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Platelet activation is found in inflammatory conditions and implicated in the pathogenesis of chronic medical conditions, such as atherosclerosis, coronary vascular disease, cerebrovascular disease, and diabetes mellitus (DM). HbA1c is inversely related to vitamin D25 levels in individuals with and without DM. This study aimed to determine the relation between platelet aggregation, vitamin D and HbA1c among healthy individuals and those with Type 2 DM (T2DM). The direct effect of vitamin D1, 25 (calcitriol) on platelet aggregation was also investigated. The study included four groups: A. normoglycemic Control group: HbA1c<5.7%; B. Pre-diabetes (DM): 5.7% ≥ HbA1c ≤ 6.4%; C. DM on aspirin therapy: HbA1c>6.4%(+)Asp.; and D. DM not on aspirin therapy: HbA1c > 6.4%(-)Asp. Platelet aggregation was tested with and without calcitriol or saline pre-treatment, using collagen or adenosine diphosphate (ADP) as agonists. Platelet aggregation was higher in DM(-)Asp group compared to normoglycemic and DM(+)Asp, and higher, but not significant compared to pre-DM. The entire study population exhibited negative correlation between HbA1c and serum concentration of vitamin D25. Excluding DM(+)Asp, aggregation induced by collagen was significantly higher in patients with insufficient (<76 nmol/L) vitamin D25 compared to sufficient (≥76 nmol/L) vitamin D25. In this cohort, a negative correlation was found between serum concentrations of vitamin D25 and collagen-induced percent maximum (%max) aggregation and area under curve (AUC) aggregation. In the DM(-)Asp group, collagen-induced aggregation was reduced by approximately 25% after calcitriol treatment. Calcitriol decreased ADP-induced aggregation of control and DM(+)Asp groups to approximately 85% of saline treatment. We conclude that glycemic control is inversely associated with high platelet aggregation and low vitamin D25 levels. This elevated aggregation could be regulated by a novel, direct effect of calcitriol, indicating a beneficial effect of vitamin D on vascular complications related to diabetes. We offer a possible non-genomic mechanism for the vitamin D/Vitamin D receptor (VDR) pathway.
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Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Agregación Plaquetaria/efectos de los fármacos , Vitamina D/farmacología , Anciano , Aspirina/farmacología , Biomarcadores , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/farmacología , Recuento de Plaquetas , Pruebas de Función PlaquetariaRESUMEN
INTRODUCTION: Critical laboratory results require prompt reporting to the attending physician, as they may indicate that a patient is in a life-threatening condition. Although this important subject has been covered in many publications, it needs more attention from our healthcare organizations, which have no official policy on the subject. Matching expectations between the doctor and the laboratory needs to be better defined. PURPOSE: The aim of this work was to inform the community of doctors and laboratories about the multiple problems concerning the reporting of critical laboratory results, to create a platform for exchanging views and ideas, and to build an extensive infrastructure for developing a unified plan to address this important issue. METHODS: We present the results of four years of experience of reporting critical laboratory values at the Meir Medical Center Laboratories. The idea leading this work was to present the relatively low rate of critical results reported by the laboratories in 2010, sharing the problems discovered while investigating the situation in depth, and presenting the solutions that enabled us to obtain the desired results within four years. RESULTS: Gradual implementation of these improvements resulted in critical value reporting increasing from 55% in 2010 to 95% currently. CONCLUSION: We suggest a model for improving critical laboratory values reporting based on our 4-year experience, which emphasizes: (1) The importance of selecting proper tests and values for critical results; (2) The significance of using technology and computerized measures to support the process; and (3) Developing quick procedures for monitoring and controlling the process.
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Técnicas de Laboratorio Clínico/normas , Laboratorios de Hospital/normas , Política Organizacional , Centros Médicos Académicos , Humanos , Israel , Laboratorios de Hospital/organización & administración , Médicos/organización & administración , Garantía de la Calidad de Atención de SaludRESUMEN
The ability to sequence nucleic acids at an unprecedented pace and decreased costs using massive parallel sequencing (MPS) strongly affects biomedical research. Here we applied MPS for the detection of rare, clinically relevant mutations in a chronic myeloid leukaemia (CML) patient. Tyrosine kinase inhibitors revolutionized CML therapy but in some patients the disease progresses due to resistance-conferring mutations. MPS was applied herein to monitor such mutations in BCR-ABL1 transcripts at different time points. The large volume of sequencing data increases sensitivity compared to direct sequencing and allows detection of marginally represented and previously uncharacterized mutations. We detected changes in the frequency of mutated clones including the emergence and disappearance of the resistance-associated ABL1 T315I mutation. We also observed correlation in appearance of adjacent mutations, and exploited this observation to demonstrate the existence of mutated clones at the time of diagnosis. A tool is provided for detection of low frequency single nucleotide variants/mutations from deep coverage MPS data, applicable to clinical translation of advanced sequencing technologies.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mieloide de Fase Crónica/genética , Mutación/genética , Adulto , Análisis Mutacional de ADN/métodos , Resistencia a Antineoplásicos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Ataxia-telangiectasia (A-T), an autosomal recessive disorder is characterized by progressive neurodegeneration, immunodeficiency, sensitivity to ionizing radiation, and predisposition to cancer, especially to lymphoid malignancies. A-T variant is characterized by a milder clinical phenotype and is caused by missense or leaky splice site mutations that produce residual ataxia telangiectasia mutated (ATM) kinase activity. Lymphoid malignancy can precede the diagnosis of A-T, particularly in young children with mild neurological symptoms. We studied a consanguineous family with four A-T variant patients, three of them developed T-ALL at a young age before the diagnosis of A-T was established. ATM mutation analysis detected two new missense mutations both within exon 12: c.1514T>C and c.1547T>C. All four patients are homozygous for the two mutations, while their parents are heterozygous for the mutations. ATM protein level was low in all patients and the response to the radiomimetic agent, neocarzinostatin, was reduced. Leukemic presentation in a young age in three members of consanguineous family led to the identification of a new missense mutation in the ATM gene. The diagnosis of A-T or A-T variant should be considered in children with neurological abnormalities who develop T-ALL at a young age.
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Antibióticos Antineoplásicos/administración & dosificación , Proteínas de la Ataxia Telangiectasia Mutada/genética , Ataxia Telangiectasia , Mutación Missense , Leucemia-Linfoma Linfoblástico de Células Precursoras , Cinostatina/administración & dosificación , Adulto , Factores de Edad , Ataxia Telangiectasia/diagnóstico , Ataxia Telangiectasia/tratamiento farmacológico , Ataxia Telangiectasia/genética , Niño , Preescolar , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
Imatinib (IM) is the current first line treatment for chronic myeloid leukemia (CML). However, the disease will progress in the majority of patients pausing IM. IFN-α may intensify the response and increase the percentage of patients maintaining remission after IM cessation. Eleven patients with stable (≥ 2 years) complete cytogenetic responses (CCyR) on IM therapy were recruited to the study. They were administered Peg-IFN-α for 9 months before and for 3 months following IM discontinuation. During the 12 months of Peg-IFN-α therapy the remission status improved in five (45%) of the patients. Six (55%) of the patients experienced cytogenetic relapses at a median period of 8 months (range 2-33) after IM withdrawal. All six patients regained CCyR following IM restart. With a median follow up of 47 months (range 35-50), five (45%) out of the 11 studied patients maintain cytogenetic response off IM therapy. The role of Peg-IFN-α in patients pausing IM is to be further evaluated.
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Antineoplásicos/uso terapéutico , Antivirales/uso terapéutico , Interferón-alfa/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Privación de Tratamiento , Adulto , Benzamidas , Demografía , Femenino , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Spontaneous remission in 2 children with myelofibrosis, one with megakaryocytic acute myeloblastic leukemia and t(1;22) (with recurrence later) and one with Down syndrome and GATA1 mutation (permanent), are described. One had sepsis and was treated with antibiotics and blood products, whereas the other received only blood products. Remission was spontaneous, without chemotherapy treatment. Possible explanations for these outcomes include immunologic response to sepsis by a leukemia-specific T-cell response or the release of various cytokines, such as tumor necrosis factor and interleukin-2, during infections. Natural killer and cytotoxic T cells transfused with blood products might have also triggered an immune response.
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Leucemia Megacarioblástica Aguda/fisiopatología , Mielofibrosis Primaria/fisiopatología , Remisión Espontánea , Enfermedad Aguda , Preescolar , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Humanos , Lactante , Leucemia Megacarioblástica Aguda/inmunología , Masculino , Mielofibrosis Primaria/inmunologíaRESUMEN
Chronic myeloid leukemia (CML) is a clonal hematological disease that represents 15-20% of all adult leukemia cases. The study and treatment of CML has contributed pivotal advances to translational medicine and cancer therapy. The discovery that a single chromosomal abnormality, the Philadelphia (Ph) chromosome, is responsible for the etiology of this disease was a milestone for treating and understanding CML. Subsequently, CML became the first disease for which allogeneic bone marrow transplantation is the treatment of choice. Currently, CML is one of the few diseases where treatment targeted against the chromosomal abnormality is the sole frontline therapy for newly diagnosed patients. The use of directed therapy for CML challenged disease monitoring during treatment and led to the development of definitions that document response and predict relapse sooner than the former routine methods. These methods relied on classical cytogenetics through molecular cytogenetics (FISH) and, finally, on molecular monitoring assays. This review discusses the laboratory tools used for diagnosing CML, for monitoring during treatment, and for assessing remission or relapse. The advantages and disadvantages of each test, the common definition of response levels, and the efforts to standardize molecular monitoring for CML patient management are discussed.
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Técnicas de Laboratorio Clínico/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Monitoreo Fisiológico/métodos , Citogenética/métodos , Proteínas de Fusión bcr-abl/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , MutaciónRESUMEN
Cellular interactions with microenvironmental components are critical in multiple myeloma (MM) and impede effective disease treatment. Membranal-embedded tetraspanins, associated with metastasis suppression, are underexpressed in MM. We aimed to investigate the consequences of CD81/CD82 tetraspanins over-expression in MM cell lines. CAG and RPMI 8226 were transfected with pEGFP-N1/C1 fusion vectors of CD81/CD82. Employing flow cytometry, immunocytochemistry, and activity assays we assessed transfected cells for: morphology, survival, death, caspases, cell cycle, proliferation, oxidative stress, adhesion, motility and invasion. Overexpressed CD81/CD82 pEGFP-N1 vectors reduced survival without elevation of pre-G1 or AnnexinV+/7AAD- and independently of caspases. Decreased Ki67 and elevated intracellular glutathione were detected. No perturbations in cell cycle distribution were observed. The pEGFP-C1 vectors of CD81/CD82 caused reduction of MM cell adherence with/without fibronectin, insulin-like growth factor (IGF)-I, and matrigel. They also reduced cell motility and attenuated invasion potential, expressed by reduced secreted MMP-9 activity. These novel findings delineate the significance of CD81/CD82 expression to MM cell survival and their negative effects on cell adhesion, motility, and invasion thus, supporting their role as tumor metastasis suppressors.
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Supervivencia Celular/fisiología , Proteínas de la Membrana/metabolismo , Mieloma Múltiple/patología , Animales , Ciclo Celular/fisiología , Proliferación Celular , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Invasividad Neoplásica , Tetraspaninas , Células Tumorales CultivadasRESUMEN
BACKGROUND: A study was designed to identify the source of fever in a patient with post-polycythemia myelofibrosis, associated with clonal Janus Kinase 2 (JAK2) mutation involving duplication of exon 12. The patient presented with 1-2 day long self-limited periodic episodes of high fever that became more frequent as the hematologic disease progressed. METHODS: After ruling out other causes for recurrent fever, analysis of the pyrin encoding Mediterranean fever gene (MEFV) was carried out by Sanger sequencing in peripheral blood DNA samples obtained 4 years apart, in buccal cells, laser dissected kidney tubular cells, and FACS-sorted CD3-positive or depleted mononucleated blood cells. Hematopoeitc cells results were validated by targeted deep sequencing. A Sanger sequence based screen for pathogenic variants of the autoinflammatory genes NLRP3, TNFRSF1A and MVK was also performed. RESULTS: A rare, c.1955G>A, p.Arg652His MEFV gene variant was identified at negligible levels in an early peripheral blood DNA sample, but affected 46 % of the MEFV alleles and was restricted to JAK2-positive, polymorphonuclear and CD3-depleted mononunuclear DNA samples obtained 4 years later, when the patient experienced fever bouts. The patient was also heterozygous for the germ line, non-pathogenic NLRP3 gene variant, p.Q705K. Upon the administration of colchicine, the gold standard treatment for familial Mediterranean fever (FMF), the fever attacks subsided. CONCLUSIONS: This is the first report of non-transmitted, acquired FMF, associated with a JAK2 driven clonal expansion of a somatic MEFV exon 10 mutation. The non-pathogenic germ line NLRP3 p.Q705K mutation possibly played a modifier role on the disease phenotype.
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Proteínas del Citoesqueleto/genética , Fiebre Mediterránea Familiar/genética , Janus Quinasa 2/genética , Mutación , Policitemia Vera/genética , Mielofibrosis Primaria/genética , Biopsia , Células Clonales , Colchicina/uso terapéutico , Exones/genética , Fiebre Mediterránea Familiar/diagnóstico , Fiebre Mediterránea Familiar/tratamiento farmacológico , Femenino , Supresores de la Gota/uso terapéutico , Humanos , Persona de Mediana Edad , Policitemia Vera/diagnóstico , Policitemia Vera/tratamiento farmacológico , Polimorfismo de Longitud del Fragmento de Restricción , Mielofibrosis Primaria/diagnóstico , Mielofibrosis Primaria/tratamiento farmacológico , PirinaRESUMEN
Extreme swing of phosphor from severe hyperphosphatemia to severe hypophosphatemia in a patient with blast crisis of myeloid origin was the result of imbalance between massive apoptosis of leukemic cells in the context of spontaneous tumor lysis syndrome and massive production of leukemic cells with only 1% of blast in peripheral blood. The mutated p53 protein suggested acting as oncogene in the presented case and possibly affecting phosphor status.
RESUMEN
BACKGROUND AND OBJECTIVES: The treatment of choice for the pregnant woman with CML has not been defined. Exposure to imatinib while pregnant may cause serious fetal malformations and interferon-alpha is sometimes associated with side effects. Furthermore, little is known of the possibility that BCR/ABL-positive cells might be passed to the fetus and the role of the treatment given to the pregnant mother. DESIGN AND METHODS: Detection of BCR-ABL transcripts in the peripheral blood of the mother, the newborn and the cord blood was performed by quantitative real time PCR and FISH. RESULTS: A patient with CML diagnosed at the beginning of pregnancy was treated with leukapheresis at 31 weeks of gestation until delivery without any untoward effects. Since no tyrosine kinase inhibitor was administered BCR-ABL transcripts contamination of the cord blood and peripheral blood of the newborn was a reasonable concern. In practice no transcripts were detected in the cord blood or in the peripheral blood of the newborn at birth, at 1 month or 3 at months of age despite the fact that throughout her pregnancy and on the day of delivery the mother had 90% BCR/ABL positive cells in her blood. INTERPRETATION AND CONCLUSIONS: Leukapheresis does not eliminate the malignant clone; however the absence of BCR-ABL transcripts in the peripheral blood of the neonate and in the cord blood supports the view that transmission of CML to a fetus is improbable even if the mother's treatment during pregnancy is suboptimal.
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Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , ARN Neoplásico/sangre , Adulto , Femenino , Sangre Fetal , Humanos , Recién Nacido , Leucaféresis , Intercambio Materno-Fetal , Madres , Embarazo , Complicaciones Neoplásicas del EmbarazoRESUMEN
Myeloma cell interface with microenvironmental components is critical to cell growth and survival and perceived as a major obstacle for effective disease treatment. Hence, molecules that facilitate cell-cell and cell-ECM interactions are particularly important. We have previously shown that re-expression of membranal microdomain organizers, tetraspanins CD81 and CD82, caused myeloma cell death. Herein we demonstrate that the anti-myeloma effect of CD81/CD82 involves a down-regulation of Akt, activation of FoxO transcription factors and a decrease in active mTOR and mTOR/rictor. We go on to show in a breast cancer cell line model that Akt dependent cells are more sensitive to the tetraspanin overexpression. Moreover, expression of a constitutively active Akt increased survival of CD81/CD82 transfected myeloma cell lines. Akt and mTOR afford attractive therapeutic targets in cancer yet, due to pathways' interactions, inhibitors of mTOR frequently activate Akt and vise versa. Our results demonstrate co-repression of both by CD81/CD82 implying that tetraspanins may serve as "switches" modulating pathways rather than blocking a single factor and constitute a promising therapeutic strategy in Akt dependent pathological conditions. The possibility that the tetraspanins affect metabolic control is broached as well.
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Antígenos CD/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteína Kangai-1/metabolismo , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antígenos CD/genética , Supervivencia Celular , Células Cultivadas , Proteína Forkhead Box O1 , Humanos , Proteína Kangai-1/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mieloma Múltiple/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , Tetraspanina 28RESUMEN
Multiple myeloma (MM) cell interactions with their microenvironment modulate acquired drug resistance and disease progression. Indeed, reported aberrant gene methylation underscores the possible role of epigenetic events in MM's molecular profile. Membranal tetraspanins are often inversely correlated with cancer prognosis and metastasis, however mutations were unidentified hitherto. Their promoter characteristics and frequent down-regulation conform to transcriptional silencing by chromatin remodeling. We delineated the baseline expression of select tetraspanins in MM cell lines (RPMI 8226, U266, ARP1, ARK, CAG and EBV transformed ARH77) and fresh bone marrow samples (n = 9) for the first time and determined reduced expression of CD9, CD81 and absence of CD82. Thus, we aimed to assess their promoter methylation status. Indeed, we established CD9, CD81 and CD82 promoter methylation in MM cell lines employing methyl-specific-PCR of bisulfite modified G-DNA and PCR of G-DNA digested with methylation-sensitive restriction enzyme (Hin6I). Re-transcription of assayed genes in the cell lines following de-methylation [5-aza-2'-deoxycytidine (5-aza-dC)] confirmed the mechanistic significance of methylation to their regulation. Combined de-methylation and de-acetylation [Trichostatin A (TsA)] induced synergistic elevation of CD82 mRNA. We conclude that chromatin remodeling contributes to tetraspanin silencing in MM.
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Antígenos CD/biosíntesis , Ensamble y Desensamble de Cromatina , Metilación de ADN , Glicoproteínas de Membrana/biosíntesis , Mieloma Múltiple/genética , Línea Celular Tumoral , Regulación hacia Abajo , Silenciador del Gen , Humanos , Mieloma Múltiple/patología , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasAsunto(s)
Aberraciones Cromosómicas , Leucemia Mielomonocítica Crónica/complicaciones , Mielofibrosis Primaria/complicaciones , Trombocitemia Esencial/complicaciones , Adulto , Examen de la Médula Ósea , Análisis Citogenético , Humanos , Leucemia Mielomonocítica Crónica/genética , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genéticaRESUMEN
Thalidomide (Thd), a potent teratogen, was shown to have therapeutic potential in cancer, primarily in multiple myeloma (MM), yet its mechanism of action has not been elucidated. It was recently suggested that its teratogenicity is derived from interference in expression of genes regulated by GC-rich promoters by blocking the binding of SP1 transcription factor to its motif. We explored the validation of the proposed model by focusing on potential molecular targets associated with MM pathogenesis. Cell lines RPMI 8226, U266, and ARH-77 were exposed for 24 h to racemic Thd and analyzed for apoptosis, membranal expression of CD29 and CD63, transcript level of hTERT, CD63, and IGFI-R (characterized by GC-rich motifs) and telomerase activity. Analysis of an hTERT core promoter reporter gene expression [enhanced green fluorescent protein (EGFP)] in transiently transfected RPMI 8226 incubated with racemic and steric (+/-)-enantiomers of Thd was performed. A consistent reduction ( approximately 10-40%) in transcript levels of all three assayed genes in all three cell lines was demonstrated in the presence of racemic Thd. Significant reduction of EGFP was demonstrated in cells transfected with hTERT reporter gene and treated with racemic and (S)-Thd. Our results show that Thd's antimyeloma activity can be ascribed to the same mechanism responsible for its teratogenic effect and that the inhibition of GC-rich promoter genes is mostly attributed to the S-racemate. Indeed, this selectivity delineates GC-rich promoter genes as a unique group eligible for specific drug targeting.
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Secuencia Rica en GC/efectos de los fármacos , Mieloma Múltiple/patología , Regiones Promotoras Genéticas/efectos de los fármacos , Talidomida/farmacología , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Unión al ADN , Secuencia Rica en GC/genética , Humanos , Inmunosupresores/farmacología , Integrina beta1/metabolismo , Plasmacitoma , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Tetraspanina 30 , Células Tumorales CultivadasRESUMEN
Phosphatidylserine's (PS) membranal distribution is associated with an expanding variety of biological processes. We studied the relevance of preliminarily exposed membranal PS levels to cellular effects of cytotoxic agents. PBL of normal controls (n = 18) and patients with doxorubicin-treated breast carcinoma (n = 27) or 5'-fluorouracil-treated colorectal cancer (n = 32) were assayed before and after drug infusion. Membranal expression levels of PS, adhesion molecules (CD18, CD11a-c, CD63) and Fas-R of leukocyte subtypes were assessed by flow cytometer. Statistical analysis was implemented. Our results demonstrate external expression of PS on all leukocyte subpopulations despite non-apoptotic light scatter characteristics. Several distinct features were observed of which the more prominent were: leukocyte subtypes each display characteristic PS levels; cancer patients' PBL display higher preliminary PS levels than normal controls in all cell groups; and existence of negative correlations between initial membranal PS levels and drug-induced changes in its expression. Our findings underscore the complex involvement of PS in PBL apoptosis and possibly drug resistance.