testing with any influenza vaccine containing ovalbumin (3). Previous studies indicate that even patients with significant egg allergy can be safely vaccinated in a 2-dose protocol when the preparation contains <1.2 lg/ml ovalbumin (4). The aim of our study was to evaluate influenza vaccine samples and select the lowest ovalbumin-containing lots for administration to egg-allergic patients. A highly sensitive competitive ELISA was developed to measure ovalbumin levels as low as 1 ng/ml. We measured and compared egg-white protein concentrations of several flu vaccine lots from 1997 to 2010 to identify lots that were safe for administration in egg-allergic patients. Based on the results of our studies, over the last several years, we have administered influenza vaccine to patients, known to be sensitive to hen’s egg, without adverse events. Since the 1997–1998 flu season, 67 lots of vaccine have been tested, demonstrating manufacturer-to-manufacturer, as well as lot-to-lot variability (Fig. 1). In brief, Immulon 4HBX medium binding (Thermo Fisher Scientific Inc. Waltham, MA) plates were coated with Sigma Ovalbumin Grade VII (St. Louis, MO, USA) overnight at a concentration of 3000 ng/cm. Standard curve dilutions from 1 to 30,000 ng/ml and sample serial dilutions from 1:2 to 1:32 were prepared. Aliquots containing standard curve and vaccine sample dilutions were incubated with rabbit anti-ovalbumin (Millipore, Billerica, MA, USA) antibody solution at concentration of 3.3 nm for 2 h at 37 C. These reaction mixtures were then transferred to the plates previously coated with ovalbumin and blocked with 2% Bovine Serum Albumin (BSA) in Phosphate-buffered sailne with Tween 20 at room temperature and incubated for 1 h at 31 C. Following thorough washing, the plates were incubated with detecting goat anti-rabbit IgG – Peroxidase antibody (Sigma, St Louis, MO, USA) for 1 h at room temperature, washed and developed with SureBlue TMB 1-Component Microwell Peroxidase Substrate (KPL, Gaithesburg, MD, USA). The results were read on a Spectra Max ELISA reader and analyzed with SoftMaxPro v5 software, Molecular Devices Inc., Sunnyvale, CA. The brands and lots with the lowest level of

A new Luminex-based peptide assay to identify reactivity to baked, fermented, and whole milk

The majority of children with cows milk allergy (CMA) tolerate baked milk. However, reactivity to fermented milk products such as yogurt/cheese has not been previously evaluated. We sought to determine whether children with CMA could tolerate yogurt/cheese and whether a patients IgE and IgG4‐binding pattern to milk protein epitopes could distinguish clinical reactivity.

Partially Hydrolyzed Whey Formula Intolerance in Cow's Milk Allergic Patients

class, may differ in immune reactivity only for the characteristics of the side chains. Thus, the reactivity of the immune system to one compound of one drug class may result in unexpected cross-reactivity to similar compounds. This crossreactivity of the immune system with similar drugs depends on the structural affinity and precursor frequency of drug-specific T (or B) cells. Drugs, which may differ in only an OH group, might be result more cross-reactive than drugs which are different in a longer side chain (9). Cross-reactivity in IgEmediated reactions is more problematic, since in sensitized individuals, small amounts of drugs (which normally behave as haptens) may induced a cross-link drug-specific IgE on mast cells by hapten-carrier complexes. Therefore, acute symptoms reflecting cross-reactivity may arise even if the amount of drug is very low and the level of cross-reactivity is limited and restricted to a small part of drug-specific IgE. Other studies would be needed about possible cross-reactivity of midazolam with other drugs or other substances occurring in nature (10).This case also confirms that skin reactivity persists for a longer period than circulating IgE, and is therefore more sensitive. In conclusion, this case demonstrates that midazolam can induce hypersensitivity reactions. Accordingly, we advise that this possibility should be considered when midazolam is administered during perioperative period in pediatric patients even if the clinical history is not indicative.