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1.
J Vis Exp ; (168)2021 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-33645553

RESUMO

Many human neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.


Assuntos
Células Sanguíneas/citologia , Neurônios/citologia , Anticorpos/química , Técnicas de Cultura de Células , Desdiferenciação Celular , Diferenciação Celular/fisiologia , Separação Celular , Células Cultivadas , Reagentes para Ligações Cruzadas/química , Glicoproteínas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Humanos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neurônios/ultraestrutura
2.
Methods Mol Biol ; 2241: 15-25, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486724

RESUMO

The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress® Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Eosinófilos/fisiologia , Sangue/metabolismo , Células Sanguíneas/citologia , Centrifugação com Gradiente de Concentração/métodos , Eosinófilos/metabolismo , Citometria de Fluxo/métodos , Humanos , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Receptores de IgG/imunologia
3.
Methods Mol Biol ; 2241: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486725

RESUMO

Eosinophils are granulocytes involved mainly in allergic inflammation and parasitic responses and constitute 1-5% of the circulating leukocytes in human healthy subjects. New immunotherapies targeting eosinophils have been developed and evaluated recently, and the availability of animal models that could mimic human eosinophil responses is important to consider. Differences in eosinophil biology and pathogenesis between humans and murine models have limited their utility in some settings. Isolation of viable eosinophils from rhesus macaque blood suitable for ex vivo and in vitro experimentation could provide a valuable tool for the study of eosinophil-targeted therapies and for the exploration of eosinophilic associated responses. Here, a new technique for the isolation of human eosinophils from rhesus macaque blood by negative selection from whole blood is described.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Eosinófilos/fisiologia , Animais , Sangue/metabolismo , Células Sanguíneas/citologia , Centrifugação com Gradiente de Concentração/métodos , Eosinófilos/metabolismo , Citometria de Fluxo/métodos , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Macaca mulatta/imunologia , Receptores de IgG/imunologia
4.
Methods Mol Biol ; 2241: 49-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486727

RESUMO

Flow cytometry is a critical tool that can be employed to detect unique cells and to isolate cells from tissues based on their antigen profiles. While mouse eosinophils can be readily detected by one or more distinct antigen profiles, many of these strategies do not result in accurate eosinophil counts. We present here our basic protocol, which permits quantitative detection of eosinophils and isolation of eosinophils from bone marrow, spleen, and lung tissue of allergen-challenged wild-type and unchallenged IL5 transgenic mice. With small protocol variations, eosinophils can be isolated from small intestines and muscle tissue, the latter from infiltrates characteristic of muscular dystrophy (mdx) mice.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Citometria de Fluxo/métodos , Alérgenos/imunologia , Animais , Sangue/metabolismo , Células Sanguíneas/citologia , Medula Óssea/imunologia , Células da Medula Óssea/citologia , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Feminino , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Receptores de IgG/imunologia , Baço/citologia , Baço/imunologia
5.
Nat Med ; 27(1): 45-48, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33432173

RESUMO

We integrated ubiquity, mass and lifespan of all major cell types to achieve a comprehensive quantitative description of cellular turnover. We found a total cellular mass turnover of 80 ± 20 grams per day, dominated by blood cells and gut epithelial cells. In terms of cell numbers, close to 90% of the (0.33 ± 0.02) × 1012 cells per day turnover was blood cells.


Assuntos
Células Sanguíneas/citologia , Células Epiteliais/citologia , Senescência Celular , Humanos
6.
J Vis Exp ; (166)2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33346187

RESUMO

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.


Assuntos
Células Sanguíneas/citologia , Metais/farmacologia , Peptídeos/metabolismo , Próteses e Implantes , Animais , Anti-Inflamatórios/farmacologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Células Sanguíneas/efeitos dos fármacos , Antígeno CD47/metabolismo , Adesão Celular/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Microscopia de Fluorescência , Monócitos/citologia , Monócitos/efeitos dos fármacos , Polietilenoimina/química , Coelhos , Ratos , Espectrometria de Fluorescência
7.
J Vis Exp ; (162)2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32894265

RESUMO

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 °C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.


Assuntos
Células Sanguíneas/metabolismo , Prótese Vascular , Materiais Revestidos Biocompatíveis/química , Hemodinâmica/fisiologia , Teste de Materiais/métodos , Células Sanguíneas/citologia , Coagulação Sanguínea , Proteínas do Sistema Complemento/metabolismo , Humanos , Teste de Materiais/normas , Modelos Biológicos , Plasma/metabolismo , Ativação Plaquetária , Cloreto de Polivinila/química
8.
Proc Natl Acad Sci U S A ; 117(26): 14779-14789, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32561645

RESUMO

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.


Assuntos
Contagem de Células Sanguíneas/métodos , Microscopia Ultravioleta/métodos , Imagem Molecular/métodos , Contagem de Células Sanguíneas/instrumentação , Células Sanguíneas/classificação , Células Sanguíneas/citologia , Desenho de Equipamento , Humanos , Microscopia Ultravioleta/instrumentação , Imagem Molecular/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito
9.
Sci Rep ; 10(1): 7237, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350358

RESUMO

Persistent and saturated oxygen distribution from perfusion media (i.e., blood, or cell culture media) to cells within cell-dense, metabolically-active biofabricated tissues is required to keep them viable. Improper or poor oxygen supply to cells within the tissue bulk severely limits the tissue culturing potential of many bioreactors. We added an oxygenator module to our modular FABRICA bioreactor in order to provide stable oxygenation to biofabricated tissues during culture. In this proof of concept study of an oxygenated and perfused bioreactor, we characterized the oxygenation of water, cell culture medium, and human blood in the FABRICA as functions of augmenting vacuum (air inlet) pressure, perfusion (volumetric flow) rate, and tubing/oxygenator components. The mean oxygen levels for water and cell culture media were 27.7 ± 2.1% and 27.6 ± 4.1%, respectively. The mean oxygen level for human blood was 197.0 ± 90.0 mmHg, with near-physiologic levels achieved with low-permeability PharMed tubing alone (128.0 ± 14.0 mmHg). Hematologic values pre- and post-oxygenation, respectively were (median ± IQR): Red blood cell: 6.0 ± 0.5 (106/µL) and 6.5 ± 0.4 (106/µL); Hemoglobin: 17.5 ± 1.2 g/dL and 19.2 ± 3.0 g/dL; and Hematocrit: 56.7 ± 2.4% and 61.4 ± 7.5%. The relative stability of the hematologic parameters indicates that blood function and thus blood cell integrity were maintained throughout oxygenation. Already a versatile research tool, the now oxygenated FABRICA provides easy-to-implement, in vivo-like perfusion and stable oxygenation culture conditions in vitro semi-independently of one another, which means the bioreactor has the potential to serve as a platform for investigating the behavior of 3D tissue models (regardless of biofabrication method), performing drug toxicity-testing, and testing pharmaceutical efficacy/safety.


Assuntos
Bioimpressão , Reatores Biológicos , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Técnicas de Cultura de Células , Impressão Tridimensional , Meios de Cultura/química , Humanos , Água/química
11.
PLoS Biol ; 18(3): e3000643, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176686

RESUMO

Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are by individual molecules such as cytokines carried by blood, by neural transmission, or, in more severe pathologies, by the entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs), but the parameters that determine their uptake are unknown. Using varied animal models that stimulate neuronal activity by peripheral inflammation, optogenetics, and selective proteasome inhibition of dopaminergic (DA) neurons, we show that the transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object placement. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.


Assuntos
Células Sanguíneas/citologia , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo , Inflamação/metabolismo , Animais , Células Sanguíneas/metabolismo , Encéfalo/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Feminino , Hipocampo/fisiologia , Inflamação/induzido quimicamente , Ácido Caínico/farmacologia , Lipopolissacarídeos/toxicidade , Masculino , Camundongos Transgênicos , Optogenética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Técnicas Estereotáxicas , Ubiquitina/metabolismo
12.
Arch Virol ; 165(1): 207-214, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776677

RESUMO

Bovine leukemia virus (BLV) infects cattle worldwide and causes B-cell lymphoma in cattle. BLV has been identified in human breast and lung cancer and in blood, but the association of BLV and human cancer is controversial. In this study, we investigated the existence of BLV in 145 Japanese human blood cell lines and 54 human cancer cell lines, using a new highly sensitive PCR assay that can amplify even one copy of BLV using LTR primers different from those in previous studies on BLV provirus in breast cancer. All samples were found negative for BLV provirus, suggesting that BLV is unlikely to infect humans.


Assuntos
Células Sanguíneas/virologia , Linhagem Celular Tumoral/virologia , Vírus da Leucemia Bovina/isolamento & purificação , Zoonoses/diagnóstico , Adulto , Idoso , Animais , Células Sanguíneas/citologia , Linhagem Celular , Linhagem Celular Tumoral/citologia , Feminino , Humanos , Japão , Vírus da Leucemia Bovina/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Sequências Repetidas Terminais , Adulto Jovem , Zoonoses/virologia
13.
Clin Chim Acta ; 501: 72-82, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31778674

RESUMO

OBJECTIVES: Standardized criteria guaranteeing harmonized interpretation among morphologists in the provision of morphology results represent an important tool to be adopted for risk management and patient safety. Aim of this work is to assess agreement among morphologists in the microscopic evaluation of the peripheral blood smear. METHODS: 17 morphologists participating in the external quality assessment (EQA) program individually evaluated the blood smear and recorded the results using a personal username and password. Agreement among operators was evaluated. RESULTS: The overall agreement rate in microscopic differential was 95% in 2016 and 97% in 2017 (acceptance limit 90%), with 6/120 and 4/120 incongruent results, respectively. The agreement for the diagnostic hypothesis was satisfactory with a full agreement being reached in 5 out of 16 cases. CONCLUSIONS: The creation of a tool to assess the agreement of readers providing morphological evaluations is a valuable step forward in ensuring patient safety and quality laboratory medicine.


Assuntos
Técnicas de Laboratório Clínico/normas , Segurança do Paciente/normas , Garantia da Qualidade dos Cuidados de Saúde/normas , Células Sanguíneas/citologia , Humanos
14.
Mol Biol Rep ; 47(1): 1-10, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31813129

RESUMO

Bone marrow mononuclear cells (BM-MNCs) transplantation has evolved as a promising experimental treatment in various regenerative therapy fields, especially in clinical hematopoietic stem cells transplantation (HSCT). In vitro methods have mainly been used to study the pre-clinical kinetics of BM-MNCs in mice after transplantation. And it is difficult to monitor the dynamic homing of BM-MNCs in living mice. The present study obtained the kinetics of transplanted BM-MNCs in the peripheral blood (PB) and the dynamic homing of BM-MNCs in the BM in living mice by a combination of in vivo flow cytometry (IVFC) and calvarium intravital microscopy. We found out that BM-MNCs were cleared rapidly from the PB and mainly localized to various hematopoietic tissues after transplantation. The number of BM-MNCs in the PB decreased over time accompanied by an increase in the BM indeed after transplantation. In addition, a lower number of BM-MNCs were found home to calvaria than long bone, probably indicating long bone marrow might also be an important hematopoietic organ. Clinical studies will benefit from non-invasive measurements to monitor the dynamic homing of transplanted cells. Our pre-clinical kinetics of BM-MNCs in living mice will have important clinical guiding significance in HSCT and other regenerative therapy fields.


Assuntos
Células da Medula Óssea/fisiologia , Transplante de Medula Óssea , Movimento Celular , Rastreamento de Células/métodos , Citometria de Fluxo/métodos , Microscopia Intravital/métodos , Animais , Células Sanguíneas/citologia , Medula Óssea/metabolismo , Quimiotaxia de Leucócito/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membro Posterior , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Fisiológica
15.
IEEE J Biomed Health Inform ; 24(1): 160-170, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892256

RESUMO

Cell classification, especially that of white blood cells, plays a very important role in the field of diagnosis and control of major diseases. Compared to traditional optical microscopic imaging, hyperspectral imagery, combined with both spatial and spectral information, provides more wealthy information for recognizing cells. In this paper, a novel blood cell classification framework, which combines a modulated Gabor wavelet and deep convolutional neural network (CNN) kernels, named as MGCNN, is proposed based on medical hyperspectral imaging. For each convolutional layer, multi-scale and orientation Gabor operators are taken dot product with initial CNN kernels. The essence is to transform the convolutional kernels into the frequency domain to learn features. By combining characteristics of Gabor wavelets, the features learned by modulated kernels at different frequencies and orientations are more representative and discriminative. Experimental results demonstrate that the proposed model can achieve better classification performance than traditional CNNs and widely used support vector machine approaches, especially as training small-sample-size situations.


Assuntos
Células Sanguíneas/classificação , Células Sanguíneas/citologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Redes Neurais de Computação , Algoritmos , Técnicas Citológicas/métodos , Humanos , Análise de Ondaletas
16.
J Pharmacol Toxicol Methods ; 101: 106664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31837438

RESUMO

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.


Assuntos
Contagem de Células/métodos , Citometria de Fluxo/métodos , Potencial da Membrana Mitocondrial/fisiologia , Reticulócitos/citologia , Reticulócitos/fisiologia , Animais , Células Sanguíneas/citologia , Células da Medula Óssea/citologia , Contagem de Eritrócitos/métodos , Eritropoese , Camundongos , Coloração e Rotulagem
17.
Mater Sci Eng C Mater Biol Appl ; 106: 110298, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753336

RESUMO

Cancer is a leading cause of mortality worldwide. Cell membrane-coated nanocarriers actively targeting tumor sites are known to circumvent the limitations of conventional treatments and nanosized drug delivery systems. Cell membrane-coated nanocarriers can evade the immune system and can target tumors, thereby exhibiting a prolonged circulation time, enhancing tumor accumulation, increasing cancer therapeutic efficacy, and facilitating tumor imaging in vivo. Numerous studies have focused on cell membrane-coated nanocarriers homing to tumors. The use of these biomimetic nanocarriers in combination with photothermal or photodynamic cancer therapy have received increasing attention. This review discusses various sources of cell membranes, which have been harnessed previously in this field and highlights the mechanism underlying the targeting action of these nanocarriers and the method of their extraction, along with the applications of biomimetic cell membrane-coated nanocarriers in cancer phototherapy and diagnosis. Finally, this review discusses prospects in methods to resist cancer metastasis.


Assuntos
Membrana Celular/química , Portadores de Fármacos/química , Nanopartículas/química , Animais , Bactérias/metabolismo , Materiais Biomiméticos/química , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Parede Celular/química , Humanos , Linfócitos/citologia , Linfócitos/metabolismo
18.
Sci Rep ; 9(1): 15735, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672997

RESUMO

Complex immune dysregulation is a hallmark of sepsis. The occurring phases of immunosuppression and hyperinflammation require rapid detection and close monitoring. Reliable tools to monitor patient's immune status are yet missing. Currently, microRNAs are being discussed as promising new biomarkers in sepsis. However, no suitable internal control for normalization of miRNA expression by qPCR has been validated so far, thus hampering their potential benefit. We here present the first evaluation of endogenous controls for miRNA analysis in human sepsis. Novel candidate reference miRNAs were identified via miRNA microArray. TaqMan qPCR assays were performed to evaluate these microRNAs in T-cells and whole blood cells of sepsis patients and healthy controls in two independent cohorts. In T-cells, U48 and miR-320 proved suitable as endogenous controls, while in whole blood cells, U44 and miR-942 provided best stability values for normalization of miRNA quantification. Commonly used snRNA U6 exhibited worst stability in all sample groups. The identified internal controls have been prospectively validated in independent cohorts. The critical importance of housekeeping gene selection is emphasized by exemplary quantification of imuno-miR-150 in sepsis patients. Use of appropriate internal controls could facilitate research on miRNA-based biomarker-use and might even improve treatment strategies in the future.


Assuntos
Células Sanguíneas/metabolismo , MicroRNAs/metabolismo , Sepse/patologia , Linfócitos T/metabolismo , Biomarcadores/metabolismo , Células Sanguíneas/citologia , Estudos de Casos e Controles , Humanos , Estudos Retrospectivos , Sepse/genética , Linfócitos T/citologia
19.
Nature ; 574(7778): 365-371, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31597962

RESUMO

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.


Assuntos
Feto/citologia , Hematopoese , Fígado/citologia , Fígado/embriologia , Células Sanguíneas/citologia , Microambiente Celular , Feminino , Feto/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Fígado/metabolismo , Tecido Linfoide/citologia , Análise de Célula Única , Células-Tronco/metabolismo
20.
Stem Cell Res Ther ; 10(1): 305, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31623690

RESUMO

BACKGROUND: Within the last years, the interest in physical exercise as non-invasive stimulus influencing circulating hematopoietic stem and progenitor cell (CPC) concentrations has constantly grown. Cell estimates are often derived by determining the subgroup of CPC as percent lymphocytes (LYM) or mononuclear cells (MNC) via flow cytometry and back calculation over whole blood (WB) cell counts. However, results might depend on the used cell isolation technique and/or gating strategy. We aimed to investigate MNC loss and apoptosis during the flow cytometry sample preparation process preceded by either density gradient centrifugation (DGC) or red blood cell lysis (RBCL) and the potential difference between results derived from back calculation at different stages of cell isolation and from WB. METHODS: Human blood was subjected to DGC and RBCL. Samples were stained for flow cytometry analysis of CPC (CD34+/CD45dim) and apoptosis analysis (Annexin V) of MNC and CPC subsets. MNC and LYM gating strategies were compared. RESULTS: Both DGC as well as RBCL yielded comparable CPC concentrations independent of the gating strategy when back calculated over WB values. However, cell loss and apoptosis differed between techniques, where after DGC LYM, and monocyte (MONO) concentrations significantly decreased (p < 0.01 and p < 0.05, respectively), while after RBCL LYM concentrations significantly decreased (p < 0.05) and MONO concentrations increased (p < 0.001). LYM apoptosis was comparable between techniques, but MONO apoptosis was higher after DGC than RBCL (p < 0.001). CONCLUSIONS: Investigated MNC counts (LYM/MONO ratio) after cell isolation and staining did not always mimic WB conditions. Thus, final CPC results should be corrected accordingly, especially when reporting live CPC concentrations after DGC; otherwise, the CPC regenerative potential in circulation could be biased. This is of high importance in the context of non-invasively induced CPC mobilization such as by acute physical exercise, since these cell changes are small and conclusions drawn from published results might affect further applications of physical exercise as non-invasive therapy.


Assuntos
Células Sanguíneas/citologia , Células-Tronco/citologia , Antígenos CD34/metabolismo , Contagem de Células Sanguíneas/métodos , Células Sanguíneas/metabolismo , Contagem de Células/métodos , Separação Celular/métodos , Exercício Físico/fisiologia , Citometria de Fluxo/métodos , Humanos , Antígenos Comuns de Leucócito/metabolismo , Células-Tronco/metabolismo
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