Marija Gavrovic-jankulovic - Academia.edu (original) (raw)

Papers by Marija Gavrovic-jankulovic

Phytochemistry, Oct 1, 2013

Plant proteinase inhibitors are considered important defense molecules against insect and pathoge... more Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality.

Molecular Nutrition & Food Research, Dec 9, 2011

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No i... more In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.

Scientific Reports

This study explored humoral and cellular responses to anti-SARS-CoV-2 BNT162b2 mRNA vaccine in br... more 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+...

Biosensors

This paper aims to develop an amperometric, non-enzymatic sensor for detecting and quantifying UA... more 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 de...

Applied Biochemistry and Biotechnology, 2022

International Journal of Molecular Sciences, 2021

Diagnostic evaluation of specific antibodies against the SARS-CoV-2 virus is mainly based on spik... more 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 pr...

Biomolecules, 2019

Impairment of the intestinal barrier is one of the key events in the initiation of the sensitizat... more 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 o...

Journal of Trace Elements in Medicine and Biology, 2019

Highlights 1. Toxic and essential trace metals in papillary thyroid carcinoma were investigated. ... more Highlights 1. Toxic and essential trace metals in papillary thyroid carcinoma were investigated. 2. The role of cadmium in the etiology of PTC has been proposed.

Trends in Food Science & Technology, 2019

Background: Before introducing proteins from new or alternative dietary sources into the market, ... more Background: Before introducing proteins from new or alternative dietary sources into the market, a compressive risk assessment including food allergic sensitization should be carried out in order to ensure their safety. We have recently proposed the adverse outcome pathway (AOP) concept to structure the current mechanistic understanding of the molecular and cellular pathways evidenced to drive IgE-mediated food allergies. This AOP framework offers the biological context to collect and structure existing in vitro methods and to identify missing assays to evaluate sensitizing potential of food proteins. Scope and approach: In this review, we provide a state-of-the-art overview of available in vitro approaches for assessing the sensitizing potential of food proteins, including their strengths and limitations. These approaches are structured by their potential to evaluate the molecular initiating and key events driving food sensitization. Key findings and conclusions: The application of the AOP framework offers the opportunity to anchor existing testing methods to specific building blocks of the AOP for food sensitization. In general, in vitro methods evaluating mechanisms involved in the innate immune response are easier to address than assays addressing the adaptive immune response due to the low precursor frequency of allergen-specific T and B cells. Novel ex vivo culture strategies may have the potential to become useful tools for investigating the sensitizing potential of food proteins. When applied in the context of an integrated testing strategy, the described approaches may reduce, if not replace, current animal testing approaches.

TrAC Trends in Analytical Chemistry, 2017

Use of foodomics, mostly proteomic and genomic based methods, for study of allergens in food is p... more Use of foodomics, mostly proteomic and genomic based methods, for study of allergens in food is presented. Immunological methods and nucleic acid-based methods are still most frequently used for diagnosis of allergies and for qualitative and quantitative determination of food allergens. They are sensitive, and can be used for the determination of allergens in trace concentrations. However, lack of specificity and cross-reaction of some antibodies can still be a relevant source of bias. The epitopes of protein allergens with posttranslational modifications and their changes originated during food processing cannot be traced by use of nucleic acid-based strategies. Recent developments of both antibody and nucleic acid-based biosensors, their miniaturization and increasing application of nanotechnology, significantly supported further use of both strategies. Regarding accuracy, reliability and sensitivity, mass spectrometry-based methods bring important advantage over both above presented strategies. Furthermore, the increasing use of mass spectrometry (MS) is discussed. Combined with proper sample preparation, liquid chromatography (LC) and/or different electrophoretic methods, targeted approach in mass spectrometry-based allergen analysis brings an additional strategic advance. However, MS is still rarely used for high-throughput analyses and detection and quantification of allergens for the reasons of price and relatively long time necessary for analysis. Recent developments of new high-resolution instruments are encouraging and enable development in the direction of a highthroughput strategy. Consequently, fast, very sensitive, reliable and accurate detection and quantification of allergens in highly complex samples such as food matrices, and the use of MS in routine determination of allergens can be reached in near future.

Drug Discovery Today: Disease Models, 2015

Measurement of protein transport across the intestinal barrier might be a relevant approach in al... more Measurement of protein transport across the intestinal barrier might be a relevant approach in allergenicity risk assessment. Traditionally, studies on protein transport, were performed using stable cell lines cultured as a monolayer. One of the major advantages of these models is their relatively low price and easy handling. However, monolayers lack a physiologically relevant environment (presence of other cell-types and a mucus layer), which may have an effect on transport characteristics and thus correct prediction of protein allergenicity. This paper summarizes the most widely used epithelial models and discusses their benefits and limitations for measuring protein transport and allergic sensitization to food.

PLOS ONE, 2017

We demonstrated that a recombinant banana lectin (rBanLec), which structural characteristics and ... more We demonstrated that a recombinant banana lectin (rBanLec), which structural characteristics and physiological impacts highly resemble those reported for its natural counterparts, binds murine peritoneal macrophages and specifically modulates their functional characteristics. By using rBanLec in concentrations ranging from 1 μg to 10 μg to stimulate resident (RMs) and thioglycollate-elicited (TGMs) peritoneal macrophages from BALB/c and C57BL/ 6 mice, we have shown that effects of rBanLec stimulation depend on its concentration but also on the functional status of macrophages and their genetic background. rBanLec, in a positive dose-dependent manner, promotes the proliferation of TGMs from both BALB/c and C57BL/6 mice, while its mitogenic influence on RMs is significantly lower (BALB/c mice) or not detectable (C57BL/6 mice). In all peritoneal macrophages, irrespective of their type and genetic background, rBanLec, in a positive dose dependent manner, enhances the secretion of IL-10. rBanLec stimulation of RMs from both BALB/c and C57BL/6 resulted in a positive dose-dependent promotion of proinflammatory phenotype (enhancement of NO production and IL-12 and TNFα secretion, reduction of arginase activity). Positive dosedependent skewing toward proinflammatory phenotype was also observed in TGMs from C57BL/6 mice. However, the enhancement of rBanLec stimulation promotes skewing of TGMs from BALB/c mice towards anti-inflammatory profile (reduction of NO production and IL-12 secretion, enhancement of arginase activity and TGFβ and IL-4 secretion). Moreover, we established that rBanLec binds oligosaccharide structures of TLR2 and CD14 and that blocking of signaling via these receptors significantly impairs the production of TNFα and NO in BALB/c macrophages. Since the outcome of rBanLec stimulation depends on rBan-Lec concentration as well as on the functional characteristics of its target cells and their genetic background, further studies are needed to investigate its effects under physiological and specific pathological conditions.

Pediatric Allergy and Immunology, 2016

BackgroundNon‐immediate reactions to beta‐lactam antibiotics (BL) occur more than one hour after ... more BackgroundNon‐immediate reactions to beta‐lactam antibiotics (BL) occur more than one hour after drug administration, and the most common manifestations are maculopapular exanthemas and delayed‐appearing urticaria and/or angioedema. Infections can lead to skin eruptions and mimic drug hypersensitivity reactions (DHR), if a drug is taken at the same time. The most of children are labeled as ‘drug allergic’ after considering only the clinical history.ObjectiveTo diagnose/detect a hypersensitivity or an infection which mimic DHR in children with non‐immediate reactions to BLMethodsA prospective survey was conducted in a group of 1026 children with histories of non‐immediate reactions to BL by performing patch tests, skin tests, and in case of negative results, drug provocation tests (DPTs). In 300 children, a study was performed to detect infections by viruses or Mycoplasma pneumoniae.ResultsUrticaria and maculopapular exanthemas were the most reported non‐immediate reactions. Only 76 ...

Journal of Biotechnology, 2015

Group I grass pollen allergens are major contributors to grass pollen-related seasonal allergic r... more Group I grass pollen allergens are major contributors to grass pollen-related seasonal allergic rhinitis, and as such a primary target for allergen specific immunotherapy. In this study the potential therapeutic role of oral application of Lactobacillus plantarum WCFS1, directing cell wall attachment of the recombinant Fes p1 allergen, from Festuca pratensis was tested in a mouse model of Fes p1 allergy. For surface expression of Fes p1 allergen in L. plantarum WCFS1 pSIP system with inducible expression was used. Balb/c mice were sensitized with Fes p1 protein in alum and subsequently received live recombinant L. plantarum orally. Antibody levels (IgE, total IgG, IgG1, IgG2a, and IgA) were determined by ELISA. Differential eosinophil count in peripheral blood was performed. Reduced peripheral blood eosinophilia and increased serum IgG2A levels was detected in both groups which received live L. plantarum orally. Specific serum IgA levels were increased only in mice treated with the recombinant bacteria. Oral application of L. plantarum WCFS1 has a beneficial therapeutic effect in a mouse model of Fes p1 allergy. Cell surface expression of Fes p1 allergen potentiates this phenomenon in an allergen specific way.

Protein Expression and Purification, 2013

Vojnotehnicki glasnik, 2011

This overview summarizes the application of enzymes in the manufacture and design of biofuel cell... more This overview summarizes the application of enzymes in the manufacture and design of biofuel cells and biosensors. The emphasis will be put on the protein engineering techniques used for improoving the properties of enzymes such as nanobiocatalysts, e.g. immobilization orientation, stability, activity and efficiency of electron transfer between immobilized enzymes and electrodes. Some possible applications in the military and some future designs of these electric devices will be discussed as well.

Pediatric Allergy and Immunology, 2012

Background: Multiple drug hypersensitivity (MDH) has been defined as a hypersensitivity to two or... more Background: Multiple drug hypersensitivity (MDH) has been defined as a hypersensitivity to two or more chemically different drugs. Two types of MDH have been reported: the first one, which develops to different drugs administered simultaneously and the second type, in which sensitizations develop sequentially. In children, studies which diagnose MDH on the basis of positive allergologic tests to 2 or more chemically different drugs are lacking. Methods: We conducted a prospective study evaluating children with histories of MDH by skin tests, patch tests, serum-specific IgE assays, and drug provocation tests. Results: A MDH was diagnosed in 7 (2.5%) of the 279 children evaluated who completed the study. The responsible drugs were b-lactams (penicillins and cephalosporins) in 5 episodes, ibuprofen and anticonvulsants in 3, and erythromycin, fentanyl, methylprednisolone, and cotrimoxazole in 1. Sensitivity to 2 chemically different drugs was diagnosed in 6 children and to 3 drugs in 1 child. Two of the 7 children presented the first type of MDH, whereas 5 displayed the second one. Conclusions: MDH can occur in children, even to drugs other than antibiotics. It is crucial to evaluate children with histories of MDH using both in vivo and in vitro allergologic tests, including challenges. In fact, such approach allows the physician to confirm the diagnosis of MDH in a small percentage of children with histories of MDH, as well as to rule it out in the great majority of them.

Srpski arhiv za celokupno lekarstvo, 2012

Introduction. In spite of the wide usage of corticosteroids for the treatment of a plethora of di... more Introduction. In spite of the wide usage of corticosteroids for the treatment of a plethora of diseases, sometimes they can induce immediate hypersensitivity reactions, which are however uncommon. Case Outline. We report a case of immediate allergic reaction induced by intravenous methylprednisolone given before operation for surgical repair of an arm contracture as a sequel of burns, which the child had tolerated a month before. Six weeks later the patient repeated the anaphylactic reaction during skin testing to methylprednisolone. In addition, basophile activation test with methylprednisolone (BAT) was positive. Conclusion. This case report describes a patient who experienced intraoperative anaphylaxis and anaphylactic reaction induced by skin testing. This is the first report on induction of both anaphylactic reactions by methylprednisolone in the same child. Clinical findings, positive BAT and positive skin tests with methylprednisolone imply that the child developed type-I hyp...

Chemical Industry, 2013

Convenient source of lipases for science and industry is yeast Candida rugosa. Crude preparation ... more Convenient source of lipases for science and industry is yeast Candida rugosa. Crude preparation of Candida rugosa lipase (CRL) consists of several extracellular lipases. Isoenzyme profile depends on the culture or fermentation conditions. All isoforms are coded by lip pseudogene family; they are monomers of 534 amino acids and molecular weight of about 60 kDa. They share the same catalytic mechanism and interfacial mode of activation. Isoenzymes differ in isoelectric points, post-translational modifications, substrate specificity and hydrophobicity. The presence of different lipase isoforms and other substances (i. e. inhibitors) in crude preparation leads to lack of their productivity in biocatalytic reactions. Purification of specific isoform improves its overall performance and stability. This paper provides an overview of different methods for purification of CRL isoenzymes up to date, their advantages and disadvantages.

Srpski arhiv za celokupno lekarstvo, 2010

Introduction. Ingestion is the principal route for food allergens to trigger allergic reaction in... more Introduction. Ingestion is the principal route for food allergens to trigger allergic reaction in atopic persons. However, in some highly sensitive patients severe symptoms may develop upon skin contact and by inhalation. The clinical spectrum ranges from mild facial urticaria and angioedema to life-threatening anaphylactic reactions. Outline of Cases. We describe cases of severe anaphylactic reactions by skin contact, induced by kissing in five children with prior history of severe anaphylaxis caused by food ingestion. These cases were found to have the medical history of IgE mediated food allergy, a very high total and specific serum IgE level and very strong family history of allergy. Conclusion. The presence of tiny particles of food on the kisser's lips was sufficient to trigger an anaphylactic reaction in sensitized children with prior history of severe allergic reaction caused by ingestion of food. Allergic reaction provoked with food allergens by skin contact can be a ri...