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1.
Clin. transl. oncol. (Print) ; 24(9): 1755–1763, septiembre 2022.
Artigo em Inglês | IBECS | ID: ibc-JHG-508

RESUMO

IntroductionRegulatory T Cells (Tregs) play an important role in carcinogenesis and tumor immunoediting by preventing the development of effective antitumor immunity. Several reports showed that circulating Tregs are increased in patients with solid tumors, including lung cancer. Treg population could be categorized into “naive,” “effector,” and “memory” subtypes, bearing potential unique functions. However, the data regarding the prognostic impact of these Tregs subtypes is limited in lung cancer. The aim of this study was to investigate the frequency of different circulating Tregs subtypes in lung cancer and their correlation with clinical outcomes.MethodsWe analyzed the frequency of circulating CD4, CD8 and, Tregs lymphocytes in 66 patients with lung cancer and 32 healthy controls using flow cytometry. Circulating Tregs subtypes: naïve (CD3+ , CD4+ , CCR4+ , CD25+ and CD127low, CD45RO−), memory (CD3+ , CD4+ , CCR4+ , CD25+ and CD127low, CD45RO+) and the expression of the activation marker HLA-DR were correlated with overall survival.ResultsThe percentage and the absolute number of total, memory and activated Tregs was significantly higher in lung cancer patients than healthy controls. Patients with a Tregs percentage higher than 5.4% and higher than 20% of HLA-DR + Tregs had worse overall survival than those with lower levels.ConclusionsCirculating Tregs and activated Tregs are a potential prognostic factor in patients with lung cancer treated with conventional therapy and could be considered a predictive biomarker in patients not eligible for immune blockade treatments. Additionally, it will be interesting to study these Tregs subsets for immune treatments in future clinical trials. (AU)


Assuntos
Humanos , Citometria de Fluxo , Neoplasias Pulmonares/patologia , Linfócitos T Reguladores , Prognóstico , Pacientes
2.
Cytometry A ; 101(8): 614-616, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35915877
3.
Anal Chim Acta ; 1221: 340151, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35934381

RESUMO

The drug-induced diverse response among patients is a severe problem for improving hemorheological character. However, there is no validated method for personalized therapy to the best of our knowledge. Here, we apply a gravity-driven deformability cytometry platform (GD-DCP) to profile the drug response of the red cell deformability (RCD) at the single-cell level using pentoxifylline (PTX) as a model drug, the effect of different concentrations of PTX (0, 2, 20, 200 µg mL-1, the clinical dosage of PTX is 20 µg mL-1) on RCD in patients with cardiovascular disease was explored. Based on the GD-DCP, about 38 and 56% of the acute phase of acute myocardial infarction (AMI) patients in the acute phase and coronary heart disease (CHD) patients respond positively to PTX, respectively, indicating that PTX has a strong patient dependency on RCD. Moreover, RCD is observed to be significantly inversely correlated with the activation of membrane protein kinase C (PKC) as well as the concentration of Ca2+ (both P < 0.001). The results of animal experiments show that the protective effects of PTX on myocardial ischemia rats have substantial individual variation, too. It is noted that the effect of PTX is highly consistent between RCD in vitro and in vivo outcomes (blood viscosity, myocardial injury, and electrocardiogram (ECG)) in the same rat. All these new findings suggest that the GD-DCP is a promising method that uses deformability in vitro as one of the important criteria in personalized medicine, and our study provides unique insight into the individual-dependent mechanisms of PTX for improving RCD.


Assuntos
Microfluídica , Pentoxifilina , Animais , Viscosidade Sanguínea , Deformação Eritrocítica/fisiologia , Eritrócitos/metabolismo , Citometria de Fluxo , Pentoxifilina/metabolismo , Pentoxifilina/farmacologia , Ratos
4.
Front Endocrinol (Lausanne) ; 13: 865807, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35937843

RESUMO

Background: Recurrent implantation failure (RIF) is a challenge during assisted reproductive technology (ART). In the present study, potential diagnostic biomarkers for the immune status of peripheral blood lymphocyte subsets in patients with RIF were analyzed, with the aim of identifying novel biomarkers that may predict RIF. Methods: A total of 41 participants, including 21 women with RIF and 20 fertile controls, were included in the present study. Functional analysis was performed and the cytokine status of natural killer (NK), T, CD8+ T, T helper (Th), and γδ T cells which are lymphocyte subsets in peripheral blood was measured using flow cytometry. Binary logistic regression analysis adjusted for T follicular helper 1 (Tfh1), Tfh2, Tfh17, and early NK cells was performed to determine the relationship between the peripheral blood lymphocyte subsets and RIF. Potential diagnostic biomarkers were assessed by logistic regression analysis and receiver operating characteristic curves. Results: There were significantly more Tfh1, Tfh17, and NK cells in the RIF group compared with the control group (all P < 0.05). However, the percentage of T, regulatory T (Tregs), and Tfh2 cells, as well as early inhibitory NK cells, was significantly lower in the RIF group compared with the control group (all P < 0.05). Following logistics regression analysis, Treg, Tfh17, and early inhibitory NK cells exhibited significant differences between the two groups. Combination diagnosis using these 3 biomarkers had a higher area under the curve of 0.900 (95% confidence interval: 0.808-0.992, P < 0.001) in the RIF group compared with that in the control group. Conclusion: T, Tregs, Tfh1, Tfh2, Tfh17, NK cells, and early inhibitory NK cells may play important regulatory roles in embryo implantation. The combination of 3 molecular markers (Treg, Tfh17, and early inhibitory NK cells) could provide a high diagnostic value for women with RIF, thus providing novel potential biomarkers for RIF in ART. The present findings could provide a reference either for the clinical treatment of patients with RIF or for future large, well-designed studies.


Assuntos
Subpopulações de Linfócitos , Linfócitos T Reguladores , Biomarcadores , Feminino , Citometria de Fluxo , Humanos , Contagem de Linfócitos
5.
Methods Mol Biol ; 2538: 275-284, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35951306

RESUMO

Bacterial functional amyloids are remarkable examples of how amyloid aggregation can be kept under control and even leveraged to perform diverse biological processes. In this context, it is highly relevant to understand how amyloidogenesis is modulated by relevant factors, including key amino acids promoting or preventing aggregation. This chapter describes a methodology to identify critical residues for amyloid formation in bacterial proteins, based on mutant construction guided by bioinformatics prediction, their expression in bacteria, and their analysis by flow cytometry. Additionally, we describe a simple downstream analysis of selected mutants to assess their in vitro aggregation properties upon protein purification. We applied the proposed methodology to identify critical residues modulating the aggregation of the antimicrobial peptide microcin E492, a well-studied model of bacterial amyloids.


Assuntos
Amiloide , Proteínas de Bactérias , Amiloide/química , Bactérias/metabolismo , Citometria de Fluxo , Mutagênese Sítio-Dirigida
6.
Methods Cell Biol ; 171: 173-195, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35953200

RESUMO

Flow cytometry is a powerful technology that allows not only multiparameter quantitative data analysis at single cell resolution but also simultaneous cell separation of different populations of interest at high speed. It has been rapidly employed in biological research and clinical diagnostics. This technology has enabled the thorough understanding of murine hematopoiesis, especially the physiology of surface marker-defined hematopoietic stem and progenitor cell populations. The isolation of these populations has been well established over the last three decades with a large consensus among leading laboratories. Here, we describe a detailed methodology protocol of two different state-of-the-art approaches to isolate bone marrow cells and purify hematopoietic stem and progenitor cells via flow cytometry. Different gating schemes are introduced to identify well-defined populations of murine hematopoietic stem and multipotent progenitor cells.


Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Medula Óssea/metabolismo , Células da Medula Óssea , Separação Celular , Citometria de Fluxo/métodos , Camundongos
7.
Nat Commun ; 13(1): 4622, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941135

RESUMO

Clinical recommendations for Acute Myeloid Leukemia (AML) classification and risk-stratification remain heavily reliant on cytogenetic findings at diagnosis, which are present in <50% of patients. Using comprehensive molecular profiling data from 3,653 patients we characterize and validate 16 molecular classes describing 100% of AML patients. Each class represents diverse biological AML subgroups, and is associated with distinct clinical presentation, likelihood of response to induction chemotherapy, risk of relapse and death over time. Secondary AML-2, emerges as the second largest class (24%), associates with high-risk disease, poor prognosis irrespective of flow Minimal Residual Disease (MRD) negativity, and derives significant benefit from transplantation. Guided by class membership we derive a 3-tier risk-stratification score that re-stratifies 26% of patients as compared to standard of care. This results in a unified framework for disease classification and risk-stratification in AML that relies on information from cytogenetics and 32 genes. Last, we develop an open-access patient-tailored clinical decision support tool.


Assuntos
Leucemia Mieloide Aguda , Análise Citogenética , Citometria de Fluxo/métodos , Humanos , Quimioterapia de Indução/métodos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Neoplasia Residual
8.
J Vis Exp ; (185)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35969098

RESUMO

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter. Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels. Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.


Assuntos
Vesículas Extracelulares , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Dióxido de Silício , Coloração e Rotulagem
9.
Mikrochim Acta ; 189(9): 331, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35969307

RESUMO

Flow cytometry has become an indispensable tool for counting, analyzing, and sorting large cell populations in biological research and medical practice. Unfortunately, it has limitations in the analysis of non-spherically shaped cells due to the variation of their alignment with respect to the flow direction and, hence, the optical interrogation axis, resulting in unreliable cell analysis. Here, we present a simple on-chip acoustofluidic method to fix the orientation of ellipsoidal cells and focus them into a single, aligned stream. Specifically, by generating acoustic standing waves inside a 100 ⋅ 100 µm square-shaped microchannel, we successfully aligned and focused up to 97.7% of a population of Euglena gracilis (an ellipsoidal shaped microalgal species) cells in the center of the microchannel with high precision at a volume rate of 25 to 200 µL min-1. Uniform positioning of ellipsoidal cells is essential for making flow cytometry applicable to the investigation of a greater variety of cell populations and is expected to be beneficial for ecological studies and aquaculture.


Assuntos
Euglena gracilis , Acústica , Citometria de Fluxo/métodos
10.
Proc Natl Acad Sci U S A ; 119(34): e2206208119, 2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-35969754

RESUMO

Although glioblastoma multiforme (GBM) is not an invariably cold tumor, checkpoint inhibition has largely failed in GBM. In order to investigate T cell-intrinsic properties that contribute to the resistance of GBM to endogenous or therapeutically enhanced adaptive immune responses, we sorted CD4+ and CD8+ T cells from the peripheral blood, normal-appearing brain tissue, and tumor bed of nine treatment-naive patients with GBM. Bulk RNA sequencing of highly pure T cell populations from these different compartments was used to obtain deep transcriptomes of tumor-infiltrating T cells (TILs). While the transcriptome of CD8+ TILs suggested that they were partly locked in a dysfunctional state, CD4+ TILs showed a robust commitment to the type 17 T helper cell (TH17) lineage, which was corroborated by flow cytometry in four additional GBM cases. Therefore, our study illustrates that the brain tumor environment in GBM might instruct TH17 commitment of infiltrating T helper cells. Whether these properties of CD4+ TILs facilitate a tumor-promoting milieu and thus could be a target for adjuvant anti-TH17 cell interventions needs to be further investigated.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos , Citometria de Fluxo , Glioblastoma/patologia , Humanos , Linfócitos do Interstício Tumoral
11.
Elife ; 112022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35920626

RESUMO

Single-cell technologies (RNA-sequencing, flow cytometry) are critical tools to reveal how cell heterogeneity impacts developmental pathways. The placenta is a fetal exchange organ, containing a heterogeneous mix of mesenchymal cells (fibroblasts, myofibroblasts, perivascular, and progenitor cells). Placental mesenchymal stromal cells (pMSC) are also routinely isolated, for therapeutic and research purposes. However, our understanding of the diverse phenotypes of placental mesenchymal lineages, and their relationships remain unclear. We designed a 23-colour flow cytometry panel to assess mesenchymal heterogeneity in first-trimester human placentae. Four distinct mesenchymal subsets were identified; CD73+CD90+ mesenchymal cells, CD146+CD271+ perivascular cells, podoplanin+CD36+ stromal cells, and CD26+CD90+ myofibroblasts. CD73+CD90+ and podoplanin + CD36+ cells expressed markers consistent with cultured pMSCs, and were explored further. Despite their distinct ex-vivo phenotype, in culture CD73+CD90+ cells and podoplanin+CD36+ cells underwent phenotypic convergence, losing CD271 or CD36 expression respectively, and homogenously exhibiting a basic MSC phenotype (CD73+CD90+CD31-CD144-CD45-). However, some markers (CD26, CD146) were not impacted, or differentially impacted by culture in different populations. Comparisons of cultured phenotypes to pMSCs further suggested cultured pMSCs originate from podoplanin+CD36+ cells. This highlights the importance of detailed cell phenotyping to optimise therapeutic capacity, and ensure use of relevant cells in functional assays.


Assuntos
Dipeptidil Peptidase 4 , Células-Tronco Mesenquimais , Adapaleno/metabolismo , Biomarcadores/metabolismo , Antígeno CD146/genética , Antígeno CD146/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Dipeptidil Peptidase 4/metabolismo , Feminino , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , Fenótipo , Placenta/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Antígenos Thy-1/metabolismo
12.
J Vis Exp ; (185)2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35913198

RESUMO

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated ß-galactosidase (SA-ß-gal), the DNA synthesis marker 5-ethynyl-2'-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (γH2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.


Assuntos
Senescência Celular , Neoplasias , Biomarcadores , Senescência Celular/fisiologia , Dano ao DNA , Citometria de Fluxo , Humanos , Neoplasias/patologia , Microambiente Tumoral , beta-Galactosidase/genética
13.
Sci Rep ; 12(1): 11180, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35778474

RESUMO

To improve the understanding of the complex biological process underlying the development of non-alcoholic steatohepatitis (NASH), 3D imaging flow cytometry (3D-IFC) with transmission and side-scattered images were used to characterize hepatic stellate cell (HSC) and liver endothelial cell (LEC) morphology at single-cell resolution. In this study, HSC and LEC were obtained from biopsy-proven NASH subjects with early-stage NASH (F2-F3) and healthy controls. Here, we applied single-cell imaging and 3D digital reconstructions of healthy and diseased cells to analyze a spatially resolved set of morphometric cellular and texture parameters that showed regression with disease progression. By developing a customized autoencoder convolutional neural network (CNN) based on label-free cell transmission and side scattering images obtained from a 3D imaging flow cytometer, we demonstrated key regulated cell types involved in the development of NASH and cell classification performance superior to conventional machine learning methods.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Inteligência Artificial , Citometria de Fluxo , Humanos , Imageamento Tridimensional , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Prognóstico
14.
Biochem Med (Zagreb) ; 32(2): 020713, 2022 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35799989

RESUMO

Introduction: Point-of-care (POC) platelet function tests are faster and easier to perform than in-depth assessment by flow cytometry. At low platelet counts, however, POC tests are prone to assess platelet function incorrectly. Lower limits of platelet count required to obtain valid test results were defined and a testing method to facilitate comparability between different tests was established. Materials and methods: We assessed platelet function in whole blood samples of healthy volunteers at decreasing platelet counts (> 100, 80-100, 50-80, 30-50 and < 30 x109/L) using two POC tests: impedance aggregometry and in-vitro bleeding time. Flow cytometry served as the gold standard. The number of platelets needed to reach 50% of the maximum function (ED50) and the lower reference limit (EDref) were calculated to define limits of test validity. Results: The minimal platelet count required for reliable test results was 100 x109/L for impedance aggregometry and in-vitro bleeding time but only 30 x109/L for flow cytometry. Comparison of ED50 and EDref showed significantly lower values for flow cytometry than either POC test (P value < 0.05) but no difference between POC tests nor between the used platelet agonists within a test method. Conclusion: Calculating the ED50 and EDref provides an effective way to compare values from different platelet function assays. Flow cytometry enables correct platelet function testing as long as platelet count is > 30 x109/L whereas impedance aggregometry and in-vitro bleeding time are inconsistent unless platelet count is > 100 x109/L.


Assuntos
Agregação Plaquetária , Trombocitopenia , Plaquetas , Citometria de Fluxo , Humanos , Contagem de Plaquetas , Testes de Função Plaquetária/métodos , Sistemas Automatizados de Assistência Junto ao Leito
15.
Biosensors (Basel) ; 12(7)2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35884246

RESUMO

This article first reviews scientific meanings of single-cell analysis by highlighting two key scientific problems: landscape reconstruction of cellular identities during dynamic immune processes and mechanisms of tumor origin and evolution. Secondly, the article reviews clinical demands of single-cell analysis, which are complete blood counting enabled by optoelectronic flow cytometry and diagnosis of hematologic malignancies enabled by multicolor fluorescent flow cytometry. Then, this article focuses on the developments of optoelectronic flow cytometry for the complete blood counting by comparing conventional counterparts of hematology analyzers (e.g., DxH 900 of Beckman Coulter, XN-1000 of Sysmex, ADVIA 2120i of Siemens, and CELL-DYN Ruby of Abbott) and microfluidic counterparts (e.g., microfluidic impedance and imaging flow cytometry). Future directions of optoelectronic flow cytometry are indicated where intrinsic rather than dependent biophysical parameters of blood cells must be measured, and they can replace blood smears as the gold standard of blood analysis in the near future.


Assuntos
Testes Hematológicos , Microfluídica , Contagem de Células Sanguíneas , Citometria de Fluxo , Testes Hematológicos/métodos , Análise de Célula Única
16.
Methods Mol Biol ; 2537: 51-62, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35895258

RESUMO

Alternative splicing of pre-mRNAs expands the coding abilities of genomes by generating distinct transcription variants from individual genes. It contributes to the marvelous complexity of the transcriptome in neurons. Given the differential expression of alternative splicing regulators and diversity in alternative splicing programs in neuronal subpopulations, it is urgent and necessary to develop methods to efficiently isolate diverse subgroups of neurons and analyze their transcriptomic diversity. Here, we describe a protocol to isolate RNA from specific neuronal types using a fluorescence-activated cell sorting (FACS)-based method to analyze alternative splicing events in a cell type-specific manner. The method is universally applicable to analyze alternative splicing in fluorescent protein-labeled neuronal types. It was optimized to preserve the transcription state and improve efficiency in cell suspension purification. With our protocol, fluorescent protein-labeled neurons could be efficiently purified. The transcription states suitable for gene expression and alternative splicing analysis could be well-preserved.


Assuntos
Processamento Alternativo , Neurônios , Citometria de Fluxo , Neurônios/metabolismo , RNA/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA
17.
Int J Mol Sci ; 23(14)2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35886877

RESUMO

Multiple myeloma results from the clonal proliferation of abnormal plasma cells (PCs) in the bone marrow (BM). In this study, the cell surface expression markers (CD) on atypical PCs (detected by multiparametric flow cytometry (MFC)) were correlated with copy number alterations (CNAs) in the genome (detected by multiplex ligation-dependent probe amplification (MLPA)) to assess their impact on prognosis in newly diagnosed MM patients. Statistically significant results were obtained when different stages of PC maturation (classified based on CD19 and CD81 expression) were associated with CD117 expression and identified CNAs. In the intermediately differentiated PC group (CD19(-) CD81(+)), patients who didn't express CD117 had a lower median progression free survival (PFS) (p = 0.024). Moreover, within this group, patients with less than three adverse CNAs, which harbor CD117, had a better outcome with a PFS of more than 48 months compared with 19 months (p = 0.008). Considering all the results, our study suggested the need to integrate both the CD markers and copy number alterations to evaluate the prognosis of newly diagnosed multiple myeloma patients.


Assuntos
Mieloma Múltiplo , Medula Óssea/metabolismo , Variações do Número de Cópias de DNA/genética , Citometria de Fluxo , Humanos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Prognóstico
18.
Int J Mol Sci ; 23(14)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35887287

RESUMO

Trophoblast cell-surface antigen 2 (Trop-2) is a tumor-associated antigen that is connected with the development of various tumors and has been identified as a promising target for tumor immunotherapy. To date, the immunotherapy against Trop-2 mainly relies on the specific targeting by monoclonal antibody in antibody-drug conjugate (ADC). Alternatively, the single domain antibodies of nanobodies (Nbs) possesses unique properties such as smaller size, better tissue penetration, etc., to make them good candidates for tumor targeting. Thus, it was proposed to develop anti-Trop-2 Nbs for tumor targeting in this study. Generally, three consecutive rounds of bio-panning were performed against immobilized recombinant Trop-2, and yielded three Nbs (Nb60, Nb65, and Nb108). The affinity of selected Nbs was determined in the nanomolar range, especially the good properties of Nb60 were verified as a promising candidate for tumor labeling. The binding to native Trop-2 was confirmed by flow cytometry against tumor cells. The inhibitory effects of the selected Nbs on tumor cell proliferation and migration were confirmed by wound healing and Transwell assay. The clear localization of the selected Nbs on the surface of tumor cells verified the potent labeling efficiency. In conclusion, this study provided several Nbs with the potential to be developed as targeting moiety of drug conjugates.


Assuntos
Imunoconjugados , Neoplasias , Anticorpos de Domínio Único , Anticorpos Monoclonais , Citometria de Fluxo , Humanos , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico
19.
J Biomol Tech ; 33(1)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35837003

RESUMO

Cells isolated using electrostatic cell sorters are subsequently evaluated in a variety of in vitro and in vivo applications. Thus, manipulations to the cells during the pre- and post-sort processing as well as when the cells are being analyzed by and passing through the sorter fluidics has the potential to affect the experimental results. There are many variables to consider when seeking to preserve cellular integrity and function during the cell-sorting process. A previous study by the Association of Biomolecular Resource Facilities Flow Cytometry Research Group (FCRG) investigated downstream effects on different cell types as a function of sorting variables such as pressure, nozzle size, and temperature. This multisite study revealed site-to-site variability based on differential gene expression when the same cell type and sort conditions were used. These results indicated the possibility that environmental factors such as the presence of contaminants in the sorter fluidics could exhibit effects on downstream molecular assays (ie, endotoxins or RNases). In the study described here, the FCRG sought to better understand how sorters are maintained and evaluated for contaminants such as bacteria, endotoxin, and RNases. In addition, the efficacy of an endotoxin decontamination method was evaluated. The results demonstrated that the majority of sorters in shared resource laboratories are free of RNase activity and bacteria; however, many are contaminated with endotoxin. The efficacy of a hydrogen peroxide cleaning procedure was tested and found to exhibit only a short-term effectiveness in eliminating endotoxin contamination.


Assuntos
Infertilidade , Laboratórios , Separação Celular/métodos , Endotoxinas/genética , Citometria de Fluxo/métodos , Humanos
20.
Sci Rep ; 12(1): 12166, 2022 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842449

RESUMO

Despite introduction of biological disease modifying anti-rheumatic drugs (DMARDs) for Rheumatoid arthritis (RA) treatment, therapeutic strategies do not always lead to disease control and remission. Hence, a more efficient patient stratification and monitoring biomarkers and tools are needed to enable a more personalized medicine. We used a whole blood based functional flow cytometry assay to characterize immune cells from RA patients (treated or not), healthy donors and psoriatic arthritis (PsA) patients according to their responses to LPS and/or anti-TNFα (infliximab, IFX). Activation marker expression was measured using a 10-color flow cytometry panel following a no-wash protocol. Naïve-to-treatment RA patients had a stronger inflammatory profile in comparison to healthy donors at basal level. Higher expression of activation markers (CD69 and/or CD11b) on NK, B cells and granulocytes and lower expression of the adhesion molecule CD62L were measured on monocytes, granulocytes and B cells. After LPS, naïve RA patients' cells were less capable of regulating CD69, CD11b, CD16 or CD62L showing impaired activation capabilities. Upon LPS and IFX co-incubation, hierarchical clustering analysis showed different profiles between cohorts. We believe that this whole blood-based approach should further be assessed for RA patient characterization as it provides new perspectives for stratification and/or monitoring.


Assuntos
Antirreumáticos , Artrite Psoriásica , Artrite Reumatoide , Antirreumáticos/uso terapêutico , Artrite Psoriásica/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Biomarcadores/metabolismo , Citometria de Fluxo , Humanos , Lipopolissacarídeos/farmacologia , Pesquisa Translacional Biomédica
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