RESUMEN
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.
Asunto(s)
Antígeno de Maduración de Linfocitos B , Mieloma Múltiple , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/mortalidad , Mieloma Múltiple/terapia , Antígeno de Maduración de Linfocitos B/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Anciano , Femenino , Adulto , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Resultado del Tratamiento , Receptores Quiméricos de Antígenos/uso terapéutico , Recurrencia , Anciano de 80 o más Años , Anticuerpos Monoclonales HumanizadosRESUMEN
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.
Asunto(s)
Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Mieloma Múltiple , Humanos , Células Plasmáticas/patología , Supervivencia Celular , Amiloidosis/genética , Amiloidosis/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/genética , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/patología , Amiloide/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Mieloma Múltiple/patologíaRESUMEN
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.
Asunto(s)
Mieloma Múltiple , Receptores Quiméricos de Antígenos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Linfocitos T , AnticuerposRESUMEN
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.
Asunto(s)
Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Mieloma Múltiple , Receptores Quiméricos de Antígenos , Humanos , Estudios de Factibilidad , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/tratamiento farmacológico , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/etiología , Inmunoterapia Adoptiva/efectos adversos , Mieloma Múltiple/tratamiento farmacológicoRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Paraproteinemias/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Diferencial , Femenino , Pruebas Hematológicas/métodos , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Paraproteinemias/sangreRESUMEN
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.
RESUMEN
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.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Amiloidosis/tratamiento farmacológico , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Anciano , Amiloidosis/etiología , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunoglobulinas/metabolismo , Masculino , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/mortalidad , Estadificación de Neoplasias , Pronóstico , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Plaquetas/citología , Técnicas de Cultivo de Célula/métodos , Megacariocitos/citología , Células Madre Pluripotentes/citología , Animales , Plaquetas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Colágeno/farmacología , Ensayo de Unidades Formadoras de Colonias , Embrión de Mamíferos/citología , Células Nutrientes/citología , Células Nutrientes/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Megacariocitos/efectos de los fármacos , Metilcelulosa/farmacología , Ratones , Activación Plaquetaria/efectos de los fármacos , Ploidias , Células Madre Pluripotentes/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Endurance, marathon-type exertion is known to induce adverse changes in the immune system. Increased airway hyper-responsiveness and airway inflammation are well documented in endurance athletes and endurance exercise is considered a major risk factor for asthma in elite athletes. Yet, the mechanisms underlying this phenomenon are still to be deduced. We studied the effect of strenuous endurance exercise (marathon and half-ironman triathlon) on CD4+ lymphocyte sub-populations and on the balance between effector and regulatory CD4+ lymphocytes in the peripheral blood of trained athletes, Endurance exercise induced a significant increase in Th17 cells and a sustained decrease in peripheral blood regulatory T cells (Tregs). While interleukin (IL)-2 levels remained undetectable, post-race serum IL-6 and transforming growth factor (TGF) ß levels were significantly elevated. Treg levels in sedentary controls' decreased in vitro after incubation with athletes' post-exercise serum, an effect that was attenuated by supplements of IL-2 or anti IL-6 neutralizing antibodies. Our data suggest that exercise-induced changes in serum cytokine levels promote alterations in Tregs and Th17 cell populations, which may divert the subtle balance in the immune system towards inflammation. This may explain allergic and autoimmune phenomena previously reported in endurance athletes and contribute to our understanding of exercise-related asthma.
Asunto(s)
Ejercicio Físico/fisiología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Adulto , Linfocitos T CD4-Positivos/metabolismo , Citocinas/sangre , Femenino , Humanos , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Mieloma Múltiple/genética , Mieloma Múltiple/patología , FN-kappa B/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Apoptosis/genética , Ciclo Celular/genética , División Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Deleción Cromosómica , Cromosomas Humanos Par 13 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Mieloma Múltiple/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⺠cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⺠Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⺠and CD45⺠cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Megacariocitos/citología , Adenosina Difosfato/farmacología , Antígenos CD/metabolismo , Plaquetas/metabolismo , Diferenciación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Células Nutrientes , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Megacariocitos/efectos de los fármacos , Factor Plaquetario 4/genética , Poliploidía , Receptores de Trombopoyetina/genética , Factores de TiempoRESUMEN
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.
Asunto(s)
Reprogramación Celular/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/fisiología , FN-kappa B/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Diferenciación Celular/fisiología , Línea Celular Tumoral , Células Cultivadas , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Regulación hacia Abajo , Fibroblastos/citología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoquímica , Células Madre Pluripotentes Inducidas/citología , Interferón gamma/inmunología , Interferón gamma/farmacología , Análisis por Micromatrices , FN-kappa B/biosíntesis , FN-kappa B/genética , FN-kappa B/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/inmunología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor de Transcripción ReIA/biosíntesis , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIA/metabolismo , Microglobulina beta-2/biosíntesis , Microglobulina beta-2/inmunologíaRESUMEN
The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogren's syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9(-/-) mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-κB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogren's syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-κB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9(-/-) mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogren's disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-κB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-κB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogren's syndrome and may be applicable to additional autoimmune syndromes.
Asunto(s)
FN-kappa B/metabolismo , Transducción de Señal , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Animales , Biomarcadores/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/patología , Ratones , Saliva/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Glándulas Salivales/fisiopatología , Síndrome de Sjögren/fisiopatología , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/deficienciaRESUMEN
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.
Asunto(s)
Impresión Genómica/genética , Células Madre Pluripotentes Inducidas/metabolismo , Metilación de ADN/genética , Metilación de ADN/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Proteínas de Homeodominio/fisiología , Mesodermo/citología , Mesodermo/metabolismo , Animales , Células 3T3 BALB , Western Blotting , Cadherinas/genética , Cadherinas/metabolismo , Diferenciación Celular/genética , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Endodermo/citología , Citometría de Flujo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Ratones , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Diferenciación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Células Madre Embrionarias/citología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Hematopoyesis/fisiología , Factor de Células Madre/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/farmacología , Células Cultivadas , Combinación de Medicamentos , Sinergismo Farmacológico , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Línea Primitiva/metabolismo , Factor de Células Madre/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
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.
Asunto(s)
Citometría de Flujo/métodos , Colorantes Fluorescentes/análisis , Leucemia Linfocítica Crónica de Células B/diagnóstico , Proteína Tirosina Quinasa ZAP-70/análisis , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfocitos B/metabolismo , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/biosíntesis , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Solubilidad , Tasa de Supervivencia , Resultado del Tratamiento , Proteína Tirosina Quinasa ZAP-70/biosíntesisRESUMEN
To study the role of the stress-induced "readthrough" acetylcholinesterase splice variant, AChE-R, in thrombopoiesis, we used transgenic mice overexpressing human AChE-R (TgR). Increased AChE hydrolytic activity in the peripheral blood of TgR mice was associated with increased thrombopoietin levels and platelet counts. Bone marrow (BM) progenitor cells from TgR mice presented an elevated capacity to produce mixed (GEMM) and megakaryocyte (Mk) colonies, which showed intensified labeling of AChE-R and its interacting proteins RACK1 and PKC. When injected with bacterial lipopolysaccharide (LPS), parent strain FVB/N mice, but not TgR mice, showed reduced platelet counts. Therefore, we primed human CD34+ cells with the synthetic ARP26 peptide, derived from the cleavable C-terminus of AChE-R prior to transplantation, into sublethally irradiated NOD/SCID mice. Engraftment of human cells (both CD45+ and CD41+ Mk) was significantly increased in mice that received ARP26-primed CD34+ human cells versus mice that received fresh nonprimed CD34+ human cells. Moreover, ARP26 induced polyploidization and proplatelet shedding in human MEG-01 promegakaryotic cells, and human platelet engraftment increased following ex vivo expansion of ARP26-treated CD34+ cells as compared to cells expanded with thrombopoietin and stem cell factor. Our findings implicate AChE-R in thrombopoietic recovery, suggesting new therapeutic modalities for supporting platelet production.