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Allergenicity assessment of Cor a 8 from raw and roasted hazelnut upon oral-gastric digestion phase of INFOGEST protocol
No full textCor a 8 is a relevant allergen that can cause severe allergic reactions. It is a 115 amino acid protein with a molecular mass of 9 kDa and is a member of the non-specific lipida transfer protein family. This allergen is resistant to high temperatures, pH changes, gastric and intestinal enzymes. The main route of exposure is through ingestion. In order to examine its resistance to digestion, we have applied a popular 1.0 INFOGEST protocol [1], specialized for the complete food, which in vitro mimics physiologically relevant conditions of oral-gastric-intestinal digestion. The aim of this study was to compare Cor a 8 resistance to gastric digestion, from both, raw and roasted hazelnuts, before and upon pepsin (gastric) digestion. Stability of the Cor a 8 protein was investigated by simulation of oral and gastric digestion phases, performed with ground raw and roasted hazelnut kernels. Hazelnut proteins were extracted from the digestion mixture and analyzed by 1D and 2D SDS-PAGE, while raw and roasted Cor a 8 western blots were probed with specific anti-Cor a 8 antibodies in 1D and 2D immunoblots. The electrophoretic patterns of the raw and roasted extracts were similar. 1D SDS PAGE profiles demonstrated high stability of Cor a 8 against enzymatic treatments. Control samples of Cor a 8 from raw and roasted hazelnut extracts migrated as a single band at around 12 kDa in 1D immunoblot. However, in case of roasted hazelnut, the protein showed a slightly lower capacity to bind specific anti-Cor a 8 antibody, as compared to raw hazelnut extract. In 2D immunoblot, with higher resolution, specific antibody binding was decting a significant and noticeable smear in the basic region indicating a range of different protein variants. This was more pronounced detectable in the case of roasted sample upon digestion, pointing to a mix of variants in this allergen batch. It has been suggested that the allergenicity of the Cor a 8 is almost insensitive to temperature. The allergen is stable even after digestion and roasting processes up to 140˚C. We hypothesize that a lipid-rich food matrix delays extraction of proteins, thereby delaying their gastrointestinal digestion, which may affect allergen sensitizing capacity and clinical symptoms
BanLec-eGFP Chimera as a Tool for Evaluation of Lectin Binding to High-Mannose Glycans on Microorganisms
Get PDFFluorescently 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 microorganism
Allergome of oral-gastric in vitro digest of roasted hazelnut shows stronger IgE binding compared to the raw counterpart
Get PDFBackground: In vitro pepsin digestion is important factor when assessing protein foodallergenicity. Roasted hazelnut is more common in human nutrition than a raw hazelnut;however, all studies were focused on Cor a 9 allergen obtained from a raw hazelnut. Thereare only two studies employing in vitro INFOGEST digestion harmonized protocol onhazelnut with its full matrix. The aim of this study was to assess immunoreactivity of rawand roasted hazelnut gastric digests and to compare secondary/tertiary structure of Cor a 9allergen purified from these two sources.Methods: Digestion resistant protein fragments were analysed by 1D/2D electrophoresis.Following digestion, IgE binding from patients’ pooled sera and by specific antibodies, wereassessed in ELISA and immunoblot. CD spectroscopy was applied for Cor a 9 structuralanalyses.Results: Cor a 11 and acidic forms of Cor a 9 were more prone to pepsin proteolysis, yettheir large fragments survived partially. Cor a 8 was protected by lipids, retaining capabilityto bind its specific antibody. Roasting did not significantly affect secondary structure of themost abundant hazelnut allergen, Cor a 9.Conclusion: Roasting of hazelnut seems to boost IgE binding derived from pooled sera ofhazelnut allergic patients with oral-gastric allergen digests.Related to Book of Abstracts: [https://www.cost-infogest.eu/content/download/4051/35805/file/V-ICFD%20Book%20of%20Abstracts.pdf]Related to the record of lecture: [https://www.youtube.com/watch?v=Dj0_1fyY724
Effects of water-filtered infrared A and visible light (wIRA/VIS) radiation on heat- and stress-responsive proteins in the retina and cornea of guinea pigs
Get PDFWater-filtered infrared A and visible light (wIRA/VIS), shown to reduce chlamydial infections in vitro and in vivo, might represent an innovative therapeutic approach against trachoma, a neglected tropical disease caused by ocular infection with the bacterium C. trachomatis. In this in vivo study, we assessed the impact of wIRA radiation in combination with VIS (wavelength range 595–1400 nm, intensity 2100 W/m2) on the retina and cornea in a guinea pig animal model of inclusion conjunctivitis. We investigated the effects 19 days after wIRA/VIS irradiation by comparing a single and double wIRA/VIS treatment with a sham control. By immunolabeling and western blot analyses of critical heat- and stress-responsive proteins, we could not detect wIRA/VIS-induced changes in their expression pattern. Also, immunolabeling of specific retinal marker proteins revealed no changes in their expression pattern caused by the treatment. Our preclinical study suggests wIRA/VIS as a promising and safe therapeutic tool to treat ocular chlamydial infections.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_639
Supplementary material for: Popović, M.; Stojanović, M.; Veličković, Z.; Kovačević, A.; Miljković, R.; Mirković, N.; Marinković, A. D. Characterization of Potential Probiotic Strain, L. Reuteri B2, and Its Microencapsulation Using Alginate-Based Biopolymers. International Journal of Biological Macromolecules 2021, 183, 423–434. https://doi.org/10.1016/j.ijbiomac.2021.04.177.
Get PDFPreparation of materials for encapsulation (Materials, Laboratory isolation of ricinoleic acid, Laboratory preparation of starch maleate monoester, Characterization, Statistical analysis). Additional results (Antimicrobial activity, Optimization of encapsulation yield, Size distribution of alginate beads in acidic conditions). additional referencesThe supplementary material for the article: Popović, M., Stojanović, M., Veličković, Z., Kovačević, A., Miljković, R., Mirković, N.,& Marinković, A. (2021). Characterization of potential probiotic strain, L. reuteri B2, and its microencapsulation using alginate-based biopolymers. International Journal of Biological Macromolecules, Elsevier, 183, 423-434. [https://doi.org/10.1016/j.ijbiomac.2021.04.177]Published version of the article: [http://intor.torlakinstitut.com/handle/123456789/626]The peer-reviewed version of the article: [http://intor.torlakinstitut.com/handle/123456789/628
Supplementary material for: Frohns, A.; Stojanović, M.; Barisani-Asenbauer, T.; Kuratli, J.; Borel, N.; Inić-Kanada, A. Effects of Water-Filtered Infrared A and Visible Light (WIRA/VIS) Radiation on Heat- and Stress-Responsive Proteins in the Retina and Cornea of Guinea Pigs. Journal of Photochemistry and Photobiology B: Biology 2021, 224, 112306. https://doi.org/10.1016/j.jphotobiol.2021.112306.
Get PDFFigure S1. Structure of guinea pig retina and cornea. Nuclear staining with DAPI of guinea pig retina and cornea. In the retina, three nuclear layers (ONL-outer nuclear layer, INL-inner nuclear layer, and GCL-ganglion cell layer) are present. In the ONL, the photoreceptor cells are located, with their outer segments (OS) projecting into the adjacent retinal pigment epithelium (RPE). The INL exhibit nuclei of horizontal-, bipolar-, Müller glia, and amacrine cells. The cornea consists of an epithelium (EP), the stroma (S) containing keratinocytes, and the endothelium (EN); Figure S2. Western blots of HSPs in the guinea pig retina and cornea 19 days after wIRA/VIS treatment. Exemplary western blots of HSP40, HSP60, HSP70, and HSP90 in the retina and cornea. Comparison of non-irradiated (no wIRA/VIS) tissues with ones subjected to single (wIRA/VIS 1×) or sequential (wIRA/VIS 2×) irradiation. The constitutively expressed protein vinculin served as a loading control; Figure S3. Localization of Vinculin in the guinea pig retina and cornea 19 days after wIRA/VIS treatment. Immunolabeling of Vinculin in retinal layers (OS-outer segments, ONL-outer nuclear layer, INL-inner nuclear layer, and GCL-ganglion cell layer) and corneal layers (EP-epithelium, S-Stroma, and EN-endothelium). Comparison of non-irradiated (no wIRA/VIS) tissues with ones subjected to single (wIRA/VIS 1×) or sequential (wIRA/VIS 2×) irradiation. Inserts in cornea images show magnified sections of EP and EN. Scale bars = 10 μm retina, 20 μm cornea; Figure S4. Western blots of stress-responsive proteins in the guinea pig retina and cornea 19 days after wIRA/VIS treatment. Exemplary western blots of Akt, pAkt, pPTEN, pcRaf, Erk1/2, and pErk1/2 in the retina and cornea. Comparison of non-irradiated (no wIRA/VIS) tissues with ones subjected to single (wIRA/VIS 1×) or sequential (wIRA/VIS 2×) irradiation. The constitutively expressed protein vinculin served as a loading control.Supplementary material for: [https://doi.org/10.1016/j.jphotobiol.2021.112306]Related to the published version: [https://intor.torlakinstitut.com/handle/123456789/617
Colonization of vancomycin-resistant Enterococcus spp. strains in hospitals setting – phenotipic and genotipic characterization of isolated strains and risk factors for colonization
Get PDFDetekcija fekalne kolonizacije vankomicin-rezistentnim Enterococcus spp. (VRE) sojevima ubolničkoj sredini ima veliki značaj u nadzoru nad bolničkom infekcijom s obzirom da je poznato da VREkolonizacija najčešće prethodi VRE infekciji. Ovo istraživanje predstavlja prvo epidemiološkomikrobiološkoistraživanje o fekalnoj kolonizaciji VRE sojevima kod hospitalizovanih pacijenata naodelјenjima sa povišenim rizikom za nastanak VRE kolonizacije.CILJEVI: Ciljevi ovog istraživanja su bili: (1) utvrđivanje učestalosti fekalne kolonizacije VREsojevima kod hospitalizovanih pacijenata na odelјenjima sa povišenim rizikom za nastanak kolonizacije;(2) identifikacija faktora rizika za fekalnu VRE kolonizaciju; (3) ispitivanje fenotipskih i genotipskihkarakteristika izolovanih VRE sojeva i utvrđivanje klonalne povezanosti i klonalne diseminacije VREsojeva.MATERIJAL I METODE: Istraživanje je uključilo 268 ispitanika sa šest odeljenja u tri univerzitetskebolnice u Beogradu. Za ispitivanje faktora rizika (FR) korišćena je multivarijantna logistička regresija.Karakterizacija VRE izolata obuhvatila je određivanje profila rezistencije primenom automatizovanogBD Phoenix™ sistema, molekularnu identifikaciju detekcijom vrsno specifičnih gena (ddlE. faecium, ddlE.faecalis), detekciju gena rezistencije na glikopeptidne antimikrobne lekove (vanA, vanB, vanC1, vanC2/C3), detekciju gena rezistencije na kvinupristin-dalfopristin (Q-D) (vatD, vatE, vgbA, ermB1),detekciju gena za faktore virulencije (esp, hyl, efaA, asa1, gelE, cpd) i MLVA analizu (eng. MultiplelocusVariable-number tandem repeat analysis). Ispitivanje sposobnosti formiranja biofilma rađeno jemetodom u mikrotitracionoj ploči.REZULTATI: Učestalost fekalne VRE kolonizacije je iznosila 28,7%. Nezavisni prediktori za nastanakVRE kolonizacije među ispitanicima hospitalizovanim na kliničkim odeljenjima sa povišenim rizikomza nastanak VRE kolonizacije bili su hospitalizacija na kliničkim odeljenjima, hospitalizacija preuzorkovanja duža od tri dana, primena cefalosporina i primena flourohinolona. U odnosu na odeljenja zahemodijalizu, boravak na odeljenjima za gerijatriju povećao je rizik za VRE kolonizaciju 6,5 puta,boravak u jedinicama intenzivnog lečenja 5 puta, a boravak na hemato-onkološkim odeljenjima 4,7 puta.U odnosu na ispitanike koji su hospitalizovani 48 sati pre uzorkovanja stolice na VRE, ispitanicihospitalizovani 3-7 dana pre uzorkovanja imali su 5,6 puta veći rizik za VRE kolonizaciju, ispitanicihospitalizovani 8-15 dana pre uzorkovanja 5,5 puta veći rizik za VRE kolonizaciju, dok su ispitanicihospitalizovani duže od 16 dana pre uzorkovanja imali 8,4 puta veći rizik za VRE kolonizaciju. Primenacefalosporina povećala je rizik za VRE kolonizaciju 2,2 puta, a primena flourohinolona 1,8 puta. Sviizolovani VRE sojevi su bili multirezistentni vanA Enterococcus faecium (VRfm) sojevi sa sledećimgenima za faktore virulencije (procenat sojeva s naznačenim genom je prikazan u zagradi): ermB-1(38,9%), esp (84%), efaA (71,2%), hyl (54,5%), asa1 (23,4%), gelE i cpd (11,6%) genima. Sposobnostproizvodnje biofilma pokazalo je 20,8% izolata. Analiza genetske srodnosti izolata otkrila je heterogenupopulaciju VRE izolata raspoređenih u 13 klastera sa 25 jedinstvenih MLVA profila (MT). VREfm sojevikoji su pripadali najvećim klasterima su bili dispergovani po različitim bolničkim odeljenjima. SRB3 jeoznačen kao osnivač populacije. SRB3 i SRB9 su označeni kao najbliži srodnici MT-1, a SRB16 kaonajbliži srodnik MT-159 klona...INTRODUCTION: Intestinal carriage of vancomycin-resistant Enterococcus spp. (VRE) often precedes VRE infection and it could be of key importance in infection control. Our study represents the first epidemiological-microbiological study on VRE intestinal carriage in at-risk inpatients in high-risk departments for VRE colonization. AIMS: The aims of this study were: (1) to determine the frequency of VRE intestinal carriage among high-risk inpatients in Serbia in wards with an increased risk for VRE colonization; (2) identification of risk factors for VRE intestinal carriage; (3) examination of phenotypic and genotypic characteristics of isolated VRE strains and determination of clonal relatedness and clonal dissemination of circulating VRE strains. MATERIALS AND METHODS: The study population included 268 inpatients from six hospital departments at 3 university hospitals in Belgrade. To examine risk factors (RF) multivariate analysis was used. Characterization of the isolated VRE stains was performed with BD Phoenix system. Genotypic identification (ddlE. faecium, ddlE. faecalis), glycopeptide (vanA, vanB, vanC1, van C2/C3) and quinupristin– dalfopristin (Q–D) resistance probing (vatD, vatE, vgbA, ermB1), virulence gene detection (esp, hyl, efaA, asa1, gelE, cpd) and MLVA (Multiple-locus Variable-number tandem repeat analysis) were performed by molecular genetic methods. Biofilm formation was evaluated with the microtiter plate method. RESULTS: VRE carriage prevalence among at-risk patients was 28.7%. Independent predictors of VRE colonization among at-risk inpatients for VRE colonization were hospitalization in clinical wards, hospitalization longer than three days before sampling, use of cephalosporins and fluoroquinolones. Compared to the hemodialysis departments, stay in the geriatric departments increased the risk of VRE colonization 6.5 times, stay in intensive care units increased the risk 5 times, and a stay in the hematooncology departments 4.7 times. Compared to inpatients who were hospitalized 48 hours before stool sampling for VRE, inpatients hospitalized 3-7 days before sampling had 5.6-fold higher risk for VRE colonization, inpatients hospitalized 8–15 days prior to sampling had 5.5-fold higher risk for VRE colonization, while inpatients hospitalized longer than 16 days prior to sampling had 8.4-fold higher risk for VRE colonization. The use of cephalosporins increased the risk of VRE colonization 2.2 times and the use of fluoroquinolones 1.8 times. All VRE strains were vanA positive multidrug-resistant Enterococcus faecium (VRfm), harboring ermB-1 (38.9%), esp (84%), efaA (71.2%), hyl (54.5%), asa1 (23.4%), gelE and cpd (11.6%) genes. The ability of biofilm production was detected in 20.8%. Genetic relatedness of the isolates revealed 13 clusters and 25 unique MLVA (MT) profiles. VREfm strains belonging to the largest clusters were dispersed among different hospital departments. SRB3 was labeled as group founder. SRB3 and SRB9 were labeled as subgroup founders of MT-1 clone and SRB16 as subgroup founder of MT-159 clone. CONCLUSION: The obtained prevalence of VRE intestinal carriage among high-risk inpatients in Serbia is higher than European average. Prolonged hospitalization in high-risk departments and administration of cephalosporins and fluoroquinolones were the main risk factors for VRE colonization. High percentage of multidrug- resistance VREfm, with the ability of biofilm production and with various virulence genes that might affect the pathogenicity of the strains, as well as the emergence of resistance to Q–D which has never been licensed for clinical use in our country, are of particular concern. The illicit ABSTRACT usage of antibiotics in animal farming could be implicated. MLVA analysis revealed 29 genotypes, of which 25 were new. MLVA could exclude cross transmission among inpatients. Identified VREfm genotypes were phylogenetically closly related to the most common MTs causing VRE nosocomial infections in the world
Enterocytes in Food Hypersensitivity Reactions
Get PDFFood hypersensitivity reactions are adverse reactions to harmless dietary substances, whose causes are hidden within derangements of the complex immune machinery of humans and mammals. Until recently, enterocytes were considered as solely absorptive cells providing a physical barrier for unwanted lumen constituents. This review focuses on the enterocytes, which are the hub for innate and adaptive immune reactions. Furthermore, the ambiguous nature of enterocytes is also reflected in the fact that enterocytes can be considered as antigen-presenting cells since they constitutively express major histocompatibility complex (MHC) class II molecules. Taken together, it becomes clear that enterocytes have an immense role in maintaining oral tolerance to foreign antigens. In general, the immune system and its mechanisms underlying food hypersensitivity are still unknown and the involvement of components belonging to other anatomical systems, such as enterocytes, in these mechanisms make their elucidation even more difficult. The findings from studies with animal models provide us with valuable information about allergic mechanisms in the animal world, while on the other hand, these models are used to extrapolate results to the pathological conditions occurring in humans. There is a constant need for studies that deal with this topic and can overcome the glitches related to ethics in working with animals
Sexual dimorphism in CX3CR1/Nrf2/HO‐1 spinal cord axis affects therapeutic efficacy of β‐adrenoceptor blockade in EAE rats
No full textOur previous studies showed more severe EAE in male compared with female adult rats, and moderating effect of propranolol‐induced β‐adrenoceptor blockade on EAE in female rats through stimulation of Nrf2/HO‐1 signalling pathway in spinal cord microglia. This study was designed to examine putative sexual dimorphism in Nrf2/HO‐1 signalling pathway and CX3CL1, as one of its activators. Propranolol treatment beginning from the appearance of the first clinical signs of EAE mitigated the disease severity in rats of both sexes, but its effect was more prominent in males. This correlated with more prominent effect of propranolol on the expression of CX3CL1 in spinal cord tissue, CX3CR1 on microglial surface, and Nrf2/HO‐1 in spinal cord microglia in males. Consistently, the proportion of CX3CR1‐expressing microglia and CX3CR1 density on their surface increased more prominently in males. Consistently, propranolol increased the proportion of IL‐10/TGF‐β‐producing microglia and microglia expressing CD163, molecule highlighting ramified microglia with neuroprotective properties in damaged tissue, to a greater extent in males. Additionally, propranolol increased phagocyting capacity of microglia to a greater extent in males. Moreover, propranolol more prominently decreased the frequency of blood‐borne myeloid cells and highly pathogenic IL‐17+ T‐cells, coexpressing GM‐CSF/IFN‐γ, in male rat spinal cord. This correlated with greater reducing effect of propranolol on the spinal cord tissue expression of CCL2/CCL19/CCL21 chemokines in males. The study as a whole indicates that sexual dimorphism in CX3CR1/Nrf2/HO‐1 spinal cord axis could contribute to greater severity of EAE in male rats, and sexually dimorphic action of substances affecting its signalling capacity.6th European Congress ofImmunology 1‐4 September 2021, Virtual meeting, Abstracts of ECI 2021Abstracts: [https://doi.org/10.1002/eji.202170200
Intestinal carriage of vancomycin-resistant Enterococcus spp. among high-risk patients in university hospitals in Serbia: first surveillance report
Get PDFBackground: The screening for intestinal carriage of vancomycin-resistant Enterococcus spp. (VRE) among high risk patients in the Balkan region and molecular epidemiology of VRE is insufficiently investigated, yet it could be of key importance in infection control. The aim of this study was to provide baseline data on VRE intestinal carriage among high-risk patients in Serbian university hospitals, to determine the phenotypic/genotypic profiles of the isolated VRE, to obtain knowledge of local resistance patterns and bridge the gaps in current VRE surveillance.
Methods: The VRE reservoir was investigated using stool samples from 268 inpatients. Characterization of isolated VRE stains consisted of BD Phoenix system, genotypic identification, glycopeptide and quinupristin-dalfopristin (Q-D) resistance probing, virulence gene (esp, hyl, efaA, asa1, gelE, cpd) detection and MLVA. Biofilm formation was evaluated by the microtiter plate method.
Results: VRE carriage prevalence among at-risk patients was 28.7%. All VRE strains were vanA positive multidrug-resistant Enterococcus faecium (VRfm), harboring ermB-1 (38.9%), esp (84%), efaA (71.2%), hyl (54.5%), asa1 (23.4%), gelE and cpd (11.6%) each. Ability of biofilm production was detected in 20.8%. Genetic relatedness of the isolates revealed 13 clusters, heterogeneous picture and 25 unique MTs profiles.
Conclusion: The obtained prevalence of VRE intestinal carriage among high-risk inpatients in Serbia is higher than the European average, with high percentage of multidrug resistance. The emergence of resistance to Q-D is of particular concern. Close monitoring of pattern of resistance and strict adherence to specific guidelines are urgently needed in Serbia