983 research outputs found
Sort by
Changes in the composition of rat peritoneal cells and their response to stimulation by selected gut microbiota during colitis
No full textBACKGROUND Deregulation of the immune response to microbiota or pathogens, and increased intestinal permeability have been proposed as disease-driving mechanisms in colitis. Since peritoneal macrophages guard the sterility of peritoneal cavity from bacterial leakage from the gut, it is plausible to assume that peritoneal macrophages are involved in colitis development. OBJECTIVES The objective was to investigate changes in the composition of peritoneal cells and their response to stimulation by selected gut microbiota during colitis. METHODS Seven days following induction of colitis with intrarectal instillation of ethanol or trinitrobenzenesulfonic acid (TNBS, 10mg/kg or 40mg/kg), peritoneal cells of Dark Agouti (DA) rats were isolated and subjected to flow cytometry. The amount of IL-6 and TNF-α produced by adherent cells was determined by ELISA following in vitro stimulation with LPS and commensal E.coli and Enteroccocus spp.
RESULTS
Instillation of ethanol or TNBS (10 and 40 mg/kg) increased the proportion of CD11bintCD4low monocytes
and decreased the proportion of resident CD163+MHCIIIo macrophages and CD163-MHCIIhi macrophage/dendritic cells.
In vitro treatment with Enterococcus spp. was superior over LPS and E.coli in increasing macrophage TNF-α release in all
but saline-injected control rats. In vitro treatment with E.coli exceeded the level of LPS stimulation in
inducing macrophage IL-6 release in saline- and ethanol-injected rats. It may be concluded that changes in the
composition of peritoneal cells during colitis and, subsequently, their selectively altered response to gut commensals
may perpetuate or modulate inflammation during disease developmen
Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells
Get PDFBackground: Although still considered a safer alternative to classical cigarettes, growing body of work points toharmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect ofe-cigarettes needs to be investigated in more detail considering their widespread use.Methods: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, withand without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cel-lular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performedvia high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry.Results: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however itnegatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletionin total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteinswere upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of post-translational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data areavailable via ProteomeXchange with identifier PXD032071.Conclusions: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMsalterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact
Differences in mouse strains determine the outcome of Der p 2 allergy induction protocols
No full textIn 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
Retrospective Analysis of Six Years of Acute Flaccid Paralysis Surveillance and Polio Vaccine Coverage Reported by Italy, Serbia, Bosnia and Herzegovina, Montenegro, Bulgaria, Kosovo, Albania, North Macedonia, Malta, and Greece
Get PDFHere we analyzed six years of acute flaccid paralysis (AFP) surveillance, from 2015 to 2020, of 10 countries linked to the WHO Regional Reference Laboratory, at the Istituto Superiore di Sanità, Italy. The analysis also comprises the polio vaccine coverage available (2015–2019) and enterovirus (EV) identification and typing data. Centralized Information System for Infectious Diseases and Laboratory Data Management System databases were used to obtain data on AFP indicators and laboratory performance and countries’ vaccine coverage from 2015 to 2019. EV isolation, identification, and typing were performed by each country according to WHO protocols. Overall, a general AFP underreporting was observed. Non-Polio Enterovirus (NPEV) typing showed a high heterogeneity: over the years, several genotypes of coxsackievirus and echovirus have been identified. The polio vaccine coverage, for the data available, differs among countries. This evaluation allows for the collection, for the first time, of data from the countries of the Balkan area regarding AFP surveillance and polio vaccine coverage. The need, for some countries, to enhance the surveillance systems and to promote the polio vaccine uptake, in order to maintain the polio-free status, is evident
Supplementary information for the article: Ljubic, V.; Milosevic, M.; Cvetkovic, S.; Stojanovic, M.; Novovic, K.; Dinic, M.; Popovic, M. The New Exopolysaccharide Produced by the Probiotic Strain L. Reuteri B2: Extraction, Biological Properties, and Possible Application for Ni2+ Ion Removal from the Contaminated Water. Biomass Conversion and Biorefinery 2022. https://doi.org/10.1007/s13399-022-03292-5.
Get PDFUsing the universal primers (UNI16Sfw and UNI16Srev) [1], the representative isolates were identified by 16S rDNA sequencing. Amplification was carried out in a thermal cycler (Applied Biosystems, ThermoFisher Scientific) and DNA fragments were amplified as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles consisting of denaturation at 94 °C for 1 min, annealing at 55°C for 1 min, and polymerization at 72°C for 1 min, and a final extension at 72°C for 7 min. The expected length was 1549 bp. Aliquots (5 µl) of the amplified products were subjected to electrophoresis in 1% agarose gel (ThermoFisher Scientific) in TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.2). Gels were stained with ethidium bromide (500 ng/mL) and visualized under UV light (BioDoc Analyze). All amplicons were eluted and purified using GeneJet PCR Purification Kit (ThermoScientific) by following the manufacturer`s protocol. The PCR products that we obtained were sequenced by the Macrogen Sequencing Service (Macrogen, Amsterdam, The Netherlands) and analyzed by using BLAST algorithm (http://www.ncbi.nlm.nih.gov/index.html). Selected isolates were identified as follows: isolate B2 - Lacotbacillus reuteri, isolate H10 - Lactobacillus murinus, and isolate J7 - Klebsiella oxytoca [2]. The most numerous colonies belong to isolate B2, hence it was chosen for further characterization as a potential source for exopolysaccharide (EPS) production.Related to the published version: [https://intor.torlakinstitut.com/handle/123456789/631]Supplementary material for: [https://doi.org/10.1007/s13399-022-03292-5
Development of SARS-CoV-2 N-protein specific capture ELISA
No full textTačna dijagnoza ljudi sa sumnjom na infekciju SARS-CoV-2 je od suštinskog zna aja zasuzbijanje globalnog širenja COVID-19. Prisustvo SARS-CoV-2 može se otkriti RT-PCRom (otkriva RNK virusa) ili detekcijom prisustva virusnih antigena u biološkim te nostimaELISA-om ili sli nom tehnikom koje koriste antitela razvijena u životinjama. Cilj studijeje bio uspostavljanje kvantitativnog testa koji se zasniva na koriš enju poliklonskih serumaza rutinsko odre ivanje koncentracije SARS-CoV-2 nukleokapsidnog proteina merenjamapsorbancije u standardnoj mikrotitarskoj plo ici sa 96 bunara. Za potrebe razvoja testaproizveden je rekombinantni N-protein i koriš en za proizvodnju antiseruma u miševima ize evima. Proizvedeni antiserumi su pre iš eni i odre en im je titar. Poliklonskiantiserumivisokog afiniteta specifi ni za N-protein koriš eni su za razvoj ELISA testa specifi nog zaovaj protein. Test se zasniva na koriš enju poliklonskih seruma miševa koji su adheriranina dno bunara mikrotitarske plo ice za hvatanje N-proteina iz uzorka. Razli itekoncentracije rekombinantnog N-proteina su koriš ene za standardnu krivu zakvantifikaciju proteina. N-protein vezan za antitela miševa je detektovan ze jimpoliklonskim serumom i anti-ze jim antitelom povezanim sa enzimom koji obezbe ujespektrofotometrijsko merenje. Uspešno smo razvili prototip ELISA testa za kvantifikacijuN-proteina sa granicom detekcije u opsegu od ng/mL. Prose na vrednost LOD za prototipELISA testa za detekciju N-proteina je 9,2 ng/mL, dok je prose na vrednost LOQ10,2 ng/mL. Pokazali smo da su proizvedeni poliklonski antiserumi pogodni za detekcijuN-proteina sa sli nim ili boljim afinitetom i specifi noš u od komercijalnih antitela.Štaviše, prototip ELISA testa se može koristiti sa zadovoljavaju om pouzdanoš u zakvantifikaciju N-proteina u uzorcima bogatim proteinima, poput ljudskih seruma.The accurate diagnosis of people with suspected infection with the SARS-CoV-2 isessential to curb the global spread of COVID-19. The presence of SARS-CoV-2 can bedetected by RT-PCR (it detects RNA of the virus) or by the presence of viral antigens inbiological fluids in ELISA or similar techniques using antibodies developed in animals.The aim of the study was the establishment of a quantitative polyclonal sera-based test forroutine measurement of the concentration of SARS CoV-2 nucleocapsid protein usingabsorbance measurement in a standard 96-well microtiter plate. For the purposes of the testdevelopment, recombinant N protein was produced and used for the production of miceand rabbit antisera. Produced antisera were purified and titer was determined. High-affinitypolyclonal N-protein specific antisera were used for N-protein specific ELISA testdevelopment. The test is based on mice polyclonal sera adhered to microtiter plate bottomfor the capture of the N protein from the specimen. Various concentrations of therecombinant N-protein were used to generate a standard curve for protein quantification.The N-protein bound to the mice antibodies was detected with rabbit polyclonal sera andanti-rabbit antibody coupled to an enzyme that provides spectrophotometric measurement.We have successfully developed the prototype ELISA for the quantification of N-proteinwith the detection limit being in the range of ng/mL. The average LOD value for theprototype ELISA was determined to be 9.2 ng/mL, while the average LOQ value was10.2 ng/mL. We have demonstrated that produced polyclonal antisera are suitable for thedetection of N-protein with affinity and specificity similar to, or better than commercialantibodies. Furthermore, the prototype ELISA can be used with satisfactory confidence forquantification of the N-protein in protein-rich samples, similar to human sera.Poster: [https://intor.torlakinstitut.com/handle/123456789/871
Comparison of Enzyme-Linked Lectin Sorbent Assay and Flow Cytometry for Profiling Microbial Glycans
Get PDFThe 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 information: [https://hdl.handle.net/21.15107/rcub_intor_642
The effect of non-specific binding of Pd(II) complexes with N-heteroaromatic hydrazone ligands on the protein structure
No full textPreviously, the cytotoxic actions of five Pd(II) complexes with bidentate N-heteroaromatic chelators (complexes 1–5) on a palette of several cancercell lines were investigated. However, the results of the cytotoxic activity didnot correlate with the hydrophobic character of the complexes. To gain furtherinsight into the structure–activity relationship, essential for the design of novelpotential drugs, other factors, such as non-specific interactions with cellularproteins, have to be taken into account. To explore the potential non-specificinfluence of the complexes on protein structures, ovalbumin (OVA) waschosen as a model system to mimic cellular non-specific crowding environments with high protein concentrations. A Fourier-transform infrared spectroscopy study implied that the binding of 3 and 4 led to only moderate alternations in the secondary structures of the protein, without the possibility to penetrate into hydrophobic core of the protein and disruption of protein native fold.Contrary, the effect of complex 5 on OVA secondary structures was concentration-dependent. While the lower concentration of complex 5 had no effecton OVA structure, a doubled concentration of complex 5 led to complete disruption of the content native-like secondary structures. The concentration-dependent effect of complex 5 on the changes in secondary structures and considerable increase in the exposure of OVA hydrophobic surfaces to water maybe related to a potential crosslinking that leads to OVA aggregation.Supplementary material: [https://intor.torlakinstitut.com/handle/123456789/1015
West Nile virus in the Republic of Serbia—Diagnostic performance of five serological tests in dog and horse sera
Get PDFWest Nile virus (WNV) is a zoonotic mosquito-borne virus classified as family Flaviviridae and genus Flavivirus. The first WNV outbreak in humans in the Republic of Serbia was recorded in 2012. Equids and dogs can show clinical symptoms after WNV infection and are often used as sentinels. This study aimed to (i) give insight into seropositivity for WNV in clinically healthy dog and horse sera in different regions of Serbia and (ii) compare diagnostic value of ‘in-house’ and commercially available indirect immunofluorescence (IFA) and enzyme-linked immunoassay (ELISA) tests to ‘gold standard’ virus neutralization test (VNT). Due to cross-reactivity, sera were tested for Usutu virus and tick-borne encephalitis virus in VNT based on the epidemiological data of field presence. Blood sera of dogs (n = 184) and horses (n = 232) were collected from 2011 to 2013. The seropositivity was confirmed by VNT in 36.9 % tested dog sera and 34.9% tested horse sera with highest positivity in regions near two big rivers, while in four dog and seven horse sera, positivity resulted from Usutu virus infection. Comparative results of diagnostic tests in dogs ranged from 18.7 % seropositivity by ‘in-house’ ELISA to 31.9% by commercially available ELISA. In horses, seropositivity ranged from 36.2% by ‘in-house’ IFA to 32.5% by commercially available IFA and from 26.3% by ‘in-house’ IgG ELISA to 20.9% by commercially available ELISA. There were no statistically significant differences according to the McNemar test between ‘in-house’ and commercially available IFA and ELISA test in horse sera, while the same was not true for two ELISAs used in dog sera (χ2 = 8.647, p = .003). Established seropositivity in dogs and horses was in accordance with the epidemiological situation and WNV spread in the Republic of Serbia and proven Usutu virus co-circulation. ‘In-house’ tests remain a valuable tool in early diagnostics of WNV.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_648
A herd immunity to rubella virus in selected geographical regions
Get PDFSince 2017, the incidence rate of rubella in the Russian Federation has been below 1 case per million total population. In addition, no circulation of endemic strains of the rubella virus is recorded evidencing about achieving infection elimination phase. In modern conditions, it is important to constantly monitor the level of herd immunity to the rubella virus to identify epidemically significant population groups, especially in countries lacking rubella vaccination or featured with insufficient disease control. Purpose: to study herd immunity to the rubella virus in selected countries in Eurasia and Africa. Materials and methods. Between 2018 and 2021, 15,594 samples of blood sera were tested for IgG and IgM antibodies to the rubella virus from subjects of different ages obtained from regional measles and rubella surveillance centers in the Northwestern Federal District (NWFD) of the Russian Federation, the Republic of Serbia, South Vietnam, and the Republic of Guinea. The “Anti-Rubella Virus ELISA (IgМ)” and “Anti-Rubella Virus ELISA (IgG)” (Euroimmun, Germany) test kits were used. Statistical data processing was carried out using the MS Excel, Prizm 5.0 (GraphPad Software Inc.), and Statistica 8.0 (StatSoft Inc.) software package. Results. During the observation period (2018–2020) the population seroprevalence of the to the rubella virus in the NWFD of the Russian Federation was 96.6–97.7% and fluctuated slightly both in separate years and among individual age groups evidencing about high coverage of rubella vaccination. In the Republic of Serbia conducting two-fold immunization against rubella the overall seroprevalence rate was lower than in the Russian Federation and comprising 86.8%. The minimum number of IgGpositive sera was recorded in the 2–4-year-old age group pointing to the shortcomings of routine vaccination. In South Vietnam, children aged 1–3 years (41.9%) predominated among those recovering from rubella, i.e. the age cohort that should be protected by vaccination at the age of 18 months. No rubella vaccination is carried out in Guinea. The total proportion of seropositive individuals was 75%; herd immunity to the rubella virus was established mainly among children and adolescents, reaching 90% only in the older age group. 30% of unprotected subjects of the most active reproductive age were identified among the females surveyed in Guinea. Conclusion. Insufficient herd immunity to the rubella virus, identified in a number of countries, may contribute to the maintenance of the infectious process and the spread of infection. Globalization contributes to the virus importation into regions being at the stage of measles and rubella elimination. The results obtained suggest about a need to continue efforts aimed at maintaining epidemiological wellbeing regarding rubella in diverse countries of the world.Начиная с 2017 г. в Российской Федерации показатель заболеваемости краснухой находится на уровне ниже 1 случая на 1 млн населения. Также отсутствует циркуляция эндемичных штаммов вируса краснухи. Это свидетельствует о достижении фазы элиминации инфекции. В современных условиях важным является постоянный мониторинг уровня коллективного иммунитета к вирусу краснухи для выявления эпидемически значимых групп населения, особенно в странах, где вакцинация против краснухи не проводится или контроль недостаточен. Цель исследования: изучение коллективного иммунитета к вирусу краснухи в ряде стран Евразии и Африки. Материалы и методы. В период с 2017 по 2021 г. на IgG- и IgM-антитела к вирусу краснухи исследовано 15 594 образца сывороток крови лиц разного возраста, полученные из региональных центров по надзору за корью и краснухой в СЗФО РФ, Республике Сербия, в Южном Вьетнаме, в Гвинейской Республике. Использовали ИФА тест-наборы «Anti-Rubella Virus ELISA IgМ» и «Anti-Rubella Virus ELISA (IgG)» (Euroimmun, Германия). Статистическая обработка результатов проводилась с помощью пакета программ MS Excel, Prizm 5.0 (GraphPadSoftware Inc.), Statistica 8.0 (StatSoft Inc.). Результаты. В СЗФО РФ за период наблюдения серопревалентность населения к вирусу краснухи составляла 96,6–97,7% и колебалась незначительно как по отельным годам, так и среди отдельных возрастных групп, что свидетельствуют о высоком охвате вакцинацией против краснухи. В Республике Сербия общий показатель серопревалентности оказался ниже, чем в РФ, и составил 86,8%. Наименьшее количество IgG-положительных сывороток регистрировали в возрастной группе 2–4 года, что говорит о недостатках плановой вакцинации. В Южном Вьетнаме среди переболевших краснухой преобладали дети в возрасте 1–3 года (41,9%), то есть та группа, которая должна быть максимально защищена плановой прививкой против краснухи в 18 месяцев. В Гвинее специфическая профилактика краснухи не проводится. Общая доля серопозитивных лиц составила 75%, коллективный иммунитет к вирусу краснухи формировался, в основном, среди детей и подростков, достигая 90% лишь в старшей возрастной группе. Среди обследованных женщин Гвинеи выявлено 30% незащищенных лиц наиболее активного репродуктивного возраста. Заключение. Недостаточный уровень коллективного иммунитета к вирусу краснухи, выявленный в ряде стран, может способствовать распространению инфекции, а условия глобализации — импортированию вируса в регионы, находящиеся на этапе элиминации кори и краснухи. Полученные результаты свидетельствуют о необходимости продолжения усилий, направленных на поддержание эпидемиологического благополучия в отношении краснухи в разных странах мира