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Lactobacillus reuteri alleviate the severity of chemicallyinduced colitis in mice
No full textInflammatory bowel diseases (IBDs) are associated with reduced diversity and decreased
contribution of probiotic bacteria within the comensal microbiota. A range of probiotic
bacteria, including the genera Lactobacillus, could be protective in IBDs due to their
capacity to trigger anti-inflammatory mechanisms 1. The aim of the study was to explore
immunomodulatory impact of probiotic bacteria. Lactobacillus reuteri (LR) and its
contribution to the alleviation of severity of a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-
induced colitis in mice. Colitis was induced in outbred Intor Swiss:Albino mice by a single
administration of TNBS dissolved in 50% ethanol (day 0). LR in phosphate buffer solution
(5x106 CFU/mL, p.o.) was given daily, 7 days prior and/or 7 days after day 0 (n=10 per
treatment). Mice subjected to the induction of colitis without LR treatment were referent.
A significant reduction in disease severity was noticed only in group treated by LR prior
and upon colitis induction. The alleviation of disease severity correlated with lessening of
local infiltration of leukocytes, a decrease in local inflammatory response
(myeloperoxidase activity, production of superoxide ions, IL-6, a nd T NFα), a nd a n
increase in local production of IL-10. Presented results show that LR triggers a regulatory
mechanism which alleviates severity of TNBS-induced colitis in mice, implying that it is
worth further evaluation in prevention and treatment of IBDs
MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation
No full textThe MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms
Supplementary information for the article: Kosanović, D.; Dyas, M.; Grogan, H.; Kavanagh, K. Differential Proteomic Response of Agaricus Bisporus and Trichoderma Aggressivum f. Europaeum to Bacillus Velezensis Supernatant. European Journal of Plant Pathology 2021, 160 (2). https://doi.org/10.1007/s10658-021-02252-5.
Get PDFTable S1. Bacterial species used in this study with amplicon deposition number obtained from NCBI GeneBank; Figure S1. 16S rRNA gene analysis; Figure S2. Specific protein analysis- gyrB gene analysis; Figure S3a. Zone of inhibition test on NA plates104 T. aggressivum conidia per plate vs 10 μl of bacterial overnight culture incubated on 30°C; Figure S3b. Zone of inhibition test on YMEA and NA plates 104 T. aggressivum conidia per plate vs 10 μl of bacterial overnight culture incubated on 25°C; Figure S4. The effect of 25%v/v bacterial supernatants on T. aggressivum growth was assessed. B. subtilis R8.3 supernatant was found to inhibit growth of T. aggressivum by 37% and that was the maximum effect compared to other bacterial supernatants; Figure S5. PCA analysis. □ - Control group and ∆ - T. aggresivum + B. velezensis SN group; Figure S6. Protein abundance similarities of two sample group (T. aggressivum control and T. aggressivum treated with B. velezensis SN) based on hierarchical clustering; Figure S7; Figure S8. PCA analysis. □ - Control group and ∆ - A. bisporus + B. velezensis SN group.Related to the published version: [https://intor.torlakinstitut.com/handle/123456789/613]Supplementary material for: [https://doi.org/10.1007/s10658-021-02252-5
The effect of influenza vaccine immunization on natural antibodies
Get PDFNatural, polyreactive, low-affinity antibodies are known to play an important role not only
in the immediate defense against pathogens, but also in shaping the acquired immune response.
On the other hand, antigen specific, high-affinity antibodies can affect the balance of natural
antibodies and lead to autoimmune diseases. In this study we have analyzed the changes that occur
in the IgM and IgG pool of natural antibodies after immunization with split or whole virion
influenza vaccine. For this purpose, “in-house” developed ELISAs were used. The subjects were
divided, according to the vaccination status, into those who had been immunized with the
influenza vaccine in previous years and those who had been immunized for the first time. The
analysis indicated that the pool of natural antibodies was not impaired by the immunization,
evidenced by the lack of changes in any of the groups, and that certain fluctuations were induced
in order to maintain the homeostasis of the immune system
Effects of extraction conditions on proteins' profiles of Tenebrio molitor
Get PDFEdible insects are recommended as a future food because of many reasons. The nutritional value of edible insects is one of criteria for the selection of seven most promising species among which is Tenebrio molitor. The objective of this study was to examine the effects of different flour Tenebrio molitor. Twelve different extraction conditions were set up in which three parameters were combined: pH extraction solutions (6, 8 and 12.5), temperature (30 °C and 60 °C), and ultrasound (US). Shotgun proteomics of trypsin digests profiled protein isolates. The highest protein yield was in extractions at pH 12.5. The temperature elevation and US application significantly increased the yield of isolated proteins at pH 12.5 but their solubility at the pH 7.4 was lower compared to isolates at pH 6 and 8. 1D-SDS-PAGE showed marked differences in protein profiles on various extraction conditions, with highest number of the distinctive bands at pH 8 at 30 °C. Shotgun proteomics showed that extraction condition at pH 12.5, on 30 °C has the highest numbers of different proteins, however, among the top 20 abundant proteins are chitin-associated proteins, allergens and proteinases, while at pH 8 these proteins are not enriched. Highly basic extraction significantly contributes to protein hydrolysis while application of US contributes to the protein cross-linking and this effect is more prominent at high temperatures.Book of Abstract
Blokada β-adrenergičkih receptora pojačava imunoregulatorna/imunoprotektivna svojstva mikroglije: ispitivanje na modelu eksperimentalnog autoimunskog encefalomijelitisa
No full textPretpostavlja se da disfunkcija simpatičkog nervnog sistema
doprinosi razvoju multiple skleroze i eksperimentalnog autoimunskog
encefalomijelitisa (EAE). Imajući u vidu važnost mikroglije za
razvoj/rezoluciju neuroinflamacije, ispitivan je imunomodulatorni
potencijal glavnog simpatičkog medijatora noradrenalina korišćenjem
pacovskog modela EAE-a. Rezultati su pokazali da tretman propranololom,
neselektivnim blokatorom β-adrenergičkih receptora, u efektorskoj
fazi EAE-a smanjuje težinu bolesti. Ovo je koreliralo sa povećanom
zastupljenošću mikroglije koja eksprimira CX3CR1, ključan molekul za
njenu imunomodulatornu/neuroprotektivnu aktivnost, i njenom pojačanom
ekspresijom Nrf2 gena, kao i gena za hem oksigenazu-1, koja se smatra
efektorskim molekulom anti-inflamatornog CX3CR1/Nrf2 signalnog
puta. Istraživanja in vitro pokazala su da aktivacija β-adrenergičkih
receptora dovodi do nishodne regulacije ekspresije Nrf2 i putem
nezavisnim od CX3CR1. Shodno prethodnim rezultatima, propranolol
je povećao fagocitnu sposobnost mikroglije, što je koreliralo sa
povećanjem površinske ekspresije anti-inflamatornih markera CD163
i CD83. Povećanje ekspresije hem oksigenaze-1 praćeno je porastom
zastupljenosti mikroglije koja sintetiše IL-10, a smanjenjem udela one
koja eksprimira proinflamatorne citokine IL-1β i IL-23. Propranolol
je smanjio i ekspresiju MCP-1/CCL2 u kičmenoj moždini. Konsekutivno,
infiltracija kičmene moždine mijeloidnim ćelijama i CD4+ T-ćelijama
bila je smanjena kod pacova tretiranih propranololom. U skladu sa svim
prethodnim, propranolol je limitirao i reaktivaciju/proliferaciju
CD4+ T-limfocita i njihovu diferencijaciju u izuzetno patogene IL17+IFN-γ+GM-CSF+ ćelije. Studija ukazuje da noradrenalin, delujući
posredstvom β-adrenergičkih receptora, podstiče neuroinflamaciju
u EAE-u tako što moduliše ekspresiju Nrf2 u ćelijama mikroglije.
Takođe, ona predstavlja moguću polaznu osnovu za buduća translaciona
farmakološka istraživanja u cilju dizajniranja novih pristupa u lečenju
multiple skleroze
Lactobacillus rhamnosus Affects Rat Peritoneal Cavity Cell Response to Stimulation with Gut Microbiota: Focus on the Host Innate Immunity
Get PDFGut microbiota contribute to shaping the immune repertoire of the host, whereas probiotics may exert beneficial effects by modulating immune responses. Having in mind the differences in both the composition of gut microbiota and the immune response between rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains, we investigated if intraperitoneal (i.p.) injection of live Lactobacillus rhamnosus (LB) may influence peri-toneal cavity cell response to invitro treatments with selected microbiota in the rat strain-dependent manner. Peritoneal cavity cells from AO and DA rats were lavaged two (d2) and seven days (d7) following i.p. injection with LB and tested for NO, urea, and H2O2 release basally, or upon invitro stimulation with autologous E.coli and Enterococcus spp. Whereas the single i.p. injection of LB nearly depleted resident macrophages and increased the proportion of small inflammatory macrophages and monocytes on d2 in both rat strains, greater proportion of MHCIIhiCD163− and CCR7+ cells and increased NO/diminished H2O2 release in DA compared with AO rats suggest a more intense inflammatory prim-ing by LB in this rat strain. Even though E.coli- and/or Enterococcus spp.-induced rise in H2O2 release invitro was abrogated by LB in cells from both rat strains, LB prevented microbiota-induced increase in NO/urea ratio only in cells from AO and augmented it in cells from DA rats. Thus, the immunomodulatory properties may not be constant for particular probiotic bacteria, but shaped by innate immunity of the host
Characterization of potential probiotic strain, L. reuteri B2, and its microencapsulation using alginate-based biopolymers
Get PDFIn this study, Lactobacillus reuteri B2was isolated fromthe feces of C57BL/6 mice and assessed on probiotic activity.L. reuteri B2was identified by 16S rDNA sequencing, which the cell viability in acidic conditions at pH 2.0was64% after 2 h, and in the presents of 0.30% of the bile salts, after 6 h, was 37%. Antimicrobial assay with L. reuteri B2showed maximumdiameters against Klebsiela oxytoca J7 (12.5±0.71mm).Wefurther hypothesized if L. reuteriB2 strain in the free form can survive all conditions in the gastrointestinal tract (GIT) then the utilization of theappropriate biomaterials would ameliorate its stability and viability in GIT. L. reuteri B2 was microencapsulatedinto sodium alginate-(Na-alg) and different content of Na-alg and sodium maleate (SM) beads. Characterizationmaterials enveloped their thermal characteristics (TGA/DTA analysis) and structure using: scanning electron microscopy(SEM), FTIR, and particle size distribution. The high survival rate of L. reuteri B2 at lowpH from2.0 to 4.0and in the presence of the bile salts, at concentrations up to 0.30%, was obtained. L. reuteri B2 showed strong antimicrobialactivity and the best protection microencapsulated with Na-alg + SM in simulated gastric juices(SGJ).The peer-reviewed version: [http://intor.torlakinstitut.com/handle/123456789/628]Supplementary material: [https://hdl.handle.net/21.15107/rcub_intor_627
Characterisation of the interaction of Pseudomonas putida and Pseudomonas tolaasii with Trichoderma aggressivum
No full textGreen mould disease is caused by Trichoderma aggressivum which colonizes mushroom compost and reduces yield. Two Pseudomonas species are associated with mushroom compost: Pseudomonas putida, which stimulates mushroom pinning, and Pseudomonas tolaasii which has a negative effect on crop production. The aim of this work was to characterize T. aggressivum – Pseudomonas interactions as these may be important factors in the development of green mould disease. P. tolaasii supernatant inhibited growth by 57% but P. putida stimulated growth of T.aggressivum by 44%. Tolaasin production was identified in P. tolaasii cultures with a peak at 96 h. Fluorescent microscopy examination of T. aggressivum hyphae revealed that exposure to P. tolaasii supernatant decreased mycelial formation while increasing the abundance of conidia. Label free proteomic analysis of changes in the abundance of T. aggressivum proteins indicated that exposure to P. tolaasii supernatant lead to an oxidative stress response and catabolic enzyme activation (mitochondrial import inner membrane translocase complex (5.7-fold), oxidoreductase (5.2-fold), glucoamylase (5.1-fold)). Exposure of T. aggressivum to P. putida supernatant lead to an increase in the abundance of proteins associated with growth and development (structural constituents of ribosome (20-fold), H/ACA ribonucleoprotein complex subunit (18-fold), DNA binding and nucleosome assembly protein (5.3-fold), and prefoldin (5-fold)). These results indicate that exposure to P. putida can stimulate the growth of T. aggressivum and this interaction may be an important factor in increasing green mould disease in mushroom crops and so reducing yield.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_641
Water-filtered Infrared A and visible light (wIRA/VIS) treatment reduces Chlamydia caviae-induced ocular inflammation and infectious load in a Guinea pig model of inclusion conjunctivitis
No full textTrachoma is a devastating neglected tropical disease caused by Chlamydia trachomatis and the leading global cause of infectious blindness. Although antibiotic treatment against trachoma is efficient (SAFE strategy), additional affordable therapeutic strategies are of high interest. Water-filtered infrared A and visible light (wIRA/VIS) irradiation has proven to reduce chlamydial infectivity in vitro and ex vivo. The aim of this study was to evaluate whether wIRA/VIS can reduce chlamydial infection load and/or ocular pathology in vivo, in a guinea pig model of inclusion conjunctivitis. Guinea pigs were infected with 1 x 10(6) inclusion-forming units/eye of Chlamydia caviae via the ocular conjunctiva on day 0. In infected animals, wIRA/VIS irradiation (2100 W/m(2)) was applied on day 2 (single treatment) and on days 2 and 4 (double treatment) post-infection (pi). wIRA/VIS reduced the clinical pathology score on days 7 and 14 pi and the conjunctival chlamydial load on days 2, 4, 7, and 14 pi in comparison with C. caviae-infected, not irradiated, controls. Furthermore, numbers of chlamydial inclusions were decreased in wIRA/VIS treated C. caviae-infected guinea pigs on day 21 pi compared to C. caviae-infected, non-irradiated, controls. Double treatment with wIRA/VIS (days 2 and 4 pi) was more efficient than a single treatment on day 2 pi. wIRA/VIS treatment did neither induce macroscopic nor histologic changes in ocular tissues. Our results indicate that wIRA/VIS shows promising efficacy to reduce chlamydial infectivity in vivo without causing irradiation related pathologies in the follow-up period. wIRA/VIS irradiation is a promising approach to reduce trachoma transmission and pathology of ocular chlamydial infection