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1.
J Biomed Opt ; 29(Suppl 2): S22702, 2025 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38434231

RESUMO

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.


Assuntos
Técnicas Histológicas , Microscopia , Animais , Citometria de Fluxo , Processamento de Imagem Assistida por Computador
2.
Commun Biol ; 7(1): 674, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824207

RESUMO

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm-2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.


Assuntos
Mecanotransdução Celular , Neoplasias da Próstata , Animais , Humanos , Camundongos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/imunologia , Masculino , Células Dendríticas/imunologia , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala/métodos , Estresse Mecânico , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Citometria de Fluxo/métodos , Canais Iônicos
3.
J Extracell Vesicles ; 13(6): e12463, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38868945

RESUMO

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.


Assuntos
Biomarcadores , Vesículas Extracelulares , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Biomarcadores/metabolismo , Citometria de Fluxo/métodos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/análise , Células Cultivadas , Antígenos CD/metabolismo
4.
PLoS One ; 19(6): e0304286, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38865329

RESUMO

BACKGROUND: A rapid screening test for urinary tract infections (UTIs) in children is needed to avoid unnecessary cultures and provide prompt reports to make appropriate clinical decisions. We have evaluated for the first time the performance of the Sysmex UF-5000 flow cytometer as a screening tool for UTIs in children. METHODS: This study included 4445 pediatric patients, with urinary sediment and urine culture data collected from January 2020 to September 2023. The Sysmex UF-5000 analyzer was utilized to measure urine white blood cell (WBC) and bacteria (BACT), with the findings being compared to the culture results. RESULTS: At ≥ 104 colony-forming unit (CFU)/mL, 513 samples were culture-positive (400 samples presented 104-105 CFU/mL, and 113 demonstrated ≥ 105 CFU/mL bacterial growth). Optimal indicators for positive cultures were BACT counts of 92.2/µL (AUC: 0.944) and WBC counts of 40.8/µL (AUC:0.863). False negative rate were 0.9% when using a 7.8 bacteria/µL cut-off and avoiding unnecessary cultures in 28.1%. The UF-5000 has a higher consistency rate for Gram-negative (GN) bacteria (90.3%) than Gram-positive (GP) bacteria (86.8%). For samples with 105 CFU/mL, UF-5000's Bacteria -Information flags showed superior concordance for samples with 104-105 CFU/mL bacteria. CONCLUSIONS: Screening pediatric urine cultures with the UF-5000 showed potential application value in identifying negative cultures and significant bacterial growth, although performance may vary depending on the study population. Furthermore, detecting Gram typing aids in guiding early clinical empirical medication, particularly for UTIs caused by GN bacteria.


Assuntos
Tomada de Decisão Clínica , Infecções Urinárias , Humanos , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia , Criança , Pré-Escolar , Feminino , Masculino , Lactente , Contagem de Leucócitos , Citometria de Fluxo/métodos , Adolescente , Urinálise/métodos , Urinálise/instrumentação , Programas de Rastreamento/métodos , Recém-Nascido
5.
Tuberk Toraks ; 72(2): 152-166, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38869208

RESUMO

Introduction: : Immune responses against Coronavirus (SARS-CoV-2) may be highly complex. It has been suggested that T-cell fatigue develops due to continuous stimulation of T-cells by SARS-CoV-2 in Coronavirus disease-2019 (COVID-19). It was aimed to assess peripheral lymphocyte subsets and T-cell exhaustion in various clinical courses of the disease in patients diagnosed with COVID-19. Materials and Methods: This study included 150 patients who were assigned into the "mild-to-moderate disease" group, or "severe disease" group based on their clinical and laboratory characteristics. Peripheral lymphocyte subsets and T-cell exhaustion markers [programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3)] were determined in the peripheral blood using flow cytometry. Result: Mean (±SD) age was 53.3 ± 14.5 years, and female to male ratio was 55/95. In the mild-to-moderate disease (MMD) group, 55 patients had pneumonia and 20 patients had COVID-19 without pneumonia. In the severe disease (SD) group, 43 patients had severe pneumoniae and 32 patients were in critical condition. Lymphocyte counts were less than 1.0 x 109/L in 69.3% of the patients in the SD group, and the difference between the MMD group and SD group was statistically significant (p= 0.001). Total T cells, CD4+ and CD8+ T-cell counts were significantly lower in the SD group vs. MMD group (p< 0.001, p< 0.001, p< 0.001, respectively). PD-1 expression by CD8+ and CD4 T+ cells was higher (p= 0.042, p= 0.029, respectively) and Tim-3 expression from CD4 T+ cells was lower (p= 0.000) in the SD group vs. MMD group. Serum IFN-γ levels were not statistically different in the MMD and SD groups (p= 0.2). Conclusions: T-cell counts may be significantly reduced along with an increased expression of the T-cell exhaustion marker PD-1 in severe COVID-19, but Tim-3 expression was not increased in our study patients.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/imunologia , COVID-19/sangue , COVID-19/complicações , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , SARS-CoV-2/imunologia , Idoso , Receptor Celular 2 do Vírus da Hepatite A/sangue , Índice de Gravidade de Doença , Receptor de Morte Celular Programada 1/sangue , Subpopulações de Linfócitos/imunologia , Contagem de Linfócitos , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo , Exaustão das Células T
7.
Invest Ophthalmol Vis Sci ; 65(6): 17, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38861275

RESUMO

Purpose: N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE. Methods: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, ß-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography. Results: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo. Conclusions: Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.


Assuntos
Homeostase , Camundongos Endogâmicos C57BL , Fatores de Transcrição Otx , Fator de Transcrição PAX6 , Proteínas de Ligação a RNA , Epitélio Pigmentado da Retina , Epitélio Pigmentado da Retina/metabolismo , Animais , Camundongos , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Homeostase/fisiologia , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Otx/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Fagocitose/fisiologia , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Tomografia de Coerência Óptica , Eletrorretinografia , Células Cultivadas
8.
Transl Psychiatry ; 14(1): 254, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38866753

RESUMO

Depression is a prevalent and incapacitating condition with a significant impact on global morbidity and mortality. Although the immune system's role in its pathogenesis is increasingly recognized, there is a lack of comprehensive understanding regarding the involvement of innate and adaptive immune cells. To address this gap, we conducted a multicenter case-control study involving 121 participants matched for sex and age. These participants had either an active (or current) major depressive episode (MDE) (39 cases) or a remitted MDE (40 cases), including individuals with major depressive disorder or bipolar disorder. We compared these 79 patients to 42 healthy controls (HC), analyzing their immunological profiles. In blood samples, we determined the complete cell count and the monocyte subtypes and lymphocyte T-cell populations using flow cytometry. Additionally, we measured a panel of cytokines, chemokines, and neurotrophic factors in the plasma. Compared with HC, people endorsing a current MDE showed monocytosis (p = 0.001), increased high-sensitivity C-reactive protein (p = 0.002), and erythrocyte sedimentation rate (p = 0.003), and an altered proportion of specific monocyte subsets. CD4 lymphocytes presented increased median percentages of activation markers CD69+ (p = 0.007) and exhaustion markers PD1+ (p = 0.013) and LAG3+ (p = 0.014), as well as a higher frequency of CD4+CD25+FOXP3+ regulatory T cells (p = 0.003). Additionally, patients showed increased plasma levels of sTREM2 (p = 0.0089). These changes are more likely state markers, indicating the presence of an ongoing inflammatory response during an active MDE. The Random Forest model achieved remarkable classification accuracies of 83.8% for MDE vs. HC and 70% for differentiating active and remitted MDE. Interestingly, the cluster analysis identified three distinct immunological profiles among MDE patients. Cluster 1 has the highest number of leukocytes, mainly given by the increment in lymphocyte count and the lowest proinflammatory cytokine levels. Cluster 3 displayed the most robust inflammatory pattern, with high levels of TNFα, CX3CL1, IL-12p70, IL-17A, IL-23, and IL-33, associated with the highest level of IL-10, as well as ß-NGF and the lowest level for BDNF. This profile is also associated with the highest absolute number and percentage of circulating monocytes and the lowest absolute number and percentage of circulating lymphocytes, denoting an active inflammatory process. Cluster 2 has some cardinal signs of more acute inflammation, such as elevated levels of CCL2 and increased levels of proinflammatory cytokines such as IL-1ß, IFNγ, and CXCL8. Similarly, the absolute number of monocytes is closer to a HC value, as well as the percentage of lymphocytes, suggesting a possible initiation of the inflammatory process. The study provides new insights into the immune system's role in MDE, paving the ground for replication prospective studies targeting the development of diagnostic and prognostic tools and new therapeutic targets.


Assuntos
Citocinas , Transtorno Depressivo Maior , Imunofenotipagem , Monócitos , Humanos , Feminino , Masculino , Estudos de Casos e Controles , Transtorno Depressivo Maior/imunologia , Transtorno Depressivo Maior/sangue , Adulto , Pessoa de Meia-Idade , Citocinas/sangue , Citocinas/imunologia , Monócitos/imunologia , Transtorno Bipolar/imunologia , Transtorno Bipolar/sangue , Inflamação/imunologia , Inflamação/sangue , Antígenos CD/sangue , Antígenos CD/imunologia , Citometria de Fluxo
9.
Platelets ; 35(1): 2358244, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38845541

RESUMO

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.


Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.


Assuntos
Plaquetas , Trombocitemia Essencial , Trombose , Humanos , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/complicações , Plaquetas/metabolismo , Masculino , Feminino , Trombose/metabolismo , Trombose/etiologia , Pessoa de Meia-Idade , Idoso , Citometria de Fluxo/métodos , Ativação Plaquetária , Estudos de Casos e Controles , Adulto
10.
Front Immunol ; 15: 1402724, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38835783

RESUMO

Background and objective: Acute ischemic stroke (AIS) is a leading cause of mortality, severe neurological and long-term disability world-wide. Blood-based indicators may provide valuable information on identified prognostic factors. However, currently, there is still a lack of peripheral blood indicators for the prognosis of AIS. We aimed to identify the most promising prognostic indicators and establish prognostic models for AIS. Methods: 484 subjects enrolled from four centers were analyzed immunophenotypic indicators of peripheral blood by flow cytometry. Least absolute shrinkage and selection operator (LASSO) regression was applied to minimize the potential collinearity and over-fitting of variables measured from the same subject and over-fitting of variables. Univariate and multivariable Cox survival analysis of differences between and within cohorts was performed by log-rank test. The areas under the receiving operating characteristic (ROC) curves were used to evaluate the selection accuracy of immunophenotypic indicators in identifying AIS subjects with survival risk. The prognostic model was constructed using a multivariate Cox model, consisting of 402 subjects as a training cohort and 82 subjects as a testing cohort. Results: In the prospective study, 7 immunophenotypic indicators of distinct significance were screened out of 72 peripheral blood immunophenotypic indicators by LASSO. In multivariate cox regression, CTL (%) [HR: 1.18, 95% CI: 1.03-1.33], monocytes/µl [HR: 1.13, 95% CI: 1.05-1.21], non-classical monocytes/µl [HR: 1.09, 95% CI: 1.02-1.16] and CD56high NK cells/µl [HR: 1.13, 95% CI: 1.05-1.21] were detected to decrease the survival probability of AIS, while Tregs/µl [HR:0.97, 95% CI: 0.95-0.99, p=0.004], BM/µl [HR:0.90, 95% CI: 0.85-0.95, p=0.023] and CD16+NK cells/µl [HR:0.93, 95% CI: 0.88-0.98, p=0.034] may have the protective effect. As for indicators' discriminative ability, the AUC for CD56highNK cells/µl attained the highest of 0.912. In stratification analysis, the survival probability for AIS subjects with a higher level of Tregs/µl, BM/µl, CD16+NK cells/µl, or lower levels of CD56highNK cells/µl, CTL (%), non-classical monocytes/µl, Monocytes/µl were more likely to survive after AIS. The multivariate Cox model showed an area under the curve (AUC) of 0.805, 0.781 and 0.819 and 0.961, 0.924 and 0.982 in the training and testing cohort, respectively. Conclusion: Our study identified 7 immunophenotypic indicators in peripheral blood may have great clinical significance in monitoring the prognosis of AIS and provide a convenient and valuable predictive model for AIS.


Assuntos
Citometria de Fluxo , Imunofenotipagem , AVC Isquêmico , Humanos , Feminino , Masculino , AVC Isquêmico/sangue , AVC Isquêmico/mortalidade , AVC Isquêmico/diagnóstico , AVC Isquêmico/imunologia , Citometria de Fluxo/métodos , Prognóstico , Idoso , Pessoa de Meia-Idade , Estudos Prospectivos , Biomarcadores/sangue , Idoso de 80 Anos ou mais
11.
BMC Infect Dis ; 24(1): 567, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38844850

RESUMO

This study investigates the longitudinal dynamic changes in immune cells in COVID-19 patients over an extended period after recovery, as well as the interplay between immune cells and antibodies. Leveraging single-cell mass spectrometry, we selected six COVID-19 patients and four healthy controls, dissecting the evolving landscape within six months post-viral RNA clearance, alongside the levels of anti-spike protein antibodies. The T cell immunophenotype ascertained via single-cell mass spectrometry underwent validation through flow cytometry in 37 samples. Our findings illuminate that CD8 + T cells, gamma-delta (gd) T cells, and NK cells witnessed an increase, in contrast to the reduction observed in monocytes, B cells, and double-negative T (DNT) cells over time. The proportion of monocytes remained significantly elevated in COVID-19 patients compared to controls even after six-month. Subpopulation-wise, an upsurge manifested within various T effector memory subsets, CD45RA + T effector memory, gdT, and NK cells, whereas declines marked the populations of DNT, naive and memory B cells, and classical as well as non-classical monocytes. Noteworthy associations surfaced between DNT, gdT, CD4 + T, NK cells, and the anti-S antibody titer. This study reveals the changes in peripheral blood mononuclear cells of COVID-19 patients within 6 months after viral RNA clearance and sheds light on the interactions between immune cells and antibodies. The findings from this research contribute to a better understanding of immune transformations during the recovery from COVID-19 and offer guidance for protective measures against reinfection in the context of viral variants.


Assuntos
COVID-19 , Citometria de Fluxo , Leucócitos Mononucleares , RNA Viral , SARS-CoV-2 , Humanos , COVID-19/imunologia , COVID-19/sangue , COVID-19/virologia , Leucócitos Mononucleares/virologia , Leucócitos Mononucleares/imunologia , SARS-CoV-2/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , RNA Viral/sangue , Adulto , Estudos Longitudinais , Análise de Célula Única/métodos , Células Matadoras Naturais/imunologia , Anticorpos Antivirais/sangue , Imunofenotipagem , Idoso
12.
Vet Res ; 55(1): 69, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38822400

RESUMO

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.


Assuntos
Linfócitos T CD4-Positivos , Doenças dos Bovinos , Citometria de Fluxo , Interferon gama , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Bovinos , Paratuberculose/imunologia , Paratuberculose/diagnóstico , Paratuberculose/microbiologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium avium subsp. paratuberculosis/fisiologia , Interferon gama/metabolismo , Citometria de Fluxo/veterinária , Citometria de Fluxo/métodos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Linfócitos T CD4-Positivos/imunologia , Biomarcadores
13.
PLoS One ; 19(6): e0304789, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38829858

RESUMO

Malaria is a deadly disease that is transmitted through mosquito bites. Microscopists use a microscope to examine thin blood smears at high magnification (1000x) to identify parasites in red blood cells (RBCs). Estimating parasitemia is essential in determining the severity of the Plasmodium falciparum infection and guiding treatment. However, this process is time-consuming, labor-intensive, and subject to variation, which can directly affect patient outcomes. In this retrospective study, we compared three methods for measuring parasitemia from a collection of anonymized thin blood smears of patients with Plasmodium falciparum obtained from the Clinical Department of Parasitology-Mycology, National Reference Center (NRC) for Malaria in Paris, France. We first analyzed the impact of the number of field images on parasitemia count using our framework, MALARIS, which features a top-classifier convolutional neural network (CNN). Additionally, we studied the variation between different microscopists using two manual techniques to demonstrate the need for a reliable and reproducible automated system. Finally, we included thin blood smear images from an additional 102 patients to compare the performance and correlation of our system with manual microscopy and flow cytometry. Our results showed strong correlations between the three methods, with a coefficient of determination between 0.87 and 0.92.


Assuntos
Malária Falciparum , Microscopia , Parasitemia , Plasmodium falciparum , Humanos , Plasmodium falciparum/isolamento & purificação , Parasitemia/diagnóstico , Parasitemia/sangue , Parasitemia/parasitologia , Malária Falciparum/diagnóstico , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Estudos Retrospectivos , Microscopia/métodos , Eritrócitos/parasitologia , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Citometria de Fluxo/métodos
14.
Rev Sci Instrum ; 95(6)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38829217

RESUMO

Blood testing using flow cytometry is a common and rapid method for the initial screening and diagnosis of patients. Measurements are often combined with other scientific techniques, and analyzed samples are commonly diluted and discarded afterward. When the sample is recollected instead, sample dilution is a challenge when the sample is intended or needed for additional measurements. Therefore, it is advantageous to recollect the undiluted sample. In order to enable measurements of the same undiluted sample aliquot, we designed and constructed a purpose-built flow cytometer. Our instrument employs syringes, acoustic focusing, and an open fluidics system to recollect and reuse the unadulterated sample. The cytometer is compact, which reduces sample consumption. It acquires forward, sideward, and fluorescence signals, offering opportunities for diverse measurement approaches. In particular, our cytometer has been designed to be ready for additional downstream analysis of cells, e.g., applying mass spectrometry, magnetic resonance spectroscopy, or other analytical tools. This study presents results on instrument performance, a comparison with a cytometer that uses standard hydrodynamic focusing, and a proof of concept for multiple measurements.


Assuntos
Citometria de Fluxo , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Humanos , Desenho de Equipamento
15.
Gan To Kagaku Ryoho ; 51(5): 579-581, 2024 May.
Artigo em Japonês | MEDLINE | ID: mdl-38881073

RESUMO

A 72-year-old male was referred with a 2-week history of diplopia. Following magnetic resonance imaging, an area of abnormal signal intensity was observed along the lateral ventricle, without any unusual findings at other sites. Cerebrospinal fluid cytology revealed abnormal lymphocytes with atypia, which were positive for CD20 and light-chain restriction, as detected by surface marker analysis, leading to a diagnosis of primary meningeal B-cell lymphoma. The patient underwent chemoradiotherapy and achieved a remission. While meningeal lymphoma is a rare occurrence, pathological tissue biopsy is considered the gold-standard diagnostic method. However, obtaining a biopsy sample from the tumor site can be challenging. In this case report, cytology and flow cytometry played a vital role in the diagnosis of meningeal lymphoma.


Assuntos
Citometria de Fluxo , Neoplasias Meníngeas , Humanos , Masculino , Idoso , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/patologia , Linfoma de Células B/diagnóstico , Linfoma de Células B/patologia , Linfoma de Células B/diagnóstico por imagem , Quimiorradioterapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imageamento por Ressonância Magnética , Citologia
16.
J Vis Exp ; (207)2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38884484

RESUMO

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.


Assuntos
Galinhas , Citometria de Fluxo , Vírus da Doença Infecciosa da Bursa , Piroptose , Citometria de Fluxo/métodos , Animais , Vírus da Doença Infecciosa da Bursa/imunologia , Linhagem Celular
17.
J Vis Exp ; (207)2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38884489

RESUMO

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry. In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.


Assuntos
Citometria de Fluxo , Microglia , Sinapses , Animais , Microglia/citologia , Microglia/metabolismo , Camundongos , Sinapses/fisiologia , Sinapses/química , Citometria de Fluxo/métodos , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/análise , Sinaptossomos/metabolismo , Encéfalo/citologia
18.
Sci Rep ; 14(1): 13344, 2024 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-38858477

RESUMO

Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.


Assuntos
Anticorpos Monoclonais , Citocinas , Citometria de Fluxo , Animais , Citometria de Fluxo/métodos , Camundongos , Humanos , Anticorpos Monoclonais/imunologia , Citocinas/metabolismo , Camundongos Knockout , Linhagem Celular , Células Th1/imunologia , Células Th1/metabolismo
19.
Cells ; 13(11)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38891094

RESUMO

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.


Assuntos
Infecções por Vírus Epstein-Barr , Citometria de Fluxo , Leucócitos , Humanos , Citometria de Fluxo/métodos , Masculino , Feminino , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/virologia , Criança , Leucócitos/imunologia , Herpesvirus Humano 4/imunologia , Adolescente , Adulto , Mononucleose Infecciosa/imunologia , Mononucleose Infecciosa/sangue , Mononucleose Infecciosa/virologia , Monócitos/imunologia , Monócitos/virologia , Monócitos/metabolismo , Pré-Escolar , Neutrófilos/imunologia , Doença Aguda , Estudos Retrospectivos , Adulto Jovem
20.
Bioanalysis ; 16(8): 179-220, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38899739

RESUMO

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.


Assuntos
Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Vacinas , Humanos , Biomarcadores/análise , Vacinas/imunologia , Citometria de Fluxo , Bioensaio/métodos , União Europeia , Brancos
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