RESUMO
Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused substantial morbidity and mortality. Despite the availability of efficacious vaccines, new variants with reduced sensitivity to vaccine-induced protection are a troubling new reality. The Ad26.COV2.S vaccine is a recombinant, replication-incompetent human adenovirus type 26 vector encoding a full-length, membrane-bound severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein in a prefusion-stabilized conformation. This review discusses the immunogenicity and efficacy of Ad26.COV2.S as a single-dose primary vaccination and as a homologous or heterologous booster vaccination. Ad26.COV2.S elicits broad humoral and cellular immune responses, which are associated with protective efficacy/effectiveness against SARS-CoV-2 infection, moderate to severe/critical COVID-19, and COVID-19-related hospitalization and death, including against emerging SARS-CoV-2 variants. The humoral immune responses elicited by Ad26.COV2.S vaccination are durable, continue to increase for at least 2-3 months postvaccination, and involve a range of functional antibodies. Ad26.COV2.S given as a heterologous booster to mRNA vaccine-primed individuals markedly increases humoral and cellular immune responses. The use of Ad26.COV2.S as primary vaccination and as part of booster regimens is supporting the ongoing efforts to control and mitigate the COVID-19 pandemic.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Ad26COVS1 , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Humanos , Pandemias/prevenção & controle , SARS-CoV-2 , Vacinas Sintéticas , Vacinas de mRNARESUMO
BACKGROUND: The Ad26.COV2.S vaccine is a recombinant, replication-incompetent human adenovirus type 26 vector encoding full-length severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein in a prefusion-stabilized conformation. METHODS: In an international, randomized, double-blind, placebo-controlled, phase 3 trial, we randomly assigned adult participants in a 1:1 ratio to receive a single dose of Ad26.COV2.S (5×1010 viral particles) or placebo. The primary end points were vaccine efficacy against moderate to severe-critical coronavirus disease 2019 (Covid-19) with an onset at least 14 days and at least 28 days after administration among participants in the per-protocol population who had tested negative for SARS-CoV-2. Safety was also assessed. RESULTS: The per-protocol population included 19,630 SARS-CoV-2-negative participants who received Ad26.COV2.S and 19,691 who received placebo. Ad26.COV2.S protected against moderate to severe-critical Covid-19 with onset at least 14 days after administration (116 cases in the vaccine group vs. 348 in the placebo group; efficacy, 66.9%; adjusted 95% confidence interval [CI], 59.0 to 73.4) and at least 28 days after administration (66 vs. 193 cases; efficacy, 66.1%; adjusted 95% CI, 55.0 to 74.8). Vaccine efficacy was higher against severe-critical Covid-19 (76.7% [adjusted 95% CI, 54.6 to 89.1] for onset at ≥14 days and 85.4% [adjusted 95% CI, 54.2 to 96.9] for onset at ≥28 days). Despite 86 of 91 cases (94.5%) in South Africa with sequenced virus having the 20H/501Y.V2 variant, vaccine efficacy was 52.0% and 64.0% against moderate to severe-critical Covid-19 with onset at least 14 days and at least 28 days after administration, respectively, and efficacy against severe-critical Covid-19 was 73.1% and 81.7%, respectively. Reactogenicity was higher with Ad26.COV2.S than with placebo but was generally mild to moderate and transient. The incidence of serious adverse events was balanced between the two groups. Three deaths occurred in the vaccine group (none were Covid-19-related), and 16 in the placebo group (5 were Covid-19-related). CONCLUSIONS: A single dose of Ad26.COV2.S protected against symptomatic Covid-19 and asymptomatic SARS-CoV-2 infection and was effective against severe-critical disease, including hospitalization and death. Safety appeared to be similar to that in other phase 3 trials of Covid-19 vaccines. (Funded by Janssen Research and Development and others; ENSEMBLE ClinicalTrials.gov number, NCT04505722.).
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Imunogenicidade da Vacina , Ad26COVS1 , Adolescente , Adulto , Idoso , Doenças Assintomáticas/epidemiologia , COVID-19/epidemiologia , COVID-19/mortalidade , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/imunologia , Método Duplo-Cego , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Gravidade do Paciente , Modelos de Riscos Proporcionais , Adulto JovemRESUMO
Clinical trial data and real-world evidence suggest that the AS04-adjuvanted vaccine targeting human papillomavirus types 16 and 18 (AS04-HPV-16/18) vaccine provides nearly 90% protection against cervical intraepithelial neoplasia grade 3 or higher irrespective of type, among women vaccinated before sexual debut. This high efficacy is not fully explained by cross-protection. Although AS04-HPV-16/18 vaccination does not affect clearance of prevalent infections, it may accelerate clearance of newly acquired infections. We pooled data from 2 large-scale randomized controlled trials to evaluate efficacy of the AS04-HPV-16/18 vaccine against clearance of nontargeted incident infections. Results of our analysis do not suggest an effect in expediting clearance of incident infections.
Assuntos
Infecções por Papillomavirus , Vacinas contra Papillomavirus/imunologia , Neoplasias do Colo do Útero , Adjuvantes Imunológicos , Costa Rica/epidemiologia , Método Duplo-Cego , Feminino , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Humanos , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/prevenção & controle , Resultado do Tratamento , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologiaRESUMO
BACKGROUND: The AS04-adjuvanted HPV16/18 (AS04-HPV16/18) vaccine provides excellent protection against targeted human papillomavirus (HPV) types and a variable degree of cross-protection against others, including types 6/11/31/33/45. High efficacy against any cervical intraepithelial neoplasia grade 3 or greater (CIN3+; >90%) suggests that lower levels of protection may exist for a wide range of oncogenic HPV types, which is difficult to quantify in individual trials. Pooling individual-level data from two randomized controlled trials, we aimed to evaluate AS04-HPV16/18 vaccine efficacy against incident HPV infections and cervical abnormalities . METHODS: Data were available from the Costa Rica Vaccine Trial (NCT00128661) and Papilloma Trial Against Cancer in Young Adults trial (NCT00122681), two large-scale, double-blind randomized controlled trials of the AS04-HPV16/18 vaccine. Primary analyses focused on disease-free women with no detectable cervicovaginal HPV at baseline. RESULTS: A total of 12 550 women were included in our primary analyses (HPV arm = 6271, control arm = 6279). Incidence of 6-month persistent oncogenic and nononcogenic infections, excluding known and accepted protected types 6/11/16/18/31/33/45 (focusing on 34/35/39/40/42/43/44/51/52/53/54/56/58/59/66/68/73/70/74), was statistically significantly lower in the HPV arm than in the control arm (efficacy = 9.9%, 95% confidence interval [CI] = 1.7% to 17.4%). Statistically significant efficacy (P < .05) was observed for individual oncogenic types 16/18/31/33/45/52 and nononcogenic types 6/11/53/74. Efficacy against cervical abnormalities (all types) increased with severity, ranging from 27.7% (95% CI = 21.7% to 33.3%) to 58.7% (95% CI = 34.1% to 74.7%) for cytologic outcomes (low-grade squamous intraepithelial neoplasia lesion or greater, and high-grade squamous intraepithelial neoplasia lesion or greater, respectively) and 66.0% (95% CI = 54.4% to 74.9%) to 87.8% (95% CI = 71.1% to 95.7%) for histologic outcomes (CIN2+ and CIN3+, respectively). Comparing Costa Rica Vaccine Trial and Papilloma Trial Against Cancer in Young Adults results, there was no evidence of heterogeneity, except for type 51 (efficacy = -28.6% and 20.7%, respectively; two-sided P = .03). CONCLUSIONS: The AS04-HPV16/18 vaccine provides some additional cross-protection beyond established protected types, which partially explains the high efficacy against CIN3+.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Adolescente , Adulto , Costa Rica/epidemiologia , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Vacinas contra Papillomavirus/química , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Estudos Retrospectivos , Resultado do Tratamento , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia , Adulto Jovem , Displasia do Colo do Útero/epidemiologia , Displasia do Colo do Útero/prevenção & controle , Displasia do Colo do Útero/virologiaRESUMO
BACKGROUND: Substantial heterogeneity has been reported in efficacy against high-grade cervical intraepithelial neoplasia (CIN) irrespective of HPV type in phase III results for bivalent and quadrivalent human papillomavirus virus (HPV) vaccines (AS04-HPV and qHPV). Real-world data recently confirmed a very high overall impact of AS04-HPV, supporting the validity of the observed heterogeneity. To explore the reasons for heterogeneous efficacy, we assessed vaccine impact on high-grade lesions not caused by vaccine types. RESEARCH METHODS: We extracted case counts of CIN lesions containing (1) at least one vaccine HPV type, (2) at least one vaccine HPV type and a high-risk non-vaccine type (co-infections) and (3) no vaccine types (non-vaccine or no high-risk HPV types). Based on these, Phase III cross-protective efficacies were estimated with exclusion (3) and with inclusion (2 and 3) of co-infections. RESULTS: Cross-protective efficacy of AS04-HPV against CIN3 lesions ranges from 81.3% (95%CI: 34.7;96.5) (excluding co-infections) to 88.5% (95%CI:62.4;97.8) (including co-infections). For qHPV the efficacy ranges from -58.7% (95%CI: -180.5;8.5) (excluding co-infections) to 13.1% (95%CI: -39.0;45.9) (including co-infections). CONCLUSIONS: Heterogenous overall efficacy against CIN3 between AS04-HPV and qHPV is driven by differential efficacy against lesions that do not contain vaccine types, which may be related to the impact of different adjuvants on the immune response.
Assuntos
Papillomaviridae/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Ensaios Clínicos Fase III como Assunto , Feminino , Seguimentos , Humanos , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Adulto Jovem , Displasia do Colo do Útero/prevenção & controleRESUMO
Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein-coupled receptors and has important functions in cell migration and proliferation. This study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant downregulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression, and induced chemotaxis in all tested human leukemic cell lines and human CD34(+) progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80%, demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.
Assuntos
Movimento Celular/genética , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide/genética , Diester Fosfórico Hidrolases/genética , Tirosina Quinase 3 Semelhante a fms/genética , Doença Aguda , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Células K562 , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Diester Fosfórico Hidrolases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Sequências de Repetição em Tandem/genéticaRESUMO
A shift from conventional cytology to a molecular approach could improve cervical cancer screening. This proof-of-concept study aims to develop a high-content imaging platform for the simultaneous detection of multiple biomarkers for cervical disease. Liquid-based cytology (LBC) samples were used to optimize a dual ProExC/Ki-67 immunofluorescence staining protocol for SurePath-fixed cells. The simultaneous and automated detection of these biomarkers was performed using the BD Pathway 435 system. The ability of high-content imaging to detect dysplastic cervical cells was assessed using keratinocytes spiked with immunopositive SiHa cells and a high-grade squamous intraepithelial lesion (HSIL) LBC sample. The percentages of Ki-67- and ProExC-immunopositive objects correlated significantly with the percentages of spiked SiHa cells. The dysplastic cells of the HSIL sample could be detected using high-content cell analysis. In conclusion, high-content imaging allows the simultaneous and automated detection of Ki-67- and ProExC-immunopositive dysplastic cells in LBC specimens.
Assuntos
Detecção Precoce de Câncer/métodos , Displasia do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diagnóstico por Imagem/métodos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Ensaios de Triagem em Larga Escala , Humanos , Queratinócitos/metabolismo , Antígeno Ki-67/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/metabolismo , Padrões de Referência , Coloração e RotulagemRESUMO
OBJECTIVE: Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD-positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition. MATERIALS AND METHODS: We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34(+) hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD. RESULTS: We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor-1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD-positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area-forming cells compared to FLT3-wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area-forming cells and proliferation of FLT3-ITD-positive cells, but did not affect FLT3-wild-type cells-highlighting the critical interaction between CXCR4 and FLT3-ITD. CONCLUSION: CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD-positive AML.
Assuntos
Células da Medula Óssea/patologia , Leucemia Mieloide/patologia , Receptores CXCR4/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Doença Aguda , Animais , Benzilaminas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Ciclamos , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/farmacologia , Humanos , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Fosforilação , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
OBJECTIVE: The Notch signaling pathway has been shown to play a role in bone marrow-derived stromal cell differentiation, however, the precise outcome of Notch activation remains controversial. The aim of this study was to evaluate the effect of Notch signaling in primary human bone marrow-derived stromal cells (hBMSCs). MATERIALS AND METHODS: hBMSCs were transduced to >90% with lentiviral vectors containing either human notch1 intracellular domain (NICD), jagged1, or dominant negative mastermind1. Cells were exposed to adipogenic and osteogenic differentiation stimuli and differentiation was quantified by oil red or alizarin red staining, alkaline phosphatase liver/bone/kidney (ALPL) activity and expression of adipogenic or osteogenic marker genes. RESULTS: NICD and jagged1 transgene-expressing hBMSCs demonstrated enhanced mineralization, nodule formation, and ALPL activity in osteogenic differentiation media. These findings correlated with increased gene expression of bone morphogenetic protein 2 and ALPL. In contrast, NICD or jagged1 transgene expression strongly inhibited adipocyte formation and reduced peroxisome proliferator-activated receptor-gamma, fatty acid binding protein 4, and adiponectin precursor gene expression. Co-overexpression of dominant negative mastermind1 and NICD or jagged1 led to a partial rescue of the differentiation phenotypes. In addition, high endogenous jagged1 expression levels were observed in hBMSCs samples with strong ALPL activity compared to a group of samples with low ALPL activity. CONCLUSION: In summary, our data suggest that induction of Notch signaling enhances the osteogenic differentiation of hBMSCs while inhibiting the adipogenic fate.
Assuntos
Adipócitos/citologia , Células da Medula Óssea/citologia , Diferenciação Celular , Receptores Notch/metabolismo , Transdução de Sinais , Células Estromais/citologia , Fosfatase Alcalina/metabolismo , Sequência de Bases , Células da Medula Óssea/metabolismo , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Primers do DNA , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Serrate-Jagged , Células Estromais/metabolismoRESUMO
Stem cell migration is largely regulated by the chemokine SDF-1 and its receptor CXCR4. In the present study, we analyzed the effect of protein on SDF-1 dependent chemotaxis using CXCR4 expressing primary CD34+ hematopoietic progenitor cells for transwell migration assays. We show that migration towards SDF-1 is abolished in the absence of protein, while addition of serum albumin rescues SDF-1 dependent migration. Acid hydrolyzation or tryptic digest of protein eliminates its migration supporting effect, showing that the intact protein is necessary. We demonstrate that gradients of human serum albumin (HSA) that are physiologically present in vivo between human plasma and interstitial fluid (bone marrow) greatly influence SDF-1 dependent migration of hematopoietic progenitor cells. While SDF-1 dependent migration is strongly enhanced in the presence of a HSA gradient from 4% (plasma) towards 1% (interstitial fluid), reversion of the protein concentrations inhibits SDF-1 dependent chemotaxis. Furthermore, migration is induced to lower serum albumin concentrations in the presence of equal SDF-1 concentration, while albumin gradients in the absence of SDF-1 have no effect. Our results suggest that physiological gradients of serum albumin between blood and bone marrow support SDF-1 dependent homing of hematopoietic progenitor cells to the stem cell niche.
Assuntos
Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Albumina Sérica/farmacologia , Animais , Bovinos , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peso Molecular , Fosforilação/efeitos dos fármacosRESUMO
Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 mum within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before CXCR4 mRNA transfection enhanced SDF-1alpha-directed migration of human BMSCs by 50%. Implantation of SDF-1alpha-releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo.
Assuntos
Materiais Biomiméticos/farmacologia , Células da Medula Óssea/citologia , Quimiocina CXCL12/metabolismo , Quimiotaxia/efeitos dos fármacos , Receptores CXCR4/metabolismo , Células Estromais/metabolismo , Alicerces Teciduais/química , Animais , Humanos , Camundongos , Camundongos SCID , Microscopia Eletrônica de Varredura , Porosidade/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/transplante , TransfecçãoRESUMO
The CXC chemokine receptor 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1) regulate egress and homing of hematopoietic stem cells. Activation of sphingosine-1-phosphate (S1P) receptors (S1P(1-5)) modulates chemokine-induced migration of lymphocytes and hematopoietic stem cells. To analyze the influence of S1P(1) on SDF-1-dependent chemotaxis and trafficking, we overexpressed S1P(1) in CD34+ mobilized peripheral blood progenitor cells (PBPCs). Using a gamma-retroviral vector, transgene overexpression was achieved in more than 90% of target cells. S1P(1) transgene positive PBPCs showed enhanced chemotaxis towards S1P. S1P(1) overexpression resulted in reduced CXCR4 surface expression levels and strong inhibition of SDF-1-dependent ERK1/2 phosphorylation and Ca(2+) flux. Furthermore, SDF-1-dependent migration of S1P(1) overexpressing PBPCs or Jurkat cells was reduced up to 10-fold. Sublethally irradiated NOD/SCID mice were transplanted with 6-day cultured PBPCs overexpressing either S1P(1)-IRES-GFP or GFP alone. Screening for GFP positive human cells in the mouse bone marrow 20h after transplantation revealed an eightfold reduction in bone marrow homing of S1P(1) transgene expressing cells. Our data suggest that S1P(1) acts as an inhibitor of CXCR4-dependent migration of hematopoietic cells to sites of SDF-1 production.
Assuntos
Antígenos CD34/imunologia , Quimiocina CXCL12/imunologia , Quimiotaxia/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Lisofosfolipídeos/farmacologia , Receptores CXCR4/imunologia , Receptores de Lisoesfingolipídeo/imunologia , Esfingosina/análogos & derivados , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Adesão Celular/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células Jurkat , Camundongos , Camundongos SCID , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Esfingosina/farmacologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
We present a general, entirely PCR-based strategy to construct mRNAs coding for green fluorescent protein (GFP) fusion proteins from a cDNA pool. We exemplify our approach for the chemokine receptor CXCR4. mRNA transfection of the PCR-generated fusion of CXCR4-GFP into K562 cells or primary mesenchymal stem cells (MSCs) resulted in excellent viability (> 90%) with more than 90% of target cells expressing easily detectable CXCR4-GFP for > 72 h. The fusion protein was localized in the plasma membrane and was rapidly internalized upon incubation with the CXCR4 ligand stromal cell-derived factor-1 (SDF-1). Transwell migration experiments showed significantly increased migration of CXCR4-GFP mRNA-transfected MSCs toward a gradient of SDF-1, demonstrating that mRNA-mediated chemokine receptor overexpression allows for transient initiation of chemotaxis. The presented strategy to construct a PCR-based fluorescent fusion protein can be generally applied to other genes of interest to study their function by simple overexpression and easy detection in primary cells.
Assuntos
Células-Tronco Mesenquimais/citologia , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Engenharia Tecidual/métodos , Células da Medula Óssea/metabolismo , Movimento Celular , Quimiocina CXCL12/metabolismo , Quimiotaxia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células K562 , Ligantes , Modelos Genéticos , Reação em Cadeia da Polimerase , TransfecçãoRESUMO
Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus.