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BACKGROUND: Allergen extracts are the primary tool for diagnosis and treatment of allergic diseases. In the United States, most allergen extracts are non-standardized. More sophisticated analytical approaches are needed to characterize these products and enable manufacturers and regulators to better determine potency. OBJECTIVE: To expand the multiple reaction monitoring (MRM) assay for an in-depth characterization of German cockroach (GCr; Blattella germanica) allergen extracts. METHODS: We applied advanced liquid chromatography (LC) and mass spectrometry (MS) techniques including MRM. The expanded LC/MRM-MS method was optimized to measure known GCr allergens and their isoforms/variants in commercial extracts and environmental samples. We performed isoform-specific allergen measurements in multiple extracts from four commercial sources and extracts prepared using environmental samples from urban homes. To investigate causes of heterogeneity, we examined over 30 extraction process variables. RESULTS: Evaluation of the commercial extracts confirmed the variability of production lots and commercial sources. Commonly used defatting and extraction protocols yielded extracts with comparable allergen profiles and content. However, the identity and quality of source materials was a major contributor to variability. In comparing commercial GCr extracts to environmental samples, relative quantities of Bla g 1, Bla g 2, Bla g 3, Bla g 4 and Bla g 11 were similar, while Bla g 5, Bla g 6, Bla g 7 and Bla g 8 were present in the environmental samples but largely absent for the commercial extracts. CONCLUSIONS AND CLINICAL RELEVANCE: LC/MRM-MS can be used to measure all known GCr allergens in commercial allergen extracts and environmental samples. Significant differences exist between allergen profiles of commercial extracts and the profiles of environmental samples from dwellings. This analytical platform can serve as a template to achieve better product characterization of similarly complex products.
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Alérgenos/química , Blattellidae/química , Mezclas Complejas/química , Proteínas de Insectos/química , Animales , Cromatografía Liquida , Espectrometría de MasasRESUMEN
OBJECTIVE: To review the manufacturing procedures of food allergen extracts and applicable regulatory requirements from government agencies, potential approaches to standardization, and clinical application of these products. The effects of thermal processing on allergenicity of common food allergens are also considered. DATA SOURCES: A broad literature review was conducted on the natural history of food allergy, the manufacture of allergen extracts, and the allergenicity of heated food. Regulations, guidance documents, and pharmacopoeias related to food allergen extracts from the United States and Europe were also reviewed. STUDY SELECTIONS: Authoritative and peer-reviewed research articles relevant to the topic were chosen for review. Selected regulations and guidance documents are current and relevant to food allergen extracts. RESULTS: Preparation of a food allergen extract may require careful selection and identification of source materials, grinding, defatting, extraction, clarification, sterilization, and product testing. Although extractions for all products licensed in the United States are performed using raw source materials, many foods are not consumed in their raw form. Heating foods may change their allergenicity, and doing so before extraction may change their allergenicity and the composition of the final product. CONCLUSION: The manufacture of food allergen extracts requires many considerations to achieve the maximal quality of the final product. Allergen extracts for a select number of foods may be inconsistent between manufacturers or unreliable in a clinical setting, indicating a potential area for future improvement.
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Alérgenos/metabolismo , Alérgenos/uso terapéutico , Desensibilización Inmunológica/métodos , Hipersensibilidad a los Alimentos/diagnóstico , Hipersensibilidad a los Alimentos/terapia , Alérgenos/inmunología , Reacciones Cruzadas/inmunología , Hipersensibilidad a los Alimentos/inmunología , Calor , Humanos , Instalaciones Industriales y de Fabricación/normasRESUMEN
T cell development proceeds via discrete stages that require both gene induction and gene repression. Transcription factors direct gene repression by associating with corepressor complexes containing chromatin-remodeling enzymes; the corepressors NCOR1 and NCOR2 recruit histone deacetylases to these complexes to silence transcription of target genes. Earlier work identified the importance of NCOR1 in promoting the survival of positively-selected thymocytes. Here, we used flow cytometry and single-cell RNA sequencing to identify a broader role for NCOR1 and NCOR2 in regulating thymocyte development. Using Cd4-cre mice, we found that conditional deletion of NCOR2 had no effect on thymocyte development, whereas conditional deletion of NCOR1 had a modest effect. In contrast, Cd4-cre x Ncor1f/f x Ncor2f/f mice exhibited a significant block in thymocyte development at the DP to SP transition. Combined NCOR1/2 deletion resulted in increased signaling through the T cell receptor, ultimately resulting in elevated BIM expression and increased negative selection. The NF-κB, NUR77, and MAPK signaling pathways were also upregulated in the absence of NCOR1/2, contributing to altered CD4/CD8 lineage commitment, TCR rearrangement, and thymocyte emigration. Taken together, our data identify multiple critical roles for the combined action of NCOR1 and NCOR2 over the course of thymocyte development.
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Inducing a strong and specific immune response is the hallmark of a successful vaccine. Nanoparticles have emerged as promising vaccine delivery devices to discover and elicit immune responses. Fine-tuning a nanoparticle vaccine to create an immune response with specific antibody and other cellular responses is influenced by many factors such as shape, size, and composition. Peptide amphiphile micelles are a unique biomaterials platform that can function as a modular vaccine delivery system, enabling control over many of these important factors and delivering payloads more efficiently to draining lymph nodes. In this study, the modular properties of peptide amphiphile micelles are utilized to improve an immune response against a Group A Streptococcus B cell antigen (J8). The hydrophobic/hydrophilic interface of peptide amphiphile micelles enabled the precise entrapment of amphiphilic adjuvants which were found to not alter micelle formation or shape. These heterogeneous micelles significantly enhanced murine antibody responses when compared to animals vaccinated with nonadjuvanted micelles or soluble J8 peptide supplemented with a classical adjuvant. The heterogeneous micelle induced antibodies also showed cross-reactivity with wild-type Group A Streptococcus providing evidence that micelle-induced immune responses are capable of identifying their intended pathogenic targets.
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Delivery system design and adjuvant development are crucially important areas of research for improving vaccines. Peptide amphiphile micelles are a class of biomaterials that have the unique potential to function as both vaccine delivery vehicles and self-adjuvants. In this study, peptide amphiphiles comprised of a group A streptococcus B cell antigen (J8) and a dialkyl hydrophobic moiety (diC16) were synthesized and organized into self-assembled micelles, driven by hydrophobic interactions among the alkyl tails. J8-diC16 formed cylindrical micelles with highly α-helical peptide presented on their surfaces. Both the micelle length and secondary structure were shown to be enhanced by annealing. When injected into mice, J8-diC16 micelles induced a strong IgG1 antibody response that was comparable to soluble J8 peptide supplemented with two classical adjuvants. It was discovered that micelle adjuvanticity requires the antigen be a part of the micelle since separation of J8 and the micelle was insufficient to induce an immune response. Additionally, the diC16 tail appears to be non-immunogenic since it does not stimulate a pathogen recognition receptor whose agonist (Pam3Cys) possesses a very similar chemical structure. The research presented in this paper demonstrates the promise peptide amphiphile micelles have in improving the field of vaccine engineering.