IntroductionImmunoglobulin A (IgA) is mostly considered as a non-inflammatory regulator at mucosal areas. However, previous work of our group showed that IgA can also be involved in disease pathology, because it provides a potent stimulus to activate neutrophils after crosslinking of surface CD89 (FcaRI), resulting in chronic inflammation and tissue damage. IgA (auto)antibodies and neutrophils are key players in various diseases, including blistering skin diseases and rheumatoid arthritis. Therefore, we generated an array of anti-CD89 monoclonal antibodies (mAbs) for therapeutic targeting of CD89. The biological activity of newly developed anti-human CD89 mAbs and their potential therapeutic capacity were investigated.MethodsHuman neutrophils were isolated from heparinized healthy donor blood. The ability of anti-CD89 mAbs to bind human neutrophils was investigated by flow cytometry. Furthermore, the capacity of these anti-CD89 mAbs to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and migration was studied. To this end, neutrophils were pre-incubated with/without anti-CD89 mAbs after which they were stimulated with IgA-coated beads. The amount of phagocytosed beads, NET release and migrated neutrophils were subsequently analysed. In parallel, chemoattractant leukotriene B4 and lactoferrin (as a measure for degranulation) release were determined. Finally, the therapeutic potential of our prototypic anti-CD89 mAb clone 10E7 was in vivo tested in anti-mouse collagen XVII human IgA-treated transgenic CD89 mice, a preclinical model for autoimmune linear IgA bullous disease (LABD).ResultsOur results show that all generated anti-CD89 mAbs bound surface CD89 on neutrophils. Although these anti-CD89 mAbs bind to different epitopes on EC1 of CD89, they all have the capacity to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and neutrophil migration. Moreover, IgA mediated leukotriene B4 and lactoferrin release are decreased in supernatant from anti-CD89 mAbs-treated neutrophils. Finally, anti-CD89 mAb clone 10E7, that was selected based on its selective binding profile on tissue micro arrays, reduced anti-mouse collagen XVII hIgA-induced neutrophil influx in an in vivo linear IgA bullous disease (LABD) mice model.ConclusionThis study clearly indicates that our newly developed anti-CD89 mAbs inhibited IgA-induced neutrophil activation and reduced anti-autoantigen IgA-induced neutrophil influx in vivo, supporting further clinical development for the treatment of LABD.

INTRODUCTION:Hypoallergenic formulas prepared from hydrolyzed cow's milk proteins are often used for the management of cow's milk allergy (CMA) in infants. In this study, both in vitro assays and an in vivo mouse model for CMA were used to assess the sensitizing and allergenic potential of a newly developed, extensive whey hydrolysate (eWH).METHODS:Gel permeation chromatography was used to characterize the molecular weight distribution of the peptides. Residual antigenicity was measured using a beta-lactoglobulin ELISA as well as with immunoblotting using anti-beta-lactoglobulin (BLG) and anti-alpha-lactalbumin antibodies. In vitro residual allergenicity was assessed using huFcεRIα-RBL-2H3 cells sensitized with anti-bovine BLG human IgE. In vivo sensitizing and allergenic potential was assessed in a CMA mouse model by measuring the acute allergic skin response, anaphylactic shock score, body temperature, serum mMCP-1, whey-specific IgE, and cytokines.RESULTS:There was no in vitro residual antigenicity and allergenicity observed of the eWH. Mice sensitized with eWH showed no acute allergic skin reaction after challenge with whey, confirmed by an absence of whey-specific IgE and anaphylactic symptoms and decrease in body temperature and mMCP-1 levels.CONCLUSIONS:Results from our in vitro and in vivo translational approach to assess sensitization capacity and residual allergenicity indicate that the newly developed eWH is safe for use in CMA infants. This was subsequently confirmed in a clinical study in which this eWH was tolerated by more than 90% (with 95% confidence) of infants or children with confirmed CMA.

Background: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. Objective: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. Methods: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. Results: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. Conclusions: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

Background: beta-lactoglobulin (BLG) is one of the major cow’s milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity. Primary objective: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS). Secondary objective: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells. Methods: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used. Results: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)—K47, K60, K75, K77, K91, K138 and K141—have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding (p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells (p < 0.0001). Conclusions and clinical relevance: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow’s milk allergens provided that the process is well-controlled.

Abstract Background Food allergy is a common disease that is caused by intake of allergen-containing foods such as milk, eggs, peanuts, and wheat. Systemic anaphylaxis is a severe hypersensitive allergic reaction resulting from degranulation of mast cells or basophils after crosslinking of their surface high affinity IgE receptors (Fce-RI) by allergen-specific IgE and allergens. In this study, we developed a novel human mast cell/basophil-engrafted mouse model that recapitulates systemic anaphylaxis by milk’s major allergen beta-lactoglobulin (BLG). Method Human CD34+ hematopoietic stem cells were transferred into NOG (nonTg) or NOG hIL-3/GM-CSF Tg mice. After 14–16 weeks, mature human basophils and mast cells increased in Tg mice, and bovine BLG specific human IgE was intravenously injected in these humanized mice, followed by either intravenous or oral bovine BLG protein exposure, 1 day later. Subsequently, allergic reactions were monitored by measuring body temperature, serum histamine level, and anaphylaxis scoring. Furthermore, the adrenaline agent, ‘epinephrine’ was used to investigate whether the allergic symptoms could be suppressed. Results After bovine BLG exposure, body temperature in Tg mice, but not in nonTg mice, gradually decreased within 10 minutes and 80 % of Tg mice died within 60 minutes. Serum histamine levels and anaphylaxis scores in Tg mice were markedly increased compared to nonTg mice after 30 minutes BLG challenge. These allergic symptoms were significantly inhibited by epinephrine treatment in Tg mice. Discussion The current hIL-3/GM-CSF Tg mouse model could possibly be used to develop novel anaphylaxis drugs in food allergy.

Food allergy is a common disease caused by intake of allergen-containing foods, such as milk, eggs, peanuts and wheat. Systemic anaphylaxis is a severe hypersensitive allergic reaction resulting from degranulation of mast cells or basophils after cross-linking of surface high-affinity IgE receptors (Fc epsilon-RI) with allergen-specific IgE and allergens. In this study, we developed a novel human mast cell/basophil-engrafted mouse model that recapitulates systemic anaphylaxis triggered by beta-lactoglobulin (BLG), a major allergen found in cow's milk. Human CD34(+) hematopoietic stem cells were transferred into NOG (non-Tg) or NOG hIL-3/hGM-CSF transgenic (Tg) mice. After 14-16 weeks, bovine BLG-specific human IgE was intravenously injected into humanized mice, followed by intravenous or oral bovine BLG exposure 1 day later. Body temperature in Tg, but not in non-Tg, mice gradually decreased within 10 min, and 80% of Tg mice died within 1 h by intravenous BLG exposure. Serum histamine levels and anaphylaxis scores in Tg mice were markedly increased compared to non-Tg mice. Furthermore, these allergic symptoms were significantly inhibited by epinephrine treatment of the Tg mice. Therefore, the current NOG hIL-3/hGM-CSF Tg mouse model may be useful for development of novel anaphylaxis drugs for treatment of food allergies and for safety assessment of low-allergenicity extensively hydrolyzed cow's milk whey protein-based infant formulas.

Improving the safety of subcutaneous immunotherapy (SCIT) for food allergy is necessary to reduce side effects and achieve long-term tolerance. We determined the effect of dietary supplementation with 1% non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) on safety and efficacy of SCIT using a peanut allergy mouse model.

Current clinical food allergy guidelines recommend an extensively hydrolyzed formula (EHF) as the first-line treatment in nonbreastfed infants with cow's milk protein allergy (CMPA).1, 2 This recommendation is based on the assumption that EHF is tolerated by at least 90% of infants with CMPA.3 Several studies have reported allergic reactions to EHF.4, 5 This includes the Dutch EuroPrevall cohort study which achieved adequate symptom control in <50% of EHF-treated infants with CMPA.4 We hypothesized that the observed residual allergenicity was due to insufficient milk protein hydrolysis and/or contamination with milk allergens.3, 6 In order to better understand the observed variability in clinical efficacy, we aimed to characterize a representative sample of marketed EHF with regard to their peptide molecular weight (MW) profile, content of residual immunogenic cow's milk proteins or peptides, and in vitro allergenicity. Between 2014 and 2018, we collected samples (cans) of 76 commercially available whey- and casein-based EHF (EHF-W and EHF-C) products positioned for the management of CMPA, from 9 manufacturers. To determine possible between- and within-batch variation, samples from different production batches, as well as multiple cans of the same batch, were analyzed when available; Table S1. Product samples were coded and blinded for analysis. Peptide size distribution analysis was performed by size-exclusion, high-pressure liquid chromatography. As a surrogate marker for potential allergenicity, an arbitrary cutoff of >1200 Da was chosen (equivalent to the MW of 10-12 amino acids). Immunogenic peptides or proteins (IPP) derived from bovine beta-lactoglobulin (BLG) and casein were quantified by high-sensitivity ELISA (Euroclone Spa, Pero, Italy). In addition, a subset of 9 EHF products with a range of percentages of peptides with a MW > 1200 Da was assessed for residual BLG-induced in vitro allergenicity, using a humanized rat basophilic leukemia cell degranulation assay. This assay was developed with IgE directed against “allergenic” immunodominant regions/epitopes on bovine BLG, also recognized by serum IgE from CMPA infants.7 A detailed description of the laboratory methods is provided in Table S2. Characterization of the MW profiles found that 89%-100% of EHF peptides were <2400 Da; Table 1. Three clusters were observed for the content of IPP with a MW > 1200 Da: <5% (Group 1; n = 14), 5%-15% (Group 2; n = 12), and > 15% (Group 3; n = 7); Figure 1. All EHF-C analyzed were in Group 1. There was variability in the content of peptides <240 Da (6 to 38%) and <600 Da (37 to 88%); Table 1. For some products (W1, W21, and C12), significant MW profile differences were noticed between or within batches; Table S3. Residual BLG-derived IPP were detected in 4 of 12 (33%) EHF-C and 18 of 21 (86%) EHF-W products. Four EHF-W products (W1, W2, W3, and W4) showed residual BLG-IPP exceeding the limit of quantification (0.01mg/kg) by 20-fold, including one product with an IPP content of >2000 times the quantification limit; Table 1. One of 12 (8%) and 2 of 21 (10%) EHF-C and EHF-W tested positive for casein-derived IPP, respectively. Two samples showed significant between- and within-batch variation for both casein- and BLG-IPP contents (C12 and W1). Three further EHF-W products (W2, W3, and W4) displayed noticeable between- and within-batch variation for residual BLG-IPP content; Figure S1. A positive relationship between residual BLG-IPP content and the percentage of peptides >1200 Da was found (R2 = 0.65); Figure S2A. An inverse association was demonstrated for peptides with a MW < 240 Da (R2 = 0.89); data not shown. For the subset of 9 EHF (2 EHF-C and 7 EHF-W), BLG-induced RBL cell degranulation levels varied depending on the content of peptides >1200 Da; Table 1. No BLG-induced in vitro allergenicity was detected for the 3 samples in Group 1 (C1, C4, and W7). The remaining 6 EHF-W in Groups 2 and 3 induced a dose-dependent degranulation with a calculated residual allergenicity ranging from 272 to 1881 µg BLG/g protein. These exploratory data suggest a close relationship between residual BLG-induced in vitro allergenicity and the content of peptides with a MW > 1200 Da (R2 = 0.79); Figure S2B. We characterized the physicochemical profiles of a representative sample of marketed EHF-W and EHF-C products. There was significant variability in the MW profile of peptides, residual BLG- and casein-IPP contents, and in vitro allergenicity, with significant batch-to-batch or within-batch variation observed for some products. These findings are in keeping with earlier studies demonstrating significant heterogeneity among marketed EHF regarding their peptide composition and clinical safety.5, 8, 9 The enzymatic hydrolysis and heat treatment used during manufacturing of EHF are designed to disrupt the vast majority of allergenic epitopes. The final product safety of an EHF relies on multiple processes, including effective protein hydrolysis, the removal of residual allergenic peptides or proteins by ultrafiltration (for some products) and ongoing quality management. The significant residual BLG- or casein-derived IPP found in some EHF products suggests incomplete hydrolysis and/or contamination during manufacturing. Furthermore, the significant batch-to-batch or within-batch variation observed may be due to inadequate quality management for some EHF products. The content of peptides with a MW > 1200 Da appeared to closely correlate with both the residual BLG-IPP content and BLG-induced in vitro allergenicity. The percentage of peptides with a MW > 1200 Da may therefore provide a useful reference for comparison of the residual allergenicity between EHF products. This is particularly relevant for EHF-W because the globular, three-dimensional structure of whey proteins renders the final peptide profile highly dependent on hydrolysis conditions. By contrast, caseins are more easily hydrolyzed. Our report has several limitations. Firstly, the products analyzed represent a selection of commercially available EHF. Secondly, our findings only apply to the product characteristics at the time of sampling, and recipes or quality management standards may have changed since. Importantly, the clinical implications of our findings are at this stage uncertain, and further studies are needed. Despite these limitations, our survey highlights the need for a more meaningful definition of EHF products. Efforts should be made to standardize analytical methods for residual allergen detection, improve quality control measures during EHF manufacturing, and define minimum clinical evidence requirements for product safety. The authors wish to thank Niels de Jong (Bioceros BV, The Netherlands) for his expertise and technical support on the humanized RBL cell degranulation assay, as well as Michaël Affolter (Nestlé Research, Switzerland) for scientific guidance. The contribution to the market analysis by Doreen Benardout (Nestlé Health Science, Switzerland) and Peter Fryer (Nestlé Nutrition, Australia) is also gratefully acknowledged. This study was sponsored by Nestlé Health Science, Switzerland. The authors declare the following potential conflicts of interest: SN, AJ, MK, and RH are salaried employees of Nestlé Health Science, Switzerland, which sponsored the study. The other authors have no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Scope During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β‐lactoglobulin (BLG), in their interactions with cells crucially involved in allergy. Methods and results BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T‐cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco‐2 monolayer. Uptake of glycated BLG by bone marrow–derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor‐mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4+ T‐cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG‐specific IgE sensitized basophil cells. Conclusions This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy.

In formula-fed infants, extensively hydrolyzed formula (EHF) is considered the first-line treatment of cow's milk protein allergy (CMPA).1 The American Academy of Pediatrics (AAP) defines an EHF to be 'hypoallergenic' if at least 90% of infants with CMPA tolerate it under double-blind, placebo-controlled conditions.2 The protein hydrolysis process for EHF has traditionally involved the use of porcine pancreatic enzymes. This article is protected by copyright. All rights reserved.

The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High‐throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress‐induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D‐N) reduced the binding of IgG to the low‐affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short‐term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in‐process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H+-ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V1 and the integral membrane V0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction.

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models.

Insulin resistance is an important risk factor for the development of several cardiac pathologies, thus advocating strategies for restoring insulin sensitivity of the heart in these conditions. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), mainly eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), have been shown to improve insulin sensitivity in insulin-sensitive tissues, but their direct effect on insulin signaling and metabolic parameters in the myocardium has not been reported previously. The aim of this study was therefore to examine the ability of EPA and DHA to prevent insulin resistance in isolated rat cardiomyocytes. Primary rat cardiomyocytes were made insulin resistant by 48 h incubation in high insulin (HI) medium. Parallel incubations were supplemented by 200 μM EPA or DHA. Addition of EPA or DHA to the medium prevented the induction of insulin resistance in cardiomyocytes by preserving the phosphorylation state of key proteins in the insulin signaling cascade and by preventing persistent relocation of fatty acid transporter CD36 to the sarcolemma. Only cardiomyocytes incubated in the presence of EPA, however, exhibited improvements in glucose and fatty acid uptake and cell shortening. We conclude that ω-3 PUFAs protect metabolic and functional properties of cardiomyocytes subjected to insulin resistance-evoking conditions.

Blocking the interaction of CD40 with its ligand CD154 is a desirable goal of therapies for preventing and/or ameliorating autoimmune diseases and transplant rejection. CD154-blocking mAbs used in human clinical trials resulted in unanticipated vascular complications, leading to heightened interest in the therapeutic potential of antagonist mAbs specific for human CD40. Abs that do not require physical competition with CD154 to inhibit CD40 signaling have particular therapeutic promise. In this study, we demonstrate that the antagonist anti-human CD40 mAb PG102 fails to trigger CD40-mediated activation, as well as impairs CD154-mediated CD40 activation, via a distinct nonstimulatory CD40 signaling mechanism. PG102 did not induce early CD40-induced signaling events, and it inhibited early kinase and transcription factor activation by CD154 or agonist anti-CD40 mAbs. However, PG102 stimulated normal CD40-mediated TNFR-associated factor (TRAF)2 and TRAF3 degradation. PG102 induced the formation of a CD40 signaling complex that contained decreased amounts of both TRAF2 and TRAF3 and TRAF2-associated signaling proteins. Additionally, PG102-induced CD40 signaling complexes failed to recruit TRAF6 to detergent-insoluble membrane fractions. Fab fragments of PG102, while retaining CD40 binding, did not induce TRAF degradation, nor could they inhibit CD154-stimulated B cell signaling, indicating that CD40 aggregation is required for the signaling inhibition induced by PG102. The antagonistic impact of PG102 on CD40 signaling reveals that the manner of CD40 ligation can determine sharply different outcomes for CD40 signaling and suggests that such information can be used to therapeutically manipulate these outcomes.

Background Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. Objective An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Methods Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. Results After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. Conclusion Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

An increased cardiac fatty acid supply and increased sarcolemmal presence of the long-chain fatty acid transporter CD36 are associated with and contribute to impaired cardiac insulin sensitivity and function. In the present study we aimed at preventing the development of insulin resistance and contractile dysfunction in cardiomyocytes by blocking CD36-mediated palmitate uptake. Insulin resistance and contractile dysfunction were induced in primary cardiomyocytes by 48 h incubation in media containing either 100 nM insulin (high insulin; HI) or 200 μM palmitate (high palmitate; HP). Under both culture conditions, insulin-stimulated glucose uptake and Akt phosphorylation were abrogated or markedly reduced. Furthermore, cardiomyocytes cultured in each medium displayed elevated sarcolemmal CD36 content, increased basal palmitate uptake, lipid accumulation and decreased sarcomere shortening. Immunochemical CD36 inhibition enhanced basal glucose uptake and prevented elevated basal palmitate uptake, triacylglycerol accumulation and contractile dysfunction in cardiomyocytes cultured in either medium. Additionally, CD36 inhibition prevented loss of insulin signalling in cells cultured in HP, but not in HI medium. In conclusion, CD36 inhibition prevents lipid accumulation and lipid-induced contractile dysfunction in cardiomyocytes, but probably independently of effects on insulin signalling. Nonetheless, pharmacological CD36 inhibition may be considered as a treatment strategy to counteract impaired functioning of the lipid-loaded heart.