RESUMEN
INTRODUCTION: The extension of quantitative flow cytometric studies to the erythroid lineage in patients with suspected myelodysplastic syndrome has prompted a reassessment of cell surface antigen expression during normal erythropoiesis. Erythropoiesis in normal and pathologic bone marrows was studied to determine the expected antigenic relationships of maturing erythroid cells. METHODS: A total of 200 bone marrow specimens were evaluated by multidimensional flow cytometry (MDF). Samples were prepared using either NH4 Cl lysis or Ficoll density gradient separation. RESULTS: Normal erythroid development is described as a two-step process observable with the intensity relationships between CD235a, CD71, CD45, CD105, CD34, CD117, and CD36. The variability of these intensities (CV) was determined. A comparison of processing techniques determined lysis is the optimal analytic technique for the analysis of early-stage erythroid cells. Nucleic acid staining with DRAQ5 revealed that Ficoll allows for the analysis of reticulocytes and mature erythrocytes otherwise eliminated by lysis. CONCLUSION: These data demonstrate while lysis alters the light scatter characteristics of erythroid precursors, it did not alter quantitative antigen expression or nucleic acid content. The expected variability in antigen intensities is defined. These studies provide a basis for a comparison of erythroid development between normal individuals and those with erythroid dysplasia associated with myelodysplastic syndromes.
Asunto(s)
Eritropoyesis/fisiología , Citometría de Flujo/métodos , Antígenos CD/metabolismo , Médula Ósea , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Ciclo Celular , Diferenciación Celular , Humanos , Inmunofenotipificación/métodos , Manejo de Especímenes/métodos , Manejo de Especímenes/normasRESUMEN
OBJECTIVE: To compare the sealing ability of ProRoot mineral trioxide aggregate (MTA) to the sealing ability of EndoSequence Bioceramic Root Repair Material (ES-BCRR) putty using a bacterial leakage model. METHOD AND MATERIALS: Root canals of 60 single-rooted extracted teeth were enlarged to an apical diameter of 0.5 mm using EndoSequence files. The apical 3 mm of each root was sectioned at 90 degrees to the long axis of the root. An ultrasonic surgical tip was used to prepare a 3-mm deep root-end preparation in all teeth. Teeth were equally divided into four groups: Group 1, MTA; Group 2, ES-BCRR putty; Group 3, positive control, gutta-percha without sealer; Group 4, negative control, sealed with wax and nail varnish. Prepared teeth were kept moist for 48 hours to allow for initial setting of the materials. After ethylene oxide sterilization, the teeth were suspended in sterilized vials containing 3% phenol lactose broth and inoculated with Enterococcus faecalis through the occlusal access openings. The samples were observed daily for leakage to a maximum of 28 days. Chi-square and Fisher exact tests were used to compare the experimental groups and an alpha level of significance was set at P = .05. RESULTS: In the ES-BCRR group 93% of samples leaked, compared to only 20% of samples in the MTA group. There was a significant difference in leakage between the experimental groups (P < .0001). Also there were no significant differences between the negative control group and MTA group and between the positive control group and ES-BCRR group (P = 1.00). CONCLUSION: Samples in the ES-BCRR group leaked significantly more than samples in the MTA group.
Asunto(s)
Filtración Dental/prevención & control , Materiales de Obturación del Conducto Radicular , Obturación del Conducto Radicular , Compuestos de Aluminio , Compuestos de Calcio , Fosfatos de Calcio , Distribución de Chi-Cuadrado , Filtración Dental/microbiología , Combinación de Medicamentos , Enterococcus faecalis , Humanos , Óxidos , Preparación del Conducto Radicular , SilicatosRESUMEN
Somatic mutations and genomic alterations are frequent events in the clonal evolution of hematologic malignancies. Recent studies have reported copy neutral loss of heterozygosity (LOH) for the mismatched human leukocyte antigen (HLA) haplotype in patients relapsed after haploidentical hematopoietic cell transplantation (HCT) for a hematologic malignancy. Herein, we report 15 cases of somatic mutations in the HLA genes of patients with a variety of hematologic diseases, including acute myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and non-Hodgkin's lymphoma, encountered at our institute over the past decade. While two of the cases were identified in patient relapse specimens collected post-HCT, 13 cases were found in peripheral blood specimens submitted for HLA typing prior to transplantation. Ten patients exhibited acquired LOH for all or part of one HLA haplotype. Five other cases involved somatic mutations in the nucleotide sequences of common HLA-A or HLA-B alleles. Since they are not systematically evaluated prior to HCT, acquired mutations in HLA genes are likely under reported. Beyond the implications for accurate HLA typing and donor selection, alternations that result in the loss of HLA expression may allow escape from immune surveillance and adversely impact transplant outcome.
Asunto(s)
Antígenos HLA-A/genética , Antígenos HLA-B/genética , Neoplasias Hematológicas/genética , Leucemia/genética , Linfoma no Hodgkin/genética , Adulto , Niño , Haplotipos , Trasplante de Células Madre Hematopoyéticas , Prueba de Histocompatibilidad , Humanos , Pérdida de Heterocigocidad , Persona de Mediana Edad , Mutación , Polimorfismo GenéticoRESUMEN
Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Citometría de Flujo/normas , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/metabolismo , Guías de Práctica Clínica como Asunto/normas , Médula Ósea/metabolismo , Médula Ósea/patología , Citometría de Flujo/métodos , Humanos , Inmunofenotipificación , Agencias Internacionales , Síndromes Mielodisplásicos/inmunología , Pronóstico , Estándares de Referencia , Sociedades CientíficasRESUMEN
This study was designed to determine the safety of a nonmyeloablative regimen in patients with primary immunodeficiency disorders (PID) who had infections, organ dysfunction or other risk factors that precluded conventional hematopoietic cell (HC) transplant. Fourteen patients received HLA-matched related (n=6) or unrelated (n=8) HC grafts from marrow (n=8), peripheral blood mononuclear cells (n=5) or umbilical cord blood (n=1), either without conditioning (n=1), or after 200 cGy total body irradiation alone (n=3) or with 90 mg/m2 fludarabine (n=10). All patients were given postgrafting immunosuppression with mycophenolate mofetil and cyclosporine. Mixed (n=5) or full (n=8) donor chimerism was established in 13 patients, and one patient rejected the graft. Eight patients developed acute grade III (n=1) and/or extensive chronic GVHD (n=8). With a median follow-up of 4.9 (range, 0.7-8.1) years, the 3-year overall survival, event-free survival and transplant-related mortality were 62, 62 and 23%, respectively. Correction of immune dysfunction was documented in 8 of 10 patients with stable donor engraftment. These preliminary results indicated that this approach was associated with stable donor engraftment and a low incidence of early mortality and, thus, can be considered for certain high-risk patients with PID. However, there was a risk of GVHD, which is an undesirable outcome for this group of patients.
Asunto(s)
Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Síndromes de Inmunodeficiencia/terapia , Adolescente , Adulto , Causas de Muerte , Niño , Preescolar , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/epidemiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/mortalidad , Prueba de Histocompatibilidad , Humanos , Inmunosupresores/uso terapéutico , Lactante , Selección de Paciente , Proyectos Piloto , Análisis de Supervivencia , Sobrevivientes , Quimera por Trasplante , Acondicionamiento Pretrasplante , Irradiación Corporal TotalRESUMEN
BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Leucemia Mieloide Aguda/inmunología , Antígenos CD , Linfocitos T CD4-Positivos/metabolismo , Antígeno CTLA-4 , Técnicas de Cultivo de Célula , Proliferación Celular , Medios de Cultivo Condicionados , Citotoxicidad Inmunológica/efectos de los fármacos , Células Dendríticas/citología , Citometría de Flujo , Humanos , Interferón gamma/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/patología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Receptores de Interleucina-2/metabolismo , Células Tumorales CultivadasRESUMEN
We analyzed the safety and efficacy of Mylotarg (gemtuzumab ozogamicin, an antibody-targeted chemotherapy consisting of a humanized anti-CD33 antibody linked to calicheamicin, a potent antitumor antibiotic) in the treatment of 101 patients > or =60 years of age with acute myeloid leukemia (AML) in untreated first relapse in three open-label trials. Mylotarg is administered as a 2-h intravenous infusion at 9 mg/m(2) for two doses with 14 days between doses. The overall remission rate was 28%, with complete remission (CR) in 13% of patients and complete remission with incomplete platelet recovery (CRp) in 15%. Median survival was 5.4 months for all patients and 14.5 months and 11.8 months for patients achieving CR and CRp, respectively. CD33 antigen is present on normal hematopoietic progenitor cells; thus, an expected high incidence of grade 3 or 4 neutropenia (99%) and thrombocytopenia (99%) was observed. The incidences of grade 3 or 4 elevations of bilirubin and hepatic transaminases were 24% and 15%, respectively. There was a low incidence of grade 3 or 4 mucositis (4%) and infections (27%) and no treatment-related cardiotoxicity, cerebellar toxicity, or alopecia. Mylotarg is an effective treatment for older patients with CD33-positive AML in first relapse and has acceptable toxicity.
Asunto(s)
Aminoglicósidos , Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Inmunotoxinas/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Supervivencia sin Enfermedad , Femenino , Gemtuzumab , Humanos , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/mortalidad , Leucemia Mieloide/patología , Masculino , Persona de Mediana Edad , Monitoreo Fisiológico , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Pronóstico , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Tasa de SupervivenciaRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, binds to several cell-surface receptors with distinct functions (agonistic receptors 1 and 2 [TRAIL-R1, TRAIL-R2]; decoy receptors 3 and 4 [TRAIL-R3, TRAIL-R4]). Expression and function was characterized in patients with myelodysplastic syndromes (MDSs). While normal marrow showed negligible expression of TRAIL and receptors (except TRAIL-R3), TRAIL and all receptors were constitutively expressed in MDS marrow. Following TRAIL exposure, MDS marrow showed significant increases in apoptosis, whereas normal marrow, except for a subset of CD34+ precursors, did not (P =.012). Marrow from 21 patients with MDS was then propagated in long-term cultures in the presence or absence of TRAIL. While in advanced MDS (refractory anemia with excess blasts in transformation [RAEB-T] and tAML [MDS transformed into AML]), colony numbers decreased in the presence of TRAIL (63.0% +/- 10.4% of untreated group [100%]), numbers increased in patients with RA or RAEB (160.2% +/- 90.5% of untreated group). TRAIL eliminated preferentially clonally abnormal cells as identified by chromosomal markers. Thus, TRAIL and receptor expression differed significantly between normal and MDS marrow, and TRAIL modulated in vitro hemopoiesis in MDS dependent upon disease stage but not, to a detectable extent, in normal marrow.
Asunto(s)
Hematopoyesis , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/biosíntesis , Síndromes Mielodisplásicos/metabolismo , Receptores del Factor de Necrosis Tumoral/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Aneuploidia , Apoptosis , Proteínas Reguladoras de la Apoptosis , Western Blotting , Células de la Médula Ósea/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Células Cultivadas/metabolismo , Aberraciones Cromosómicas , Ensayo de Unidades Formadoras de Colonias , Proteínas Ligadas a GPI , Regulación de la Expresión Génica , Factores de Crecimiento de Célula Hematopoyética/farmacología , Humanos , Hibridación Fluorescente in Situ , Leucina Zippers , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , ARN Mensajero/biosíntesis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/genética , Miembro 10c de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ligando Inductor de Apoptosis Relacionado con TNF , Receptores Señuelo del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
PURPOSE: Three open-label, multicenter trials were conducted to evaluate the efficacy and safety of single-agent Mylotarg (gemtuzumab ozogamicin; CMA-676; Wyeth Laboratories, Philadelphia, PA), an antibody-targeted chemotherapy agent, in patients with CD33-positive acute myeloid leukemia (AML) in untreated first relapse. PATIENTS AND METHODS: The study population comprised 142 patients with AML in first relapse with no history of an antecedent hematologic disorder and a median age of 61 years. All patients received Mylotarg as a 2-hour intravenous infusion, at a dose of 9 mg/m(2), at 2-week intervals for two doses. Patients were evaluated for remission, survival, and treatment-emergent adverse events. RESULTS: Thirty percent of patients treated with Mylotarg obtained remission as characterized by 5% or less blasts in the marrow, recovery of neutrophils to at least 1,500/microL, and RBC and platelet transfusion independence. Although patients treated with Mylotarg had relatively high incidences of myelosuppression, grade 3 or 4 hyperbilirubinemia (23%), and elevated hepatic transaminase levels (17%), the incidences of grade 3 or 4 mucositis (4%) and infections (28%) were relatively low. There was a low incidence of severe nausea and vomiting (11%) and no treatment-related cardiotoxicity, cerebellar toxicity, or alopecia. Many patients received Mylotarg on an outpatient basis (38% and 41% of patients for the first and second doses, respectively). Among the 142 patients, the median total duration of hospitalization was 24 days; 16% of patients required 7 days of hospitalization or less. CONCLUSION: Administration of the antibody-targeted chemotherapy agent Mylotarg to patients with CD33-positive AML in first relapse induces complete remissions with what appears to be a favorable safety profile.
Asunto(s)
Aminoglicósidos , Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Inmunotoxinas/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/efectos adversos , Antibacterianos/farmacología , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Supervivencia sin Enfermedad , Europa (Continente)/epidemiología , Femenino , Gemtuzumab , Humanos , Inmunotoxinas/efectos adversos , Inmunotoxinas/farmacología , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/mortalidad , Masculino , Persona de Mediana Edad , Análisis Multivariante , América del Norte/epidemiología , Pronóstico , Recurrencia , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Tasa de SupervivenciaRESUMEN
RATIONALE AND OBJECTIVES: Most radiographic contrast media are hyperosmotic and able to shrink cells with which they are in contact. The authors studied cell volume control in rabbit proximal renal tubules after incubation with three contrast media: iohexol, ioxaglate, and iodixanol. MATERIALS AND METHODS: Proximal renal tubules were isolated from rabbit kidneys. The tubules were exposed to Ringer solutions containing 5% vol/vol iohexol (final osmolality, 330 mOsm), ioxaglate (323 mOsm), iodixanol (305 mOsm), or mannitol (control solutions with identical osmolalities), and tubule volumes were monitored. After 2 hours of incubation, the tubules were stimulated with a hyposmotic Ringer solution (165 mOsm). Three groups of 10 experiments were performed. RESULTS: All solutions induced cell shrinkage (8.3%+/-3.8 [standard error] to 15.4%+/-0.5), which was completely or partly reversible in most experiments (volume increase, 44.8%+/-14.7 to 149.9%+/-107.3) but not those with iohexol and iodixanol. With exposure to the hyposmotic solution, the cells swelled by 11.0%+/-1.8 to 39.7%+/-4.8. In general, the tubules that had been exposed to the most hyperosmotic solution swelled the most. Those exposed to contrast media showed less swelling than the mannitol-exposed controls. In all control experiments, the cells exhibited a gradual shrinkage (43.6%+/-28.5 to 87.0%+/-13). This regulatory response was partly inhibited in tubules exposed to iohexol (39.9%+/-15.8 shrinkage) or iodixanol (8.9%+/-15.8) and completely inhibited in those exposed to ioxaglate. Iohexol and ioxaglate exposure also led to a decrease in water permeability. CONCLUSION: Exposure to hyperosmotic contrast medium tends to induce prolonged cell shrinkage, decrease the water permeability of the cellular plasma membranes, and compromise the ability to regulate cellular volume. These changes seem to reflect both the hyperosmolality of the solutions and their inherent chemical properties.
Asunto(s)
Medios de Contraste/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Femenino , Yohexol/farmacología , Ácido Yoxáglico/farmacología , Túbulos Renales Proximales/citología , Masculino , Concentración Osmolar , Conejos , Factores de Tiempo , Ácidos Triyodobenzoicos/farmacologíaRESUMEN
PURPOSE: We compared multidimensional flow cytometry (MDF) with morphology in evaluating early marrow response to induction chemotherapy in pediatric ALL. METHODS: Chemotherapy response was determined by standard morphology or by MDF assessed by residual leukemic cell percentage remaining in the marrow on days 7, 14, and 28 of induction. Bone marrow response was classified as M3 (>25% leukemic blasts) or M1/M2 (< or = 25% leukemic blasts). Multidimensional flow cytometry evaluation was compared with that of standard morphology. Available day-7 and day-14 marrow slides were also reevaluated by a single pathologist without patients' clinical information. RESULTS: Of 46 day-7 specimens, eight (17%) had discordant MDF and morphologic results (P < 0.001), including six classified as M3 by morphology but were M1/M2 by MDF, and two were classified as M3 by MDF but were M1/M2 by morphology. Of 24 day-14 bone marrow specimens, five (20.5%) were discordant (P < 0.001), including two classified as M3 by morphology but were M1/M2 by MDF, and three were classified as M3 by MDF but were M1/M2 by morphology. Reevaluation of the blinded day-7 and day-14 marrow slides yielded discordance between repeated pathology readings of 11% (P < 0.001) and 6% (P = 0.04), respectively. CONCLUSION: Our data show significant discordance between the morphologic and MDF evaluation of early marrow response. Early response to therapy is a significant prognostic indicator in pediatric acute lymphoblastic leukemia and is used to alter subsequent treatment; thus, precise assessment of response is important. A larger comparison of MDF with morphology for the evaluation of early response, including correlation with clinical outcome, is warranted.
Asunto(s)
Examen de la Médula Ósea/métodos , Citometría de Flujo/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/patología , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Valor Predictivo de las Pruebas , Riesgo , Sensibilidad y Especificidad , Método Simple Ciego , Resultado del TratamientoRESUMEN
The complexity of cell populations requires careful selection of reagents to detect cells of interest and distinguish them from other types. Additional reagents are frequently used to provide independent criteria for cell identification. Two or three monoclonal antibodies in combination with forward and right-angle light scatter generate a data set that is difficult to visualize because the data must be represented in four- or five-dimensional space. The separation between cell populations provided by the multiple characteristics is best visualized by multidimensional analysis using all parameters simultaneously to identify populations within the resulting hyperspace. Groups of cells are distinguished based on a combination of characteristics not apparent in any usual two-dimensional representation of the data.
Asunto(s)
Sistemas de Administración de Bases de Datos/normas , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Inmunofenotipificación/métodos , Sistemas de Información/normas , Animales , Biología Computacional/métodos , Computadores , Interpretación Estadística de Datos , Humanos , Inmunofenotipificación/instrumentación , Luz , Lenguajes de Programación , Dispersión de Radiación , Programas InformáticosRESUMEN
RATIONALE AND OBJECTIVES: Most radiographic contrast media (CM) are hyperosmotic and pose an osmotic threat to cells they are in contact with. To study these effects at the cellular level, cell volume regulatory mechanisms were observed in proximal renal tubules following exposure to the CM iohexol, ioxaglate, and iodixanol. MATERIALS AND METHODS: Isolated renal tubules from trout (Salmo trutta) were exposed to 5% vol/vol iohexol (326 mOsm), ioxaglate (314 mOsm), or iodixanol (300 mOsm) or mannitol (to achieve the same osmolalities), and cell volume changes were observed videometrically. RESULTS: Iohexol and ioxaglate solutions induced a rapid shrinkage (12%-13%) not followed by cell volume regulation. Without CM (same osmolality), the cells shrank 11% but then showed a 77%-88% volume recovery. This reswelling was inhibited by 55% with the Na+, K+, Cl- symporter inhibitor bumetanide (50 micromol/L). Iodixanol did not significantly affect cell volume. Tubules preincubated with CM or mannitol were then stimulated with a hypoosmotic Ringer solution (160 mOsm) resulting in a 26%-36% cellular volume increase. Compared with results of experiments without mannitol and CM, preexposure to iohexol or ioxaglate almost completely inhibited the expected regulatory shrinkage phase, while previous exposure to hyperosmotic solutions with mannitol reduced the shrinkage response by 40%-53%. CONCLUSION: In this system, the hyperosmotic iohexol and ioxaglate cause cell shrinkage followed by an impaired cell volume regulatory response. Exposure to these two CM also inhibits cell volume regulation on hypoosmotic stimulation. The isosmotic iodixanol has no such effects. These changes appear to some extent to be a result of the CM's degree of hyperosmolality, but this property alone does not explain these findings.
Asunto(s)
Agua Corporal/metabolismo , Medios de Contraste/metabolismo , Túbulos Renales/metabolismo , Trucha/metabolismo , Animales , Tamaño de la Célula/efectos de los fármacos , Yohexol/metabolismo , Ácido Yoxáglico/metabolismo , Túbulos Renales/citología , Ósmosis , Estadísticas no Paramétricas , Ácidos Triyodobenzoicos/metabolismoRESUMEN
Children with neurofibromatosis type 1 (NF1) carry germline mutations in one allele of the NF1 gene and are predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML). Disruption of the remaining NF1 allele can be found in malignant cells. Flow cytometric cell sorting techniques to isolate the malignant cell populations and molecular genetic methods to assay for somatic loss of the normal NF1 allele were used to study an unusual child with NF1 and JMML who subsequently had T-cell lymphoma. The data show that malignant JMML and lymphoma cells share a common loss of genetic material involving the normal NF1 gene and approximately 50 Mb of flanking sequence, suggesting that the abnormal T-lymphoid and myeloid populations were derived from a common precursor cell. These data support the hypothesis that JMML can arise in a pluripotent hematopoietic cell.
Asunto(s)
Leucemia Mielomonocítica Aguda/etiología , Leucemia Mielomonocítica Aguda/genética , Linfoma de Células T/etiología , Linfoma de Células T/genética , Células Madre Neoplásicas/patología , Proteínas del Tejido Nervioso/genética , Diferenciación Celular , Preescolar , Compensación de Dosificación (Genética) , Humanos , Leucemia Mielomonocítica Aguda/patología , Linfoma de Células T/patología , Masculino , Neoplasias Primarias Secundarias , Neurofibromina 1Asunto(s)
Antígenos de Neoplasias/análisis , Glicoproteínas/análisis , Leucemia Mieloide/inmunología , Síndromes Mielodisplásicos/inmunología , Células Madre Neoplásicas/inmunología , Péptidos/análisis , Antígeno AC133 , Antígenos CD , Antígenos CD34/análisis , Antígenos de Neoplasias/genética , Epítopos/análisis , Epítopos/inmunología , Citometría de Flujo , Expresión Génica , Regulación Leucémica de la Expresión Génica , Glicoproteínas/genética , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Péptidos/genéticaRESUMEN
The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis factor (TNF)-alpha, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-alpha and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-alpha levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-alpha were mononuclear (non-stromal) cells which appeared to produce TNF-alpha at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-alpha or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction.
Asunto(s)
Células de la Médula Ósea/patología , Hematopoyesis , Síndromes Mielodisplásicos/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Adhesión Celular , Separación Celular , Células Cultivadas , Niño , Preescolar , Citogenética , Femenino , Hematopoyesis/genética , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/clasificación , Síndromes Mielodisplásicos/genética , Células del Estroma/patologíaRESUMEN
A vexing problem after hematopoietic cell transplantation (HCT) for leukemia is assessing the biologic significance of low numbers of cells "suspicious" for relapse seen in morphologic review of peripheral blood smears (PBSs). In 27 patients, in apparent hematologic remission after HCT for leukemia, we studied the nature of such cells in PBSs to the endpoint of leukemic relapse by using multidimensional flow cytometry (MDF) on blood or bone marrow aspirates. Based on abnormal cytometric maturational patterns, +/- cell sorting of blasts with fluorescence in situ hybridization with informative markers, we differentiated benign recovering myeloid and lymphoid precursors from leukemic cells. In 17 patients, blasts were characterized by MDF as normal early hematopoietic precursors, lymphoblasts, or NK cells. Of these patients, 16 remained in remission for at least 42 days. In 10 patients, blasts were characterized by MDF as a malignant immunophenotype; 9 relapsed within 10 days and 1 relapsed 280 days after a graft-vs-leukemia effect. MDF status was strongly associated with a 90 x probability of relapse even after adjusting for other potential variables. Morphologic triggered MDF characterization of peripheral blasts is a powerful and rapid tool for distinguishing immature regenerative forms from early leukemic relapse.
Asunto(s)
Crisis Blástica/etiología , Citometría de Flujo , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Adolescente , Adulto , Separación Celular , Niño , Preescolar , Femenino , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Leucemia/terapia , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
A 50-year-old woman who was retrospectively diagnosed with an early asymptomatic myelodysplastic syndrome (MDS) served as a haemopoietic stem cell donor for her HLA-identical sister who had chemotherapy-refractory non-Hodgkin's lymphoma. The MDS of the donor was classified as refractory anaemia (RA) and cytogenetically characterized by deletion of the long arm of chromosome 20 [del(20q)]. Donor cell engraftment in marrow and peripheral blood was analysed over a period of 5 months after transplant using conventional cytogenetics, fluorescence in situ hybridization, and variable number of tandem repeats. Neutrophil counts >0.5 x 109/l and platelet counts >20 x 109/l were reached promptly on days 12 and 24, respectively. Throughout the period of observation the percentage of cells with the del(20q) abnormality in the recipient's marrow and peripheral blood was comparable to the proportion of these cells in the donor. These data indicate that the abnormal clone was capable of homing to the marrow, proliferating, differentiating, and therefore contributing to haemopoiesis in a relatively efficient manner. This implies that MDS progenitor cells may not have homing and growth deficiencies, a finding that has particular relevance for autologous transplantation in MDS patients where tumour cells potentially contaminate the graft.