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1.
Allergy ; 79(7): 1893-1907, 2024 07.
Artigo em Inglês | MEDLINE | ID: mdl-38573073

RESUMO

BACKGROUND: Extracellular vesicles (EVs) have been implicated in the pathogenesis of asthma, however, how EVs contribute to immune dysfunction and type 2 airway inflammation remains incompletely understood. We aimed to elucidate roles of airway EVs and their miRNA cargo in the pathogenesis of NSAID-exacerbated respiratory disease (N-ERD), a severe type 2 inflammatory condition. METHODS: EVs were isolated from induced sputum or supernatants of cultured nasal polyp or turbinate tissues of N-ERD patients or healthy controls by size-exclusion chromatography and characterized by particle tracking, electron microscopy and miRNA sequencing. Functional effects of EV miRNAs on gene expression and mediator release by human macrophages or normal human bronchial epithelial cells (NHBEs) were studied by RNA sequencing, LC-MS/MS and multiplex cytokine assays. RESULTS: EVs were highly abundant in secretions from the upper and lower airways of N-ERD patients. N-ERD airway EVs displayed profoundly altered immunostimulatory capacities and miRNA profiles compared to airway EVs of healthy individuals. Airway EVs of N-ERD patients, but not of healthy individuals induced inflammatory cytokine (GM-CSF and IL-8) production by NHBEs. In macrophages, N-ERD airway EVs exhibited an impaired potential to induce cytokine and prostanoid production, while enhancing M2 macrophage activation. Let-7 family miRNAs were highly enriched in sputum EVs from N-ERD patients and mimicked suppressive effects of N-ERD EVs on macrophage activation. CONCLUSION: Aberrant airway EV miRNA profiles may contribute to immune dysfunction and chronic type 2 inflammation in N-ERD. Let-7 family miRNAs represent targets for correcting aberrant macrophage activation and mediator responses in N-ERD.


Assuntos
Anti-Inflamatórios não Esteroides , Vesículas Extracelulares , Macrófagos , MicroRNAs , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , MicroRNAs/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Anti-Inflamatórios não Esteroides/efeitos adversos , Citocinas/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Ativação de Macrófagos/imunologia , Ativação de Macrófagos/genética , Adulto
2.
Ecotoxicol Environ Saf ; 262: 115147, 2023 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-37343485

RESUMO

Nanoplastics smaller than 1 µm accumulate as anthropogenic material in the food chain, but only little is known about their uptake and possible effects on potentially strongly exposed cells of the small intestine. The aim of the study was to observe the uptake of 100 nm polystyrene nanoplastics into a non-tumorigenic small intestine cell culture model (IPEC-J2 cells) and to monitor the effects on cell growth and gene regulation, compared to a 100 nm non-plastic silica nanoparticle reference. The intracellular uptake of both types of nanoparticles was proven via (confocal) fluorescence microscopy and complemented with transmission electron microscopy. Fluorescence microscopy showed a growth phase-dependent uptake of nanoparticles into the cells, hence further experiments included different time points related to epithelial closure, determined via electric cell substrate impedance sensing. No retardations in epithelial closure of cells after treatment with polystyrene nanoparticles could be found. In contrast, epithelial cell closure was partly negatively influenced by silica nanoparticles. An increased production of organic nanoparticles, like extracellular vesicles, was not measurable via nanoparticle tracking analysis. An assessment of messenger RNA by next generation sequencing and subsequent pathway analysis revealed that the TP53 pathway was influenced significantly by the polystyrene nanoparticle treatment. In both treatments, dysregulated mRNAs were highly enriched in the NOTCH signaling pathway compared to the non-particle control.

3.
Am J Hematol ; 96(3): 338-353, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33326140

RESUMO

Autologous blood doping refers to the illegal re-transfusion of any quantities of blood or blood components with blood donor and recipient being the same person. The re-transfusion of stored erythrocyte concentrates is particularly attractive to high-performance athletes as this practice improves their oxygen capacity excessively. However, there is still no reliable detection method available. Analyzing circulating microRNA profiles of human subjects that underwent monitored autologous blood transfusions seems to be a highly promising approach to develop novel biomarkers for autologous blood doping. In this exploratory study, we randomly divided 30 healthy males into two different treatment groups and one control group and sampled whole blood at several time points at baseline, after whole blood donation and after transfusion of erythrocyte concentrates. Hematological variables were recorded and analyzed following the adaptive model of the Athlete Biological Passport. microRNA profiles were examined by small RNA sequencing and comprehensive multivariate data analyses, revealing microRNA fingerprints that reflect the sampling time point and transfusion volume. Neither individual microRNAs nor a signature of transfusion-dependent microRNAs reached superior sensitivity at 100% specificity compared to the Athlete Biological Passport (≤11% 6 h after transfusion versus ≤44% 2 days after transfusion). However, the window of autologous blood doping detection was different. Due to the heterogenous nature of doping, with athletes frequently combining multiple medications in order to both gain a competitive advantage and interfere with known testing methods, the true applicability of the molecular signature remains to be validated in real anti-doping testings.


Assuntos
Identificação Biométrica/métodos , Dopagem Esportivo , MicroRNAs/sangue , Transfusão de Sangue Autóloga , Dopagem Esportivo/prevenção & controle , Índices de Eritrócitos , Ferritinas/sangue , Hematócrito , Hemoglobinas/análise , Humanos , Ferro/sangue , Estudos Longitudinais , Masculino , Análise Multivariada , RNA-Seq , Receptores da Transferrina/sangue , Sensibilidade e Especificidade , Transferrina/análise
4.
BMC Res Notes ; 17(1): 154, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38840260

RESUMO

OBJECTIVE: The IPEC-J2 cell line is used as an in vitro small intestine model for swine, but it is also used as a model for the human intestine, presenting a relatively unique setting. By combining electric cell-substrate impedance sensing, with next-generation-sequencing technology, we showed that mRNA gene expression profiles and related pathways can depend on the growth phase of IPEC-J2 cells. Our investigative approach welcomes scientists to reproduce or modify our protocols and endorses putting their gene expression data in the context of the respective growth phase of the cells. RESULTS: Three time points are presented: (TP1) 1 h after medium change (= 6 h after seeding of cells), (TP2) the time point of the first derivative maximum of the cell growth curve, and a third point at the beginning of the plateau phase (TP3). Significantly outstanding at TP1 compared to TP2 was upregulated PLEKHN1, further FOSB and DEGS2 were significantly downregulated at TP2 compared to TP3. Any provided data can be used to improve next-generation experiments with IPEC-J2 cells.


Assuntos
Proliferação de Células , Perfilação da Expressão Gênica , RNA Mensageiro , Animais , Linhagem Celular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Perfilação da Expressão Gênica/métodos , Proliferação de Células/genética , Intestino Delgado/metabolismo , Intestino Delgado/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Transcriptoma/genética
5.
Nanoscale Adv ; 5(6): 1691-1705, 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36926576

RESUMO

BACKGROUND: Extracellular vesicles (EV) are cell-derived vesicles released by all cells in health and disease. Accordingly, EVs are also released by cells in acute myeloid leukemia (AML), a hematologic malignancy characterized by uncontrolled growth of immature myeloid cells, and these EVs likely carry markers and molecular cargo reflecting the malignant transformation occurring in diseased cells. Monitoring antileukemic or proleukemic processes during disease development and treatment is essential. Therefore, EVs and EV-derived microRNA (miRNA) from AML samples were explored as biomarkers to distinguish disease-related patterns ex vivo or in vivo. METHODOLOGY: EVs were purified from serum of healthy (H) volunteers and AML patients by immunoaffinity. EV surface protein profiles were analyzed by multiplex bead-based flow cytometry (MBFCM) and total RNA was isolated from EVs prior to miRNA profiling via small RNA sequencing. RESULTS: MBFCM revealed different surface protein patterns in H versus AML EVs. miRNA analysis showed individual as well as highly dysregulated patterns in H and AML samples. CONCLUSIONS: In this study, we provide a proof-of-concept for the discriminative potential of EV derived miRNA profiles as biomarkers in H versus AML samples.

6.
Methods Mol Biol ; 2504: 113-133, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35467283

RESUMO

Urine bears high potential for serving as biomarker repository for renal and urinary tract associated disorders. Besides various metabolites and salts, urine carries extracellular vesicles (EVs)-a heterogeneous group of cell-derived mediators comprising proteins, lipids, and nucleic acids such as microRNAs (miRNAs). Particularly, EV-derived miRNA biomarkers have already been identified for numerous disorders such as sepsis, various blood and solid cancer entities, respiratory and renal diseases. However, study results are often incomparable due to poorly reported EV separation and miRNA isolation protocols and emphasize the need for standardization and reproducibility. To ensure valid EV-derived miRNA biomarker findings from urine, a step-by-step protocol compliant with the "Minimal Information for Studies of Extracellular Vesicles" (MISEV) is outlined in the following paragraphs. Actually, an immunoaffinity-based EV separation method followed by EV characterization, quantification, and normalization, as well as consecutive miRNA isolation and miRNA profiling by small RNA sequencing, are described.


Assuntos
Vesículas Extracelulares , MicroRNAs , Sistema Urinário , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Sistema Urinário/metabolismo
7.
Front Mol Biosci ; 9: 1030749, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36589227

RESUMO

Introduction: MicroRNAs have a significant role in the regulation of the transcriptome. Several miRNAs have been proposed as potential biomarkers in different malignancies. However, contradictory results have been reported on the capability of miRNA biomarkers in cancer detection. The human biological clock involves molecular mechanisms that regulate several genes over time. Therefore, the sampling time becomes one of the significant factors in gene expression studies. Method: In the present study, we have tried to find miRNAs with minimum fluctuation in expression levels at different time points that could be more accurate candidates as diagnostic biomarkers. The small RNA-seq raw data of ten healthy individuals across nine-time points were analyzed to identify miRNAs with stable expression. Results: We have found five oscillation patterns. The stable miRNAs were investigated in 779 small-RNA-seq datasets of eleven cancer types. All miRNAs with the highest differential expression were selected for further analysis. The selected miRNAs were explored for functional pathways. The predominantly enriched pathways were miRNA in cancer and the P53-signaling pathway. Finally, we have found seven miRNAs, including miR-142-3p, miR-199a-5p, miR-223-5p, let-7d-5p, miR-148b-3p, miR-340-5p, and miR-421. These miRNAs showed minimum fluctuation in healthy blood and were dysregulated in the blood of eleven cancer types. Conclusion: We have found a signature of seven stable miRNAs which dysregulate in several cancer types and may serve as potential pan-cancer biomarkers.

8.
Cancer Gene Ther ; 29(11): 1600-1615, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35477770

RESUMO

Extracellular Vesicles (EVs) are membranous vesicles produced by all cells under physiological and pathological conditions. In hematological malignancies, tumor-derived EVs might reprogram the bone marrow environment, suppress antileukemic immunity, mediate drug resistance and interfere with immunotherapies. EVs collected from the serum of leukemic samples might correlate with disease stage, drug-/immunological resistance, or might correlate with antileukemic immunity/immune response. Special EV surface protein patterns in serum have the potential as noninvasive biomarker candidates to distinguish several disease-related patterns ex vivo or in vivo. EVs were isolated from the serum of acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL) patients, and healthy volunteers. EVs were characterized by transmission electron microscopy and fluorescence nanoparticle tracking analysis, and EV surface protein profiles were analyzed by multiplex bead-based flow cytometry to identify tumor- or immune system-related EVs of AML, ALL, CLL, and healthy samples. Aiming to provide proof-of-concept evidence and methodology for the potential role of serum-derived EVs as biomarkers in leukemic versus healthy samples in this study, we hope to pave the way for future detection of promising biomarkers for imminent disease progression and the identification of potential targets to be used in a therapeutic strategy.


Assuntos
Vesículas Extracelulares , Neoplasias Hematológicas , Leucemia Linfocítica Crônica de Células B , Leucemia Mieloide Aguda , Humanos , Citometria de Fluxo , Vesículas Extracelulares/metabolismo , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/terapia , Biomarcadores/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Proteínas de Membrana/metabolismo
9.
Adv Drug Deliv Rev ; 174: 348-368, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33964356

RESUMO

Extracellular vesicles (EVs) are membranous nanovesicles secreted from living cells, shuttling macromolecules in intercellular communication and potentially possessing intrinsic therapeutic activity. Due to their stability, low immunogenicity, and inherent interaction with recipient cells, EVs also hold great promise as drug delivery vehicles. Indeed, they have been used to deliver nucleic acids, proteins, and small molecules in preclinical investigations. Furthermore, EV-based drugs have entered early clinical trials for cancer or neurodegenerative diseases. Despite their appeal as delivery vectors, however, EV-based drug delivery progress has been hampered by heterogeneity of sample types and methods as well as a persistent lack of standardization, validation, and comprehensive reporting. This review highlights specific requirements for EVs in drug delivery and describes the most pertinent approaches for separation and characterization. Despite residual uncertainties related to pharmacodynamics, pharmacokinetics, and potential off-target effects, clinical-grade, high-potency EV drugs might be achievable through GMP-compliant workflows in a highly standardized environment.


Assuntos
Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos/métodos , Vesículas Extracelulares/metabolismo , Animais , Comunicação Celular/fisiologia , Humanos , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Ácidos Nucleicos/administração & dosagem , Proteínas/administração & dosagem
10.
Methods Mol Biol ; 2065: 23-38, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578685

RESUMO

MicroRNAs (miRNAs), a class of small non-coding RNAs that modulate gene expression at the post-transcriptional level, are attractive targets in many academic and diagnostic applications. Among them, assessing miRNA biomarkers in minimally invasive liquid biopsies was shown to be a promising tool for managing diseases, particularly cancer. The initial screening of disease-relevant transcripts is often performed by high-throughput next-generation sequencing (NGS), in here RNA sequencing (RNA-Seq). After complex processing of small RNA-Seq data, differential gene expression analysis is performed to evaluate miRNA biomarker signatures. To ensure experimental validity, biomarker candidates are commonly validated by an orthogonal technology such as reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). This chapter outlines in detail the material and methods one can apply to reproducibly identify miRNA biomarker signatures from blood total RNA. After screening miRNA profiles by small RNA-Seq, resulting data is validated in compliance with the "Minimum Information for Publication of Quantitative Real-Time PCR Experiments" (MIQE) guidelines.


Assuntos
MicroRNA Circulante/sangue , Técnicas de Diagnóstico Molecular/métodos , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real/normas , Estudos de Validação como Assunto , Biomarcadores/sangue , MicroRNA Circulante/isolamento & purificação , Interpretação Estatística de Dados , Biblioteca Gênica , Voluntários Saudáveis , Humanos , Biópsia Líquida/métodos , Técnicas de Diagnóstico Molecular/normas , Reprodutibilidade dos Testes
11.
PLoS One ; 15(1): e0227648, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935258

RESUMO

Sample collection, processing, storage and isolation methods constitute pre-analytic factors that can influence the quality of samples used in research and clinical practice. With regard to biobanking practices, a critical point in the sample's life chain is storage, particularly long-term storage. Since most studies examine the influence of different temperatures (4°C, room temperature) or delays in sample processing on sample quality, there is only little information on the effects of long-term storage at ultra-low (vapor phase of liquid nitrogen) temperatures on biomarker levels. Among these biomarkers, circulating miRNAs hold great potential for diagnosis or prognosis for a variety of diseases, like cancer, infections and chronic diseases, and are thus of high interest in several scientific questions. We therefore investigated the influence of long-term storage on levels of eight circulating miRNAs (miR-103a-3p, miR-191-5p, miR-124-3p, miR-30c-5p, miR-451a, miR-23a-3p, miR-93-5p, miR-24-3p, and miR-33b-5p) from 10 participants from the population-based cohort study KORA. Sample collection took place during the baseline survey S4 and the follow-up surveys F4 and FF4, over a time period spanning from 1999 to 2014. The influence of freeze-thaw (f/t) cycles on miRNA stability was also investigated using samples from volunteers (n = 6). Obtained plasma samples were profiled using Exiqon's miRCURYTM real-time PCR profiling system, and repeated measures ANOVA was used to check for storage or f/t effects. Our results show that detected levels of most of the studied miRNAs showed no statistically significant changes due to storage at ultra-low temperatures for up to 17 years; miR-451a levels were altered due to contamination during sampling. Freeze-thawing of one to four cycles showed an effect only on miR-30c-5p. Our results highlight the robustness of this set of circulating miRNAs for decades of storage at ultra-low temperatures and several freeze-thaw cycles, which makes our findings increasingly relevant for research conducted with biobanked samples.


Assuntos
Congelamento/efeitos adversos , Manejo de Espécimes/métodos , Tempo , Adulto , Bancos de Espécimes Biológicos/normas , Biomarcadores/sangue , MicroRNA Circulante/sangue , MicroRNA Circulante/genética , MicroRNA Circulante/isolamento & purificação , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , MicroRNAs/sangue , MicroRNAs/genética , MicroRNAs/isolamento & purificação , Estabilidade de RNA , Transcriptoma/genética
12.
Biomol Detect Quantif ; 17: 100089, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31194192

RESUMO

Small extracellular vesicles (EVs) are 50-200 nm sized mediators in intercellular communication that reflect both physiological and pathophysiological changes of their parental cells. Thus, EVs hold great potential for biomarker detection. However, reliable purification methods for the downstream screening of the microRNA (miRNA) cargo carried within urinary EVs by small RNA sequencing have yet to be established. To address this knowledge gap, RNA extracted from human urinary EVs obtained by five different urinary EV purification methods (spin column chromatography, immunoaffinity, membrane affinity, precipitation and ultracentrifugation combined with density gradient) was analyzed by small RNA sequencing. Urinary EVs were further characterized by nanoparticle tracking analysis, Western blot analysis and transmission electron microscopy. Comprehensive EV characterization established significant method-dependent differences in size and concentration as well as variances in protein composition of isolated vesicles. Even though all purification methods captured enough total RNA to allow small RNA sequencing, method-dependent differences were also observed with respect to library sizes, mapping distributions, number of miRNA reads and diversity of transcripts. Whereas EVs obtained by immunoaffinity yielded the purest subset of small EVs, highly comparable with results attained by ultracentrifugation combined with density gradient, precipitation and membrane affinity, sample purification by spin column chromatography indicated a tendency to isolate different subtypes of small EVs, which might also carry a distinct subset of miRNAs. Based on our results, different EV purification methods seem to preferentially isolate different subtypes of EVs with varying efficiencies. As a consequence, sequencing experiments and resulting miRNA profiles were also affected. Hence, the selection of a specific EV isolation method has to satisfy the respective research question and should be well considered. In strict adherence with the MISEV (minimal information for studies of extracellular vesicles) guidelines, the importance of a combined evaluation of biophysical and proteomic EV characteristics alongside transcriptomic results was clearly demonstrated in this present study.

13.
Int J Oncol ; 55(4): 925-937, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432155

RESUMO

In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor ß receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF­ß signaling. This pathway can alter the expression of coding and non­coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post­transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116­TGFBR2 MSI cell line model enables the doxycycline (dox)­inducible reconstituted expression of TGFBR2 in an isogenic background (­dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2­dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2­dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2­deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR­381­3p in TGFBR2­deficient MSI tumor cells and their secreted EVs.


Assuntos
Neoplasias Colorretais/genética , Vesículas Extracelulares/genética , MicroRNAs/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Neoplasias Colorretais/metabolismo , Doxiciclina/farmacologia , Vesículas Extracelulares/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Instabilidade de Microssatélites , Mutação , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo
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