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Autoaggregation, the ability to self-aggregate, is widespread among both Gram-positive and Gram-negative bacteria. The functional role of aggregation is not fully understood, but it is believed to be involved in the adaptation of bacteria to environmental conditions (PMID: 31294207). One interesting class of compounds responsible for the aggregation of lactic acid bacteria is aggregation factors—surface high-molecular-weight proteins rich in threonine and lysine (PMID: 30027759). Recently, our research group discovered a new strain of Streptococcus thermophilus in the mountainous regions of Serbia, exhibiting an aggregation phenotype. Aggregation phenotype was confirmed visually and using microscopy. Complete genome of Agg+ strain was sequenced using NGS and a gene encoding a potential aggregation factor, which was named aggS was identified. The predicted threonine (12.5%) and lysine (10.5%) rich protein contains 2367 amino acids, with an average molecular weight of 255986.63 Da. AggS also contains two cysteine residues,whereas previously well-described aggregation factors of this type did not contain any cysteine residues. The predicted protein includes an N-terminal YSIRK-like signal sequence and an LPXTG cell wall anchor domain. It has 6 Mucin binding domain repeats alternating with 6 Mub B2-like domain repeats. Additionally, we found a region resembling an ice-binding domain. Given that these bacteria endure prolonged periods of low temperatures, it can be speculated that this surface membrane protein also helps the bacteria withstand freezing. The fact that the alignment using BLASTp revealed AggS to be most closely related to an uncharacterised protein from the genome of Lactococcus garvieae, along with the discovery of a transposase gene sequence upstream of the gene, suggests that the aggregation factor was likely acquired through horizontal gene transfer. We plan to clone it into a shuttle vector and investigate the aggregation phenotype using a heterologou

that can most closely evaluate the biological activity of the drug of interest. For testing the potency of antivenom, these are the methods used to assess cytotoxicity. The aim of this study was to evaluate the most commonly used cytotoxicity methods for determining the protective potency of the antivenom Viekvin, which neutralizes Vipera ammodytes venom. Material and methods: The selected methods are based on different biological mechanisms: MTT assay, based on the activity of cell oxidoreductase enzymes; crystal violet staining, based on the degree of cell adhesion; trypan blue staining, based on cell membrane permeability, and propidium iodide staining, based on measurement of nucleic acids of dead cells. The pro-apoptotic effect of the venom was also determined with annexin V staining. Results: The IC50 value of V. ammodytes venom obtained by these methods was very similar, while the EC50 values differed significantly. Conclusions: We concluded that the choice of the method used to measure the anticytotoxic antivenom potency depends on the immunogenicity of the venom components that cause cell death; for each venom/antivenom pair, it is necessary to select the appropriate assay separately, and at present, none of the standard cytotoxic methods can be universally applied to determine antivenom potency.

Aging is closely related to the main aspects of multiple sclerosis (MS). The average age of the MS population is increasing and the number of elderly MS patients is expected to increase. In addition to neurons, N-methyl-D-aspartate receptors (NMDARs) are also expressed on non-neuronal cells, such as immune cells. The aim of this study was to investigate the role of NMDARs in experimental autoimmune encephalomyelitis (EAE) in young and aged rats. Memantine, a non-competitive NMDAR antagonist, was administered to young and aged Dark Agouti rats from day 7 after immunization. Antagonizing NMDARs had a more favourable effect on clinical disease, reactivation, and apoptosis of CD4+ T cells in the target organ of aged EAE rats. The expression of the fractalkine receptor CX3CR1 was increased in memantine-treated rats, but to a greater extent in aged rats. Additionally, memantine increased Nrf2 and Nrf2-regulated enzymes’ mRNA expression in brain tissue. The concentrations of superoxide anion radicals, malondialdehyde, and advanced oxidation protein products in brain tissue were consistent with previous results. Overall, our results suggest that NMDARs play a more important role in the pathogenesis of EAE in aged than in young rats.

Aging is closely related to the main aspects of multiple sclerosis (MS). The average age ofthe MS population is increasing and the number of elderly MS patients is expected to increase. Inaddition to neurons,N-methyl-D-aspartate receptors (NMDARs) are also expressed on non-neuronalcells, such as immune cells. The aim of this study was to investigate the role of NMDARs inexperimental autoimmune encephalomyelitis (EAE) in young and aged rats. Memantine, a non-competitive NMDAR antagonist, was administered to young and agedDark Agoutirats from day7 after immunization. Antagonizing NMDARs had a more favourable effect on clinical disease,reactivation, and apoptosis of CD4+T cells in the target organ of aged EAE rats. The expression ofthe fractalkine receptor CX3CR1 was increased in memantine-treated rats, but to a greater extent inaged rats. Additionally, memantine increased Nrf2 and Nrf2-regulated enzymes’ mRNA expressionin brain tissue. The concentrations of superoxide anion radicals, malondialdehyde, and advancedoxidation protein products in brain tissue were consistent with previous results. Overall, our resultssuggest that NMDARs play a more important role in the pathogenesis of EAE in aged than in young rats.

Introduction: In recent decades, many efforts have been made in laboratories to develop alternative methods that would replace animal tests or reduce the number of animals used. In this sense, the methods for measuring cytotoxicity have proven to be very suitable substitutes for testing toxicity. As each individual substance being tested has its own specificities, it is necessary to carefully select a specific, appropriate cytotoxic method. This is especially important if the investigated substance is complex, such as animal venom. Also, antivenoms are drugs for which it is necessary to correctly determine the protective power.Methods: This study aims to evaluate most commonly methods for evaluating citotoxiciy of Vipera ammodytes venom and protective potency of appropriate antivenom. For this purpose, methods based on different biological mechanisms: MTT enzyme assay, Crystal Violet (CV), Trypan Blue (TB) and Propidium Iodide (PI) staining, were examined on Vero cells. The proapoptotic effect of the venom was also determined with AnexinV staining.Results: The IC50 value of V. ammodytes venom evaluated by MTT, CV, TB, PI (%) methods was similar: 62.5, 55, 66, 87 g/ml, respectively, and the most suitable methods for testing the cytotoxicity of V. ammodytes venom were methods based on the measurement of living cells, which were MTT and CV staining. The same methods were used to test the anticytotoxic potency of the antivenom and each method confirmed significant anticytotoxic potential, but the EC50values obtained by those methods varied significantly.Conclusion: Choice of the method used to measure the anticytotoxic antivenom potency depends on the immunogenicity of the venom components that cause cell death.

Inflammation is a redistribution of immune cells, providing a more efficient elimination of the inflammatory offense. However, it is not limited to local microenvironment. In this study, the interaction of the effect of BCG priming and peritoneal inflammation on the remote inflammatory milieu of infected lung was investigated. Young male AO rats infected with Mycoplasma spp. were s.c. injected with BCG (3x105 CFU) or saline, and 7 days later received an i.p. injection of 7ml of thioglycollate (TG) or saline. Up to 7 days after TG injection, a broncho-alveolar lavage (BAL) was performed, and cells were analysed for their surface marker expression and NO production. Infected rats had a high percentage of HIS48HiCD11bHi neutrophils. BCG priming didn’t alter BAL cells phenotype, while TG injection increased the proportion of MHCII+CD11blow activated alveolar macrophages (aAMFs) on day 7. However, the BCG+TG group showed significant changes – percentage of HIS48HiCD11bHi neutrophils decreased from day 3, the share of aAMFs increased from day 5 and the share of MHCII+CD11b-AMFs increased on days 3-5. However, the percentage of B220+FSClow B lymphocytes were increased from day 1. Production of NO from BAL fluid cells was low in all groups. We conclude that BCG vaccination likely increased the number of circulating B lymphocytes, while TG-induced peritoneal inflammation potentially prevented their entry into the peritoneal cavity, forcing them into permissive tissues, such as lungs.

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.

SDS-PAGE showing: Lane 1 – purified banana lectin – BanLec; Lane 2 – chimera of banana lectin and enhanced green fluorescent protein BanLec-eGFP; Lane 3 – MW – molecular weight markers; Table 1. Repeatability of flow cytometric detection of different quantities of BL-eGFP binding to C. albicans; Table 2. Repeatability of ELLSA detection of different quantities of BanLec-B binding to C. albicans; Table 3. Repeatability of flow cytometric detection of different quantities of RCA120-FITC binding to L. casei DG; Table 4. Repeatability of ELLSA detection of different quantities of RCA120-B binding to L. casei DG; Table 5. Reproducibility of BanLec-B binding to 21 different microorganisms. Experiments were done in three different laboratories, with preparing new plates each time; Fig. 1 Determining linearity of ELLSA method, by measuring binding between C.albicans and different quantity of BanLec-B; Fig. 2 Determining linearity of flow cytometry, by measuring binding between C.albicans and different quantity of BanLec-eGFP; Fig. 3 Determining linearity of ELLSA method, by measuring binding between L.casei DG and different quantity of RCA120; Fig. 4 Determining linearity of flow cytometry, by measuring binding between L.casei DG and different quantity of RCA120.

BACKGROUND A variety of commensal bacterial taxa elicit serum IgA responses resulting in protection against polymicrobial sepsis, whereas anti-commensal IgG may have deleterious role in gastrointestinal and systemic inflammation. OBJECTIVES The aim was to determine the systemic levels of specific antibodies directed to autologous E.coli in rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains during colitis. METHODS Rats were intrarectally injected with ethanol or trinitrobenzenesulfonic acid (TNBS, 10 or 40mg/kg) whereas controls received saline in the same manner. Sera were tested for the level of anti-E.coli antibodies of IgG1, IgG2a, IgG2b and IgA classes by ELISA. RESULTS Both rat strains developed colitis, but the degree of colon necrosis and hyperemia were slightly greater in DA than in AO rats and the survival rate was significantly lower in DA relative to AO rats. Among saline-treated controls, the levels of IgG1, IgG2a, and IgG2b anti-E.coli antibodies were comparable between rat strains, whereas the amounts of IgA were significantly higher in DA compared to AO rats. Development of colitis significantly increased the amount of anti-E.coli antibodies of IgA and IgG2a classes in sera of AO rats, and those of IgG2a and IgG2b classes in sera of DA rats. Hence, mostly beneficial role of IgA and probably deleterious role of IgG2b antibodies directed to commensal E.coli during colitis may be suggested, whereas the role of anti-E.coli antibodies of IgG2a classes remains intriguing.

BCG vaccination induces a memory-like response in innate immune cells known as trained immunity. In this study, we investigated the modification of innate immune cells by BCG vaccination in acute peritoneal inflammation. We induced peritonitis with thioglycollate (TG) in young Albino Oxford male rats which were immunised s.c. with a BCG vaccine (BCG group) or saline (control group) 7 days prior. Peritoneal cells were examined for 7 days after TG injection by flow cytometry, and for NO production and peroxidase activity. Prior in vivo BCG priming altered TG-elicited peritoneal lavage cells as following: increased in vitro LPS and BCG stimulated NO production from total cells compared to adherent cells (day 1); increased cell number (days 3 and 5); increased percentage of inflammatory monocytes (SSCmidCD43lowCD11bmid) and eosinophils (SSCHihiS48+CD43hi), and a higher level of surface CD11b expression on CD163+ macrophages (day 5); increased in vitro LPS and BCG stimulated peroxidase activity (days 5 and 7); and increased percentage of CD163+MHCII+ cells (day 7). On day 7, cells from both experimental groups showed no production of NO in response to in vitro stimulation. We conclude that BCG vaccination had a substantial effect on the acute phase of sterile inflammation, which may lead to the later observed phenotypic and functional changes that could be seen as accelerated resolution of inflammation and possibly point to trained immune response.