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1.
Front Immunol ; 12: 655122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408743

RESUMO

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.


Assuntos
Edição de Genes/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Receptores de Interleucina-6/genética , Linfócitos T Reguladores/metabolismo , Buffy Coat/citologia , Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Voluntários Saudáveis , Humanos , Imunoterapia Adotiva/métodos , Cultura Primária de Células , RNA Guia/genética , Fatores de Tempo
2.
Transfusion ; 61(2): 546-556, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33345368

RESUMO

BACKGROUND: Cryopreserved platelets show a reduced recovery and viability after freezing and thawing including several ultrastructural and phenotypic deteriorations compared with liquid-stored platelets. It is suggested that using Controlled-Rate Freezing (CRF) can reduce variability and optimize the functionality profile for cells. The objective of the study is to compare cellular, metabolic, phenotypic and functional effects on platelets after cryopreservation using different freezing rate protocols. STUDY DESIGN AND METHODS: To evaluate the possible effects of different freezing rate protocols a two-experimental study comparing diverse combinations was tested with a pool and split design. Uncontrolled freezing of platelets in materials with different thermal conductivity (metal vs cardboard) was evaluated in experiment 1. Experiment 2 evaluated uncontrolled vs a controlled-rate freezing protocol in metal boxes. All variables were assessed pre and post cryopreservation. RESULTS: Directly after thawing, no major differences in platelet recovery, LDH, ATP, Δψ, CD62P, CD42b, platelet endothelial cell adhesion molecule and sCD40L were seen between units frozen with different thermal conductivity for temperature. In contrast, we observed signs of increased activation after freezing using the CRF protocol, reflected by increased cell surface expression of CD62P, PAC-1 binding and increased concentration of LDH. Agonist induced expression of a conformational epitope on the GPIIb/IIIa complex and contribution to blood coagulation in an experimental rotational thromboelastometry setup were not statistically different between the groups. CONCLUSION: The use of a uncontrolled freezing rate protocol is feasible, creating a platelet product comparable to using a controlled rate freezing equipment during cryopreservation of platelets.


Assuntos
Buffy Coat/citologia , Plaquetas , Preservação de Sangue/métodos , Criopreservação/métodos , Difosfato de Adenosina/farmacologia , Coagulação Sanguínea , Plaquetas/química , Plaquetas/citologia , Plaquetas/fisiologia , Ligante de CD40/farmacologia , Separação Celular , Sobrevivência Celular , Centrifugação , Colágeno/farmacologia , Criopreservação/instrumentação , Dimetil Sulfóxido , Humanos , Fatores Imunológicos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Refrigeração/instrumentação , Condutividade Térmica , Tromboelastografia
3.
PLoS Genet ; 16(11): e1009090, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33147208

RESUMO

Interferon ß (IFN-ß) is a cytokine that induces a global antiviral proteome, and regulates the adaptive immune response to infections and tumors. Its effects strongly depend on its level and timing of expression. Therefore, the transcription of its coding gene IFNB1 is strictly controlled. We have previously shown that in mice, the TRIM33 protein restrains Ifnb1 transcription in activated myeloid cells through an upstream inhibitory sequence called ICE. Here, we show that the deregulation of Ifnb1 expression observed in murine Trim33-/- macrophages correlates with abnormal looping of both ICE and the Ifnb1 gene to a 100 kb downstream region overlapping the Ptplad2/Hacd4 gene. This region is a predicted myeloid super-enhancer in which we could characterize 3 myeloid-specific active enhancers, one of which (E5) increases the response of the Ifnb1 promoter to activation. In humans, the orthologous region contains several single nucleotide polymorphisms (SNPs) known to be associated with decreased expression of IFNB1 in activated monocytes, and loops to the IFNB1 gene. The strongest association is found for the rs12553564 SNP, located in the E5 orthologous region. The minor allele of rs12553564 disrupts a conserved C/EBP-ß binding motif, prevents binding of C/EBP-ß, and abolishes the activation-induced enhancer activity of E5. Altogether, these results establish a link between a genetic variant preventing binding of a transcription factor and a higher order phenotype, and suggest that the frequent minor allele (around 30% worldwide) might be associated with phenotypes regulated by IFN-ß expression in myeloid cells.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/imunologia , Interferon beta/genética , Células Mieloides/metabolismo , Alelos , Animais , Buffy Coat/citologia , Células Cultivadas , Humanos , Interferon beta/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Sci Rep ; 10(1): 20312, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219265

RESUMO

Diagnostic leukapheresis (DLA) enables to sample larger blood volumes and increases the detection of circulating tumor cells (CTC) significantly. Nevertheless, the high excess of white blood cells (WBC) of DLA products remains a major challenge for further downstream CTC enrichment and detection. To address this problem, we tested the performance of two label-free CTC technologies for processing DLA products. For the testing purposes, we established ficollized buffy coats (BC) with a WBC composition similar to patient-derived DLA products. The mimicking-DLA samples (with up to 400 × 106 WBCs) were spiked with three different tumor cell lines and processed with two versions of a spiral microfluidic chip for label-free CTC enrichment: the commercially available ClearCell FR1 biochip and a customized DLA biochip based on a similar enrichment principle, but designed for higher throughput of cells. While the samples processed with FR1 chip displayed with increasing cell load significantly higher WBC backgrounds and decreasing cell recovery, the recovery rates of the customized DLA chip were stable, even if challenged with up to 400 × 106 WBCs (corresponding to around 120 mL peripheral blood or 10% of a DLA product). These results indicate that the further up-scalable DLA biochip has potential to process complete DLA products from 2.5 L of peripheral blood in an affordable way to enable high-volume CTC-based liquid biopsies.


Assuntos
Dispositivos Lab-On-A-Chip , Leucaférese/instrumentação , Neoplasias/diagnóstico , Células Neoplásicas Circulantes , Buffy Coat/citologia , Linhagem Celular Tumoral , Humanos , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Neoplasias/sangue
5.
Nat Commun ; 11(1): 4498, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908142

RESUMO

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy.


Assuntos
Macrófagos/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Idoso , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Anilidas/farmacologia , Anilidas/uso terapêutico , Biópsia , Buffy Coat/citologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Quimioterapia Adjuvante , Técnicas de Cocultura , Intervalo Livre de Doença , Humanos , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/prevenção & controle , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Intervalo Livre de Progressão , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/terapia , Procedimentos Cirúrgicos Robóticos , Transdução de Sinais/imunologia , Análise de Célula Única , Células THP-1 , Compostos de Tosil/farmacologia , Compostos de Tosil/uso terapêutico
6.
PLoS One ; 15(8): e0237795, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833989

RESUMO

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.


Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Buffy Coat/citologia , Diferenciação Celular , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Técnicas de Cocultura , Voluntários Saudáveis , Humanos , Microscopia Intravital , Macrófagos/citologia , Macrófagos/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Monócitos/fisiologia , Cultura Primária de Células
7.
Methods Mol Biol ; 2163: 57-62, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32766965

RESUMO

Cultured human mast cells are a useful tool for research into innate immune responses as well as allergic mechanisms. Mast cells cultured from peripheral blood can provide information on immune mechanisms of known, selected individuals. With the method presented here, eight million mast cells can be cultured from ca. one million stem cells purified from one unit (450 mL) of human peripheral blood. Culture with IgE and IL4 optimizes an allergic phenotype of the mast cells.


Assuntos
Hipersensibilidade/imunologia , Mastócitos/citologia , Células-Tronco de Sangue Periférico/citologia , Fenótipo , Cultura Primária de Células/métodos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Buffy Coat/citologia , Células Cultivadas , Meios de Cultura/química , Humanos , Hipersensibilidade/sangue , Imunidade Inata , Imunoglobulina E/imunologia , Imunoglobulina E/farmacologia , Interleucina-4/imunologia , Interleucina-4/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Células-Tronco de Sangue Periférico/efeitos dos fármacos , Células-Tronco de Sangue Periférico/imunologia
8.
PLoS One ; 15(7): e0234150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614830

RESUMO

To investigate a Florida manatee (Trichechus manatus latirostris) mortality event following a red tide bloom in Southwest Florida, an RNA sequencing experiment was conducted. Gene expression changes in white blood cells were assessed in manatees rescued from a red tide affected area (n = 4) and a control group (n = 7) using RNA sequencing. The genes with the largest fold changes were compared between the two groups to identify molecular pathways related to cellular and disease processes. In total, 591 genes (false discovery rate <0.05) were differentially expressed in the red tide group. Of these, 158 were upregulated and 433 were downregulated. This suggests major changes in white blood cell composition following an exposure to red tide. The most highly upregulated gene, Osteoclast associated 2C immunoglobulin-like receptor (OSCAR), was upregulated 12-fold. This gene is involved in initiating the immune response and maintaining a role in adaptive and innate immunity. The most highly downregulated gene, Piccolo presynaptic cytomatrix protein (PCLO), was downregulated by a factor of 977-fold. This gene is associated with cognitive functioning and neurotransmitter release. Downregulation of this gene in other studies was associated with neuronal loss and neuron synapse dysfunction. Among the cellular pathways that were most affected, immune response, including inflammation, wounds and injuries, cell proliferation, and apoptosis were the most predominant. The pathway with the most differentially expressed genes was the immune response pathway with 98 genes involved, many of them downregulated. Assessing the changes in gene expression associated with red tide exposure enhances our understanding of manatee immune response to the red tide toxins and will aid in the development of red tide biomarkers.


Assuntos
Perfilação da Expressão Gênica , Proliferação Nociva de Algas , Trichechus manatus/fisiologia , Animais , Buffy Coat/citologia , Florida , Ontologia Genética , Sistema Imunitário , Leucócitos/metabolismo , Toxinas Marinhas/envenenamento , Redes e Vias Metabólicas/genética , Neurotoxinas/envenenamento , Oxocinas/envenenamento , Envenenamento/sangue , Envenenamento/reabilitação , Envenenamento/veterinária , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Transcriptoma , Trichechus manatus/sangue , Trichechus manatus/genética , Trichechus manatus/imunologia
9.
BMC Cancer ; 20(1): 566, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32552799

RESUMO

BACKGROUND: Only 10-30% of oesophageal and rectal adenocarcinoma patients treated with neoadjuvant chemoradiotherapy have a complete pathological response. Inflammatory and angiogenic mediators in the tumour microenvironment (TME) may enable evasion of anti-tumour immune responses. METHODS: The TME influence on infiltrating dendritic cells (DCs) was modelled by treating immature monocyte-derived DCs with Tumour Conditioned Media (TCM) from distinct gastrointestinal sites, prior to LPS-induced maturation. RESULTS: Cell line conditioned media from gastrointestinal cell lines inhibited LPS-induced DC markers and TNF-α secretion. TCM generated from human tumour biopsies from oesophageal, rectal and colonic adenocarcinoma induced different effects on LPS-induced DC markers - CD54, CD80, HLA-DR, CD86 and CD83 were enhanced by oesophageal cancer; CD80, CD86 and CD83 were enhanced by rectal cancer, whereas CD54, HLA-DR, CD86, CD83 and PD-L1 were inhibited by colonic cancer. Notably, TCM from all GI cancer types inhibited TNF-α secretion. Additionally, TCM from irradiated biopsies inhibited DC markers. Profiling the TCM showed that IL-2 levels positively correlated with maturation marker CD54, while Ang-2 and bFGF levels negatively correlated with CD54. CONCLUSION: This study identifies that there are differences in DC maturational capacity induced by the TME of distinct gastrointestinal cancers. This could potentially have implications for anti-tumour immunity and response to radiotherapy.


Assuntos
Neoplasias do Colo/imunologia , Células Dendríticas/imunologia , Neoplasias Esofágicas/imunologia , Neoplasias Retais/imunologia , Microambiente Tumoral/imunologia , Biópsia , Buffy Coat/citologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Meios de Cultivo Condicionados/metabolismo , Células Dendríticas/metabolismo , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Humanos , Lipopolissacarídeos/imunologia , Terapia Neoadjuvante/métodos , Cultura Primária de Células , Neoplasias Retais/patologia , Neoplasias Retais/terapia , Evasão Tumoral
10.
Sci Rep ; 10(1): 6234, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32277133

RESUMO

The protozoan Giardia lamblia is the most common cause of parasitic gastrointestinal infection worldwide. The parasite developed sophisticated, yet not completely disclosed, mechanisms to escape immune system and growth in the intestine. To further understand the interaction of G. lamblia with host immune cells, we investigated the ability of parasites to modulate the canonical activation of mouse macrophages (Raw 264.7 cell line) and human monocyte-derived macrophages triggered by the TLR4 agonist, lipopolysaccharide (LPS). We observed that G. lamblia impairs LPS-evoked pro-inflammatory status in these macrophage-like cells through inhibition of cyclooxygenase-2 and inducible nitric oxide synthase expression and subsequent NO production. This effect was in part due to the activity of three G. lamblia proteases, a 135 kDa metalloprotease and two cysteine proteases with 75 and 63 kDa, that cleave the p65RelA subunit of the nuclear factor-kappa B (NF-κB). Moreover, Tnf and Ccl4 transcription was increased in the presence of the parasite. Overall, our data indicates that G. lamblia modulates macrophages inflammatory response through impairment of the NF-κB, thus silencing a crucial signaling pathway of the host innate immune response.


Assuntos
Giardia lamblia/imunologia , Giardíase/imunologia , Interações Hospedeiro-Parasita/imunologia , Macrófagos/imunologia , Fator de Transcrição RelA/metabolismo , Animais , Buffy Coat/citologia , Giardíase/parasitologia , Voluntários Saudáveis , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Peptídeo Hidrolases/metabolismo , Cultura Primária de Células , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Células RAW 264.7 , Fator de Transcrição RelA/análise
11.
Proc Natl Acad Sci U S A ; 117(16): 9042-9053, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32241891

RESUMO

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Interferon Tipo I/genética , Proteínas de Membrana Transportadoras/metabolismo , RNA não Traduzido/metabolismo , Infecções Respiratórias/imunologia , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adulto , Idoso , Buffy Coat/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/sangue , Interferon Tipo I/imunologia , Macrófagos , Masculino , Pessoa de Meia-Idade , Fosforilação/genética , Cultura Primária de Células , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/metabolismo , RNA não Traduzido/sangue , RNA não Traduzido/genética , RNA-Seq , Infecções Respiratórias/sangue , Infecções Respiratórias/microbiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Adulto Jovem
12.
Sci Rep ; 10(1): 6488, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32300208

RESUMO

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn's disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4+ Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4+ Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6+ Th17- were compared to CCR6neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (ß-galactosidase activity, p16Ink4a- and p21 expression) and cytokines production. CCR6+ Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6+Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6+Th17 cells, therefore potential targets for therapeutic purposes.


Assuntos
Senescência Celular/imunologia , Fumar Cigarros/imunologia , Células Th17/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Buffy Coat/citologia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Fumar Cigarros/efeitos adversos , Fumar Cigarros/sangue , Citocinas/metabolismo , Voluntários Saudáveis , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Receptores CCR6/metabolismo , Fumaça/efeitos adversos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(14): 8055-8063, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193343

RESUMO

HIV-1 particles incorporate various host transmembrane proteins in addition to viral Env glycoprotein during assembly at the plasma membrane. In polarized T cells, HIV-1 structural protein Gag localizes to the plasma membrane of uropod, a rear-end protrusion. Notably, uropod transmembrane proteins PSGL-1 and CD43 cocluster specifically with Gag assembling at the plasma membrane even in cells that do not form uropods. Recent reports have shown that expression of either PSGL-1 or CD43 in virus-producing cells reduces the infectivity of progeny virions and that HIV-1 infection reduces the cell surface expression of these proteins. However, the mechanisms for both processes remain to be determined. In this study, we found that virion incorporation of PSGL-1 and CD43 closely correlates with diminished virion infectivity. PSGL-1 and CD43 inhibited virus attachment to CD4+ cells irrespective of the presence of Env. These proteins also inhibited virion attachment to CD4- lymphoid organ fibroblastic reticular cells that mediate transinfection of CD4+ T cells. Consistent with the possibility that highly extended extracellular domains of these proteins physically block virus-cell attachment, the inhibitory effect of PSGL-1 required its full-length ectodomain. HIV-1 encoding Gag mutants that are defective in either coclustering with these host proteins or ESCRT-dependent particle release failed to reduce PSGL-1 on surface of infected cells. This study reveals an anti-HIV-1 mechanism that suppresses virus-cell attachment and a previously unappreciated process of HIV-1-mediated down-regulation of host antiviral proteins, both of which likely require virion incorporation of these proteins.


Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Interações Hospedeiro-Patógeno/genética , Leucossialina/genética , Glicoproteínas de Membrana/genética , Vírion/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Buffy Coat/citologia , Regulação para Baixo , Técnicas de Inativação de Genes , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Voluntários Saudáveis , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mutação , Domínios Proteicos/genética , Linfócitos T/imunologia , Montagem de Vírus/genética , Montagem de Vírus/imunologia , Ligação Viral , Replicação Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
14.
J Immunotoxicol ; 17(1): 10-20, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31909636

RESUMO

Mucosal-associated invariant T-cells (MAIT) can react to metabolites of the vitamins riboflavin and folate which are produced by the human gut microbiota. Since several studies showed that the pesticide chlorpyrifos (CPF) and glyphosate (GLP) can impair the gut microbiota, the present study was undertaken to investigate the impact of CPF and GLP treatment on the metabolism of gut microbiota and the resulting bacteria-mediated modulation of MAIT cell activity. Here, Bifidobacterium adolescentis (B. adolescentis), Lactobacillus reuteri (L. reuteri), and Escherichia coli (E. coli) were treated with CPF (50-200 µM) or GLP (75-300 mg/L) and then used in MAIT cell stimulation assays as well as in vitamin and proteome analyses. All three bacteria were nonpathogenic and chosen as representatives of a healthy human gut microflora. The results showed that E. coli activated MAIT cells whereas B. adolescentis and L. reuteri inhibited MAIT cell activation. CPF treatment significantly increased E. coli-mediated MAIT cell activation. Treatment of B. adolescentis and L. reuteri with CPF and GLP weakened the inhibition of MAIT cell activation. Riboflavin and folate production by the test bacteria was influenced by CPF treatment, whereas GLP had only minor effects. Proteomic analysis of CPF-treated E. coli revealed changes in the riboflavin and folate biosynthesis pathways. The findings here suggest that the metabolism of the analyzed bacteria could be altered by exposure to CPF and GLP, leading to an increased pro-inflammatory immune response.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Herbicidas/toxicidade , Inseticidas/toxicidade , Ativação Linfocitária/efeitos dos fármacos , Células T Invariantes Associadas à Mucosa/imunologia , Bifidobacterium adolescentis/efeitos dos fármacos , Bifidobacterium adolescentis/imunologia , Bifidobacterium adolescentis/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/imunologia , Buffy Coat/citologia , Clorpirifos/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Escherichia coli/metabolismo , Ácido Fólico/análise , Ácido Fólico/biossíntese , Microbioma Gastrointestinal/imunologia , Glicina/análogos & derivados , Glicina/toxicidade , Voluntários Saudáveis , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lactobacillus reuteri/efeitos dos fármacos , Lactobacillus reuteri/imunologia , Lactobacillus reuteri/metabolismo , Ativação Linfocitária/imunologia , Proteômica , Riboflavina/análise , Riboflavina/biossíntese
15.
Transfus Clin Biol ; 27(1): 10-17, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31812494

RESUMO

OBJECTIVE: The objective of this study was to compare the activity and biological function of leukocytes isolated using apheresis platelet leukoreduction system chambers (LRSC), whole blood leukoreduction filters (LRF), and leukocytes in unfiltered peripheral whole blood (WB). METHODS: Peripheral blood mononuclear cells (PBMCs) and granulocytes were obtained by density gradient centrifugation using recovery filters and WB. Flow cytometry was used to detect the activity, phenotype, and apoptosis ratio of each cell subtype. RESULTS: The proportion of lymphocytes obtained from PBMCs was similar when using the two different filters as compared to traditional isolation; however, there were significant differences between the monocytes and granulocytes. The phenotypic frequency of lymphocytes was similar, but the apoptosis rate of lymphocytes from the two filters was slightly higher. Additionally, monocytes isolated via the three sources were able to be induced into dendritic cells expressing specific molecules; Granulocytes isolated from the LRF showed a lower purity and a higher level of apoptosis than granulocytes isolated from the WB. CONCLUSION: Compared with WB, the PBMCs isolated from the filters used in our blood center had no statistical difference in their activity and biological function, but they did differ in the proportion and quantity of monocytes and granulocytes. Our results show that the two filters can be used as an alternative method to collect leukocytes, which solves the problem of an insufficient blood supply for clinical and basic science research. Thus, these filters have significant value beyond their practical use in clinics.


Assuntos
Granulócitos/citologia , Procedimentos de Redução de Leucócitos/instrumentação , Leucócitos Mononucleares/citologia , Apoptose , Buffy Coat/citologia , Separação Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Células Dendríticas/citologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Procedimentos de Redução de Leucócitos/métodos , Contagem de Linfócitos , Plaquetoferese/instrumentação , Plaquetoferese/métodos
16.
Transfusion ; 60(3): 454-459, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31782799

RESUMO

BACKGROUND AND OBJECTIVES: Cryopreservation provides an option for long-term storage of platelet concentrates. While platelets are usually frozen as soon as practical after collection (within 2 days), the ability to freeze units at a later stage of the shelf life may improve inventory management. As such, the aim of this study was to determine the impact of freezing platelets approaching expiry (Day 5/6). MATERIALS AND METHODS: Two ABO-matched buffy coat-derived platelets (30% plasma/70% platelet additive solution) were pooled and split to produce matched pairs (n = 8 pairs). Platelets were frozen on Day 1 after collection (cryopreserved platelets [CPPs]) or Day 5 or 6 (expired-CPPs) at -80°C with 5% to 6% dimethyl sulfoxide. In vitro platelet quality was tested before freezing and after thawing and reconstitution in plasma. RESULTS: The majority of prefreeze parameters were equivalent for all platelet units (Day 1 vs. Day 5 or 6). Expired-CPPs had a higher mean postthaw platelet recovery (82 ± 4%) compared to CPPs (75 ± 4%; p = 0.0021). Cryopreservation resulted in a loss of surface glycoproteins (glycoprotein (GP) Ibα, GPIIb, GPVI), an increase in activation markers (phosphatidylserine and P-selectin) and microparticle release, compared to unfrozen platelets. However, the cryopreservation-induced changes were equivalent in CPPs and expired-CPPs. Functionality was measured by thromboelastography and was similar between expired-CPPs (R-time: 5.3 ± 0.3) and CPPs (R-time: 5.4 ± 0.5; p = 0.7094). CONCLUSION: The phenotype and functional profile of platelets frozen at expiry were similar to platelets frozen 1 day following collection. These data suggest that expired platelets may represent a suitable starting material for cryopreservation.


Assuntos
Plaquetas/citologia , Congelamento , Buffy Coat/citologia , Criopreservação/métodos , Humanos
17.
J Immunol Methods ; 478: 112715, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31809709

RESUMO

Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species.


Assuntos
Antígenos Virais/imunologia , Linfócitos B/imunologia , Separação Celular/métodos , Imunofluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Memória Imunológica , Animais , Buffy Coat/citologia , Separação Celular/instrumentação , Citomegalovirus/imunologia , Estudos de Viabilidade , Imunofluorescência/instrumentação , Corantes Fluorescentes/química , Vacinas contra Hepatite B/administração & dosagem , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Hibridomas , Imunogenicidade da Vacina , Camundongos , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Coloração e Rotulagem , Toxoide Tetânico/imunologia , Vacinação , Fluxo de Trabalho
18.
Vox Sang ; 115(1): 94-102, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31709567

RESUMO

BACKGROUND AND OBJECTIVES: Platelet transfusion is a standard medical therapy used to treat several bleeding disorders. However, a critical drawback is the dependency on donor-derived platelets, which leads to concerns like insufficient availability and immunological complications. In vitro platelet production from hematopoietic progenitor cells (CD34) may represent a reasonable solution. MATERIALS AND METHODS: CD34+ cells were isolated from either buffy coat or peripheral blood and compared in terms of platelet production in vitro. The number and the quality of magnetically isolated CD34+ cells and their capability to differentiate into mature megakaryocytes were investigated using flow cytometry. Additionally, the functionality of megakaryocytes in term of in vitro platelet production was tested. RESULTS: Similar purity and quantity of CD34+ cells was found after their isolation from both cell sources. In contrast, after 6 days of culture, enhanced number of CD34+ cells isolated from buffy coat compared with peripheral blood was observed (5·3 x 106 vs. 3·0 x 106, respectively). Interestingly, despite a comparable nuclear maturation phenotype, the yield of platelets released from buffy coat-derived megakaryocytes was significantly higher than from peripheral blood cells (platelet yield pro MK: 7·2 vs. 2·7, respectively). Importantly, platelets produced from buffy coat-derived cells could be activated by agonists. CONCLUSION: Haematopoietic progenitor cells isolated from buffy coat have increased yield of platelets released from mature megakaryocytes and enhanced in vitro functionality, compared with peripheral blood-derived cells. Our study, suggests that buffy coat, obtained during blood donation processing, might be a promising source of megakaryocytes for in vitro platelet production.


Assuntos
Buffy Coat/citologia , Doadores de Sangue , Plaquetas , Células-Tronco Hematopoéticas/fisiologia , Megacariócitos , Citometria de Fluxo , Hematopoese , Humanos
19.
Cytometry A ; 95(11): 1178-1190, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31692248

RESUMO

Cytometry by time-of-flight (CyTOF) has emerged as a high-throughput single cell technology able to provide large samples of protein readouts. Already, there exists a large pool of advanced high-dimensional analysis algorithms that explore the observed heterogeneous distributions making intriguing biological inferences. A fact largely overlooked by these methods, however, is the effect of the established data preprocessing pipeline to the distributions of the measured quantities. In this article, we focus on randomization, a transformation used for improving data visualization, which can negatively affect multivariate data analysis methods such as dimensionality reduction, clustering, and network reconstruction algorithms. Our results indicate that randomization should be used only for visualization purposes, but not in conjunction with high-dimensional analytical tools. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.


Assuntos
Algoritmos , Citometria de Fluxo/métodos , Leucócitos Mononucleares/citologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Buffy Coat/citologia , Buffy Coat/metabolismo , Análise por Conglomerados , Humanos , Leucócitos Mononucleares/metabolismo , Análise Multivariada , Redes Neurais de Computação , Distribuição Aleatória , Análise de Célula Única , Linfócitos T/citologia , Linfócitos T/metabolismo
20.
Vox Sang ; 114(8): 876-883, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31625187

RESUMO

BACKGROUND AND OBJECTIVES: There is no standard methodology for post-thaw sample preparation for viability analysis of umbilical cord blood units (CBU). A common challenge faced by CB bank is for their product to meet the post-thaw cell viability threshold for CD45+ cells set at 40% by NetCord-FACT. The objective of this work was to improve the post-thaw staining method to maximize CD45+ cell viability so that clinically valuable samples meet the NetCord-FACT threshold criteria for CD45+ and CD34+ cell viabilities. MATERIALS AND METHODS: Samples of CBU buffy coats and CBU segments were thawed and taken for staining. Various parameters were evaluated on CD45+ and CD34+ cell viability as measured by 7-actinomycin D (7-AAD) staining. RESULTS: The results revealed that initiating the staining at 20 min post-thaw instead of 30, shortening the red cell lysis treatment, or performing lysis on ice and removing this step all together, all improved the viability of CD45+ cells. Using CBU segments, it was shown that the most effective approach in increasing the viability of CD45+ cells was the complete omission of red cell lysis step. However, removal of the lysis step can create technical artefacts during flow cytometry acquisition that results in an underestimation of the viability of CD34+ cells. This can be avoided and CD34+ cell viability restored with additional thresholding on CD45 signal. CONCLUSION: CB CD45+ cells are sensitive to red cell lysis treatment post-thaw; omission of this step provides the best viability and ultimately better reflects the quality of cells used for transplantation.


Assuntos
Criopreservação/métodos , Sangue Fetal/citologia , Antígenos Comuns de Leucócito/metabolismo , Antígenos CD34/genética , Antígenos CD34/metabolismo , Buffy Coat/citologia , Buffy Coat/metabolismo , Sobrevivência Celular , Criopreservação/normas , Sangue Fetal/metabolismo , Humanos , Antígenos Comuns de Leucócito/genética
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