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Michalickova, D, Minic, R, Kotur-Stevuljevic, J, Andjelkovic, M, Dikic, N, Kostic-Vucicevic, M, Slanar, O, and Djordjevic, B. Changes in parameters of oxidative stress, immunity, and behavior in endurance athletes during a preparation period in winter.J Strength Cond Res34(10): 2965-2973, 2020-The current study monitored markers of immunological and oxidative status in 9 male elite endurance athletes: V?o(2)max: 68 +/- 11 ml center dot kg(-1)center dot min(-1), age: 24 +/- 2.5 years, and training loads: 128 +/- 21 metabolic equivalents-h center dot wk(-1)during a 3-month preparation period in winter (January-March). Self-rated state of moods evaluation (by Profile of Mood States questionnaire) was performed, and blood samples were collected at the beginning and end of the study. Spectrophotometric methods and enzyme-linked immunosorbent assay were used for parameters' determination. The level of concanavalin A (ConA)-stimulated interferon-gamma (IFN-gamma) from peripheral blood mononuclear cells (PBMCs) was increased (562 [147-852] vs. 1,097 [451-1842] pg center dot ml(-1),p= 0.013). Also, the level of transforming growth factor-1 (TGF-beta 1) in serum was elevated (2.5 [1.4-5.1] vs. 7.2 [4.9-8.2] ng center dot ml(-1),p= 0.015). There was no change in the level of peptidoglycan (PGN)-stimulated interleukin (IL)-10 from PBMCs. There were no significant changes in PBMCs proliferation/viability on stimulation with ConA and PGN during the study. No changes in superoxide dismutase, prooxidative-antioxidative balance, total oxidant status (TOS), and thiobarbituric acid reactive substances were observed along the study. Total antioxidant status (TAS) was increased (910 +/- 174 vs. 1,090 +/- 102 mu mol center dot L-1,p= 0.018), and activity of paraoxonase (PON1) was decreased (523 +/- 295 vs. 335 +/- 183 U center dot L-1,p= 0.003) at the end of the study. Advanced oxidation protein products were increased (25 +/- 7.9 vs. 42 +/- 7.6 mu mol center dot L-1,p= 0.011). The self-rated sense of vigor significantly declined (20 +/- 2.1 vs. 14 +/- 3.4,p= 0.045). In conclusion, 3 months of regular training in winter induced prominent changes in cytokines, biomarkers of oxidative stress, and antioxidative enzyme activity. These changes might increase susceptibility of athletes to disease and muscle damage and consequently lead to performance reduction.

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.

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.

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.

The MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms.

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.

The study explored influence of biological sex on development of humoral immune response to seasonal trivalent whole inactivated virus (WIV) and split virus (SV) influenza vaccines in outbred Swiss mouse model. To this end, mice of both sexes were immunized with WIV (WIV mice) and SV vaccines (SV mice) and examined for specific antibody response. Irrespective of sex, total IgG and neutralizing antibody responses to distinct virus strains were weaker in SV than in WIV mice. In WIV mice of both sexes, irrespective of strain specificity, IgG isotype response was dominated by IgG2a antibodies, while in SV mice nearly equal representation of IgG2a and IgG1 antibodies was found. The analyses of sex differences showed higher titers of H1N1-specific and both H1N1- and H3N2-specific total IgG and neutralizing antibodies in female WIV and SV mice, respectively. Additionally, sexual dimorphism in IgG subclass profile depended on vaccine type. Specifically, compared with males, in females WIV shifted IgG2a/IgG1 antibody ratio towards IgG2a isotype on the account of weaker IgG1 response, whereas in SV mice, irrespective of virus strain, IgG2a and IgG1 isotypes were equally represented in both sexes. These findings indicate the vaccine type-dependent sex bias in antibody response to inactivated influenza vaccines.

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.

Immunoglobulins, either natural or induced, represent powerful means for eliminating microbial threats. For decades, vaccines have been used which function by inducing antibodies for the successful elimination of microbes, preventing disease and mortality. Polyclonal immunoglobulin products from human plasma were used for the first time to treat immune deficiency during the 1950s. Nowadays, the administration of immunoglobulin, either intravenous or subcutaneous, is the cornerstone of the treatment of primary immunodeficiency affecting the humoral immune system. The therapy can help people with weakened or underdeveloped immune system fight off infections. This is also the treatment of choice for certain autoimmune and neurological diseases. The polyclonal antibody preparation consists of both natural antibodies and antigen-induced antibodies and reflects the history of antigen experience of the donor population. Natural antibodies are germline-encoded, polyspecific, low-affinity antibodies that also have a regulatory role in maintaining the immune system homeostasis. Antibody preparations, enriched for certain desired specificities, or hyperimmune human globulins are used for prophylaxis and treatment of infectious diseases with a high mortality rate. It was shown that lymphocytes from survivors of viral infections, without available vaccines, could be used for protective monoclonal antibodies production. This chapter provides insight into the current knowledge of the usage of antibody preparations, efficacy and mechanism of action, with respect to specific diseases, including the treatment of infectious diseases and future prospects.

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.

BACKGROUND Group 1 and group 2 allergens from house dust mite are the major elicitors of respiratory allergic diseases and the main candidates for immunotherapy. RESULTS The potential therapeutic role of a chimera composed of recombinant Der p 2 (D2) linked to Influenza A virus hemagglutinin 1 (H1) for intranasal application was created, expressed and tested in a mouse model. H1D2 and D2 were produced by genetic engineering in E. coli and their primary structure was confirmed by mass fingerprint. Both antigens preserved IgE reactivity in immunoblot with serum from seven house dust mite allergic persons. Balb/c mice were sensitized with D2 allergen in alum and subsequently received H1D2 or D2, intranasally. The reduced levels of serum D2 specific IgE, together with the increased serum specific IgG and IgA were detected in both groups which received H1D2 and D2 intranasally. A higher level of effector CD4+CD25+ spleen lymphocytes was found only in the group of mice which received i.n. H1D2. CONCLUSION H1D2 chimera can have a therapeutic potential in Der p 2 allergic persons as dual vaccine which, beside protective allergen specific, can provide protective antibodies against Influenza A virus hemagglutinin 1.

Western life style, and high calorie diet in particular is causing major health problems such as insulin resistance, hepatic steatosis and heart disease in the modern age. High fat diet (HFD) induces similar changes in mice, such as increased body weight, hypercholesterolemia and accumulation of triglycerides in the liver. These changes can be ameliorated by the administration of some Lactobacillus species. The focus of this study was to analyze the fatty acid content of liver, heart and brain tissues of mice fed HFD and administered with either Lactobacillus plantarum WCFS1 or Lactobacillus rhamnosus LA68, and to analyze the fatty acid content of these organs after a two months washout period. The fatty acid composition of mouse liver tissue changed significantly due to probiotic administration during a 12 weeks HFD regime and active Lactobacillus administration had a slightly reversing effect toward the standard mouse diet group, but after the washout period these changes disappeared. The fatty acid composition of the heart and brain tissues was significantly changed in the HFD regime but probiotic administration had no significant influence on the fatty acid profile of these two organs. Upon the 8 weeks washout period the only remaining beneficial effect was the significantly lower mouse weight in the supplemented groups compared to the HFD group.

Aims The study examined the influence of sex and mouse strain on germinal center (GC) reaction and antibody responses to seasonal split trivalent influenza vaccine (TIV). Main methods C57BL/6 and BALB/c mice of both sexes were immunized with TIV and examined for specific antibody response by ELISA. Splenic T follicular regulatory (Tfr), T follicular helper (Tfh) and GC B cells are detected by flow cytometry. The proliferative response of splenocytes, and concentrations of IFN-γ and IL-4 upon restimulation with vaccine antigens were examined by 7-AAD staining and ELISA, respectively. Key findings BALB/c mice developed more robust IgG responses to vaccine type A antigens than their sex-matched C57BL/6 counterparts, while that to B antigen did not differ between strains. In both strains IgG responses against type A vaccine antigens were greater in females than in males. The greater IgG responses correlated with lower splenic Tfr/Tfh and Tfr/GC B cell ratios and greater vaccine antigen-specific proliferative responses of CD4+ and B cells in splenocyte cultures. In both mouse strains IgG2a(c)/IgG1 ratios were comparable between sexes, but lower in BALB/c than in C57BL/6 mice indicating a shift in Th1/Th2 balance towards Th2 response in BALB/c ones. Consistently, splenocytes from BALB/c mice produced more IL-4 and less IFN-γ than those from C57BL/6 mice. Significance The study indicated that magnitude of humoral response to influenza type A haemagglutinins depends on mouse strain and sex, and thereby set background for the vaccination strategies taking into account biological sex, and in a longterm perspective individual differences in immune reactivity.

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].

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. (C) 2015 Elsevier B.V. All rights reserved.

An outbred mouse model was used to determine if antibody response to immunization with whole-virus trivalent inactivated influenza vaccine (TIV) differs between the sexes. The antibody response was examined one (serum titer of IgM antibodies), and three and six weeks post-immunization (serum titer of neutralizing and total IgG antibodies and IgG subclass profile). Compared with male in female mice was found (i) the more robust IgM response against all influenza strains included in TIV and (ii) more vigorous neutralizing antibody and total IgG responses against H1N1 influenza virus at both the examined time points post-immunization. The total IgG antibody response against H3N2 and B influenza viruses was comparable between female and male mice three weeks post-immunization, but significantly greater in female mice six weeks post-immunization. The neutralizing antibody response against H3N2 and B influenza viruses did not significantly differ between sexes at both the examined points post-immunization. Finally, three weeks post-immunization subclass profile of IgG specific to the influenza strains included in TIV differed between female and male mice, reflecting the lower titer of IgG1 antibodies in female ones, so that IgG2a (contributing mainly to the total IgG) to IgG1 ratio in mice of this sex was shifted toward the former. In agreement with this shift, compared with male mice, Th1/Th2 balance in female mice was shifted toward Th1, as shown by ELISPOT. Collectively, the results showed influenza virus strain-dependent sexual dimorphism in the magnitude, dynamics and characteristics of antibody response in outbred mice immunized with TIV. (C) 2015 Elsevier Ltd. All rights reserved.

In this study, two Lactobacillus strains (L. rhamnosus LA68 and L. plantarum WCFS1) were evaluated for their effects on high fat diet induced pathology in mice. The aim was to determine whether the administration of lactic acid bacteria had beneficial effects on ameliorating pathology. C57BL/6 mice fed a high fat diet were orally administered with the Lactobacillus strains. Both the metabolic and immunological parameters were analyzed. The administration of both of the strains had beneficial effects on mouse weight, serum cholesterol, TNF-alpha levels and liver histology. LA68 lowered the total cholesterol and HDL levels more prominently, whereas WCFS1 was more potent in lowering the TG and LDL levels. Leptin and adiponectin levels were increased in all experimental groups to different extents. The administration of L. plantarum WCFS1 led to a marked increase in leptin levels, as well as an increase in CD3+CD4+ and CD3+CD8+ cells, and a decrease of CD25+ cells, and had a lowering effect on IL-6 production and cell metabolic activity. In conclusion, active administration of both Lactobacillus strains had a positive effect on HFD-induced pathology. Although both of the tested strains had beneficial effects, oral administration of WCFS1 increased leptin levels and had a more prominent immunomodulatory effect, which should be taken into consideration in case of humane usage.

Recent reports suggest that the metabolic activity of the enteric microbiota may influence the fatty acid composition of the host tissue. There are many studies dealing with the influence of lactobacilli on various pathological conditions, and some of the effects are strain-specific. This study was designed to test the effects of a particular Lactobacillus strain, Lactobacillus rhamnosus LA68 on fatty acid composition of the liver and the brain of C57BL/6 mice in the absence of an underlying pathological condition. Female mice were supplemented with live L. rhamnosus LA68 bacteria for the duration of 1 month. Serum biochemistry was analyzed and liver and brain fatty acid composition was assessed by gas-liquid chromatography. Significant changes in liver and brain fatty acid composition were detected. In the liver tissue we detected an increase in palmitoleic acid (p = 0.038), while in the brain compartment we found an increase in palmitic (p = 0.042), stearic (p = 0.017), arachidonic acid (p = 0.009) and docosahexaenoic acid (p = 0.004) for control versus experimental group. These results show discrete changes caused by LA68 strain consumption. Even short duration of administration of LA68 influences the fatty acid composition of the host which adds to the existing knowledge about Lactobacillus host interaction, and adds to the growing knowledge of metabolic intervention possibilities.