rBet v 1a-BanLecwt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors.

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT.

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.

Differences in mouse strains determine the outcome of Der p 2 allergy induction protocols.

In vivo animal models can provide worthy information on various aspects of asthma mechanism and pathogenesis. The genetic predisposition and phenotype of mice may affect the immune response itself. Here we compare the early immune response to Der p 2 or HDM allergen extract upon injection and inhalation in BALB/c and C57BL/6 mice. Female C57BL/6 and BALB/c mice were immunized with Der p 2 allergen subcutaneously followed by inhalation of Der p 2 or HDM extract. After challenge, the mice were euthanized; blood, bronchoalveolar lavage (BAL), spleens and lungs were collected. Cells from BAL were identified by May-Grünwald Giemsa staining and lung leukocyte populations were analyzed by flow cytometry. Serum antibody levels of Der p 2 specific IgE, IgG, IgG1 and IgG2a were assessed by ELISA, and cytokine secretion (IL-4, IFN-γ and IL-10) was evaluated upon stimulation with Der p 2 or HDM extract. The Th2 immune response was confirmed by elevated allergen-specific immunoglobulin E (IgE) and the allergic reaction was evidenced by infiltration of eosinophils and/or neutrophils into BAL. We found that BALB/c mice were inefficient in integrating local with systemic immune response, evidenced by almost no IgG or IgE production upon one subcutaneous injection and subsequent inhalation of Der p 2 allergen; also, the bronchoalveolar lavage infiltrate in these mice consisted of neutrophil infiltration, unlike C57BL/6 mice in which eosinophilic infiltrate predominated. The differences between BALB/c and C57BL/6 mice strains could be exploited for generating different types of responses to the Der p 2 allergen.

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.

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.

Comparison of Enzyme-Linked Lectin Sorbent Assay and Flow Cytometry for Profiling Microbial Glycans.

The surface of microorganisms is covered with carbohydrates, which makes them unique, self-sustaining glycan probes. Lectins are able to bind to these probes, and this interaction can be exploited for selecting microorganisms or novel lectins. To examine lectin-microorganism interactions, we have previously developed an enzyme-linked lectin sorbent assay (ELLSA) with whole bacterial cells. To further test the validity of this methodology, here we compare it with flow cytometry. For this purpose, we used biotinylated recombinantly produced lectin from Musa acuminata (BanLec), this lectin's recombinantly produced chimera with green fluorescent protein (BanLec-eGFP) and a lectin from Ricinus communis (RCA120), both biotinylated and FITC labeled. Parallel testing showed equivalent results for the two methods, in terms of the presence or absence of binding, with signal intensity yielding high Pearson correlation coefficient of 0.8 for BanLec and 0.95 for RCA120. The ELLSA method demonstrated multiple advantages, such as reliability and convenience for high-throughput analysis; it also required less lectin and yielded more consistent results. As such, ELLSA proved to be a useful tool for profiling microbial glycan structures or testing novel lectins.

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.

IgM and IgG Immunoreactivity of SARS-CoV-2 Recombinant M Protein.

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.

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.

BanLec-eGFP Chimera as a Tool for Evaluation of Lectin Binding to High-Mannose Glycans on Microorganisms.

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 ß-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.

Alteration of Trace Elements in Multinodular Goiter, Thyroid Adenoma, and Thyroid Cancer.

Modest progress has been made in understanding the role of trace elements as endocrine disruptors. The aim of this study was to examine whether there is a change in the content of trace elements in thyroid disease, as well as whether the ratio of elements could be considered a blood marker for thyroid disease. In addition, this study examined the influence of biological and clinical/pathological parameters on the elemental profile. Blood samples from patients diagnosed with multinodular goiter (MNG), thyroid adenoma (TA), and thyroid cancer (TC) were examined and compared with control samples using chemometric analysis. The concentrations of essential (Mn, Co, Cu, Zn, Se) and toxic elements (Ni, As, Cd, Pb, U) were determined by ICP-MS. This study showed for the first time that the content of Mn, Co, Ni, Cu, Zn, Se, and Pb in pathological blood samples was significantly lower compared to the control, while opposite results were obtained for As, Cd, and U. Based on the classification model, the most important trace metals for discrimination of MNG and TC from the control group (CG) were Co and Zn, while Co, Zn, and Mn influenced the distinction of CG from TA. Moreover, it was found that Cu/Zn and U/Se ratios had significantly increased values in pathological blood samples leading to the possibility of establishing new circulating screening markers. These findings can represent significant translational information since these diseases are widespread and the diagnostic procedure is still difficult in many cases.

The first insight into the trace element status of human adrenal gland accompanied by elemental alterations in adrenal adenomas.

BACKGROUND: The baseline status of trace metals in adrenal tissue is unresolved, while the elemental profile for any adrenal pathology has not been examined so far. This study aimed to determine the baseline status of important toxic (Ni, As, Cd, Pb, Th, U) and essential trace elements (Mn, Co, Cu, Zn, Se) in healthy adrenal tissues (HATs) as well as to examine whether there are alterations in the elemental composition of adenomatous adrenal tissues (AATs). Furthermore, this study aimed to find potential trace metals that could play a role in the pathogenesis of adrenal adenoma (AA). METHODS: The study included 45 patients diagnosed with AA. Impacts of relevant parameters such as gender, age, smoking habits and nodular sizes were considered. All samples were subjected to microwave digestion and the trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: This is the first study that provided an insight into the elemental status of HATs. It was also shown that AATs had altered trace metal contents. Compared to HATs, the most significant findings were related to the high content of essential (Cu, Mn, Se, Zn) and Pb as a non-essential metal. Although gender, age and smoking habits had a modest effect on metal profiles, the most significant alterations were related to the nodular diameter above 4 cm, indicating that the growth of benign tumor could influence changes in elemental composition. CONCLUSION: For the first time the baseline contents of essential and toxic trace metals in HATs were determined. The results of this study may highlight the role of toxic and essential trace metals in AAs and could provide new insights into the molecular basis of pathophysiological changes caused by the hazardous effects of trace metals on adrenal structure and function.

Significance of arsenic and lead in Hashimoto's thyroiditis demonstrated on thyroid tissue, blood, and urine samples.

Our previous investigation showed significantly increased arsenic (As) content in thyroid tissue samples of patients with Hashimoto's thyroiditis (HT). This research aimed to extend previous findings and provide reliable insight into the close relationship between As and other trace elements with HT by considering a greater number of thyroid tissue samples, accompanied by blood and urine samples. The essential trace elements for thyroid homeostasis (Mn, Cu, Zn, Se) and the main threatening toxic trace elements (Ni, As, Pb, Cd, U) was analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Relevant parameters that could affect the concentration of trace elements were considered. This research showed that there was a difference in the elemental profile between HT and control samples. The most important findings were related to the elevated As and Pb content in the thyroid tissue and HT blood samples. The obtained negative correlations between As and Pb with Se may explain the antagonistic effect of As and Pb on the extrusion of essential Se from the HT tissue. The reduced Se content in the blood and its increased content in urine samples may further confirm this hypothesis and explain the lack of Se in HT. Furthermore, the reported results may highlight the unresolved molecular basis of HT and could indicate the role of trace element effects on thyroid homeostasis.

Assessment of trace metal alterations in the blood, cerebrospinal fluid and tissue samples of patients with malignant brain tumors.

The pathogenesis of malignant brain tumors (MBTs) should be better understood due to the evident association between prolonged exposure to metals and increased risk of MBTs. The present research aimed to find trace metals that could contribute to the pathogenesis of MBTs. Essential trace elements (Mn, Co, Zn, Cu, Se) and relevant toxic metals (Al, Ni, As, Sr, Cd, Ce, Pt, Pb, U) in the serum, cell fraction (CF), cerebrospinal fluid (CSF) and cancerous tissue (CT) samples of MBT patients were analyzed. The results were compared with sex- and age-matched control groups. For the first time, this research showed that elemental profiles of serum, CF, CSF and CT samples in MBT patients were significantly altered compared to the appropriate controls, as well as that higher contents of trace elements (particularly Mn, Se, and Pb) could be involved in the pathogenesis of MBTs. However, the most noticeable change found was the elevated U content, indicating its considerable role as a major cerebral discriminator of the presence/absence of MBTs. The U/Se ratio could be considered as an appropriate blood marker in diagnostic MBT evaluation. The reported results could contribute to better understanding of the poorly understood pathogenesis of MBTs. Furthermore, the reported results could highlight a molecular basis for the pathophysiological changes caused by the hazardous effects of trace metals on brain homeostasis.

Impact of Tree Pollen Distribution on Allergic Diseases in Serbia: Evidence of Implementation of Allergen Immunotherapy to Betula verrucosa.

Background and objectives: The relationship between air pollen quantity and the sensitization of allergic patients is crucial for both the diagnosis and treatment of allergic diseases. Weather conditions influence the distribution of allergenic pollen and increases in pollen concentration may negatively affect the health of allergic patients. The aim of this study was to analyze the implementation of allergen immunotherapy with regard to air pollen concentration. Material and Methods: Here we examined the relationship between Betula air pollen concentration and the usage of Betula verrucosa allergen immunotherapy in Serbia. Examination covered the period from 2015 to 2018. Measurement of airborne pollen concentration was performed with Lanzoni volumetric pollen traps. The evidence of the usage of sublingual allergen immunotherapy (SLIT) was gathered from patients with documented sensitization to specific pollen. Results: During this period tree pollens were represented with 58% ± 21% of all measured air pollen species, while Betula pollen represented 15% ± 8% of all tree pollens. Betula pollination peaked in April. Allergen immunotherapy to Betula verrucosa in Serbia is entirely conducted as sublingual immunotherapy and represents 47.1% ± 1.4% of issued tree pollen SLIT. The use of pollen SLIT increased by 68% from 2015 to 2018, with an even greater increase in usage recorded for Betula SLIT-80%. Conclusions: This analysis shows a clear causative relationship between pollination and the type/prevalence of applied allergen immunotherapy. Information about the flowering seasons of allergenic plants is very important for people who suffer from allergy, for clinical allergologists, as well as for governing authorities. The presented data is of practical importance to the proper timing of immunotherapy initiation and of importance for urban landscaping. The obtained data can be the starting point for the instatement of a thorough epidemiological study and the inclusion of Serbia on the pollen map of Europe.

ELLSA based profiling of surface glycosylation in microorganisms reveals that ß-glucan rich yeasts' surfaces are selectively recognized with recombinant banana lectin.

The surface of microorganisms is covered with polysaccharide structures which are in immediate contact with receptor structures on host's cells and antibodies. The interaction between microorganisms and their host is dependent on surface glycosylation and in this study we have tested the interaction of plant lectins with different microorganisms. Enzyme-linked lectin sorbent assay - ELLSA was used to test the binding of recombinant Musa acuminata lectin - BL to 27 selected microorganisms and 7 other lectins were used for comparison: Soy bean agglutinin - SBA, Lens culinaris lectin - LCA, Wheat germ agglutinin - WGA, RCA120 - Ricinus communis agglutinin, Con A - from Canavalia ensiformis, Sambucus nigra agglutinin - SNA I and Maackia amurensis agglutinin - MAA. The goal was to define the microorganisms' surface glycosylation by means of interaction with the selected plant lectins and to make a comparison with BL. Among the tested lectins most selective binding was observed for RCA120 which preferentially bound Lactobacillus casei DG. Recombinant banana lectin showed specific binding to all of the tested fungal species. The binding of BL to Candida albicans was further tested with fluorescence microscopy and flow cytometry and it was concluded that this lectin can differentiate ß-glucan rich surfaces. The binding of BL to S. boulardii could be inhibited with ß-glucan from yeast with IC50 1.81 µg mL-1 and to P. roqueforti with 1.10 µg mL-1. This unique specificity of BL could be exploited for screening purposes and potentially for the detection of ß-glucan in solutions.

Reference values for trace essential elements in the whole blood and serum samples of the adult Serbian population: significance of selenium deficiency.

This study was aimed to determine reference values (RVs) for the manganese (Mn), copper (Cu), zinc (Zn), and selenium (Se) in the whole blood (B) and serum (S) samples of the Serbian population. Blood specimens were collected from healthy persons (n = 295; women/men ratio = 149/146; mean age: 42 ± 2 years). The RVs were calculated as lower limit (LL) and upper limit (UL) of the 95% confidence interval (CI) and were expressed as percentiles (P) in the range from P2.5 to P97.5. The influences of sex, age, and smoking habits on element profiles were considered. It was found that the contents of B-Cu and S-Cu were higher in women, while the contents of B-Zn and S-Zn were higher in men. Both trace elements were significantly increased in a group of persons above 40 when compared to a younger persons (≤ 40 years). According to smoking habits, increased content was found only for S-Mn in the nonsmoker's group (p < 0.05). Comparing our results to the results reported in other population groups worldwide, the Serbian population had significantly reduced content of Se in both types of samples. This finding could highlight the deficiency of Se in the investigated Serbian population and could contribute to the better understanding of the molecular basis for the increased incidence of thyroid and other diseases in which selenium plays a key role.

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.

Evaluation of trace metals in thyroid tissues: Comparative analysis with benign and malignant thyroid diseases.

Evaluation of trace metals at level of solid tissue can provide better information than blood or urine and, therefore, could highlight the role of metals in the etiology of organ-specific disease. The current study aimed to establish the baseline content of four essential (Mn, Cu, Zn, Se) and four toxic metals (As, Cd, Pb, U) in the healthy thyroid tissues (HTTs) by considering sex, age and smoking habits. A further aim was to examine whether differences in the content of metals exist in regard to the thyroid diseases, such as benign tumor (BT), Hashimoto's thyroiditis (HT), multinodular goiter (MNG) and thyroid cancer (TC). A total number of investigated tissue samples were 423. All metals were quantified by inductively coupled plasma-mass spectrometry (ICP-MS). It was found that the content of Cu and U was higher in HTTs of women, while the content of Zn was higher in HTTs of men. Increased content of Zn and decreased content of U was found in the group of HTTs above 50 years compared to a younger group (<50 years). Increased content of Cd, Pb and U distinguish smokers from the non-smokers. In comparison with other population groups worldwide, investigated Serbian population had up to 15 times reduced content of Se. Despite the difference in metal's profile according to biological variables, this study also demonstrated, for the first time, that each thyroid disease has its unique metal's profile. The most altered metal's content was found in tissues with HT. Contrarily, the greatest similarity in metal's content with HTTs was found in BT tissues. Based on the increased content, metal's that dominantly discriminated HTTs from the HT, MNG and TC was As, Pb and Cd, respectively. Reported results could highlight the role of toxic and essential trace metals in the not very well clarified etiology of thyroid diseases and, moreover, could provide a molecular basis for pathophysiological changes of metal's hazardous effects on thyroid health at the tissue level.

Cadmium as main endocrine disruptor in papillary thyroid carcinoma and the significance of Cd/Se ratio for thyroid tissue pathophysiology.

BACKGROUND: The etiology of papillary thyroid carcinoma (PTC) is unknown and some literature data support the hypothesis that heavy metals, as endocrine disrupters, could play a major role in the pathogenesis of thyroid cancer. This study aimed to estimate the content of selected toxic and essential trace metals (Mn, Co, Ni, Cu, Zn, As, Se, Cd, Pb, Th, and U), as well as the selected ratio's (Cu/Zn and Cd/Se) in the malignant thyroid tissues according to sex, age, smoking habits, familial history of any thyroid disease, pathohistological (PH) types of PTC, tumor size, the existence of a thyroid capsular invasion, intrathyroid tumor dissemination, retrosternal thyroid growth, and TNM progress of PTC. METHODS: The study included 66 patients with PTC (women/men ratio = 46/20, mean age: 54 ± 14 years). A comparative analysis was made by collecting the healthy thyroid tissues (HTTs) of the same patients, making the total number of samples 132. All trace metals were quantified by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Metals that significantly separated papillary thyroid tissues (PTTs) from the HTTs were Cd, U and Se (p < 0.05). The obtained negative correlation between Cd and Se in the PTTs could explain extrusion of essential Se caused by increased content of Cd. Only Cd had an influence on the retrosternal thyroid growth, while the essential metals (Mn, Co, and Zn) had an influence on thyroid capsular invasion. CONCLUSION: It was found that Cd act as the main endocrine disrupter, which could highlight its role in the etiology of PTC. Considering that the Cd/Se ratio significantly separated two studied groups and had an influence on the retrosternal thyroid growth, its altered content could contribute to the better understanding of the molecular basis for pathophysiological changes in the PTC.