Targeting NF-κB Signaling: Selected Small Molecules Downregulate Pro-Inflammatory Cytokines in Both Food Allergen and LPS-Induced Inflammation.

Although inflammation is primarily a protective response guarding the human body, it can result in a variety of chronic diseases such as allergies, auto-immune, cardiovascular diseases, and cancer. In NF-κB-mediated inflammation, many small molecules and food compounds characterized as nutraceuticals have shown positive effects associated with immunomodulatory properties. We investigated the effects of selected bioactive small molecules, commonly found in food components, vanillyl alcohol (VA) and lauric acid (LA), on different cell lines exposed to pro-inflammatory stimuli, lipopolysaccharide (LPS), and the food allergen actinidin (Act d 1). Pro-inflammatory cytokines were downregulated in response to both VA and LA, and this downregulation was caused by a decrease in the activation of the NF-κB pathway and the translocation of p65, the pathway's major component. Small nutraceutical molecules, VA and LA, showed not only inhibition of the pro-inflammatory cytokines, but also inhibition of the NF-κB activation, and reduced translocation of the p65 component. The present study may contribute to the therapeutic use of these molecules for various inflammatory diseases, which have in common an increased expression of pro-inflammatory cytokines and NF-κB-mediated inflammation.

A new approach for activation of the kiwifruit cysteine protease for usage in in-vitro testing.

Actinidin (Act d 1), a highly abundant cysteine protease from kiwifruit, is one of the major contributors to the development of kiwifruit allergy. Many studies have focused on the optimization of Act d 1 purification and its role in the development of food allergies. Testing on cell culture monolayers is a common step in the elucidation of food allergen sensitization. In the case of cysteine proteases, an additional activation step with L-cysteine is required before the testing. Hence, we aimed to evaluate whether L-cysteine already present in commonly used cell culture media would suffice for Act d 1 activation. Successfully activated Act d 1 (98.1% of proteolytic activity, as compared to L-cysteine activated Act d 1) was further tested in two commonly used 2D model systems (Caco-2 and HEK293 cells) to evaluate its role on the mRNA expression of cytokines involved in the innate immunity (IL-1ß, IL-6, TNFα, TSLP). Furthermore, the contribution of Act d 1 in the promotion of inflammation through regulation of inducible nitric oxide synthase (iNOS) mRNA expression was also examined. These results demonstrate that activation of cysteine proteases can be achieved without previous enzyme incubation in L-cysteine -containing solution. Act d 1 incubated in cell culture medium was able to modulate gene expression of pro-inflammatory cytokines when tested on two model systems of the epithelial barrier.

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions.

BACKGROUND: The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease--actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs). METHODS: Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry. RESULTS: Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 µg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 µg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin. CONCLUSION: Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice. GENERAL SIGNIFICANCE: In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.

Effect of malondialdehyde on the ovalbumin structure and its interactions with T84 epithelial cells.

BACKGROUND: Protein oxidation can occur as a consequence of lipid peroxidation during food processing. The aim of this work was to explore the effect of malondialdehyde (MDA) modification of ovalbumin (OVA) on its interaction with T84 intestinal cells. METHODS: Molecular dynamics simulation was employed for the prediction of MDA modification in the OVA, while introduced structural changes were evaluated by measurement of carbonyl group content, fluorescence spectra, MS/MS analysis, and IgE reactivity. Effects of MDA modified OVA on T84 epithelial cells were analyzed by gene expression for pro-inflammatory cytokines and protein secretion. RESULTS: Out of 9 predicted, five modified Lys residues were confirmed by MS/MS analysis: 51TQINKVVR58, 85DILNQITKPNDVYSFSLASR104, 111YPILPEYLQCVKELYR126, 187AFKDEDTQAMPFR199, 277KIKVYLPR284, and 278IKVYLPR284. The introduced MDA modifications influenced profile of IgE reactivity to OVA. Treatment of T84 epithelial cells with OVA and OVA modified with 1mM MDA, induced up-regulation of pro-inflammatory cytokines (IL-1ß, IL-25, IL-33, TSLP and TNFα), while OVA modification with 10mM MDA induced down regulation of the cytokine expression profile, except for IL-1ß. OVA and OVA modified with 1mM MDA induced secretion of epithelial cells specific cytokine IL-33. CONCLUSIONS: This finding indicated that OVA and its MDA modified form have the potential to trigger the innate immunity by inducing up-regulation and secretion of pro-allergenic IL-33 in T84 intestinal epithelial cells. GENERAL SIGNIFICANCE: Interactions of ovalbumin and its MDA modified form with intestinal epithelial cells can induce a specific immunological priming necessary for the downstream activation of innate immunity.

Detection of humoral and cellular immune response to anti-SARS-CoV-2 BNT162b2 vaccine in breastfeeding women and naïve and previously infected individuals.

This study explored humoral and cellular responses to anti-SARS-CoV-2 BNT162b2 mRNA vaccine in breastfeeding women and naïve and seropositive individuals in the first six months after vaccination.Sixty-one volunteers vaccinated with two doses of the BNT162b2 mRNA vaccine were enrolled in the study. In-house developed ELISA was used for the quantification of SARS-CoV-2 RBD-specific antibodies. Cell surface marker expression and intracellular IFN-γ analysis were carried out by flow cytometry. The concentrations of IFN-γ, IL-6 and TNF were determined by ELISA. A significant rise in anti-RBD IgG antibody levels was observed 14 days after the first vaccine dose (p < 0.0001) in serum and milk. The expression of CD28 on CD4+ T cells was significantly higher compared to baseline (p < 0.05). There was a significant increase (p ≤ 0.05) in B cell lymphocyte subset after revaccination, and increased percentage of CD80+ B cells. The expression of IFN-γ in peripheral blood lymphocytes, CD3+ T cells and serum was significantly increased (p < 0.05). No significant difference in immune response was observed between breastfeeding women and other study participants. The anti-SARS-CoV-2 BNT162b2 mRNA vaccine-induced measurable and durable immune response in breastfeeding women and in naïve and previously infected individuals.

Levels of non-essential trace metals and their impact on placental health: a review.

According to recent research, even low levels of environmental chemicals, particularly heavy metals, can considerably disrupt placental homeostasis. This review aims to explore the profile of non-essential trace metals in placental tissues across the globe and to specify trace metal(s) that can be candidates for impaired placental health. Accordingly, we conducted an extensive survey on relevant databases of peer-reviewed papers published in the last two decades. Among a considerable number of non-essential trace metals, arsenic (As), lead (Pb), cadmium (Cd), and mercury (Hg) were identified as the most detrimental to placental health. Comparative analysis showed remarkable differences in placental levels of these trace metals worldwide. Based on current data reported across the globe, a median (min-max) range from 0.55 to 15 ng/g for placental As levels could be deemed safe. The placental Cd and Pb levels were markedly higher in smokers than in non-smokers. Occupationally exposed pregnant women had several orders of magnitude higher Cd, Pb, and Hg levels in placental tissues than non-occupationally exposed women. Also, we concluded that even low-level exposure to As, Cd, Pb, and Hg could be deleterious to proper fetal development. This review implies the need to reduce exposure to non-essential trace metals to preserve placental health and prevent numerous poor pregnancy outcomes. Overall, the information presented is expected to help plan future fundamental and applied investigations on the placental toxicity of As, Cd, Pb, and Hg.

A six-month study of anti-SARS-CoV-2 BNT162b2 mRNA vaccination: A comparative analysis of essential trace elements and anti-RBD IgG sera levels.

BACKGROUND: Although essential trace elements (ETEs) play pivotal roles in life-supporting biochemical processes, their function in innate and adaptive immunity has not been fully elucidated, particularly during immunization. Furthermore, the association between anti-SARS-CoV-2 specific IgG antibodies and ETE levels with vaccine responsiveness has not been investigated. METHODS: The present study explored the status of ETEs (Mn, Cu, Zn, and Se) in sera of healthy women before and after vaccination with the anti-SARS-CoV-2 BNT162b2 mRNA vaccine in a follow-up period of six months. The main aim was to explore links between ETE levels and IgG antibodies produced against Spike glycoprotein's Receptor-Binding Domain (RBD). RESULTS: A recombinant protein of SARS-CoV-2 comprising the receptor binding domain was successfully expressed in HEK-293 T cells. The purified protein was suitable for producing a sensitive antibody detection assay for human serum and monitored seropositivity, indicating a transient response with peak anti-SARS-CoV-2 IgG levels 2 months after vaccination. In parallel to increasing antibody titers, serum concentrations of Cu, Mn, and Se were not affected by vaccination, and concentrations remained relatively constant at the different sampling times during the 6-month observation period. Total serum Zn concentrations were slightly elevated when compared between the first and last sampling dates. Overall, no consistent effects of vaccination on any of the three trace elements analyzed in our study were observed. CONCLUSION: Vaccination of adult healthy female volunteers with an mRNA vaccine was not associated with consistent changes in serum trace element concentrations over a six-month observation period.

La(OH)3 Multi-Walled Carbon Nanotube/Carbon Paste-Based Sensing Approach for the Detection of Uric Acid-A Product of Environmentally Stressed Cells.

This paper aims to develop an amperometric, non-enzymatic sensor for detecting and quantifying UA as an alert signal induced by allergens with protease activity in human cell lines (HEK293 and HeLa). Uric acid (UA) has been classified as a damage-associated molecular pattern (DAMP) molecule that serves a physiological purpose inside the cell, while outside the cell it can be an indicator of cell damage. Cell damage or stress can be caused by different health problems or by environmental irritants, such as allergens. We can act and prevent the events that generate stress by determining the extent to which cells are under stress. Amperometric calibration measurements were performed with a carbon paste electrode modified with La(OH)3@MWCNT, at the potential of 0.3 V. The calibration curve was constructed in a linear operating range from 0.67 µM to 121 µM UA. The proposed sensor displayed good reproducibility with an RSD of 3.65% calculated for five subsequent measurements, and a low detection limit of 64.28 nM, determined using the 3 S/m method. Interference studies and the real sample analysis of allergen-treated cell lines proved that the proposed sensing platform possesses excellent sensitivity, reproducibility, and stability. Therefore, it can potentially be used to evaluate stress factors in medical research and clinical practice.

MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation.

The MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms.

Recombinant Bet v 1-BanLec chimera modulates functional characteristics of peritoneal murine macrophages by promoting IL-10 secretion.

Allergen-specific immunotherapy (AIT) is a desensitizing treatment for allergic diseases that corrects the underlined pathological immune response to innocuous protein antigens, called allergens. Recombinant allergens employed in the AIT allowed the production of well-defined formulations that possessed consistent quality but were often less efficient than natural allergen extracts. Combining recombinant allergens with an adjuvant or immunomodulatory agent could improve AIT efficacy. This study aimed to perform structural and functional characterization of newly designed recombinant chimera composed of the Bet v 1, the major birch pollen allergen, and Banana Lectin (BanLec), TLR2, and CD14 binding protein, for the application in AIT. rBet v 1-BanLec chimera was designed in silico and expressed as a soluble fraction in Escherichia coli. Purified rBet v 1-BanLec (33.4 kDa) retained BanLec-associated biological activity of carbohydrate-binding and preserved IgE reactive epitopes of Bet v 1. The chimera revealed secondary structures with predominant ß sheets. The immunomodulatory capacity of rBet v 1-BanLec tested on macrophages showed changes in myeloperoxidase activity, reduced NO production, and significant alterations in the production of cytokines when compared to both rBanLec and rBet v 1. Comparing to rBet v 1, rBet v 1-BanLec was demonstrated to be more efficient promoter of IL-10 production as well as weaker inducer of NO production and secretion of pro-inflammatory cytokines TNFα, and IL-6. The ability of rBet v 1-BanLec to promote IL-10 in together with the preserved 3D structure of Bet v 1 part implies that the construct might exert a beneficial effect in the allergen-specific immunotherapy.

The Kiwifruit Allergen Act d 1 Activates NF-κB Signaling and Affects mRNA Expression of TJ Proteins and Innate Pro-Allergenic Cytokines.

Impairment of the intestinal barrier is one of the key events in the initiation of the sensitization process in food allergy. The aim of this study was to explore the effects of kiwifruit allergen Act d 1 on intestinal permeability and tight junction protein (TJP) gene expression in vivo and to explore its potential to activate the NF-ĸB signaling pathway and to regulate expression of epithelial pro-allergenic cytokines. Influences of Act d 1 on TJP gene expression and pro-allergenic cytokines in the mouse intestine was analyzed by qPCR upon allergen administration by oral gavage. The effect on the in vivo intestinal permeability was assessed in ELISA by measuring the translocation of ß-lactoglobulin (BLG) into circulation. The capacity of Act d 1 to activate the NF-ĸB pathway was tested in HEK293 cells by fluorescent microscopy and flow cytometry. Administration of Actinidin (Act d 1) increased intestinal permeability to the BLG. This was accompanied by changes in gene expression of TJP mRNA and pro-allergenic cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) compared to the control. Act d 1 reduced TEER of the HEK293 monolayer, was positive in an NF-ĸB-reporter HEK293 cell assay, and induced secretion of TSLP. These findings shed more light on the molecular events in the sensitization process of kiwifruit but possibly also of other protease food allergens.

Employment of proteomic and immunological based methods for the identification of catalase as novel allergen from banana.

Diagnostic reagents based on food allergen extracts often lack sufficient sensitivity. The introduction of well characterized food allergens in molecular allergy diagnosis has been recognized as valid approach to circumvent unstandardized allergen extracts. Banana fruit (Musa acuminata) is a well-established allergen source which besides six characterized allergens, contains unidentified IgE reactive proteins whose clinical relevance remains undefined. By employment of a combinatorial peptide ligand library (CPLL) methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis, a novel allergen from banana fruit was detected in banana as catalase. A recombinant homologue of natural catalase was produced, isolated and biochemically characterized. The recombinant protein showed IgE reactivity in 7 out of 13 tested patients with suspected allergy to banana in immunoblot. Novel banana fruit allergens should be added as components to allergen-microarrays for the diagnosis and the monitoring of banana allergy. SIGNIFICANCE: By employment of CPLL methodology with 2-D PAGE, mass spectrometric and 2-D immunoblot analysis catalase from banana fruit is identified as a novel allergen, with proposed designation as Mus a 7. IgE reactive recombinant Mus a 7 was produced and should be included in a component-resolved allergy diagnosis.