RESUMO
OBJECTIVES: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. METHODS: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. RESULTS: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). CONCLUSIONS: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation.
Assuntos
Biomarcadores Tumorais/genética , Células da Medula Óssea/metabolismo , Lectinas Tipo C/genética , Leucemia Mieloide Aguda/genética , Mutação , Células Mieloides/metabolismo , Receptores Mitogênicos/genética , Antígenos CD34/genética , Antígenos CD34/imunologia , Biomarcadores Tumorais/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Análise Citogenética , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Imunofenotipagem , Lectinas Tipo C/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Células Mieloides/patologia , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Cultura Primária de Células , Receptores Mitogênicos/imunologiaRESUMO
BACKGROUND: Antiangiogenic-targeting agents have low response rates in patients with nonpancreatic neuroendocrine tumors (NETs). Nintedanib is an oral antiangiogenic agent that has inhibitory effects on the fibroblast growth factor receptor, which is highly expressed in NETs. The authors hypothesized that nintedanib would be active in patients with nonpancreatic NETs. METHODS: Patients with advanced, grade 1 or 2, nonpancreatic NETs who were receiving a stable dose of somatostatin analogue were enrolled. Nintedanib was administered at a dose of 200 mg twice daily in 28-day cycles. The primary endpoint was progression-free survival (PFS) at 16 weeks. RESULTS: Thirty-two patients were enrolled, and 30 were evaluable for the primary outcome. Most had radiographic disease progression within 12 months before enrollment. The 16-week PFS rate was 83%, and the median PFS and overall survival were 11.0 months and 32.7 months, respectively. Nintedanib was well tolerated and delayed deterioration in quality of life. The baseline serotonin level had a strong, positive correlation with activated but exhausted T cells. CONCLUSIONS: Nintedanib is active in nonpancreatic NETs. The immunosuppressive effect of serotonin should be targeted in future clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Indóis/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Tumores Neuroendócrinos/tratamento farmacológico , Idoso , Inibidores da Angiogênese/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Progressão da Doença , Feminino , Humanos , Indóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neovascularização Patológica/patologia , Intervalo Livre de Progressão , Somatostatina/administração & dosagem , Somatostatina/efeitos adversos , Resultado do TratamentoRESUMO
OBJECTIVE: To assess the effect of aspirin use in low-risk pregnancy on: (1) pregnancy-associated plasma protein-A (PAPP-A) and placental-like growth factor (PLGF); (2) urinary albumin-to-creatinine ratio (ACR) and blood pressure; (3) fetal growth parameters; and (4) placental histopathology. STUDY DESIGN: This secondary analysis from the T rial of low-dose aspirin with an E arly S creening T est for preeclampsia and growth restriction randomized controlled trial was based on low-risk nulliparous women randomized at 11 weeks to (1) aspirin 75 mg; (2) no aspirin; and (3) aspirin based on the preeclampsia Fetal Medicine Foundation screening test. At baseline, women underwent assessment of blood pressure, PAPP-A, PLGF, and ACR, repeated 9 to 10 weeks postaspirin, in addition to fetal growth assessment. Gross and histopathological placental analyses were performed in line with Amsterdam criteria. RESULTS: A total of 445 subjects were included (aspirin n = 163 [36.6%]; no aspirin n = 282 [63.4%]). Although the fetal-to-placental weight ratio was significantly greater in the aspirin group (7.5 [±1.3] vs. 7.3 [±1.4], p = 0.045), as was change in ultrasound assessed estimated fetal weight from second to third trimesters (1,624.5 g [±235.1] vs. 1,606.2 [±189.4], p = 0.042), this was invalidated by the lack of a difference in birth weight. Aspirin did not significantly impact on change in serum or urine preeclampsia biomarkers, maternal blood pressure, or placental histopathology. CONCLUSION: Aspirin use in low-risk pregnancy does not appear to impact on preeclampsia biomarkers, fetal growth, or placental pathology.
Assuntos
Aspirina/farmacologia , Biomarcadores , Desenvolvimento Fetal/efeitos dos fármacos , Doenças Placentárias/diagnóstico , Pré-Eclâmpsia/diagnóstico , Adulto , Albuminúria , Aspirina/administração & dosagem , Biomarcadores/sangue , Biomarcadores/urina , Creatinina/urina , Feminino , Humanos , Placenta/patologia , Fator de Crescimento Placentário/sangue , Gravidez , Proteína Plasmática A Associada à Gravidez/análise , Ultrassonografia Pré-NatalRESUMO
The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater effect on the IRF8 nuclear localization versus total fluorescence assessment. The data thus indicate that IRF8 can have cytoplasmic presence and that during disease its nuclear-cytoplasmic distribution may be altered, which may provide a basis for potential myeloid defects during certain pathologies.
Assuntos
Carcinoma/genética , Núcleo Celular/genética , Citoplasma/genética , Hematopoese/genética , Fatores Reguladores de Interferon/genética , Neoplasias Renais/genética , Anticorpos/farmacologia , Carcinoma/imunologia , Carcinoma/patologia , Estudos de Casos e Controles , Diferenciação Celular , Núcleo Celular/imunologia , Núcleo Celular/ultraestrutura , Citoplasma/imunologia , Citoplasma/ultraestrutura , Citometria de Fluxo/métodos , Expressão Gênica , Hematopoese/imunologia , Humanos , Citometria por Imagem/métodos , Fatores Reguladores de Interferon/antagonistas & inibidores , Fatores Reguladores de Interferon/imunologia , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Células Mieloides , Peptídeos/farmacologia , Coloração e Rotulagem/métodosRESUMO
MHC-multimers are reagents used for the detection and enumeration of antigen-specific T cells (ASTs). These reagents exploit the mechanism by which T cell receptors (TCR) on cytotoxic CD8 T cells recognize specific antigens in the context of a major histocompatibility complex (MHC) molecule during antigen presentation. MHC-multimers are fluorescently-labeled dextran polymers that carry MHC Class I molecules and peptide sequences that can be modified to represent specific cognate sequences of the antigen of interest with dextramers having a 10-fold multiplicity of the MHC/peptide combination within a single multimer. Since the binding of antigen-specific dextramers mimics antigen presentation to the TCR, the present study sought to determine whether this TCR engagement on the AST was sufficient to elicit a functional T cell response. The effect of binding of CMV specific dextramers on the activation of the NFAT signal transduction cascade was assessed in peripheral blood from bone marrow transplant recipients previously determined to be positive for CMV-ASTs (CASTs). NFAT activation was quantified by measuring nuclear translocation of NFAT1 in CD8+ CASTs and CD8+ non-CASTs by imaging flow cytometry. Our results demonstrate that an increase in the nuclear localization of NFAT1 was detectable in the CASTs following the CMV-dextramer binding and could be observed as early as 10min post-exposure. The NFAT1 activation correlated with a downstream functional response in the form of interferon gamma production. Sample preparation, temperature, and duration of dextramer exposure were important parameters affecting the dextramer-induced NFAT activation with 2h exposure in whole blood at room temperature being the optimal of the conditions tested. Intra- and inter-individual heterogeneity was observed with regards to the NFAT activation in the CASTs. Importantly, no effect of the dextramers was observed in the CD8+ non-CASTs, and therefore dextramer negative cell populations. Exposure to PMA/ionomycin following dextramer exposure resulted in a homogeneous NFAT activation in both the dextramer-positive but NFAT1 nonresponsive CAST and non-CAST cells. Thus, the data demonstrate that binding of antigen-specific dextramers to ASTs specifically results in activation of NFAT, that the NFAT activation correlates with a downstream functional response and that the response can be heterogeneous. This functional parameter may provide insight to the issue whether enumeration alone of ASTs is a sufficient parameter to assess an individual's immune status against a specific antigen.
Assuntos
Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Complexo Principal de Histocompatibilidade , Fatores de Transcrição NFATC/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Apresentação de Antígeno , Citomegalovirus/imunologia , Citometria de Fluxo/instrumentação , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Humanos , Citometria por Imagem/instrumentação , Interferon gama/farmacologia , Ionomicina/farmacologia , Leucemia/imunologia , Leucemia/patologia , Leucemia/terapia , Ativação Linfocitária/efeitos dos fármacos , Fatores de Transcrição NFATC/agonistas , Fatores de Transcrição NFATC/genética , Ficoeritrina/química , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/genética , Coloração e Rotulagem/métodos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/patologia , Acetato de Tetradecanoilforbol/farmacologia , TransplantadosRESUMO
The aim of this prospective, observational study was to investigate the impact of gestational weight gain (GWG) among euglycaemic obese pregnant women on maternal and foetal metabolic parameters and neonatal outcome. Total GWG was recorded for 101 obese, non-diabetic women with a singleton pregnancy. At 28 weeks of gestation, fasting maternal blood samples were analysed for glucose, insulin, c-peptide and lipids. Cord bloods were collected at delivery for analysis of glucose, c-peptide and lipids. GWG (mean ± SD =10.9 ± 5.5 kg) was greatest among those of younger age and lower body mass index and 58% of women exceeded the Institute of Medicine GWG recommendations of 5-9 kg for obese pregnancy. GWG was significantly positively associated with increased risk of birthweight >4 kg, cord c-peptide levels and inversely associated with cord total cholesterol. This study identified that higher GWG in obese pregnancy may increase the risk of macrosomia and neonatal hyperinsulinaemia, within a euglycaemic maternal cohort. Impact statement Excess gestational weight gain (GWG) and maternal obesity frequently co-occur with adverse consequences for maternal and neonatal health; however, little is known of the underlying biological pathways which may be affected to contribute to adverse outcomes. Greater understanding of the biological mechanisms involved may help guide future studies to develop targeted interventions for more effective clinical outcomes. This study identified that higher GWG among obese pregnant women resulted in foetal hyperinsulinaemia even in the absence of maternal hyperglycaemia, potentially representing a biological pathway for larger birthweight babies. These results may highlight the need for more intensive dietary and lifestyle interventions among obese women who would not normally receive additional counselling beyond standard antenatal care if not diagnosed with glucose intolerance in pregnancy.
Assuntos
Obesidade/fisiopatologia , Complicações na Gravidez/fisiopatologia , Aumento de Peso , Adulto , Peso ao Nascer , Glicemia/análise , Índice de Massa Corporal , Peptídeo C/sangue , Jejum , Feminino , Sangue Fetal/metabolismo , Humanos , Insulina/sangue , Lipídeos/sangue , Obesidade/sangue , Gravidez , Complicações na Gravidez/sangue , Estudos ProspectivosRESUMO
OBJECTIVE: Probiotics are live microorganisms that may confer health benefits on the host. Recent trials of probiotic use among healthy pregnant women demonstrate potential for improved glycemic control. The aim of this study was to investigate the effects of a probiotic capsule intervention on maternal metabolic parameters and pregnancy outcome among women with gestational diabetes. STUDY DESIGN: This double-blind placebo-controlled randomized trial recruited pregnant women with a new diagnosis of gestational diabetes or impaired glucose tolerance following a 3-hour 100-g glucose tolerance test. Women were randomized to a daily probiotic (Lactobacillus salivarius UCC118) or placebo capsule from diagnosis until delivery. Fasting blood samples were collected at baseline and 4-6 weeks after capsule commencement for analysis of glucose, insulin, c-peptide, and lipids. The primary outcome was difference in fasting glucose postintervention, first analyzed on an intention-to-treat basis and followed by per-protocol analysis that excluded women commenced on pharmacological therapy (insulin or metformin). Secondary outcomes were changes in insulin, c-peptide, homeostasis model assessment and lipids, requirement for pharmacological therapy, and neonatal anthropometry. RESULTS: Of 149 women recruited and randomized, there were no differences between the probiotic and placebo groups in postintervention fasting glucose (4.65 ± 0.49 vs 4.65 ± 0.53 mmol/L; P = 373), requirement for pharmacological therapy (17% vs 14%; P = .643), or birthweight (3.57 ± 0.64 vs 3.60 ± 0.57 kg; P = .845). Among 100 women managed with diet and exercise alone, fasting plasma glucose decreased significantly within both the probiotic (4.76 ± 0.45 to 4.57 ± 0.42 mmol/L; P < .001) and placebo (4.85 ± 0.58 to 4.58 ± 0.45 mmol/L; P < .001) groups, but the levels between groups did not differ (P = .316). The late gestation-related rise in total and low-density lipoprotein (LDL) cholesterol was attenuated in the probiotic vs the placebo group (+0.27 ± 0.48 vs +0.50 ± 0.52 mmol/L total cholesterol, P = .031; +0.08 ± 0.51 vs +0.31 ± 0.45 mmol/L LDL cholesterol, P = .011). No differences were noted between groups in other metabolic parameters or pregnancy outcome. CONCLUSION: A probiotic capsule intervention among women with abnormal glucose tolerance had no impact on glycemic control. The observed attenuation of the normal pregnancy-induced rise in total and LDL cholesterol following probiotic treatment requires further investigation, particularly in this obstetric group at risk of future metabolic syndrome.
Assuntos
Diabetes Gestacional/terapia , Lactobacillus , Probióticos/uso terapêutico , Adulto , Biomarcadores/sangue , Glicemia/metabolismo , Peptídeo C/sangue , Colesterol/sangue , Diabetes Gestacional/sangue , Método Duplo-Cego , Feminino , Humanos , Recém-Nascido , Insulina/sangue , Análise de Intenção de Tratamento , Gravidez , Resultado da Gravidez , Resultado do TratamentoRESUMO
In the field of transplantation, flow cytometry serves a well-established role in pre-transplant crossmatching and monitoring immune reconstitution following hematopoietic stem cell transplantation. The capabilities of flow cytometers have continuously expanded and this combined with more detailed knowledge of the constituents of the immune system, their function and interaction and newly developed reagents to study these parameters have led to additional utility of flow cytometry-based analyses, particularly in the post-transplant setting. This review discusses the impact of flow cytometry on managing alloantigen reactions, monitoring opportunistic infections and graft rejection and gauging immunosuppression in the context of solid organ transplantation.
Assuntos
Citometria de Fluxo , Rejeição de Enxerto/imunologia , Transplante de Células-Tronco Hematopoéticas , Tolerância Imunológica , Isoantígenos/imunologia , Infecções Oportunistas/imunologia , Transplante de Órgãos , Animais , Separação Celular , Rejeição de Enxerto/prevenção & controle , Histocompatibilidade/imunologia , Teste de Histocompatibilidade , Humanos , Monitorização Imunológica/métodosRESUMO
We identified unique molecular heterogeneity of CD79 of human B cell antigen receptor (BCR) that may open a new approach to the ongoing CD79b-targeted therapy of B cell tumors. The primary purpose of the present study is to gain new information valuable for the enhanced CD79-targeted therapy. The molecular heterogeneity of CD79 was identified by sequential immunoprecipitation of BCR by use of anti-CD79b monoclonal antibody (mAb) SN8 and anti-CD79a mAb SN8b. SN8 is the antibody component of polatuzumab vedotin, an anti-CD79b antibody drug conjugate, that has been widely used for therapy of diffuse large B-cell lymphoma (DLBCL). The sequential immunoprecipitation shows that anti-CD79b mAb will be able to react only with a subgroup of CD79 molecules while anti-CD79a mAb will react with another subgroup of CD79 molecules; CD79 is a disulfide-linked heterodimer of CD79a and CD79b. Therapeutic study of SCID mice bearing human B-cell tumor shows synergistic potentiation by co-targeting CD79b and CD79a. Furthermore, simultaneous targeting of PD-1 strongly potentiates CD79a/CD79b-targeted therapy of B cell tumors. Flow cytometry analyses of CD79a/CD79b on malignant B cells of patients may provide a method for selection of the candidate patients for the CD79a/CD79b dual targeting therapy.
Assuntos
Linfoma Difuso de Grandes Células B , Receptores de Antígenos de Linfócitos B , Animais , Camundongos , Humanos , Camundongos SCID , Linfócitos B , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Transdução de SinaisRESUMO
The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Correpressor 1 de Receptor Nuclear/metabolismo , Neoplasias da Próstata/genética , Receptores de Calcitriol/metabolismo , Apoptose/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Calcitriol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Imunoprecipitação da Cromatina , Epigênese Genética , Humanos , Masculino , Correpressor 1 de Receptor Nuclear/genética , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Calcitriol/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Nuclear factor of activated T cells (NFAT) is a family of transcription factors involved in regulating the immune response. The canonical NFAT pathway is calcium-dependent and upon activation, NFAT is dephosphorylated by the phosphatase, calcineurin. This results in its translocation from the cytoplasm to the nucleus and transcription of downstream target genes that include the cytokines IL-2, IL-10, and IFNγ. Calcineurin inhibitors including tacrolimus inhibit the NFAT pathway and are used as immunosuppressants in transplant settings to prevent graft rejection. There is, as yet, no direct means to monitor tacrolimus pharmacodynamics. In this study, a rapid, quantitative, image cytometry-based measurement of nuclear translocation of NFAT1 is used to evaluate NFAT activation in T cells and its tacrolimus-induced inhibition. A strong dose-dependent correlation between NFAT1 inhibition and tacrolimus dose is demonstrated in vitro. Time kinetic analysis of NFAT1 inhibition in plasma from stable renal transplant recipients before and after an in vivo dose with tacrolimus correlated with the expected pharmacokinetic profile of tacrolimus. This was further corroborated by analysis of patients' autologous CD4 and CD8 T cells. This is the first report to show that the measurement of NFAT1 activation potential by nuclear translocation can be used as a direct, sensitive, reproducible and quantitative pharmacodynamic readout for tacrolimus action. These results, and the rapid turnaround time for this assay, warrant its evaluation in a larger clinical setting to assess its role in therapeutic drug monitoring of calcineurin inhibitors.
Assuntos
Núcleo Celular/metabolismo , Imunossupressores/farmacologia , Fatores de Transcrição NFATC/metabolismo , Tacrolimo/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ionóforos de Cálcio/farmacologia , Citometria de Fluxo , Rejeição de Enxerto/prevenção & controle , Humanos , Imunossupressores/farmacocinética , Imunossupressores/uso terapêutico , Interferon gama/sangue , Ionomicina/farmacologia , Células Jurkat , Transplante de Rim , Transporte Proteico , Reprodutibilidade dos Testes , Tacrolimo/farmacocinética , Tacrolimo/uso terapêutico , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Epigênese Genética , Próstata/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Calcitriol/farmacologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Retroalimentação Fisiológica , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Próstata/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
Hypoxic conditions preserve the multipotency and self-renewing capacity of murine bone marrow and human cord blood stem cells. Blood samples stored in sealed blood gas tubes become hypoxic as leukocytes metabolize and consume oxygen. Taken together, these observations suggest that peripheral blood stem cell (PBSC) samples stored under airtight conditions become hypoxic, and that the stem cells contained may undergo qualitative or quantitative changes. This study aimed to determine the effect of storage for 8 hours in a sealed system on PBSC samples. Granulocyte colony-stimulating factor-mobilized PBSC samples were collected prospectively from 9 patients with myeloma or amyloidosis prior to apheresis, followed by measurement of CO2, O2, hydrogen ion (pH), lactate, and glucose concentrations in the blood and immunophenotyping of stem cell and multipotent progenitor cell populations before and after 8 hours of storage in sealed blood collection tubes. Blood concentrations of O2 and glucose and pH measurements were significantly decreased, whereas concentrations of CO2 and lactate were significantly increased after storage. Significantly higher concentrations of CD34+ cells (552 ± 84 cells/106 total nucleated cells [TNCs] versus 985 ± 143 cells/106 TNCs; P = .03), CD34+CD38- cells (98 ± 32 cells/106 TNCs versus 158 ± 52 cells/106 TNCs; P = .03), CD34+CD38+ cells (444 ± 92 cells/106 TNCs versus 789 ± 153 cells/106 TNCs; P = .03), and CD34+CD38-CD45RA-CD90+ cells (55 ± 17 cells/106 TNCs versus 89 ± 25 cells/106 TNCs; P = .02) were detected after 8 hours of storage. The changes in concentrations of CD34+CD38+ cells and CD34+ cells were inversely associated with the change in glucose concentration (P = .003 and P < .001, respectively) and positively associated with the change in lactate concentration (P = .01 and P <.001, respectively) after 8 hours of airtight storage. Storage of PBSC samples in a sealed, airtight environment is associated with microenvironmental changes consistent with hypoxia and increased concentrations of immunophenotypically defined stem cells. These results may have clinical implications with regard to the collection and processing of stem cell products and warrant confirmation with functional and mechanistic studies.
Assuntos
Células-Tronco de Sangue Periférico , Humanos , Animais , Camundongos , Células-Tronco de Sangue Periférico/metabolismo , Dióxido de Carbono , Antígenos CD34/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Antígenos Thy-1/metabolismo , Moléculas de Adesão Celular , LactatosRESUMO
BACKGROUND: Presence of cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) predicts the effectiveness of cancer immunotherapies. The ability of toll-like receptor 3 (TLR3) ligands, interferons (IFNs) and COX2 inhibitors to synergistically induce CTL-attracting chemokines (but not regulatory T cell (Treg)-attractants) in the TME, but not in healthy tissues, observed in our preclinical studies, suggested that their systemic application can reprogram local TMEs. METHODS: Six evaluable patients (33-69 years) with metastatic triple-negative breast cancer received six doses of systemic chemokine-modulating (CKM) regimen composed of TLR3 ligand (rintatolimod; 200 mg; intravenous), IFN-α2b (20 MU/m2; intravenous) and COX2 inhibitor (celecoxib; 2×200 mg; oral) over 2 weeks. The predetermined primary endpoint was the intratumoral change in the expression of CTL marker, CD8α, in the post-CKM versus pre-CKM tumor biopsies. Patients received follow-up pembrolizumab (200 mg, intravenously, every 3 weeks), starting 3-8 days after completion of CKM. RESULTS: Post-CKM biopsies showed selectively increased CTL markers CD8α (average 10.2-fold, median 5.5-fold, p=0.034) and granzyme B (GZMB; 6.1-fold, median 5.8-fold, p=0.02), but not FOXP3 (Treg marker) relative to HPRT1 expression, resulting in the increases in average CD8α/FOXP3 ratio and GZMB/FOXP3 ratio. CKM increased intratumoral CTL-attractants CCL5 and CXCL10, but not Treg-attractants CCL22 or CXCL12. In contrast, CD8+ T cells and their CXCR3+ subset showed transient decreases in blood. One clinical response (breast tumor autoamputation) and three stable diseases were observed. The patient with clinical response remains disease free, with a follow-up of 46 months as of data cut-off. CONCLUSIONS: Short-term systemic CKM selectively increases CTL numbers and CTL/Treg ratios in the TME, while transiently decreasing CTL numbers in the blood. Transient effects of CKM suggest that its simultaneous application with checkpoint blockade and other forms of immunotherapy may be needed for optimal outcomes.
Assuntos
Neoplasias da Mama , Linfócitos T Citotóxicos , Humanos , Feminino , Linfócitos T Citotóxicos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Neoplasias da Mama/patologia , Receptor 3 Toll-Like/metabolismo , Microambiente Tumoral , Ligantes , Interferon-alfa/metabolismo , Fatores de Transcrição Forkhead/metabolismoRESUMO
Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects.
Assuntos
Aneuploidia , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/patologia , Algoritmos , Feminino , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/genética , Limite de Detecção , Masculino , Modelos Biológicos , Reprodutibilidade dos Testes , Análise de Célula ÚnicaRESUMO
BACKGROUND: Studies in T-cell acute lymphoblastic leukemia (T-ALL) have shown that leukemic blast populations may display immunophenotypic heterogeneity. In the clinical setting, evaluation of measurable residual disease during treatment and follow-up is highly dependent on knowledge of the diversity of blast subsets. Here, we set out to evaluate whether variation in expression of the blast marker, TdT, in T-ALL blasts could correspond to differences in morphometric features. METHODS: We investigated diagnostic bone marrow samples from six individual T-ALL patients run in parallel on imaging flow cytometry (IFC) and conventional flow cytometry (CFC). RESULTS: Guided by the imagery available in IFC, we identified distinct TdTneg and TdTpos subpopulations with apparent differences in internal complexity. As TdTneg blasts predominantly displayed very low forward scatter (FSC) on CFC, these subsets were initially excluded from routine analysis as debris, elements of small diameter, apoptotic, and/or dead cells. However, IFC-based morphometric analyses demonstrated that cell size and shape of TdTneg blasts were comparable to the TdTpos cells and without morphometric apoptotic hallmarks, supporting that the TdTneg subpopulation corresponded to T-ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdTneg FSCvery-low cells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdTneg FSCvery-low and TdTpos FSCint subsets. CONCLUSION: We highlight this finding as knowledge of phenotypic heterogeneity is of crucial importance in the clinical setting for delineation and quantification of blast subpopulations of potential biological relevance. We argue that the IFC imagery may allow for visual verification and improvement of applied gating strategies.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Doença Aguda , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Hibridização in Situ Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Linfócitos TRESUMO
The nuclear factor kappa B (NF-κB) pathway, which regulates many cellular processes including proliferation, apoptosis, and survival, has emerged as an important therapeutic target in cancer. Activation of the NF-κB transcription factor is associated with nuclear translocation of the p65 component of the complex. Conventional methods employed to determine nuclear translocation of NF-κB either lack statistical robustness (microscopy) or the ability to discern heterogeneity within the sampled populations (Western blotting and Gel Shift assays). The ImageStream platform combines the high image content information of microscopy with the high throughput and multiparameter analysis of flow cytometry which overcomes the aforementioned limitations of conventional assays. It is demonstrated that ImageStream assessment of receptor-mediated (TNFα) and drug (Daunorubicin, DNR)-induced NF-κB translocation in leukemic cell lines correlates well with microscopy analysis and Western blot analysis. It is further demonstrated that ImageStream cytometry enables quantitative assessment of p65 translocation in immunophenotypically defined subpopulations; and that this assessment is highly reproducible. It is also demonstrated that, quantitatively, the DNR-induced nuclear translocation of NF-κB correlates well with a biological response (apoptosis). We conclude that the ImageStream has the potential to be a powerful tool to evaluate NF-κB /p65 activity as a determinant of response to therapies designed to target aberrant NF-κB signaling activities.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citometria de Fluxo/métodos , Microscopia Confocal/métodos , Fator de Transcrição RelA , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Daunorrubicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/agonistas , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In humans, parity without breastfeeding increases risk of estrogen receptor-negative (ER-) breast cancer and is associated with hypermethylation of FOXA1, a pioneer factor regulating lineage commitment of mammary gland luminal progenitor cells. We postulate that pregnancy-associated repression of FOXA1 results in the accumulation of aberrant, differentiation-arrested luminal progenitor cells which, following additional genetic and epigenetic insults, may give rise to ER- tumors. Consistent with this hypothesis, we show that deletion of Foxa1 in the mouse mammary gland results in a two-fold increase in the proportion of luminal progenitor cells and a reduction in mammary gland epithelial cells that stain positive for ER. These results provide compelling support for the notion that reduced Foxa1 expression is sufficient to alter mammary gland luminal cell fate determination in vivo, which could be a mechanism linking parity with ER- breast cancer.
RESUMO
PURPOSE: We evaluated the antitumor efficacy of cetuximab in combination with pembrolizumab in patients with RAS wild-type (RASwt), metastatic colorectal adenocarcinoma (mCRC). PATIENTS AND METHODS: In this phase Ib/II study, cetuximab was combined with pembrolizumab in patients with RASwt mCRC with ≥ one prior line of therapy for advanced disease. We analyzed baseline on-treatment tumor tissues for changes in the tumor microenvironment (TME), using flow cytometry and multispectral immunofluorescence. RESULTS: Forty-four patients were evaluable for efficacy. The study was negative for the primary efficacy endpoint [overall response rate: 2.6%, 6-month progression-free survival (PFS): 31%; P = 0.52]. Median PFS was 4.1 months [95% confidence interval (CI): 3.9-5.5 months]. No increase in adverse effects was identified. We observed favorable immunomodulation with 47% increase in the number of intratumoral CTLs posttreatment (P = 0.035). These changes were more pronounced in patients with tumor shrinkage (P = 0.05). The TME was characterized by high numbers of TIM3+ and CTLA4+ cells; there were few activated OX40+ cells. PD-L1 expression was higher in pretreatment tumor cells from metastatic sites versus primary tumor samples (P < 0.05). Higher numbers of PD-L1+ tumor cells at baseline were associated with tumor shrinkage (P = 0.04). Analysis of immune populations in the blood demonstrated decreases in PD-1+ memory effector cells (P = 0.04) and granulocytic myeloid-derived suppressor cells (P = 0.03), with simultaneous increases in CD4+/CTLA4+ cells (P = 0.01). CONCLUSIONS: The combination of cetuximab and pembrolizumab is inactive in patients with RASwt mCRC, despite its partial local immunologic efficacy. Further development of immuno-oncology combinations with enhanced efficacy and/or targeting additional or alternative immune checkpoints merits investigation.