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There is growing interest in the role of extracellular vesicles (EVs) in neonatal pathology. This study aimed to characterise circulating EVs following preterm birth. This single-centre prospective observational study included cord and postnatal plasma from preterm (n = 101) and full-term infants (n = 66). EVs were analysed using nanoparticle tracking analysis, flow cytometry, proteomics and procoagulant activity assay. We found changes in the concentration, size, cellular origin and proteomic content of circulating EVs in preterm infants during perinatal adaptation. To understand if these changes were related to prematurity or normal adaptation to extrauterine life, they were also investigated in term infants. There was a dramatic increase in the concentration of small and large EVs on Day 3 in the preterm group; specific subsets of platelet (CD42b+ and CD62P+), endothelial (VEGFR2) and tissue factor EVs were elevated. Differentially expressed proteins relating to haemostasis, pulmonary physiology and immunity were identified between Day 1 and 3 in preterm infants. These changes have never previously been described in a large cohort of preterm infants and differ from healthy term infants. These findings have major implications for future neonatal EV studies, particularly the timing of sample collection. Further work is required to understand the clinical implications of this unique EV profile following preterm birth.
- MeSH
- extracelulární vezikuly * metabolismus MeSH
- fyziologická adaptace * MeSH
- lidé MeSH
- novorozenec nedonošený * krev MeSH
- novorozenec MeSH
- prospektivní studie MeSH
- proteomika metody MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- pozorovací studie MeSH
The parasitic protozoan Entamoeba histolytica secretes extracellular vesicles (EVs), but so far little is known about their function in the interaction with the host immune system. Infection with E. histolytica trophozoites can lead to formation of amebic liver abscesses (ALAs), in which pro-inflammatory immune responses of Ly6Chi monocytes contribute to liver damage. Men exhibit a more severe pathology as the result of higher monocyte recruitment and a stronger immune response. To investigate the role of EVs and pathogenicity in the host immune response, we studied the effect of EVs secreted by low pathogenic EhA1 and highly pathogenic EhB2 amebae on monocytes. Size and quantity of isolated EVs from both clones were similar. However, they differed in their proteome and miRNA cargo, providing insight into factors potentially involved in amebic pathogenicity. In addition, EVs were enriched in proteins with signaling peptides compared with the total protein content of trophozoites. Exposure to EVs from both clones induced monocyte activation and a pro-inflammatory immune response as evidenced by increased surface presentation of the activation marker CD38 and upregulated gene expression of key signaling pathways (including NF-κB, IL-17 and TNF signaling). The release of pro-inflammatory cytokines was increased in EV-stimulated monocytes and more so in male- than in female-derived cells. While EhA1 EV stimulation caused elevated myeloperoxidase (MPO) release by both monocytes and neutrophils, EhB2 EV stimulation did not, indicating the protective role of MPO during amebiasis. Collectively, our results suggest that parasite-released EVs contribute to the male-biased immunopathology mediated by pro-inflammatory monocytes during ALA formation.
- MeSH
- amébový absces jater imunologie parazitologie MeSH
- cytokiny metabolismus MeSH
- entamébóza imunologie parazitologie MeSH
- Entamoeba histolytica * imunologie patogenita genetika MeSH
- extracelulární vezikuly * imunologie metabolismus MeSH
- lidé MeSH
- monocyty * imunologie parazitologie MeSH
- signální transdukce * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Colorectal cancer (CRC) ranks as the second most prevalent malignancy globally, highlighting the urgent need for more effective diagnostic and therapeutic strategies, as well as a deeper understanding of its molecular basis. Extensive research has demonstrated that cells actively secrete extracellular vesicles (EVs) to mediate intercellular communication at both proximal and distal sites. In this study, we conducted a comprehensive analysis of the RNA content of small extracellular vesicles (sEVs) secreted into the culture media of five frequently utilised CRC cell lines (RKO, HCT116, HCT15, HT29, and DLD1). RNA sequencing data revealed significant insights into the RNA profiles of these sEVs, identifying nine protein-coding genes and fourteen long non-coding RNA (lncRNA) genes that consistently ranked among the top 30 most abundant across all cell lines. Notably, the genes found in sEVs were highly similar among the cell lines, indicating a conserved molecular signature. Several of these genes have been previously documented in the context of cancer biology, while others represent novel discoveries. These findings provide valuable insights into the molecular cargo of sEVs in CRC, potentially unveiling novel biomarkers and therapeutic targets.
- MeSH
- extracelulární vezikuly * metabolismus genetika MeSH
- HCT116 buňky MeSH
- kolorektální nádory * genetika patologie metabolismus MeSH
- lidé MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- regulace genové exprese u nádorů MeSH
- RNA dlouhá nekódující genetika MeSH
- sekvenční analýza RNA MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: The objective of this work was to gather an international consensus group to propose a global definition and diagnostic approach of laryngopharyngeal reflux (LPR) to guide primary care and specialist physicians in the management of LPR. METHODS: Forty-eight international experts (otolaryngologists, gastroenterologists, surgeons, and physiologists) were included in a modified Delphi process to revise 48 statements about definition, clinical presentation, and diagnostic approaches to LPR. Three voting rounds determined a consensus statement to be acceptable when 80% of experts agreed with a rating of at least 8/10. Votes were anonymous and the analyses of voting rounds were performed by an independent statistician. RESULTS: After the third round, 79.2% of statements (N = 38/48) were approved. LPR was defined as a disease of the upper aerodigestive tract resulting from the direct and/or indirect effects of gastroduodenal content reflux, inducing morphological and/or neurological changes in the upper aerodigestive tract. LPR is associated with recognized non-specific laryngeal and extra-laryngeal symptoms and signs that can be evaluated with validated patient-reported outcome questionnaires and clinical instruments. The hypopharyngeal-esophageal multichannel intraluminal impedance-pH testing can suggest the diagnosis of LPR when there is >1 acid, weakly acid or nonacid hypopharyngeal reflux event in 24 h. CONCLUSION: A global consensus definition for LPR is presented to improve detection and diagnosis of the disease for otolaryngologists, pulmonologists, gastroenterologists, surgeons, and primary care practitioners. The approved statements are offered to improve collaborative research by adopting common and validated diagnostic approaches to LPR. LEVEL OF EVIDENCE: 5 Laryngoscope, 134:1614-1624, 2024.
- MeSH
- elektrická impedance MeSH
- laryngofaryngeální reflux * diagnóza MeSH
- larynx * MeSH
- lidé MeSH
- monitorování jícnového pH MeSH
- otorinolaryngologové MeSH
- průzkumy a dotazníky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Helicobacter pylori infection is the major risk factor associated with the development of gastric cancer. Currently, administration of standard antibiotic therapy combined with probiotics and postbiotics has gained significant attention in the management of H. pylori infection. In this work, the immunomodulatory effects of Lactobacillus crispatus-derived extracellular vesicles (EVs) and cell-free supernatant (CFS) were investigated on H. pylori-induced inflammatory response in human gastric adenocarcinoma (AGS) cells. L. crispatus-derived EVs were isolated by ultracentrifugation and physically characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Furthermore, the protein content of L. crispatus-derived EVs was also evaluated by SDS-PAGE. Cell viability of AGS cells exposed to varying concentrations of EVs and CFS was assessed by MTT assay. The mRNA expression of IL-1β, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR. ELISA was used for the measurement of IL-8 production in AGS cells. In addition, EVs (50 μg/mL) and CFS modulated the H. pylori-induced inflammation by downregulating the mRNA expression of IL-1β, IL-6, IL-8, and TNF-α, and upregulating the expression of IL-10, and TGF-ß genes in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with L. crispatus-derived EVs and CFS. In conclusion, our observation suggests for the first time that EVs released by L. crispatus strain RIGLD-1 and its CFS could be recommended as potential therapeutic agents against H. pylori-triggered inflammation.
- MeSH
- antiflogistika farmakologie MeSH
- cytokiny * metabolismus genetika MeSH
- epitelové buňky * mikrobiologie MeSH
- extracelulární vezikuly * metabolismus chemie imunologie MeSH
- Helicobacter pylori * genetika MeSH
- infekce vyvolané Helicobacter pylori mikrobiologie imunologie MeSH
- kultivační média speciální farmakologie MeSH
- Lactobacillus metabolismus fyziologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- probiotika farmakologie MeSH
- viabilita buněk účinky léků MeSH
- zánět mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
A case study on Sitagliptin drug products and Sitagliptin/Metformin drug products concerning contamination with N-nitrosamines was performed using two newly developed analytical methods for determination of N-nitroso-triazolopyrazine (NTTP; 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine) and its precursor triazolopyrazine (3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine). The method for determination of triazolopyrazine was previously unpublished, the method for determination of NTTP was published only for analysis of active pharmaceutical ingredient Sitagliptin and not the drug forms. Solving the N-nitrosamine contamination is requested by regulatory authorities all over the world and thus is vital for all pharmaceutical companies. The solution always requires a sensitive analytical method. Both newly developed methods use liquid chromatography coupled with mass spectrometry (single quadrupole analyzer in case of triazolopyrazine and triple quadrupole analyzer in case of NTTP). Separation of triazolopyrazine was achieved on a column Acquity CSH C18 using a mobile phase consisting of aqueous ammonium formate buffered at pH 4.2 and acetonitrile. Detection was performed using positive electrospray and selected ion monitoring at m/z 193. Separation of NTTP was achieved on a column Acquity HSS T3 using a mobile phase consisting of 0.1 % formic acid in water and methanol. Detection was performed using positive electrospray and multiple reaction monitoring at transitions m/z 222.15→42.05 (collision energy 17 eV) and m/z 222.15→192.15 (collision energy 11 eV). Two issues specific to NTTP and triazolopyrazine previously not described in scientific literature were successfully troubleshooted. Spontaneous degradation of Sitagliptin to triazolopyrazine and methyl (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoate was solved by using N,N-dimethylformamide as sample solvent during development of the method for quantitation of triazolopyrazine. A bad peak shape of NTTP due to the presence of rotamers of NTTP was successfully troubleshooted by increasing column temperature. Both methods were used during an optimization study of manufacturing of Sitagliptin and Sitagliptin/Metformin drug products. The goal of the study was to decrease NTTP content in the final drug product under the strict legislative limit set by Federal Drug Agency. The efficacy of several solutions was proven, but could not be fully disclosed due to Intellectual Property Protection policy of Zentiva. Instead, a brief review of recently published strategies to cope with N-nitrosamine contamination is presented.
Extracelulární vezikuly (EV) jsou transportní váčky derivované ze zdrojové buňky do extracelulárního prostředí. Představují nový pilíř mezibuněčné komunikace, neboť přenáší nukleové kyseliny, proteiny a různé signální molekuly, které jsou chráněny před degradací v extracelulárním prostředí a posléze jsou uvolněny fúzí vezikuly s cílovou buňkou. Transportní mechanismus je zajištěn povrchovými strukturami účastnícími se buněčné adheze. Je všeobecně známé, že všechny buněčné organismy jsou schopné tvořit EV. Většina lidských buněk je schopna produkovat EV, díky tomu je možná jejich detekce ve všech tělesných kompartmentech. EV při svém objevu byly vnímány jako nepotřebné odpadní váčky a stály na okraji zájmů. Zásluhou nově popsaným mechanismům transportu biologicky aktivních molekul je známo, že se EV účastní celé řady homeostatických mechanismů. V infekčním lékařství je nejvíce studována oblast modulace imunitní odpovědi, kdy jsou vnímány jako potenciální biomarkery, neboť jejich produkce či nesený obsah může být alterován za patologických stavů. U mikrobů stojí v popředí interakce na úrovni patogen-patogen a patogen-hostitel. Další možností je potenciální využití EV jako transportních lékových systémů a nových cílů farmakoterapie.
Extracellular vesicles (EVs) are mother cell derived transport units released into the extracellular environment. They are a new pillar of intercellular communication as they carry nucleic acids, proteins, and other signalling molecules, protecting them from degradation in the extracellular environment until fusion of the vesicle with the target cell. The transport mechanism relies on surface structures involved in cell adhesion. It is well known that all cellular organisms are capable of producing EVs. Most human cells have this capability, and EVs can be detected in all body compartments. At the time of their discovery, EVs were considered as useless waste vesicles of marginal interest. Thanks to the newly described transport mechanisms of biologically active molecules, EVs are currently known to participate in a variety of homeostatic mechanisms. In infectious diseases, the most studied area is the modulation of the immune response, where they are seen as potential biomarkers, as their production or the content they carry can be altered under pathological conditions. For microbes, interactions at the pathogen-pathogen and pathogen-host level are at the forefront of attention. EVs also have potential for use as drug delivery systems and novel targets for pharmacotherapy.
- MeSH
- biologické markery metabolismus MeSH
- exozómy klasifikace mikrobiologie MeSH
- extracelulární vezikuly * fyziologie klasifikace mikrobiologie MeSH
- fyziologie bakterií MeSH
- fyziologie virů MeSH
- mezibuněčná komunikace genetika MeSH
- transportní vezikuly fyziologie klasifikace mikrobiologie MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFβ1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators. Video Abstract.
OBJECTIVE: To evaluate the effect of iatrogenic menopause on the physiology of the vagina of the ewe and to evaluate if vaginal changes in ewes can be translated to women with genitourinary syndrome of menopause (GSM). METHODS: Preclinical research with Dohne Merino ewes. Iatrogenic menopause was induced by bilateral ovariectomy (OVX). Animals were randomized for surgery, blinded for allocation and outcome assessment. Differences between groups were determined by linear regression analyses at 5 months after OVX. Outcome measures were vaginal epithelial thickness, pH, vaginal maturation value, vaginal maturation index, epithelial glycogen accumulation, content of elastin fibers, collagen, and vascularity. RESULTS: OVX ewes (n = 20) showed epithelial thinning of the vaginal wall from 146 μm to 47 μm (mean, P < 0.001). Furthermore, epithelial glycogen accumulation and vascularity of the vaginal wall significantly decreased (43% and 23%, respectively) as compared with the control group (no intervention; n = 5). No significant differences were found for other outcome measures. CONCLUSION: This study established the ewe as a suitable large animal model for GSM. Furthermore, the similar relevant outcomes in humans and ewes hold great value for future translational research for the evaluation and optimization of different treatment modalities for GSM.
- MeSH
- glykogen MeSH
- iatrogenní nemoci MeSH
- lidé MeSH
- menopauza * MeSH
- modely u zvířat MeSH
- ovarektomie škodlivé účinky MeSH
- ovce MeSH
- vagina * chirurgie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The observation that aging is regulated by microRNAs (miRNA) and at the same time represents the greatest risk factor for Alzheimer's disease (AD), prompted us to examine the circulating miRNA network in AD beyond aging. We here show that plasma miRNAs in aging are downregulated and predicted to be preferentially targeted to the extracellular vesicle (EV) content. In AD, miRNAs are further downregulated, display altered proportions of motifs relevant to their loading into EVs and secretion propensity, and are forecast to be found exclusively in EVs. The circulating miRNA network in AD, therefore, reflects pathological exacerbation of the aging process whereby physiological suppression of AD pathology by miRNAs becomes insufficient.
- MeSH
- Alzheimerova nemoc * genetika MeSH
- extracelulární vezikuly * MeSH
- lidé MeSH
- mikro RNA * genetika MeSH
- stárnutí genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
