Prolaktin a cirkulující monocyty - význam a funkce v patogenezi T1DM. Studie in vitro

Úvod: Diabetes mellitus 1. typu (T1DM) je autoimunitní onemocnění charakterizované absolutním deficitem inzulínu. Na patogenezi onemocnění se mimo jiné podílí mechanismy vrozené imunity a jsou zapojeny také monocyty. Ty lze na základě exprese CD14 a CD16 povrchových markerů klasifikovat do tří subtypů s odlišnou funkcí. Monocyty na svém povrchu kromě dalších markerů exprimují prolaktinový receptor (PRLR) a toll-like receptory (TLR), které indukují zánětlivé reakce, a navíc produkují mimohypofyzární prolaktin (PRL), jenž ovlivňuje imunitní odpověď. Cílem práce bylo sledovat vliv exogenního PRL na imunitní reakce monocytů a pokusit se odhalit jeho možnou úlohu v patogenezi T1DM. Materiál a metody: In vitro kultivace a stimulace monocytů 10 zdravých kontrol a 10 pacientů s T1DM. Jako stimulační agens byly použity PRL ve dvou dávkách a/nebo lipopolysacharid (LPS). Pro sledování hladin mRNA cytokinů TNF-α, IL-6, IRF-1, FOS byla celková RNA izolovaná z monocytů získaných imunomagnetickou separací z periferní krve kvantifikována pomocí Real Time PCR. Exprese vybraných povrchových markrů CD14, CD16 a PRLR byla detekována pomocí průtokové cytometrie. Pro detekci polymorfismů Asp299Gly a Thr399Ile v genu TLR4 jsme použili metodu PCR-RFLP s enzymy Nco1 a Hinf1. Závěr: Získaná data naznačují, že PRL...Introduction: Type 1 diabetes mellitus (T1DM) is characterized by an absolute deficiency of the insulin-producing beta cells of the islets of Langerhans in the pancreas. Among mechanisms that lead to pathogenesis of T1DM, innate immunity including key cells monocytes are involved. Based on expression of CD14 and CD16 surface markers, monocytes are classified into three subtypes with different functions. In addition to other markers, monocytes express on their surface prolactin receptor (PRLR) and toll-like receptors (TLR), which induce inflammatory responses, and produce extrapituitary hormone prolactin (PRL) that affects immune response. The aim of thesis was to study an effect of exogenous prolactin on the immune responses of monocytes and to try to detect its possible role in the pathogenesis of T1DM. Material and Methods: In vitro cultivation and stimulation of monocytes derived from 10 patients with T1DM and 10 healthy controls. As stimulating agents were used PRL and/or lipopolysacharide (LPS). For determination of mRNA levels of the studied cytokines (TNF-α, IL-6, FOS, IRF-1), total RNA isolated from monocytes acquired by immunomagnetic separation has been quantified by using Real Time PCR. The expression of surface markers (CD14, CD16, PRLR) was detected by flow cytometry. For detection of...Department of Anthropology and Human GeneticsKatedra antropologie a genetiky člověkaPřírodovědecká fakultaFaculty of Scienc

Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid

The importance of salicylic acid (SA) in the signal transduction pathway of plant disease resistance has been well documented in many incompatible plant–pathogen interactions, but less is known about signalling in compatible interactions. In this type of interaction, tomato plants have been found to accumulate high levels of 2,5-dihydroxybenzoic acid (gentisic acid, GA), a metabolic derivative of SA. Exogenous GA treatments induce in tomato plants a set of PR proteins that differ from those induced by salicylic acid. While SA accumulates in tomato plants mainly as 2-O-β-D-glucoside, GA has only been found as 5-O-β-D-xyloside. To characterize this step of the GA signalling pathway further, the present work focuses on the study of the GA-conjugating activity in tomato plants. A gentisate glycosyltransferase (GAGT) cDNA has been isolated and overexpressed in Pichia pastoris, and GA-conjugating activity was confirmed by detecting the xylosylated GA. The purified plant protein is highly specific for GA, showing no activity toward many other phenolic compounds, including SA. In addition, it shows an outstanding selectivity for UDP-xylose as the sugar donor, which differentiates this enzyme from most glycosyltransferases. Both the GA-conjugating activity and the corresponding mRNA show a strong, rapid, and transient induction upon treatment of tomato plants with GA or SA. Furthermore, its expression is rapidly induced by compatible infections. However, neither the gene nor the activity seems to respond to incompatible infections or wounding. The unique properties of this new glycosyltransferase suggest a specific role in regulating the free GA levels in compatible plant–pathogen interactions

Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications

[EN] Although plant expression systems used for production of therapeutic proteins have the advantage of being scalable at a low price, the downstream processing necessary to obtain pure therapeutic molecules is as expensive as for the traditional Chinese hamster ovary (CHO) platforms. However, when edible plant tissues (EPTs) are used, there is no need for exhaustive purification, because they can be delivered orally as partially purified formulations that are safe for consumption. This economic benefit is especially interesting when high doses of recombinant proteins are required throughout the treatment/prophylaxis period, as is the case for antibodies used for oral passive immunization (OPI). The secretory IgA (SIgA) antibodies, which are highly abundant in the digestive tract and mucosal secretions, and thus the first choice for OPI, have only been successfully produced in plant expression systems. Here, we cover most of the up-todate examples of EPT-produced pharmaceuticals, including two examples of SIgA aimed at oral delivery. We describe the benefits and drawbacks of delivering partially purified formulations and discuss a number of practical considerations and criteria to take into account when using plant expression systems, such as subcellular targeting, protein degradation, glycosylation patterns and downstream strategies, all crucial for improved yield, high quality and low cost of the final product.The authors would like to thank Annick Bleys for assistance with the manuscript preparation. P.J. would like to express gratitude towards the Spanish Ministry of Economy and Competiveness for her FPU fellowship and towards the International Society for Plant Molecular Farming for their generous bursaries for attending the PBVAB 2015. This work was supported by grants from Research Foundation Flanders (FWO project G0C9714N), from the European Commission (H2020-MSCA-IF-2014 Proposal 658701-ImmunoFarm) and from the Spanish Ministry of Economy and Competiveness (Plan Nacional I+D Grant BIO2013-42193R).Juarez, P.; Virdi, V.; Depicker, A.; Orzáez Calatayud, DV. (2016). Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications. Plant Biotechnology Journal. 14(9):1791-1799. https://doi.org/10.1111/pbi.12541S1791179914

Prolactin and circulating monocytes - their significance and function in pathogenesis T1DM. Study in vitro

Introduction: Type 1 diabetes mellitus (T1DM) is characterized by an absolute deficiency of the insulin-producing beta cells of the islets of Langerhans in the pancreas. Among mechanisms that lead to pathogenesis of T1DM, innate immunity including key cells monocytes are involved. Based on expression of CD14 and CD16 surface markers, monocytes are classified into three subtypes with different functions. In addition to other markers, monocytes express on their surface prolactin receptor (PRLR) and toll-like receptors (TLR), which induce inflammatory responses, and produce extrapituitary hormone prolactin (PRL) that affects immune response. The aim of thesis was to study an effect of exogenous prolactin on the immune responses of monocytes and to try to detect its possible role in the pathogenesis of T1DM. Material and Methods: In vitro cultivation and stimulation of monocytes derived from 10 patients with T1DM and 10 healthy controls. As stimulating agents were used PRL and/or lipopolysacharide (LPS). For determination of mRNA levels of the studied cytokines (TNF-α, IL-6, FOS, IRF-1), total RNA isolated from monocytes acquired by immunomagnetic separation has been quantified by using Real Time PCR. The expression of surface markers (CD14, CD16, PRLR) was detected by flow cytometry. For detection of..

Prolactin and circulating monocytes - their significance and function in pathogenesis T1DM. Study in vitro

Introduction: Type 1 diabetes mellitus (T1DM) is characterized by an absolute deficiency of the insulin-producing beta cells of the islets of Langerhans in the pancreas. Among mechanisms that lead to pathogenesis of T1DM, innate immunity including key cells monocytes are involved. Based on expression of CD14 and CD16 surface markers, monocytes are classified into three subtypes with different functions. In addition to other markers, monocytes express on their surface prolactin receptor (PRLR) and toll-like receptors (TLR), which induce inflammatory responses, and produce extrapituitary hormone prolactin (PRL) that affects immune response. The aim of thesis was to study an effect of exogenous prolactin on the immune responses of monocytes and to try to detect its possible role in the pathogenesis of T1DM. Material and Methods: In vitro cultivation and stimulation of monocytes derived from 10 patients with T1DM and 10 healthy controls. As stimulating agents were used PRL and/or lipopolysacharide (LPS). For determination of mRNA levels of the studied cytokines (TNF-α, IL-6, FOS, IRF-1), total RNA isolated from monocytes acquired by immunomagnetic separation has been quantified by using Real Time PCR. The expression of surface markers (CD14, CD16, PRLR) was detected by flow cytometry. For detection of..

Analysis of Nickel-Binding Proteins from Various Animal Sera

Nickel-binding proteins play an important role in the biological processes and can also be utilized in several fields of biotechnology. This study was focused on analysing the nickel-binding proteins from the blood sera of humans (Homo sapiens), cattle (Bos taurus), sheep (Ovis aries), red deer (Cervus elaphus), mouflon (Ovis orientalis), fallow deer (Dama dama), horses (Equus ferus caballus), pigs (Sus scrofa domesticus), wildboars (Sus scrofa), brown bears (Ursus arctos) and pheasants (Phasianus colchicus). The presence of higher abundance proteins in the blood serum, such as albumins, may mask the detection of lower abundance proteins. The samples were depleted from these higher abundance proteins to facilitate the detection of those with lower abundance. For the characterization of these proteins, nickel cations bound to tetradentate ligand nitrilotriacetic acid(Ni-NTA)immobilized on agarose beads were incubated with animal sera to capture nickel-binding proteins and subsequently the proteins were eluted and fractionated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed a set of nickel-binding proteins with various molecular weights within different animal species. A unique ~42 kDa nickel-binding protein in the brown bear serum, which was not present in any of the other species, was further characterized and identified by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS). This protein was identified as ahaptoglobin-like protein. This result may provide some valuable clue for the physiological difference in the metal binding proteins in the serum of Ursus arctos and other animals