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Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa
No full textAdhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed
Phenotypic and functional characteristics of splenocytes in tetanus toxoid-hyperimmunized Balb/c mice is influenced by the context of tetanus toxoid application
Get PDFPurpose/Objective: The hyperimmunization with tetanus toxoid
(TTd) induces protective TTd-specific as well as autoreactive b2-glycoprotein I (b2GPI)-specific immune responses in BALB/c mice. The
overall immune response characteristics, especially its pathogenic potential, depended on adjuvants applied prior and in combination with TTd. Beside structural homology between TTd and b2GPI, tolerance
toward b2GPI could be impaired by adjuvants acting as polyclonal
stimulators. In order to clarify the impact of adjuvants, phenotypic and
functional analyses of immune system cells within spleen were done
upon immunization completion.
Materials and methods: Non- or CFA-pretreated BALB/c mice were
immunized with TTd (3 · 100 lg/dose; 2-week intervals) mixed with
alum or 2.5M glycerol. Ex vivo analyses of CD3, CD4, CD8, CD19, CD
25, CD27 and mIgM expression on age-matched control and
immunized mice’s splenocytes were done by flow cytometry. Changes
in TLR2, TLR4 and TLR9 expression were assessed indirectly, by
measuring cytokine production, following in vitro stimulation of
splenocytes with appropriate agonist.
Results: TTd-immunization diminished CD27 expression on T cells
implying on their differentiation into potent effector cells. T cell
activation (increase in CD25 expression and the raise of percentage of
CD4+ CD8+ CD3+
) and B cell activation (rise in percentage of
CD19+ CD25+ cells and the increase of mIgM density) occurred in all
immunized mice, being more intensive in CFA-pretreated groups.
Irrespective to the applied immunization protocol, statistically significant rise in abundance of CD4- CD8- cells (often cited as cells having
suppressive potential) within T cell pool was registered too. Differences
in cytokines production (IL4, IL10, IFNc) registered upon in vitro
stimulation with peptidoglycan, LPS and CpG ODN implied on
context-dependant modulation of TLR2, TLR4 and TLR9 expression
on splenocytes.
Conclusions: TTd-hyperimmunization promoted concomitant rise in
abundance of activated cells and the cells that have suppressive
potential. This could be regarded as an attempt of the system to retain
control. Imbalance in percentages and activities between activated cells
and those having suppressive potential, highly influenced by the
context of TTd application, is most likely the cause for the observed
pathology appearance after TTd hyperimmunization
Green tea catechins of food, supplements facilitate pepsin digestion of major food allergens, but hampers their digestion if oxidized by phenol oxidase
No full textThe in vitro gastric digestion of several food allergens (beta-lactoglobulin (BLG), alpha-lactalbumin (LA) and peanut allergens (PE)) in the presence of a catechin-enriched polyphenol extract of green tea (GTC), oxidized polyphenols and phenol oxidase processed food allergens and GTC was investigated. Pepsin-resistant proteins, such as BLG, major peanut allergens, Ara h 1 and Ara h 2, degrade faster in the presence of catechin-enriched green tea polyphenols. Phenol oxidase polymerized GTC affected adversely protein digestion of BLG and LA, but not digestion of PE proteins. Protecting effect of polyphenols correlated well with the ability of proteins to form insoluble complexes with oxidized catechins. Cross-linking of proteins and polyphenols further extended the half-lives of BLG and LA in the in vitro digestion by pepsin. Catechin-enriched green tea polyphenols of food supplements facilitate pepsin digestion of major food allergens, but hamper their digestion if oxidized and polymerized by phenol oxidase. (c) 2012 Elsevier Ltd. All rights reserved
Isolation of Klebsiella pneumoniae Producing NDM-1 Metallo-beta-Lactamase from the Urine of an Outpatient Baby Boy Receiving Antibiotic Prophylaxis
No full textC Role of macrophages in pathophysiology
No full textIt етра тле that KM exerts ap fics рon ит of the res immune ои: The aim of the present Study was
to clarify whether ageing influences phenotype characteristics of the resident and thioglycollate-elicited rat peritoneal macrophages
and their functional capacities.
Frequency of Е02+ cells was considerably reduced in resident peritoneal exudate cells obtained from old animals (17 months)
when compared to young (2 months) and adult animals (8 months). The expression of ED2 and CD14 molecules on ED2+ cells also
gradually decreased during ageing. In addition, LPS-elicited NO production and TNF-a were decreased in macrophages acquired from
adult and old rats, respectively.
In contrast, the frequency of ED2+ cells did not change during ageing in thioglycollate-elicited peritoneal macrophages, whereas
density of ED2 marker on these cells was markedly increased in old animals when compared to young and adult animals. Induction
of thioglycollate peritonitis also led to the rise of CD16+ cells within population of peritoneal cells in adult and old animals when
compared to young rats, while density of this marker on ED2+ cells gradually decreased during ageing. Phagocytic potential of macrophages was also decreased during ageing.
Overall, these data suggest that functional capabilities of rat macrophages are affected by senescence and that it could be a
consequence of observed age-related changes in expression of ED2, CD14 and CD16 markers on macrophages.
(Supported by Ministry of Science, Serbia, Grant 175050
Catecholaminergic signalling through thymic nerve fibres, thymocytes and stromal cells is dependent on both circulating and locally synthesized glucocorticoids
Get PDFGlucocorticoids have been shown to modulate the expression of noradrenaline metabolizing enzymes and beta(2)- and alpha(1B)-adrenoceptors in a tissue- and cell- specific manner. In the thymus, apart from extensive sympathetic innervation, a regulatory network has been identified that encompasses catecholamine-containing non-lymphoid and lymphoid cells. We examined a putative role of adrenal- and thymus-derived glucocorticoids in modulation of rat thymic noradrenaline levels and adrenoceptor expression. Seven days postadrenalectomy, the thymic levels of mRNAs encoding tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase-A and, consequently, noradrenaline were decreased. Catecholamine content was diminished in autofluorescent nerve fibres (judging by the intensity of fluorescence) and thymocytes (considering HPLC measurements of noradrenaline and the frequency of tyrosine hydroxylase-positive cells), while it remained unaltered in non-lymphoid autofluorescent cells. In addition, adrenalectomy diminished the thymocyte expression of beta(2)- and alpha(1B)-adrenoceptors at both mRNA and protein levels. Administration of ketoconazole (an inhibitor of glucocorticoid synthesis/action; 25 mg kg(-1) day(-1), s.c.) to glucocorticoid-deprived rats increased the thymic levels of tyrosine hydroxylase, dopamine beta-hydroxylase and, consequently, noradrenaline. The increased intensity of the autofluorescent cell fluorescence in ketoconazole-treated rats indicated an increase in their catecholamine content, and suggested differential glucocorticoid-mediated regulation of catecholamines in thymic lymphoid and non-lymphoid cells. In addition, ketoconazole increased the thymocyte expression of alpha(1B)-adrenoceptors. Thus, this study indicates that in the thymus, as in some other tissues, glucocorticoids not only act in concert with cateholamines, but they may modulate catecholamine action by tuning thymic catecholamine metabolism and adrenoceptor expression in a cell-specific manner. Additionally, the study indicates a role of thymus-derived glucocorticoids in this modulation
Aluminum excytotoxicity and neuroautotoimmunity: The role of the brain expression of CD32+ (FcγRIIa), ICAM-1+ and CD3ε in aging
No full textIn the central nervous system (CNS) microglia are crucial for the defense of the brain against invading microorganisms, formation of tumors, and damage following trauma [1]. However, uncontrolled activation of these cells may have deleterious outcomes [2] through activation of Fcγ and the complement 3 receptors and the induction of an adaptive immune reaction [3]. Proteins contributing to this reaction are the intercellular adhesion molecule-1 (ICAM-1) [3] and CD3 molecules, among others. Both can be expressed on the glia cells before cytokine release and may facilitate an autoimmune inflammatory reaction in the brain. Round microglial cells among the pyramidal cells of the hippocampus with increased expression of CD32+ (FcγIIa) and near the site of injection of aluminum were detected immunohistochemically and indicate microglial activation at the site of aluminum injury. ICAM-1+ immunoreactivity significantly increased in the hippocampus and in the choroids plexus, indicating increased inflammation in the brain as well as increased CD3ε+ expression in the hippocampus and non-MHC-restricted T cytotoxicity after aluminum injection. The pattern of expression of CD32+ (FcγIIa receptor) near the site of aluminum injection indicates that microglia may play a phagocytic role at the site of aluminum-induced excitotoxicity in the brain. Significant expression of ICAM-1+ and CD3ε + immunoreactive cells with the clusters of ICAM-1+ in the choroid plexus suggests a consequently neurotoxic autoimmune reaction induced by microglial hyperactivation in the injured brain
End-point effector stress mediators in neuroimmune interactions: their role in immune system homeostasis and autoimmune pathology
No full textMuch evidence has identified a direct anatomical and functional link between the brain and the immune system, with glucocorticoids (GCs), catecholamines (CAs), and neuropeptide Y (NPY) as its end-point mediators. This suggests the important role of these mediators in immune system homeostasis and the pathogenesis of inflammatory autoimmune diseases. However, although it is clear that these mediators can modulate lymphocyte maturation and the activity of distinct immune cell types, their putative role in the pathogenesis of autoimmune disease is not yet completely understood. We have contributed to this field by discovering the influence of CAs and GCs on fine-tuning thymocyte negative selection and, in particular, by pointing to the putative CA-mediated mechanisms underlying this influence. Furthermore, we have shown that CAs are implicated in the regulation of regulatory T-cell development in the thymus. Moreover, our investigations related to macrophage biology emphasize the complex interaction between GCs, CAs and NPY in the modulation of macrophage functions and their putative significance for the pathogenesis of autoimmune inflammatory diseases
One-step method for isolation and purification of native beta-lactoglobulin from bovine whey
No full textBACKGROUND: The major whey protein beta-lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one-step method for the isolation and purification of BLG while preserving its native structure. RESULTS: BLGwas purified fromdefattedwheyobtainedfromrawcow's milkbyanionexchangechromatography. Protein purity and identitywere determined using reverse phase high-performance liquid chromatography andmass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLGwere compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme-linked immunosorbent assay inhibition. CONCLUSION: Theproposedmethodis veryuseful for the rapid preparationofBLGsuitable for studying antigenicandmolecular characteristics of this protein, aswell as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mgof BLG from a single run using a small column (2.5 cmx20 cm) of diethylaminoethyl Sephadex and has potential for scaling up. (C) 2011 Society of Chemical IndustryThis is the peer-reviewed version of the article: (1) Stojadinović, M. M.; Burazer, L. M.; Ercili-Cura, D.; Sancho, A.; Buchert, J.; Ćirković-Veličković, T.; Stanić-Vučinić, D. One-Step Method for Isolation and Purification of Native Beta-Lactoglobulin from Bovine Whey. Journal of the Science of Food and Agriculture 2012, 92 (7), 1432–1440. [https://doi.org/10.1002/jsfa.4722].Published version: [https://intor.torlakinstitut.com/handle/123456789/361
Production, characterization and applications of a tetanus toxin specific monoclonal antibody T-62
No full textTetanus neurotoxin (TeNT) represents a potent toxin that binds to its receptors on neurons and inhibits the release of neurotransmitters. Additionally, its fragments are used to transport pharmacological substances to neuronal cell bodies. The main objective of this study was the development of a suitable model system to study internalization of the TeNT. We have produced a monoclonal antibody (MoAb) specific for TeNT by hybridoma technology, after immunization of BALB/c mice with tetanus toxoid, and have named it T-62. The immunochemical characteristics of MoAb T-62 were tested using ELISA, PAGE and immunoblotting. Finally, we have used an immunohistochemical method to detect specific binding of MoAb T-62 to TeNT bound to PC 12 cells. Our results show that MoAb T-62 is highly specific for TeNT, even when it is bound to its receptor, and that it could be of considerable importance in studies regarding fundamental research on TeNT receptors, intracellular transport of TeNT, as well as retrograde transport of pharmaceutical substances and non-invasive delivery of polypeptides through the blood brain barrier. In addition, MoAb T-62 is an invaluable tool in TeNT vaccine production as it can be used for the detection of reverse toxicity, which could drastically reduce the need to use animals in these experiments. (C) 2011 Elsevier GmbH. All rights reserved