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OBJECTIVES: To investigate laboratory and bone marrow findings that can help predict a diagnosis of hemophagocytic lymphohistiocytosis (HLH) for patients who have demonstrated hemophagocytes (HPCs) in the bone marrow. METHODS: A total of 57 cases from 48 patients with HPCs present on bone marrow examination were included. The numbers and morphologic characteristics of HPCs with ingested nucleated cells (nHPC) were counted. Pertinent medical history, relevant laboratory values, and flow cytometry data at the time of bone marrow biopsy were collected. RESULTS: A total of 24 patients fulfilled diagnostic criteria for HLH, and the remaining 24 patients did not. By using HLH-2004 cutoffs, only hypertriglyceridemia (≥265 mg/dL) was significantly associated with HLH diagnosis. The HLH cases more frequently had nHPC-ingesting granulocytic cells (gHPC) (75.9% vs 24.1%, P = .009). The percentage of gHPC to all nHPC was also significantly higher in HLH cases (median, 15.4% vs 0%; P = .0002). Both triglyceride level (area under the curve [AUC] = 0.88, P < .0001) and gHPC percentage (AUC = 0.81, P = .0005) were significant in predicting HLH diagnosis. Finally, no overt immunophenotypic abnormality was noted for 19 HLH cases with available flow cytometry data. CONCLUSIONS: The presence of hypertriglyceridemia and more frequent gHPC has predictive value for HLH diagnosis in patients with bone marrow HPC.
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Hipertrigliceridemia , Linfohistiocitosis Hemofagocítica , Humanos , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/patología , Médula Ósea/patología , Examen de la Médula Ósea , Hipertrigliceridemia/diagnóstico , Hipertrigliceridemia/patología , BiopsiaRESUMEN
OBJECTIVES: This study compares the effectiveness of an interactive e-learning module with a traditional text-based method for teaching peripheral blood smear analysis. METHODS: Pathology trainees at Accreditation Council for Graduate Medical Education residency programs were asked to participate. Participants completed a multiple-choice test on peripheral blood smear findings. Trainees were randomized into completing an e-learning module or a PDF reading exercise with the same educational content. Respondents rated their experience and completed a postintervention test composed of the same questions. RESULTS: In total, 28 participants completed the study; 21 improved their score in the posttest (mean, 21.6 correct answers) compared with the pretest (19.8; P < .001). This improvement was seen in both the PDF (n = 19) and interactive (n = 9) groups, with no difference in performance between the 2 groups. Trainees with less clinical hematopathology experience showed a trend of having the largest performance improvement. Most participants completed the exercise within 1 hour, rated the exercise as easy to navigate, were engaged, and reported learning new information about peripheral blood smear analysis. All participants indicated that they would likely complete a similar exercise in the future. CONCLUSIONS: This study suggests that e-learning is an effective tool for hematopathology education and equivalent to traditional narrative-based methods. This module could easily be incorporated into a curriculum.
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Instrucción por Computador , Internado y Residencia , Humanos , Instrucción por Computador/métodos , Educación de Postgrado en Medicina/métodos , Curriculum , Evaluación EducacionalRESUMEN
OBJECTIVE: Obesity has emerged as a prominent risk factor for multiple serious disease states, including a variety of cancers, and is increasingly recognized as a primary contributor to preventable cancer risk. However, few studies of leukemia have been conducted in animal models of obesity. This study sought to characterize the impact of obesity, diet, and sex in a murine model of acute promyelocytic leukemia (APL). METHODS: Male and female C57BL/6J.mCG+/PR mice, genetically predisposed to sporadic APL development, and C57BL/6J (wild type) mice were placed on either a high-fat diet (HFD) or a low-fat diet (LFD) for up to 500 days. RESULTS: Relative to LFD-fed mice, HFD-fed animals displayed increased disease penetrance and shortened disease latency as indicated by accelerated disease onset. In addition, a diet-responsive sex difference in APL penetrance and incidence was identified, with LFD-fed male animals displaying increased penetrance and shortened latency relative to female counterparts. In contrast, both HFD-fed male and female mice displayed 100% disease penetrance and insignificant differences in disease latency, indicating that the sexual dimorphism was reduced through HFD feeding. CONCLUSIONS: Obesity and obesogenic diet promote the development of APL in vivo, reducing sexual dimorphisms in disease latency and penetrance.
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Leucemia Promielocítica Aguda , Caracteres Sexuales , Animales , Dieta Alta en Grasa/efectos adversos , Femenino , Leucemia Promielocítica Aguda/complicaciones , Leucemia Promielocítica Aguda/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , PenetranciaRESUMEN
OBJECTIVES: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a group of rare and heterogeneous hematopoietic disorders that frequently present a diagnostic challenge. Here we present our institutional experience with next-generation sequencing (NGS), together with morphologic, flow cytometric, and cytogenetic evaluation, in the diagnosis of MDS/MPN, with particular emphasis on MDS/MPN unclassifiable (MPN-U). METHODS: We evaluated the morphologic, flow cytometric, cytogenetic, and molecular characteristics of all MDS/MPN cases that underwent NGS at our institution between April 2016 and February 2019. RESULTS: Thirty-seven cases of MDS/MPN were identified, including 14 cases of MDS/MPN-U. Ninety-seven percent harbored mutations and immunophenotypic aberrancies (36/37), while only 38% had cytogenetic abnormalities (12/32). The MDS/MPN-U group had the highest rate of myeloblast phenotypic abnormalities and had a high mutation rate of approximately 2.7 mutated genes per case, most commonly in JAK2, SRSF2, and ASXL1. CONCLUSIONS: No single ancillary study was abnormal in every case, but all cases had at least one abnormal finding, demonstrating the usefulness of a multiparameter approach to the diagnosis of MDS/MPN. Although a few specific mutations were found exclusively in MDS/MPN-U and JAK2 mutations were most prevalent, larger studies are needed to determine whether MDS/MPN-U has a mutational "fingerprint," which may aid in diagnosis and targeted therapy.
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Enfermedades Mielodisplásicas-Mieloproliferativas/diagnóstico , Trastornos Mieloproliferativos/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Citogenética , Femenino , Citometría de Flujo , Células Precursoras de Granulocitos/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Mutación , Tasa de Mutación , Enfermedades Mielodisplásicas-Mieloproliferativas/clasificación , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/patología , Trastornos Mieloproliferativos/clasificación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Hematopathology fellowship education has grown in complexity as patient-centered treatment plans have come to depend on integration of clinical, morphologic, immunophenotypic, molecular, and cytogenetic variables. This complexity is in competition with the need for timely hematopathology care with stewardship of patient, laboratory, and societal resources. Accreditation Council for Graduate Medical Education Milestones provide a guidance document for hematopathology training, but fellows and their educators are in need of a simple framework that allows assessment and feedback of growth toward independent hematopathology practice. Entrustable professional activities provide one such framework, and herein, we provide proposed Hematopathology Fellowship Entrustable Professional Activities based on review of pertinent guidelines and literature, with multiple rounds of expert and stakeholder input utilizing a modified mini-Delphi approach. Ten core entrustable professional activities deemed essential for graduating hematopathology fellows were developed together with skills and knowledge statements, example scenarios, and corresponding Accreditation Council for Graduate Medical Education Milestones. Application of these entrustable professional activities in program design, fellow evaluation, and decisions regarding level of supervision is discussed with consideration of benefits and barriers to implementation. These entrustable professional activities may be used by hematopathology fellowship directors and faculty to provide fellows with timely constructive feedback, determine entrustment decisions, provide the Clinical Competency Committee with granular data to support Milestone evaluations, and provide insight into areas of potential improvement in fellowship training. Fellows will benefit from a clear roadmap to independent hematopathology practice with concrete and timely feedback.
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OBJECTIVES: We investigated the usefulness of a custom-designed 31-gene next-generation sequencing (NGS) panel implemented on a routine basis for the evaluation of low-grade lymphoproliferative disorders (LPDs). METHODS: In total, 147 blood, bone marrow, and tissue specimens were sequenced, including 81% B-cell, 15% T-cell, and 3% natural killer (NK)-cell neoplasms. RESULTS: Of the cases, 92 (63%) of 147 displayed at least one pathogenic variant while 41 (28%) of 147 had two or more. Low mutation rates were noted in monoclonal B-cell lymphocytoses and samples with small T- and NK-cell clones of uncertain significance. Pathogenic molecular variants were described in specific disorders and classified according to their diagnostic, prognostic, and potential therapeutic value. Diagnostically, in addition to confirming the diagnosis of 15 of 15 lymphoplasmacytic lymphomas, 10 of 12 T large granular lymphocytic leukemias, and 2 of 2 hairy cell leukemias (HCLs), the panel helped resolve the diagnosis of 10 (62.5%) of 16 challenging cases lacking a specified diagnosis based on standard morphology, phenotype, and genetic analysis. CONCLUSIONS: Overall, implementation of this targeted lymphoid NGS panel as part of regular hematopathology practice was found to be a beneficial adjunct in the evaluation of low-grade LPDs.
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Leucemia de Células Pilosas/diagnóstico , Leucemia Linfocítica Granular Grande/diagnóstico , Linfoma de Células B/diagnóstico , Trastornos Linfoproliferativos/diagnóstico , Macroglobulinemia de Waldenström/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/patología , Médula Ósea/patología , Femenino , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Asesinas Naturales/patología , Leucemia de Células Pilosas/patología , Leucemia Linfocítica Granular Grande/patología , Linfoma de Células B/patología , Trastornos Linfoproliferativos/patología , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Análisis de Secuencia de ADN , Macroglobulinemia de Waldenström/patologíaRESUMEN
OBJECTIVES: Although morphologic dysplasia is not typically considered a feature of CCUS, we have consistently observed low-level bone marrow (BM) dysplasia among CCUS patients. We sought to determine whether sub-diagnostic BM dysplasia in CCUS patients is associated with other clinico-pathologic findings of myelodysplastic syndrome (MDS). METHODS: We identified 49 CCUS patients, 25 with sub-diagnostic dysplasia (CCUS-D), and 24 having no dysplasia (CCUS-ND). We compared the clinical, histologic, and laboratory findings of CCUS-D and CCUS-ND patients to 49 MDS patients, including blood cell counts, BM morphology, flow cytometry, cytogenetics, and results of next-generation sequencing. RESULTS: No statistically significant differences were observed between CCUS-D and CCUS-ND patients in the degree of cytopenias, BM cellularity, myeloid-to-erythroid ratio, or the presence of flow cytometric abnormalities. However, compared to CCUS-ND, CCUS-D patients exhibited increased mutations in myeloid malignancy-associated genes, including non-TET2/DNMT3A/ASXL1 variants, spliceosome (SF3B1, SRSF2, ZRSR2, or U2AF1) variants, and IDH2/RUNX1/CBL variants. CCUS-D patients were also enriched for higher variant allele frequencies and co-mutation of TET2/DNMT3A/ASXL1 with other genes. CONCLUSIONS: CCUS-D patients exhibit a molecular (but not clinical) profile more similar to MDS patients than CCUS-ND, suggesting CCUS-D may represent a more immediate precursor to MDS and may warrant closer clinical follow-up.
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Síndromes Mielodisplásicos/diagnóstico , Pancitopenia/diagnóstico , Anciano , Anciano de 80 o más Años , Biomarcadores , Biopsia , Médula Ósea , Evolución Clonal , Hematopoyesis Clonal , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/etiología , Pancitopenia/sangre , Pancitopenia/etiología , FenotipoRESUMEN
OBJECTIVES: At a discussion on molecular/cytogenetic education for hematopathology fellows at the 2018 Society for Hematopathology Program Directors Meeting, consensus was that fellows should understand basic principles and indications for and limitations of molecular/cytogenetic testing used in routine practice. Fellows should also be adept at integrating results of such testing for rendering a final diagnosis. To aid these consensus goals, representatives from the Society for Hematopathology and the Association for Molecular Pathology formed a working group to devise a molecular/cytogenetic curriculum for hematopathology fellow education. CURRICULUM SUMMARY: The curriculum includes a primer on cytogenetics and molecular techniques. The bulk of the curriculum reviews the molecular pathology of individual malignant hematologic disorders, with applicable molecular/cytogenetic testing for each and following the 2017 World Health Organization classification of hematologic neoplasms. Benign hematologic disorders and bone marrow failure syndromes are also discussed briefly. Extensive tables are used to summarize genetics of individual disorders and appropriate methodologies. CONCLUSIONS: This curriculum provides an overview of the current understanding of the molecular biology of hematologic disorders and appropriate ancillary testing for their evaluation. The curriculum may be used by program directors for training hematopathology fellows or by practicing hematopathologists.
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Curriculum , Educación de Postgrado en Medicina , Patología Clínica/educación , Patología Molecular/educación , Becas , Hematología , HumanosRESUMEN
BACKGROUND: Neoplasms derived from plasmacytoid dendritic cells (PDCs) are currently divided into two broad categories: mature PDC proliferations associated with myeloid neoplasms (MPDMN) and blastic plasmacytoid dendritic cell neoplasm (BPDCN); only BPDCN is recognized in the WHO 2016 classification of hematopoietic neoplasms. We present seven patients with high grade myeloid neoplasms (MNs), mostly acute leukemias, having a spectrum of PDC differentiation and not fitting with MPDMN or BPDCN. METHODS: We analyzed seven MN cases having increased myeloblasts and prominent CD56-negative PDC proliferations comprising 5-26% of bone marrow or blood cellularity as measured by flow cytometry. The cases included five acute myeloid leukemia (three FAB M4 subtype, two unclassified), one mixed phenotype acute leukemia, and one case of unclassified MN. RESULTS: Six cases demonstrated immunophenotypic evidence of PDC differentiation from leukemic blasts, based on variable expression of CD34, CD45, CD123, and CD304 by the leukemic cells. Four cases had circulating PDC populations in blood. None of the cases met clinical or pathologic criteria for BPDCN. Morphologic review was available for four acute leukemia cases and demonstrated either nodular or interstitial infiltrates of PDCs. All cases had an aggressive clinical course, and three cases had FLT3 ITD mutation. CONCLUSIONS: These cases demonstrate that high grade MNs, in particular AML, can exhibit PDC differentiation, with or without monocytic differentiation, in a manner distinct from MPDMN or BPDCN. The existence of MNs with immature PDC proliferations suggests that there is a broader spectrum of PDC-associated neoplasms than currently recognized. © 2019 International Clinical Cytometry Society.
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Células Dendríticas/patología , Neoplasias Hematológicas/patología , Leucemia Mieloide Aguda/patología , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/patología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Femenino , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación/métodos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE. The purpose of this article is to provide for radiologists an overview of the radiologic, clinical, and pathologic features of hemophagocytic lymphohistiocytosis. CONCLUSION. Hemophagocytic lymphohistiocytosis is a rare, life-threatening syndrome characterized by abnormal, excessive activation of the immune system. Imaging plays an important role in determining the extent of involvement of hemophagocytic lymphohistiocytosis. Knowledge of this entity, including its imaging, clinical, and pathologic findings, is critical to facilitate timely diagnosis.
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Linfohistiocitosis Hemofagocítica/diagnóstico por imagen , Humanos , Linfohistiocitosis Hemofagocítica/terapia , RadiologíaRESUMEN
OBJECTIVES: We investigated the frequency and pattern of detection of therapeutic monoclonal antibodies (t-mAbs) daratumumab and elotuzumab by routine serum protein electrophoresis (SPE) and immunofixation (IF) in treated patients with myeloma. METHODS: Detection of t-mAb was assessed in 22 patients by retrospective review of SPE/IF ordered prior to, during, and after 26 individual courses of therapy. RESULTS: t-mAb was distinguishable from M-protein in 16 of 26 courses, with daratumumab detected in nine of nine and elotuzumab in six of seven patients. t-mAb was detected on first follow-up SPE/IF in 12 patients, with earliest detection 7 days after therapy initiation and latest detection 70 days after therapy. t-mAb persisted throughout induction therapy in most patients, with loss of detection during maintenance daratumumab. CONCLUSIONS: When distinguishable from M-protein, t-mAbs are detectable in 93% of treated patients as soon as 7 days after the initial dose and are consistently observed throughout induction therapy, warranting increased monitoring and careful interpretation of SPE/IF.
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Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales/sangre , Electroforesis de las Proteínas Sanguíneas , Proteínas de Mieloma/análisis , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/sangre , Antineoplásicos Inmunológicos/uso terapéutico , Reacciones Falso Positivas , Humanos , Mieloma Múltiple/tratamiento farmacológicoAsunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Proteínas de Mieloma/metabolismo , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos Clínicos Fase III como Asunto , Humanos , Masculino , Mieloma Múltiple/diagnóstico por imagen , Mieloma Múltiple/metabolismo , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
Epstein-Barr virus-associated mucocutaneous ulcer (EBV-MCU) is a recently characterized entity that falls under the spectrum of EBV-lymphoproliferative disorders. First described in 2010 by Dojcinov et al, it is an EBV-driven localized proliferation of B cells, occurring in mucocutaneous tissues including the skin, the oropharynx, and the gastrointestinal tract of immunosuppressed patients in the absence of an intact T-cell repertoire. Typically, it has been described in elderly patients with age-related immunosenescence and patients who are on immunosuppressive therapy. However, only 2 cases have been reported in pediatric, adolescent, and young adult age groups, with all these patients manifesting after solid organ transplant. To the best of our knowledge there are no case reports of EBV-MCU occurring in association with hematologic malignancy. Here, we present a case of EBV-MCU in a young adult patient with T-cell acute lymphoblastic leukemia. Our report serves to promote awareness among clinicians regarding this newly described and extremely rare clinical entity in young immunosuppressed patients. In addition, we highlight the importance of accurate diagnosis to prevent overtreatment of this indolent, often self-resolving disease that has a significant clinicopathologic overlap with other aggressive forms of EBV-lymphoproliferative disorders that require more intensive therapy.
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Herpesvirus Humano 4 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/virología , Úlcera Cutánea/virología , Adulto , Tratamiento Conservador , Infecciones por Virus de Epstein-Barr/complicaciones , Humanos , Huésped Inmunocomprometido , Trastornos Linfoproliferativos/virología , Úlcera Cutánea/complicacionesRESUMEN
Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3ß) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.
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Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/inmunología , Aminofenoles/química , Aminofenoles/farmacología , Animales , Microambiente Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Inmunoterapia , Molécula 1 de Adhesión Intercelular/metabolismo , Maleimidas/química , Maleimidas/farmacología , Ratones , FN-kappa B/metabolismo , Tiadiazoles/química , Tiadiazoles/farmacología , Factor de Necrosis Tumoral alfaRESUMEN
Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2(flox/flox) Lyz2(Cre/Cre) mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice was enriched for CD11b+ myeloid cells, CD11b(hi) Gr-1(hi) neutrophils, Lin- c-Kit+ Sca-1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.
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Células de la Médula Ósea/citología , Sistema de Señalización de MAP Quinasas , Macrófagos/citología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Eliminación de Gen , Regulación de la Expresión Génica , Granulocitos/citología , Granulocitos/metabolismo , Hematopoyesis , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Monocitos/metabolismoRESUMEN
The use of natural killer (NK) cells as cell therapy against acute leukemia is an active area of investigation. The optimal source of cytotoxic NK cells for therapeutic use is presently unknown. With funds from the National Blood Foundation, the author's lab has developed in vitro culture systems that use the Notch receptor ligand Delta4 for the differentiation and expansion of functional NK cells from CD34+ cord blood hematopoietic progenitor cells. These Notch-induced NK (N-NK) cells display a predominantly immature, CD56(bright) surface phenotype, with expression of activating receptors important for leukemia cell recognition and killing, but with an absence of inhibitory receptors that bind major histocompatibility complex (MHC) class I, making them free of restriction by self-MHC. They are capable of directly killing hematopoietic tumor cell lines and primary leukemia cells in vitro. Thus, cytotoxic, HLA-independent N-NK cells may represent a novel cell therapy for hematopoietic malignancy.
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Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Asesinas Naturales/citología , Receptores Notch/fisiología , Transducción de Señal/fisiología , Antígenos CD34/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Citotoxicidad Inmunológica , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Leucemia/terapia , Microesferas , Receptores KIR/análisisRESUMEN
Notch receptor signaling is required for T cell development, but its role in natural killer (NK) cell development is poorly understood. We compared the ability of the 5 mammalian Notch ligands (Jagged1, Jagged2, Delta1, Delta3, or Delta4) to induce NK cell development from human hematopoietic progenitor cells (HPCs). CD34(+) HPCs were cultured with OP9 stromal cell lines transduced with 1 of the Notch ligands or with OP9 stromal cells alone, in the presence of IL-7, Flt3L, and IL-15. Differentiation and expansion of CD56(+)CD3(-) cells were greatly accelerated in the presence of Jagged2, Delta-1, or Delta-4, versus culture in the absence of ligand or in the presence of Jagged1 or Delta3. At 4 weeks, cultures containing Jagged2, Delta1, or Delta4 contained 80% to 90% NK cells, with the remaining cells being CD33(+) myelogenous cells. Notch-induced NK (N-NK) cells resembled CD56(bright) NK cells in that they were CD16(-), CD94(-), CD117(+), and killer immunoglobulin-like receptors (KIR(-)). They also expressed NKp30, NKp44, NKp46, 2B4, and DNAM-1, with partial expression of NKG2D. The N-NK cells displayed cytotoxic activity against the K562 and RPMI-8226 cell lines, at levels similar to activated peripheral blood (PB) NK cells, although killing of Daudi cells was not present. N-NK cells were also capable of interferon (IFN)-gamma secretion. Thus, Notch ligands have differential ability to induce and expand immature, but functional, NK cells from CD34(+) HPCs. The use of Notch ligands to generate functional NK cells in vitro may be significant for cellular therapy purposes.
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Antígenos CD34/inmunología , Células Madre Hematopoyéticas/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/inmunología , Animales , Antígenos CD34/biosíntesis , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Antígeno CD56/biosíntesis , Antígeno CD56/inmunología , Diferenciación Celular/inmunología , Línea Celular , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intracelular , Proteína Jagged-2 , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Ligandos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Receptores Notch/inmunología , Células del Estroma/citología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Transducción GenéticaRESUMEN
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyoma virus. Electron microscopy and immunohistochemistry are the traditional methods of confirming the presence of the virus in brain biopsies from these patients. We studied the brain biopsies from 7 patients with PML and 6 patients without PML with chromogenic in situ hybridization (CISH) for the JC polyoma virus using a commercially available probe. The biopsies from the patients with the PML cases were proven to contain the JC polyoma virus by traditional and molecular methods. The CISH findings were compared with the known state of infection. All (7/7) of the biopsies from patients with PML were positive for the presence of polyoma virus by CISH, whereas the biopsies from patients without PML were uniformly negative. CISH seems to be a useful tool for the detection of the JC virus in brain biopsies from patients with PML, and is more accessible because a commercial probe is available.