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Comparative study between virus neutralisation testing and other serological methods detecting anti-SARS-CoV-2 antibodies in Europe, 2021
Get PDFOne consequence of the ongoing coronavirus disease pandemic was the rapid development of both in-house and commercial serological assays detecting anti-SARS-CoV-2 antibodies, in an effort to reliably detect acute and past SARS-CoV-2 infections. It is crucial to evaluate the quality of these serological tests and consequently the sero-epidemiological studies that are performed with the respective tests. Here, we describe the set-up and results of a comparative study, in which a laboratory contracted by the European Centre for Disease Prevention and Control offered a centralised service to EU/EEA Member and pre-accession Member States to test representative serum specimens with known serological results, with the gold standard technique (virus neutralisation tests) to determine the presence of neutralising antibodies. Laboratories from 12 European countries shared 719 serum specimens with the contractor laboratory. We found that in-house serological tests detecting neutralising antibodies showed the highest percent agreement, both positive and negative, with the virus neutralisation test results. Despite extensive differences in virus neutralisation protocols neutralisation titres showed a strong correlation. From the commercial assays, the best positive percent agreement was found for SARS-CoV-2 IgG (sCOVG) (Siemens - Atellica IM Analyzer). Despite lower positive percent agreement of LIAISON SARS-CoV-2 TrimericS IgG kit (Diasorin Inc.), the obtained results showed relatively good correlation with neutralisation titres. The set-up of this study allowed for high comparability between laboratories and enabled laboratories that do not have the capacity or capability to perform VNTs themselves. Given the variety of in-house protocols detecting SARS-CoV-2 specific neutralising antibodies, including the virus strain, it could be of interest to select reference isolates for SARS-CoV-2 diagnostic to be made available for interested EU Member States and pre-accession countries
Adrenoceptors as potential target for add-on immunomodulatory therapy in multiple sclerosis
No full textThis review summarizes recent findings related to the role of the sympathetic nervous system (SNS) in pathogenesis of multiple sclerosis (MS) and its commonly used experimental model – experimental autoimmune encephalomyelitis (EAE). They indicate that noradrenaline, the key end-point mediator of the SNS, acting through β-adrenoceptor, has a contributory role in the early stages of MS/EAE development. This stage is characterized by the SNS hyperactivity (increased release of noradrenaline) reflecting the net effect of different factors, such as the disease-associated inflammation, stress, vitamin D hypovitaminosis, Epstein-Barr virus infection and dysbiosis. Thus, the administration of propranolol, a non-selective β-adrenoceptor blocker, readily crossing the blood-brain barrier, to experimental rats before the autoimmune challenge and in the early (preclinical/prodromal) phase of the disease mitigates EAE severity. This phenomenon has been ascribed to the alleviation of neuroinflammation (due to attenuation of primarily microglial activation/proinflammatory functions) and the diminution of the magnitude of the primary CD4+ T-cell autoimmune response (the effect associated with impaired autoantigen uptake by antigen presenting cells and their migration into draining lymph nodes). The former is partly related to breaking of the catecholamine-dependent self-amplifying microglial feed-forward loop and the positive feedback loop between microglia and the SNS, leading to down-regulation of the SNS hyperactivity and its enhancing influence on microglial activation/proinflammatory functions and the magnitude of autoimmune response. The effects of propranolol are shown to be more prominent in male EAE animals, the phenomenon important as males (like men) are likely to develop clinically more severe disease. Thus, these findings could serve as a firm scientific background for formulation of a new sex-specific immune-intervention strategy for the early phases of MS (characterized by the SNS hyperactivity) exploiting anti-(neuro)inflammatory and immunomodulatory properties of propranolol and other relatively cheap and safe adrenergic drugs with similar therapeutic profile.This is the peer-reviewed version of the article: Pilipović, I.; Stojić-Vukanić, Z.; Leposavić, G. Adrenoceptors as potential target for add-on immunomodulatory therapy in multiple sclerosis. Pharmacology & Therapeutics 2023, 243, 108358. [https://doi.org/10.1016/j.pharmthera.2023.108358]
Seasonal and inter-seasonal RSV activity in the European Region during the COVID-19 pandemic from autumn 2020 to summer 2022
Get PDFBackground The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in early 2020 and subsequent implementation of public health and social measures (PHSM) disrupted the epidemiology of respiratory viruses. This work describes the epidemiology of respiratory syncytial virus (RSV) observed during two winter seasons (weeks 40–20) and inter-seasonal periods (weeks 21–39) during the pandemic between October 2020 and September 2022. Methods Using data submitted to The European Surveillance System (TESSy) by countries or territories in the World Health Organization (WHO) European Region between weeks 40/2020 and 39/2022, we aggregated country-specific weekly RSV counts of sentinel, non-sentinel and Severe Acute Respiratory Infection (SARI) surveillance specimens and calculated percentage positivity. Results for both 2020/21 and 2021/22 seasons and inter-seasons were compared with pre-pandemic 2016/17 to 2019/20 seasons and inter-seasons. Results Although more specimens were tested than in pre-COVID-19 pandemic seasons, very few RSV detections were reported during the 2020/21 season in all surveillance systems. During the 2021 inter-season, a gradual increase in detections was observed in all systems. In 2021/22, all systems saw early peaks of RSV infection, and during the 2022 inter-seasonal period, patterns of detections were closer to those seen before the COVID-19 pandemic. Conclusion RSV surveillance continued throughout the COVID-19 pandemic, with an initial reduction in transmission, followed by very high and out-of-season RSV circulation (summer 2021) and then an early start of the 2021/22 season. As of the 2022/23 season, RSV circulation had not yet normalised
Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom
No full textIntroduction: In recent decades, many efforts have been made in laboratories to develop alternative methods that would replace animal tests or reduce the number of animals used. In this sense, the methods for measuring cytotoxicity have proven to be very suitable substitutes for testing toxicity. As each individual substance being tested has its own specificities, it is necessary to carefully select a specific, appropriate cytotoxic method. This is especially important if the investigated substance is complex, such as animal venom. Also, antivenoms are drugs for which it is necessary to correctly determine the protective power.Methods: This study aims to evaluate most commonly methods for evaluating citotoxiciy of Vipera ammodytes venom and protective potency of appropriate antivenom. For this purpose, methods based on different biological mechanisms: MTT enzyme assay, Crystal Violet (CV), Trypan Blue (TB) and Propidium Iodide (PI) staining, were examined on Vero cells. The proapoptotic effect of the venom was also determined with AnexinV staining.Results: The IC50 value of V. ammodytes venom evaluated by MTT, CV, TB, PI (%) methods was similar: 62.5, 55, 66, 87 g/ml, respectively, and the most suitable methods for testing the cytotoxicity of V. ammodytes venom were methods based on the measurement of living cells, which were MTT and CV staining. The same methods were used to test the anticytotoxic potency of the antivenom and each method confirmed significant anticytotoxic potential, but the EC50values obtained by those methods varied significantly.Conclusion: Choice of the method used to measure the anticytotoxic antivenom potency depends on the immunogenicity of the venom components that cause cell death.Preprint:[http://dx.doi.org/10.2139/ssrn.4374567
Multistep Approach Points to Compounds Responsible for the Biological Activity and Safety of Hydrolates from Nine Lamiaceae Medicinal Plants on Human Skin Fibroblasts
Get PDFAs byproducts of essential oil distillation, hydrolates are used in natural cosmetics/biomedicine due to their beneficial skin effects. However, data on their safety with relevant biological targets, such as human skin cells, are scarce. Therefore, we have tested nine hydrolates from the Lamiaceae family with skin fibroblasts that are responsible for extracellular collagenous matrix builds. Thyme, oregano, and winter savoury hydrolates showed several times higher total phenolics, which correlated strongly with their radical scavenging and antioxidative capacity; there was no correlation between their viability profiles and the reducing sugar levels. No proteins/peptides were detected. All hydrolates appeared safe for prolonged skin exposure except for 10-fold diluted lavender, which showed cytotoxicity (~20%), as well as rosemary and lavandin (~10%) using viability, DNA synthesis, and cell count testing. Clary sage, oregano, lemon balm, and thyme hydrolates (10-fold diluted) increased fibroblast viability and/or proliferation by 10–30% compared with the control, while their viability remained unaffected by Mentha and winter savoury. In line with the STITCH database, increased viability could be attributed to thymol presence in oregano and thyme hydrolates in lemon balm, which is most likely attributable to neral and geranial. The proliferative effect of clary sage could be supported by alpha-terpineol, not linalool. The major volatile organic compounds (VOCs) associated with cytotoxic effects on fibroblasts were borneol, 1,8-cineole, and terpinene-4-ol. Further research with pure compounds is warranted to confirm the roles of VOCs in the observed effects that are relevant to cosmetic and wound healing aspects
Evaluation of the immunomodulatory potential of chimera Bv1a-BLwt and its mutants on the co-culture model system
Get PDFAllergen immunotherapy (AIT) is currently the only disease-modifying treatment for
allergies. Pre-clinical models for the evaluation of novel therapeutics are crucial for
ensuring their efficacy and safety. While cell culture models are cost-effective and
efficient, they cannot fully replicate the cellular interactions in vivo. Therefore, it is
essential to use more sophisticated model systems, such as co-cultures, to assess the
potential of new therapeutics more accurately. Immunomodulatory protein banana lectin
(BLwt) is an attractive candidate for adjuvant in AIT. Its mutant BLH84T was developed to
reduce its potential mitogenicity. The aim of this study was the development of the coculture model system for testing the immunomodulatory effect of chimeras composed of
the major birch pollen allergen (Bv1a) and BLwt (Bv1a-BLwt, Cwt), the hypoallergenic
isoform of Bv1a (Bv1l) and BLH84T (Bv1l-BLH84T, C1 and BLH84T-Bv1l, C2). Chimeric
structures were designed in silico, fully minimized, and relaxed without van der Waals
atomic clashes. Afterward, proteins were successfully expressed in Escherichia coli and
purified by IMAC yielding around 0.4 mg per 1L of expression medium. The IgE binding
capacity was assessed using ELISA inhibition with birch pollen allergic patients’ sera.
Caco-2 intestinal epithelial cells and THP-1 differentiated macrophages were used for the
co-culture model system development. After protein application on the apical side of the
co-culture, the integrity of the epithelial monolayer was not disturbed. The
immunomodulatory potential of antigens was tested by measuring the gene expression
levels for pro- and anti-inflammatory cytokines in both cell lines from co-culture. The
obtained results indicate that the best anti-inflammatory response was favored after
treatment with Cwt. Additionally, to further confirm the immunomodulatory effect of the
recombinant chimeras, PBMCs obtained from individuals allergic to birch pollen were
employed and treated with recombinant proteins. Only after treatment with Cwt, PBMCs
secreted the anti-inflammatory cytokine IL-10. Obtained results suggest that Cwt chimera
could have a therapeutic effect in AIT in birch pollen allergy
Antioxidant Properties of Protein-Rich Plant Foods in Gastrointestinal Digestion-Peanuts as Our Antioxidant Friend or Foe in Allergies
Get PDFThermally processed peanuts are ideal plant models for studying the relationship between allergenicity and antioxidant capacity of protein-rich foods, besides lipids, carbohydrates and phytochemicals. Peanut is highly praised in the human diet; however, it is rich in allergens (>75% of total proteins). One-third of peanut allergens belong to the products of genes responsible for the defence of plants against stress conditions. The proximate composition of major peanut macromolecules and polyphenols is reviewed, focusing on the identity and relative abundance of all peanut proteins derived from recent proteomic studies. The importance of thermal processing, gastrointestinal digestion (performed by INFOGEST protocol) and their influence on allergenicity and antioxidant properties of protein-rich plant food matrices is elaborated. Antioxidant properties of bioactive peptides from nuts were also considered. Moreover, there are no studies dealing simultaneously with the antioxidant and allergenic properties of protein- and polyphenol-rich foods, considering all the molecules that can significantly contribute to the antioxidant capacity during and after gastrointestinal digestion. In summary, proteins and carbohydrates are underappreciated sources of antioxidant power released during the gastrointestinal digestion of protein-rich plant foods, and it is crucial to decipher their antioxidant contribution in addition to polyphenols and vitamins before and after gastrointestinal digestion.The article processing charge (APC) was financed through the joint effort of all authors by providing their reviewer’s vouchers
Comparative digestion of thermally treated vertebrates and invertebrates allergen pairs in real food matrix
No full textThe digestion stability of allergen pairs, tropomyosin, TM (fish and seafood allergen), and myosin light chain, MLC (chicken meat allergen) is compared among vertebrates and invertebrates in raw and cooked food matrix under standardized simulated in vitro gastrointestinal (GI) digestion. SDS-PAGE followed by multiple TM and MLC-specific antibodies in semidry WB revealed pepsin resistance of invertebrate TMs (abalone, oyster, shrimp) under diet-relevant conditions (raw, cooked). Vertebrate TMs (chicken, pork, beef) were less stable to digestion except that the raw chicken TM was observed pepsin resistant (not diet-relevant). Vertebrate (chicken) MLC was thermally stable. A new 28 kDa protein bound to anti-MLC antibody in cooked chicken and pork; could be the aggregates of MLC. Raw shrimp MLC showed pepsin resistance among invertebrates. A good correlation was observed between combined resistance of TM and MLC to GI digestion following the diet-relevant thermal treatment and reported protein allergenicity among vertebrates and invertebrates.This is the peer-reviewed version of the article: [(1) Khulal, U.; Stojadinović, M. M.; Prodić, I.; Rajković, A.; Ćirković-Veličković, T. Comparative Digestion of Thermally Treated Vertebrates and Invertebrates Allergen Pairs in Real Food Matrix. Food Chemistry 2023, 405, 134981. https://doi.org/10.1016/j.foodchem.2022.134981].Published version: [https://intor.torlakinstitut.com/handle/123456789/766
Dobijanje rekombinantnog imunogenog fragmenta proteina nukleokapsida SARS-CoV-2 virusa za proizvodnju reagenasa i dijagnostičkih testova na novi korona virus
No full textNovi korona virus (SARS CoV-2) koji se pojavio u Vuhanu 2019. godine pripada grupi jednolančanih RNK virusa [1]. Predstavlja novi infektivni agens za humanu populaciju i veoma je brzo detektovan u velikom broju zemalja. Uzročnik je respiratornih infekcija koje mogu da budu praćene i veoma teškom kliničkom slikom. Brzo širenje, odsustvo imuniteta na ovaj virus i odsustvo pouzdanih testova za detekciju virusa u trenutku izbijanja pandemije su bolest izazvanu ovim virusom brzo pretvorili u zdravstveni i društveni problem najvišeg prioriteta na globalnom nivou. Iako su najveće biotehnološke kompanije ubrzano počele sa razvojem i masovnom proizvodnjom dijagnostičkih testova i vakcina, njihova dostupnost u trenucima najveće potražnje je i dalje nedovoljna, a cene istih su limitirajući faktor za bolju kontrolu bolesti i širenja pandemije [2]. Razvoj sopstvenih i održiva proizvodnja testova i vakcina za COVID-19 su od velikog društvenog značaja. Važan preduslov za održivu proizvodnju testova je dostupnost rekombinantnih antigena virusa i mogućnost proizvodnje istih na velikoj skali za potrebe proizvodnje domaćih testova. Ovim tehničkim rešenjem se opisuje dobijanje dva ključna antigena novog korona virusa rekombinantnom tehnologijom i njihova primena u serološkom ELISA testu koji proizvodi Institut za primenu nuklearne energije, INEP, kao i za dobijanje reagenasa za detekciju antigena novog korona virusa (specifičnih antitela). U prvoj fazi, optimizovane su sekvence proteina koje su podigle osetljivost postojećih seroloških testova. Inovativnost našeg pristupa se ogleda i u razrađenim eksperimentalnim protokolima za dobijanje rekombinantnih proteina nukleokapsida na velikoj skali, kao i u solubilnoj formi, što olakšava postupak prečišćavanja. Izbor fragmenta nukleokapsida koji se heterologo eksprimira u solubilnoj formi, a specifično detektuje antitela i generiše jak imuni odgovor tokom imunizacije životinja (imunogenost) na osnovu pregleda poznatih epitopskih sekvenci je ključna inovacija ovog tehničkog rešenja. Ovo je prvi primer uspešno primenjenog rekombinatnog proteina proizvedenog u Srbiji u dijagnostičkom testu koji je registrovankod Agencije za lekove i medicinska sredstva Republike Srbije (broj rešenja 515-02-02370-21-002), a koji je primenu našao i na međunarodnom nivou.Tehničko rešenje je priznato u kategoriji M82 - novo tehničko rešenje (metoda) primenjeno na nacionalnom nivou. U prilogu se nalazi odluka Matičnog naučnog odbora za biotehnologiju i poljoprivredu
Long read sequencing – the next level in genomic research
No full textLong read or third-generation sequencing produces reads from 1 kb to several Mb in length with preserved epigenetic marks, at the single-molecule level and in real-time. Single-molecule real-time sequencing (PacBio) and protein nanopore sequencing (Oxford Nanopore Technologies) are available technologies. PacBio technology is based on monitoring the nucleotide incorporation by a single DNA polymerase molecule in real time using fluorescence as a surrogate marker. PacBio HiFi reads are ~15 kb in length with >99.9% accuracy. Oxford Nanopore sequencing infers nucleotide sequence from the changes in ion current intensity while DNA passes through a stochastic sensor – a protein nanopore. It can sequence DNA fragments ranging in five orders of magnitude (20 bp to several Mb), with duplex read accuracy >99.9% when using R10.4.1 nanopores. Innovations such as a miniature device of the palm-size with a price 99,9%. Oxford Nanopore tehnologija izvodi sekvencu nukleotida iz promena u intenzitetu jonske struje dok DNK prolazi kroz stohastički senzor – proteinsku nanoporu.Može sekvencirati fragmente DNK u rasponu od pet redova veličina (20 bp do nekoliko Mb) sa tačnošću dupleks očitavanja >99,9% kada se koriste R10.4.1 nanopore. Sa elektronskim „čitanjem” nukleinskih kiselina, inovacije kao što su minijaturni uređaj veličine dlana sa cenom <1000 dolara, sekvenciranje na terenu, digitalno obogaćivanje ciljnih sekvenci (adaptivno uzorkovanje) i direktno sekvenciranje RNK, postali su stvarnost. Sekvenciranje dugih fragmenata omogućilo je kompletiranje sekvence genoma čoveka, objavljivanje drafta ljudskog pangenoma i ubrzalo je sekvenciranje genoma eukariota. Od uvođenja metode 2011. godine sekvencirano je ~1000 od 1065 genoma deponovanih u NCBI bazi. Puni potencijal metode u izučavanju transkriptoma i epigenoma biće vidljiv u godinama koje slede. Sekvenciranje dugih fragmenata postaje osnova precizne medicine efikasne za sve ljudske populacije i očuvanja biodiverziteta, i zavredelo je da bude metoda 2022. godine prema časopisu Nature Methods