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Infecciones por Actinomycetales/veterinaria , Artritis Infecciosa/veterinaria , Enfermedades de los Caballos/microbiología , Cojera Animal/microbiología , Osteítis/veterinaria , Rhodococcus equi/aislamiento & purificación , Infecciones por Actinomycetales/terapia , Animales , Antibacterianos/administración & dosificación , Artritis Infecciosa/tratamiento farmacológico , Artritis Infecciosa/microbiología , Bacteriemia/tratamiento farmacológico , Bacteriemia/veterinaria , Eritromicina/administración & dosificación , Femenino , Miembro Anterior/microbiología , Enfermedades de los Caballos/diagnóstico por imagen , Enfermedades de los Caballos/terapia , Caballos , Cojera Animal/diagnóstico por imagen , Cojera Animal/terapia , Osteítis/tratamiento farmacológico , Osteítis/microbiología , Radiografía , Reperfusión/veterinaria , Tennessee , Resultado del TratamientoRESUMEN
Genospheres are cationic lipid-nucleic acid nanoparticles prepared by the assembly of the lipids and nucleic acids from an aqueous/organic liquid monophase that independently dissolves the components, where the resultant particles are homogeneously sized (70-110 nm), with efficiently incorporated and protected DNA. In the present study, we demonstrate pH-dependent modulation of the Genosphere surface charge using pH-titratable lipids. By incorporation of the lipids with titratable anionic or imidazole headgroups, Genospheres with neutral or anionic surface charge at neutral pH were produced and compared for cellular uptake and transfection of a reporter gene (luciferase) in culture of breast cancer cells. The extent of particle-cell association was also studied by fluorescent microscopy and quantified by cytofluorometery. The effects of Genosphere surface modification with poly(ethylene glycol) (molecular weight 2000) at low (0.5 mol %) and high (5 mol %) grafting densities, as well as the effects of HER2-receptor-directed targeting by an internalizable anti-HER2 scFv F5, linked via PEG spacer, were also studied. Inclusion in the Genosphere formulation of pH-titratable lipids CHEMS (cholesteryl hemisuccinate), CHIM (1-(3-(cholesteryloxycarbonylamino)propyl)imidazole), or DSGG (1,2-distearoyl-sn-glycero-3-hemiglutarate) rendered the particles surface-charge neutral or slightly anionic at neutral pH, and cationic at mildly acidic pH, as shown by zeta-potential measurements. In HER2-targeted systems, transfection activity and target specificity with HER2-overexpressing SKBR-3 breast cancer cells were dependent on Genosphere surface charge and PEGylation. The highest target specificity correlated with low cationic charge at neutral pH, while incorporation of 5 mol % PEG-lipid had only minor effects on Genosphere-cell association, internalization, and transfection activity. The implications of this work for potential in vivo applications are discussed.
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Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Polietilenglicoles/química , Receptor ErbB-2/inmunología , Anticuerpos/química , Ingeniería Biomédica , Humanos , Modelos Biológicos , Sensibilidad y Especificidad , Propiedades de Superficie , Transfección , Células Tumorales CultivadasRESUMEN
Effective, reproducible, and scalable methods for DNA-lipid assembly are important for the success of non-viral vectors in in vivo gene therapy. We hypothesized DNA-lipid assembly would be optimal if started from a liquid monophase where both DNA and lipids separately form molecular or micellar solutions prior to mixing, without preexisting condensed lipid phases, thus allowing DNA-lipid assembly under conditions close to equilibrium. Previously, we found that mixing plasmid DNA, 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), cholesterol and a cationic lipid, 1, 2-dioleoyl-3-(trimethylammonio) propane (DOTAP) in 50% (v/v) aqueous ethanol spontaneously produced an optically transparent solution. Upon ethanol removal, DNA-lipid nanoparticles (Genospheres) were formed. For comparison with well-known technologies, different DNA-lipid particles were prepared by interaction of plasmid DNA and stable or ethanol-destabilized lipid vesicles by combining the components in water or 30% (v/v) aqueous ethanol, respectively. Among the three studied DNA-lipid assembly methods, only Genospheres combined the properties of small size (less than or around 100 nm), high incorporation of both lipid and DNA, high degree of DNA protection (dye accessibility 5-12%), a narrow distribution of particle density and when immuno-targeted, the highest transfection efficiency in HER2-overexpressing cells in vitro. We conclude that the Genosphere assembly methodology offers advantages for the development of effective, scalable and targetable non-viral gene delivery vectors.
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Lípidos/análisis , Nanoestructuras/análisis , Fosfatidilcolinas/análisis , Secuencia de Bases , ADN/análisis , Indicadores y Reactivos , Oligodesoxirribonucleótidos/química , Compuestos Orgánicos , Fosfatidilcolinas/genética , Plásmidos , Receptor ErbB-2/genética , Solventes , AguaRESUMEN
We describe the assembly of a cationic lipid-nucleic acid nanoparticle from a liquid monophase containing water and a water miscible organic solvent where both lipid and DNA components are separately soluble prior to their combination. Upon removal of the organic solvent, stable and homogenously sized (70-100 nm) lipid-nucleic acid nanoparticles (Genospheres) were formed. The low accessibility (<15%) of the nanoparticle-encapsulated DNA to a DNA intercalating dye indicated well-protected nucleic acids and high DNA incorporation efficiencies. It was demonstrated that Genospheres could be stably stored under a variety of conditions including a lyophilized state where no appreciable increase in particle size or DNA accessibility was observed following reconstitution. Finally, Genospheres were made target-specific by insertion of an antibody-lipopolymer (anti-HER2 scFv (F5)-PEG-DSPE) conjugate into the particle. The target specificity (>100-fold) in HER2 overexpressing SK-BR-3 breast cancer cells was dependent on the degree of PEGylation, where the incorporation of high amounts of PEG-lipid on the particle surface (up to 5 mol%) had only a minor effect on the transfection activity of the targeted Genospheres. In summary, this work describes a novel, readily scalable method for preparing highly stable immunotargeted nucleic acid delivery vehicles capable of achieving a high degree of specific transfection activity.
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ADN/administración & dosificación , Terapia Genética/métodos , Región Variable de Inmunoglobulina/genética , Nanotecnología/métodos , Receptor ErbB-2/inmunología , Portadores de Fármacos , Marcación de Gen , Terapia Genética/instrumentación , Humanos , Liposomas , Microscopía Electrónica , Nanoestructuras , Fosfatidiletanolaminas , PolietilenglicolesRESUMEN
Adhesion of Cryptosporidium parvum and Giardia lamblia to four materials of different surface charge and hydrophobicity was investigated. Glass beads were used with and without three polymer coatings: aminosilines (A0750), fluorosilines (T2494), an amino cationic polymer. Surface charge density and hydrophobicity of the beads were characterized by measuring the zeta potential (ZP) and the contact angle, respectively. Adhesion was derived from batch experiments where negatively charged (oo)cysts were mixed with the beads and recovery was determined by counting (oo)cysts remaining in suspension using a flow cytometer. Experimental results clearly show that adhesion to solid surfaces of C. parvum is different from G. lamblia. Adhesion of C. parvum to positively charged, hydrophilic beads (82% recovery relative to control) indicated that surface charge was the more important factor for C. parvum, dominating any hydrophobic effects. Adhesion of G. lamblia cysts to negatively charged, hydrophobic beads (0% recovery relative to control) indicated that although hydrophobicity and surface charge both played a role in the adhesion of G. lamblia to solid surfaces, hydrophobicity was more important than surface charge.
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Cryptosporidium parvum/fisiología , Giardia lamblia/fisiología , Animales , Cryptosporidium parvum/citología , Giardia lamblia/citología , Interacciones Hidrofóbicas e Hidrofílicas , Oocistos/fisiología , Polímeros/química , Propiedades de SuperficieRESUMEN
The effects of prostaglandin E2, forskolin, and phorbol 12-myristate 13-acetate on cell proliferation, cell surface antigen expression, vitamin D-24-hydroxylase activity and vitamin D receptor (VDR) expression have been studied in an adherent variant (Ad-HL60) of the human HL60 promyelomonocytic leukemia cell line. Ad-HL60 cells have a more differentiated phenotype than the nonadherent HL60 cells from which they were derived and, unlike the parent cell line, constitutively express vitamin D-24-hydroxylase activity. Treatment of Ad-HL60 cells with 1 microM PGE2 resulted in a decrease in the rate of cell proliferation (cell numbers were approximately 23% of control values after 72 h treatment), a change in expression of leukocyte surface antigens (decreased CD13 and CD14, increased CD11b and CD49d expression), an increase in the synthesis of 24,25-dihydroxyvitamin D3 from substrate 25-hydroxyvitamin D3 (control 5.76 +/- 0.17, 72 h PGE2-treated cells 12.10 +/- 1.90 pmol/h/10(6) cells), and an increase in receptors for the active metabolite of vitamin D, 1 alpha,25-dihydroxyvitamin D3, from 3910 to 11285 receptors per cell in control and 7-day treated cells, respectively. Prostaglandin E2 may be acting via a mechanism involving cyclic AMP in these cells, as we have also demonstrated that 10 microM forskolin, an adenylate cyclase activator, has similar effects. Phorbol 12-myristate 13-acetate had little effect on any of the parameters measured in this cell line.
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Sistema Enzimático del Citocromo P-450/metabolismo , Dinoprostona/farmacología , Receptores de Calcitriol/metabolismo , Esteroide Hidroxilasas/metabolismo , 24,25-Dihidroxivitamina D 3/metabolismo , Antígenos CD/análisis , Antígenos CD/metabolismo , Calcitriol/metabolismo , División Celular/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/metabolismo , Citometría de Flujo , Células HL-60 , Humanos , Fenotipo , Acetato de Tetradecanoilforbol/farmacología , Vitamina D3 24-HidroxilasaRESUMEN
OBJECTIVE: This study was designed to investigate whether 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3), produced by activated synovial fluid macrophages, promotes its own catabolism by upregulating vitamin D-24-hydroxylase (24-OHase) in synovial fibroblasts through a vitamin D receptor (VDR) mediated mechanism. METHODS: Synovial macrophages and fibroblasts were derived from patients with rheumatoid arthritis. Expression of VDR and 24-OHase mRNAs was determined using in situ hybridisation. Vitamin D hydroxylase activity was determined by incubating cells with [3H]-25-(OH)D3, or [3H]-1,25-(OH)2D3, and metabolite synthesis quantified using high performance liquid chromatography. RESULTS: 1, 25-(OH)2D3 increased expression of mRNA for both VDR and 24-OHase in fibroblasts by approximately threefold over 24 hours. 1,25-(OH)2D3 increased fibroblast 24-OHase activity, yielding 24-hydroxylated, and more polar, metabolites. In co-culture, fibroblasts were able to catabolise macrophage derived 1,25-(OH)2D3. CONCLUSIONS: 1, 25-(OH)2D3 is produced by macrophages in vitro at biologically relevant concentrations and can increase its own catabolism by synovial fibroblasts; this effect is probably mediated via upregulation of both synovial fibroblast VDR and 24-OHase.
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Artritis Reumatoide/metabolismo , Comunicación Autocrina , Calcitriol/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Activación de Macrófagos/fisiología , Esteroide Hidroxilasas/metabolismo , Membrana Sinovial/metabolismo , Calcifediol/farmacología , Calcitriol/farmacología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/genética , Fibroblastos/metabolismo , Humanos , Hibridación in Situ , Macrófagos/metabolismo , ARN Mensajero/análisis , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Esteroide Hidroxilasas/genética , Vitamina D3 24-HidroxilasaRESUMEN
One of 16 human small cell lung cancer cell lines examined was shown to synthesize a metabolite resembling 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The NCI H82 line converted 25-hydroxyvitamin D3 (25OHD3) into a compound indistinguishable from 1,25-(OH)2D3 in 3 different high performance liquid chromatography systems. Electron impact mass spectra for the trimethylsilylethers of the metabolite and authentic 1,25-(OH)2D3 were indistinguishable. Binding to an anti-1,25-(OH)2D3 antibody was identical for the metabolite and authentic 1,25-(OH)2D3, whereas administration to rats in vivo caused equivalent stimulation of calcium transport measured in vitro in duodenal sacs. Activity of the H82 1 alpha-hydroxylase appears to be substrate dependent and is not stimulated by PTH, suggesting that it is similar to the enzyme expressed by activated macrophages and other cell types at extrarenal sites. Inhibition by ketoconazole indicates that, like the renal and extrarenal enzymes, the H82 enzyme is cytochrome P450 dependent. These data indicate that the H82 small cell lung cancer cell line constitutively expresses 25-hydroxyvitamin D3-1 alpha-hydroxylase and can synthesize 1,25-(OH)2D3.
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Calcitriol/biosíntesis , Carcinoma de Células Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Calcifediol/metabolismo , Calcitriol/farmacología , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Cetoconazol/farmacología , Espectrometría de Masas , Hormona Paratiroidea/farmacología , Receptores de Calcitriol/metabolismo , Células Tumorales CultivadasRESUMEN
1. Cyclosporin A, an immunosuppressive drug used to treat psoriasis, stimulates renal synthesis of 1,25-dihydroxyvitamin D in rats. 1,25-Dihydroxyvitamin D can also reduce the activity of psoriasis, and in the present study we have examined the possibility that cyclosporin A mediates some of its actions in psoriasis by renal or extra-renal production of 1,25-dihydroxyvitamin D. 2. Treatment of 12 psoriatic patients with cyclosporin A (5 mg day-1 kg-1) for 3 months significantly improved the psoriasis activity and severity index and reduced glomerular filtration rate, but serum 1,25-dihydroxyvitamin D levels were not changed. However, 1-3 months after stopping cyclosporin A treatment, an increase in the psoriasis activity and severity index score was accompanied by a small, but significant, increase in serum 1,25-dihydroxyvitamin D concentration. Plasma 1,25-dihydroxyvitamin D levels in rats gavaged with cyclosporin A (15 mg day-1 kg-1 for 2 weeks) were significantly increased compared with controls, but a lower dose of cyclosporin A (2.4 mg day-1 kg-1) had no effect. Renal 25-hydroxyvitamin D-24-hydroxylase activity in rat kidney homogenates was not different between control and cyclosporin A-treated rats. Renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity was not detectable in these homogenates. Extra-renal production of 1,25-dihydroxyvitamin D by activated macrophages isolated from the synovial fluid of patients with inflammatory arthritis was reduced after incubation with cyclosporin A (0.1-10 mumol/l) for 30 h or 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)
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Calcitriol/metabolismo , Ciclosporina/uso terapéutico , Psoriasis/tratamiento farmacológico , Animales , Artritis/metabolismo , Calcitriol/biosíntesis , Calcitriol/sangre , Células Cultivadas , Ciclosporina/farmacología , Tasa de Filtración Glomerular/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Psoriasis/sangre , Ratas , Ratas Wistar , Líquido Sinovial/metabolismoRESUMEN
High serum concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D] can occur with hypercalcemia in malignant lymphoma. We have investigated the potential for abnormal vitamin D metabolism by giving a single oral dose of 25-hydroxyvitamin D (25OHD) in 10 lymphoma patients (8 Hodgkin's and 2 T-cell) and 7 controls. Serum 25OHD increased similarly in both groups (peak concentrations, 114.1 +/- 9.5 vs. 123.9 +/- 9.6 nmol/L). In controls, serum calcium and PTH did not change after treatment [calcium, 2.31 +/- 0.02 and 2.33 +/- 0.02 mmol/L (mean +/- SEM); PTH, 21.6 +/- 4.0 and 25.4 +/- 4.3 ng/L] 1,25-(OH)2D increased within the normal range from [median (range)] 81 (48-125) to 117 (91-156) pmol/L. In lymphoma patients, serum calcium increased from 2.29 +/- 0.04 to 2.40 +/- 0.06 mmol/L (P = 0.03), PTH decreased from 12.9 +/- 2.6 to 8.0 +/- 1.9 ng/L (P = 0.06), and one patient became hypercalcemic (2.92 mmol/L). Serum 1,25-(OH)2D became supranormal in 6 lymphoma patients; the group median rose from 74.5 (46-180) to 151 (120-487) pmol/L; this peak response differed from that in the controls (P = 0.019). Lymph node and spleen cells from a patient with T-cell lymphoma synthesized [3H]1,25-(OH)2D3 from [3H] 25OHD3 in vitro. The data suggest that abnormal production of 1,25-(OH)2D in lymphoma may be more common than previously recognized given an adequate supply of precursor 25OHD and provide further evidence for the extrarenal synthesis of 1,25-(OH)2D in this condition.
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Calcitriol/biosíntesis , Linfoma/metabolismo , Calcifediol/metabolismo , Calcio/sangre , Humanos , Hormona Paratiroidea/sangreRESUMEN
Phorbol 12-myristate 13-acetate (100 nM), a potent protein kinase C and macrophage activator, has a biphasic affect on 25(OH)D3-1 alpha-hydroxylase activity in synovial fluid macrophages from arthritis patients. After 5 h, 1 alpha, 25(OH)D3 synthesis fell from 5.2 +/- 0.1 to 1.6 +/- 0.2 pmol/h per 10(6) cells, however, after 24 h and 48 h, synthesis increased to 17.4 +/- 0.3 and 22.3 +/- 1.4 pmol/h per 10(6) cells, respectively. Although an independent short-term mechanism is suggested, protein kinase C may promote macrophage activation, thus increasing long-term 25(OH)D3-1 alpha-hydroxylase expression. Intracellular calcium and cAMP are unlikely to activate the enzyme, since 0.1 microM of the calcium ionophore, A23187, and 1 mM dibutyryl-cAMP inhibited synthesis by 87% and 79%, respectively, after 24 h.
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Artritis/metabolismo , Calcitriol/biosíntesis , Macrófagos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Artritis Gotosa/metabolismo , Artritis Reumatoide/metabolismo , Bucladesina/farmacología , Calcimicina/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Macrófagos/efectos de los fármacos , Líquido Sinovial/citologíaRESUMEN
OBJECTIVE: We wished to assess the effect of changes in the plasma concentration of 1,25-dihydroxyvitamin D on the plasma elimination half-time for 25-hydroxyvitamin D in man. DESIGN: The turnover of 25-hydroxyvitamin D in plasma was investigated after intravenous doses of the radioactively labelled metabolite had been given to a group of patients (n = 17) with disorders of bone and mineral metabolism before and after oral treatment with calcium or 1,25-dihydroxyvitamin D. PATIENTS: Seven patients with post-menopausal osteoporosis, five with hypoparathyroidism, three with hypophosphataemic osteomalacia, one with renal osteodystrophy and one patient with coeliac disease were studied. MEASUREMENTS: Intravenous injections of 3H-labelled 25-hydroxyvitamin D were given and plasma elimination half-time assessed over periods of 4-14 days during which frequent measurements of plasma calcium, phosphate, parathyroid hormone, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were made. Changes in the plasma elimination half-time for 3H-25-hydroxyvitamin D before and after treatment with calcium and 1,25-dihydroxyvitamin D were evaluated by non-parametric statistical analysis. RESULTS: The elimination half-time for 3H-25-hydroxyvitamin D in plasma was significantly shortened by raising the circulating concentration of 1,25-dihydroxyvitamin D. Conversely, in a patient with intestinal malabsorption of calcium, the metabolic clearance of 3H-25-hydroxyvitamin D was prolonged when the concentration of 1,25-dihydroxyvitamin D in plasma was decreased by suppressing secondary hyperparathyroidism with large calcium supplements. In the longer-term studies (n = 10) there was a highly significant inverse relation (r = -0.88, P < 0.001) between the change in the plasma concentration of 1,25-dihydroxyvitamin D and the induced change in the elimination half-time of 3H-25-hydroxyvitamin D. There was also a significant correlation (r = 0.66, p < 0.0025) between the observed fall in the plasma concentration of unlabelled 25-hydroxyvitamin D and the predicted fall calculated from the measured value for the half-time of the 3H-labelled metabolite. In acute studies in patients with post-menopausal osteoporosis (n = 7), enhanced metabolic inactivation of 3H-25-hydroxyvitamin D was detectable within 24 hours of oral administration of 1,25-dihydroxyvitamin D. CONCLUSIONS: The effect of 1,25-dihydroxyvitamin D on the catabolism of 25-hydroxyvitamin D can contribute to the development of vitamin D deficiency in many clinical disorders. When the natural supply of vitamin D is limited by sunlight deprivation, a sustained increase in the plasma concentration of 1,25-dihydroxyvitamin D due to primary or secondary hyperparathyroidism will lead to accelerated depletion of vitamin D stores.
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Calcitriol/sangre , Deficiencia de Vitamina D/sangre , Anciano , Anciano de 80 o más Años , Calcifediol/sangre , Calcio/sangre , Femenino , Estudios de Seguimiento , Semivida , Humanos , Hipoparatiroidismo/sangre , Masculino , Persona de Mediana Edad , Osteomalacia/sangre , Osteoporosis Posmenopáusica/sangreRESUMEN
Previous work has shown that renal metabolism of 25-dihydroxyvitamin D3 (25(OH)D3) to the active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is stimulated by prostaglandin E2 and inhibited by acetylsalicylate (aspirin). As prostaglandins are primary inflammatory mediators and synovial fluid macrophages are known to synthesise 1,25(OH)2D3 in vitro, the effects of prostaglandin E1, prostaglandin E2, and aspirin on the metabolism of 25(OH)D3 by cells cultured from synovial fluid of patients with inflammatory arthritis were investigated. Most cultures contained non-proliferating macrophages which formed 1,25(OH)2D3; however, two of 13 cultures contained colonies of rapidly proliferating fibroblast-like cells which formed 24,25(OH)2D3 (24,25(OH)2D3). Prostaglandin E1 and prostaglandin E2 (0.01-10 mumol/l) induced marked inhibition of 1,25(OH)2D3 synthesis (up to 94%) in a dose dependent manner after preincubations of 24 hours but not over straightforward six hour incubations. Exposure of macrophages to aspirin (1 mumol/l-1 mmol/l) for 24 hours did not affect 1,25(OH)2D3 synthesis unless the cells had been pretreated with lipopolysaccharides, in which instance 1 mM aspirin increased 1,25(OH)2D3 synthesis. Lipopolysaccharide is a macrophage activating factor which stimulates macrophages to form 1,25(OH)2D3, and it also induces prostaglandin synthesis which would be inhibited by aspirin. Taken together these results suggest that prostaglandin E1 and prostaglandin E2 synthesised by macrophages may act in an autocrine manner to attenuate the ability of macrophage activating factors, such as lipopolysaccharide, to stimulate 1,25(OH)2D3 synthesis. Prostaglandins synthesised by other inflammatory cells may also inhibit 1,25(OH)2D3 synthesis in a paracrine manner. In contrast, prostaglandin E2 and aspirin had limited effects on fibroblast 24,25(OH)2D3 synthesis. This study shows that the effects of prostaglandin E1, prostaglandin E2, and aspirin in macrophages contrast with those previously reported for the renal 25(OH)D3-1alpha-hydroxylase, where prostaglandin E2 stimulated and aspirin inhibited enzyme activity. These results further emphasise that synthesis of 1,25(OH)2D3 in non-renal sites is independently regulated, which is consistent with it having an immunological role at a local level rather than playing a part in systemic calcium homeostasis.
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Alprostadil/farmacología , Artritis/metabolismo , Calcitriol/biosíntesis , Dinoprostona/farmacología , Líquido Sinovial/metabolismo , Alprostadil/metabolismo , Aspirina/metabolismo , Aspirina/farmacología , Calcitriol/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión , Dinoprostona/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Articulación de la Rodilla/metabolismo , Macrófagos/metabolismoRESUMEN
Differential metabolism of 25-hydroxyvitamin D3 (25(OH)D3) has been shown for macrophages and fibroblast-like cells (possibly synoviocytes) cultured for two to 50 days after isolation from the synovial fluid of 12 patients with various forms of inflammatory arthritis. Macrophages synthesised the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the synthesis of which was increased by bacterial lipopolysaccharide, a known macrophage activating factor. In contrast, fibroblast-like cells formed 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3), synthesis of which was stimulated by 1,25(OH)2D3 and inhibited by lipopolysaccharide. The synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 by macrophages and fibroblast-like cells respectively was inhibited by ketoconazole, indicating that both hydroxylases are dependent on cytochrome P-450. Mean (SEM) synovial fluid and serum 25(OH)D3 concentrations were 16.7 (1.7) and 22.2 (2.6) ng/ml and those of 1,25(OH)2D3 were 29.4 (4.8) and 43.3 (4.0) pg/ml respectively. In most cases concentrations were lower in synovial fluid than in paired serum samples, but in two patients 1,25(OH)2D3 concentrations were greater in synovial fluid than in serum, suggesting local synthesis within the affected joints.
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Artritis/metabolismo , Calcifediol/metabolismo , Fibroblastos/metabolismo , Macrófagos/metabolismo , Líquido Sinovial/metabolismo , 24,25-Dihidroxivitamina D 3/análisis , 24,25-Dihidroxivitamina D 3/biosíntesis , Adulto , Anciano , Artritis/sangre , Calcitriol/análisis , Calcitriol/biosíntesis , Células Cultivadas , Femenino , Humanos , Cetoconazol/farmacología , Lipopolisacáridos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
The constitutive expression of 25-hydroxyvitamin D3-24-hydroxylase (25-(OH)D3-24-hydroxylase) activity has been studied in an adherent variant (Ad-HL60) of the human promyelomonocytic leukaemia cell line HL60. The Ad-HL60 cells have a more differentiated phenotype than the non-adherent cells from which they were derived, and synthesized 1.88 +/- 0.07 (+/- S.E.M.) pmol 24,25-(OH)2D3/h per 10(6) cells following culture in RPMI-1640 medium containing less than 0.02 nM 1 alpha,25-(OH)2D3. They also synthesized 1.66 +/- 0.05 pmol 24,25-(OH)2D3/h per 10(6) cells following culture in 1 alpha,25(OH)2D3-free medium supplemented with 1 g bovine serum albumin/l instead of 10% serum. In contrast, non-adherent HL60 cells required exposure to 10-100 nM 1 alpha,25-(OH)2D3 to induce equivalent 24,25-(OH)2D3 synthesis. The 25-(OH)D3-24-hydroxylase expressed by Ad-HL60 cells had an apparent Michaelis constant of 1 microM and maximal rate of 20 pmol/h per 10(6) cells with substrate concentrations from 0.012 to 1.2 microM/incubation (5-500 ng/ml). Furthermore, 24,25-(OH)2D3 synthesis was inhibited in a dose-dependent manner by ketoconazole (0.01-10 microM), suggesting that the enzyme is cytochrome P-450 dependent. Ad-HL60 cells expressed approximately 3500 specific receptors for 1 alpha,25-(OH)2D3/cell with a dissociation constant of 40 pM. Following exposure to 0.1-100 nM 1 alpha,25-(OH)2D3, Ad-HL60 cell proliferation was significantly inhibited compared with controls grown in medium containing less than 0.02 nM 1 alpha,25-(OH)2D3 for 96 h. Expression of 25-(OH)D3-24-hydroxylase was also inhibited in a dose- and time-dependent manner; however, expression of nonspecific esterase was not induced. Both of these findings are contrary to those previously demonstrated for non-adherent HL60 cells, whereas the dose-dependent inhibition of cell proliferation by 1 alpha,25-(OH)2D3 occurs in both adherent and non-adherent phenotypes. These observations on Ad-HL60 cells represent the first description of a cell type in which 1 alpha,25-(OH)2D3 appears to inhibit 25-(OH)D3-24-hydroxylase activity. The Ad-HL60 cells also constitutively metabolized 1 alpha,25-(OH)2D3 in a manner consistent with formation of 1 alpha,25-(OH)2D3 without previous exposure to 1 alpha,25-(OH)2D3. In contrast, many other cell types, including non-adherent HL60 cells, require exposure to 1 alpha,25-(OH)2D3 to induce metabolism of 1 alpha,25-(OH)2D3 to 1 alpha,24,25-(OH)3D3, a reaction that represents the initial step for catabolism of 1 alpha,25-(OH)2D3 to calcitroic acid.
Asunto(s)
Calcitriol/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Esteroide Hidroxilasas/metabolismo , Células Tumorales Cultivadas , Adhesión Celular , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , Humanos , Cetoconazol/farmacología , Leucemia Promielocítica Aguda , Esteroide Hidroxilasas/antagonistas & inhibidores , Vitamina D3 24-HidroxilasaRESUMEN
The extrarenal synthesis of 1,25-dihydroxyvitamin D [1,25-(OH)2D] is a characteristic of activated macrophages and has been demonstrated to occur in vitro in synovial fluid macrophages from patients with inflammatory arthritis. To examine whether such synthesis occurs in vivo, 19 patients with rheumatoid arthritis, 5 patient controls, and 5 healthy controls received a challenge oral dose of 250 micrograms 25-hydroxyvitamin D3 (25-OHD3) and the serum 1,25-(OH)2D3 response was measured. The median rise in serum 1,25-(OH)2D3 was significantly greater (22 pg/ml) in the rheumatoid patients compared to either of the control groups (8 pg/ml), although the increase in precursor 25-OHD3 was similar in all groups. The serum 1,25-(OH)2D concentration did not rise above the normal upper limit in any of the control subjects but exceeded the normal range in 8 of the rheumatoid patients. Extrarenal 1,25-(OH)2D synthesis is substrate dependent, unlike renal 1 alpha-hydroxylation, which is homeostatically controlled. Excessive 1,25-(OH)2D3 synthesis in the rheumatoid group on raising the 25-OHD3 concentration is indicative of nonrenal production of the hormonal metabolite. Further evidence for substrate-dependent extrarenal synthesis came from measurements of 25-OHD and 1,25-(OH)2D in paired serum and synovial fluid samples from 19 patients with inflammatory arthritis, including 15 with rheumatoid arthritis. Synovial fluid 1,25-(OH)2D was usually present at a lower concentration than serum 1,25(OH)2D, with which it was strongly correlated (Kendall's R = 0.46, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Artritis Reumatoide/metabolismo , Calcitriol/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/complicaciones , Calcifediol/administración & dosificación , Calcifediol/metabolismo , Calcitriol/sangre , Femenino , Humanos , Riñón/metabolismo , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Osteoporosis/etiología , Líquido Sinovial/metabolismo , Proteína de Unión a Vitamina D/metabolismoRESUMEN
We have examined the ability of blood-derived monocytes and macrophages isolated from a patient with alveolar rhabdomyosarcoma and hypercalcaemia, to form 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3). Adherent monocyte-macrophage cells incubated with 25(OH)D3 over the initial 2 days in culture synthesized 1.9 pmol 24,25(OH)2D3/h/incubation (representing 0.63 pmol/h/10(6) cells), whereas macrophages synthesized 1.03 and 1.15 pmol 1 alpha,25(OH)2D3/h/incubation after 1 and 4 weeks in culture respectively. In a further experiment synthesis of 1 alpha,25(OH)2D3 by long-term cultured macrophages fell from 2.25 to 0.04 pmol/h/incubation following exposure to 10 nM 1 alpha,25(OH)2D3 for 7 days, whereas 24,25(OH)2D3 synthesis was induced (0.46 pmol/h/incubation). The vitamin D3 metabolites were identified by co-chromatography with authentic 24,25(OH)2D3 or 1 alpha,25(OH)2D3 in three different high-performance liquid chromatography systems. Serum 1 alpha,25(OH)2D3 in the patient was markedly suppressed at 5 pg/ml (normal 20-50 pg/ml) indicating that raised 1 alpha,25(OH)2D3 was not the cause of the hypercalcaemia, but rather, that raised calcium may have suppressed renal 1 alpha,25(OH)2D3 synthesis. Administration of APD (3-amino-1-hydroxypropylidine-1,1-bisphosphonate) corrected the hypercalcaemia in the patient suggesting that increased bone resorption was responsible for the raised calcium. The results of this study show for the first time that immature blood derived monocyte-macrophage cells can synthesize 24,25(OH)2D3 before they mature into macrophages able to synthesize 1 alpha,25(OH)2D3.
Asunto(s)
24,25-Dihidroxivitamina D 3/metabolismo , Calcifediol/metabolismo , Calcitriol/metabolismo , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Rabdomiosarcoma/metabolismo , 24,25-Dihidroxivitamina D 3/biosíntesis , Calcifediol/biosíntesis , Células Cultivadas , Cromatografía Líquida de Alta Presión , Humanos , Hipercalcemia/sangreRESUMEN
The ability to synthesise [3H]1,25(OH)2D3 was studied in spleen cells incubated in short-term primary culture with [3H]25(OH)D3 which were isolated from two patients with idiopathic myelofibrosis and from one normal subject. Formation of a metabolite co-chromatographing with authentic 1,25(OH)2D3 on three different high-performance liquid chromatography systems was observed for cells from all three patients. [3H]1,25(OH)2D3 synthesis was 0.37 and 1.6 fmol/10(6) cells/incubation for cells with a density below that of lymphocyte separating media (1.077 g/ml) for the two myelofibrotic patients respectively and 0.15 fmol/10(6) cells/incubation for the normal subject. The most likely cell type capable of this synthesis were those of the monocyte-macrophage lineage which would have been present in abnormally high numbers in the patients with myelofibrosis. However, the exact identity of the cell-type responsible could not be determined because of the heterogeneity of cell types present. The observation that spleen cells from two patients with myelofibrosis and from a normal accident victim could metabolise [3H]25(OH)D3 to its active form [3H]1,25(OH)2D3 suggests a possible role for this metabolite in spleen haematopoiesis.
Asunto(s)
Calcitriol/biosíntesis , Mielofibrosis Primaria/metabolismo , Bazo/metabolismo , Calcitriol/análisis , Cromatografía Líquida de Alta Presión , Humanos , Técnicas In Vitro , Valores de Referencia , Conteo por Cintilación , Espectrofotometría Ultravioleta , Bazo/citologíaRESUMEN
Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([ 3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1 alpha,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12 x 10(6) cells/incubation. The optimum substrate concentration for its synthesis was 125 nM, giving an apparent Michaelis constant of 360 nM. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nM 1 alpha,25-(OH)2D3 for 4 days synthesized 2.17 +/- 0.07 (S.E.M.) pmol 24,25-(OH)2D3/10(6) cells per h.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
24,25-Dihidroxivitamina D 3/metabolismo , Calcifediol/metabolismo , Sistema Enzimático del Citocromo P-450 , Cetoconazol/farmacología , Esteroide Hidroxilasas/metabolismo , Carboxilesterasa , Hidrolasas de Éster Carboxílico/metabolismo , Diferenciación Celular , División Celular , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Leucemia Promielocítica Aguda , Vitamina D3 24-HidroxilasaRESUMEN
Synthesis of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown in cells from knee joint synovial fluid of 20 patients with inflammatory rheumatoid disease, reactive or psoriatic arthritis, or gout, all of which had high synovial fluid cell counts, and by cells from a patient with aseptic necrosis of a femoral condyle after short term (less than 24 hours) or long term (seven days) primary culture. Cells from 18 patients with inflammatory arthritis, five of which had low synovial fluid cell counts and cells from six patients with osteoarthritis were unable to synthesise this metabolite from 25-hydroxyvitamin D3 (25(OH)D3). Macrophages are believed to be the cells responsible for synthesising 1,25(OH)2D3 because these were significantly more numerous in samples that formed 1,25(OH)2D3; they were also the predominant cell type present in the aseptic necrosis sample and the only cell type present in preparations maintained for one week in monolayer culture.