RESUMO
Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.
Assuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Mieloma Múltiplo , Humanos , Plasmócitos/patologia , Sobrevivência Celular , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Amiloide/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Mieloma Múltiplo/patologiaRESUMO
Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR T) therapy shows remarkable efficacy in patients with relapsed and/or refractory (R/R) multiple myeloma (MM). HBI0101, a novel second generation optimized anti- BCMA CAR T-cell therapy, was developed in an academic setting. We conducted a phase I dose-escalation study of HBI0101 (cohort 1: 150x106 CAR T cells, n=6; cohort 2: 450x106 CAR T cells, n=7; cohort 3: 800x106 CAR T cells, n=7) in 20 heavily pre-treated R/R MM patients. Grade 1-2 cytokine release syndrome (CRS) was reported in 18 patients (90%). Neither grade 3-4 CRS nor neurotoxicity of any grade were observed. No dose-limiting toxicities were observed in any cohort. The overall response rate (ORR), (stringent) complete response (CR/sCR), and very good partial response rates were 75%, 50%, and 25%, respectively. Response rates were dose-dependent with 85% ORR, 71% CR, and 57% minimal residual disease negativity in the high-dose cohort 3. Across all cohorts, the median overall survival (OS) was 308 days (range 25-466+), with an estimated OS of 55% as of June 27th (data cut-off). The median progression-free survival was 160 days, with 6 subjects remaining progression free at the time of data cut-off. Our findings demonstrate the manageable safety profile and efficacy of HBI0101. These encouraging data support the decentralization of CAR T production in an academic setting, ensuring sufficient CAR T supply to satisfy the increasing local demand. Clinicaltrials.gov NCT04720313.
Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Linfócitos T , AnticorposRESUMO
OBJECTIVE: Daratumumab is a promising new antimyeloma agent. We report a single center "real-world" series of multiple myeloma (MM) and amyloidosis (AL) patients treated with daratumumab. METHODS: Forty-one patients were included: 7 second-line MM, 30 heavily pretreated (median number of therapies of 5) advanced MM, and 4 with AL. RESULTS: Second-line patients and advanced AL showed high rate of durable overall responses. However, advanced MM patients had a dismal prognosis with an overall response rate (ORR) of 36%, and a short median progression-free and overall survival of 2.3 and 6.6 months, respectively. Responses were particularly poor in patients with extramedullary plasmacytomas. Neither the addition of another agent to daratumumab nor changing to the next line of therapy produced significant durable responses in this patient population. Flow cytometry analysis demonstrated that CD38 expression level was not predictive of response. We show that CD38 expression dynamics by a commercially available anti-CD38 antibody after daratumumab administration was hindered by competitive binding of daratumumab. CONCLUSIONS: Responses to daratumumab and combinations in patients with advanced MM, particularly with extramedullary disease, are low and short-lived, stressing the administration of this agent should be early in the course of the disease.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Amiloidose/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Idoso , Amiloidose/etiologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imunoglobulinas/metabolismo , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/mortalidade , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida , Resultado do TratamentoRESUMO
ABSTRACT: HBI0101 is an academic chimeric antigen receptor T-cell (CART)-targeted to B-cell maturation antigen (BCMA) for the treatment of relapsed and refractory multiple myeloma (R/RMM) and light chain amyloidosis. Herein, we present the phase 1b/2 results of 50 heavily pretreated patients with R/RMM dosed with 800 × 106 CART cells. Inclusion criteria were relatively permissive (i.e., performance status and baseline organ function) and consequently, approximately half of the enrolled patients would have been ineligible for pivotal clinical trials. The median time elapsed from patient enrollment until CART delivery was 25 days (range, 14-65). HBI0101-related toxicities included grade 1 to 3 cytokine release syndrome, grade 3 to 4 hematologic toxicities, and grade 1 to 2 immune effector cell-associated neurotoxicity syndrome. Responses were achieved in 90% of the patients, 56% achieved stringent and complete response, and 70% reached a minimal residual disease negativity. Within a median follow-up of 12.3 months, the median progression-free survival (PFS) was 11.0 months (95% confidence interval [CI], 6.2-14.6), and the overall survival was not reached (95% CI, 13.3 to not reached). Multivariable analysis on patient/disease and CART-related characteristics revealed that high-risk cytogenetic, extramedullary disease, and increased number of effector-memory T cells in CART products were independently associated with inferior PFS. In conclusion, comprehensive analyses of the parameters affecting the response to CART therapy are essential for improving patients' outcome. This trial was registered at www.ClinicalTrials.gov as #NCT04720313.
Assuntos
Antígeno de Maturação de Linfócitos B , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Idoso , Feminino , Adulto , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Resultado do Tratamento , Receptores de Antígenos Quiméricos/uso terapêutico , Recidiva , Idoso de 80 Anos ou mais , Anticorpos Monoclonais HumanizadosRESUMO
Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , NF-kappa B/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Apoptose/genética , Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Deleção Cromossômica , Cromossomos Humanos Par 13 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mieloma Múltiplo/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The immunogenicity of human pluripotent stem cells plays a major role in their potential use in the clinic. We show that, during their reprogramming, human-induced pluripotent stem (iPS) cells downregulate expression of human leukocyte antigen (HLA)-A/B/C and ß2 microglobulin (ß2M), the two components of major histocompatibility complex-I (MHC-I). MHC-I expression in iPS cells can be restored by differentiation or treatment with interferon-gamma (IFNγ). To analyze the molecular mechanisms that regulate the expression of the MHC-I molecules in human iPS cells, we searched for correlation between the expression of HLA-A/B/C and ß2M and the expression of transcription factors that bind to the promoter of these genes. Our results show a significant positive correlation between MHC-I expression and expression of the nuclear factors, nuclear factor kappa B 1 (NFκB1) and RelA, at the levels of RNA, protein and was confirmed by chromatin binding. Concordantly, we detected robust levels of NFκB1 and RelA proteins in the nucleus of somatic cells but not in the iPS cell derived from them. Overexpression of NFκB1 and RelA in undifferentiated pluripotent stem cells led to induction in expression of MHC-I, whereas silencing NFκB1 and RelA by small hairpin RNA decreased the expression of ß2M after IFNγ treatment. Our data point to the critical role of NFκB proteins in regulating the MHC-I expression in human pluripotent stem cells.
Assuntos
Reprogramação Celular/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/fisiologia , NF-kappa B/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Regulação para Baixo , Fibroblastos/citologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Interferon gama/imunologia , Interferon gama/farmacologia , Análise em Microsséries , NF-kappa B/biossíntese , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/imunologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Microglobulina beta-2/biossíntese , Microglobulina beta-2/imunologiaRESUMO
Multiple myeloma (MM) progression is dependent on its interaction with the bone marrow microenvironment and the immune system and is mediated by key surface antigens. Some antigens promote adhesion to the bone marrow matrix and stromal cells, while others are involved in intercellular interactions that result in differentiation of B-cells to plasma cells (PC). These interactions are also involved in malignant transformation of the normal PC to MM PC as well as disease progression. Here, we review selected surface antigens that are commonly used in the flow cytometry analysis of MM for identification of plasma cells (PC) and the discrimination between normal and malignant PC as well as prognostication. These include the markers: CD38, CD138, CD45, CD19, CD117, CD56, CD81, CD27, and CD28. Furthermore, we will discuss the novel marker CD24 and its involvement in MM. The bioactivity of each antigen is reviewed, as well as its expression on normal vs. malignant PC, prognostic implications, and therapeutic utility. Understanding the role of these specific surface antigens, as well as complex co-expressions of combinations of antigens, may allow for a more personalized prognostic monitoring and treatment of MM patients.
RESUMO
Surface antigens are commonly used in flow cytometry assays for the diagnosis of multiple myeloma (MM). Some of these are directly involved in MM pathogenesis or interactions with the microenvironment, but most are used for either diagnostic or prognostic purposes. In a previous study, we showed that in-vitro, CD24-positive plasma cells exhibit a less tumorigenic phenotype. Here, we assessed the prognostic importance of CD24 expression in patients newly diagnosed with MM as it correlates to their clinical course. Immunophenotyping by flow cytometry of 124 patients uniformly treated by a bortezomib-based protocol was performed. The expression of CD24, CD117, CD19, CD45, and CD56 in bone marrow PCs was tested for correlations to clinical parameters. None of the CD markers correlated with the response rates to first-line therapy. However, patients with elevated CD24+ expression on their PCs at diagnosis had a significantly longer PFS (p = 0.002) and OS (p = 0.044). In contrast, the expression of CD117, CD56, or CD45 was found to have no prognostic value; CD19 expression was inversely correlated with PFS alone (p < 0.001) and not with OS. Thus, elevated CD24 expression on PCs appears to be strongly correlated with survival and can be used as a single-surface antigenic prognostic factor in MM.
RESUMO
PURPOSE: AL amyloidosis (AL) treatments are generally based on those employed for multiple myeloma. Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T (CART)-cell therapy, already approved for multiple myeloma, might be too toxic for patients with AL. EXPERIMENTAL DESIGN: Here we describe the ex vivo applicability of a novel in-house, academic anti-BCMA CAR construct on AL primary cells, as well as the safety and efficacy in 4 patients with relapsed/refractory (RR) primary AL, treated in a phase I clinical trial (NCT04720313). RESULTS: Three had MAYO stage IIIa cardiac involvement at enrollment. The treatment proved relatively safe, with a short and manageable grade 3 cytokine release syndrome evident in 2 patients and no neurotoxicity in any. Cardiac decompensations, observed in 2 patients, were also short and manageable. The overall hematologic response and complete response rates were observed in all patients with an organ response evident in all four. Within a median follow-up period of 5.2 (2.5-9.5) months, all 4 patients maintained their responses. CONCLUSIONS: BCMA-CART cells provide a first proof-of-concept that this therapy is safe enough and highly efficacious for the treatment of patients with advanced, RR AL.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina , Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Estudos de Viabilidade , Amiloidose de Cadeia Leve de Imunoglobulina/tratamento farmacológico , Amiloidose de Cadeia Leve de Imunoglobulina/etiologia , Imunoterapia Adotiva/efeitos adversos , Mieloma Múltiplo/tratamento farmacológicoRESUMO
INTRODUCTION: AL amyloidosis (AL) is a malignant form of plasma cell dyscrasia (PCD). It is insidious, and its end-organ damage can mimic that of common diseases. At diagnosis, routine tests for monoclonal protein are insufficient for the differential diagnosis. We hypothesized that Hevylite® (HLC) isotype patterns may help discriminate between AL and benign PCD states. METHODS: Serum samples of patients with a high clinical suspicion of AL were prospectively tested for IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ concentrations and ratios using Hevylite® assays in a blinded manner. The results were correlated with the final diagnosis. RESULTS: Of the 99 samples analyzed, 46 were newly diagnosed AL, and the majority, 38 (82.6%), presented with suppression of at least one HLC isotype. Of the 53 benign PCD patients, 36 (67.9%) presented with elevation of at least one HLC isotype. By multivariate analysis, Hevylite® was the best independent test predictor of AL amyloidosis. HLC suppression had an odds ratio (OR) of 14.591, and elevation an OR of 10.149, and thus were significant variables in the diagnosis and exclusion of AL. Furthermore, patients with both HLC suppression, together with no elevation, had an OR of 316.69 to be diagnosed with AL rather than a benign PCD. CONCLUSIONS: Hevylite® HLC analysis for Ig isotypes patterns offers an effective non-invasive tool in the evaluation of patients with high suspicion of AL and may assist further explorative decisions for diagnosis.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina/diagnóstico , Paraproteinemias/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Testes Hematológicos/métodos , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Amiloidose de Cadeia Leve de Imunoglobulina/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Paraproteinemias/sangueRESUMO
Genomic imprinting is an epigenetic phenomenon whereby genes are expressed in a monoallelic manner, which is inherited either maternally or paternally. Expression of imprinted genes has been examined in human embryonic stem (ES) cells, and the cells show a substantial degree of genomic imprinting stability. Recently, human somatic cells were reprogrammed to a pluripotent state using various defined factors. These induced pluripotent stem (iPS) cells are thought to have a great potential for studying genetic diseases and to be a source of patient-specific stem cells. Thus, studying the expression of imprinted genes in these cells is important. We examined the allelic expression of various imprinted genes in several iPS cell lines and found polymorphisms in four genes. After analyzing parent-specific expression of these genes, we observed overall normal monoallelic expression in the iPS cell lines. However, we found biallelic expression of the H19 gene in one iPS cell line and biallelic expression of the KCNQ10T1 gene in another iPS cell line. We further analyzed the DNA methylation levels of the promoter region of the H19 gene and found that the cell line that showed biallelic expression had undergone extensive DNA demethylation. Additionally we studied the imprinting gene expression pattern of multiple human iPS cell lines via DNA microarray analyses and divided the pattern of expression into three groups: (a) genes that showed significantly stable levels of expression in iPS cells, (b) genes that showed a substantial degree of variability in expression in both human ES and iPS cells, and (c) genes that showed aberrant expression levels in some human iPS cell lines, as compared with human ES cells. In general, iPS cells have a rather stable expression of their imprinted genes. However, we found a significant number of cell lines with abnormal expression of imprinted genes, and thus we believe that imprinted genes should be examined for each cell line if it is to be used for studying genetic diseases or for the purpose of regenerative medicine.
Assuntos
Impressão Genômica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genéticaRESUMO
The Mixl1 gene encodes a homeodomain transcription factor that is required for normal mesoderm and endoderm development in the mouse. We have examined the consequences of enforced Mixl1 expression during mouse embryonic stem cell (ESC) differentiation. We show that three independently derived ESC lines constitutively expressing Mixl1 (Mixl1(C) ESCs) differentiate into embryoid bodies (EBs) containing a higher proportion of E-cadherin (E-Cad)(+) cells. Our analysis also shows that this differentiation occurs at the expense of hematopoietic mesoderm differentiation, with Mixl1(C) ESCs expressing only low levels of Flk1 and failing to develop hemoglobinized cells. Immunohistochemistry and immunofluorescence studies revealed that Mixl1(C) EBs have extensive areas containing cells with an epithelial morphology that express E-Cad, FoxA2, and Sox17, consistent with enhanced endoderm formation. Luciferase reporter transfection experiments indicate that Mixl1 can transactivate the Gsc, Sox17, and E-Cad promoters, supporting the hypothesis that Mixl1 has a direct role in definitive endoderm formation. Taken together, these studies suggest that high levels of Mixl1 preferentially allocate cells to the endoderm during ESC differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Proteínas de Homeodomínio/fisiologia , Mesoderma/citologia , Mesoderma/metabolismo , Animais , Células 3T3 BALB , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Endoderma/citologia , Citometria de Fluxo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismoRESUMO
Multiple myeloma (MM) is an incurable neoplasm characterized by infiltration of malignant plasma cells (PCs). Recently, the tumor microenvironment has become of great interest in MM as it known to be involved in progression and metastasis of the disease. CD24, is an adhesion molecule expressed during B cell maturation, is down regulated through the cells differentiation into PCs. Though the role of CD24 in solid cancers is well defined, its role in MM remains unknown. We aimed to understand the involvement of CD24 in MM by up-regulating its expression on MM cell lines by co-culturing the cells with bone marrow stromal cell (BMSCs). We then studied the differences between CD24+ and CD24- MM cells and found that CD24+ MM cells presented a less tumorigenic phenotype by impaired capability to migrate and to create colonies as compared with CD24- MM cells. Furthermore, there were significantly more apoptotic cells in the CD24+ fraction. Additionally, the CD24+ cells also upregulated CXCR4 expression. The decrease tumorigenicity correlated with a "more normal" PC immunophenotype in patients with MM and correlated with CD45 expression and a stronger expression of CXCR4. In summary, we found the expression of CD24 on PCs to correlate with attenuated tumorigenicity.
RESUMO
We have utilized a serum- and stromal cell-free "spin embryoid body (EB)" differentiation system to investigate the roles of four growth factors, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), and basic fibroblast growth factor (FGF2), singly and in combination, on the generation of hematopoietic cells from human embryonic stem cells (HESCs). Of the four factors, only BMP4 induced expression of genes that signaled the emergence of the primitive streak-like population required for the subsequent development of hematopoietic mesoderm. In addition, BMP4 initiated the expression of genes marking hematopoietic mesoderm and supported the generation of hematopoietic progenitor cells at a low frequency. However, the appearance of robust numbers of hematopoietic colony forming cells and their mature progeny required the inclusion of VEGF. Finally, the combination of BMP4, VEGF, SCF, and FGF2 further enhanced the total yield of hematopoietic cells. These data demonstrate the utility of the serum-free spin EB system in dissecting the roles of specific growth factors required for the directed differentiation of HESCs toward the hematopoietic lineage.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias/citologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Hematopoese/fisiologia , Fator de Células-Tronco/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/farmacologia , Células Cultivadas , Combinação de Medicamentos , Sinergismo Farmacológico , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Linha Primitiva/metabolismo , Fator de Células-Tronco/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
BACKGROUND: ZAP-70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP-70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP-70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories. METHODS: Intracellular ZAP-70 levels in CLL and normal B cells were assessed by molecules of equivalent soluble fluorochrome (MESF), employing Quantum FITC MESF calibration beads to establish a standard curve relating channel value to fluorescence intensity in MESF units and the QuickCal v. 2.2 program (www.bangslabs.com) and clinical relevance of the data was determined. RESULTS: The average ZAP-70 expression value in the CD19(+)/CD5(+) cells from 35 CLL patients was 103,701 MESF when compared with 12,621 MESF in B cells from 20 normal blood samples. "Low" and "high" ZAP-70 CLL subgroups were defined. Patients with "high ZAP-70 MESF" CLL had a shorter time to disease progression (P = 0.0005) and a more advanced clinical stage (P = 0.0018) when compared with patients in the "low ZAP-70 MESF" CLL subgroup. CONCLUSIONS: This quantitative analysis method can be employed to obtain a more specific and highly accurate assessment of ZAP-70 levels in CLL cells. The method can easily be standardized, in any routine flow laboratory, thereby improving reproducibility and reliability of ZAP-70 analysis.
Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Leucemia Linfocítica Crônica de Células B/diagnóstico , Proteína-Tirosina Quinase ZAP-70/análise , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/biossíntese , Progressão da Doença , Feminino , Seguimentos , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Solubilidade , Taxa de Sobrevida , Resultado do Tratamento , Proteína-Tirosina Quinase ZAP-70/biossínteseRESUMO
Human pluripotent stem cells (hPSC) have the potential to produce any tissue type in the body and thus represent a source of cells for regenerative medicine. Here we have shown that human platelets can be produced from embryonic or induced pluripotent stem cells in a defined culture system. We describe a serum- and feeder-free culture system that enabled the generation of megakaryocyte (Mk) progenitors and functional platelets from hPSCs. After 13 days the differentiated population included precursor cells that formed colonies containing differentiated Mks, and after 20 days these Mks were able to fragment into platelet-like particles that were functional. This protocol represents an important step towards the generation of human platelets for therapeutic use.
Assuntos
Plaquetas/citologia , Técnicas de Cultura de Células/métodos , Megacariócitos/citologia , Células-Tronco Pluripotentes/citologia , Animais , Plaquetas/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Ensaio de Unidades Formadoras de Colônias , Embrião de Mamíferos/citologia , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Humanos , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Metilcelulose/farmacologia , Camundongos , Ativação Plaquetária/efeitos dos fármacos , Ploidias , Células-Tronco Pluripotentes/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: Nonsteroidal anti-inflammatory drugs reduce the risk of colorectal cancer. The cyclooxygenase (COX) pathway of arachidonic acid metabolism is an important target for nonsteroidal anti-inflammatory drugs. Increased expression of COX-2 was recently shown to be an important step in the multistep process of colorectal cancer carcinogenesis. The new COX-2-specific inhibitors offer the benefit of cancer protection without the gastrointestinal toxicity reported for the old drugs. The purpose of this study was to compare the growth effects of two specific COX-2 inhibitors, celecoxib (Pfizer, Inc., New York, NY), and rofecoxib (Merck, White House Station, NJ) in normal and transformed enterocytes. EXPERIMENTAL DESIGN: Cultures of normal rat intestinal epithelial cell line, IEC-18, vector control cells, c-K-ras, c-K-ras-bak, and antisense-bak derivatives were treated with different dosages of celecoxib (0-60 micro M) and rofecoxib (0-20 micro M). Cell cycle analysis and apoptosis were assessed by fluorescence-activated cell sorting analysis. Protein expression was assessed by Western blot analysis and caspases 3 and 8 activities by ELISA. RESULTS: Celecoxib inhibited cell growth and induced apoptosis in a time- and dose-dependent manner. IEC18 parental cells were two to four times more resistant to celecoxib than ras, ras-bak, and antisense bak transformed cells that overexpress the COX-2 protein. The induction of apoptosis by celecoxib involved the caspase pathways. Rofecoxib, up to its maximal concentration of 20 micro M, did not inhibit cell growth or induce apoptosis. CONCLUSIONS: Celecoxib may prove to be a very efficient component in the prevention and treatment of gastrointestinal tumors because it inhibits the growth of cancerous cells without affecting the growth of normal cells.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Transformada/efeitos dos fármacos , Enterócitos/efeitos dos fármacos , Lactonas/farmacologia , Sulfonamidas/farmacologia , Animais , Caspase 3 , Caspase 8 , Caspases/metabolismo , Celecoxib , Linhagem Celular Transformada/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Enterócitos/citologia , Enterócitos/metabolismo , Ensaio de Imunoadsorção Enzimática , Genes ras/fisiologia , Técnicas In Vitro , Pirazóis , Ratos , SulfonasRESUMO
OBJECTIVE: Umbilical cord blood (CB) provides an alternative source of hematopoietic progenitor cells for transplantation; however, prolonged thrombocytopenia remains a major obstacle due to the low numbers of megakaryocyte progenitor (Mk-prog) cells and their subsequent delayed engraftment. In this study, we improved techniques for enrichment, cryopreservation, and ex vivo expansion of Mk-prog cells from CB. MATERIALS AND METHODS: CB mononuclear cells (MNC) were isolated and Mk-prog enriched by sedimentation on gelatin followed by centrifugation with Ficoll-Hypaque and cryopreserved. The capacity of MNC to produce Mk-prog cells, assessment of CD34(+) and Mk-prog expansion in liquid culture, and analysis of the cell populations by flow cytometry were studied in cryopreserved separated CB and compared to whole CB and freshly separated samples. RESULTS: Excellent viability of greater than 85% was maintained after cryopreservation of separated CB. The number of colony-forming Mk-prog, myeloid, and erythroid progenitor cells did not decrease with cryopreservation. Flow cytometric analysis of cryopreserved cells revealed significant removal of the residual red blood cells while maintaining complete recovery of CD34(+), CD41(+) (Mk), myeloid, and T and B cells compared to noncryopreserved CB cells. There was no difference in the ability of separated cryopreserved MNC CB cells to be expanded in short-term liquid cultures. CONCLUSIONS: The conditions defined here for cryopreservation of gelatin/Ficoll-Hypaque separated CB, followed by ex vivo expansion of MNC, allowed complete recovery of proliferating CD41(+), CD34(+), Mk-prog cells, and other hematopoietic progenitors. Mk-prog cell expansion just before the scheduled transplantation is easily applicable by this technically simple and economical procedure that requires only an aliquot of red cell cell-depleted MNC to be separated from the CB unit before cryopreservation.
Assuntos
Preservação de Sangue , Separação Celular/métodos , Criopreservação , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Megacariócitos/citologia , Transplante de Células-Tronco , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas/citologia , Ensaio de Unidades Formadoras de Colônias , Diatrizoato , Ficoll , Citometria de Fluxo , Gelatina , Células-Tronco Hematopoéticas/classificação , Humanos , Recém-NascidoRESUMO
OBJECTIVE: Hematopoietic stress responses involve increases in leukocyte and platelet counts, implying the existence of stress responsive factors that modulate hematopoiesis. Acetylcholinesterase (AChE) is expressed in mammalian neurons and hematopoietic cells. In brain, it responds to stress by mRNA overexpression and alternative splicing, yielding the rare stress-associated "readthrough" AChE-R variant protein. This led us to explore the hematopoietic involvement of AChE-R and its cleavable C-terminal peptide ARP. MATERIALS AND METHODS: AChE mRNA variants were labeled in CD34(+) hematopoietic progenitor cells by in situ hybridization. ARP expression was detected by multicolor flow cytometry. Bromo-deoxyuracil incorporation and viable cell counts served to evaluate the proliferative effects of ARP and suppressive effects of the AChE antisense oligonucleotide AS1 on CD34(+) cells. RESULTS: The distal enhancer, proximal promoter, and first intron of the human AChE gene include consensus binding sites for hematopoietically active and stress-induced transcription factors. CD34(+) cells from human cord blood were found to express all three variant AChE mRNAs, having different intracellular distributions. ARP was found in 5 to 15% of adult peripheral blood, bone marrow, and fetal CD34(+) cells (both committed CD38(+) and uncommitted CD38(-)) and in acute myeloid leukemia blasts. Externally supplied ARP by itself facilitated the proliferation of CD34(+) cells in an antisense suppressible manner. When combined with early-acting cytokines, ARP enhanced survival and expansion of CD34(+) cells up to 28 days in culture. CONCLUSIONS: Our findings support ARP, the C-terminal peptide of AChE-R, as a new hematopoietic growth factor that may promote the myelopoietic expansion and thrombopoiesis characteristic of stress and may be used to enhance the efficiency of ex vivo expansion for bone marrow transplantation.
Assuntos
Acetilcolinesterase/genética , Divisão Celular/fisiologia , Variação Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Estresse Fisiológico/genética , Adulto , Antígenos CD/sangue , Antígenos CD34/sangue , Sangue Fetal/citologia , Feto/fisiologia , Citometria de Fluxo , Substâncias de Crescimento/farmacologia , Humanos , Imunofenotipagem , Hibridização In Situ , Recém-Nascido , Oligodesoxirribonucleotídeos Antissenso , Fragmentos de Peptídeos/farmacologia , Estresse Fisiológico/enzimologiaRESUMO
Three principal features of mammalian stress responses are that they span peripheral and CNS changes, modify blood cell composition and activities, and cover inter-related alterations in a large number of gene products. The finely tuned spatiotemporal regulation of these multiple events suggests the hierarchic involvement of modulatory neurotransmitters and modified process(es) in the pathway of gene expression that together would enable widely diverse stress responses. We report evidence supporting the notion that acetylcholine (ACh) acts as a stress-response-regulating transmitter and that altered ACh levels are variously associated with changes in the alternative splicing of pre-mRNA transcripts in brain neurons and peripheral blood cells. We used acetylcholinesterase (AChE) gene expression as a case study and developed distinct probes for its alternative splice variants at the mRNA and protein levels. In laboratory animals and human-derived cells, we found stress-induced changes in the alternative splicing patterns of AChE pre-mRNA, which attributes to this gene and its different protein products diverse stress responsive functions that are associated with the enzymatic and noncatalytic properties of AChE. Together, these approaches provide a conceptually unified view of the studied pathways for controlling stress responses in brain and blood.