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Noncovalent interactions of bovine alpha-lactalbumin with green tea polyphenol, epigalocatechin-3-gallate
No full textBovine alpha-lactalbumin (ALA) is an important Ca-binding protein of milk. Epigallocatechin-3-gallate (EGCG) is the major and the most biologically active catechin of green tea, which has the highest binding affinity to whey proteins due to galloyl functional group. In this study experimental and computational methods were used to investigate noncovalent interactions of EGCG and ALA. Binding affinity of EGCG for ALA, determined by fluorescence quenching analysis, was in the range described for complexes of EGCG and other dietary proteins, and lower than affinity of some phenolic compounds to ALA. Based on circular dichroism and Fourier transform infrared spectroscopy spectra, binding of EGCG change ALA conformation inducing alpha-helix to beta-structures transition. The isothermal titration calorimetry results suggest that the binding of EGCG to ALA is enthalpically favorable. The docking analysis shows that EGCG binds in the hydrophobic pocket at the entrance of cleft between alpha-helical and beta-sheetrich domains and includes residues of aromatic cluster II. Uptake of ALA by monocytes proceeds at a slower rate in the presence of EGCG suggesting that EGCG binding may impair uptake of ALA by antigen-presenting cells. ALA, being of low cost and widely available protein, can serve as suitable delivery system for EGCG, as well as for food fortification with this bioactive catechin. (C) 2016 Elsevier Ltd. All rights reserved
Uticaj starenja na polni dimorfizam u CD4+ limfocitima posredovanoj neuroinflamaciji
No full textCilj studije je bio da se ispita; 1) uticaj starenja na polni dimorfizam u Tć limfoniime
posredovanoj neuroinflamaciji i 2) ćelijski i molekulski substrat koji stoji u njegovoj
osnovi. Dark Agouti pacovi uzrasta 3 i 22-24 meseca su imunizovani homogenatom kičmene
moždine. Suprotno nalazu kod mladih pacova, kod kojih su mužjaci pokazivali teže
kliničke manifestacije neuroinflamacije, kod starih pacova njene manifestacije su bile
lakše kod mužjaka u odnosu na ženke. U induktivnoj fazi bolesti, u kulturama
mononuklearnih ćelija drenirajućeg limfnog čvora mužjaka je: 1) nezavisno od uzrasta,
uočena manja proliferacija TA limfocita i manja produkcija 1-17 i (2EM-u, ključnih citokina
za razvoj neuroinflamacije, u odgovoru na stimulaciju mijelin baznim proteinom i 2)
pokazano da iza ovog fenomena stoje različiti mehanizmi (veća zastupljenost
sr4'EohR3"S025" ćelija i njihov veći supresivni kapacitet kod starih pacova) kod životinja
različitog uzrasta. Nezavisno od uzrasta, teža klinička slika neuroinflamacije je
koincidirala sa: 1) izolacijom većeg broja mononuklearnih ćelija iz kičmene moždine; 2)
više reaktivisanih Tć, što se moglo povezati sa seksualnim dimorfizmom u transmigraciji
Tć ćelija i, moguće, proinflamatornih monocita kod mladih pacova, i efikasnijom
aktivacijom mikroglije kod starih pacova; 3) većom ekspresijom T117-polarišućih citokina
i, sledstveno, većom zastupljenosti 1117 limfocita, posebno onih 1-17ČEM-u" fenotipa u inflamatornom _infiltratu, što je koreliralo sa većom ekspresijom M-S5E u maononuklearnim ćelijama. na osnovu rezultata, može se zaključiti da starenje: (1) značajno
smanjuje osetljivost na indukciju Tć limfocitima posredovane neuroinflamacije i težinu
njenih kliničkih manifestacija kod oba pola i (2) dovodi do inverzije polnog dimorfizma u n= neuroinflamacije i njenih kliničkih manifestacija, pre svega usled inverzije g dimorfizma u karakteristikama mikrosredine ciljnog organa
Karakterizacija Intor:Swiss soja albino miševa donetog u Institut za virusologiju, vakcine i serume - Torlak početkom XX veka
Get PDFThe Institute of Virology, Vaccines and Sera Torlak was established in 1927, while the first vaccine was produced in the Institute in 1930. Vaccines production implies using experimental animals, including mice, in in-process controls. The laboratory mice which have been in use in Torlak Institute from the very beginning belong to Swiss albino outbred stock. This stock, which has been in use for more than 80 years contains a large number of mice maintained at all times, was recently named Intor:Swiss. Biological characteristics of Intor:Swiss stock, are presented in this paper for the first time. Taking into account the presented characteristics, the Institute Torlak's Swiss mice are suitable for use in pharmaceutical studies, vaccine development research and basic research, as well as in toxicological studies. The publication of data on the Intor:Swiss mice represents a contribution to the international scientific community, since it offers the possibility for obtaining an additional outbred mouse stock for research.Institut za Virusologiju, vakcine i serume Torlak, osnovan je 1927., a prva vakcina u Institutu proizvedena je 1930. Proizvodnja vakcina je složen proces koji između ostalog podrazumeva i korišćenje eksperimentalnih životinja u kontroli samog procesa. Laboratorijski miševi koji su od samog početka bili u upotrebi u Institutu Torlak, pripadaju Swiss albino outbred soju. Ova kolonija je u upotrebi više od 80 godina i sve vreme se sastoji od velikog broja jedinki što omogućava očuvanje genetske raznolikosti, pa samim tim i outbred karakteristika. Ovi miševi su odnedavno registrovani pod imenom Intor:Swiss, i njihove biološke osobine su u ovom radu prikazane po prvi put. Swiss miševi Instituta Torlak pogodni su za upotrebu u farmaceutskim studijama, za razvojno istraživanje vakcina, osnovna istraživanja i toksikološka ispitivanja. Zbog svega navedenog Intor:Swiss miševi predstavljaju još jedan pogodan animalni model za ispitivanje lekova i vakcina
Recombinantly produced banana lectin isoform promotes balanced pro-inflammatory response in the colon
No full textRecombinant banana lectin isoform (rBanLec) attaches specifically to the mucosal surface, crosses the epithelial barrier and then directly affects the immune response in mouse colon. Structural characteristics, specificity and physiological impacts of rBanLec reported until now highly resemble those of its natural counterpart. Here, we demonstrated that a dose dependent stimulation of the colon with rBanLec skewed the immune response towards Th1/Th17 direction and this effect was counterbalanced by the rise in IL-10 production. Qualitative and quantitative characteristics of the established cytokine network were dependent on the applied rBanLec concentration. In addition, rBanLec enhanced local NO production and myeloperoxidase activity and promoted an increase in local IgA and IgG production. Stimulation with rBanLec can be beneficial in prevention of pathologies raised due to inappropriate cell-mediated immune response as well as in prevention of the pathogen invasion via the colon. (C) 2015 Elsevier Ltd. All rights reserved
Carrageenan - a natural inhibitor of ocular chlamydial infection in vitro and in vivo
No full textPurpose : Ocular infection with Chlamydia trachomatis (Ct) is the leading cause of infectious blindness. As Ct infects via extracellular elementary bodies (EB), we suggest that carrageenan, a natural extract from red seaweed that binds virus particles, might physically bind EB and thereby prevent their attachment to epithelial cells. We tested the hypothesis that carrageenan inhibits Ct infection in vitro using an experimental ocular infection model and in vivo using our guinea pig model.
Methods : Confluent monolayers of human conjunctival epithelial (HCjE) cells were inoculated with Ct serovar B in the presence or absence of carrageenan. Cells were cultured for 48 hours, then fixed and stained with α-Chlamydia LPS antibody and visualized with fluorescent microscopy. Hartley strain guinea pigs were treated either with placebo or with 0.06 mg per eye of carrageenan for 2h before infecting with 1x104 IFU of Chlamydia caviae (3 animals per group). The palpebral and bulbar conjunctivae were evaluated for erythema, edema, and exudation on days 4, 7, 14, and 21.
Results : HCjE cells treated with 1.2 mg/ml carrageenan showed minimal infection (mean of 326±10.94 IFU), with a 7-fold reduction compared to placebo treated cells (mean of 2403±89.47 IFU, p=0.001). In the guinea pigs the pathology score was significantly reduced in the group pre-treated with carrageenan at all time points (p=0.05).
Conclusions : Carrageenan reduced the absolute number of infected cells in vitro and the pathology in vivo, suggesting it should be investigated further as treatment and/or prophylaxis for Ct infection
Infectious dose and repeated infections are key factors influencing immune response characteristics in guinea pig ocular chlamydial infection
No full textThe aim of this study was to determine whether infectious dose of Chlamydia caviae after repeated infections influences the immunological responses and subsequent clearance of pathogen at the ocular surface of guinea pigs. Animals were infected three times via the conjunctiva at six- and twelve-week intervals by applying either 1 x 10(4) or 1 x 10(6) inclusion-forming units (IFUs) of C. caviae. Ocular pathology, infection course, C. caviae-specific serum IgG levels and their capacity to bind and neutralize infection ex vivo were assessed. Animals infected with 1 x 10(4) IFUs had completely diminished ocular infection and pathology after the 2nd infection with increased levels of C. caviae-specific serum IgG and their effective capacity to bind and neutralize C. caviae. Only partial protection was observed in animals infected with 1 x 10(6) IFUs after the 2nd and 3rd infections. Our findings show that full protection was observed in animals repeatedly infected with the lower dose. The lower dose appeared not to compromise the host immune system, thereby enabling fast clearance of the pathogen and the establishment of competent neutralizing antibodies. (C) 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved
Efficacy of Sublingual Immunotherapy with Dermatophagoides Pteronyssinus: A Real-life Study
Get PDFSublingual allergen immunotherapy (SLIT) is considered to be safer and more convenient than subcutaneus immunotherapy. SLIT trials with house dust mites involving patients with allergic rhinitis (AR) and asthma reported discordant results. The aim of the study was to investigate the clinical efficacy and safety of SLIT with Dermatophagoides pteronyssinus (D.pt) extract produced in Serbia and patient's satisfaction through open-label trial. Adult patients with allergic rhinitis were randomized into two groups: one received drugs and SLIT, while other received only drugs. Symptom score (SS), medication score (MS) and cumulative score (CS), skin prick tests (SPT) and serum level of D. pt specific IgE were assessed. One year after, the patients were re-evaluated. In total, 61 patients were enrolled in the study, but 52 of them were analyzed at the end of the year. CS (29.3%, p lt 0.001) and MS (54.3%, p lt 0.05) reduced significantly in the SLIT group. There was a significant improvement of MS and CS in the SLIT compared to control group (p lt 0.001 and p lt 0.05 respectively). There was no significant improvement of SS as well as specific slgE. Patients in the SLIT group were more satisfied with treatment (p lt 0.001). The incidence of mild adverse reaction was 38.4%. Specific lgG was not done. One year SLIT with D. pt extract was clinically efficient treatment in AR patients
Soj kao determinanta uticaja starenja na autoimunsku neuroinflamaciju kod pacova
No full textPodaci o uticaju starenja na autoimunsku neuroinflamaciju, posebno
oni vezani za značaj sojnih razlika, su veoma oskudni. Naša ispitivanja
na modelu eksperimentalnog autoimunskog encefalomijelitisa (EAE) su
pokazala da starenje kod osetljivog DA soja pacova smanjuje osetljivost na
indukciju bolesti (koja je akutnog monofaznog toka) i njenu težinu, a kod
relativno rezistentnog AO soja dovodi do razvoja blagog, ali protrahovanog
neurološkog deficita, što implicira da su sojne razlike u autoimunskoj
neuroinflamaciji uzrastno specifične. Sledstveno, ispitivani su celularni
i molekularni mehanizmi koji stoje u osnovi ovih fenomena. Pokazano je da
je efikasnost generisanja neuroantigen-specifičnih CD4+ limfocita u
drenirajućem limfnom čvoru starih AO pacova bila manja nego kod DA pacova,
što se moglo pripisati većoj zastupljenosti CD4+ regulatornih limfocita
i „defektu“ u ekspresiji CD25 subjedinice receptora za IL-2. Osim toga,
nađene su sojne razlike u efikasnosti mehanizama koji regulišu zadržavanje
neuroantigen-specifičnih ćelija u slezini, kinetici ekspresije hemokina
(CHCL12 i CCL2) koji regulišu migraciju neuroantigen-specifičnih
patogenih CD4+ limfocita u kičmenu moždinu tokom razvoja bolesti, kao i
razlike u zastupljenosti i odnosu subpopulacija regulatornih CD4+ i CD8+
T limfocita i efektorskih Th17 limfocita u smislu veće zastupljenosti
regulatornih T limfocita CD8+IL-10+ fenotipa i IL-17+IL-10+ ćelija u
okviru subpopulacije Th17 limfocita kod AO pacova, što bi, prema podacima
dobijenim u drugim modelima ove bolesti, moglo da se poveže sa protrahovanim
trajanjem neuroloških simptoma kod pacova ovog soja. U celini, rezultati
ukazuju da bi sojne razlike u intrinzičnom funkcijskom kapacitetu CD4+
limfocita i odnosu različitih subpopulacija regulatornih T limfocita i
efektorskih Th17 limfocitamogle biti odgovorne za razlike u kliničkom
ispoljavanju EAE-a kod starih pacova
Strain-dependent response to stimulation in middle-aged rat macrophages: A quest after a useful indicator of healthy aging
No full textRats of Albino Oxford (AO) strain in our animal facility exhibit a longer average healthy life span than rats of Dark Agouit (DA) strain. Since chronic activation of macrophages contributes to chronic low level inflammation common in older age, elucidation of the changes in middle-aged rats could be useful in prevention of unbalanced inflammatory response in advanced age. We have analysed the phenotype of unelicited and thioglycollate-elicited peritoneal macrophages from young and middle-aged DA and AO rats and tested functions of these cells following stimulation with lipopolysaccharide (LPS) in vitro. Unelicited cells from middle-aged DA rats produced higher amounts of proinflammatory mediators interleukin-6 (IL-6) and nitric oxide (NO), but have a diminished response to LPS stimulation then cells from young rats, in spite of increased frequency of TLR4- and CD14-expressing mature macrophages. Injection of thioglycollate robustly increased overall cytokine production in young rats' macrophages, while diminishing their response to LPS stimulation. In middle-aged DA rats injection of thioglycollate diminished IL-6 production, but increased it in response to LPS stimulation. Quite the contrary to DA rats, the macrophages from middle-aged AO rats have released diminished levels of TNF-alpha, and NO, whereas urea production was strongly increased, when compared to the macrophages from young rats. Although the thioglycollate injection has increased the proportion of CD86(+)MHCII(+) mature macrophages in young rats, and percentages of activated TLR4(+) macrophages in both age groups of AO rats, it has not affected the cytokine production in young rats' macrophages, and the TNF-alpha production in middle-aged rats' macrophages. Moreover, the injection of thioglycollate has robustly increased the production of urea in macrophages derived from both age groups of AO rats. Although middle-aged rats of both strains were healthy during experiment, differences between the inflammatory responses of peritoneal macrophages of middle-aged rats of these strains might be one of the contributing factors defining their health in their advanced age. Development of strategies for the prevention of undesirable inflammatory changes in the elderly would benefit from the prospective study of the middle-aged. (C) 2016 Elsevier Inc. All rights reserved
Optimization and Validation of ELISA for Pre-Clinical Trials of Influenza Vaccine
No full textTesting of every new vaccine involves investigation of its immunogenicity, which is based on monitoring its ability to induce specific antibodies in animals. The fastest and most sensitive method used for this purpose is enzyme-linked immunosorbent assay (ELISA). However, commercial ELISA kits with whole influenza virus antigens are not available on the market, and it is therefore essential to establish an adequate assay for testing influenza virus-specific antibodies. We developed ELISA with whole influenza virus strains for the season 2011/2012 as antigens and validated it by checking its specificity, accuracy, linearity, range, precision, and sensitivity. The results show that we developed high-quality ELISA that can be used to test immunogenicity of newly produced seasonal or pandemic vaccines in mice. The pre-existence of validated ELISA enables shortening the time from the process of vaccine production to its use in patients, which is particularly important in the case of a pandemic