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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. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4888]

Increased interest in microbiota calls for the thorough analysis of antibody reactivity to different microorganisms. As salivary IgA represents the first line of defence against microorganisms contacting mucosal surfaces, we explored the binding and specificity of salivary IgA by testing the binding of purified, FITC-labelled salivary IgA to different microorganisms in flow cytometry and conclude that this kind of analysis enables the differentiation of species/strains with high IgA binding capacity, which should be corroborated on a larger sample size. Further we compare, with in-house ELISA, the binding of polyclonal salivary IgA with the binding of polyclonal serum IgA from the same individuals to whole microbial cells and to purified microbial components. High correlations were obtained in total salivary IgA binding to Lactobacillus rhamnosus and Escherichia coli, very distant bacterial species, as well as to isolated bacterial components (r = .70–.97). The binding of total salivary IgA resembled the binding of both salivary IgA1 and IgA2, with IgA2 predominating. For serum polyclonal IgA repertoire, substantially higher specificity was obtained. Serum IgA binding to E. coli correlated best with serum IgA binding to lipopolysaccharide (r = .86), and serum IgA against L. rhamnosus correlated best with the anti-peptidoglycan IgA levels (r = .88). We have also detected that total serum IgA response is governed by either IgA1 or IgA2 response, depending on the nature of the antigen/s. We conclude that steady state salivary IgA repertoire, unlike serum IgA repertoire, consists of polyreactive antibodies with innate specificity, questioning its capacity to select resident microbiota.

S. pneumoniae is an important human pathogen which has a polysaccharide capsule with virulent properties. This work aims to estimate the titres of S. pneumoniae specific IgG and IgA isotypes, with respect to age and sex. An in-house whole bacterial cell ELISA was used for the determination of relative levels and endpoint titres of IgG subclasses and IgA1 subclass specific for S. pneumoniae serogroup 1, and to quantify specific IgG1 and IgG2 levels. Significantly lower anti-pneumococcus IgG1 titres were found in older individuals, which was more pronounced in men. Lower IgG2 titres were detected in men over 50 years of age, in comparison to women under 50 years of age. The levels of IgG3 and IgG4 did not differ between different sex and age groups. Lower IgA1 levels were detected in male individuals in both age groups in comparison to females under 50 years of age. The levels of IgG1 showed a moderate correlation with IgG4 in younger individuals of both sexes (r = 0.61 in men and 0.63 in women) which was not noted in the older age group. We highlight the deficiency in humoral immunity in older people, especially male and suggest immunization of this population with pneumococcal vaccines.

Background: The aim of this study was to compare serum sclerostin concentrations in patients with thyroid dysfunction with euthyroid control subjects and to assess the relationship between sclerostin and markers of bone metabolism (osteocalcin and beta-cross-laps). Methods: The study included 30 patients with thyroid dysfunction (hypothyroidism, hyperthyroidism and subclinical hyperthyroidism) and ten euthyroid controls. Free thyroxine (FT4) was measured by radioimmunoassay, while thyroid stimulating hormone (TSH) concentration was determined immunoradiometrically. We used an ELISA kit to determine the sclerostin level. The electrochemiluminescence method was applied for measuring the bone markers. Results: Sclerostin levels were significantly lower in hypothyroid patients (p=0.009) and significantly elevated in hyperthyroid patients (p=0.008) compared to control values. Hyperthyroid patients also had higher sclerostin than patients with subclinical hyperthyroidism (p=0.013). Sclerostin concentrations were negatively correlated with TSH levels (r=-0.746, p lt 0.001), but positively with FT4 (r=0.696, p lt 0.001). Moreover, sclerostin was positively associated with osteocalcin (r=0.605, p=0.005) and beta-cross-laps levels (r=0.573, p=0.008) in all thyroid patients. Conclusions: Serum sclerostin is significantly affected in subjects with thyroid dysfunction. Both sclerostin and thyroid status affect bone homeostasis, which is reflected through the significant correlations with osteocalcin and beta-cross-laps. Uvod: Cilj ove studije bio je da uporedimo serumske koncentracije sklerostina kod pacijenata sa disfunkcijom štitaste žlezde u odnosu na eutiroidne kontrolne ispitanike, i da procenimo odnos između sklerostina i markera koštanog metabolizma (osteokalcina i beta-cross-lapsa) u istoj populaciji. Metode: Studijom je obuhvaćeno 30 pacijenata sa disfunkcijom štitaste žlezde (hipotireozom, hipertireozom i supkliničkom hipertireozom) i 10 eutiroidnih kontrola. Slobodni tiroksin (FT4) meren je radioimunološkom metodom, dok je koncentracija tireostimulišućeg hormona (TSH) određivana imunoradiometrijski. Za merenje nivoa sklerostina koristili smo ELISA-test. Metoda elektrohemiluminiscencije primenjena je za merenje koncentracije koštanih markera. Rezultati: Nivo sklerostina bio je značajno niži kod bolesnika sa hipotireozom (p = 0,009) odnosno značajno viši kod bolesnika sa hipertireozom (p = 0,008) u poređenju sa vrednostima kod eutiroidnih kontrolnih ispitanika. Pacijenti sa hipertireozom su takođe imali statistički značajno viši nivo sklerostina u odnosu na bolesnike sa supkliničkom hipertireozom (p = 0,013). Pokazana je negativna korelacija koncentracije sklerostina i TSH (r = -0,746, p lt 0,001), odnosno pozitivna korelacija sa FT4 (r = 0,696, p lt 0,001) kod pacijenata sa tireoidnom disfunkcijom. Štaviše, sklerostin pozitivno korelira sa koncentracijom osteokalcina (r = 0,605, p = 0,005) i beta-cross-lapsa (r = 0,573, p = 0,008) kod ovih pacijenata. Zaključak: Serumski nivoi sklerostina izmenjeni su kod osoba sa disfunkcijom štitaste žlezde. Sklerostin zajedno sa tireoidnim statusom utiče na koštani metabolizam, što se ogleda u njegovoj značajnoj korelaciji sa osteokalcinom i beta-cross-lapsom.

Natural, polyreactive, low-affinity antibodies are known to play an important role not only in the immediate defense against pathogens, but also in shaping the acquired immune response. On the other hand, antigen specific, high-affinity antibodies can affect the balance of natural antibodies and lead to autoimmune diseases. In this study, we have analyzed the changes that occur in the IgM and IgG pool of natural antibodies after immunization with split or whole virion influenza vaccine. For this purpose, "in-house" developed ELISAs were used. The subjects were divided, according to the vaccination status, into those who had been immunized with the influenza vaccine in previous years and those who had been immunized for the first time. The analysis indicated that the pool of natural antibodies was not impaired by the immunization, evidenced by the lack of changes in any of the groups, and that certain fluctuations were induced in order to maintain the homeostasis of the immune system.

The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical immunochemistry assay based on the specific bond between the antigen and the antibody. The application of this test has significantly changed the practice of medical laboratories in which it is used for detection and quantification of molecules such as hormones, peptides, antibodies, and proteins. Various technical variants of this test can detect antigen (native or foreign) or antibody, determine the intensity of the immune response whether pathological or not; the type of induced immune response as well as the innate immunity potential; and much more. These capabilities, as well as the high sensitivity and robustness of the test and a small price, make it possible to quickly and reliably diagnose diseases in most laboratories. Besides, ELISA is a test that is also used in veterinary medicine, toxicology, allergology, food industry, etc. Despite the fact that it has existed for almost 50 years, different ELISA tests with different technical solutions are still being developed, which improves and expands the application of the this exceptional test. The aim of this chapter is to empower the rider to optimize, standardize and validate an enzyme linked immunosorbent assay.

During time, both professionals and general public became aware of the importance of animal welfare. This term not only covers endangered wild animal species, animals used in food industry, pets and experimental animals, but also animals used in production of biologics. The implementation of the 3R concept (Replacement, Reduction and Refinement) is especially important in this type of production. In this article, we describe for the first time the low dose, low volume and multi-site immunization protocol, as well as appropriate ELISA we developed for production of European anti-viper (V. ammodytes, long horned) antivenom in horses, which can help to significantly improve the welfare of the used animals.

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% &plusmn, 21% of all measured air pollen species, while Betula pollen represented 15% &plusmn, 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% &plusmn, 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&mdash, 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.

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, RCA(120) - 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 RCA(120) 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 mu g mL(-1) and to P. roqueforti with 1.10 mu g mL(-1). This unique specificity of BL could be exploited for screening purposes and potentially for the detection of ss-glucan in solutions.

Aim To investigate the immunomodulatory potential of a chimera composed of the receptor-binding domain of hemagglutinin 1 (H1s) from Influenza virus and Der p 2 (D2) allergen for allergen-specific immunotherapy of house-dust mite allergy (HDM). Main methods: H1sD2 chimera and D2 allergen were produced by genetic engineering in E. coli. Recombinant antigens were extracted from inclusion bodies by urea, then refolded and purified by immobilized-metal affinity chromatography (IMAC). Purity was verified by 2D-PAGE and secondary structures were assessed by CD spectroscopy. IgE reactivity of H1sD2 and D2 was tested in western blot with sera from 8 persons with clinical history of HDM allergy. Immunogenicity of H1sD2 and D2 were analyzed in Balb/c mice. Cytokine profile was analyzed by ELISA after stimulation of mouse spleen cells with H1sD2 and D2. Leukocyte population abundance of cells isolated from spleen and lymph node was assessed by flow cytometry. Key findings: Purified recombinant proteins H1sD2 (42 kDa) and D2 (15 kDa) revealed well defined secondary structures, and preserved IgE reactive epitopes. Analysis of supernatants of mouse spleen cells after stimulation with H1sD2 and D2, revealed a qualitatively different cytokine profile from H1sD2 immunized mouse cells (increase in IL10). CD8+ cells were decreased in the lymph node of D2 immunized mice, whereas H1sD2 immunization led to an increase of CD8+ cells in both the lymph node and the spleen. Significance: H1sD2 chimera attenuates Der p 2-inherent Th2 response and directs the immune response toward Th1 and Treg phenotype. This is the peer-reviewed version of the article: Mrkić, I., Minic, R., Popović, D., Zivkovic, I.,& Gavrović-Jankulović, M. (2018). Newly designed hemagglutinin-Der p 2 chimera is a potential candidate for allergen specific immunotherapy. Life SciencesPergamon-Elsevier Science Ltd, Oxford., 213, 158-165. [https://doi.org/10.1016/j.lfs.2018.10.036] Published version: [https://cer.ihtm.bg.ac.rs/handle/123456789/2380]

The goal of this work was to elucidate similarities between microorganisms from the perspective of the humoral immune system reactivity in professional athletes. The reactivity of serum IgG of 14 young, individuals was analyzed to 23 selected microorganisms as antigens by use of the in house ELISA. Serum IgM and IgA reactivity was also analyzed and a control group of sex and age matched individuals was used for comparison. The obtained absorbance levels were used as a string of values to correlate the reactivity to different microorganisms. IgM was found to be the most cross reactive antibody class, Pearson's r = 0.7 +/- 0.92, for very distant bacterial species such as Lactobacillus and E. coli. High correlation in IgG levels was found for Gammaproteobacteria and LPS (from E. coli) (r = 0.77 for LPS vs. P. aeruginosa to r = 0.98 for LPS vs. E. coli), whereas this correlation was lower in the control group (r = 0.49 for LPS vs. P. aeruginosa to r = 0.66 for LPS vs. E. coli). The correlation was also analyzed between total IgG and IgG subclasses specific for the same microorganism, and IgG2 was identified as the main subclass recognising different microorganisms, as well as recognising LPS. Upon correlation of IgG with IgA for the same microorganism absence of or negative correlation was found between bacteria-specific IgA and IgG in case of Lactobacillus and Staphylococcusgeni, whereas correlation was absent or positive for Candida albicans, Enterococcusfaecalis, Streptococcus species tested in professional athletes. Opposite results were obtained for the control group. Outlined here is a simple experimental procedure and data analysis which yields functional significance and which can be used for determining the similarities between microorganisms from the aspect of the humoral immune system, for determining the main IgG subclass involved in an immune response as well as for the analysis of different target populations.

To test the influence of probiotic supplementation on humoral immune response, a double-blind, placebo-controlled trial was conducted. Thirty athletes (24 males and 6 females, females: (V)over dotO(2)max 38.2 +/- 4.9 ml.kg(-1).min(-1), age 23.2 +/- 1.4 years; males: (V)over dotO(2)max 57.5 +/- 9.2 ml.kg(-1).min(-1), age 24.0 +/- 2.4 years, mean +/- SD) were randomized either to the probiotic group (Lactobacillus helveticus Lafti L10, 2 x 10(10) colony-forming units) or to the placebo group. Serum and saliva samples were collected at the baseline and after 14 weeks. Total and specific antibacterial antibody levels of IgM, IgG, and IgA classes were determined for different bacteria in the serum, and in saliva, total and specific antibacterial IgA levels were examined. Total IgM was elevated in both probiotic (18%, 15-20%; mean, 90% confidence interval; p = 0.02) and placebo group (35%, 22-47%; p = 0.02), without observed differences in changes between the groups. No significant changes in IgM levels specific for tested bacteria were found. Total IgG level was constant in both groups. A significant (16%, 22.8 to 35%, p = 0.04) reduction of anti-Enterococcus faecalis IgG was noted in the placebo group, in comparison with the probiotic group. There was a substantial decrease in total IgA level in the placebo group, when measured either in serum (15%, 12-18%, p = 0.04) or in saliva (35%, -1.4 to 53%, p = 0.03). Significantly reduced levels of serum anti-lactic acid bacteria IgA antibodies in the placebo group compared with the probiotic group were detected for Lactobacillus rhamnosus LA68 (24%, 5.8-42%, p = 0.02) and for L. rhamnosus LB64 (15%, 2.7-27%, p = 0.02). Probiotic administration could have beneficial effects on systemic humoral and mucosal immune responses. This is peer-reviewed version of the following article: Michalickova, D. M.; Kostic-Vucicevic, M. M.; Vukasinovic-Vesic, M. Dj.; Stojmenovic, T. B.; Dikic, N. V.; Andjelkovic, M. S.; Djordjevic, B. I.; Tanaskovic, B. P.; Minic, R. D. Lactobacillus Helveticus Lafti L10 Supplementation Modulates Mucosal and Humoral Immunity in Elite Athletes: A Randomized, Double-Blind, Placebo-Controlled Trial. J. Strength Cond. Res. 2017, 31 (1), 62–70. [https://doi.org/10.1519/JSC.0000000000001456]

Tuberculosis (TB) remains among the most deadly diseases in the world. The only available vaccine against tuberculosis is the bacille Calmette-Guérin (BCG) vaccine, which does not ensure full protection in adults. There is a global urgency for the development of an effective vaccine for preventing disease transmission, and it requires novel approaches. We are exploring the use of lactic acid bacteria (LAB) as a vector for antigen delivery to mucosal sites. Here, we demonstrate the successful expression and surface display of a Mycobacterium tuberculosis fusion antigen (comprising Ag85B and ESAT-6, referred to as AgE6) on Lactobacillus plantarum . The AgE6 fusion antigen was targeted to the bacterial surface using two different anchors, a lipoprotein anchor directing the protein to the cell membrane and a covalent cell wall anchor. AgE6-producing L. plantarum strains using each of the two anchors induced antigen-specific proliferative responses in lymphocytes purified from TB-positive donors. Similarly, both strains induced immune responses in mice after nasal or oral immunization. The impact of the anchoring strategies was reflected in dissimilarities in the immune responses generated by the two L. plantarum strains in vivo . The present study comprises an initial step toward the development of L. plantarum as a vector for M. tuberculosis antigen delivery. IMPORTANCE This work presents the development of Lactobacillus plantarum as a candidate mucosal vaccine against tuberculosis. Tuberculosis remains one of the top infectious diseases worldwide, and the only available vaccine, bacille Calmette-Guérin (BCG), fails to protect adults and adolescents. Direct antigen delivery to mucosal sites is a promising strategy in tuberculosis vaccine development, and lactic acid bacteria potentially provide easy, safe, and low-cost delivery vehicles for mucosal immunization. We have engineered L. plantarum strains to produce a Mycobacterium tuberculosis fusion antigen and to anchor this antigen to the bacterial cell wall or to the cell membrane. The recombinant strains elicited proliferative antigen-specific T-cell responses in white blood cells from tuberculosis-positive humans and induced specific immune responses after nasal and oral administrations in mice.

A randomized, double-blind, placebo-controlled study was conducted to evaluate if Lactobacillus helveticus Lafti L10 (Lallemand Health Solutions, Montreal, Que., Canada) supplementation during 14 weeks in winter can influence the duration, severity, and incidence of upper respiratory tract illness (URTI), as well as to monitor different immune parameters in the population of elite athletes. Before and after the treatment, cardiopulmonary testing and self-rated state of moods evaluation (by Profile of Mood States questionnaire) were performed and blood samples were collected. Thirty-nine elite athletes were randomized either to the placebo (n = 19) or the probiotic (n = 20) group. The probiotic group received L. helveticus Lafti L10, 2 x 10(10) Colony Forming Units. Lafti L10 significantly shortened the URTI episode duration (7.25 +/- 2.90 vs. 10.64 +/- 4.67 days, p = 0.047) and decreased the number of symptoms in the probiotic group (4.92 +/- 1.96 vs. 6.91 +/- 1.22, p = 0.035). Severity and incidence of URTI did not differ between the treatments. There were no significant changes in leukocyte subpopulation abundance, transforming growth factor-beta serum levels, level of interleukin-10 secreted from peptidoglican stimulated peripheral blood mononuclear cells (PBMCs), interferon-gamma level secreted from concanavalin A-stimulated PBMCs or viability/proliferation of PBMCs upon antigen stimulation. Group effect for CD4+/CD8+ ratio was significant (F-[1,F-37] = 6.99, p = 0.020, eta(2) = 0.350); this difference was not significant at baseline, but was evident after 14 weeks (p = 0.02). A significant interaction effect was noted for self-rated sense of vigor (F-[1,F-37] = 11.76, p = 0.009, eta(2) = 0.595). Self-rated sense of vigor increased in the probiotic group (18.5 +/- 4.1 vs. 21.0 +/- 2.6, p = 0.012). Probiotic strain Lafti L10 can be a beneficial nutritional supplement for the reduction of URTI length in elite athletes. This is the peer-reviewed version of the following article: Michalickova, D.; Minić, R.; Dikić, N.; Anđelković, M.; Kostić-Vučićević, M.; Stojmenović, T.; Nikolić, I.; Đorđević, B. Lactobacillus Helveticus Lafti L10 Supplementation Reduces Respiratory Infection Duration in a Cohort of Elite Athletes: A Randomized, Double-Blind, Placebo-Controlled Trial. Applied Physiology Nutrition and Metabolism 2016, 41 (7), 782–789. [https://doi.org/10.1139/apnm-2015-0541].