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1.
Int Immunol ; 33(3): 183-189, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33027513

ABSTRACT

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 (Fcε-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 ß-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.


Subject(s)
Anaphylaxis/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Immunoglobulin E/immunology , Lactoglobulins/immunology , Milk Hypersensitivity/immunology , Anaphylaxis/mortality , Animals , Basophils/immunology , Cattle , Disease Models, Animal , Epinephrine/therapeutic use , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Histamine/blood , Humans , Interleukin-3/genetics , Interleukin-3/metabolism , Mast Cells/immunology , Mice , Mice, Inbred NOD , Mice, Transgenic
2.
J Allergy Clin Immunol ; 147(4): 1420-1429.e7, 2021 04.
Article in English | MEDLINE | ID: mdl-32926878

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/pharmacology , Complement C2/antagonists & inhibitors , Complement Inactivating Agents/pharmacology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/pharmacokinetics , Calcium , Complement Activation/drug effects , Complement C2/analysis , Complement C2/metabolism , Complement Inactivating Agents/blood , Complement Inactivating Agents/pharmacokinetics , Epitope Mapping , Female , Humans , Hydrogen-Ion Concentration , Macaca fascicularis , Male
3.
Clin Mol Allergy ; 17: 7, 2019.
Article in English | MEDLINE | ID: mdl-30988664

ABSTRACT

BACKGROUND: 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. METHODS: After sensitization, mice received a scFOS/lcFOS or control diet for the rest of the study. To study safety of SCIT, mice were dosed with a single subcutaneous injection of peanut extract (PE) or PBS. To study efficacy, mice were dosed subcutaneously (SCIT, 3 times/week) with PE or PBS for 3 weeks. Hereafter, acute allergic skin responses, anaphylactic shock symptoms and body temperature were assessed. To study the mechanism in vitro, the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) line was sensitized with an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation. RESULTS: scFOS/lcFOS reduced anaphylaxis caused by a single PE SCIT dose. scFOS/lcFOS alone also reduced the acute allergic skin response. Moreover, scFOS/lcFOS supplementation resulted in lower MMCP-1 levels in serum after PE SCIT dose compared to control diet, while antibody levels were not affected by the diet. In vitro incubation with scFOS/lcFOS at 0.5% suppressed the degranulation of IgE-sensitized RBL cells. However, dietary supplementation with scFOS/lcFOS did not improve the efficacy of SCIT. CONCLUSIONS: We show that scFOS/lcFOS diet improves the safety of SCIT, as evidenced by lower anaphylactic responses without compromising the efficacy in a mouse model for peanut allergy. This effect is likely to result from the suppression of mast cell effector function.

4.
J Immunol ; 194(9): 4319-27, 2015 May 01.
Article in English | MEDLINE | ID: mdl-25795759

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/metabolism , CD40 Antigens/metabolism , Signal Transduction , Antibodies, Monoclonal/pharmacology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CD40 Antigens/antagonists & inhibitors , CD40 Ligand/metabolism , Cell Line , Humans , Lymphocyte Activation/immunology , Protein Binding , Proteolysis , Signal Transduction/drug effects , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism
5.
Am J Physiol Cell Physiol ; 308(4): C297-307, 2015 Feb 15.
Article in English | MEDLINE | ID: mdl-25472960

ABSTRACT

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.


Subject(s)
Cardiotonic Agents/pharmacology , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/pharmacology , Energy Metabolism/drug effects , Insulin Resistance , Insulin/pharmacology , Myocytes, Cardiac/drug effects , Animals , CD36 Antigens/metabolism , Cells, Cultured , Docosahexaenoic Acids/metabolism , Eicosapentaenoic Acid/metabolism , Glucose/metabolism , Male , Myocardial Contraction/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Phosphorylation , Protein Transport , Rats, Inbred Lew , Sarcolemma/drug effects , Sarcolemma/metabolism , Signal Transduction/drug effects , Time Factors
8.
Biochem J ; 448(1): 43-53, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22780108

ABSTRACT

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.


Subject(s)
CD36 Antigens/physiology , Insulin Resistance/physiology , Myocytes, Cardiac/metabolism , Palmitates/metabolism , Animals , Biological Transport , Calcium Signaling/drug effects , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Diabetic Cardiomyopathies/etiology , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/prevention & control , Fatty Acids/metabolism , Glucose/metabolism , Insulin/pharmacology , Male , Mitochondria, Heart/metabolism , Myocardial Contraction , Myocytes, Cardiac/drug effects , Palmitates/pharmacology , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Inbred Lew , Sarcolemma/metabolism , Sarcomeres/ultrastructure , Signal Transduction/drug effects , Triglycerides/metabolism
9.
Front Immunol ; 14: 1118539, 2023.
Article in English | MEDLINE | ID: mdl-37081893

ABSTRACT

Introduction: Immunoglobulin 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. Methods: Human 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). Results: Our 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. Conclusion: This 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.


Subject(s)
Autoimmunity , Immunoglobulin A , Animals , Mice , Lactoferrin/metabolism , Leukotriene B4/metabolism , Inflammation
10.
Foods ; 11(14)2022 Jul 07.
Article in English | MEDLINE | ID: mdl-35885248

ABSTRACT

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.

11.
Nutrients ; 13(6)2021 Jun 15.
Article in English | MEDLINE | ID: mdl-34203636

ABSTRACT

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.


Subject(s)
Lactoglobulins/analysis , Milk Hypersensitivity , Milk/chemistry , Allergens/analysis , Animals , Cattle , Female , Humans , Immunoglobulin E/analysis , Immunoglobulin G , Lactose/analysis , Maillard Reaction , Mast Cells , Milk Proteins/analysis , Whey , Whey Proteins/analysis
12.
Biochim Biophys Acta ; 1770(5): 839-46, 2007 May.
Article in English | MEDLINE | ID: mdl-17321686

ABSTRACT

Several pulmonary pathologies, like cystic fibrosis (CF), are characterized by hypersecretion and stasis of tenacious mucus. Bacterial glycosidases are known to degrade mucins but their use as mucolytic agents is questionable. The observation that bacterial chitinases degrade mucins and the recent discovery of human chitinases, which have been proposed to be involved in the genesis of asthma, prompted us to evaluate the mucolytic properties of human derived chitinases. The effect of these human chitinases, and bacterial chitinases (positive control), on the viscoelasticity of CF sputa and on the electrophoretic mobility of human mucins was tested. Commercial bacterial chitinase drastically degraded CF sputum, while human derived chitinases did not. Accordingly, the commercial bacterial chitinase was found to degrade mucins, whereas recombinant human chitinases did not. A thorough analysis of the commercial chitinase elucidated that contaminating proteases and also nucleases assisted in the mucolytic effect. Indeed, recombinant bacterial chitinases very slightly reduced the viscoelasticity of CF sputum, but they caused a significant degradation of the CF sputum when they were combined with proteases. In conclusion, this work shows that recombinant human and recombinant bacterial chitinases have no or very low mucolytic activities, respectively. The observed mucolytic properties of commercial bacterial chitinase are due to a synergistic effect between chitinolytic and proteolytic enzymes at one hand and at the other hand also due to the presence of contaminating nucleases.


Subject(s)
Bacteria/enzymology , Bacteria/metabolism , Chitinases/metabolism , Mucins/genetics , Mucins/metabolism , Cell Line, Tumor , Chitinases/genetics , Colorectal Neoplasms/enzymology , Cystic Fibrosis/metabolism , Deoxyribonucleases/metabolism , Drug Contamination , Drug Interactions , Elasticity , Gallbladder/enzymology , Humans , Mucin 5AC , Mucin-2 , Peptide Hydrolases/metabolism , Recombinant Proteins/metabolism , Sputum/enzymology , Sputum/metabolism , Viscosity
13.
Mol Nutr Food Res ; 62(17): e1800341, 2018 09.
Article in English | MEDLINE | ID: mdl-30004175

ABSTRACT

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 ß-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.


Subject(s)
Allergens/chemistry , Lactoglobulins/chemistry , Lactoglobulins/immunology , Milk Hypersensitivity/immunology , Allergens/immunology , Allergens/pharmacokinetics , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Caco-2 Cells , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Endocytosis/drug effects , Endocytosis/physiology , Female , Food Handling , Humans , Lactoglobulins/pharmacokinetics , Lysosomes/drug effects , Lysosomes/metabolism , Maillard Reaction , Mice, Inbred C3H , Milk/chemistry , Milk/immunology
14.
J Endocrinol ; 192(2): 289-99, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17283229

ABSTRACT

Adiponectin and, especially, its oligomeric complex composition have been suggested to be critical in determining insulin sensitivity. Pro-inflammatory cytokines play an important role in the development of insulin resistance in obesity and associated diseases. Therefore, we investigated the effect of long-term exposure of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta, and interferon (IFN)-gamma on total insulin-sensitizing adiponectin secretion and adiponectin complex formation from human adipocytes. In parallel, adipocyte delipidation and leptin production levels were monitored. The present study demonstrates that TNF-alpha, IL-1beta, and IFN-gamma dose and time dependently suppressed total adiponectin secretion within 7 days (60, 70, and 35% reduction respectively). IL-6 was also able to reduce (50%) adiponectin production, although only in combination with exogenous soluble IL-6 receptors (sIL-6R). However, the oligomeric distribution (high, middle, and low molecular weight (HMW) complexes) of secreted adiponectin was not altered by any of these cytokines. All studied pro-inflammatory cytokines resulted in delipidation and reduction of lipid-laden adipocyte numbers. Despite this reduction of lipid-laden adipocytes, TNF-alpha, IL-6/sIL-6R, and IL-1beta stimulated leptin release. Our data indicate that (i) long-term pro-inflammatory cytokine exposure downregulates total adiponectin secretion from delipidizing adipocytes and (ii) pro-inflammatory cytokines are not important regulators of adipocyte-derived adiponectin oligomerization. Hence, their individual contribution to low expression of HMW adiponectin found in insulin-resistant conditions seems unlikely. Furthermore, delipidizing adipocytes and preadipocytes are active leptin producers when stimulated by TNF-alpha, IL-6/sIL-6R, and IL-1beta.


Subject(s)
Adipocytes/metabolism , Adiponectin/metabolism , Cytokines/pharmacology , Adipocytes/drug effects , Adult , Analysis of Variance , Azo Compounds , Cell Proliferation , Cells, Cultured , Coloring Agents , Depression, Chemical , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Insulin Resistance , Interferon-gamma/pharmacology , Interleukin-1beta/pharmacology , Interleukin-6/pharmacology , Leptin/metabolism , Lipid Metabolism , Middle Aged , Protein Isoforms/metabolism , Receptors, Interleukin-6/metabolism , Subcutaneous Fat , Time Factors , Tumor Necrosis Factor-alpha/pharmacology
15.
FEBS Open Bio ; 7(10): 1557-1574, 2017 10.
Article in English | MEDLINE | ID: mdl-28979843

ABSTRACT

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.

16.
Diabetes ; 66(6): 1521-1534, 2017 06.
Article in English | MEDLINE | ID: mdl-28302654

ABSTRACT

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.


Subject(s)
CD36 Antigens/metabolism , Endosomes/drug effects , Glucose/metabolism , Heart/drug effects , Insulin Resistance , Myocardial Contraction/drug effects , Myocardium/metabolism , Myocytes, Cardiac/drug effects , Palmitates/pharmacology , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Animals , Blotting, Western , Carbon Radioisotopes , Cells, Cultured , Deoxyglucose/metabolism , Diet, High-Fat , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration , Induced Pluripotent Stem Cells , Male , Myocytes, Cardiac/metabolism , Myosin Heavy Chains/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred Lew , Triglycerides/metabolism , Tritium , Troponin T/genetics
17.
Toxicol In Vitro ; 33: 29-34, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26921666

ABSTRACT

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 ß-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.


Subject(s)
Allergens/immunology , Cell Degranulation , Immunoglobulin E/immunology , Lactoglobulins/immunology , Mast Cells/physiology , Receptors, IgE/immunology , Animals , Biological Assay , Cell Line , Humans , Laboratories , Milk/immunology , Reproducibility of Results
18.
Transplantation ; 78(10): 1429-38, 2004 Nov 27.
Article in English | MEDLINE | ID: mdl-15599306

ABSTRACT

Clinical trials designed to achieve tolerance in humans by selectively antagonizing one of the T-cell costimulatory pathways, CD40-CD40L or CD80/CD86-CD28, are pending. However, simultaneous blockade of both pathways synergistically prevented graft rejection and successfully induced donor-specific tolerance in animal models. Synergism is also supported in human T-cells in vitro following anti-CD86 mAb and anti-CD40 mAb blockade. Therefore, in our view the most promising clinical strategy would be to antagonize both CD40 and CD86. Fast clinical entrance of this anti-CD86 and anti-CD40 bidirectional concept is highly facilitated by a single molecule approach. In the present study, a single bispecific fusion protein was constructed that specifically binds human CD40 and CD86 and which combines the antagonistic activities of both anti-CD40 and anti-CD86 humanized mAb. The anti-CD40/86 fusion protein showed tolerance inducing potential as it prevented both allogeneic T-cell expansion and generation of cytotoxic effector T cells and induced anergic antigen specific regulatory T cells. These data provide proof of concept in successfully combining the antagonistic activity of two humanized mAb with great clinical potential in transplantation and autoimmunity, in one single molecule.


Subject(s)
Antibodies, Bispecific/immunology , Antigens, CD/immunology , CD40 Antigens/immunology , Immune Tolerance/immunology , Membrane Glycoproteins/immunology , T-Lymphocytes/immunology , B-Lymphocytes/immunology , B7-2 Antigen , Binding Sites , Cells, Cultured , Humans , Immunoglobulin Fab Fragments/immunology , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Lymphocytes/immunology , Recombinant Fusion Proteins/immunology
19.
PLoS One ; 9(8): e106025, 2014.
Article in English | MEDLINE | ID: mdl-25153680

ABSTRACT

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 ß-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. METHODS: Mice were immunized with bovine ß-lactoglobulin, and subsequently the variable domains of dissimilar anti-ß-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-ß-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa ß-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-ß-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on ß-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 ß-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.


Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin E/immunology , Lactoglobulins/immunology , Whey/immunology , Allergens/immunology , Animals , Caseins/immunology , Cattle , Cell Line , Child , Female , Humans , Mice , Mice, Inbred BALB C , Milk Hypersensitivity/immunology , Protein Hydrolysates/immunology , Rats , Receptors, IgE/immunology , Reproducibility of Results , Whey Proteins/immunology
20.
Acta Histochem ; 113(8): 839-43, 2011 Dec.
Article in English | MEDLINE | ID: mdl-20950842

ABSTRACT

CD36 is the receptor for long chain fatty acids (LCFA), and is expressed in lingual taste cells from rodents. In these animals, CD36 has been proposed to play an important role in oral detection of LCFA, and subsequently, determines their dietary fat preference. Humans also seem to detect LCFA in the oral cavity, however, information on the molecular mechanism of this human orosensory LCFA recognition is currently lacking. The aim of our study was to investigate whether CD36 is also expressed in lingual human and porcine taste buds cells. Using fluorescence immunohistochemistry, apical CD36 expression was revealed in human and porcine taste bud cells from circumvallate and foliate papillae. These data suggest CD36 as the putative orosensory receptor for dietary LCFA in human, and, therefore, may be involved in our preference for fatty foods.


Subject(s)
CD36 Antigens/analysis , Taste Buds/immunology , Animals , CD36 Antigens/metabolism , Dietary Fats/metabolism , Fatty Acids/metabolism , Fluorescent Antibody Technique , Humans , Swine , Taste Buds/cytology , Taste Buds/metabolism , Tongue/cytology , Tongue/metabolism
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