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Sexual dimorphism in mechanisms controlling development of CD4+ T cell response in collagen-induced arthritis
No full textIntroduction: Considering sex bias in rheumatoid arthritis prevalence, influence of biological sex on the disease development in Dark Agouti rat collagen II (CII)−induced arthritis (CIA) model of the human disease was examined. Methods: Sex bias in CD4+ T cell responses in inguinal (draining the site of immunization in preclinical CIA) and popliteal (draining inflamed joints at the peak of CIA) lymph nodes (LNs) and mechanisms controlling their development were examined using flow cytometry and/or ELISA/qRT−PCR. Results: In both inguinal and popliteal LNs greater number of CD4+CD25+Foxp3− cells, presumably activated effector T cells, was found in females compared with males, and they exhibited greater CII−specific proliferation. Consistently, more IL−17+, IFN−γ+ and IL−17+IFN−γ+ T cells were retrieved from both inguinal and popliteal female rat LNs. Moreover, more GM−CSF+ and IL−17+IFN−γ+GM−CSF+ T cells were retrieved from female compared with male rat popliteal LNs. On the other hand, lower frequency of PD−1+ cells among CD4+CD25+Foxp3+ regulatory T cells (Tregs) from female popliteal and inguinal LNs suggested lower suppressive capacity of their Tregs. Additionally, from female rat popliteal LNs fewer Tregs were recovered. Furthermore, the number of regulatory LN B10 cells was lower in females. Moreover, compared with males, in females was shifted LN INF−γ+/IL−4+ T−cell ratio towards the former, and accordingly serum CII−specific IgG2a/IgG1 antibody ratio was shifted towards pathogenic IgG2a antibodies. Conclusion: The study suggests that a less efficient control of (auto)immune Th1/Th17 cell responses during CIA development contributes to sex bias in the susceptibility to CIA
Effects of chitosan and chitosan Nacetylcysteine solutions on conjunctival epithelial cells
No full textAim of the study: Chitosan, a partially deacetylated polysaccharide derived from chitin, and chitosan-N-acetylcysteine (C-NAC), a thiolated chitosan, both show enhanced retention times on the ocular surface when compared to other polymers commonly used in eye drops. To evaluate these compounds as adjuvants for ocular drug delivery or uptake of topically administered conjunctival vaccines, biochemical characteristics of both polymers were investigated in vitro and in vivo.
Methods:
Human conjunctival epithelial (HCjE) cells were used to investigate biocompatibility of buffered chitosan and C-NAC containing formulations. Cellular uptake was studied using fluorescein-isothiocyanate (FITC)-labelled polymers. Transepithelial electrical resistance (TEER) measurements were performed to determine effects of chitosan on tight junctions of stratified HCjE cells in vitro. In vivo uptake of topically applied chitosan into conjunctival epithelial cells was investigated in guinea pigs.
Results:
Minimal effects on cell viability were seen with both compounds after application for 30 min in a concentration of 0.1%. In vitro uptake into HCjE cells was only observed with the chitosan containing solution. An effect on tight junctions was demonstrated by significantly (P < .05) decreasing TEER levels 60 min after incubation with chitosan. In vivo, FITC-labelled chitosan was detected within guinea pig conjunctival epithelial cells 120 min after topical administration. No adverse effects were observed
Profiling of microorganism-binding serum antibody specificities in professional athletes
Get PDFThe 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
Influence of peanut matrix on stability of allergens in gastric-simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion-resistant peptides
No full textBackground: Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion-resistant peptides (SDRPs; lt 10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated. Objective: The aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of SDRPs released by pepsin digestion of whole peanut grains. Methods: Two-dimensional gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. Results: Ara h 2 and Ara h 6 remained mostly intact, and SDRPs from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified SDRPs from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. Conclusion and Clinical Relevance: Peanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut.Peer-reviewed manuscript: [http://intor.torlakinstitut.com/handle/123456789/608
Influence of peanut matrix on stability of allergens in gastric-simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion-resistant peptides
Get PDFBackground: Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion-resistant peptides (SDRPs; lt 10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated. Objective: The aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of SDRPs released by pepsin digestion of whole peanut grains. Methods: Two-dimensional gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. Results: Ara h 2 and Ara h 6 remained mostly intact, and SDRPs from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified SDRPs from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. Conclusion and Clinical Relevance: Peanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut.This is the peer-reviewed version of the following article: Prodić, I.; Stanić-Vučinić, D.; Apostolović, D.; Mihailović, J.; Radibratović, M.; Radosavljević, J.; Burazer, L.; Milcić, M.; Smiljanić, K.; van Hage, M.; Ćirković-Veličković, T. Influence of Peanut Matrix on Stability of Allergens in Gastric-Simulated Digesta: 2S Albumins Are Main Contributors to the IgE Reactivity of Short Digestion-Resistant Peptides. Clinical and Experimental Allergy 2018, 48 (6), 731–740. [https://doi.org/10.1111/cea.13113]
Mcc-like gene cluster from Streptococcus equinus
No full textMicrocin C-like compounds (McC) are a family of peptidenucleotide antibiotics, with some of having additional post-translational modifications. Bioinformatic searches revealed mcc-like
gene clusters in a diverse range of Gram-negative and Grampositive bacteria. One of these discovered clusters is from S. equinus. There are four genes in the cluster: mccBSeq, mccASeq,
mccE2S and mccCSeq. A 19 amino-acid peptide precursor molecule is encoded by mccASeq. The N-terminal domain of MccBSeq
demonstrates an ability to attach a terminal cytidine monophosphate to the cognate precursor peptide MccASeq. The C-terminal
domain of MccE2SSeq is a carboxy-S-adenosyl-L-methionine
(cx-SAM) synthetase. MccSSeq is responsible for the synthesis of
cx-SAM in the presence of SAM and prephenic acid. Next, the
C-terminal domain of MccBSeq uses cx-SAM as a donor of a carboxymethyl group for the modification reaction of the cytidine
residue of MccASeq-cytidylate. The entire biosynthetic pathway
of the carboxymethylated peptide-cytidylate (MccASeq-cxCMP)
from S. equinus was reconstructed in vitro. However, this compound did not demonstrate any antimicrobial activity against
E. coli. MccCSeq is a member of the superfamily of membrane
transport proteins, and is responsible for microcin export from
producing cells. The N-terminal domain of MccE2SSeq is a predicted acetyltransferase, and may possibly play a role in selfimmunity to produced microcin. To test this, cells overexpressing
MccE2
Seq from S. equinus were obtained, and conferred immunity
to McC from both E. coli and Y. pseudotuberculosis. This work
was supported by Russia Science Foundation RSF 16-14-10356
Cooperative binding of anti-tetanus toxin monoclonal antibodies: Implications for designing an efficient biclonal preparation to prevent tetanus toxin intoxication
No full textOligoclonal combinations of several monoclonal antibodies (MAbs) are being considered for the treatment of various infectious pathologies. These combinations are less sensitive to antigen structural changes than individual MAbs; at the same time, their characteristics can be more efficiently controlled than those of polyclonal antibodies. The main goal of this study was to evaluate the binding characteristics of six biclonal equimolar preparations (BEP) of tetanus toxin (TeNT)-specific MAbs and to investigate how the MAb combination influences the BEPs' protective capacity. We show that a combination of TeNT-specific MAbs, which not only bind TeNT but also exert positive cooperative effects, results in a BEP with superior binding characteristics and protective capacity, when compared with the individual component MAbs. Furthermore, we show that a MAb with only partial protective capacity but positive effects on the binding of the other BEP component can be used as a valuable constituent of the BEP. (C) 2018 Elsevier Ltd. All rights reserved
Safety and immunogenicity of a seasonal trivalent inactivated split influenza vaccine: a phase I randomized clinical trial in healthy Serbian adults
Get PDFThis study was a phase I double-blind, randomized, placebo-controlled trial to evaluate the safety and immunogenicity of a Serbian-produced seasonal trivalent split, inactivated influenza vaccine in healthy adults. The vaccine was manufactured in eggs by the Torlak Institute of Virology, Vaccines and Sera, Belgrade, Serbia and contained A/H1N1, A/H3N2 and B viruses. The clinical trial took place at the Clinical Center of Serbia in Belgrade. Sixty healthy volunteers, aged 18-45years, were enrolled in the trial. On the day of immunization, volunteers were randomly assigned to receive either a single dose of the trivalent seasonal influenza vaccine (15g of hemagglutinin per strain) or placebo (phosphate-buffered saline). Subjects were monitored for adverse events through a clinical history and physical examination, and blood was taken for testing at screening and on day 8 to assess vaccine safety. Serum samples obtained before and 21days after immunization were tested for influenza antibody titers using hemagglutination-inhibition (HAI) and microneutralization (MN) tests. No serious adverse events were reported. Pain and tenderness at the injection site were the most commonly reported symptoms in both vaccine and placebo groups. Overall, serum HAI responses of fourfold or greater magnitude were observed to H1, H3, and B antigen in 80%, 75%, and 70% of subjects, respectively. Seroprotection rates as measured by HAI were also high (100%, 100% and 86.67%, respectively, for H1, H3 and B). Thus, Torlak's seasonal trivalent influenza vaccine was not associated with adverse events, was well-tolerated and immunogenic. It should be further evaluated in clinical trials to provide sufficient safety and immunogenicity data for licensing in Serbia
Association of strategic management with vaccination in the terms of globalization
Get PDFGlobalization is having an ever growing impact on the field of vaccine production and distribution in the world and domestically. In this article we examine the impact of taking a strategic approach to vaccination programmes by all the relevant actors: WHO, UNICEF, national immunization programmes, and vaccine manufacturers and distributors. The review of the relevant literature indicates that there are commonalities to the worldwide vaccination programmes. A comparative analysis of various vaccination strategies recommended by WHO and the immunization calendars of certain European countriesis made as well as an analysis of the Serbian vaccination programme. New and more expensive vaccines will continue to appear on the market in increasingly short periods of time
Deep and quantitative profiling of PTMs in ecologically preserved and polluted pollen proteomes of timothy grass reveals predominant source of contamination
Get PDFObjective: to create method for unrestrictive deep, relative quantification of post translationalmodifications (PTMs) within different proteomes. Pollution field studies of bio indicators such aspollen are valuable because of realistic situation of target contamination, however they carry thegreat extent of uncertainty in attributing and delineating the polluting effect from multiple sources.Holistic research platform focusing on comprehensively characterized and quantified PTMs ofcomparable bio-indicator proteomes could help and overcome these obstacles of field pollutionstudies.Material and Methods: Scanning electron and light microscopy assessed surface and sub pollenparticle (SPP) releasing features of timothy grass (TG) pollen. Inductively coupled atomic emissionspectrometry revealed metal elemental content in pollen while in solution trypsin digested pollenproteomes analysed with high resolution Orbitrap mass tandem spectrometry and PEAKS Suite 8.5brought quantitative information on protein expression level and its PTM profiling.Results: TG polluted pollen samples (P2) collected along regional road and chemical plant,exposed to air contaminants from road traffics and chemical plants showed 4.5 times higher SPPreleasing capacity, with notable surface changes, as well as significantly higher contents of Mn, Hgand Cd. Antioxidative enzymes (oxidoreductases, superoxide dismutases and peroxidases),including actin, were upregulated several times in polluted sample compared to ecologicallypreserved pollen (P1). While the level of spontaneous and physiological PTMs includingmethylation, acetylation, deamidation and formylation, was similar without significant changes inP1 and P2 pollens, oxidative PTMs including oxidation of Met, Lys, His, Pro and HNE and hexoseadducts showed several times higher and significant increase in abundancy of P2 compared to P1.PTMs connected to road traffic such as tyrosine nitration were very rare and low abundant.Conclusion: Results suggest prominent role of chemical pollution compared to effect of road trafficpollution, with primary consequences from oxidative properties of mercury (Hg) and cadmium(Cd).Book of Abstract