Bovine ß-lactoglobulin-induced passive systemic anaphylaxis model using humanized NOG hIL-3/hGM-CSF transgenic mice.

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.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2.

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.

Non-digestible oligosaccharides scFOS/lcFOS facilitate safe subcutaneous immunotherapy for peanut allergy.

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.

Consistency and sources of divergence in recommendations on screening with questionnaires for presently experienced health problems or symptoms: a comparison of recommendations from the Canadian Task Force on Preventive Health Care, UK National Screening Committee, and US Preventive Services Task Force.

BACKGROUND: Recently, health screening recommendations have gone beyond screening for early-stage, asymptomatic disease to include "screening" for presently experienced health problems and symptoms using self-report questionnaires. We examined recommendations from three major national guideline organizations to determine the consistency of recommendations, identify sources of divergent recommendations, and determine if guideline organizations have identified any direct randomized controlled trial (RCT) evidence for the effectiveness of questionnaire-based screening. METHODS: We reviewed recommendation statements listed by the Canadian Task Force on Preventive Health Care (CTFPHC), the United Kingdom National Screening Committee (UKNSC), and the United States Preventive Services Task Force (USPSTF) as of 5 September 2016. Eligible recommendations focused on using self-report questionnaires to identify patients with presently experienced health problems or symptoms. Within each recommendation and accompanying evidence review we identified screening RCTs. RESULTS: We identified 22 separate recommendations on questionnaire-based screening, including three CTFPHC recommendations against screening, eight UKNSC recommendations against screening, four USPSTF recommendations in favor of screening (alcohol misuse, adolescent depression, adult depression, intimate partner violence), and seven USPSTF recommendations that did not recommend for or against screening. In the four cases where the USPSTF recommended screening, either the CTFPHC, the UKNSC, or both recommended against. When recommendations diverged, the USPSTF expressed confidence in benefits based on indirect evidence, evaluated potential harms as minimal, and did not consider cost or resource use. CTFPHC and UKNSC recommendations against screening, on the other hand, focused on the lack of direct evidence of benefit and raised concerns about harms to patients and resource use. Of six RCTs that directly evaluated screening interventions, five did not report any statistically significant primary or secondary health outcomes in favor of screening, and one trial reported equivocal results. CONCLUSIONS: Only the USPSTF has made any recommendations for screening with questionnaires for presently experienced problems or symptoms. The CTFPHC and UKNSC recommended against screening in all of their recommendations. Differences in recommendations appear to reflect differences in willingness to assume benefit from indirect evidence and different approaches to assessing possible harms and resource consumption. There were no examples in any recommendations of RCTs with direct evidence of improved health outcomes.

Induction of an altered CD40 signaling complex by an antagonistic human monoclonal antibody to CD40.

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.

Marine omega-3 fatty acids prevent myocardial insulin resistance and metabolic remodeling as induced experimentally by high insulin exposure.

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.

CD36 inhibition prevents lipid accumulation and contractile dysfunction in rat cardiomyocytes.

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.

Antagonizing FcαR1 (CD89) as treatment in IgA-mediated chronic inflammation and autoimmunity.

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.

Discovery of regulators of receptor internalization with high-throughput flow cytometry.

We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the ß2-adrenergic receptor (ß2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged ß2ARs, all 33 known ß2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

An In Vitro and In Vivo Translational Research Approach for the Assessment of Sensitization Capacity and Residual Allergenicity of an Extensive Whey Hydrolysate for Cow's Milk-Allergic Infants.

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.

Prevalence and duration of anti-SARS-CoV-2 antibodies in healthcare workers.

INTRODUCTION: Knowledge of the seroprevalence and duration of antibodies against SARS-CoV-2 was needed in the early phases of the COVID-19 pandemic and is still necessary for policy makers and healthcare professionals. This information allows us to better understand the risk of reinfection in previously infected individuals. METHODS: We investigated the prevalence and duration of detectable antibodies against SARS-CoV-2 in sequentially collected samples from 379 healthcare professionals. RESULTS: SARS-CoV-2 seroprevalence at inclusion was 5.3% (95% confidence interval (CI): 3.3-8.0%) and 25% of seropositive participants reverted during follow-up. At the end of follow-up, the calculated probability of having detectable antibodies among former seropositive participants was 72.2% (95% CI: 54.2-96.2%). CONCLUSION: Antibodies against SARS-CoV-2 were detectable in a subset of infected individuals for a minimum of 39 weeks. FUNDING: The assays performed at Rigshospitalet were developed with financial support from the Carlsberg Foundation (CF20-0045) and the Novo Nordisk Foundation (NFF205A0063505 and NNF20SA0064201). TRIAL REGISTRATION: The study was registered with the Danish National Committee on Health Research Ethics (H-20022312).

Partner attachment as a predictor of long-term response to treatment with couples therapy.

Vulnerability factors such as insecure attachment may have a lasting effect on the outcome of couples therapy, even long after discharge from treatment. Given that attachment has never been examined as an outcome predictor for couples therapy in the long term, the authors studied its effect on outcome during and after couples therapy. This prospective study included 71 inpatients participating in group couples therapy who the authors measured at baseline, immediately posttreatment at 2 months, and at 8 and 20 months, regarding two outcomes: problem-solving capacity (using the Interactional Problem Solving Questionnaire) and psychopathology (using the 90-item Symptom Check List). At baseline, the authors measured partner attachment (using the Experiences in Close Relationships Questionnaire). Mixed model analyses showed that attachment-related dysfunctional working models of self and others predicted less improvement in psychopathology (p = .04) and problem-solving capacity (p = .01), respectively. Special attention to insecure attachment in couples therapy may therefore prove valuable in terms of outcome in the long run.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

Integrin activation is an essential component of SARS-CoV-2 infection.

SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's ß subunit, we demonstrate that talin-mediated signaling is essential for productive infection.

Integrin activation is an essential component of SARS-CoV-2 infection.

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2 R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn 2+ , which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2 R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2 R18 with basal state integrins, but is ineffective against Mn 2+ -activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2 R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand binding function of integrins via a talin dependent mechanism and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα 13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα 13 binding to the cytoplasmic tail of an integrin's ß subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.

Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Limited Lactosylation of Beta-Lactoglobulin from Cow's Milk Exerts Strong Influence on Antigenicity and Degranulation of Mast Cells.

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.

Mucolytic activity of bacterial and human chitinases.

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.