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Biomedical subjects

R O Brady

Publications and source records attributed to R O Brady.

At least 19 recordsLinked to original sources
PubMed1992

Type C Niemann-Pick disease: a murine model of the lysosomal cholesterol lipidosis accumulates sphingosine and sphinganine in liver.

We have determined the levels of free sphingoid bases in livers of normal and cholesterol lipidotic Niemann-Pick type C mice. Hepatic sphingosine and sphinganine levels in affected mice (593 pmol/mg protein) were elevated more than 20-fold when compared to levels in age-matched normal mice (26 pmol/mg protein). Upon fractionation of mutant liver homogenates by differential centrifugation, most of the sphingoid bases sedimented with beta-hexosaminidase in the 9000 x g pellet. Co-sedimentation of sphingoid bases with a lighter beta-hexosaminidase peak in Percoll gradients suggests that these bases accumulate in lipid laden lysosomes. A cytosolic sphinganine kinase is the first enzyme in the degradative pathway of sphingoid base metabolism. Activity of this enzyme was partially deficient in crude mutant liver cytosolic extracts due to the presence of an inhibitory substance. Following molecular sieving of mutant cytosolic extracts on Sepharose 4B, sphinganine kinase, with normal levels of activity, was resolved from a complex higher-molecular-weight inhibitor fraction. The Km values for either sphinganine or ATP-Mg substrates with partially purified sphinganine kinase from normal and mutant mouse liver extracts, were similar. These findings indicate that accumulation of free sphingoid bases is not due to a direct inherent deficiency in the catalytic activity of sphinganine kinase. The possible cause and effect relationship between the accumulation of these endogenous hydrophobic amines and the lesion in intracellular cholesterol trafficking in Niemann-Pick type C disease is discussed.

Animals↗
PubMed1992

Deficiencies in sex-regulated expression and levels of two hepatic sterol carrier proteins in a murine model of Niemann-Pick type C disease.

Hepatic sterol carrier protein-2 (SCP2) and sterol carrier protein-X (SCPx) levels in normal and in mutant Niemann-Pick Type C mice were determined by immunoblotting with antiserum against rat SCP2. A 14-kDa protein (SCP2) was detected in the cytosol fraction and a 58-kDa protein (SCPx) was found in both cytosolic and organellar fractions. Expression of hepatic SCPx protein was developmentally regulated in a sex-specific pattern. The amounts of organelle-associated SCPx increased 4-fold during sexual development of normal males but decreased dramatically during development of normal females. Levels of hepatic SCP2 increased much less dramatically during sexual maturation of normal males and females. Adult Niemann-Pick Type C mice were deficient in both hepatic SCPx and SCP2. The deficit in SCPx in affected males reflected a failure to increase hepatic SCPx levels during sexual maturation. In affected males SCPx remained at levels found in immature mice. Affected male and female mice were also unable to maintain levels of hepatic SCP2. The level of SCP2 was near normal in affected immature males and subnormal in affected immature females. During sexual maturation hepatic SCP2 declined in affected animals.

Aging↗
PubMed1992

Niemann-Pick type-C disease: deficient intracellular transport of exogenously derived cholesterol.

NPC disease is an autosomal recessive neurovisceral storage disorder. A pleiotropic array of secondary enzymatic and storage abnormalities has in the past obscured a cohesive understanding of the underlying metabolic basis of this disorder. Recent findings, reviewed in this report, demonstrate that NPC disease is a cholesterol lipidosis resulting from defective intracellular cholesterol transport. The sequence of cellular events characteristic of NPC is 1) deficient intracellular transport of exogenously derived cholesterol resulting in retarded induction of cellular cholesterol homeostatic regulation; 2) accumulation of cholesterol in lysosomes; and 3) secondary cellular effects. Retarded esterification of exogenous cholesterol and accumulation of unesterified cholesterol in lysosomes is tightly coupled to the primary defect and serves as the basis for biochemical diagnosis of NPC.

Antigens, CD↗
PubMed1992

Quantitative chemical shift imaging of vertebral bone marrow in patients with Gaucher disease.

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.

Body Water↗
PubMed1992

Gaucher disease: abdominal MR imaging findings in 46 patients.

Abdominal magnetic resonance imaging findings were reviewed in 46 patients with Gaucher disease. All patients had hepatosplenomegaly at the time of initial imaging. Splenic nodules were present in 14 patients (30%) and varied in signal intensity. These nodules were isointense on T1-weighted and hypointense on T2-weighted images. Splenic infarcts were seen in 15 patients (33%), and four of these patients (9%) also had subcapsular fluid collections. Both nodules and infarcts were present in the spleen in four patients (9%). Pathologic correlation was performed with specimens from two patients who underwent partial splenectomy. Focal areas of abnormal signal intensity were noted in the liver in nine patients (20%). They were either stellate or segmental, and may represent fibrotic septa with ischemic changes associated with aggregates of Gaucher cells. No changes were noted in the kidneys or abdominal lymph nodes.

Adolescent↗
PubMed1992

Quantitative imaging of Gaucher disease.

Twenty-three patients with type 1 Gaucher disease were evaluated with a battery of quantitative imaging techniques. Plain radiographs were used to measure cortical thickness and Erlenmeyer flask deformity. Xenon-133 uptake was measured in scans of the lower extremities. Dual-energy quantitative computed tomography was used for calculation of trabecular bone and bone marrow fat content in the spine and long bones. Magnetic resonance (MR) imaging was performed to evaluate disease extent and three-dimensional splenic volume. MR images were also used to provide quantitative measurements of each vertebral fat fraction. Each imaging modality was correlated with the others as well as with the clinical history of skeletal complications and the hematocrit and acid phosphatase activity. There was a strong relationship between splenic volume and disease severity as measured clinically and with laboratory testing. The spinal fat fraction also correlated with these measures of disease severity and with the femoral fat fraction and xenon uptake. No measurement allowed discrimination of patients with from those without skeletal complications.

Adolescent↗
PubMed1992

Hypoprebetalipoproteinemia, acanthocytosis, retinitis pigmentosa, and pallidal degeneration (HARP syndrome).

We describe the clinical and laboratory studies of an 11-year-old girl with prominent orofacial dyskinesia, dystonia, and progressive dementia. Investigations revealed hypoprebetalipoproteinemia, acanthocytosis, atypical retinitis pigmentosa, and evidence of iron deposition in the pallidal nuclei. Electroneuromyography and skin and sural nerve biopsies were normal. The "eye-of-the-tiger" sign, used to describe the pallidal nuclei in Hallervorden-Spatz syndrome, was present on T2-weighted MRIs (GE Signa, 1.5 T). Phase-contrast microscopy of whole blood showed 80 to 90% acanthocytes whose morphology was confirmed by electron microscopy. High-resolution lipoprotein electrophoresis demonstrated an absence of the pre-beta fraction. This case differs phenotypically from the previous reports of Hallervorden-Spatz disease with acanthocytosis by the presence of prominent orofacial dyskinesia and abnormal serum lipoproteins.

Acanthocytes↗
PubMed1992

A clinical staging classification for type C Niemann-Pick disease.

Analysis of the temporal sequence of neurologic events, neurophysiologic abnormalities, and longevity in 36 Niemann-Pick type C patients revealed two clinical subgroups with five stages of severity within each group. Patients with a preschool onset (group I; n = 18) had a higher mortality than did patients with a school-age onset (group II; n = 18). An asymptomatic phase (stage 0) was defined by biochemical and histopathologic evidence of disease. The initial manifestations of stage 1 were a movement disorder (group I) and cognitive difficulties (group II) accompanied by impaired vertical saccadic eye movements and abnormal acoustic reflexes. Stage 2 was characterized by the sequential occurrence of vertical supranuclear gaze palsy (VSGP), cognitive difficulties, and dysarthria in group I and a movement disorder, VSGP, and dysarthria in group II. Pyramidal tract signs and abnormal brainstem auditory evoked responses defined stage 3 in both groups. Stage 4 culminated in a nonambulant, vegetative state.

Adolescent↗
PubMed1991

Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol.

A uniquely attenuated disruption of cholesterol homeostasis has been characterized in certain Niemann-Pick, type C (NP-C) fibroblasts. Uptake of LDL-cholesterol by cultured fibroblasts derived from two clinically affected brothers with this variant biochemical phenotype led to less intracellular accumulation of unesterified cholesterol than found in other typical cell lines. This limited cholesterol lipidosis in the variant NP-C cells reflected cholesterol processing errors that differed from the cellular lesions in classical NP-C cells in the following ways: (1) a more limited intracellular distribution of the excessive unesterified cholesterol; (2) shorter and more transient delays in the induction of cholesterol-mediated homeostatic responses; and (3) more efficient intracellular transport of exogenously derived cholesterol to the plasma membrane and the endoplasmic reticulum. Activation of acyl-CoA cholesterol acyltransferase (ACAT) was greater than 100-fold in both control and NP-C fibroblasts when cell cultures were preconditioned with 25-hydroxycholesterol, but the subsequent esterification of exogenous non-lipoprotein [3H]cholesterol remained deficient in all NP-C cells. In the variant NP-C cells conditioned with the oxysterol, this esterification of exogenous [3H]cholesterol was less affected than in classical NP-C cultures. The NP-C mutation affects a broad spectrum of metabolic responses related to the processing of exogenously derived cholesterol. Among this pleiotropic array of deficient responses, retarded intracellular cholesterol transport appears most closely linked to the primary mutation. This conclusion is supported by two current observations: (1) the degree to which sterol transport is affected in mutant cells in turn reflects the extent to which cholesterol-homeostatic responses are compromised; and (2) sterol transport remains deficient despite concurrent normal activation of other cellular responses, such as cholesterol esterification.

Adult↗
PubMed1991

Replacement therapy for inherited enzyme deficiency--macrophage-targeted glucocerebrosidase for Gaucher's disease.

BACKGROUND AND METHODS: Gaucher's disease, the most prevalent of the sphingolipid storage disorders, is caused by a deficiency of the enzyme glucocerebrosidase (glucosylceramidase). Enzyme replacement was proposed as a therapeutic strategy for this disorder in 1966. To assess the clinical effectiveness of this approach, we infused macrophage-targeted human placental glucocerebrosidase (60 IU per kilogram of body weight every 2 weeks for 9 to 12 months) into 12 patients with type 1 Gaucher's disease who had intact spleens. The frequency of infusions was increased to once a week in two patients (children) during part of the trial because they had clinically aggressive disease. RESULTS: The hemoglobin concentration increased in all 12 patients, and the platelet count in 7. Serum acid phosphatase activity decreased in 10 patients during the trial, and the plasma glucocerebroside level in 9. Splenic volume decreased in all patients after six months of treatment, and hepatic volume in five. Early signs of skeletal improvements were seen in three patients. The enzyme infusions were well tolerated, and no antibody to the exogenous enzyme developed. CONCLUSIONS: Intravenous administration of macrophage-targeted glucocerebrosidase produces objective clinical improvement in patients with type 1 Gaucher's disease. The hematologic and visceral responses to enzyme replacement develop more rapidly than the skeletal response.

Acid Phosphatase↗
PubMed1991

Gaucher's disease: lack of antibody response in 12 patients following repeated intravenous infusions of mannose terminal glucocerebrosidase.

An amplified ELISA has been employed for monitoring the safety of repeated intravenous infusions of modified human placental glucocerebrosidase. The enzyme infusions consisted of biweekly injections of macrophage targeted glucocerebrosidase over a 6 month duration. Serum samples collected throughout the study were assayed by use of an ELISA using alkaline phosphatase coupled to alcohol dehydrogenase for amplification. Using this protocol, 0.2-5 ng affinity purified immunoglobulin specific for glucocerebrosidase can be detected. Occasional false positives necessitate multiple repeat assays over time to accurately assess immunogenic response. Blinded ELISAs were performed on sera from both infused patients with Gaucher's disease and uninfused control patients and compared with apparent immunoglobulin concentration in 54 normal control sera. Although several samples showed apparently elevated immunoglobulin levels, repeat analyses failed to demonstrate high levels reproducibly. Furthermore, these sera were unable to neutralize enzyme or to precipitate radiolabelled enzyme, confirming the absence of antibody. Problems with high sensitivity ELISA formats are discussed.

Animals↗
PubMed1991

Neurologic complications of nonneuronopathic Gaucher's disease.

We describe eight patients with type 1 Gaucher's disease who developed neurologic complications that were secondary to systemic features of the illness. Four patients experienced neurologic difficulties because of coagulopathy, and the other four patients had involvement of the nervous system secondary to skeletal disease. Early recognition of these complications in patients with type 1 Gaucher's disease may lead to improved neurologic outcome.

Aged↗
PubMed1991

Hematologic improvement in a patient with Gaucher disease on long-term enzyme replacement therapy: evidence for decreased splenic sequestration and improved red blood cell survival.

We describe here an investigation of the hematologic response of a child with Gaucher disease to a six-year therapeutic trial of human placental mannose-terminated glucocerebrosidase. While on enzyme replacement therapy, the patient's hemoglobin and platelet count significantly increased (6.9 g/dl to 12.2 g/dl, and 39,000/microliters to 74,000/microliters, respectively). Over the same time interval his absolute reticulocyte count decreased (88.1 x 10(3)/microliters to 31.5 x 10(3)/microliters). The patient's splenic volume decreased by over 60% (2108ml to 797ml) and his bone marrow demonstrated a dramatic clearing of Gaucher cell infiltration. Serum erythropoietin levels were at the lower range of normal at points in time when he was severely anemic and did not change with improvement in hemoglobin. Coincident with the improvement in the patient's hemoglobin, in vivo survival of autologous radiolabeled RBCs increased 34%. The improved hematopoietic profile appeared to result primarily from an increase in the in vivo survival of mature hematopoietic elements secondary to a decrease in splenic sequestration. Improved marrow function secondary to the clearing of Gaucher cell infiltrates may also play a role in his improved hematologic status.

Biopsy↗
PubMed1991

Effect of dimethylsulfoxide on the proliferation and glycosaminoglycan synthesis of rat prostate adenocarcinoma cells (PAIII) in vitro: isolation and characterization of DMSO-resistant cells.

Dimethylsulfoxide (DMSO) modulates the tumorigenicity and other characteristics of some malignant cell lines in vitro. We have investigated DMSO effects on cell proliferation and glycosaminoglycan (GAG) synthesis in rat prostate adenocarcinoma (PAIII) cells in culture. DMSO inhibited cell proliferation and GAG synthesis and shedding. Cells that survived the initial exposure to 2.5% DMSO could be propagated in this concentration of the agent and were designated PAIII-DMSO resistant (PAIII-DMSOr). PAIII-DMSOr cells showed reversible indications of increased differentiation such as decreased growth rate and saturation density. Although the PAIII-DMSOr cells were grown in 2.5% DMSO, they had normal or elevated GAG content. The major GAG of both PAIII and PAIII-DMSOr cells was undersulfated heparan sulfate. Verapamil, a calcium channel blocker that reverses drug resistance in tumor cells, stimulated the growth of PAIII-DMSOr cells in the presence of 2.5% DMSO, but was inhibitory in the absence of DMSO. Growth of PAIII cells was inhibited by the differentiating agents sodium butyrate and retinoic acid and by the ionophore monensin. Interestingly, growth of PAIII-DMSOr cells in the presence of 2.5% DMSO was largely unaffected by sodium butyrate or retinoic acid. The results suggest that (1) PAIII-DMSOr cells arise from the induction of a compensation mechanism to DMSO effects in a preexisting population of cells: (2) there is a correlation between GAG synthesis and cell proliferation; and (3) further study of the verapamil effect may help elucidate the mechanism of the DMSO-induced differentiation of cancer cells.

Adenocarcinoma↗
PubMed1991

Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport.

Niemann-Pick Type C (NPC) disease is a cholesterol lipidosis resulting from defective postlysosomal cholesterol transport. In normal cells this segment of cholesterol trafficking is inhibited by treatment with either U18666A or imipramine. Other compounds are also capable of blocking postlysosomal cholesterol transport: stearylamine, RV-538, and sphinganine inhibit low-density lipoprotein-induced esterification of cholesterol and cause unesterified cholesterol to accumulate in perinuclear vesicles. These vesicles can be stained with filipin to give a staining pattern indistinguishable from that seen in NPC fibroblasts. Because all of these compounds are hydrophobic amines, we conclude that most, if not all, hydrophobic amines block the postlysosomal transport of cholesterol. These results also raise the possibility that an endogenous amine, e.g., sphinganine, may inhibit cholesterol transport in NPC.

Amines↗
PubMed1990

Type C Niemann-Pick disease: documentation of abnormal LDL processing in lymphocytes.

Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (+/- 5%) and 45% (+/- 16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.

Cell Compartmentation↗
PubMed1990

Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher disease.

Enzyme replacement has been under consideration as a therapeutic strategy for patients with Gaucher disease for more than two decades. Previous studies indicated that single injections of purified glucocerebrosidase reduced the amount of storage material in the liver. It was important to determine whether administration of exogenous enzyme on a regular basis would be of clinical benefit. We report here that weekly i.v. infusions of a macrophage-targeted preparation of human placental glucocerebrosidase in a child with type 1 Gaucher disease increased hemoglobin from 6.9 +/- 0.8 g/dl (+/- 1 SD) to 10.2 +/- 0.4 g/dl (+/- 1 SD) over a 20-week period. The platelet count also increased from a pretreatment value of 30,000 +/- 7000/mm3 (+/- 1 SD) to 54,000 +/- 11,000/mm3 (+/- 1 SD). Phagocytic activity in the spleen decreased during the period of enzyme administration, and there was radiographic evidence of skeletal improvement. These observations document objective clinical responses to enzyme supplementation in a patient with a sphingolipid storage disorder.

Bone and Bones↗