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Supplementary information for the article: Dragačević, L.; Lopandić, Z.; Gavrović-Jankulović, M.; Živković, I.; Blagojević, V.; Polović, N.; Minić, R. Comparison of Enzyme-Linked Lectin Sorbent Assay and Flow Cytometry for Profiling Microbial Glycans. Applied Biochemistry and Biotechnology 2022, 194, 2047–2060. https://doi.org/10.1007/s12010-021-03772-w.
Get PDFSDS-PAGE showing: Lane 1 – purified banana lectin – BanLec; Lane 2 – chimera of banana lectin and enhanced green fluorescent protein BanLec-eGFP; Lane 3 – MW – molecular weight markers; Table 1. Repeatability of flow cytometric detection of different quantities of BL-eGFP binding to C. albicans; Table 2. Repeatability of ELLSA detection of different quantities of BanLec-B binding to C. albicans; Table 3. Repeatability of flow cytometric detection of different quantities of RCA120-FITC binding to L. casei DG; Table 4. Repeatability of ELLSA detection of different quantities of RCA120-B binding to L. casei DG; Table 5. Reproducibility of BanLec-B binding to 21 different microorganisms. Experiments were done in three different laboratories, with preparing new plates each time; Fig. 1 Determining linearity of ELLSA method, by measuring binding between C.albicans and different quantity of BanLec-B; Fig. 2 Determining linearity of flow cytometry, by measuring binding between C.albicans and different quantity of BanLec-eGFP; Fig. 3 Determining linearity of ELLSA method, by measuring binding between L.casei DG and different quantity of RCA120; Fig. 4 Determining linearity of flow cytometry, by measuring binding between L.casei DG and different quantity of RCA120.Supplementary material for: [https://doi.org/10.1007/s12010-021-03772-w]Related to the published version: [https://intor.torlakinstitut.com/handle/123456789/621
β-Adrenoceptor Blockade Moderates Neuroinflammation in Male and Female EAE Rats and Abrogates Sexual Dimorphisms in the Major Neuroinflammatory Pathways by Being More Efficient in Males
Get PDFOur previous studies showed more severe experimental autoimmune encephalomyelitis (EAE) in male compared with female adult rats, and moderating effect of propranolol-induced β-adrenoceptor blockade on EAE in females, the effect associated with transcriptional stimulation of Nrf2/HO-1 axis in spinal cord microglia. This study examined putative sexual dimor- phism in propranolol action on EAE severity. Propranolol treatment beginning from the onset of clinical EAE mitigated EAE severity in rats of both sexes, but to a greater extent in males exhibiting higher noradrenaline levels and myeloid cell β 2 -adrenoceptor expression in spinal cord. This correlated with more prominent stimulatory effects of propranolol not only on CX3CL1/CX3CR1/Nrf2/HO-1 cascade, but also on Stat3/Socs3 signaling axis in spinal cord microglia/myeloid cells (mirrored in the decreased Stat3 and the increased Socs3 expression) from male rats compared with their female counterparts. Propranolol diminished the frequency of activated cells among microglia, increased their phagocyting/endocyting capacity, and shifted cytokine secretory profile of microglia/blood-borne myeloid cells towards an anti-inflammatory/neuroprotective phenotype. Additionally, it downregulated the expression of chemokines (CCL2, CCL19/21) driving T-cell/monocyte traf- ficking into spinal cord. Consequently, in propranolol-treated rats fewer activated CD4+ T cells and IL-17+ T cells, including CD4+IL17+ cells coexpressing IFN-γ/GM-CSF, were recovered from spinal cord of propranolol-treated rats compared with sex-matched saline-injected controls. All the effects of propranolol were more prominent in males. The study as a whole disclosed that sexual dimorphism in multiple molecular mechanisms implicated in EAE development may be responsible for greater severity of EAE in male rats and sexually dimorphic action of substances affecting them.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_649
Age and gender associated changes in immunoglobulin subclass levels specific to S. pneumoniae, serotype 1
Get PDFS. pneumoniae is an important human pathogen which has a polysaccharide capsule with virulent properties. This work aims to estimate the titres of S. pneumoniae specific IgG and IgA isotypes, with respect to age and sex. An in-house whole bacterial cell ELISA was used for the determination of relative levels and endpoint titres of IgG subclasses and IgA1 subclass specific for S. pneumoniae serogroup 1, and to quantify specific IgG1 and IgG2 levels. Significantly lower anti-pneumococcus IgG1 titres were found in older individuals, which was more pronounced in men. Lower IgG2 titres were detected in men over 50 years of age, in comparison to women under 50 years of age. The levels of IgG3 and IgG4 did not differ between different sex and age groups. Lower IgA1 levels were detected in male individuals in both age groups in comparison to females under 50 years of age. The levels of IgG1 showed a moderate correlation with IgG4 in younger individuals of both sexes (r = 0.61 in men and 0.63 in women) which was not noted in the older age group. We highlight the deficiency in humoral immunity in older people, especially male and suggest immunization of this population with pneumococcal vaccines.This is the peer‐reviewed version of the article: Knežević, S.; Kosanović, D.; Dragačević, L.; Živković, I.; Ilić, V.; Hajduković, L.; Savić, O.; Minić, R. Age and Gender Associated Changes in Immunoglobulin Subclass Levels Specific to S. Pneumoniae, Serotype 1. Comparative Immunology, Microbiology and Infectious Diseases 2022, 87. [https://doi.org/10.1016/j.cimid.2022.101834]
A combination of N-acetyl cysteine and propolisattenuates oxidative-inflammatory parametersduring COPD exacerbation
Get PDFOBJECTIVE: The aim of this study was to determine any differences in ox- idative stress and inflammation parameters in COPD patients treated with either N-acetyl cys- teine (NAC) alone or with NAC in combination with propolis (NACP). PATIENTS AND METHODS: Forty COPD pa- tients in the exacerbation phase were enrolled into the study and were treated with either NAC (NAC group; n=20) or NACP (NACP group; n=20) twice daily for one month. Redox status was de - termined by measuring superoxide anion (O 2 .– ), advanced oxidation protein products (AOPP), total oxidative status (TOS), prooxidative-anti- oxidant balance (PAB), malondialdehyde (MDA), ischemia modified albumin (IMA) and sever- al other antioxidant markers: superoxide dis- mutase (SOD), paraoxonase 1 (PON1), total sulf- hydryl groups (SHG) and total antioxidant status (TAS). Interleukins 6, 8 and 17 were measured as markers of inflammatory status. RESULTS: Both groups had similar socio-de - mographic and clinical characteristics. Af- ter treatment significantly higher SHG [0.446 (0.395-0.516) vs. 0.292 (0.270-0.325), p<0.001] and significantly lower TOS – 50.6 [49.7-53.4 vs. 73.2 (50.9-84.6), p<0.05] – and IMA [0.650 (0.629-0.682) vs. 0.709 (0.667-0.756), p<0.05] – were found in the NACP group compared to the NAC group. Factorial analysis indicated a larger oxidative stress-inflammatory load in the NAC group after treatment. CONCLUSIONS: From an oxidative stress and inflammatory status perspective, treatment with NACP was more successful than with NAC. The inclusion of propolis into therapy for COPD patients, especially those in the exacerbation phase, could prove beneficial
Corrigendum: Developing a novel resorptive hydroxyapatite-based bone substitute for over-critical size defect reconstruction: Physicochemical and biological characterization and proof of concept in segmental rabbit's ulna reconstruction (Biomedical Engineering / Biomedizinische Technik 65:4 (491-505) DOI: 10.1515/bmt-2019-0218)
No full textArticle Corrigendum to: Developing a novel resorptive hydroxyapatite-based bone substitute for over-critical size defect reconstruction: physicochemical and biological characterization and proof of concept in segmental rabbit’s ulna reconstruction was published on June 28, 2022 in the journal Biomedical Engineering / Biomedizinische TechnikLink to the corrected article: [https://intor.torlakinstitut.com/handle/123456789/542
Profiling surface glycosylation of microorganisms using plant lectins
No full textInterakcije između mikroorganizama i domaćina imaju glavnu ulogu u infekciji iprogresiji infektivnih bolesti. Upravo zbog toga postoji opravdana potreba da se identifikuju iokarakterišu sve komponente koje učestvuju u toj interakciji. Procesom glikozilacije napovršini svih mikroorganizima formira se jedinstvena arhitektura površinskih polisaharidnihstruktura i upravo ovaj proces smatra se ključnim faktorom virulencije mnogihmikroorganizama.Površinske polisaharidne strukture mikroorganizama su značajne jer ostvaruju direktankontakt sa “receptorskim” strukturama na ćelijama domaćina i varijabilnim regionima antitela,ali prvenstveno u formiranju mehanizama za izbegavanje imunskog odgovora. Upravo ovepovršinske strukture igraju važnu ulogu prilikom prepoznavanja od strane imunskog sistemadomaćina, pa se tako vakcinacija protiv nekih patogenih mikroorganizama zasniva navakcinaciji polisaharidnim komponentama kovalentno vezanim za proteinske nosače...The interaction between microorganisms and their potential host plays a major role ininfection and progression of infective diseases. To better understand those mechanisms, thereis a need to identify and characterize all the components involved in that interaction.Surface polysaccharide structures of microorganisms are unique in architecture, andthey are often key virulence factors of many microorganisms, which shield them from theimmune system and are involved in immune evasion mechanisms. In parallel, polysaccharidestructures found on the surface of microorganisms act as “receptor” structures for host cells oract as epitopes of antibacterial antibodies. This holds true, for example, for vaccination againstcertain pathogenic microorganisms which is based on polysaccharide components that arecovalently bound to protein carriers..
Recombinant Bet v 1-BanLec chimera modulates functional characteristics of peritoneal murine macrophages by promoting IL-10 secretion
Get PDFAllergen-specific immunotherapy (AIT) is a desensitizing treatment for allergic diseases that corrects the underlined pathological immune response to innocuous protein antigens, called allergens. Recombinant allergens employed in the AIT allowed the production of well-defined formulations that possessed consistent quality but were often less efficient than natural allergen extracts. Combining recombinant allergens with an adjuvant or immunomodulatory agent could improve AIT efficacy. This study aimed to perform structural and functional characterization of newly designed recombinant chimera composed of the Bet v 1, the major birch pollen allergen, and Banana Lectin (BanLec), TLR2, and CD14 binding protein, for the application in AIT. rBet v 1-BanLec chimera was designed in silico and expressed as a soluble fraction in Escherichia coli. Purified rBet v 1-BanLec (33.4 kDa) retained BanLec-associated biological activity of carbohydrate-binding and preserved IgE reactive epitopes of Bet v 1. The chimera revealed secondary structures with predominant β sheets. The immunomodulatory capacity of rBet v 1-BanLec tested on macrophages showed changes in myeloperoxidase activity, reduced NO production, and significant alterations in the production of cytokines when compared to both rBanLec and rBet v 1. Comparing to rBet v 1, rBet v 1-BanLec was demonstrated to be more efficient promoter of IL-10 production as well as weaker inducer of NO production and secretion of pro-inflammatory cytokines TNFα, and IL-6. The ability of rBet v 1-BanLec to promote IL-10 in together with the preserved 3D structure of Bet v 1 part implies that the construct might exert a beneficial effect in the allergen-specific immunotherapy
Phenomapping for classification of doxorubicin-induced cardiomyopathy in rats
No full textCardiomyopathy resistant to treatment is the most serious adverse effect of doxorubicin (dox). The mechanisms of dox-induced cardiomyopathy (DCM) have been extensively studied in dilated forms of DCM. However, efficient treatment did not emerge. The aim of the present work was to revisit the experimental model of DCM in rats, to define phenotype/s and associate them to the changes in cardiac transcriptome. Male Wistar rats equipped with radiotelemetry device, were randomized in DOX group (5 mg/0,5 mL/kg, IV dox; n = 18) and CONT group (0,5 mL/kg IV saline; n = 6). Echocardiography, autonomic spectral markers and baroreceptor reflex evaluation was performed prior to, and after treatment. Blood samples were collected at the end of experimentation. Cardiac, renal and hepatic tissues were analysed post-mortem by histology. Changes in expression of key cardiac genes affected by dox were assessed by RT-qPCR. Phenotypes were identified by clustering non-redundant features using four different algorithms averaged by evidence accumulation cluster technique. The results emphasize the existence of two major phenotypes of DCM with comparably high mortality rates: phenotype 1 characterized by, left ventricular (LV) dilatation, thinning of LV posterior wall, reduced LV ejection fraction (LVEF) and fractional shortening (LVFS), decreased HR variability (HRV), decreased baroreceptor effectiveness index (BEI) and increased NT-proBNP; and phenotype 2 with LV hypertrophy - increased LV mass, preserved LVEF, LVFS, no changes in HRV and BEI and moderate NT-proBNP increase. Both phenotypes exhibited a genetic shift to a new-born program
Treatment by Lactobacillus reuteri influences the severity of chemicaly‐induced colitis
No full textInflammatory bowel diseases (IBDs) are associated with significant alterations in composition of commensal microbiota. The main characteristics of microbiota in IBDs is low abundance
of beneficial bacteria belonging to Lactobacillus sp. and Bifidobacterium sp. The aims of our study were to explore whether daily treatment with Lactobacillus reuteri (LR; a strain
isolated from the feces of C57BL/6 mice having confirmed probiotic characteristics) could alleviate severity of an experimental, chemically‐induced, colitis, and to evaluate its impact
on local immune response at the peak of disease. Experimental colitis was induced in outbred Intor Swiss:Albino mice by a single intrarectal administration of 2,4,6‐trinitrobenzene
sulfonic acid dissolved in 50% ethanol (day 0). LR/PBS (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. Colon samples were collected on day 3 (peak of disease) for histological analyses (HE staining), evaluation of superoxide ions, IL‐6 and
TNFα productions, and MPO activity. 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 (MPO activity, production of superoxide ions, IL‐6, and TNFα), and an
increase in local production of IL‐10. Presented results show that LR triggers a regulatory mechanism which alleviates severity of experimental colitis, implying that it is worth further
evaluation in prevention and treatment of IBDs
Differential proteomic response of Agaricus bisporus and Trichoderma aggressivum f. europaeum to Bacillus velezensis supernatant
Get PDFTrichoderma aggressivum, a mycopathogen causing green mould disease, is a major problem in Agaricus bisporus cultivation due to crop loss, and resistance to chemical fungicides. There is an urgent need for novel biological ways to control mycopathogens without affecting the growth of A. bisporus. Bacteria from the mushroom-casing environment were identified and tested for antagonistic effect on T. aggressivum. Bacillus velezensis produced a large zone of inhibition and its supernatant inhibited the growth of T. aggressivum [−37%], and slightly stimulated A. bisporus growth [+2%]. Label free quantitative-proteomic (LFQ) analysis of changes in the abundance of T. aggressivum proteins following exposure to B. velezensis supernatant indicated increased abundance of proteins associated with catabolic processing of amino acids (40-fold), amino oxidase proteins (14-fold), oxidoreductase proteins (13-fold, 4-fold) and hydrolases (3-fold). Proteins that decreased in relative abundance were antioxidants (29-fold), NTF2 domain containing protein (17-fold), 60S ribosomal protein L-13 (14-fold), glucoamylase proteins (13-fold), proteasome subunit proteins (11-fold) and other ribosomal proteins (9-fold). LFQ analysis revealed that exposing A. bisporus to B. velezensis supernatant led to a decrease in: prohibitin (13-fold, 6-fold), proteasomal proteins (11-fold), cytosolic adaptor domain containing protein (5-fold), aldehyde dehydrogenase (4-fold), ribosomal proteins (4-fold), DLH domain-containing protein (4-fold) and PKS_ER domain containing protein (3-fold). The results indicate that A. bisporus was not under stress upon contact with B. velezensis. Whereas a detrimental effect of B. velezensis on T. aggressivum is shown by inhibition of growth and damage-preventing proteins and increased abundance of proteins associated with stress.Supplementary information: [https://hdl.handle.net/21.15107/rcub_intor_638