RESUMEN
OBJECTIVES: To elucidate the natural history of T-cell large granular lymphocyte (T-LGL) lymphoproliferation, we followed changes in associated fluctuating neutropenia for 3 years in an untreated patient presenting with the disease. MATERIALS AND METHODS: We report a nonlinear mathematical analysis of irregular neutrophil fluctuation, using iterative data maps, to detect long-term regulation of the neutrophil population. RESULTS: This geometric analysis indicated that variations of this sequence of neutrophil counts followed bounded deterministic dynamics around a fixed low level equilibrium, a situation similar to that previously observed for cultured mouse early bone marrow progenitor cells. CONCLUSION: These findings illustrate how the deleterious effect of T-LGL on neutrophils is balanced, over periods of years, by pulses of compensatory neutrophil production, potentially accounting for the commonly observed prolonged indolent course of the disease.
Asunto(s)
Granulocitos/fisiología , Leucemia Linfocítica Granular Grande/fisiopatología , Neutropenia/fisiopatología , Células Madre/fisiología , División Celular/fisiología , Linaje de la Célula/fisiología , Proliferación Celular , Células Clonales/patología , Células Clonales/fisiología , Femenino , Granulocitos/patología , Humanos , Leucemia Linfocítica Granular Grande/patología , Persona de Mediana Edad , Modelos Teóricos , Neutropenia/etiología , Neutropenia/patología , Dinámicas no Lineales , Células Madre/patología , Linfocitos T/patología , Linfocitos T/fisiología , Factores de TiempoRESUMEN
The proliferation rate of various cell types in vitro, including hepatoma Fao cells, displays aperiodic oscillations. The frequency of these oscillations is about one every 3-5 weeks, and there are variations in cell functions and polarity. Topological analysis has showed that these oscillations in growth rate are determined, and presumably chaotic. One characteristic of complex chaotic systems is that their dynamics can be persistently modified by a small external perturbation. We show that treatment with a single small dose of the anticancer drug methotrexate causes long-term stable alteration of the oscillatory dynamics of Fao cell proliferation. The oscillations of growth rate are shifted, and their mean level decreased according to a fractal pattern.
Asunto(s)
División Celular/efectos de los fármacos , Antagonistas del Ácido Fólico/toxicidad , Metotrexato/toxicidad , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Neoplasias Hepáticas , Dinámicas no Lineales , Oscilometría , RatasRESUMEN
Clinical and experimental observations indicate that resistance to anticancer drugs may be spontaneously reversible over time, but the mechanisms of this reversal are unknown. The resistance of cultured hepatoma cells to methotrexate (MTX) and cisplatin was followed for 9 months. Cells were exposed to three treatments: MTX 200 nM for 24 h or 15 nM continuously and cisplatin 50 microM for 2 h. We investigated the relation between the temporal pattern of cell resistance and the previously reported fluctuations in cell proliferation rate, telomere length and telomerase activity. Spontaneous major peaks in resistance to each drug fell in time windows of 2-3 months (60-70 population doublings) and were at different times for each drug. The frequency of the fluctuations in drug resistance was the same as that of variations in cell growth rate, but amplitudes were unrelated. By contrast, resistance was directly related to telomere length dynamics in the same cells. MTX resistance occurred when telomeres shortened and cisplatin resistance when they were elongated. Furthermore, peaks of resistance to the continuous treatment with MTX were observed at 350-bp intervals of mean telomere length (9.06, 9.41, and 9.76 kbp) during the two 2-month phases of telomere shortening. Statistical analysis demonstrates the sinusoidal relationship between intermittent MTX resistance and telomere length. Possibly, erosion of telomeres encroaches on periodically spaced nucleosomal proteins, defining the onset of resistance phases. This evidence that resistance of tumoral cells to anticancer drugs may be intermittent and that onset of resistance is dictated by telomere length has major implications for clinical practice.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Metotrexato/farmacología , Telómero/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Células Clonales , ADN de Neoplasias/análisis , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/ultraestructura , Ratas , Telómero/ultraestructuraRESUMEN
The proliferative activity of long-term cultured mammalian cells exhibits traits of a complex dynamic system, with a succession of spontaneous rises and falls in proliferation rate. We analyzed three successive series of proliferation data for the Fao hepatoma cell line in long-term cultures. In the three series the proliferation rate displayed apparently disordered oscillations, which each lasted about 3-5 passages, with variable amplitude and were therefore unpredictable. Such non-linear kinetics raises the major issue of whether these fluctuations are random, or determined and coordinated. We used a graphical method of analysis of the data, which demonstrated that all troughs of proliferation were mathematically related to a common value in each series. This common value was itself related to the maximum level of proliferation of the cell line. Non-linear analysis thus confirmed that the fluctuations in proliferation rate of tumoral Fao cells are, at least in part, determined. This pattern evokes chaotic dynamics and is evidence for the flexible coordination of the complex system linking positive and negative growth regulators in long-term cultured cells.
Asunto(s)
Neoplasias Hepáticas Experimentales/patología , Modelos Biológicos , Modelos Teóricos , Animales , División Celular , Ratas , Células Tumorales CultivadasRESUMEN
PURPOSE: In about 25% of patients suffering from acute lymphoblastic leukemia (ALL) treatment failures occur that are most likely due to development of resistance to methotrexate (MTX). Blasts from patients with ALL were evaluated for MTX uptake, formation of long-chain MTX polyglutamates (MTX-Glu5+6), cytotoxicity and thymidylate synthase inhibition by MTX and compared to blasts from patients with acute myelogenous leukemia (AML). METHODS: Radioactively labeled MTX-Glu(n) were analyzed by means of HPLC. Thymidylate synthase activity was measured by a tritium-release assay. Cytotoxicity was determined by trypan blue exclusion. RESULTS: In most ALL blasts (n = 9) large amounts of MTX-Glu5+6 (1.06-7.03 pmol/10(7) cells) and high cytotoxicity (43.5% 92.7%) were found, while in others small amounts of MTX-Glu5+6 (0.0-0.39 pmol/10(7) cells) caused only weak cytotoxicity (6.0% 27.9%) (n = 5, 2 relapsed patients). Resistance to MTX in blasts from AML patients (n = 5) was also caused by reduced synthesis of MTX-Glu5s+6 (0.0-0.42 pmol/10(7) cells). In contrast, some ALL blasts (n = 7, 4 relapsed patients) were able to survive MTX treatment despite large amounts of MTX-Glu5+6 (1.5-5.05 pmol/10(7) cells) and extensive thymidylate synthase inhibition. CONCLUSIONS: Since the majority of ALL patients were examined at first diagnosis, an inherent mechanism of resistance seems most likely. We propose a mechanism based on the switch of thymidylate synthesis to the salvage pathway.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Metotrexato/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Timidilato Sintasa/antagonistas & inhibidores , Adolescente , Antimetabolitos Antineoplásicos/uso terapéutico , Niño , Preescolar , Femenino , Humanos , Lactante , Leucemia Mieloide Aguda/enzimología , Masculino , Metotrexato/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , RecurrenciaRESUMEN
Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3-4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22-23 additional population doublings each. Normal embryonic fibroblast culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attrition and repair processes regulates the waves of proliferation.
Asunto(s)
División Celular , Senescencia Celular , Hígado/citología , Animales , Carcinoma Hepatocelular , Recuento de Células , Línea Celular Transformada , Ratas , Telómero , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
This study focused on the activation/differentiation of human microglial cells and astrocytes, which is a prerequisite for HIV1 replication. In vitro, IFN gamma induced a differentiation-like morphological change in embryonic microglia and astrocytes, in both primary and in purified culture. This effect was enhanced by TNF alpha which in itself had no effect. IFN gamma did not increase the low level of endogenous TNF alpha, which is not secreted by embryonic cells, but stimulated the expression of TNF alpha R1, although to a relatively low extent. IFN gamma facilitated TNF alpha response through an increase in TNF alpha R1 expression, but probably also through interaction with different intracellular signal transduction pathways.
Asunto(s)
Astrocitos/efectos de los fármacos , Interferón gamma/farmacología , Microglía/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Astrocitos/inmunología , Diferenciación Celular , Células Cultivadas , Colecalciferol/metabolismo , Humanos , Interferón gamma/inmunología , Interleucina-1/metabolismo , Interleucina-3/metabolismo , Lipopolisacáridos/metabolismo , Microglía/inmunología , Proteínas Recombinantes/inmunología , Acetato de Tetradecanoilforbol/metabolismoRESUMEN
A method for the simultaneous determination of the antifolates methotrexate and 7-hydroxymethotrexate as well as the folates 5-methyltetrahydrofolic acid and folinic acid (5-formyltetrahydrofolic acid) in serum and cerebrospinal fluid (CSF) is described. High-performance liquid chromatography with gradient elution and dual detection (ultraviolet absorption and fluorescence) was used to separate and quantitate the analytes. Serum samples containing high levels of the substances of interest and CSF samples were injected directly onto the HPLC column. For determination of low concentrations, serum samples were subjected to a solid-phase extraction method for clean-up and concentration purposes. The determination limits were 10 ng/ml for both antifolates, 100 ng/ml for folinic acid, and 0.1 ng/ml for the physiologically occurring methylated folate which is about 1/100 the serum concentration in healthy children. The suitability of the method for pharmacokinetic monitoring of high-dose methotrexate therapy combined with leucovorin rescue administered to children with acute lymphoblastic leukemia was demonstrated. Minimum values of the serum folate during treatment ranged from 0.2 to 3.1 ng/ml. Even those very low concentrations could be reliably measured.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Antagonistas del Ácido Fólico/sangre , Leucovorina/sangre , Metotrexato/análogos & derivados , Metotrexato/sangre , Tetrahidrofolatos/sangre , Calibración , Niño , Antagonistas del Ácido Fólico/líquido cefalorraquídeo , Humanos , Leucovorina/líquido cefalorraquídeo , Metotrexato/líquido cefalorraquídeo , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/líquido cefalorraquídeo , Manejo de Especímenes , Tetrahidrofolatos/líquido cefalorraquídeoRESUMEN
The incorporation of radioactivity from [1-14C]-galactose into TCA-precipitable material was determined in skin fibroblasts derived from 11 galactosemic patients deficient in galactose 1-phosphate uridyl transferase (GALT-). "R" ratios (designated the R phenotype) were defined as the ratio between [14C]galactose incorporation and [3H]leucine incorporation. Results were expressed as a percentage of the controls. In the GALT-strains this ratio varied from strain to strain, presumably depending on the efficiency of the secondary route via the UDP-galactose pyrophosphorylase pathway. In 10 GALT-patients without late serious clinical manifestations, the R phenotype varied from 37 to 57% of the control value. In the 11th patient, the R phenotype was only 20% of the control. Thus, we obtained a significantly lower R phenotype in one patient who was distinguished from the others by having very severe delayed neurological complications, although compliance to galactose-free diet was good. We suggest that, in this patient, the development of the UDP-galactose pyrophosphorylase pathway was not sufficient to ensure the availability of enough galactose for the necessary synthesis of glycoproteins and glycolipids. Thus the R phenotype may be an indicator of the risk of late neurological complications. The determination of the R phenotype of GALT-patients may therefore be valuable. However, further investigations of galactosemic patients with neurological complications are required to confirm this relationship.
Asunto(s)
Galactosa/metabolismo , Galactosemias/metabolismo , Enfermedades del Sistema Nervioso/etiología , Adolescente , Células Cultivadas , Niño , Fibroblastos/metabolismo , Galactosemias/complicaciones , Humanos , Lactante , Recién Nacido , Leucina/metabolismo , Fenotipo , Piel/citología , UTP-Hexosa-1-Fosfato Uridililtransferasa/deficienciaRESUMEN
A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.
Asunto(s)
División Celular , Transformación Celular Viral/fisiología , Animales , Antígenos Transformadores de Poliomavirus , División Celular/fisiología , Línea Celular Transformada/citología , Línea Celular Transformada/metabolismo , Tamaño de la Célula , Aberraciones Cromosómicas , Cricetinae , Humanos , Periodicidad , Ploidias , PielRESUMEN
Even in patients with normal renal function, high-dose methotrexate therapy (HDMTX) may be followed by extremely prolonged MTX elimination through alkaline diuresis is performed correctly. By inquiry in Germany, Austria and Switzerland for HDMTX infusions with MTX plasma concentration 42 h after start of exposure (MTX-42) higher than 5 mumol/l (microM), we analyzed data from 21 patients in whom impairment of renal methotrexate elimination had received 5 g/m2.24h, 3 had received 12 g/m2.4h. They presented with MTX-48 serum level between 1.7 and 1404 microM. There was no recognizable causative factor. As early signs for impaired elimination, we identified enhanced vomiting during MTX infusion in 8/21, elevated steady-state-MTX in 11/15, and a rise of serum creatinine greater than 50% in 14/16 patients in whom respective data were available. Creatinine rose to a maximum of 1.0-4.9 mg/dl within 1-4 days in 19/21 patients (accompanied by diuresis problems in only 5 patients) and normalized within 3-17 days in all but two patients. Creatinine maximum correlated weakly with MTX-48 (r = 0.34) and with extrarenal toxicity. 8 patients had normal (WHO 0-II degree), 8 other had intensified (III-IV degrees) but not critical extrarenal MTX toxicity with calcium folinate (CF) doses of 0.2-1.6 mg/kg.microM MTX q 6 h started 28-54 h after beginning of MTX exposure. 5 patients had unusual toxicity. 2 patients suffered from severe but reversible encephalopathies with CF doses of 0.05 and 1 mg/kg.microM MTX q 6 h started after 51 and 36 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tasa de Filtración Glomerular/efectos de los fármacos , Metotrexato/efectos adversos , Neoplasias/tratamiento farmacológico , Adolescente , Niño , Preescolar , Creatinina/orina , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Lactante , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Metotrexato/administración & dosificación , Metotrexato/farmacocinética , Neoplasias/orinaRESUMEN
Fructose strongly stimulates the growth of normal diploid human skin fibroblasts (SFs) and induces marked changes in their morphology and lipid accumulation. This mitogenic effect occurs despite very low fructose consumption and depends on the presence of glutamine. The cell kinetics of cultured fructose-fed human skin fibroblasts were different from those fed on glucose: in the presence of fructose a high proliferative index persisted at Day 14 of culture and the duration of the total cell cycle and of the G1 + 1/2 M and S phases was slightly shorter. The mitogenic effect of fructose on SF was largest in the presence of human serum: it was small or undetectable when fibroblasts were cultured in media supplemented with dialyzed human serum, fetal bovine serum, or serum substitutes. This suggests that serum growth factor(s) mediate the mitogenic effect of fructose. Only normal diploid human cells seem to be sensitive to this mitogenic effect of fructose: the long-term growth of normal human liver cells on fructose was slightly better or similar to that on glucose. In contrast, fructose could only support limited growth of hamster fibroblastic Nil cells and of a transformed human fibroblastic line, which grew better with glucose.
Asunto(s)
Diploidia , Fibroblastos/citología , Fructosa/farmacología , Hígado/citología , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Línea Celular Transformada , Preescolar , Cricetinae , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Glutamina/farmacología , Humanos , Lactante , Hígado/efectos de los fármacos , Hígado/fisiología , Factores de TiempoRESUMEN
PURPOSE: Pharmacokinetics, toxicity, and therapeutic efficacy of two different methotrexate (MTX) infusions for remission induction of relapsed childhood acute lymphoblastic leukemia (ALL) were investigated in a randomized multicenter trial. PATIENTS AND METHODS: Sixty patients with early bone marrow relapse received a polychemotherapy induction protocol starting with either 12 g/m2 MTX as a 4-hour infusion (high-dose [HDM]) or 1 g/m2 as a 36-hour infusion (intermediate-dose [IDM]). In HDM, leucovorin (LCV) was administered orally (12 times, 15 mg/m2 every 6 hours), beginning at hour 24. In IDM, only two doses were administered at hours 48 and 54. RESULTS: Median serum MTX concentrations during infusion were 716 mumol/L in HDM and 7.2 mumol/L in IDM. In HDM, MTX serum levels at hour 24 (median, 2.8 mumol/L) were significantly less than steady-state levels of IDM. Concentrations greater than 1 mumol/L were maintained for 36 hours with HDM and 45 hours with IDM. General tolerance to treatment was better in the HDM group. Mucosal lesions occurred significantly more often and were more severe after IDM treatment. A median treatment delay of 3 days was required in the IDM group but not in the HDM group. At day 15, complete remission (CR) was documented in 45% of IDM- and 48% of HDM-treated patients. Persistent blasts (> 5%) appeared more frequently in HDM than in IDM (35% v 19% of patients; P = NS). After completion of induction therapy, 28 of 30 patients in each group achieved CR. CONCLUSION: Both regimens produced the same remission rates. The tendency to better antileukemic activity of IDM was accompanied by more severe side effects as a consequence of long-lasting cytotoxic MTX levels. Hence, long-term infusion of IDM followed by low-dose LCV is an effective treatment for recurrent ALL.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Metotrexato/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Adolescente , Asparaginasa/administración & dosificación , Niño , Citarabina/administración & dosificación , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Infusiones Intravenosas , Masculino , Metotrexato/efectos adversos , Metotrexato/farmacocinética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prednisona/administración & dosificación , Vincristina/administración & dosificaciónRESUMEN
In skin fibroblasts of patients presenting with galactosaemia, either from galactose 1-phosphate uridyltransferase or galactokinase deficiency, a deficit in extracellular glucose utilization was observed. This deficit was constant over 3 weeks of continuous cell growth in a medium containing 5.5 mmol/L glucose as the only hexose, and homologous serum. Levels of glucose utilization by deficient skin fibroblasts were stable at about 65-70% of the glucose utilization of control normal skin fibroblasts. Cell morphology was normal, and cell growth was subnormal during this period. However, the energy provision appeared sufficient for cellular needs since cell growth in this glucose medium was observed not to depend on the presence of extracellular glutamine. In contrast, glutamine was required for growth of galactosaemic fibroblasts cultured in medium containing 5.5 mmol/L galactose. If expressed in many cell types, this impaired glucose uptake would be expected seriously to damage highly glucose-dependent tissues such as the central nervous system. This might be of relevance to the persistent neurological damage observed in many galactosaemic patients in spite of their compliance with an early strict galactose-free diet.
Asunto(s)
Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Galactosemias/metabolismo , Hexosas/metabolismo , Piel/metabolismo , Adolescente , Glucemia/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/genética , División Celular , Células Cultivadas , Niño , Preescolar , Diploidia , Fibroblastos/citología , Fibroblastos/metabolismo , Galactosa/metabolismo , Galactosa/toxicidad , Glucosa/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Lactante , Recién Nacido , Lactatos/biosíntesis , Lactatos/metabolismoRESUMEN
The activity of Glutamine Synthetase (GS) was measured during the growth of human diploid skin fibroblasts cultured for three weeks in the presence or absence of either glucose or glutamine or both. In medium free of both glucose and glutamine, a single late peak in GS activity was observed concomitantly with delayed small cell protein increment. In all media containing either glucose or glutamine or both. GS activity rose sharply during rapid cell growth, displayed a plateau, and then decreased once the cells had reached confluency. The variations in extracellular amino acid levels were also determined and were found to depend on the composition of the medium but not on the cell culture duration. These results demonstrate, for the first time as far as we know, that strong GS activity is present in rapidly growing skin fibroblasts. In contrast to many other mammalian cell types, GS activity in human skin fibroblasts appears not to be subject to regulation by extracellular glutamine. This difference may well be connected with cell differentiation.
Asunto(s)
Fibroblastos/efectos de los fármacos , Glucosa/farmacología , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/farmacología , Piel/efectos de los fármacos , Aminoácidos/metabolismo , División Celular , Células Cultivadas , Espacio Extracelular/química , Fibroblastos/enzimología , Humanos , Recién Nacido , Piel/citología , Piel/enzimologíaRESUMEN
The pharmacokinetics of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diaminomethylpteroic acid (APA), folinic acid, and 5-methyltetrahydrofolate (5-MTHF) have been studied during 21 high-dose MTX (HDMTX) infusions (5 g.m-2 in 24 h) with leucovorin (LCV) rescue, a component of the therapy of 5 children with acute lymphoblastic leukemia (ALL). The median steady-state concentration of MTX was 66 mumol.l-1. Three elimination half-lifes were determined for MTX: 1.8 h, 6.4 h and a terminal 15 h. The median systemic MTX clearance was 110 mg.m-2.min-1. The 7-OHMTX level increased during each infusion and a Cmax of 19 mumol.l-1 was achieved at the end. Its initial half-life was 5 h and the terminal half-life was 12 h. Thus, the peak serum concentration ratio of 7-OHMTX to MTX was reached 24 h after the end of the infusion at a median ratio of 8. The MTX metabolite APA was detected in concentrations less than 0.06 mumol.l-1. The median folinic acid level during rescue, 48 h after starting the infusion, was 7.0 mumol.l-1 and 18 h following the last dose of LCV it was 0.44 mumol.l-1, leading to ratios of folinic acid to MTX of 31 and 6, respectively. The median 5-MTHF level during rescue was 0.44 mumol.l-1 with a median ratio of 5-MTHF to MTX of 2. Twenty infusions with 48 h MTX levels of less than 0.5 mumol.l-1 were without marked toxicity. Only one patient with a 48 h MTX concentration of 5.5 mumol.l-1 and a ratio of 5-MTHF to MTX of 0.08 suffered from ulcerating mucositis and septicaemia despite increased and prolonged LCV rescue.
Asunto(s)
Leucovorina/farmacocinética , Leucovorina/uso terapéutico , Metotrexato/análogos & derivados , Metotrexato/farmacocinética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Niño , Preescolar , Cromatografía Líquida de Alta Presión , Semivida , Humanos , Leucovorina/administración & dosificación , Leucovorina/sangre , Tasa de Depuración Metabólica , Metotrexato/administración & dosificación , Metotrexato/sangre , Metotrexato/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Human infant skin fibroblasts and liver cells were subcultured with 250 microM PUFA (polyunsaturated fatty acid), and primary cultures of glial brain cells from new-born rats with 100 microM; oleic acid was added to controls. Minimum essential medium (MEM) supplemented with bovine serum was used as a reference. During the short-term experiment (18-24 h), control liver cells showed a regular increase in protein level, while protein increment was more rapid in linoleic and especially in arachidonic acid-treated cells, but only for the first 3 hours. During the long-term experiment (7 d), control skin fibroblasts showed a faster growth rate (increase in number of cells) than reference or fibroblasts cultured with the added PUFAs. Lipid droplets were seen in the PUFA-treated liver cells and skin fibroblasts, and ultrastructural modifications were observed in fibroblasts, but without growth rate alteration. During the long-term experiment (2 w), control glial brain cells showed faster protein increment (measuring growth rate) than PUFA-treated cells, particularly than arachidonic acid-treated cells. HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) activity, determined after 6 h (liver cells) or 1 and 2 w (brain cells) of culture, was low in controls and reference, whilst higher in PUFA-treated-cells, and was especially high in arachidonic acid-treated brain cells. The present study indicates than the high HMGR activity may correspond to cultures of cells rapidly stopped in their protein increment, and to cultures of cells showing a slow rate of proliferation. This contrasts with results obtained from in vivo experiments; it also emphasizes the high mevalonate (MVA) level as a possible sign of nutritional medium imbalance.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácidos Grasos Insaturados/farmacología , Hígado/citología , Piel/citología , Animales , Ácido Araquidónico/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Lactante , Cinética , Ácido Linoleico , Ácidos Linoleicos/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ácido Oléico , Ácidos Oléicos/farmacología , Proteínas/metabolismo , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
The combined effects of carbohydrates and glutamine were investigated in diploid strains of normal human skin fibroblasts cultured for 21 days under eight different culture conditions: hexose-free medium or medium containing D-glucose, D-galactose, or D-fructose, with or without added glutamine. Cell growth, hexose consumption, lactate production, intracellular glycogen content and extracellular amino acid levels were measured every third to fourth day. In the presence of glutamine, cells reached a higher saturation density in fructose medium than in glucose or galactose medium but per cell consumption of fructose and galactose was much less than that of glucose. Consumption of all three carbohydrates per unit cell growth exhibited three distinct phases: Days 1-3, 3-10, and 10-20, respectively. In the absence of glutamine the rate of cell growth was not altered in glucose or galactose medium, but slowed down considerably in fructose medium. Glutamine deprivation also led to changes in hexose consumption. In hexose-free media the cell growth rate at first was very slow, but rose after 2 or 3 weeks of culture. The levels of extracellular nonessential amino acids varied according to medium and growth phase. One of the most exciting findings was that human fibroblasts are able to maintain a slight excess of glutamine in all media not supplemented with glutamine and, more surprisingly, to synthesize it in a medium containing galactose and glutamine.
Asunto(s)
Glutamina/metabolismo , Hexoquinasa/metabolismo , Piel/citología , Aminoácidos/metabolismo , División Celular , Células Cultivadas , Niño , Fibroblastos/citología , Fibroblastos/metabolismo , Fructosa/metabolismo , Galactosa/metabolismo , Glucosa/metabolismo , Humanos , Lactatos/metabolismo , Piel/metabolismoRESUMEN
The localization of factor VIII procoagulant antigen (VIII:Ag) and factor VIII von Willebrand antigen (VWF:Ag) was investigated in human liver, lung, spleen, placenta and umbilical cord, by an immunoperoxidase technique using an avidin biotin complex (ABC). Positive staining for VIII:Ag was observed in the endothelial cells of liver sinusoids, veins and arteries, as well as in the endothelial cells of placenta, lung and spleen. VWF:Ag was detected in the vascular endothelial cells of all the organs explored. The staining intensity of both VIII:Ag and VWF:Ag varied in the different tissues and showed a distinctive pattern of distribution in the liver. VIII:Ag was also observed in the cytoplasm of dysplastic, foetal-like hepatocytes which infiltrated one liver specimen. Our results agree with the view that liver endothelial cells are a major site of Factor VIII (F VIII) storage and secondary release into the circulation. However, the bright staining intensity of VIII:Ag and VWF:Ag in the lung and placenta suggests that these two tissues might also be a substantial source of F VIII.
Asunto(s)
Antígenos/aislamiento & purificación , Factor VIII/inmunología , Femenino , Humanos , Inmunohistoquímica , Hígado/inmunología , Pulmón/inmunología , Placenta/inmunología , Embarazo , Bazo/inmunología , Distribución Tisular , Cordón Umbilical/inmunología , Factor de von Willebrand/inmunologíaRESUMEN
UNLABELLED: Many publications indicate the beneficial effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) in the control of coronary heart disease and diabetes, although the mechanism is not clear. Some of our previous results suggest that, in contrast to other lipids, n-6 PUFAs could have a permissive effect on carbohydrate oxidation. To check this hypothesis, we determined pyruvate dehydrogenase (PDH, decarboxylase: EC 1.2.4.1) activity in infant skin fibroblasts (ISF) incubated 6 hours in the presence of 0.25 mM linoleic (LI) or arachidonic (AR) acid, compared to oleic acid (OL) and control ISF incubated without addition of fatty acids. The four groups of cells were preincubated 36 hours either in the presence of fetal bovine serum (FBS), or in the presence of lipoprotein-deprived serum (LPDS). RESULTS: (1) When the ISF were maintained in the medium containing FBS, the two PUFAs had little inhibitory effect on PDH activity, in contrast with the effect of OL. (2) When the ISF were kept in the lipoprotein-deficient medium, PDH activity was low in controls and in the OL cells, but the addition of LI or AR increased the activity. This suggests the role of n-6 PUFAs in enhancing carbohydrate oxidation, under certain conditions.