RESUMO
Fabry disease results in a global vasculopathy leading to early-onset stroke and renal and cardiac failure. We found that random myeloperoxidase in serum and plasma was significantly elevated in 73 consecutive male patients with Fabry disease. Random serum myeloperoxidase level in men predicted the risk of a Fabry vasculopathy-related event in subsequent years. Long-term enzyme replacement therapy did not reduce myeloperoxidase level or eliminate the risk of vasculopathic events.
Assuntos
Doenças Cardiovasculares/enzimologia , Doença de Fabry/enzimologia , Doença de Fabry/genética , Peroxidase/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Criança , Doença de Fabry/complicações , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Peroxidase/genética , Valor Preditivo dos Testes , Estudos Prospectivos , Fatores de RiscoRESUMO
We identified four unrelated patients (three female, one male) aged 20 to 30 years with hypomyelination, pituitary hypogonadotropic hypogonadism, and hypodontia. Electron microscopy and myelin protein immunohistochemistry of sural nerves showed granular debris-lined clefts, expanded abaxonal space, outpocketing with vacuolar disruption, and loss of normal myelin periodicity. Reduced galactocerebroside, sphingomyelin, and GM1-N-acetylglucosamine and increased esterified cholesterol were found. This is a clinically homogeneous progressive hypomyelinating disorder. The term 4H syndrome is suggested.
Assuntos
Anodontia/patologia , Doenças Desmielinizantes/patologia , Hipogonadismo/patologia , Adulto , Anodontia/complicações , Doenças Desmielinizantes/complicações , Feminino , Humanos , Hipogonadismo/complicações , Masculino , Hipófise/patologia , Nervo Sural/patologiaRESUMO
Gaucher disease, the most common sphingolipidosis, is caused by a decreased activity of glucosylceramide beta-glucosidase, resulting in the accumulation of glucosylceramide in macrophage-derived cells known as Gaucher cells. Much of the storage material is thought to originate from the turnover of cell membranes, such as phagocytosed red and white blood cells. In this study, an in vitro model of Gaucher disease was developed by treating the murine macrophage cell line J774 with a specific inhibitor of glucosylceramide beta-glucosidase, conduritol B-epoxide, and feeding red blood cell ghosts, in order to mimic the disease state. It was found in this model system that glucosylceramide beta-glucosidase activity could be reduced to about 11-15% of the normal control level before increased storage of glucosylceramide occurred. This in vitro system allows insight into the correlation between enzyme activity and lipid storage as predicted by the theory of residual enzyme activity that was proposed by Conzelmann and Sandhoff.
Assuntos
Doença de Gaucher/diagnóstico , Doença de Gaucher/metabolismo , Glucosilceramidase/metabolismo , Inositol/análogos & derivados , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Membrana Eritrocítica/metabolismo , Doença de Gaucher/genética , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidas/metabolismo , Heterozigoto , Inositol/farmacologia , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Camundongos , Microscopia EletrônicaRESUMO
Effective gene therapy depends on the efficient transfer of therapeutic genes and their protein products to target cells. Lentiviral vectors appear promising for virus-mediated gene delivery and long-term expression in nondividing cells. The herpes simplex virus type 1 tegument protein VP22 has recently been shown to mediate intercellular transport of proteins, raising the possibility that it may be helpful in a setting where the global delivery of therapeutic proteins is desired. To investigate the effectiveness of lentiviral vectors to deliver genes encoding proteins fused to VP22, and to test whether the system is sufficiently potent to allow protein delivery from transduced cells in vitro and in vivo, fusion constructs of VP22 and the enhanced green fluorescent protein (EGFP) were prepared and delivered into target cells by using HIV-1-based lentiviral vectors. To follow the spread of VP22-EGFP to other cells, transduced COS-7 cells were coplated with a number of different cell types, including brain choroid plexus cells, human endothelial cells, H9 cells, and HeLa cells. We found that VP22-EGFP fusion proteins were transported from transduced cells to recipient cells and that such fusion proteins accumulated in the nucleus and in the cytoplasm of such cells. To determine the ability to deliver fusion proteins in vivo, we injected transduced H9 cells as well as the viral vector directly into the brain of mice. We present evidence that VP22-EGFP fusion proteins were transported effectively from lentivirus transduced cells in vivo. We also show that the VP22-EGFP fusion protein encoded by the lentivirus is transported between cells. Our data indicate that such fusion proteins are present in the nucleus and in the cytoplasm of neighboring cells. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22.
Assuntos
Vetores Genéticos , Lentivirus/genética , Proteínas Estruturais Virais/administração & dosagem , Animais , Células COS , Proteínas de Fluorescência Verde , Células HeLa , Hipocampo/metabolismo , Hipocampo/virologia , Humanos , Proteínas Luminescentes/genética , Camundongos , Proteínas Estruturais Virais/genéticaRESUMO
Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb(3); also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified alpha-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered alpha-gal A, (ii) to assess the pharmacokinetics of i.v.-administered alpha-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb(3). alpha-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified alpha-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of alpha-gal A, nine of the 10 patients had significantly reduced Gb(3) levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of alpha-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb(3) burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.
Assuntos
Doença de Fabry/enzimologia , Triexosilceramidas/metabolismo , alfa-Galactosidase/uso terapêutico , Adulto , Doença de Fabry/terapia , Humanos , Imuno-Histoquímica , Fígado/citologia , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Urinálise , alfa-Galactosidase/farmacocinéticaRESUMO
Gaucher disease is caused by insufficient activity of the enzyme glucocerebrosidase. Great benefit has been obtained through enzyme replacement therapy for patients with type 1 (non-neuronopathic) Gaucher disease. In contrast, inconsistent effects of enzyme therapy have been observed in patients with type 3 (chronic neuronopathic) Gaucher disease, and no benefit on the lethal course of the disease occurs in patients with Type 2 (acute neuronopathic) Gaucher disease. We examined the use of convection-enhanced delivery to augment the delivery and distribution of exogenous glucocerebrosidase (m.w. 63,000) to the brain by infusing it under slight hydrostatic pressure into the striatal region of rats. The enzyme was comparatively stable under these conditions. It was distributed from the site of injection toward the cerebral cortex where it became primarily localized in neurons. These findings provide considerable incentive for the exploration of intracerebral microinfusion of enzyme to the brain of patients with metabolic storage disorders involving the CNS.
Assuntos
Encéfalo/metabolismo , Glucosilceramidase/administração & dosagem , Manose/metabolismo , Neurônios/metabolismo , Animais , Barreira Hematoencefálica , Encéfalo/citologia , Doença de Gaucher/terapia , Glucosilceramidase/metabolismo , Glucosilceramidase/farmacocinética , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
We prospectively evaluated the clinical and biochemical responses to enzyme-replacement therapy (ERT) with macrophage-targeted glucocerebrosidase (Ceredase) infusions in 5 patients (age, 3.5-8.5 years) with type 3 Gaucher's disease. The patients were followed for up to 5 years. Enzyme dosage ranged from 120 to 480 U/kg of body weight/month. Systemic manifestations of the disease regressed in all patients. Neurological deficits remained stable in 3 patients and slightly improved in 1. One patient developed myoclonic encephalopathy. Cognitive deterioration occurred in 1 patient and electroencephalographic deterioration in 2. Sequential cerebrospinal fluid (CSF) samples were obtained during the first 3 years of treatment in 3 patients and were analyzed for biochemical markers of disease burden. Glucocerebroside and psychosine levels were not elevated in these specimens, whereas chitotriosidase and quinolinic acid were elevated in 2 patients. Progressive decrease in the CSF levels of these latter macrophage markers during 3 years of treatment implies a decreased number of Gaucher cells in the cerebral perivascular space. Similar changes were not observed in the patient who had a poor neurological outcome. In conclusion, ERT reverses systemic manifestations of type 3 Gaucher's disease and appears to reduce the burden of Gaucher cells in the brain-CSF compartment in some patients.
Assuntos
Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/administração & dosagem , Macrófagos/enzimologia , Anticorpos/sangue , Biomarcadores , Criança , Pré-Escolar , Eletroencefalografia , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Doença de Gaucher/diagnóstico , Doença de Gaucher/fisiopatologia , Glucosilceramidase/efeitos adversos , Glucosilceramidase/imunologia , Hexosaminidases/sangue , Hexosaminidases/líquido cefalorraquidiano , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/sangue , Interleucina-1/líquido cefalorraquidiano , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Exame Neurológico , Testes Neuropsicológicos , Nitritos/sangue , Nitritos/líquido cefalorraquidiano , Estudos Prospectivos , Psicosina/sangue , Psicosina/líquido cefalorraquidiano , Ácido Quinolínico/sangue , Ácido Quinolínico/líquido cefalorraquidiano , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/sangue , Sialoglicoproteínas/líquido cefalorraquidiano , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/líquido cefalorraquidianoRESUMO
Quantitative chemical shift magnetic resonance imaging (QCSI) is currently being utilized for measuring the extent of bone marrow involvement and its response to enzyme replacement therapy in patients with Gaucher's disease. Quantitation of the major lipid species in human bone marrow is required to accurately interpret QCSI data on bone marrow composition. The major lipid species in bone marrow specimens from normal individuals and from patients with type 1 Gaucher's disease were analyzed by thin-layer and high-pressure liquid chromatography. In normal marrow (N = 5), triglycerides were by far the most abundant lipid (278 +/- 70 mg/gm wet wt), with other non-polar lipids and phospholipids totaling less than 20 mg/gm wet weight. The concentration of glucocerebroside in normal marrow was 0.061 +/- 0.06 mg/gm wet weight. Gaucher marrow (N = 9) had dramatically lower triglyceride levels (51 +/- 53 mg/gm wet wt), and as expected, it had markedly elevated levels of glucocerebroside (7.1 +/- 3.4 mg/gm wet wt). The other major non-polar lipids and phospholipids were measured in selected specimens, but none were found that differed so profoundly between normal and Gaucher's disease. These data support a model of bone marrow alteration in Gaucher's disease in which triglyceride-rich adipocytes are progressively replaced by storage cells, leading to an overall reduction in total lipid content. This phenomenon provides an explanation for the changes in proton signal intensity observed in QCSI studies of Gaucher bone marrow.
Assuntos
Medula Óssea/química , Doença de Gaucher/metabolismo , Lipídeos/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Feminino , Doença de Gaucher/patologia , Glucosilceramidas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/análise , Triglicerídeos/análiseRESUMO
Two adults were seen with cirrhosis caused by different lipid storage diseases. A 42-yr-old woman with Niemann-Pick disease type B had marked hepatomegaly, ascites and recent variceal bleeding. Her evaluation showed chronic bilateral pulmonary infiltrates, multiple stigmata of chronic liver disease including the recent cessation of menses, diuretic-resistant sterile ascites, hepatic encephalopathy and variceal bleeding. Five percent of normal sphingomyelinase activity was measured in peripheral leukocytes. A 42-yr-old man with Gaucher's disease and a history of bilateral hip replacements presented with hepatomegaly, jaundice, refractory ascites and renal insufficiency. His evaluation showed 20% to 23% of normal glucocerebrosidase activity in peripheral leukocytes. Both patients underwent orthotopic liver transplantation with resolution of all aspects of decompensated liver function. Assessment of the underlying metabolic defect before and 6 to 14 mo after transplantation showed that after transplantation the patient with Niemann-Pick disease had above normal hepatic sphingomyelinase activity, a less-marked increase in peripheral leukocyte enzyme activity and lower than normal hepatic sphingomyelin and cholesterol content. In contrast, the patient with Gaucher's disease had only a 61% increase in hepatic glucocerebrosidase activity but had an elevated hepatic glucocerebroside content that was only 15% of the pretransplant level and decreased peripheral leukocyte enzyme levels. These findings suggest that variable relationships may exist between posttransplant hepatic and peripheral leukocyte enzyme activities in the different lipidoses, which may have implications for recurrence of glycolipid-induced liver damage.
Assuntos
Doença de Gaucher/cirurgia , Transplante de Fígado , Doenças de Niemann-Pick/cirurgia , Adulto , Biópsia , Enzimas/metabolismo , Feminino , Doença de Gaucher/patologia , Hepatectomia , Humanos , Leucócitos/enzimologia , Metabolismo dos Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/terapia , Microscopia Eletrônica , Doenças de Niemann-Pick/patologiaRESUMO
Long-term studies of a child with Gaucher disease indicated that the response to treatment with macrophage-targeted glucocerebrosidase (glucosylceramidase) is dose dependent, and that the hematologic response precedes the skeletal response.
Assuntos
Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/administração & dosagem , Abdome/patologia , Osso e Ossos/patologia , Criança , Relação Dose-Resposta a Droga , Seguimentos , Doença de Gaucher/diagnóstico , Doença de Gaucher/enzimologia , Glucosilceramidase/sangue , Humanos , Macrófagos/efeitos dos fármacos , Imageamento por Ressonância Magnética , MasculinoRESUMO
To evaluate extent of bone marrow involvement and disease severity in Gaucher patients, results of modified Dixon quantitative chemical shift imaging (QCSI) of the lumbar spine were correlated with quantitative analysis of marrow triglycerides and glucocerebrosides and with quantitative determination of splenic volume at magnetic resonance (MR) imaging. High-field-strength MR spectra of surgical marrow specimens were dominated by a single fat and a water peak, validating use of QCSI. QCSI showed average vertebral marrow fat fractions of 10% +/- 8 in Gaucher patients (normal adult averages, 29% +/- 6). Relaxation times for lipid and water approximated normal averages; bulk T1 values were significantly longer, reflecting decreased marrow fat. Glucocerebroside concentrations were higher in Gaucher marrow and inversely correlated with triglyceride concentrations. Extent of marrow infiltration determined by fat fraction measurements correlated with disease severity measured by splenic enlargement. These results show that as Gaucher cells infiltrate bone marrow and displace normal marrow adipocytes, bulk T1 increases due to the higher T1 of water compared with that of fat. QCSI provides a sensitive, noninvasive technique for evaluating bone marrow involvement in Gaucher disease.
Assuntos
Medula Óssea/química , Doença de Gaucher/metabolismo , Vértebras Lombares , Espectroscopia de Ressonância Magnética , Água Corporal/metabolismo , Doença de Gaucher/patologia , Glucosilceramidas/análise , Humanos , Lipídeos/análise , Baço/patologia , Triglicerídeos/análiseRESUMO
beta-Glucocerebrosidase, partially purified by the method of F. S. Furbish et al. (1977, Proc. Natl. Acad. Sci. USA 74, 3560-3563), was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to contain, in addition to the desired enzyme, variable amounts of a very hydrophobic contaminant (apparent Mr 45,000). Purification of the enzyme was accomplished by gel-permeation HPLC on a TSK 3000 SW column (0.7 X 60 cm). Adsorptive losses of protein on the column were minimized by using buffers containing up to 50% ethylene glycol. We have examined the effects of varying the ethylene glycol concentration on the elution times and recoveries of the two major proteins in this preparation. The high reproducibility of the individual chromatograms permitted the use of an automatic sampler and fraction collector to perform multiple, continuous runs for the purification of milligram quantities of enzyme. Multiple runs of a preparative-scale column, TSK G3000 SWG (2.15 X 60 cm), permitted gram-scale purification of beta-glucocerebrosidase without loss in efficiency of separation. Recovery of enzyme activity is greater than 94% with less than 1% contamination by other proteins. Reaction of enzyme prepared in this fashion with rabbit polyclonal antiserum or mouse monoclonal anti-beta-glucocerebrosidase shows the enzyme to be pure and not immunologically related to the 45,000 Mr contaminant. The specific activity of enzyme prepared by this means is 1.6 X 10(6) nmol/h/mg protein. Inclusion of ethylene glycol in buffers was shown to overcome hydrophobic protein interactions with TSK 3000 SW column matrices for both the soluble human lysosomal enzyme alpha-galactosidase A and the plant toxin ricin.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Etilenoglicóis , Glucosidases/isolamento & purificação , Glucosilceramidase/isolamento & purificação , Animais , Soluções Tampão , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Etilenoglicol , Glucosilceramidase/imunologia , Camundongos , Peso Molecular , Coelhos , Ricina/isolamento & purificaçãoRESUMO
Galactosylated and mannosylated liposomes were more efficient in transporting liposome-entrapped beta-glucocerebrosidase to liver compared to nonglycosylated liposomes. The enzyme entrapped to glycoside-bearing liposomes was found to be cleared at a much faster rate than that entrapped in liposomes having no sugar on their surface. Asialoorosomucoid and hydrolyzed mannan were found to inhibit both the clearance and the uptake of galactosylated and mannosylated liposomes, respectively, supporting involvement of lectin-sugar interaction. Further studies on the uptake of glucocerebrosidase by isolated liver cells revealed that the enzyme entrapped in mannosylated liposomes has much higher affinity for nonparenchymal cells whereas the assimilation of the entrapped enzyme into hepatocytes is clearly favored for liposomes having galactose on their surface.
Assuntos
Glucosidases/administração & dosagem , Glucosilceramidase/administração & dosagem , Lectinas , Lipossomos , Animais , Feminino , Humanos , Lipossomos/metabolismo , Fígado/metabolismo , Masculino , Placenta/enzimologia , Gravidez , Ratos , Ratos EndogâmicosRESUMO
Myotonic discharges in rats given 20, 25-diazacholesterol hydrochloride and fibrillation discharges in denervated rat muscle both were silenced by procaine hydrochloride, tetrodotoxin or ischemia, or potassium chloride (after initial activation). They both were activated by succinylcholine, but only the fibrillations were silenced by alpha-bungarotoxin or atropine sulfate. It is hypothesized that fibrillations and diazacholesterol-induced myotonia are mediated through mechanisms involving ionic channels, that both can be produced by activation of the junctional/nonjunctional acetylcholine receptors (or some mechanism coupled to the receptors), but that an unfettered alpha-bungarotoxin-binding portion of the acetylcholine-receptor molecule and an unblocked atropine-binding site are obligatory only for production of fibrillations.