RESUMEN
We report on a patient who developed donor-derived cutaneous T-cell lymphoma (CTCL) 4 years after successful treatment of chronic myeloid leukaemia with an allogeneic bone marrow transplant. The patient developed an eczematous rash unresponsive to topical therapy and immunosuppression. When CTCL was diagnosed in the recipient, his sibling donor had been attending his local dermatology unit with a maculosquamous rash, which proved subsequently to be mycosis fungoides. An identical pattern of donor and recipient clonality assessment and T-cell receptor gene sequencing indicated that the CTCL was probably transmitted in the bone marrow harvest. This suggests that CTCL cells circulate in the marrow at an early subclinical stage in this disease. This is the second case of donor-derived CTCL reported to date.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Micosis Fungoide/etiología , Neoplasias Cutáneas/etiología , Humanos , Masculino , Persona de Mediana Edad , Hermanos , Trasplante Homólogo/efectos adversosRESUMEN
BACKGROUND: In recent years, much progress has been made in the treatment of multiple myeloma. However, a major limitation of existing chemotherapeutic drugs is the eventual emergence of resistance; hence, the development of novel agents with new mechanisms of action is pertinent. Here, we describe the activity and mechanism of action of pyrrolo-1,5-benzoxazepine-15 (PBOX-15), a novel microtubule-targeting agent, in multiple myeloma cells. METHODS: The anti-myeloma activity of PBOX-15 was assessed using NCI-H929, KMS11, RPMI8226, and U266 cell lines, and primary myeloma cells. Cell cycle distribution, apoptosis, cytochrome c release, and mitochondrial inner membrane depolarisation were analysed by flow cytometry; gene expression analysis was carried out using TaqMan Low Density Arrays; and expression of caspase-8 and Bcl-2 family of proteins was assessed by western blot analysis. RESULTS: Pyrrolo-1,5-benzoxazepine-15 induced apoptosis in ex vivo myeloma cells and in myeloma cell lines. Death receptor genes were upregulated in both NCI-H929 and U266 cell lines, which displayed the highest and lowest apoptotic responses, respectively, following treatment with PBOX-15. The largest increase was detected for the death receptor 5 (DR5) gene, and cotreatment of both cell lines with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), the DR5 ligand, potentiated the apoptotic response. In NCI-H929 cells, PBOX-15-induced apoptosis was shown to be caspase-8 dependent, with independent activation of extrinsic and intrinsic apoptotic pathways. A caspase-8-dependent decrease in expression of Bim(EL) preceded downregulation of other Bcl-2 proteins (Bid, Bcl-2, Mcl-1) in PBOX-15-treated NCI-H929 cells. CONCLUSION: PBOX-15 induces apoptosis and potentiates TRAIL-induced cell death in multiple myeloma cells. Thus, PBOX-15 represents a promising agent, with a distinct mechanism of action, for the treatment of this malignancy.
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Apoptosis/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Mieloma Múltiple/patología , Oxazepinas/farmacología , Pirroles/farmacología , Receptores de Muerte Celular/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Regulación hacia Arriba/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Humanos , Microscopía FluorescenteRESUMEN
Patients with Hodgkin lymphoma who relapse or are refractory to first line multi-agent chemotherapy can be successfully salvaged with high dose therapy (HDT) and autologous stem cell transplant (ASCT). Twenty-six patients with relapsed or refractory Hodgkin lymphoma have been treated with HDT and ASCT at St James Hospital between 2000 and 2005. At day 100 post HDT-ASCT, 23 patients were in complete remission. This group included all 6 patients transplanted at first relapse, 8 of 9 with advanced disease and 9 of 11 with primary refractory disease. Patients treated in first relapse had the best outcome with an overall and progression free survival of 100% (median, 37 months). Patients with primary refractory disease had the poorest outcome with an overall survival of 76% (median, 28 months). All patients with primary refractory disease responsive to salvage chemotherapy were in remission at a median of 28 months. The presence of chemosensitive disease prior to transplantation was the most important determinant of outcome. PET-CT imaging is useful to assess chemosensititvity prior to HDT and thus predict which patients will do well post HDT-ASCT. No patient died of treatment related toxicity. The outcome of this patient series compares favourably with international figures.
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Antineoplásicos/uso terapéutico , Trasplante de Médula Ósea , Enfermedad de Hodgkin/tratamiento farmacológico , Trasplante de Células Madre , Trasplante Autólogo , Adolescente , Adulto , Femenino , Enfermedad de Hodgkin/mortalidad , Enfermedad de Hodgkin/terapia , Humanos , Irlanda , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/prevención & control , Inducción de Remisión , Terapia Recuperativa , Análisis de Supervivencia , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
The nitrostyrene scaffold was previously identified as a lead target structure for the development of effective compounds targeting Burkitt's lymphoma. The present study aimed to develop these compounds further in haematological malignancies, including chronic lymphocytic leukaemia (CLL). Cellular viability, flow cytometry and lactate dehydrogenase assays, amongst others, were used to examine the effects of nitrostyrene compounds on CLL cells, including a cell line representing disease with poor prognosis (HG3) and in ex vivo CLL patient samples (n=14). The results demonstrated that two representative nitrostyrene compounds potently induced apoptosis in CLL cells. The proapoptotic effects of the compounds were found to be reactive oxygen species and caspasedependent, and had minimal effects on the viability of normal donor peripheral blood mononuclear cells. Nitrostyrene compounds exhibited synergistic augmentation of apoptosis when combined with the phosphatidylinositol 3kinase inhibitor idelalisib and demonstrated potent toxicity in ex vivo CLL cells, including those cocultured with bone marrow stromal cells, making them more resistant to apoptosis (n=8). These compounds also demonstrated activity in samples from patients with poor prognostic indicators; unmutated immunoglobulin heavy chain genes, expression of CD38 and deletions in chromosomes 17p and 11q. These results suggest that this class of pharmaceutically active compounds offer potential in the treatment of CLL.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Nitrocompuestos/farmacología , Estirenos/farmacología , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Humanos , Concentración 50 Inhibidora , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , Nitrocompuestos/química , Nitrocompuestos/uso terapéutico , Cultivo Primario de Células , Pronóstico , Purinas/farmacología , Purinas/uso terapéutico , Quinazolinonas/farmacología , Quinazolinonas/uso terapéutico , Relación Estructura-Actividad , Estirenos/química , Estirenos/uso terapéuticoRESUMEN
Abnormal deposits of free iron are found on the cytoplasmic surface of red blood cell (RBC) membranes in beta-thalassemia. To test the hypothesis that this is of importance to RBC pathobiology, we administered the iron chelator deferiprone (L1) intraperitoneally to beta-thalassemic mice for 4 wk and then studied RBC survival and membrane characteristics. L1 therapy decreased membrane free iron by 50% (P = 0.04) and concomitantly improved oxidation of membrane proteins (P = 0.007), the proportion of RBC gilded with immunoglobulin (P = 0.001), RBC potassium content (P < 0.001), and mean corpuscular volume (P < 0.001). Osmotic gradient ektacytometry confirmed a trend toward improvement of RBC hydration status. As determined by clearance of RBC biotinylated in vivo, RBC survival also was significantly improved in L1-treated mice compared with controls (P = 0.007). Thus, in vivo therapy with L1 removes pathologic free iron deposits from RBC membranes in murine thalassemia, and causes improvement in membrane function and RBC survival. This result provides in vivo confirmation that abnormal membrane free iron deposits contribute to the pathobiology of thalassemic RBC.
Asunto(s)
Membrana Eritrocítica/química , Hierro/fisiología , Talasemia/etiología , Animales , Supervivencia Celular , Deferiprona , Membrana Eritrocítica/inmunología , Globinas , Quelantes del Hierro/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Piridonas/farmacología , Receptores de Antígenos de Linfocitos B/análisisRESUMEN
UNLABELLED: Essentials Treatment options are limited for refractory bleeding in acquired von Willebrand Syndrome (AVWS). Lenalidomide therapy was studied in two patients with AVWS due to monoclonal gammopathy (MG). Lenalidomide increased von Willebrand factor (VWF), lowered VWF clearance and resolved bleeding. Lenalidomide is a potential treatment option for refractory bleeding in AVWS secondary to MG. SUMMARY: Background Acquired von Willebrand syndrome (AVWS) is associated with lymphoproliferative disorders, including monoclonal gammopathy (MG) of undetermined significance (MGUS) and multiple myeloma. Patients commonly present with significant bleeding complications that are difficult to manage, owing to a markedly reduced von Willebrand factor (VWF) half-life. Objectives To investigate the use of the immunomodulatory drug lenalidomide in two patients with severe refractory bleeding caused by AVWS associated with MGs. Results In both patients, lenalidomide treatment resulted in significant clinical improvement, and marked increases in plasma VWF antigen (VWF:Ag) and VWF ristocetin cofactor levels. This normalization in plasma VWF levels was sustained for > 2 years in both patients. Furthermore, in one patient, plasma VWF levels remain normal for at least 14 months following discontinuation of lenalidomide treatment. To investigate the molecular mechanisms underlying these observations, VWF propeptide (VWFpp)/VWF:Ag ratios were analyzed to assess VWF clearance. At enrolment, plasma VWFpp/VWF:Ag ratios were significantly elevated in both patients. Importantly, lenalidomide treatment resulted in normalization of VWFpp/VWF:Ag ratios in both patients. These novel data suggest that lenalidomide functions to attenuate enhanced VWF clearance in AVWS. Interestingly, in a patient with MGUS, lenalidomide treatment was associated with a significant increase in plasma VWF levels, despite no major change in paraprotein level. Conclusions Collectively, our findings suggest that lenalidomide constitutes a novel therapeutic option for the management of AVWS associated with MG. The biological mechanism(s) through which lenalidomide causes a sustained increase in plasma VWF levels in AVWS independently of paraprotein level requires further study, but is in part modulated through inhibition of enhanced VWF clearance.
Asunto(s)
Paraproteinemias/tratamiento farmacológico , Talidomida/análogos & derivados , Enfermedades de von Willebrand/tratamiento farmacológico , Anciano , Anticoagulantes/uso terapéutico , Esquema de Medicación , Hemorragia , Humanos , Lenalidomida , Masculino , Persona de Mediana Edad , Mieloma Múltiple/inmunología , Paraproteinemias/sangre , Paraproteinemias/complicaciones , Inducción de Remisión , Talidomida/uso terapéutico , Resultado del Tratamiento , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/complicaciones , Factor de von Willebrand/uso terapéuticoRESUMEN
BACKGROUND: Sporadic Burkitt lymphoma (BL), characterised by translocation-associated C-MYC upregulation is a rare, aggressive lymphoma with a cure rate up to 90 % using the R-CODOX-M/R-IVAC (RCRI) protocol. RCRI is active in HIV-associated BL in combination with HAART. The WHO classification system defines lymphomas intermediate between DLBCL and BL, in which lymphomas with t(14;18)(q32;q21) and C-MYC-carrying translocation, i.e. 'double-hit' are included (BL-DH), and these patients are conventionally treated with RCRI. RESULT: We describe the SJH experience of 25 patients with BL, BL + HIV and BL-DH treated with RCRI between 2002 and 2011. Twelve BL patients (8M/4F), median age 49.1 years (range 20-73 years); of whom 9 had extensive disease, including 8 with marrow and 2 with CNS involvement. Eleven patients remain in remission at 80.5 months (range 37-147 months) from completion of treatment and one died of progressive BL giving an OS of 91.6 % at 1 year with no late relapses. Eight patients with BL + HIV were treated (6M/2F) with a median age 40.25 years (range 24-64). Five remain in complete remission (CR) at 65 months (range 13-109 months), three patients died, two of progressive disease and one of treatment-associated hepatotoxicity in CR. Five patients with BL-DH were included; (3M/2F), age 47.8 years (range 42-55 years); and all patients died of progressive disease, 4 on RCRI therapy and a further patient despite an allogeneic transplantation. CONCLUSION: These results confirm that RCRI is an effective treatment in adults with BL and BL + HIV and remains the gold standard against which other regimens should be compared. We confirm the poor prognosis found in BL-DH, indicating new treatment approaches are needed for this sub-group which should be identified at diagnosis by FISH analysis.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Adulto , Anciano , Terapia Antirretroviral Altamente Activa/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: High-dose treatment with autologous stem cell transplantation (ASCT) has become the standard of care for patients with myeloma below the age of 65 years. AIMS: We report an audit of 60 patients (median age: 52.5 years) who underwent ASCT in the National Bone Marrow Transplant centre in St James's Hospital in Dublin between 1997 and 2003 inclusive. METHODS: Clinical and laboratory data were retrieved from patient medical records and hospital information management systems. RESULTS: Thirty-six patients had IgG, 11 IgA, 1 IgD, 9 light chain and 3 non-secretory MM. Fifty-seven (95%) patients received anthracycline-corticosteroid combination chemotherapy prior to autografting. There was no transplant-related mortality (TRM). Complete (CR) and Partial Responses (PR) were seen in 16 (29.6%) and 29 (53.7%) of those evaluable (n = 54 (90%)). The actuarial Progression-Free (PFS) and Overall Survival (OS) rates at five years are 13% and 55% respectively. CONCLUSION: Centre outcome is comparable to published international series and supports the use of ASCT in the treatment of this malignancy.
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Mieloma Múltiple/cirugía , Trasplante de Células Madre de Sangre Periférica , Trasplante Autólogo , Resultado del Tratamiento , Anciano , Progresión de la Enfermedad , Femenino , Humanos , Irlanda , Masculino , Auditoría Médica , Persona de Mediana Edad , Mieloma Múltiple/mortalidad , Mieloma Múltiple/fisiopatología , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
We report a case of acute lymphoblastic leukaemia relapsing after allogeneic bone marrow transplantation in which the polymerase chain reaction (PCR) was used to assess chimeric status. This technique demonstrated the progressive reappearance of host cells prior to clinical relapse. The relapse was of host cell origin as shown by the presence of female (recipient) metaphases containing an abnormal chromosomal marker (iso 9q) which had also been present at initial diagnosis. The emergence of host cells in this case, detected only by PCR techniques but not by cytogenetic methods, appeared to herald overt relapse. PCR analysis provides a sensitive tool for detecting a progressive rise in host cell numbers which may predict clinical relapse.
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Trasplante de Médula Ósea/efectos adversos , Rechazo de Injerto , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Adulto , Trasplante de Médula Ósea/patología , Ciclo Celular , Quimera , Cromosomas , ADN/análisis , Femenino , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/ultraestructura , Humanos , Cariotipificación , Metafase , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/cirugíaRESUMEN
Chronic graft-versus-host disease (GVHD) refractory to standard immunosuppressive therapy remains a major cause of morbidity and mortality after allogeneic bone marrow transplantation (BMT). Thalidomide may be effective in some patients with high-risk or refractory chronic GVHD. We report a single-institution study of thalidomide in 37 BMT patients with extensive chronic GVHD refractory to standard immunosuppressive therapy. Acute GVHD occurred in 34 (91%) of patients and evolved progressively into chronic GVHD in 23 (62%) patients. Thalidomide was added to standard immunosuppressive therapy a median of 11 months (range 0-105 months) after the diagnosis of chronic GVHD. Fourteen of 37 (38%) patients responded after introduction of thalidomide (one complete, 13 partial). Ten of 21 (46%) children and four of 16 (25%) adults responded. Responses were seen in eight of 17 (47%) recipients of related donor marrow and six of 20 (30%) recipients of unrelated donor marrow. Eight of 23 (34%) patients with progressive onset of chronic GVHD showed a response. There were no deaths among the responders. The remaining 23 patients (62%) did not respond and of these only two survive, one with progressive scleroderma, and the other with bronchiolitis obliterans. Chronic GVHD with associated infection (most commonly disseminated fungal infection) was a major contributor to mortality in all cases. Overall, after initiation of thalidomide, the 2-year Kaplan-Meier survival was 41% (95% C.I. 24%-59%). We conclude that thalidomide is a useful and well-tolerated therapy for patients with previously treated refractory chronic GVHD, including those with progressive onset of chronic GVHD, recipients of unrelated donor marrow, and children. Earlier introduction of thalidomide as an adjunct to standard immunosuppressive therapy may lead to more frequent responses and possible better survival.
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Enfermedad Injerto contra Huésped/tratamiento farmacológico , Talidomida/uso terapéutico , Enfermedad Crónica , Femenino , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Masculino , Talidomida/efectos adversosRESUMEN
Hairy cell leukaemia (HCL) has distinct clinical, morphological and immunophenotypic features with no recurrent cytogenetic or molecular abnormalities reported until the recent description of the BRAF V600E mutation in patients with classical HCL. The incidence of this mutation was sought in 27 patients with either classical HCL or HCL variant by an allele-specific PCR approach and findings related to morphology, cytochemistry and immunophenotype. A high degree of correlation was noted between the presence of BRAF V600E and established diagnostic criteria in 26/27 patients with HCL/HCL variant. Detection of the BRAF V600E mutation is therefore a useful adjunct in the differential diagnosis of HCL and HCL variant and highlights the value of a multifaceted approach to the diagnosis of this malignancy.
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Leucemia de Células Pilosas/diagnóstico , Leucemia de Células Pilosas/genética , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/patología , Diagnóstico Diferencial , Femenino , Variación Genética , Histocitoquímica , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Estudios RetrospectivosAsunto(s)
Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de Células Germinales y Embrionarias/terapia , Terapia Recuperativa , Adolescente , Adulto , Carboplatino/administración & dosificación , Etopósido/administración & dosificación , Estudios de Seguimiento , Humanos , Ifosfamida/administración & dosificación , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia , Paclitaxel/administración & dosificación , Pronóstico , Estudios Prospectivos , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenAsunto(s)
Anemia Hemolítica Congénita/complicaciones , Leucemia Linfocítica Crónica de Células B/terapia , Trasplante de Células Madre , Corticoesteroides/uso terapéutico , Adulto , Humanos , Terapia de Inmunosupresión/métodos , Leucemia Linfocítica Crónica de Células B/complicaciones , Masculino , Quimera por TrasplanteAsunto(s)
Trasplante de Células Madre Hematopoyéticas/efectos adversos , Leucemia Linfocítica Crónica de Células B/terapia , Toxoplasmosis/diagnóstico , Adulto , Coroiditis/etiología , Coroiditis/patología , Coroiditis/terapia , Diagnóstico por Imagen , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Leucemia Linfocítica Crónica de Células B/complicaciones , Masculino , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/diagnóstico , Toxoplasmosis/inducido químicamente , Toxoplasmosis Cerebral/inducido químicamente , Toxoplasmosis Cerebral/diagnósticoRESUMEN
Secondary or late graft failure has been defined as the development of inadequate marrow function after initial engraftment has been achieved. We describe a case of profound marrow aplasia occurring 13 years after sibling allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) in first chronic phase. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with anti-thymocyte globulin (ATG).
Asunto(s)
Enfermedades de la Médula Ósea/etiología , Trasplante de Médula Ósea/efectos adversos , Leucemia Mielógena Crónica BCR-ABL Positiva/complicaciones , Suero Antilinfocítico/uso terapéutico , Enfermedades de la Médula Ósea/tratamiento farmacológico , Enfermedades de la Médula Ósea/patología , Trasplante de Médula Ósea/métodos , Femenino , Prueba de Histocompatibilidad , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica , Hermanos , Trasplante Homólogo , Resultado del TratamientoRESUMEN
Vasoocclusive episodes in sickle cell anemia may be initiated by adherence of erythrocytes to endothelium, one mechanism of which involves thrombospondin binding to CD36 on the red blood cell (RBC). We compared CD36 expression and its relationship to stress reticulocytosis in patients with sickle cell anemia and other chronic hemolytic disorders, including some after splenectomy. Adults with sickle cell anemia had significantly more CD36-positive cells (4.1% +/- 3.4%, mean +/- SD, n = 12) in unfractionated blood than did normal adults, who had almost none (0.13% +/- 0.15%, n = 8, p < 0.05). In density-fractionated blood, sickle samples contained significantly more CD36-positive cells (39.8% +/- 21.9%, n = 10) in the lowest density layers than did splenectomized high-reticulocyte controls (8.5% +/- 6.4%, n = 4, p < 0.05). "Stress" reticulocytes (identified by their unique morphology) were significantly more frequent in low-density layers from sickle blood (44.3% +/- 23.9%, n = 10) than from nonsplenectomized high-reticulocyte controls (10.5% +/- 14.8%, n = 5, p < 0.05). There was a strong correlation (r = 0.92) between stress reticulocyte count and number of CD36-positive cells for all patients except thalassemics, in whom CD36-positive cells were more frequent than stress reticulocytes. Flow cytometry confirmed that the maximal CD36 signal was found on immature reticulocytes. We conclude that CD36-positive stress reticulocytes occur more frequently in sickle cell anemia than in other chronic hemolytic states, even after surgical splenectomy, suggesting that this enhanced CD36-positive stress reticulocytosis does not simply reflect absent splenic function. These results explain why RBCs from high-reticulocyte control patients fail to show the significant CD36-dependent thrombospondin-mediated adhesion to endothelium that is exhibited by sickle red cells.
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Anemia de Células Falciformes/sangre , Antígenos CD36/sangre , Enfermedades Hematológicas/sangre , Recuento de Reticulocitos , Reticulocitos/química , Centrifugación por Gradiente de Densidad , Citometría de Flujo , Humanos , Microscopía Electrónica de Rastreo , Reticulocitos/ultraestructura , EsplenectomíaRESUMEN
Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr, since mRNA for kdr was detected using RT-PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.
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Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Linfocinas/farmacología , Animales , Anticuerpos Monoclonales , Bovinos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Thrombospondin (TSP), a large protein found in platelet alpha-granules (as TSP-1), mediates adhesion of sickle reticulocytes to cultured vascular endothelium. To further explore the physiologic relevance of this observation, we have measured plasma TSP levels and platelet TSP-1 content in subjects with sickle cell disease. Plasma TSP levels were similar for normal controls (mean 491 ng/ml, range 331-723) and steady-state HbSS patients (mean 536, range 333-1107) and were significantly (P = 0.012) but variably elevated for HbSS patients presenting with acute painful crisis (mean 868, range 442-2780). Some of these elevated plasma TSP levels reached those previously observed to support maximal red cell adhesion to endothelium in vitro. Compared to normals, both steady-state and in-crisis HbSS patients had significantly (P < 0.001) depressed platelet TSP-1 content (82.6 +/- 11.9, 47.1 +/- 16.0 and 45.9 +/- 20.7 ng/l0(6) platelets, respectively, mean +/- SD). HbSC disease patients, all examined during steady state, had low-normal plasma levels of TSP and either normal or depressed platelet TSP-1 content. Serial observations on three sickle cell anemia subjects indicated a probable relationship between platelet TSP-1 release, elevated plasma TSP levels, and acute vaso-occlusive episodes. These results suggest a state of ongoing release and depletion of TSP-1 from activated platelets in patients with sickle cell disease.
Asunto(s)
Anemia de Células Falciformes/sangre , Plaquetas/química , Enfermedad de la Hemoglobina SC/sangre , Glicoproteínas de Membrana/sangre , Enfermedad Aguda , Anemia de Células Falciformes/fisiopatología , Enfermedad de la Hemoglobina SC/fisiopatología , Humanos , Dolor/sangre , Dolor/fisiopatología , Activación Plaquetaria , Adhesividad Plaquetaria , Bazo/fisiopatología , TrombospondinasRESUMEN
It is well recognized that haemoglobin variants can be detected during the measurement of HbA1c by high-performance liquid chromatography (HPLC). A number of variants have been reported as compromising the quantification of HbA1c, a marker used in the assessment of glycaemic control in diabetes. We describe a case of haemoglobin Etobicoke, a rare alpha chain variant detected in an Irish diabetic during HbA1c analysis. Its identity was confirmed using a series of investigations. These included haemoglobin electrophoresis at alkaline and acid pH, isoelectric focusing and globin chain electrophoresis. Ultimately mass spectrometry isolated the mutation at position alpha 84 (F5). Haemoglobin Etobicoke, first described in Canada in 1969 has not previously been detected on HbA1c analysis. In the presence of this rare variant, HbA1c, a standard method using HPLC to assess glycaemic control in diabetes is unreliable and alternatives such as fructosamine need to be considered. HbA1c measured by automated HPLC will effectively screen populations where haemoglobin variants were not previously known. Precise identity of these variants when they are detected is crucial to the reliable interpretation of HbA1c analyses.
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Cromatografía Líquida de Alta Presión , Diabetes Mellitus Tipo 2/sangre , Hemoglobina Glucada/análisis , Hemoglobinas Anormales/análisis , Electroforesis de las Proteínas Sanguíneas , Reacciones Falso Negativas , Globinas/genética , Hemoglobinas Anormales/genética , Humanos , Hallazgos Incidentales , Irlanda , Focalización Isoeléctrica , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Monoclonal antibody P1H12 recognizes circulating endothelial cells and endothelia of all sizes of blood vessels. To identify the protein recognized by P1H12, we expressed a cDNA library in CHO cells and sequenced the cDNA from positive cells. The P1H12 sequence was identical, except at several bases, to that reported for melanoma cell surface antigen MUC18/CD146. Aggregation assays demonstrated that CD146 mediates Ca(++)-independent homotypic endothelial cell adhesion. P1H12 mAb abrogated interactions between human microvascular endothelial cells (HMVECs) but not between human umbilical vein endothelial cells (HUVECs). P1H12 mAb abrogated P1H12-positive (CHO(P1H12))-association with HMVECs or HUVECs. CD146 distribution is sparser on HUVECs than on HMVECs. These data imply that HMVECs and HUVECs express the CD146 binding partner but that CD146 is functional (or at sufficient density) only on HMVECs. HMVEC monolayers treated with soluble P1H12 mAb showed increased permeability to albumin, with accompanying changes in actin, paxillin, FAK, and caveolin distribution and changes in tyrosine phosphorylation of FAK. Stimulation with P1H12 mAb led to redistribution of NF-kappa B to the nucleus. P1H12 mAb bound to beads inhibited closure of wounded endothelial monolayers. CD146 thus joins VE-cadherin and PECAM-1 as a molecule that mediates homotypic endothelial cell adhesion. CD146 has both structural functions and signaling functions important for endothelial monolayer integrity.