Effects of actinidin protease activity on intestinal epithelial cellsʼ integrity

Gastrointestinalna barijera izgrađena je od veoma kompleksne mreže ćelija, mukusa i drugih važnih komponenti imunog sistema. Epitelni sloj ćelija gastrointestinalnog trakta predstavlja važnu komponentu urođene imunosti koja na dinamičan način održava homeostazu. Međutim, narušavanje integriteta gastrointestinalne barijere može se nepovoljno odraziti na organizam. Takav poremećaj dovodi do razvoja alergije na hranu, koja predstavlja neprimeren odgovor ćelija imunog sistema na proteine hrane. Epitelne ćelije gastrointestinalnog trakta, kao posledica kontakta sa alergenom hrane, utiču na komponente imunog sistema (smeštene u sloju lamina propria) u pravcu razvoja Th2 ćelijskog odgovora i proizvodnje specifičnih IgE antitela.Alergije na hranu nastaju kao posledica narušavanja imunološkog odgovora označenog kao oralna toleranca na proteine hrane koji podrazumeva kako proizvodnju specifičnih regulatornih ćelija tako i sintezu određenih signalnih molekula. Alergijske reakcije izazivaju širok spektar kliničkih simptoma i mogu se manifestovati od vrlo blagih reakcija do fatalnih, kao što je anafilaktički šok.Alergeni hrane koji poseduju proteaznu aktivnost u gastrointestinalnom traktu mogu da dovedu do narušavanja integriteta intestinalne barijere i time izazovu neprimerenu reakciju imunog sistema. Osim što pokazuju otpornost na proteolizu i drastične uslove digestivnog trakta, proteaze iz hrane mogu da zadrže i imunogena svojstva. Alergija na kivi je veoma rasprostranjena i značajan je uzročnik alergija na voće. Glavni alergen ploda zelenog kivija, aktinidin (Act d 1), je cistein proteaza (EC 3.4.22.14) koja je ujedno najzastupljeniji i najvažniji alergen kivija, čineći preko 50 % ukupnih solubilnih proteina kivija.U ovoj disertaciji ispitivan je efekat aktinidina i posledice njegove proteazne aktivnosti na epitelne ćelije gastrointestinalnog trakta...The gastrointestinal (GI) barrier consists of a complex network of cells, mucus and other important components of the immune system. The epithelial cell layer of the gastrointestinal tract (GIT) is an important component of innate immunity, which dynamically maintains homeostasis. Therefore, disruption of the integrity of the gastrointestinal barrier can adversely affect the body. Food allergy, an inappropriate response of immune cells to food proteins, is an example of homeostasis disruption. After contact with a food allergen, gastrointestinal epithelial cells stimulate immune cells of lamina propria to induce Th2 cell response and produce specific immunoglobulin E (IgE). Oral tolerance is a normal response of the immune system to food proteins, which includes the production of specific regulatory cells and signal molecules. Disruption of oral tolerance gives rise to food allergy with a wide array of clinical symptoms from mild to life-threatening anaphylaxis. Food allergens with protease activity can impair the integrity of the gastrointestinal barrier and thus cause an inappropriate response of the immune system. These allergens are resistant to proteolysis in the harsh environment of the digestive tract and can also be immunogenic. Allergy to kiwi is among the most common fruit allergies with worldwide prevalence. Actinidin (Act d 1), cysteine protease (EC 3.4.22.14) is the most abundant allergen of green kiwi-fruit. In this dissertation, the effect of Act d 1 proteolytic activity on gastrointestinal epithelial cells was investigated in vitro and in vivo. It is shown that the proteolytic activity of actinidin impairs the integrity of the gastrointestinal barrier in C57BL/6 mice. The same was confirmed in human intestinal epithelial Caco-2 cells. Actinidin hydrolyzes tight junction (TJ) proteins in situ (occludin, ZO 1, claudin 3 and E-cadherin) in Caco-2 cells and 2D organoids derived from murine intestinal epithelial cells. Additionally, active actinidin affects the expression of mRNA for tight junction proteins in vitro. In vivo study in mouse animal model confirmed the results from in vitro study..

Supplementary material for the article: Nikolić, J.; Nešić, A.; Kull, S.; Schocker, F.; Jappe, U.; Gavrović-Jankulović, M. Employment of Proteomic and Immunological Based Methods for the Identification of Catalase as Novel Allergen from Banana. Journal of Proteomics 2018, 175, 87–94. https://doi.org/10.1016/j.jprot.2018.01.007

Supplementary material for: [https://doi.org/10.1016/j.jprot.2018.01.007]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2117

Supplementary material for the article: Nikolić, J.; Nešić, A.; Kull, S.; Schocker, F.; Jappe, U.; Gavrović-Jankulović, M. Employment of Proteomic and Immunological Based Methods for the Identification of Catalase as Novel Allergen from Banana. Journal of Proteomics 2018, 175, 87–94. https://doi.org/10.1016/j.jprot.2018.01.007

Supplementary material for: [https://doi.org/10.1016/j.jprot.2018.01.007]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2117

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 β-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

Supplementary material for: Nešić, A., Stojsavljević, A., Jagodić, J., Čavić, M., Stefanović, A., Manojlović, D.,& Gavrović-Jankulović, M.. (2022). 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. in Journal of Trace Elements in Medicine and Biology Elsevier., 74, 127079. https://doi.org/10.1016/j.jtemb.2022.127079

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.Supplementary material for: [https://doi.org/10.1016/j.jtemb.2022.127079]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/5636

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

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