Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme α-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC-GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7-8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone.

Enzyme replacement therapy is currently the only approved treatment for Pompe disease, due to acid α-glucosidase deficiency. Clinical efficacy of this approach is variable, and more effective therapies are needed. We showed in preclinical studies that chaperones stabilize the recombinant enzyme used for enzyme replacement therapy. Here, we evaluated the effects of a combination of enzyme therapy and a chaperone on α-glucosidase activity in Pompe disease patients. α-Glucosidase activity was analyzed by tandem-mass spectrometry in dried blood spots from patients treated with enzyme replacement therapy, either alone or in combination with the chaperone N-butyldeoxynojirimycin given at the time of the enzyme infusion. Thirteen patients with different presentations (3 infantile-onset, 10 late-onset) were enrolled. In 11 patients, the combination treatment resulted in α-glucosidase activities greater than 1.85-fold the activities with enzyme replacement therapy alone. In the whole patient population, α-glucosidase activity was significantly increased at 12 hours (2.19-fold, P = 0.002), 24 hours (6.07-fold, P = 0.001), and 36 hours (3.95-fold, P = 0.003). The areas under the curve were also significantly increased (6.78-fold, P = 0.002). These results suggest improved stability of recombinant α-glucosidase in blood in the presence of the chaperone.

No abstract

Stroke is a rare disease in childhood with an estimated incidence of 1-6/100.000. It has an increasingly recognised impact on child mortality along with its outcomes and effects on quality of life of patients and their families. Clinical presentation and risk factors of paediatric stroke are different to those of adults therefore it can be considered as an indipendent nosological entity. The relative rarity, the age-related peculiarities and the variety of manifested symptoms makes the diagnosis of paediatric stroke extremely difficult and often delayed. History and clinical examination should investigate underlying diseases or predisposing factors and should take into account the potential territoriality of neurological deficits and the spectrum of differential diagnosis of acute neurological accidents in childhood. Neuroimaging (in particular diffusion weighted magnetic resonance) is the keystone for diagnosis of paediatric stroke and other investigations might be considered according to the clinical condition. Despite substantial advances in paediatric stroke research and clinical care, many unanswered questions remain concerning both its acute treatment and its secondary prevention and rehabilitation so that treatment recommendations are mainly extrapolated from studies on adult population. We have tried to summarize the pathophysiological and clinical characteristics of arterial ischemic stroke in children and the most recent international guidelines and practical directions on how to recognise and manage it in paediatric emergency.

Fabry disease (FD) is an X-linked inherited disease due to alpha-galactosidase A (alpha-Gal A) deficiency and characterized by lysosomal storage of globotriaosylceramide (Gb3) and related neutral glycosphingolipids. Storage of these substrates results in multisystem manifestations, including renal failure, cardiomyopathy, premature myocardial infarctions, stroke, chronic neuronopathic pain, gastrointestinal disturbances, and skin angiokeratoma. Enzyme replacement therapy (ERT) with recombinant human alpha-galactosidase A (rh-alpha-Gal A) is now available for the treatment of FD and in most patients results in clinical improvement or stabilization. However, ERT efficacy may vary in different tissues and its long-term effects remain to be defined. As a strategy to improve the efficacy of ERT, we tested the combination of rh-alpha-Gal A with the chaperone molecule 1-deoxynojirimycin (DGJ) in cultured FD fibroblasts with negligible residual enzyme activity. Compared to the effects of rh-alpha-Gal A alone, co-administration of DGJ and rh-alphaGal A resulted in better correction (4.8 to 16.9-fold) of intracellular alpha-Gal A activity, and increased amounts of the enzyme within the lysosomal compartment. The clearance of lyso-Gb3, one of the substrates stored in FD and a potent inhibitor of alpha-Gal A, was also significantly improved with the co-administration of DGJ and rh-alpha-Gal A. This study provides additional evidence for a synergistic effect between ERT and pharmacological chaperone therapy and supports the idea that the efficacy of combination protocols may be superior to ERT alone.