Degree of hydrolysis is a poor predictor of the sensitizing capacity of whey- and casein-based hydrolysates in a Brown Norway rat model of cow's milk allergy.

The use of infant formulas (IFs) based on hydrolyzed cow's milk proteins to prevent cow's milk allergy (CMA) is highly debated. The risk of sensitization to milk proteins induced by IFs may be affected by the degree of hydrolysis (DH) as well as other physicochemical properties of the cow's milk-based protein hydrolysates within the IFs. The immunogenicity (specific IgG1 induction) and sensitizing capacity (specific IgE induction) of 30 whey- or casein-based hydrolysates with different physicochemical characteristics were compared using an intraperitoneal model of CMA in Brown Norway rats. In general, the whey-based hydrolysates demonstrated higher immunogenicity than casein-based hydrolysates, inducing higher levels of hydrolysate-specific and intact-specific IgG1. The immunogenicity of the hydrolysates was influenced by DH, peptide size distribution profile, peptide aggregation, nano-sized particle formation, and surface hydrophobicity. Yet, only the surface hydrophobicity was found to affect the sensitizing capacity of hydrolysates, as high hydrophobicity was associated with higher levels of specific IgE. The whey- and casein-based hydrolysates exhibited distinct immunological properties with highly diverse molecular composition and physicochemical properties which are not accounted for by measuring DH, which was a poor predictor of sensitizing capacity. Thus, future studies should consider and account for physicochemical characteristics when assessing the sensitizing capacity of cow's milk-based protein hydrolysates.

Production of allergen-specific immunotherapeutic agents for the treatment of food allergy.

Allergen-specific immunotherapy (IT) is emerging as a viable avenue for the treatment of food allergies. Clinical trials currently investigate raw or slightly processed foods as therapeutic agents, as trials using food-grade agents can be performed without the strict regulations to which conventional drugs are subjected. However, this limits the ability of standardization and may affect clinical trial outcomes and reproducibility. Herein, we provide an overview of methods used in the production of immunotherapeutic agents for the treatment of food allergies, including processed foods, allergen extracts, recombinant allergens, and synthetic peptides, as well as the physical and chemical processes for the reduction of protein allergenicity. Commercial interests currently favor producing standardized drug-grade allergen extracts for therapeutic use, and clinical trials are ongoing. In the near future, recombinant production could replace purification strategies since it allows the manufacturing of pure, native allergens or sequence-modified allergens with reduced allergenicity. A recurring issue within this field is the inadequate reporting of production procedures, quality control, product physicochemical characteristics, allergenicity, and immunological properties. This information is of vital importance in assessing therapeutic standardization and clinical safety profile, which are central parameters for the development of future therapeutic agents.

Animal models of allergen-specific immunotherapy in food allergy: Overview and opportunities.

Food allergy is an adverse reaction to otherwise harmless proteins in food. The disease is a major health problem of growing concern, affecting approximately 5%-8% of young children and 2%-4% of adults. No accepted strategy exists for prevention and treatment of food allergy, and strict avoidance of the offending food is presently the only viable management option. Living with food avoidance may have a huge impact on the quality of life of food allergic patients, with daily fear of serious or even fatal reactions. The urgent need for safe and efficient food allergy treatment options has led to massive research efforts to develop and improve strategies for food allergy immunotherapeutic approaches. A first step in developing new and improved strategies of immunotherapy often involves the use of animal models. In present review, we provide an overview of animal studies of allergen-specific immunotherapy highlighting opportunities and challenges for each approach. The presented models, almost exclusively performed in mice, assess therapeutic efficacy and immunological outcomes following oral, intraperitoneal, subcutaneous, epicutaneous, and sublingual administration of native allergens, or preparations of hydrolyzed allergen, T-cell-directed peptides, or allergen with immunomodulatory adjuvants. Recently, approaches using immune cell therapy have demonstrated efficacy. Current models mainly assess anaphylaxis as the primary clinical outcome. With the increased appreciation that food allergy is a heterogeneous disease presenting different phenotypes, there is a continued need to develop new disease-relevant therapeutic models of food allergy.

Order of draw practices in venous blood sampling at clinical biochemistry departments in the Danish health care system.

BACKGROUND: Deviation in blood collection procedures is a central source of preanalytical variation affecting overall analytical and diagnostic precision. The order of draw of venous sampling is suspected to affect analytical results, in particular for coagulation analysis. Here we compare the procedures in venous blood sampling among clinical biochemistry departments to assess the uniformity of order of blood draw and adherence to international guidelines in the Danish health care system. METHODS: We collected venous order of draw procedures from 49 clinical biochemistry departments at 22 public hospitals in Denmark. Procedures were compared to the international guidelines fromthe Clinical Laboratory Standards Institute (CLSI) and World Health Organization (WHO), and assessed in relation to department ISO 15189:2012 accreditation. RESULTS: We observed seven different order of draw procedures related to citrate, serum, heparin, and EDTA tubes, and the use of discard tubes in relation to coagulation assays. 31 departments (63.3%) were found to adhere to CLSI and WHO guidelines. A majority of departments instructs the use of discard tubes before collection for coagulation assays in citrate tubes (44 departments; 89.8%). The citrate tube was the first sample tube to be drawn for most departments (35 departments; 75.5%); and the preferred order of non-citrate tubes was serum-heparin-EDTA (36 departments; 73.5%). Adherence to the CLSI and WHO guidelines was not associated with department ISO 15189:2012 accreditation (p = .57). CONCLUSIONS: Venous order of draw procedures is diverse at Danish clinical biochemistry departments and show moderate adherence to international guidelines.

JNK1, but not JNK2, is required in two mechanistically distinct models of inflammatory arthritis.

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.

TCR down-regulation controls virus-specific CD8+ T cell responses.

The CD3gamma di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3gamma di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8(+) T cells is impaired in mice with a mutated CD3gamma di-leucine-based motif. The CD3gamma mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8(+) T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3gamma-mediated TCR down-regulation in virus-specific CD8(+) T cell responses.