Biomedical subjects

Beth L Thurberg

Publications and source records attributed to Beth L Thurberg.

10 recordsLinked to original sources

PubMed2006

Characterization of pre- and post-treatment pathology after enzyme replacement therapy for Pompe disease.

In Pompe disease, a genetic deficiency of lysosomal acid alpha-glucosidase, glycogen accumulates abnormally in the lysosomes of skeletal, cardiac and smooth muscle, and contributes to clinically progressive and debilitating muscle weakness. The present study involved 8 infantile-onset Pompe patients, treated weekly with 10 mg/kg of recombinant human acid alpha-glucosidase (rhGAA). Muscle biopsies were obtained at baseline, 12 and 52 weeks post-treatment to establish an indicator of efficacy. Several histologic strategies were employed to characterize changes in pre- and post-treatment samples, including high-resolution light microscopy and digital histomorphometry, electron microscopy, capillary density and fiber type analysis, and confocal microscopy for satellite cell activation analysis. Histomorphometric analysis was performed on muscle samples to assess glycogen depletion in response to enzyme replacement therapy (ERT). The extent of glycogen clearance varied widely among these patient samples, and correlated well with clinical outcome. Low glycogen levels, mild ultrastructural damage, a high proportion of type I fibers, and young age at baseline were all features associated with good histologic response. There was no correlation between capillary density and glycogen clearance, and activated satellite cell levels were shown to be higher in post-treatment biopsies with poor histologic responses. This histopathologic study of infantile Pompe disease provides detailed insight into the cellular progression of the disease and its response to therapy while highlighting a number of methodologies which may be employed to assess regression or progression of the associated pathology. As enzyme replacement therapy becomes more prevalent for the treatment of lysosomal storage diseases, such evaluation of post-treatment pathology will likely become a more common occurrence in the daily practice of pathologists.

Capillaries↗

PubMed2006

Chinese hamster ovary cell-derived recombinant human acid alpha-glucosidase in infantile-onset Pompe disease.

OBJECTIVE: To conduct an open-label, multinational, multicenter study examining the safety and efficacy of recombinant human acid alpha-glucosidase (rhGAA) in treatment of infantile-onset Pompe disease. STUDY DESIGN: We enrolled 8 infant patients who had Pompe disease with GAA activity <1% of normal, cardiomyopathy, and hypotonia. In the 52-week initial phase, rhGAA was infused intravenously at 10 mg/kg weekly; an extension phase continued survivors' treatment with 10 to 20 mg/kg of rhGAA weekly or 20 mg/kg every 2 weeks for as long as 153 weeks. Safety measurements included adverse events, laboratory tests, and anti-rhGAA antibody titers. Efficacy evaluations included survival, ventilator use, echocardiograms, growth, and motor and cognitive function. RESULT: After 52 weeks of treatment, 6 of 8 patients were alive, and 5 patients were free of invasive ventilator support. Clinical improvements included ameliorated cardiomyopathy and improved growth and cognition. Five patients acquired new motor milestones; 3 patients walked independently. Four patients died after the initial study phase; the median age at death or treatment withdrawal for all patients was 21.7 months, significantly later than expected for patients who were not treated. Treatment was safe and well tolerated; no death was drug-related. CONCLUSION: rhGAA improved ventilator-free survival, cardiomyopathy, growth, and motor function in patients with infantile-onset Pompe disease compared with outcomes expected for patients without treatment.

Body Height↗

PubMed2005

Carbohydrate-remodelled acid alpha-glucosidase with higher affinity for the cation-independent mannose 6-phosphate receptor demonstrates improved delivery to muscles of Pompe mice.

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid alpha-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.

Aging↗

PubMed2005

Mice with elevated muscle glycogen stores do not have improved exercise performance.

Skeletal muscle glycogen is considered to be an important source of energy for contraction and increasing the level of the glucose polymer is generally thought to improve exercise performance in humans. A genetically modified mouse model (GSL30), which overaccumulates glycogen due to overexpression of a hyperactive form of glycogen synthase, was used to examine whether increasing the level of the polysaccharide enhances the ability of mice to run on a treadmill. The skeletal muscle of the GSL30 mice had large deposits of glycogen. There were no significant increases in the work performed by GSL30 mice as compared to their respective wild type littermates when exercised to exhaustion. The amount of muscle glycogen utilized by GSL30 mice, however, was greater, while the amount of liver glycogen consumed during exhaustive exercise was less than wild type animals. This result suggests that increased muscle glycogen stores do not necessarily improve exercise performance in mice.

Animals↗

PubMed2005

Exercise capacity of mice genetically lacking muscle glycogen synthase: in mice, muscle glycogen is not essential for exercise.

The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.

Animals↗

PubMed2005

Replacing acid alpha-glucosidase in Pompe disease: recombinant and transgenic enzymes are equipotent, but neither completely clears glycogen from type II muscle fibers.

Pompe disease (type II glycogen storage disease) is an autosomal recessive disorder caused by a deficiency of lysosomal acid alpha-glucosidase (GAA) leading to the accumulation of glycogen in the lysosomes primarily in cardiac and skeletal muscle. The recombinant human GAA (rhGAA) is currently in clinical trials for enzyme replacement therapy of Pompe disease. Both clinical data and the results of preclinical studies in our knockout model of this disease show that rhGAA is much more effective in resolving the cardiomyopathy than the skeletal muscle myopathy. By contrast, another form of human GAA--transgenic enzyme constitutively produced in liver and secreted into the bloodstream of knockout mice (Gaa-/-)--completely prevented both cardiac and skeletal muscle glycogen accumulation. In the experiments reported here, the transgenic enzyme was much less efficient when delivered to skeletal muscle after significant amounts of glycogen had already accumulated. Furthermore, the transgenic enzyme and the rhGAA have similar therapeutic effects, and both efficiently clear glycogen from cardiac muscle and type I muscle fibers, but not type II fibers. Low abundance of proteins involved in endocytosis and trafficking of lysosomal enzymes combined with increased autophagy in type II fibers may explain the resistance to therapy.

Animals↗

PubMed2005

High-resolution light microscopy (HRLM) and digital analysis of Pompe disease pathology.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase, responsible for the degradation of lysosomal glycogen. Absent or low levels of the enzyme leads to lysosomal glycogen accumulation in cardiac and skeletal muscle cells, resulting in progressive muscle weakness and death from cardiac or respiratory failure. Recombinant enzyme replacement and gene therapy are now being investigated as treatment modalities for this disease. A knockout mouse model for Pompe disease, induced by the disruption of exon 6 within the acid alpha-glucosidase gene, mimics the human disease and has been used to evaluate the efficacy of treatment modalities for clearing glycogen. However, for accurate histopathological assessment of glycogen clearance, maximal preservation of in situ lysosomal glycogen is essential. To improve retention of glycogen in Pompe tissues, several fixation and embedding regimens were evaluated. The best glycogen preservation was obtained when tissues fixed with 3% glutaraldehyde and postfixed with 1% osmium tetroxide were processed into epon-araldite. Preservation was confirmed by staining with the Periodic acid-Schiff's reaction and by electron microscopy. This methodology resulted in high-resolution light microscopy (HRLM) sections suitable for digital quantification of glycogen content in heart and skeletal muscle. Combining this method of tissue fixation with computer-assisted histomorphometry has provided us with what we believe is the most objective and reproducible means of evaluating histological glycogen load in Pompe disease.

Animals↗

PubMed2004

Conjugation of mannose 6-phosphate-containing oligosaccharides to acid alpha-glucosidase improves the clearance of glycogen in pompe mice.

Clinical studies of enzyme replacement therapy for Pompe disease have indicated that relatively high doses of recombinant human acid alpha-glucosidase (rhGAA) may be required to reduce the abnormal glycogen storage in cardiac and skeletal muscles. This may be because of inefficient cation-independent mannose 6-phosphate receptor (CI-MPR)-mediated endocytosis of the enzyme by the affected target cells. To address this possibility, we examined whether the addition of a high affinity ligand to rhGAA would improve its delivery to these cells. Chemical conjugation of high mannose oligosaccharides harboring mono- and bisphosphorylated mannose 6-phosphates onto rhGAA (neo-rhGAA) significantly improved its uptake characteristics by muscle cells in vitro. Infusion of neo-rhGAA into Pompe mice also resulted in greater delivery of the enzyme to muscle tissues when compared with the unmodified enzyme. Importantly, this increase in enzyme levels was associated with significantly improved clearance of glycogen ( approximately 5-fold) from the affected tissues. These results suggest that CI-MPR-mediated endocytosis of rhGAA is an important pathway by which the enzyme is delivered to the affected lysosomes of Pompe muscle cells. Hence, the generation of rhGAA containing high affinity ligands for the CI-MPR represents a strategy by which the potency of rhGAA and therefore the clinical efficacy of enzyme replacement therapy for Pompe disease may be improved.

Animals↗

PubMed2004

Monitoring the 3-year efficacy of enzyme replacement therapy in fabry disease by repeated skin biopsies.

The earliest clinical signs of Fabry disease often manifest as dermatologic disturbances such as angiokeratomata, hypohidrosis, acroparesthesias, and impaired thermal and vibration detection. These disturbances are caused by cellular globotriaosylceramide accumulation in the skin due to deficient lysosomal alpha-galactosidase A activity. In this histologic study, we analyzed pre- and post-treatment dermatologic biopsies from 58 Fabry patients enrolled in a 5 mo, Phase 3 double-blind, randomized, placebo-controlled trial followed by a 30 mo open label extension study of recombinant human alpha-galactosidase A (r-halphaGalA), administered i.v. at 1 mg per kg every 2 wk. Baseline evaluations revealed globotriaosylceramide in multiple dermal cell types (vascular endothelial cells, vascular smooth muscle cells, perineurium). Five months of r-halphaGalA treatment in the Phase 3 trial resulted in complete clearance of globotriaosylceramide from the superficial capillary endothelium in all treatment patients and in only 1 (3%) placebo patient (p<0.001). The placebo group achieved similar results after 6 mo of r-halphaGalA in the open label trial. The capillary endothelium remained free of globotriaosylceramide for up to 30 mo into the extension study among 39 of 40 (98%) patients who underwent biopsies. Globotriaosylceramide clearance from deep vascular endothelial cells was similarly robust. Vascular smooth muscle cells and perineurium demonstrated moderate clearance. These findings suggest that long-term treatment with r-halphaGalA may halt the progression of pathology and prevent the dermatologic disturbances in Fabry patients, and that periodic dermal biopsies can serve as a reliable monitor of sustained efficacy.

Adult↗

PubMed2002

Globotriaosylceramide accumulation in the Fabry kidney is cleared from multiple cell types after enzyme replacement therapy.

BACKGROUND: Fabry disease, a lysosomal storage disease caused by deficient lysosomal alpha-galactosidase A activity, is characterized by globotriaosylceramide (GL-3) accumulation in multiple cell types, particularly the vasculature, leading to end organ failure. Accumulation in the kidney is responsible for progressive decline in renal function in male patients with the classical phenotype, resulting in renal failure in their third to fifth decades of life. With the advent of recombinant protein synthesis technology, enzyme replacement therapy has become a viable alternative to dialysis or renal transplantation, previously the only available treatment options for end-stage renal disease. METHODS: The pre- and post-treatment renal biopsies were analyzed from fifty-eight Fabry patients enrolled in a Phase 3 double-blind, randomized, placebo-controlled trial followed by a six-month open label extension study of the recombinant human enzyme, alpha-galactosidase A (r-halphaGalA), administered IV at 1 mg/kg biweekly. The purpose of this investigation was to detail the pathologic changes in glycosphingolipid distribution and the pattern of post-treatment clearance in the kidney. RESULTS: Baseline evaluations revealed GL-3 accumulations in nearly all renal cell types including vascular endothelial cells, vascular smooth muscle cells, mesangial cells and interstitial cells, with particularly dense accumulations in podocytes and distal tubular epithelial cells. After 11 months of r-halphaGalA treatment there was complete clearance of glycolipid from the endothelium of all vasculature as well as from the mesangial cells of the glomerulus and interstitial cells of the cortex. Moderate clearance was noted from the smooth muscle cells of arterioles and small arteries. Podocytes and distal tubular epithelium also demonstrated evidence for decreased GL-3, although this clearance was more limited than that observed in other cell types. No evidence of immune complex disease was found by immunofluorescence despite circulating anti-r-halphaGalA IgG antibodies. CONCLUSIONS: These findings indicate a striking reversal of renal glycosphingolipid accumulation in the vasculature and in other renal cell types, and suggest that long-term treatment with r-halphaGalA may halt the progression of pathology and prevent renal failure in patients with Fabry disease.

Adolescent↗