RESUMEN
We report a case of a 2-year-old girl who was diagnosed with natural killer cell acute lymphoblastic leukemia and treated with an acute lymphoblastic leukemia chemotherapy regimen. Two months posttherapy, the disease relapsed with a myeloid immunophenotype. Complete response was then achieved with acute myeloid leukemia therapy followed by unrelated donor umbilical cord allogenic stem cell transplant. Retrospectively, reanalysis of the diagnostic specimen showed minimal myeloperoxidase expression that was called negative by conventional single parameter linear gating but better appreciated on histogram overlays. This case illustrates that even low levels of myeloperoxidase expression should be considered significant in lineage assignment in acute leukemia.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linaje de la Célula , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/patología , Recurrencia Local de Neoplasia/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Preescolar , Terapia Combinada , Diagnóstico Diferencial , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/terapia , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/terapia , Peroxidasa/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Pronóstico , Estudios Retrospectivos , Donante no EmparentadoRESUMEN
BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins' chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγc-/- (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8. CONCLUSION: NSG-cmah-/- mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.
Asunto(s)
Glicoproteínas/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1 , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/virología , Animales , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Sitios Genéticos , Infecciones por VIH/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/virología , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Noqueados , FenotipoRESUMEN
Clinical heterogeneity is a major barrier to effective treatment of chronic lymphocytic leukemia (CLL). Emerging evidence suggests that constitutive activation of various signaling pathways like mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-Erk) signaling plays a role in the heterogeneous clinical outcome of CLL patients. In this study, we have investigated the role of Sprouty (SPRY)2 as a negative regulator of receptor and nonreceptor tyrosine kinase signaling in the pathogenesis of CLL. We show that SPRY2 expression is significantly decreased in CLL cells, particularly from poor-prognosis patients compared with those from good-prognosis patients. Overexpression of SPRY2 in CLL cells from poor-prognosis patients increased their apoptosis. Conversely, downregulation of SPRY2 in CLL cells from good-prognosis patients resulted in increased proliferation. Furthermore, CLL cells with low SPRY2 expression grew more rapidly in a xenograft model of CLL. Strikingly, B-cell-specific transgenic overexpression of spry2 in mice led to a decrease in the frequency of B1 cells, the precursors of CLL cells in rodents. Mechanistically, we show that SPRY2 attenuates the B-cell receptor (BCR) and MAPK-Erk signaling by binding to and antagonizing the activities of RAF1, BRAF, and spleen tyrosine kinase (SYK) in normal B cells and CLL cells. We also show that SPRY2 is targeted by microRNA-21, which in turn leads to increased activity of Syk and Erk in CLL cells. Taken together, these results establish SPRY2 as a critical negative regulator of BCR-mediated MAPK-Erk signaling in CLL, thereby providing one of the molecular mechanisms to explain the clinical heterogeneity of CLL.
Asunto(s)
Linfocitos B/metabolismo , Proliferación Celular , Regulación Leucémica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Apoptosis/genética , Linfocitos B/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Quinasa Syk/genética , Quinasa Syk/metabolismoRESUMEN
Concerns over cell line identities and contamination have led investigators to acquire fresh stocks of HeLa CCL-2 cells, but results with the HeLa CCL-2 cells do not always reproduce results with HeLa cells that have long history in the laboratory. When used for TCID(50) assays of Coxsackievirus B3/28 (CVB3/28), HeLa CCL-2 cells returned titers for CVB3/28 that were more than ten-fold lower than titers obtained using laboratory HeLa cells. The viral cytopathic effect was less distinct in the HeLa CCL-2 cultures, suggestive of a mixed population of cells with varied susceptibility to viral cytopathic effect. Analysis of short tandem repeat markers confirmed the identities of the cell lines as HeLa. Subpopulations in the HeLa CCL-2 culture, separated easily based on the speed with which they were released by trypsin-EDTA, differed in their susceptibilities to CVB3/28 cytopathic effect, and in their expression of the Coxsackievirus and adenovirus receptor (CAR). The distinctions between Lab HeLa and HeLa CCL-2 cells were less obvious when infected with CVB3/RD, a strain selected for growth in RD cells. Results that differ among laboratories may be due to the use of HeLa cell strains with different histories, and experiments using HeLa CCL-2 available from the American Type Culture Collection are probably incapable of reproducing many of the published studies of Coxsackievirus that have used HeLa cells with laboratory-dependent histories.
Asunto(s)
Efecto Citopatogénico Viral , Enterovirus Humano B/patogenicidad , Variación Genética , Virología/métodos , Virología/normas , Enterovirus Humano B/aislamiento & purificación , Células HeLa , Humanos , Reproducibilidad de los Resultados , Carga Viral/métodos , Carga Viral/normas , Cultivo de Virus/métodos , Cultivo de Virus/normasRESUMEN
Chlorthalidone (CTD) may be superior to hydrochlorothiazide (HCTZ) in the reduction of adverse cardiovascular events in hypertensive patients. The mechanism of the potential benefit of CTD could be related to antiplatelet effects. The objective of this study was to determine if CTD or HCTZ have antiplatelet effects. This study was a prospective, double-blind, randomized, three-way crossover comparison evaluating the antiplatelet effects of CTD, HCTZ, and aspirin (ASA) in healthy volunteers. The effects of these treatments on platelet activation and aggregation were assessed using a well-established method with five standard platelet agonists. Thirty-four patients completed the three-way crossover comparing pre- and post-treatment changes in platelet activation and aggregation studies. There were statistically significant antiplatelet effects with ASA but not with CTD or HCTZ. Hypokalemia occurred in 0 (0%), 10 (30%), and 6 (18%) of the ASA, CTD, and HCTZ patients, respectively. The results of our study suggest that the benefits of CTD and HCTZ in reducing adverse cardiovascular events in patients with hypertension is not a result of an antiplatelet effect. In our study, hypokalemia with CTD was more prevalent than that reported in a large outcome trial in patients with hypertension. The clinical relevance of this finding is uncertain.
Asunto(s)
Hipertensión , Hipopotasemia , Humanos , Clortalidona/efectos adversos , Hidroclorotiazida/efectos adversos , Hipopotasemia/inducido químicamente , Hipopotasemia/epidemiología , Estudios Prospectivos , Antihipertensivos/efectos adversos , Presión Sanguínea , Diuréticos/uso terapéutico , Método Doble Ciego , Aspirina/farmacología , Aspirina/uso terapéutico , Quimioterapia CombinadaRESUMEN
BACKGROUND: Normal thymocyte precursors in secondary lymphoid organs have previously been described. It is important to recognize normal thymocyte precursors by flow cytometry to differentiate them from T-cell lymphoblastic leukemia. METHODS: A 3-year-old boy status 2 years postallogenic cardiac transplant underwent adenoidectomy to exclude post-transplant lymphoproliferative disorder. Microscopic, immunohistochemical, and flow cytometry analyses of the adenoid were performed. RESULTS: By flow cytometry, a population of CD45+(dim)/CD7+(bright)/CD3- cells were observed at 1.0% of lymphocytes. These cells expressed CD10, partial CD34 and exhibited acquisition of CD4 followed by CD8. Within the brighter CD45+ lymphocytes, a population of CD3-/CD4+/CD8+ thymocytes and a similarly sized population of CD4+/CD8+ cells exhibiting acquisition of low-density CD3 were identified. By immunostaining, clusters of TdT+/CD1a+/CD4+/CD8+ T-cells were identified in the interfollicular areas. Compared to normal thymus, thymocytes in the adenoid tissue lacked the classic CD4xCD8 winged differentiation profile but showed a normal early precursor pattern. CONCLUSIONS: Thymocytes in adenoid show a similar differentiation pattern to thymus and thymoma. However, the classic winged pattern of common thymocyte differentiation may not be readily apparent in thymocytes differentiating outside of the thymus. Recognition of the early thymocyte precursor antigen acquisition profile can be crucial to correct interpretation. © 2017 International Clinical Cytometry Society.
Asunto(s)
Tonsila Faríngea/patología , Timocitos/patología , Timoma/patología , Tonsila Faríngea/metabolismo , Antígenos CD/metabolismo , Preescolar , Citometría de Flujo/métodos , Humanos , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Timocitos/metabolismo , Timoma/metabolismoRESUMEN
OBJECTIVES: We developed a simple and effective rinsing technique (RT) of needle biopsies to produce cell suspensions for flow cytometry (FCM) and evaluated whether the RT is comparable to the conventional tissue cell suspension (TCS) technique. METHODS: We retrieved 93 needle core biopsy cases employing the RT for FCM and 25 needle biopsy cases using TCS for FCM. RESULTS: The diagnostic concordance between the FCM results and the morphologic diagnoses of both groups was compared. The diagnostic concordance was comparable in the RT group (92.6%) to the TCS group (71.4%). Furthermore, the diagnostic concordance in the RT group was associated with number of isolated cells. The diagnostic accuracy increased significantly when the cell number was above 30,000 in the RT group. CONCLUSIONS: The RT for FCM not only maximizes the tissue utilization, but also is a simple and effective method to obtain cell suspension as compared to traditional cell suspension technique. © 2018 International Clinical Cytometry Society.
Asunto(s)
Biopsia con Aguja Gruesa , Citometría de Flujo , Leucemia Linfoide/diagnóstico , Leucemia Linfoide/patología , Humanos , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: Normal thymocyte precursors in secondary lymphoid organs have previously been described. It is important to recognize normal thymocyte precursors by flow cytometry to differentiate them from T-cell lymphoblastic leukemia. METHODS: A 3-year-old boy status 2 years post-allogenic cardiac transplant underwent adenoidectomy to exclude post-transplant lymphoproliferative disorder. Microscopic, immunohistochemical, and flow cytometry analyses of the adenoid were performed. RESULTS: By flow cytometry, a population of CD45+(dim)/CD7+(bright)/CD3- cells were observed at 1.0% of lymphocytes. These cells expressed CD10, partial CD34, and exhibited acquisition of CD4 followed by CD8. Within the brighter CD45+ lymphocytes, a population of CD3-/CD4+/CD8+ thymocytes and a similarly sized population of CD4+/CD8+ cells exhibiting acquisition of low-density CD3 were identified. By immunostaining, clusters of TdT+/CD1a+/CD4+/CD8+ T-cells were identified in the interfollicular areas. Compared to normal thymus, thymocytes in the adenoid tissue lacked the classic CD4xCD8 winged differentiation profile but showed a normal early precursor pattern. CONCLUSIONS: Thymocytes in adenoid show a similar differentiation pattern to thymus and thymoma. However, the classic winged pattern of common thymocyte differentiation may not be readily apparent in thymocytes differentiating outside of the thymus. Recognition of the early thymocyte precursor antigen acquisition profile can be crucial to correct interpretation. © 2017 International Clinical Cytometry Society.
RESUMEN
We used a new method of data presentation and analysis, termed antigen mapping, to characterize recurring myeloblast phenotypic abnormalities in a series of 28 cases of myelodysplastic syndrome (MDS), including refractory anemia with ringed sideroblasts (RARS), refractory anemia with multilineage dysplasia (RCMLD), and refractory anemia with excess blasts (RAEB). Abnormal patterns of CD34 and CD117 expression were present in 50% of RARS, 68% of RCMLD, and 100% of RAEB cases. The presence of decreased myeloblast CD45 density, increased CD13 and CD34 density, and increased expression of CD11c and CD4(dim) were MDS grade-related. There was a direct relationship between the number of myeloblast phenotypic abnormalities (phenotypic score) and MDS grade. The myeloblast phenotypic scores also were correlated highly with International Prognostic Scoring System scores and risk categories. We found the antigen mapping technique to be an efficient data presentation and analysis method for the detection of MDS-associated abnormalities of antigen distribution and density.
Asunto(s)
Anemia Refractaria con Exceso de Blastos/metabolismo , Antígenos CD34/metabolismo , Médula Ósea/metabolismo , Células Precursoras de Granulocitos/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Anciano , Anciano de 80 o más Años , Anemia Refractaria con Exceso de Blastos/patología , Biomarcadores/metabolismo , Médula Ósea/patología , Mapeo Epitopo , Femenino , Células Precursoras de Granulocitos/patología , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
OBJECTIVE: To examine the effects of patients taking the direct blood coagulation factor Xa inhibitor rivaroxaban on lupus anticoagulant testing results in a clinical setting. METHODS: We reviewed the results of lupus anticoagulant testing performed over a 2-year period. Of 59 patients who met criteria for a lupus anticoagulant, 18 were taking rivaroxaban. We reviewed and compared the parameters of lupus anticoagulant testing. RESULTS: The average dilute Russell viper venom time (DRVVT) and normal plasma-mix screening results to confirmation ratios in rivaroxaban-naïve patients were 1.6 and 1.7, respectively. In the rivaroxaban group, the same parameters were 1.7 and 1.6, respectively (P = - 0.28 and 0.46, respectively). For 15 of 18 patients taking rivaroxaban, results were corrected on the confirmation steps of both tests. CONCLUSIONS: Rivaroxaban confounds lupus anticoagulant testing because the DRVVT is prolonged in these patients but it also corrects with excess phospholipid, mimicking a lupus anticoagulant. Patient medication review is critical to avoid false-positive findings and inappropriate diagnosis of antiphospholipid syndrome.
Asunto(s)
Inhibidores del Factor Xa/uso terapéutico , Reacciones Falso Positivas , Factores Inmunológicos/sangre , Inhibidor de Coagulación del Lupus/sangre , Lupus Eritematoso Sistémico/diagnóstico , Tamizaje Masivo/métodos , Rivaroxabán/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosfolípidos/análisisRESUMEN
DNA methyltransferase 3a (DNMT3A) catalyzes the formation of 5-methyl-cytosine in mammalian genomic DNA, and it is frequently mutated in human hematologic malignancies. Bi-allelic loss of Dnmt3a in mice results in leukemia and lymphoma, including chronic lymphocytic leukemia (CLL). Here, we investigate whether mono-allelic loss of Dnmt3a is sufficient to induce disease. We show that, by 16 months of age, 65% of Dnmt3a(+/-) mice develop a CLL-like disease, and 15% of mice develop non-malignant myeloproliferation. Genome-wide methylation analysis reveals that reduced Dnmt3a levels induce promoter hypomethylation at similar loci in Dnmt3a(+/-) and Dnmt3a(Δ/Δ) CLL, suggesting that promoters are particularly sensitive to Dnmt3a levels. Gene expression analysis identified 26 hypomethylated and overexpressed genes common to both Dnmt3a(+/-) and Dnmt3a(Δ/Δ) CLL as putative oncogenic drivers. Our data provide evidence that Dnmt3a is a haplo-insufficient tumor suppressor in CLL and highlights the importance of deregulated molecular events in disease pathogenesis.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Regiones Promotoras Genéticas , Animales , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasas/deficiencia , ADN Metiltransferasa 3A , Heterocigoto , Humanos , Ratones , Transcripción Genética , Transcriptoma/genéticaRESUMEN
We evaluated whole blood samples drawn from 25 healthy donors and 20 HIV-infected donors into K3EDTA and Cyto-Chex BCT blood collection tubes for CD4, CD8, and CD3 cell counts (HIV Panel). Samples collected in Cyto-Chex BCT were stored at room temperature and tested by 4-color flow cytometry at 6 h, 3 days, and 7 days after isolation for CD4, CD8, and CD3 absolute cell counts/microl and compared with samples collected in K3EDTA tubes and tested at 6 h. Regardless of donor type, the samples collected in Cyto-Chex BCT and tested on day 7 yielded results that were statistically indistinguishable (with correlation coefficients of 0.96 or greater) from samples collected in K3EDTA tubes and tested at 6 h. We conclude that whole blood samples collected in Cyto-Chex BCT are stabilized for their marker phenotype for at least 7 days after phlebotomy.
Asunto(s)
Conservación de la Sangre/métodos , Citometría de Flujo/métodos , Infecciones por VIH/inmunología , Leucocitos Mononucleares/inmunología , Adulto , Antígenos CD/sangre , Biomarcadores , Femenino , Infecciones por VIH/sangre , Humanos , Inmunofenotipificación/métodos , Leucocitos Mononucleares/química , Masculino , Persona de Mediana EdadRESUMEN
DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3a(Δ/Δ) mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3a(Δ/Δ) lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3a(Δ/Δ) mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.
Asunto(s)
Carcinogénesis/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , Linfoma de Células T/genética , Linfoma de Células T/patología , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Proliferación Celular/genética , ADN/genética , ADN Metiltransferasa 3A , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ratones , Regiones Promotoras Genéticas/genética , Transcriptoma/genética , Regulación hacia Arriba/genética , ADN Metiltransferasa 3BRESUMEN
Sterilizing irradiation of the US mail has been proposed as a method to prevent delivery of viable anthrax spores. Because newborn screening samples (bloodspots) and cyclosporine and tacrolimus specimens (whole blood) are delivered routinely through the mail, we studied whether sterilizing gamma irradiation could affect these test results. Specimens were exposed to 18 kGy gamma irradiation (100 hours x 18,000 rad/h), a "kill dose" for Bacillus pumilus spore strips. Irradiation had no significant effect on whole blood cyclosporine or tacrolimus results, but it had a degradative effect on bloodspot phenylalanine, hemoglobins, biotinidase, galactose-1-phosphate uridyltransferase, thyroxine, and thyrotropin. Such irradiation potentially could cause false-negative results for the detection of phenylketonuria and likely would lead to an increase in secondary testing for hemoglobin variants, but it is unlikely to lead to false-negative or false-positive results for the remaining newborn screening tests. These experiments cannot rule out possible greater effects by larger doses or other types of irradiation.
Asunto(s)
Análisis Químico de la Sangre/métodos , Ciclosporina/efectos de la radiación , Tamizaje Neonatal , Efectos de la Radiación , Esterilización , Tacrolimus/efectos de la radiación , Carbunco/prevención & control , Bacillus/efectos de la radiación , Ciclosporina/sangre , Estabilidad de Medicamentos , Rayos gamma , Humanos , Recién Nacido , Servicios Postales/métodos , Tacrolimus/sangreRESUMEN
OBJECTIVES: To characterize granulocyte colony-stimulating factor receptor (CD114) expression in normal (n = 20), myelodysplastic (n = 34), and chronic myelogenous leukemia (CML; n = 5) bone marrow by flow cytometry. METHODS: Clinical bone marrow samples were analyzed using CD33/CD114/CD34/CD117/CD45. CD114 density (mean fluorescence intensity) and cellular distribution were evaluated on early blasts (CD33-), late blasts (CD33+), promyelocytes, and granulocytes. RESULTS: Normal CD114 acquisition occurred on early blasts, peaked on promyelocytes, and decreased on granulocytes. Forty percent of CD34+ blasts expressed CD114 and one-third were early blasts. In myelodysplastic syndromes, altered CD114 distribution was more informative than density changes. In CML, CD114 density was significantly decreased on early blasts and expression was essentially limited to late blasts. We observed a specific blast dysmaturation pattern in CML involving CD33, CD34, and CD114 that was 83% sensitive and 100% specific in initial diagnosis. CONCLUSIONS: CD114 provides useful additional detail in phenotypic assessment of hematopoietic precursor maturation.
Asunto(s)
Médula Ósea/metabolismo , Síndromes Mielodisplásicos/metabolismo , Trastornos Mieloproliferativos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/metabolismo , Biomarcadores de Tumor/metabolismo , Médula Ósea/inmunología , Femenino , Citometría de Flujo , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/inmunología , Trastornos Mieloproliferativos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismoAsunto(s)
Anticuerpos Antivirales/inmunología , Antígenos Virales/análisis , VIH/inmunología , Serotipificación/métodos , Técnicas y Procedimientos Diagnósticos , Reacciones Falso Negativas , VIH/aislamiento & purificación , Seronegatividad para VIH , Humanos , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Cells in which the appropriate viral receptor cannot be detected may paradoxically act as a host to the virus. For example, RD cells are often considered to be non-permissive for infection with coxsackievirus and adenovirus receptor (CAR)-dependent group B coxsackieviruses (CVB), insofar as inoculated cell monolayers show little or no cytopathic effect (CPE) and immunohistological assays for CAR have been consistently negative. Supernatants recovered from RD cells exposed to CVB, however, contained more virus than was added in the initial inoculum, indicating that productive virus replication occurred in the monolayer. When infected with a recombinant CVB type 3 (CVB3) chimeric strain expressing S-Tag within the viral polyprotein, 4-11 % of RD cells expressed S-Tag over 48 h. CAR mRNA was detected in RD cells by RT-PCR, and CAR protein was detected on Western blots of RD lysates; both were detected at much lower levels than in HeLa cells. Receptor blockade by an anti-CAR antibody confirmed that CVB3 infection of RD cells was mediated by CAR. These results show that some RD cells in the culture population express CAR and can thereby be infected by CVB, which explains the replication of CAR-dependent CVB in cell types that show little or no CPE and in which CAR has not previously been detected. Cells within cultures of cell types that have been considered non-permissive may express receptor transiently, leading to persistent replication of virus within the cultured population.
Asunto(s)
Enterovirus Humano B/crecimiento & desarrollo , Receptores Virales/metabolismo , Línea Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Citosol/química , Humanos , ARN Mensajero/análisis , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/metabolismoRESUMEN
The specificity of human immunodeficiency virus type 1 (HIV-1) for human cells precludes virus infection in most mammalian species and limits the utility of small animal models for studies of disease pathogenesis, therapy, and vaccine development. One way to overcome this limitation is by human cell xenotransplantation in immune-deficient mice. However, this has proved inadequate, as engraftment of human immune cells is limited (both functionally and quantitatively) following transplantation of mature human lymphocytes or fetal thymus/liver. To this end, a human immune system was generated from umbilical cord blood-derived CD34(+) hematopoietic stem cells in BALB/c-Rag2(-/-)gamma(c)(-/-) mice. Intrapartum busulfan administration followed by irradiation of newborn pups resulted in uniform engraftment characterized by human T-cell development in thymus, B-cell maturation in bone marrow, lymph node development, immunoglobulin M (IgM)/IgG production, and humoral immune responses following ActHIB vaccination. Infection of reconstituted mice by CCR5-coreceptor utilizing HIV-1(ADA) and subtype C 1157 viral strains elicited productive viral replication and lymphadenopathy in a dose-dependent fashion. We conclude that humanized BALB/c-Rag2(-/-)gamma(c)(-/-) mice represent a unique and valuable resource for HIV-1 pathobiology studies.