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Age-dependent effects of memantine in experimental autoimmune encephalomyelitis
No full textAgeing affects N-methyl-D-aspartate receptors (NMDARs), their expression and function in
neuronal and non-neuronal cells. The aim of this study was to determine whether ageing affects
the role of NMDARs in experimental autoimmune encephalomyelitis (EAE). Memantine, a non-
competitive NMDAR antagonist was administered orally from day 7 after immunization to 3- and
24-month-old female Dark Agouti rats. The animals were sacrificed at the peak of the disease.
Spinal cord mononuclear cells were analyzed by flow cytometry. Brain tissue was collected for
biochemical analysis of redox status and RT-OCR. Memantine reduced the number, frequency,
and reactivation of CD4+ T lymphocytes and increased the relative percentage of CX3CR1-
expressing microglia in spinal cord, but to a greater extent in aged rats. Additionally, memantine
was more effective in reducing superoxide anion radical, maiondialdehyde and advanced
oxidation protein products in aged rats than in young ones. In accordance with previous findings,
NMDAR inhibition increased the nuclear factor erythroid 2-related factor 2 and home oxygenase-
I expression, to a greater extent in aged rats
Modulacija odgovora ćelija peritonealne šupljine pacova ao i da soja na stimulaciju bakterijama crevne mikrobiote in vitro Primenom probiotske bakterije Lactobacillus rhamnosus in vivo
No full textprimenjenog probiotika mogu da budu posledica razlika u kolonizaciji
ili metaboličkim promenama, direktno modulatorno delovanje bakterije
Lactobacillus rhamnosus (LB) na ćelije urođene imunosti je ispitivano
intraperitonealnim ubrizgavanjem (i.p.), ≈ 5 h 107 CFU pacovima Albino
Oxford (AO) i Dark Agouti (DA) soja, 2 i 7 dana pre izolacije ćelija
peritonealne šupljine i in vitro tretmana bakterijama crevne mikrobiote.
Iako je i.p. ubrizgavanje LB nakon 2 dana dovelo do porasta broja ćelija
peritonealne šupljine, porasta procentualne zastupljenosti malih
inflamatornih CD163-MHCIIlo makrofaga i SSCloHIS48+CD43+ monocita
i smanjenja procenta zrelih CD163+MHCII- makrofaga kod pacova oba
soja, porast procentualne zastupljenosti CD163-MHCIIhi makrofaga/
dendritskih ćelija je bio primećen samo kod pacova DA soja. Uprkos
relativnom oporavku u promeni sastava ćelija peritonealne šupljine
pacova oba soja sedmog dana nakon ubrizgavanja LB, značajno manja ukupna
zastupljenost CD163+ a značajno veća aktiviranih CCR7+ ćelija kod
pacova DA soja ukazuje na intenzivniju aktivaciju sa LB kod pacova DA
soja. Povećanje kapaciteta produkcije vodonik peroksida indukovane
bakterijama crevne mikrobiote in vitro prevenirano je u ćelijama pacova
oba soja 2 dana, a kod pacova AO soja i 7 dana nakon i.p. LB tretmana, dok
je, naprotiv, povećanje kapaciteta produkcije azot monoksida indukovano
bakterijama crevne mikrobiote potencirano u ćelijama pacova DA soja 2
dana nakon i.p. LB. Naši rezultati ukazuju na to da je imunomodulatorno
delovanje LB, odnosno mogućnost modulacije odgovora ćelija imunskog
sistema na komensale ili patogene, oblikovano genetski determinisanim
obrascima aktivacije ćelija domaćina (Pod pokroviteljstvom MPNTR
RS, Ugovor 451-03-9/2021-14/200177)
Upregulation of TgBAC(ankrd1a:EGFP) in zebrafish skeletal muscle during regeneration and in response to mechanical stretch
No full textFunctionally pleiotropic cardiac ankyrin repeat protein ANKRD1 (CARP) is predominantly expressed in the heart, where it participates in transcriptional regulation, sarcomere assembly and mechano-sensing as a component of I-band complex organized on titin N2A region. ANKRD1 expression is significantly upregulated in diseased skeletal muscle of patients with muscular dystrophy, congenital myopathy and motor neuron diseases. This gene is also involved in muscle stress response pathways initiated after acute resistance exercise, eccentric contractions, stretching or injury. Here we investigated activation of zebrafish homolog, ankrd1a gene, in stressed skeletal muscle, using transgenic line TgBAC(ankrd1a:EGFP). We detected transgene upregulation in cells in close proximity of the needle stab wound during regeneration. Subjecting larval skeletal muscle to stretching also increased transgene expression in muscle cells, corroborating stretch-responsiveness of ankrd1a observed for its mammalian homologue. Our results implicate ankrd1a in zebrafish skeletal muscle tissue repair and remodeling as a sensor of stressed muscle
Supplementary information for the article: Vasić, A.; Răileanu, C.; Körsten, C.; Vojinović, D.; Manić, M.; Urošević, A.; Nikolić, N.; Dulović, O.; Tews, B. A.; Petrović, T.; Silaghi, C.; Valčić, M.; Gligić, A. West Nile Virus in the Republic of Serbia—Diagnostic Performance of Five Serological Tests in Dog and Horse Sera. Transboundary and Emerging Diseases 2022, 69 (5), e2506–e2515. https://doi.org/10.1111/tbed.14593.
No full textSupplementary file 1. Dog and horse sera sampled from 15 locations from 2011 to 2013 for the serological study. Supplementary file 2. Seropositivity in dogs and horses in Serbia in period 2011-2013. Supplementary file 3. The results of „in-house“ and commercial ELISA tests in dog sera. Supplementary file 4. The results of „in-house“ and commercial IFA and ELISA tests in horse seraSupplementary material for: https://doi.org/10.1111/tbed.14593Related to the published version: https://intor.torlakinstitut.com/handle/123456789/62
The intriguing role of anti-commensal bacteria antibodies in sera of colitic rats
No full textBACKGROUND 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
B-cell response to seasonal influenza vaccine in mice is amenable to pharmacological modulation through β-adrenoceptor
Get PDFAims: Given that deprivation of noradrenaline acting on lymphocytes through β-adrenoceptor influences antibody response, the effects of propranolol treatment beginning two days before immunization with quadrivalent inactivated influenza vaccine (QIV) on IgG response and underlying cellular molecular mechanism in mice were investigated.
Main methods: Twenty-one days post-immunization the total QIV antigen-specific IgG titer and IgG subclass titers in sera were determined using ELISA. Additionally, the total counts of germinal centre (GC) B cells, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in draining lymph nodes (dLNs) and spleens, in vitro proliferation of interacting B cells and Th cells and IL-21 synthesis in Th cells in response to QIV antigens and/or mitogen were attested using flow cytometry analysis. In QIV antigen-stimulated dLN cell and splenocyte cultures were also measured concentrations of INF-γ and IL-4, cytokines upregulating IgG2a and IgG1 synthesis, respectively.
Key findings: Propranolol decreased the total QIV antigen-specific IgG titer. This correlated with lower GC B cell count and the shift in Tfr/Tfh cell and Tfr/GC B cell ratio towards Tfr in propranolol-treated mice compared with controls. Consistently, QIV antigen-stimulated proliferation of B cells and Th cells from propranolol-treated mice in vitro was impaired. This correlated with the lower frequency of QIV antigen-specific IL-21-producing cells among Th cells. Additionally, in propranolol-treated mice, in accordance with the changes in INF-γ/IL-4 ratio in dLN cell/splenocyte cultures, serum IgG2a/IgG1 ratio was shifted towards IgG1 reflecting decreased IgG2a response.
Significance: The study indicates that chronic propranolol treatment may impair response to QIV
Phenotypical characteristics of peritoneal macrophages of two rat strains during inflammatory response: the importance of gut microbiota representatives
Get PDFPredmet istraživanja ove doktorske disertacije je ispitivanje promena u fenotipu i aktivnosti makrofagaperitonealne šupljine Albino Oxford (AO) i Dark Agouti (DA) pacova tokom razvoja inflamatornogodgovora koji je indukovan delovanjem bakterija mikrobiote creva direktno, intraperitonealnom (i.p.)primenom, ili indirektno, tokom razvoja kolitisa, kao i ispitivanje modulacije ovih promena primenomprobiotske bakterije Lactobacillus rhamnosus (L. rhamnosus).Rezultati su pokazali da i.p. primena umrtvljenih E. coli ili Enterococcus spp. dovodi do izraženijeinflamatorne polarizacije makrofaga peritonealne šupljine DA pacova u odnosu na AO soj, dok i.p.primena L. rhamnosus ima veći potencijal da spreči funkcionalne promene do kojih dovodi in vitrostimulacija komensalnim bakterijama u makrofagima pacova AO soja. Tokom razvoja kolitisamakrofagi DA pacova produkuju više IL-6 u odnosu na AO soj, a značajno manja smrtnost AO pacovaje praćena intenzivnijom produkcijom IL-10 u makrofagima u odgovoru na dodatnu stimulacijukomensalnim bakterijama in vitro i višim nivoom antitela klase IgA specifičnih za E. coli u serumu. Ranipostnatalni oralni tretman bakterijom L. rhamnosus u DA pacovima povećava raznovrsnostBifidobacterium vrsta u fecesu, a nakon indukcije kolitisa u adultnom dobu smanjuje stepen ulceracijekolona, povećava zastupljenost rezidentnih makrofaga peritonealne šupljine i suprimira povećanjaprodukcije NO i IL-10 stimulisana komensalnim bakterijama in vitro.Nalazi ukazuju na značaj genetski determinisane predispozicije ka određenom obrascu aktivacijemakrofaga za promene u fenotipu i aktivnosti tokom razvoja inflamatornog odgovora koji je indukovandelovanjem bakterija mikrobiote creva, kao i za modulaciju ovih promena primenom probiotskebakterije L. rhamnosus.The subject of this doctoral dissertation is the study of changes in the phenotype and activity ofperitoneal cavity macrophages from Albino Oxford (AO) and Dark Agouti (DA) rats during thedevelopment of inflammatory response induced by intestinal microbiota directly, intraperitoneally (i.p.),or indirectly, through development of colitis, as well as study of the modulation of these changes usingthe probiotic bacterium Lactobacillus rhamnosus (L. rhamnosus).I.p. application of killed E. coli or Enterococcus spp. leads to a more pronounced inflammatorypolarization of the peritoneal cavity macrophages from DA rats compared the AO strain, while i.p.application of L. rhamnosus has a greater potential to prevent functional changes caused by in vitrostimulation with commensal bacteria in AO rat macrophages. During the development of colitis, DArat macrophages produce more IL-6 than the AO strain, and significantly lower AO rat mortality wasaccompanied by more intense IL-10 production in macrophages in response to additional stimulationwith commensal bacteria in vitro and higher levels of IgA-class antibodies specific to E. coli in the serum.Early postnatal oral treatment with L. rhamnosus in DA rats increased the diversity of Bifidobacteriumspecies in feces, and after induction of colitis in adulthood reduced the degree of colonic ulceration,increased the presence of resident peritoneal cavity macrophages and suppressed the increasedproduction of NO and IL-10 stimulated by commensal bacteria .The findings indicate the importance of genetically determined predisposition to a certain pattern ofmacrophage activation for changes in phenotype and activity during the development of inflammatoryresponse induced by intestinal microbiota, as well as for modulation of these changes using probioticbacterium L. rhamnosus
Cytomegalovirus infection may be oncoprotective against neoplasms of B-lymphocyte lineage: single-institution experience and survey of global evidence
Get PDFAlthough cytomegalovirus (CMV) is not considered tumorigenic, there is evidence for its oncomodulatory effects and association with hematological neoplasms. Conversely, a number of experimental and clinical studies suggest its putative anti-tumour effect. We investigated the potential connection between chronic CMV infection in patients with B-lymphocyte (B-cell) malignancies in a retrospective single-center study and extracted relevant data on CMV prevalences and the incidences of B-cell cancers the world over
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
No full textAs one of the most promising groups of microbes, lactic acid bacteria (LAB) can synthesize metabolites that can be used in different industries over the world, mainly in the pharmaceutical, food, and dairy industries. In this study, a novel exopolysaccharide was extracted and isolated from the probiotic strain Lactobacillus reuteri B2, and assessed on biological activity and its possible application as a biosorbent for the removal of Ni2+ ions from contaminated water. New exopolysaccharide was characterized using FTIR, SEM, XRD, NMR, MALDI-TOF MS, and TGA/DTG analysis. Biological assays included antioxidative activity, cytotoxic assay, and adhesion assay of L. reuteri B2 to HT29 cells. Our hypothesis was that if this exopolysaccharide is nontoxic, it can be used as a novel biomaterial for the possible application of the removal of Ni2+ ions from contaminated water. The scavenging effect of nontoxic exopolysaccharide was 76% at 2 mg/mL using ABTS assay, in biological assays, while the removal efficiency of nickel from the aqueous solution was 92.96% in biosorption study. According to these results, this exopolysaccharide can be considered a very promising biomaterial for potential application in different industries, from pharmacy to wastewater treatments.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_644
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.Abstract: [https://intor.torlakinstitut.com/handle/123456789/870