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J H Wolfe

Publications and source records attributed to J H Wolfe.

At least 19 recordsLinked to original sources
PubMed1999

Molecular basis of feline beta-glucuronidase deficiency: an animal model of mucopolysaccharidosis VII.

A family of domestic cats was found that exhibited clinical and biochemical abnormalities consistent with mucopolysaccharidosis VII, an autosomal recessive lysosomal storage disorder caused by beta-glucuronidase deficiency. beta-Glucuronidase activity was undetectable in affected cat fibroblasts and restored by retroviral gene transfer of rat beta-glucuronidase cDNA. beta-Glucuronidase mRNA was normal in affected cat testis by Northern blot analysis. Normal feline beta-glucuronidase cDNA was cloned and characterized, and amplified from affected cat fibroblasts by reverse transcription coupled polymerase chain reaction. There was a G-to-A transition in the affected cat cDNA that predicted an E351K substitution, destroyed a BssSI site, and eliminated GUSB enzymatic activity in expression studies. Multiple species comparison and the crystal structure of human beta-glucuronidase indicated that E351 is a highly conserved residue most likely essential in maintenance of the enzyme's conformation. BssSI digestion of polymerase chain reaction products amplified from genomic DNA indicated that affected cats were homozygous and cats with half-normal beta-glucuronidase activity were heterozygous for the missense mutation. Carriers identified in this manner produced affected kittens in prospective breedings, and a feline MPS VII breeding colony has been established.

Amino Acid Sequence↗
PubMed1999

Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.

Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.

Animals↗
PubMed1999

Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.

Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

Animals↗
PubMed1999

Sustained production of beta-glucuronidase from localized sites after AAV vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice.

The lysosomal storage disorders are a large group of inherited diseases that involve central nervous system degeneration. The disease in the brain has generally been refractory to treatment, which will require long-term correction of lesions dispersed throughout the central nervous system to be effective. A promising approach is somatic gene therapy but the methods have so far been inadequate because they have only achieved short-term or localized improvements. A potential approach to overcome these limitations is to obtain sustained high level expression and secretion of the missing normal enzyme from a small group of cells for export to neighboring diseased cells, which might allow the therapeutic protein to reach distal sites. We tested this in a mouse model of mucopolysaccharidosis VII (Sly disease) using an adeno-associated virus vector. After a single treatment the vector continuously produced the normal enzyme from infected cells at the injection sites. The secreted enzyme was disseminated along most of the neuraxis, resulting in widespread reversal of the hallmark pathology. An extensive sphere of correction surrounding the transduction sites was created, suggesting that a limited number of appropriately spaced sites of gene transfer may provide overlapping spheres of enzyme diffusion to cover a large volume of brain tissue.

Animals↗
PubMed1999

Improved management of infrainguinal bypass graft infection with methicillin-resistant Staphylococcus aureus.

BACKGROUND: There is considerable debate over the management of infected infrainguinal grafts. This report describes recent experience in this field and documents the change in clinical practice needed to deal with methicillin-resistant Staphylococcus aureus (MRSA). METHODS: All infected infrainguinal grafts between January 1991 and July 1997 were reviewed. In the light of the findings, clinical practice was modified considerably. A further 1 year was audited prospectively up to August 1998. RESULTS: Twenty-six patients were treated for 27 infrainguinal graft infections (25 prosthetic, two vein). Twenty were treated by complete graft excision as the initial therapy; graft preservation was attempted in six patients. Before 1995, the infecting organisms were predominantly Pseudomonas aeruginosa or methicillin-sensitive staphylococci. Subsequently all 14 patients treated up to 1997 had infection with MRSA. The overall amputation rate was 17 of 26; ten amputations were in patients with MRSA. Four patients died, all with MRSA sepsis. As a result of this experience a policy of complete isolation was adopted for all patients infected with MRSA. In the 12 months since this policy was introduced, 77 infrainguinal grafts (61 vein, 16 prosthetic) have been inserted. Two grafts (3 per cent) have become infected, necessitating graft excision and amputation. CONCLUSION: MRSA infection of an infrainguinal graft is a serious complication with high associated amputation and mortality rates. Isolation and barrier nursing appeared to contain the problem.

Adult↗
PubMed1999

Repair of ruptured thoracoabdominal aortic aneurysm is worthwhile in selected cases.

INTRODUCTION: The risks and benefits of operating on patients with ruptured thoracoabdominal aortic aneurysm (TAAA) have not been defined. The aim of the present study is to report this unit's experience with operations performed for ruptured TAAA over a 10-year period. METHODS: Interrogation of a prospectively gathered computerised database. PATIENTS: Between 1 January 1983 and 30 June 1996, 188 consecutive patients with TAAA were operated on, of whom 23 (12%) were operated for rupture. RESULTS: There were nine survivors (40%). Patients whose preoperative systolic blood pressure remained above 100 mmHg were significantly more likely to survive (4/8 vs. 13/15, p = 0.03 by Fisher's exact test). Survival was also related to Crawford type: type I (two of three survived); II (none of six); III (two of six); and IV (five of eight). All non-type II, non-shocked patients survived operation. Survivors spent a median of 28 (range 10-66) postoperative days in hospital, of which a median of 6 (range 2-24) days were spent in the intensive care unit. Survivor morbidity comprised prolonged ventilation (> 5 days) (n = 3); tracheostomy (n = 1); and temporary haemofiltration (n = 2). No survivor developed paraplegia or required permanent dialysis. CONCLUSIONS: Patients in shock with a Crawford type II aneurysm have such a poor prognosis that intervention has to be questioned except in the most favourable of circumstances. However, patients with types I, III and IV who are not shocked on presentation can be salvaged and, where possible, should be transferred to a unit where appropriate expertise and facilities are available.

Aged↗
PubMed1999

European CME.

Explore the source record for details and available documents.

Accreditation↗
PubMed1999

Accumulation of intracellular amyloid-beta peptide (A beta 1-40) in mucopolysaccharidosis brains.

To evaluate whether in vivo accumulations of heparan sulfate caused by inborn errors in the metabolism of glycosaminoglycans lead to the formation of neurofibrillary tangles and/or senile plaques, as seen in Alzheimer disease (AD), we studied postmortem brains from 9 patients, ages 1 to 42 years, with mucopolysaccharidosis (MPS). The brains of patients with Hurler's syndrome (MPS I: n = 5) and Sanfilippo's syndrome (MPS III; n = 4) as well as from caprine MPS IIID and murine MPS VII models were evaluated by thioflavine-S staining and by immunohistochemistry using antibodies directed against heparan sulfate proteoglycans, hyperphosphorylated tau, amyloid-beta peptide precursor proteins (APP), and amyloid-beta peptides (A beta [1-40], and A beta [1-42]). A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was also utilized to compare levels of total soluble and insoluble A beta (1-40) and A beta (1-42) obtained from temporal cortex of MPS patients. Although no neurofibrillary tangles, senile plaques, or tau-positive lesions were detected in any of the MPS brains studied here, antibodies directed against A beta (1-40) intensely and diffusely stained the cytoplasm of cells throughout the brains of the MPS patients and the caprine MPS model. The ELISA assay also demonstrated a significant 3-fold increase in the level of soluble A beta (1-40) in the MPS brains compared with normal control brains. Thus, at least some of the metabolic defects that lead to accumulations of glycosaminoglycans in MPS also are associated with an increase in immunoreactive A beta (1-40) within the cytoplasmic compartment where they could contribute to the dysfunction and death of affected cells in these disorders, but not induce the formation of plaques and tangles. Models of MPS may enable mechanistic studies of the role A beta and glycosaminoglycans play in the amyloidosis that is a neuropathological feature of AD.

Adolescent↗
PubMed1999

[Preliminary studies on gene therapy for lysosomal alpha-mannosidosis].

OBJECTIVE: Exploration of the feasibility of treating human lysosomal alpha-mannosidosis by gene therapy. METHODS: Retroviral vector-mediated transfer of human lysosomal alpha-mannosidase cDNA into the diseased cat skin fibroblasts. Detection of the enzymatic activity in the cells and their culture media at different pH conditions. Localization of the recombinant enzyme in the cells by chemical staining. Cross-correction of untransduced cat cells by incubation in medium containing the secreted enzyme in the absence and presence of mannose-6-phosphate or fetal calf serum. In vivo expression of the recombinant enzyme in organoids containing the vector-transduced cells. RESULTS: Among the two human cDNAs tested, only one encoded high levels of alpha-mannosidase activity in the cat cells, which shared the same pH profile and lysosomal localization with the normal enzyme. The recombinant enzyme was proportionally secreted into the medium of the cell culture and taken up by untransduced cells via mannose-6-phosphate receptor-mediated endocytosis. The uptake was partially inhibited in the presence of fetal calf serum, which was dose-dependent. In vivo, organoids containing the vector-transduced cells expressed detectable alpha-mannosidase activity for up to 6 weeks. CONCLUSION: The human cDNA was functional in the cat cells and the expression system could be used for development of gene therapy for lysosomal alpha-mannosidosis.

Animals↗
PubMed1998

Vein graft stenosis and the heparin responsiveness of human vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VMSC) proliferation is an essential component of myointimal hyperplasia, which is implicated in the failure of 30% to 50% of vascular interventions, such as coronary angioplasty and peripheral vein grafting. We have shown that cells derived from stenotic lesions in infrainguinal vein grafts were significantly more resistant than controls to growth inhibition by heparin. METHODS AND RESULTS: In a prospective study, we correlated antiproliferative responses to heparin in vitro with graft patency after 1 year. Sixty-two patients with infrainguinal vein grafts were entered into a graft surveillance program for > or = 1 year. At operation, saphenous vein segments were explanted for VSMC culture. Cell proliferation in response to fetal calf serum was later determined in the presence and absence of heparin. In 35 cell cultures, including 13 from the above-mentioned patients, [3H]heparin binding was also estimated. VSMCs from patients with patent grafts were significantly more sensitive to growth inhibition by heparin than cells from patients with stenoses (median, 54% versus 20.9%, P<0.001), and [3H]heparin binding was strongly correlated with inhibition of proliferation (r=0.81). CONCLUSIONS: Responsiveness to heparin in cultured VSMCs is a strong predictor of outcome for infrainguinal vein grafts, and reduced sensitivity to heparin is correlated with decreased heparin binding. Relative resistance to the antiproliferative action of heparin may be a marker for aberrant regulation of VSMC growth.

Adult↗
PubMed1998

Cloning of the canine beta-glucuronidase cDNA, mutation identification in canine MPS VII, and retroviral vector-mediated correction of MPS VII cells.

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease resulting from deficient activity of the lysosomal acid hydrolase beta-glucuronidase (GUSB) and has been reported in humans, mice, cats, and dogs. To characterize canine MPS VII, we have isolated and sequenced the canine GUSB cDNA from normal and affected animals. A single nucleotide substitution was detected in the GUSB cDNA derived from MPS VII dogs. This guanosine to adenine base change at nucleotide position 559 in the canine cDNA sequence causes an arginine to histidine substitution at amino acid position 166. Introduction of the G to A substitution at position 559 in a mammalian expression vector containing the normal canine GUSB cDNA nearly eliminated the GUSB enzymatic activity, demonstrating that this mutation is the cause of canine MPS VII. A retroviral vector expressing the full-length canine beta-glucuronidase cDNA corrected the deficiency in MPS VII cells.

Amino Acid Sequence↗
PubMed1998

Altered levels of urokinase on monocytes and in serum of children with AIDS; effects on lymphocyte activation and surface marker expression.

Urokinase (UK) type plasminogen activator is a serine protease produced by activated human monocytes. Despite the well-documented roles played by UK in cell-mediated immunity in healthy humans, the roles played by UK in the derangements of cell-mediated immune responses observed in HIV disease remain largely undefined. In these studies the numbers of peripheral blood lymphocytes and monocytes bearing surface UK (UK+) as well as serum levels of UK (flow microfluorimetry and ELISA, respectively) were determined in children with AIDS and in healthy HIV-negative children. The effects of exogenous UK on lymphocyte activation (cell cycle analysis using living cells) and surface marker (CD3, CD4, CD8, and CD19) expression (flow microfluorimetry using fixed cells) were also studied. Data are expressed as percent total cells. Numbers of UK+ lymphocytes in children with AIDS were similar to those observed in healthy children. In contrast, numbers of UK+ peripheral blood monocytes were dramatically decreased (> 70%) in the children with AIDS. However, serum levels of UK were increased (nearly threefold) in these children. When lymphocytes from these children were cultured with soluble UK, numbers of cells in S phase of cell cycle appeared suppressed. Incubation of fixed lymphocytes from either a child with AIDS or from a healthy child with exogenous UK appeared to increase numbers of cells expressing CD3. Incubation with UK had no effect on expression of any other surface marker (CD4, CD8, or CD19) using cells from the child with AIDS. In contrast, incubation with UK appeared to decrease (fivefold) numbers of cells expressing CD19 and increase numbers of cells expressing CD4 and CD8 only when fixed lymphocytes from a healthy HIV-negative child were used. The results suggest important roles for UK in regulation of lymphocyte surface markers in general and in CD3- and CD19-dependent lymphocyte activation pathways specifically. Furthermore, these studies add to a widening body of evidence implicating UK dysregulation in the pathogenesis of HIV disease and may point to pharmacological opportunities involving UK to delay or prevent progression of HIV infection into full-blown AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.

Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.

Animals↗
PubMed1998

Retroviral cDNA transfer to the RPE: stable expression and modification of metabolism.

PURPOSE: To determine the versatility of retroviral vector-mediated rat beta-glucuronidase cDNA expression in the normal retinal pigment epithelium (RPE) of eyes of various species and in RPE of eyes with three types of mucopolysaccharidosis (MPS types I, VI, and VII) and to evaluate the effect of multiple transductions and long-term stable expression in the RPE. METHODS: A retroviral construct containing a rat beta-glucuronidase cDNA (NTK-BGEO) was used to infect RPE cells at subconfluence. The transduced cells were selected in G418, an antibiotic toxic to normal mammalian cells. Beta-glucuronidase activity was measured in transduced cells and media, using a fluorogenic substrate. Glycosaminoglycan profiles were examined by metabolically labeling RPE with Na2(35)SO4. RESULTS: Transduced RPE cells, regardless of species or disease status, expressed rat beta-glucuronidase. The expressed enzyme restored normal levels of glycosaminoglycans in the RPE cells of homozygous MPS VII-affected dogs by metabolizing stored glycosaminoglycans. The expressed enzyme failed to metabolize stored glycosaminoglycans of MPS I and MPS VI, indicating that overexpression could not bypass the exoglycosidase restriction. Multiple transductions increased beta-glucuronidase activity several times in the cell layer and in the media. The expression was stable in vitro for at least 12 weeks. CONCLUSIONS: A retroviral vector can mediate transfer of beta-glucuronidase in various species of normal and MPS-affected RPE. The expression is stable in vitro. The metabolism of stored glycosaminoglycans in MPS needs replacement of only the deficient enzyme to reverse the storage.

Animals↗