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Food allergies mediated by specific IgE (sIgE) have a significant socioeconomic impact on society. Evaluating the IgE cross-reactivity between allergens from different allergen sources can enable the better management of these potentially life-threatening adverse reactions to food proteins and enhance food safety. A novel banana fruit allergen, S-adenosyl-L-homocysteine hydrolase (SAHH), has been recently identified and its recombinant homolog was heterologously overproduced in E. coli. In this study, we performed a search in the NCBI (National Center for Biotechnology Information) for SAHH homologs in ryegrass, latex, and kiwifruit, all of which are commonly associated with pollen-latex-fruit syndrome. In addition, Western immunoblot analysis was utilized to identify the cross-reactive IgE to banana SAHH in the sera of patients with a latex allergy, kiwifruit allergy, and ryegrass allergy. ClustalOmega analysis showed more than 92% amino acid sequence identity among the banana SAHH homologs in ryegrass, latex, and kiwifruit. In addition to five B-cell epitopes, in silico analysis predicted eleven T-cell epitopes in banana SAHH, seventeen in kiwifruit SAHH, twelve in ryegrass SAHH, and eight in latex SAHH, which were related to the seven-allele HLA reference set (HLA-DRB1*03:01, HLA-DRB1*07:01, HLA-DRB1*15:01, HLA-DRB3*01:01, HLA-DRB3*02:02, HLA-DRB4*01:01, HLA-DRB5*01:01). Four T-cell epitopes were identical in banana and kiwifruit SAHH (positions 328, 278, 142, 341), as well as banana and ryegrass SAHH (positions 278, 142, 96, and 341). All four SAHHs shared two T-cell epitopes (positions 278 and 341). In line with the high amino acid sequence identity and B-cell epitope homology among the analyzed proteins, the cross-reactive IgE to banana SAHH was detected in three of three latex-allergic patients, five of six ryegrass-allergic patients, and two of three kiwifruit-allergic patients. Although banana SAHH has only been studied in a small group of allergic individuals, it is a novel cross-reactive food allergen that should be considered when testing for pollen-latex-fruit syndrome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diagnostic evaluation of specific antibodies against the SARS-CoV-2 virus is mainly based on spike (S) and nucleocapsid (N) proteins. Despite the critical functions in virus infection and contribution to the pattern of immunodominance in COVID-19, exploitation of the most abundant membrane (M) protein in the SARS-CoV-2 serology tests is minimal. This study investigated the recombinant M protein's immunoreactivity with the sera from COVID-19 convalescents. In silico designed protein was created from the outer N-terminal part (19 aa) and internal C-terminal tail (101–222 aa) of the M protein (YP_009724393.1) and was recombinantly produced and purified. The designed M protein (16,498.74 Da, pI 8.79) revealed both IgM and IgG reactivity with serum samples from COVID-19 convalescents in Western blot. In ELISA, more than 93% (28/30) of COVID-19 sera were positive for IgM detection, and more than 96% (29/30) were positive for specific IgG detection to M protein. Based on the capacity to provoke an immune response and its strong antigenic properties, as shown here, and the fact that it is also involved in the virion entry into host cells, the M protein of the SARS-CoV-2 virus as a good antigen has the potential in diagnostic purposes and vaccine design. [ABSTRACT FROM AUTHOR]

Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in Escherichia coli. BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed β-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip®) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of Salmonella in flow cytometry, with especially pronounced binding to a Salmonella Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]