Cue polarization and representation in mouse home base behaviors.

The nature of the representation guiding spatial navigation has been investigated extensively; however, most of this work has used behavioral tasks that involved learning the location of food reward or an escape platform. In contrast, relatively few studies have focused on the spatial representation of a home base, a ubiquitous feature of open-field behavior, and its ability to be encoded relative to environmental cues. The current set of experiments investigated acquisition and retention of the location of home base establishment. In general, proximal cues anchored the position of the home base during acquisition sessions across all four experiments. Although mice established a home base during retention sessions, previous experience did not influence its position during retention sessions. These observations demonstrate that stimulus control of home base position depends on access to proximal cues. Further work is needed to determine the extent that home base establishment may provide a framework to encode goal-directed spatial behaviors.

Gata3 hypermethylation and Foxp3 hypomethylation are associated with sustained protection and bystander effect following epicutaneous immunotherapy in peanut-sensitized mice.

BACKGROUND: Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). METHODS: BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE-patch or by PPE-OIT. Another set of peanut-sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analyzed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. RESULTS: Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4th week and a significant hypomethylation of the Foxp3 promoter in CD62L+ Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. CONCLUSIONS: Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T-cell compartments with downregulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect.

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.

Performance of a polymer coated silicon microarray for simultaneous detection of food allergen-specific IgE and IgG4.

BACKGROUND: Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen-specific IgE sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless, specificity and sensitivity as well as a simultaneous detection of allergen-specific IgG4 , as a potential parameter for tolerance development, remain to be optimized. OBJECTIVE: We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen-specific IgE and IgG4 , and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intraslide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. METHODS: Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4 . A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4 -specific fluorescence on silicon microarrays. RESULTS: Allergen-specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg-specific IgE was comparable to the range of 0.1 to >100 kUA /L of the ImmunoCAP system, whereas for total cow's milk, the silicon microarray was less sensitive. Detectable allergen-specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. CONCLUSIONS: We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low-cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research.

Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

BACKGROUND: We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. OBJECTIVE: We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). METHODS: We measured specific IgA to a series of overlapping peptides in major CM allergens (αs1 -, αs2 -, ß- and κ-caseins and ß-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. RESULTS: There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in αs1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. CONCLUSION & CLINICAL RELEVANCE: These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated.

Treatment of gastric eosinophilia by epicutaneous immunotherapy in piglets sensitized to peanuts.

BACKGROUND: Eosinophilic gastrointestinal disorders (EGIDs) are hypersensitivity disorders frequently triggered by food allergy and manifested by mucosal eosinophilic infiltration at any level of the gastrointestinal tract. This study established a model of gastric eosinophilia in peanut-sensitized piglets to evaluate the efficacy of epicutaneous immunotherapy (EPIT) for its treatment. METHODS: Experiments were carried out in piglets first sensitized by three intra-peritoneal injections of peanut protein extract (PPE) with adjuvant, and then given PPE orally for 10 days, a sequence leading to gastric eosinophilia assessed by endoscopy. For 3 months, eight piglets received active EPIT, using Viaskin® loaded with PPE, applied daily on the ear, while eight received placebo EPIT (Placebo). Piglets were exposed to a second 10-day period of PPE orally. Lesions were scored by endoscopy on the last day of PPE exposure. After killing, all parts of the digestive tract were analysed by a pathologist unaware of the piglets' status. IgE response was measured, and mechanistic parameters were analysed in the spleen. RESULTS: After sensitization, a significant increase of total IgE was observed in sensitized compared to naive animals (61.1 ± 13.3 vs 27.8 ± 6 ng/mL, P < .01). Following oral intake of PPE, sensitized piglets developed moderate gastritis compared to naive piglets (1.5 vs 1.0, median score). After 3 months of immunotherapy, median IgE was significantly reduced in EPIT vs placebo piglets (61.4 ± 16.3 vs 105.9 ± 25.6 ng/mL, P < .01). Active EPIT significantly reduced gastric mucosal lesions induced by PPE oral intake (macroscopic score 0 [0-2] vs 2 [1-3], P < .01, respectively, active vs placebo) and gastric mucosa eosinophils counts (239 eosinophils/mm2 [59-645] vs 2554 eosinophils/mm2 [462-8057], P < .01, respectively active vs placebo). GATA-3, IL-5 and eotaxin mRNA expression decreased significantly after EPIT (P < .05). CONCLUSIONS: This study describes a large animal model of gastric eosinophil in peanut-sensitized piglets. Utilizing this model, we demonstrated the efficacy of EPIT in treating peanut-induced EGIDs.

B-FAHF-2 plus oral immunotherapy (OIT) is safer and more effective than OIT alone in a murine model of concurrent peanut/tree nut allergy.

BACKGROUND: Concurrent sensitization to peanut (PN) and tree nuts (TN), the most dangerous food allergies, is common. Current oral immunotherapy (OIT) is not fully satisfactory. OBJECTIVE: To determine whether the herbal formula B-FAHF-2 (BF2) ameliorates PN/TN OIT adverse reactions and enhances persistence of a tolerant state. METHODS: Concurrently sensitized PN-, walnut- (WN) and cashew (CSH)-allergic mice received 1-day PN/WN/CSH rush OIT plus 3 weeks of maintenance dosing, with or without 3 weeks prior and 3 weeks BF2 co-treatment. Anaphylactic symptom scores, core body temperatures, plasma histamine levels, basophil numbers, antigen-specific IgE, cytokine levels, and IL-4, INF-γ and Foxp3 gene promoter DNA methylation status, and their correlation with final challenge symptom scores were determined. RESULTS: BF2+OIT-treated mice experienced significantly fewer and less severe adverse reactions than OIT-only-treated mice (P<.01) during the 1-day rush OIT build-up dose phase. Both OIT-only and BF2+OIT mice showed significant desensitization (P<.01 and .001, respectively) at 1 week post-therapy challenge, being greater in BF2+OIT mice. All sham-treated and 91% of OIT-treated mice experienced anaphylaxis whereas only 21% of BF2+OIT-treated mice exhibited reactions during 5-6 weeks of dose escalation single PN and TN challenges. Greater and more persistent protection in BF2+OIT mice was associated with significantly lower plasma histamine and IgE levels, increased IFN-γ/IL-4 and IL-10/IL-4 ratios, DNA remethylation at the IL-4 promoter and demethylation at IFN-γ and Foxp3 promoters. Final challenge symptom scores were inversely correlated with IL-4 DNA methylation levels (P<.0002) and positively correlated with IFN-γ and Foxp3 gene promoter methylation levels (P<.0011) (P<.0165). CONCLUSIONS AND CLINICAL RELEVANCE: Combined BF2/OIT therapy was safer and produced longer post-treatment protection and more tolerance-prone immunological and epigenetic modifications than OIT alone. BF2/OIT may provide an additional OIT option for patients with concurrent PN/TN and other food allergies.

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

BACKGROUND: Although most of cow's 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. OBJECTIVE: We aimed to investigate differences in IgE and IgG4 antibody binding to CM epitopes between patients with persistent CM allergy (CMA) and those that naturally became tolerant. METHODS: Sera from 35 children with proven CMA (median age at inclusion of 10 months) were analyzed retrospectively; 22 patients have become tolerant (median age at tolerance acquisition of 51 months) during the study period as confirmed by a negative oral food challenge. IgE and IgG4 binding to sequential epitopes derived from five major CM proteins were measured with a peptide microarray-based immunoassay. RESULTS: At baselines, greater intensity and broader diversity of IgE and IgG4 binding have been found in children with persistent CMA beyond 5 years of age compared to patients with transient CMA. Moreover, children with transient CMA had IgE and IgG4 antibodies that more often recognized the same epitopes, compared to those with persistent CMA. From baseline to the time of tolerance development, both IgE and IgG4 binding intensity decreased significantly, particularly in areas of α-s- and ß-casein (P<.01, false discovery rate [FDR]<.1). Interestingly, differences between IgE and IgG4 binding intensity to CM peptides decreased when the patients became tolerant. CONCLUSIONS: Our results suggest that the overlap between IgE and IgG4 might be important in natural tolerance acquisition. Further studies are needed to confirm our data and can eventually lead to development of more targeted treatment of food allergy.

Precision medicine in allergic disease-food allergy, drug allergy, and anaphylaxis-PRACTALL document of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma and Immunology.

This consensus document summarizes the current knowledge on the potential for precision medicine in food allergy, drug allergy, and anaphylaxis under the auspices of the PRACTALL collaboration platform. PRACTALL is a joint effort of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma and Immunology, which aims to synchronize the European and American approaches to allergy care. Precision medicine is an emerging approach for disease treatment based on disease endotypes, which are phenotypic subclasses associated with specific mechanisms underlying the disease. Although significant progress has been made in defining endotypes for asthma, definitions of endotypes for food and drug allergy or for anaphylaxis lag behind. Progress has been made in discovery of biomarkers to guide a precision medicine approach to treatment of food and drug allergy, but further validation and quantification of these biomarkers are needed to allow their translation into practice in the clinical management of allergic disease.

Allergen immunotherapy for IgE-mediated food allergy: a systematic review and meta-analysis.

BACKGROUND: The European Academy of Allergy and Clinical Immunology (EAACI) is developing Guidelines for Allergen Immunotherapy (AIT) for IgE-mediated Food Allergy. To inform the development of clinical recommendations, we sought to critically assess evidence on the effectiveness, safety and cost-effectiveness of AIT in the management of food allergy. METHODS: We undertook a systematic review and meta-analysis that involved searching nine international electronic databases for randomized controlled trials (RCTs) and nonrandomized studies (NRS). Eligible studies were independently assessed by two reviewers against predefined eligibility criteria. The quality of studies was assessed using the Cochrane Risk of Bias tool for RCTs and the Cochrane ACROBAT-NRS tool for quasi-RCTs. Random-effects meta-analyses were undertaken, with planned subgroup and sensitivity analyses. RESULTS: We identified 1814 potentially relevant papers from which we selected 31 eligible studies, comprising of 25 RCTs and six NRS, studying a total of 1259 patients. Twenty-five trials evaluated oral immunotherapy (OIT), five studies investigated sublingual immunotherapy, and one study evaluated epicutaneous immunotherapy. The majority of these studies were in children. Twenty-seven studies assessed desensitization, and eight studies investigated sustained unresponsiveness postdiscontinuation of AIT. Meta-analyses demonstrated a substantial benefit in terms of desensitization (risk ratio (RR) = 0.16, 95% CI 0.10, 0.26) and suggested, but did not confirm sustained unresponsiveness (RR = 0.29, 95% CI 0.08, 1.13). Only one study reported on disease-specific quality of life (QoL), which reported no comparative results between OIT and control group. Meta-analyses revealed that the risk of experiencing a systemic adverse reaction was higher in those receiving AIT, with a more marked increase in the risk of local adverse reactions. Sensitivity analysis excluding those studies judged to be at high risk of bias demonstrated the robustness of summary estimates of effectiveness and safety of AIT for food allergy. None of the studies reported data on health economic analyses. CONCLUSIONS: AIT may be effective in raising the threshold of reactivity to a range of foods in children with IgE-mediated food allergy whilst receiving (i.e. desensitization) and post-discontinuation of AIT. It is, however, associated with a modest increased risk in serious systemic adverse reactions and a substantial increase in minor local adverse reactions. More data are needed in relation to adults, long term effects, the impact on QoL and the cost-effectiveness of AIT.

Identification and characterization of DC-SIGN-binding glycoproteins in allergenic foods.

BACKGROUND: DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) is a C-type lectin receptor expressed on macrophages and dendritic cells. DC-SIGN has high affinity for fucosylated glycans in several plant glycoproteins and pathogens. DC-SIGN is thought to be crucial for the development of allergic sensitization. However, the precise role of DC-SIGN in food allergy pathogenesis is not yet understood. OBJECTIVE: We sought to characterize DC-SIGN-binding glycoproteins in a panel of allergenic and non-allergenic foods. METHODS: Fluorescent-labeled peanut and soy extracts were used to test protein binding to human monocyte-derived dendritic cells (DCs) by flow cytometry. DC-SIGN-blocking assays were performed by incubating DCs with food extracts followed by staining with anti-DC-SIGN antibody. Using a DC-SIGN-Fc chimera, food extracts were tested for binding by ELISA and autoradiography. IgE immunoblotting was performed with pooled sera from food-allergic subjects. DC activation and maturation were assessed by flow cytometry. RESULTS AND CONCLUSIONS: We demonstrate that peanut agglutinin, a minor peanut allergen, is a novel ligand for DC-SIGN. Peanut agglutinin activates DCs to induce the expression of costimulatory molecules in vitro. We present a comprehensive report on the characterization of DC-SIGN-binding proteins in common allergenic foods such as peanut, soy, tree nuts, egg, and milk. Foods that rarely induce allergy, such as pine nuts, chickpea, and corn, showed no binding to DC-SIGN. Several DC-SIGN-binding proteins show reactivity in serum IgE immunoblots. We have also identified novel non-IgE-binding proteins that interact with DC-SIGN; these proteins may be important for regulating immune responses to these foods.

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.

Role of maternal elimination diets and human milk IgA in the development of cow's milk allergy in the infants.

BACKGROUND: The role of maternal avoidance diets in the prevention of food allergies is currently under debate. Little is known regarding the effects of such diets on human milk (HM) composition or induction of infant humoral responses. OBJECTIVE: To assess the association of maternal cow's milk (CM) avoidance during breastfeeding with specific IgA levels in HM and development of cow's milk allergy (CMA) in infants. METHODS: We utilized HM and infant serum samples from a prospective birth cohort of 145 dyads. Maternal serum and HM samples were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG by ELISA; 21 mothers prophylactically initiated a strict maternal CM avoidance diet due to a sibling's history of food allergy and 16 due to atopic eczema or regurgitation/vomiting seen in their infants within the first 3 months of life. Infants' sera were assessed for casein and BLG-specific IgG, IgA and IgE; CMA was confirmed by an oral food challenge. The impact of HM on BLG uptake was assessed in transcytosis assays utilizing Caco-2 intestinal epithelial cell line. RESULTS: Mothers avoiding CM had lower casein- and BLG-specific IgA in HM than mothers with no CM restriction (P = 0.019 and P = 0.047). Their infants had lower serum casein- and BLG-specific IgG(1) (P = 0.025 and P < 0.001) and BLG-specific IgG(4) levels (P = 0.037), and their casein- and BLG-specific IgA levels were less often detectable than those with no CM elimination diet (P = 0.003 and P = 0.007). Lower CM-specific IgG4 and IgA levels in turn were associated with infant CMA. Transcytosis of BLG was impaired by HM with high, but not low levels of specific IgA. CONCLUSIONS: Maternal CM avoidance was associated with lower levels of mucosal-specific IgA levels and the development of CMA in infants. CLINICAL RELEVANCE: HM IgA may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen and thereby in the prevention of CMA.

In silico prediction of Ara h 2 T cell epitopes in peanut-allergic children.

BACKGROUND: Despite the frequency and severity of peanut allergy, the only approved treatment is strict avoidance. Different types of immunotherapy with crude peanut extract are not universally effective and have been associated with relatively high adverse reaction rates. OBJECTIVE: We sought to determine whether in silico predictive algorithms were useful in identifying candidate peptides for an Ara h 2 peptide-based vaccine using peanut-allergic patients' peripheral blood mononuclear cells (PBMCs) in vitro. A human leucocyte antigen (HLA) distribution analysis was also performed. METHODS: Major histocompatibility complex (MHC)-class II-binding peptides were predicted using NetMHCIIpan-2.0 and NetMHCII-2.2 algorithms. PBMCs from 80 peanut-allergic patients were stimulated with overlapping 20-mer Ara h 2 peptides. Cell supernatant cytokine profiles were evaluated by multiplex assays. HLA-DRB1* and HLA-DQB1* typing were performed. RESULTS: Four regions of overlapping sequences induced PBMC proliferation and predominant Th2 cytokine production. HLA genotyping showed 30 different DRB1* allele specificities and eight DQ serological specificities. The in silico analysis revealed similar relevant regions and predicted identical or similar core 9-mer epitopes to those identified in vitro. If relevant peptides, as determined by either in vitro or in silico analysis (15 peptides and 9 core epitopes respectively), were used in a peptide-based vaccine, they would cover virtually all subjects in the cohort studied. CONCLUSIONS AND CLINICAL RELEVANCE: Four dominant regions in Ara h 2 have been identified, containing sequences that could serve as potential candidates for peptide-based immunotherapy. MHC-class II-based T cell epitope prediction algorithms for HLA-DR and -DQ loci accurately predicted Ara h 2 T cell epitopes in peanut-allergic subjects, suggesting their potential utility in a peptide-based vaccine design for food allergy.

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%.

Marrow stimulation improves meniscal healing at early endpoints in a rabbit meniscal injury model.

PURPOSE: To critically evaluate the effect of marrow stimulation (MS) on the extent of healing and the local biological environment after meniscal injury in ligamentously stable knees in a rabbit model. METHODS: A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in 18 New Zealand White rabbits (36 knees). In right knees (MS knees), a 2.4-mm Steinman pin was drilled into the apex of the femoral intercondylar notch and marrow contents were observed spilling into the joint. Left knees served as controls. Rabbits were killed in 3 groups (n = 6 rabbits each) at 1, 4, and 12 weeks with meniscal harvest and blinded histomorphometric and histologic evaluation using an established 3-component tissue quality score (range, 0 to 6). One-week specimens were also evaluated for the presence of proregenerative cytokines using immunohistochemistry. RESULTS: The mean proportion of the avascular zone defect bridged by reparative tissue was greater in MS knees than in controls at each endpoint (1 week, 55% v 30%, P = .02; 4 weeks, 71% v 53%, P = .047; 12 weeks, 96% v 77%, P = .16). Similarly, there was a consistent trend toward superior tissue quality scores in knees treated with MS compared with controls (1 week, 1.8 v 0.3, P = .03; 4 weeks, 4.3 v 2.8, P = .08; 12 weeks, 5.9 v 4.5, P = .21). No statistically significant differences, however, were observed at the 12-week endpoint. Increased staining for insulin-like growth factor I, transforming growth factor-ß, and platelet-derived growth factor was observed in regenerated tissue, compared with native meniscal tissue, in all specimens at 1 week. Staining density for all growth factors was similar, however, in reparative tissue of MS and control knees. CONCLUSIONS: The results of this study suggest that marrow stimulation leads to modest improvements in quality and quantity of reparative tissue bridging a meniscal defect, particularly during the early recovery period. CLINICAL RELEVANCE: Clinical evaluation of marrow stimulation techniques designed to enhance healing in isolated meniscus repair surgery may be indicated.

Presence of functional, autoreactive human milk-specific IgE in infants with cow's milk allergy.

BACKGROUND: Occasionally, exclusively breastfed infants with cow's milk allergy (CMA) remain symptomatic despite strict maternal milk avoidance. OBJECTIVE: To determine whether or not persistence of symptoms could be due to sensitization against endogenous human milk proteins with a high degree of similarity to bovine allergens. METHODS: Ten peptides representing known bovine milk IgE-binding epitopes [α-lactalbumin (ALA), ß- and κ-casein] and the corresponding, highly homologous human milk peptides were labelled with sera from 15 breastfed infants with CMA, aged 3 weeks to 12 months, and peptide (epitope)-specific IgE antibodies were assessed. Nine of the 15 breastfed infants became asymptomatic during strict maternal avoidance of milk and other major food allergens; six infants remained symptomatic until weaned. Ten older children, aged 5-15 years, with CMA were also assessed. The functional capacity of specific IgE antibodies was assessed by measuring ß-hexosaminidase release from rat basophilic leukaemia cells passively sensitized and stimulated with human and bovine ALA. RESULTS: A minimum of one human milk peptide was recognized by IgE antibodies from 9 of 15 (60%) milk-allergic infants, and the majority of older children with CMA. Genuine sensitization to human milk peptides in the absence of IgE to bovine milk was occasionally seen. There was a trend towards specific IgE being detected to more human milk peptides in those infants who did not respond to the maternal milk elimination diet than in those who did (P = 0.099). Functional IgE antibody to human ALA was only detected in infants not responding to the maternal diet. CONCLUSIONS AND CLINICAL RELEVANCE: Endogenous human milk epitopes are recognized by specific IgE from the majority of infants and children with CMA. Such autoreactive, human milk-specific IgE antibodies appear to have functional properties in vitro. Their role in provoking allergic symptoms in infants exclusively breastfed by mothers strictly avoiding dietary milk remains unclear.

Is epitope recognition of shrimp allergens useful to predict clinical reactivity?

BACKGROUND: Shrimp is a frequent cause of severe allergic reactions world-wide. Due to issues such as cross-reactivity, diagnosis of shrimp allergy is still inaccurate, requiring the need for double-blind, placebo-controlled food challenges (DBPCFC). A better understanding of the relationship between laboratory findings and clinical reactivity is needed. OBJECTIVE: To determine whether sensitization to certain shrimp allergens or recognition of particular IgE epitopes of those allergens are good biomarkers of clinical reactivity to shrimp. METHODS: Thirty-seven consecutive patients were selected with clinical histories of shrimp allergy. Skin prick test, specific IgE determinations, DBPCFC and immunoblot assays to shrimp extract were performed. IgE binding to synthetic overlapping peptides representing the sequence of the four allergens from the Pacific white shrimp (Litopenaeus vannamei) identified to date (Lit v1, Lit v2, Lit v3 and Lit v4) was analysed. RESULTS: Of 37 (46%) patients, 17 had a positive challenge to shrimp (11 children and 6 adults). By microarray, patients with positive challenges showed more intense binding to shrimp peptides than those with negative challenges. Statistically significant differences in terms of the frequency and intensity of IgE binding to some epitopes were observed between the two groups. Diagnostic efficiency was higher for individual epitopes than for proteins. Particularly, efficiency was highest for certain Lit v 1 and Lit v 2 epitopes, followed by Lit v 3 and Lit v 4 epitopes. CONCLUSION AND CLINICAL RELEVANCE: Patients with positive shrimp challenges present in general more intense and diverse epitope recognition to all four shrimp allergens. IgE antibodies to these shrimp epitopes could be used as biomarkers for prediction of clinical reactivity in subjects with sensitization to shrimp. Patients with positive shrimp challenges show more intense sensitization and more diverse epitope recognition. Several IgE-binding shrimp epitopes could be used as biomarkers for predicting clinical reactivity in subjects with sensitization to shrimp.

Efficacy, safety and immunological actions of butanol-extracted Food Allergy Herbal Formula-2 on peanut anaphylaxis.

BACKGROUND: Therapies for peanut allergy (PNA) are urgently needed. Food Allergy Herbal Formula-2 (FAHF-2) has profound therapeutic effects in a murine PNA model and is safe for food-allergic adults in clinical trials. However, the large FAHF-2 pill-load is not conducive to clinical studies in children. Thus, refining FAHF-2 to decrease pill-load is essential for the inclusion of children in clinical trials and to facilitate studying FAHF-2 as a clinically useful botanical drug. OBJECTIVES: Testing long-term efficacy and safety of a butanol-purified extract of FAHF-2 (B-FAHF-2) in a murine model of PNA, and to explore its immunological mechanisms of action. METHODS: FAHF-2 was purified by butanol extraction. C3H/HeJ mice with established PNA received the first course of B-FAHF-2 at 6 mg, twice daily for 7 weeks (PNA/B-FAHF-2) or water (PNA/sham) and were then challenged immediately after completing the treatment and six more times every 1-2 months post-treatment up to week 50. Mice then received a second course of B-FAHF-2 treatment at week 52 and were challenged at week 65. In vivo and in vitro immunological effects on T, B and mast cells were also determined. RESULTS: Butanol purification reduced the volume of the effective dose ∼5-fold. All PNA/B-FAHF-2 mice were completely protected from PN anaphylaxis until the fifth challenge after the first course of treatment, as compared with PNA/sham mice. Partial protection persisted up to 50 weeks. A second treatment course restored complete protection. B-FAHF-2 significantly suppressed Th2 cytokine, IgE and histamine levels in vivo, and showed direct inhibition of Th2, IgE-producing B cells and mast cell activation in vitro. B-FAHF-2 had a high margin of safety. CONCLUSION AND CLINICAL RELEVANCE: B-FAHF-2 produced long-lasting protection against PN anaphylaxis for approximately half of the murine life span without side-effects. B-FAHF-2 exhibited direct effects on multiple food allergy effector cells.