Enzyme-functionalized vascular grafts catalyze in-situ release of nitric oxide from exogenous NO prodrug.

Nitric oxide (NO) is an important signaling molecule in cardiovascular system, and the sustained release of NO by endothelial cells plays a vital role in maintaining patency and homeostasis. In contrast, lack of endogenous NO in artificial blood vessel is believed to be the main cause of thrombus formation. In this study, enzyme prodrug therapy (EPT) technique was employed to construct a functional vascular graft by immobilization of galactosidase on the graft surface. The enzyme-functionalized grafts exhibited excellent catalytic property in decomposition of the exogenously administrated NO prodrug. Localized and on-demand release of NO was demonstrated by in vitro release assay and fluorescent probe tracing in an ex vivo model. The immobilized enzyme retained catalytic property even after subcutaneous implantation of the grafts for one month. The functional vascular grafts were implanted into the rat abdominal aorta with a 1-month monitoring period. Results showed effective inhibition of thrombus formation in vivo and enhancement of vascular tissue regeneration and remodeling on the grafts. Thus, we create an enzyme-functionalized vascular graft that can catalyze prodrug to release NO locally and sustainably, indicating that this approach may be useful to develop new cell-free vascular grafts for treatment of vascular diseases.

Combining Schwann cell bridges and olfactory-ensheathing glia grafts with chondroitinase promotes locomotor recovery after complete transection of the spinal cord.

Numerous obstacles to successful regeneration of injured axons in the adult mammalian spinal cord exist. Consequently, a treatment strategy inducing axonal regeneration and significant functional recovery after spinal cord injury has to overcome these obstacles. The current study attempted to address multiple impediments to regeneration by using a combinatory strategy after complete spinal cord transection in adult rats: (1) to reduce inhibitory cues in the glial scar (chondroitinase ABC), (2) to provide a growth-supportive substrate for axonal regeneration [Schwann cells (SCs)], and (3) to enable regenerated axons to exit the bridge to re-enter the spinal cord (olfactory ensheathing glia). The combination of SC bridge, olfactory ensheathing glia, and chondroitinase ABC provided significant benefit compared with grafts only or the untreated group. Significant improvements were observed in the Basso, Beattie, and Bresnahan score and in forelimb/hindlimb coupling. This recovery was accompanied by increased numbers of both myelinated axons in the SC bridge and serotonergic fibers that grew through the bridge and into the caudal spinal cord. Although prominent descending tracts such as the corticospinal and reticulospinal tracts did not successfully regenerate through the bridge, it appeared that other populations of regenerated fibers were the driving force for the observed recovery; there was a significant correlation between numbers of myelinated fibers in the bridge and improved coupling of forelimb and hindlimb as well as open-field locomotion. Our study tests how proven experimental treatments interact in a well-established animal model, thus providing needed direction for the development of future combinatory treatment regimens.

[Fabry disease: diagnostic due of substitutive enzyme-therapy]. / Maladie de Fabry: du diagnostic à l'enzymothérapie substitutive.

Fabry disease is a X-linked sphingolipid storage disorder resulting from the defective activity of the lysosomal enzyme, alpha-galactosidase A. Hemizygotes develop severe multisystemic disease, dominated by renal failure and progressive neurological and cardiac involvement, causing premature death. Thirty percent of heterozygotes have severe involvement of one or several organs. With developments in molecular biology, it is now possible to produce the human recombinant enzyme alpha-galactosidase A. More than 20 patients are now treated in Switzerland.

Advances in the management of Anderson-Fabry disease: enzyme replacement therapy.

Anderson-Fabry disease (AFD) is a lysosomal storage disorder (LSD) due to alpha-galactosidase A (alpha-Gal A) deficiency and the resultant accumulation of incompletely metabolised glycosphingolipids (GSLs), primarily globotriosylceramide (Gb(3)), within various tissues. It is an X-linked multisystem disorder characterised by progressive renal insufficiency, with added morbidity from cardio- and cerebrovascular involvement, and associated with significant impact on quality of life and diminished lifespan. The disease manifests primarily in hemizygous males; however, there is increasing recognition that heterozygous (carrier) females may also develop disease-related complications, although onset among affected women may be delayed. Until recently, treatment has been limited to symptomatic management of pain and other measures to alleviate the problems associated with end-stage complications from renal, cardiac and nervous system involvement. The availability of the recombinant enzyme offers the potential of a safe and effective targeted treatment approach. At the moment, two distinct enzyme formulations are approved in Europe (and in other countries) and both continue to undergo FDA evaluation in the US. Increasing knowledge of the natural history of AFD and greater experience with enzyme therapy should enable optimal patient care. The relative rarity and complexity of AFD necessitates a multi-disciplinary team approach that may be facilitated by a centralised registry.

Lactase and placebo in the management of the irritable bowel syndrome: a double-blind, cross-over study.

A double-blind, cross-over, therapeutic, clinical trial of the efficacy of exogenous, microbial beta-D-galactosidase to reduce the symptoms of the irritable bowel syndrome (IBS) was conducted in 12 patients whose customary diets regularly included milk. Eight of the 12 subjects (67%) proved to be lactase-nonpersistent, lactose-maldigesters when challenged with a aqueous dose of 12.5 g. The study lasted 4 months, with the first month a non-intervention, control period and the latter 3 months alternating in the sequence, treatment/placebo/treatment, or placebo/treatment/placebo. When symptoms during trial months were analyzed by the cumulative sum procedure, gastrointestinal symptoms were found to be independent of lactase treatment. We found a positive temporal association of the severity of both gastrointestinal and non-gastrointestinal symptomatology. In populations with a high prevalence of lactose deficiency, IBS symptoms appear to be independent of lactose maldigestion.

Relative efficiency of yogurt, sweet acidophilus milk, hydrolyzed-lactose milk, and a commercial lactase tablet in alleviating lactose maldigestion.

The relative effectiveness of commercially available plain yogurt (Y), sweet acidophilus milk (SAM), hydrolyzed-lactose milk (HLM), a lactase tablet (LT), and whole milk (WM) was evaluated in 10 lactose-intolerant black subjects. In a 5 x 5 Latin square design, hourly breath hydrogen excretion (BHE) was measured for 5 h after the subjects consumed the above products (18 g lactose in each except HLM, which had 5 g). Mean BHE (ppm) for Y, SAM, LT, HLM, and WM were 12, 37, 29, 18, and 33, respectively. There was a significant (p less than 0.05) positive correlation of 0.808 between the symptoms reported and the mean peak BHE. However, the correlation between the symptoms and diagnosis by history was not significant. Although Y was as effective as HLM in minimizing lactose maldigestion, it was the least accepted by the subjects in sensory evaluations. Results of this study also indicate that microbial endogenous lactase in yogurt is superior to exogenous commercial lactase in alleviating lactose maldigestion.

Enzyme replacement for lactose malabsorption using a beta-D-galactosidase.

We evaluated 10 healthy symptomatic lactose malabsorbers for effect of an oral beta-D-galactosidase derived from Aspergillus oryzae (Lactrase, Kremers Urban Company, Milwaukee, WI, U.S.A.) on symptom and breath hydrogen response to challenge with 50 g lactose. Basally and at 30-min intervals for 8 h after lactose challenge, end-alveolar breath samples were collected and analyzed for hydrogen using gas chromatography. Symptoms were scored at 30 min and hourly for 8 h, rating bloating, cramps, nausea, pain, diarrhea, and flatulence. Four challenges were performed on 4 separate days with at least 3 days between challenges. The first two challenges served as baselines. Just before ingestion of 50 g powdered lactose dissolved in 200 ml water, beta-D-galactosidase capsules were given orally as a 250-mg dose for the third challenge and a 500-mg dose for challenge 4. Hydrogen excretion, quantified by using a trapezoidal method for computing area under the discontinuous curve of breath hydrogen concentration, was decreased in subjects receiving beta-D-galactosidase (base-line I, 346.0 ppm/h; baseline II, 367.2 ppm/h; 250-mg galactosidase 208.2 ppm/h; 500-mg galactosidase, 178.0 ppm/h; p less than or equal to 0.05). Other analyzed parameters of H2 excretion were also decreased. Analysis of symptom response scores showed a dose-related decrease for bloating and flatus (p less than or equal to 0.05) and no statistical difference in the other assessed symptoms. We conclude that beta-D-galactosidase from Aspergillus oryzae, when given just before ingestion of lactose by lactose malabsorbers, can produce a dose-dependent reduction (statistically significant for the 500-mg dose) in breath hydrogen excretion, bloating, and flatus.

Galantase (beta-galactosidase) treatment in pediatric practice.

The authors diagnosed lactose malabsorption by the breath hydrogen analysis in 11 premature and mature babies, in 16 infants and in 28 children between the ages of 3-18 years. All patients were treated with Galantase (beta-galactosidase). According to the results, Galantase is very effective in splitting of lactose of breast-milk, cow-milk and artificial formulas. Pathological hydrogen increase was not detected during the treatment.

Does oral enzyme replacement therapy reverse intestinal lactose malabsorption?

The effects of oral enzyme replacement therapy on breath hydrogen excretion and symptoms after milk ingestion were studied in lactase-deficient patients. Sixteen symptomatic patients underwent interval hydrogen breath tests using whole milk as substrate. Each study was repeated with the addition of 250 mg of beta-D-galactosidase derived from Aspergillus oryzae (Lactrase) given orally with the milk. Subsequently seven of those 11 patients who did not normalize their hydrogen excretion with 250 mg of Lactrase were available to be restudied with a 500-mg dose. Mean cumulative and peak hydrogen excretions were calculated for the baseline (milk alone), 250 mg, and 500 mg Lactrase groups. Significant (p less than or equal to 0.05) decreases in cumulative and peak hydrogen excretion were noted between the 500 mg Lactrase versus the baseline group, but not between the 250 mg versus baseline group. Five of the 16 (31%) symptomatic lactase-deficient patients normalized their hydrogen excretion after 250 mg of Lactrase; four of seven (57%) who had not normalized on 250 mg, normalized their hydrogen excretion with 500 mg of Lactrase. A different pattern was observed in the incidence of symptoms. Five of the nine patients (56%) whose hydrogen excretion normalized with the addition of Lactrase at either dosage became asymptomatic after milk ingestion; in addition, three patients who did not normalize their hydrogen also became asymptomatic. We conclude that oral Lactrase in sufficient dosage temporarily reverses lactose malabsorption in some patients.

Effective reduction of lactose maldigestion in preschool children by direct addition of beta-galactosidases to milk at mealtime.

We examined the efficiency of two beta-galactosidase preparations--one derived from the yeast, Kluyveromyces lactis (Lactaid), the other derived from the fungus, Aspergillus oryzae (Takamine)--to assist the in vivo digestion of lactose consumed by healthy Guatemalan preschool children. Milk prehydrolyzed by in vitro incubation with enzymes was used as the standard of reference, and the degree of incomplete digestion of lactose from 240 mL of milk was determined using the hydrogen breath test. In in vivo dose-response studies, both 3,250 neutral lactose units of Lactaid and 6,635 food and chemical codex lactose units of Takamine completely eliminated excess H2 excretion in a small sample of lactose-maldigesting subjects. When evaluated in a controlled, clinical trial setting, the same dose of Lactaid added directly to the milk at consumption produced an 82% relative reduction in H2 excretion, whereas Takamine was equally as effective as the prehydrolyzed milk. Thus, intraluminal conditions and gastrointestinal transit in the preschool child support the effective assisted digestion of milk lactose in an efficient manner and with the same enzyme to milk ratios as observed previously in adults.

Efficacy testing of beta-galactosidase with H2 breath test in patients with carbohydrate malabsorption.

The clinical efficacy and the potential side-effects of beta-galactosidase were studied in adult lactose intolerance. Various randomized oral tolerance tests were performed using lactose solution (35 g), glucose + galactose solution (17.5 + 17.5 g), native, skimmed milk and milk pretreated with beta-galactosidase. In each case, simultaneous examinations were made of the glucose concentration of capillary blood by an instrument constructed by the authors, of the H2 content of expired air as also of the subjective complaints and of the number of stools and their pH. It was established that pretreatment of milk with beta-galactosidase has a beneficial effect in adult lactose maldigestion, since it stops dyspeptic complaints and diarrhoea due to milk, it reduces the H2 content of expired air increases blood glucose concentration. Measuring the H2 content of the breath by using and instrument constructed by the authors, exact data can be obtained noninvasively, and rapidly on the degree of carbohydrate malabsorption in patients with lactose-intolerance.

Effectiveness of milk products in dietary management of lactose malabsorption.

Eleven lactose malabsorbers were studied to compare the effectiveness of commercially available products recommended for dietary treatment of lactose malabsorption. One product, a commercial lactase preparation, is added to milk for lactose hydrolysis before consumption. The other is a commercial milk product containing lactose-hydrolyzing, nonpathogenic bacteria, Lactobacillus acidophilus. Both of these products are presently recommended for management of lactose malabsorption, although such recommendations have not been validated by controlled studies. Lactose malabsorption was determined by breath H2 analyses after subjects drank four different test doses on 4 different days. The first test dose was 480 ml of low fat milk; the second was 480 ml of milk treated with a commercial lactase preparation; the third was 480 ml of a commercial L. acidophilus-containing milk; and the fourth was 480 ml of the L. acidophilus-containing milk after 1 wk of gastrointestinal exposure to this commercial bacteria-containing milk. The mean breath H2 response to the lactase-treated milk was significantly lower (p less than 0.001) than the mean response to regular milk. However, the mean breath H2 response to either of the test doses of the L. acidophilus-containing milk were not significantly different than responses to regular milk. It is concluded that the lactase-treated milk reduces breath H2 responses and symptomatic discomfort from malabsorption while the L. acidophilus-containing milk does not.

Enzyme therapy XVII: metabolic and immunologic evaluation of alpha- galactosidase A replacement in Fabry disease.

A pilot trial of enzyme replacement using splenic and plasma forms of alpha-galactosidase A was undertaken in 2 brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Partially purified preparations of alpha-galactosidase A from human spleen and plasma Cohn fraction IV-1 were prepared aseptically for in vivo administration. The disappearance of enzymatic activity from plasma, levels of circulating substrate, and potential immune response were evaluated following IV administration of 6 unentrapped doses (2,000 U/kg) of each enzyme form to the respective recipient during a 117-day period. Repeated injections were well tolerated. The circulating half-life of the splenic form was about 10 min whereas that for the plasma form was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either enzyme following successive administrations. After each dose of the splenic form, the concentration of the accumulated circulating substrate globotriaosylceramide, decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma form decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations of globotriaosylceramide gradually returned to preinfusion values by 36-72 hr. Two consecutive doses of the plasma form, administered on days 1 and 3, reduced the circulating substrate concentration to normal levels. Prior to the 6th enzyme administration, circulating substrate was stable-isotope labeled by the infusion of dideutero-glucose, and the effects of each enzyme form on circulating substrate degradation and reaccumulation were determined. The results of this study indicated that labeled (newly synthesized) substrate reaccumulated following injection of the splenic enzyme whereas both unlabeled (previously stored?) and labeled substrate reaccumulated in the circulation after administration of the plasma form. These studies demonstrated the differential disappearance kinetics of the splenic and plasma forms of alpha-galactosidase A, their differential effects on circulating substrate degradation and reaccumulation, as well as the lack of an immune response to repeated administrations of these homologous, unentrapped enzymes.

Enzyme therapy in Fabry disease: differential in vivo plasma clearance and metabolic effectiveness of plasma and splenic alpha-galactosidase A isozymes.

A pilot trial of enzyme replacement with splenic and plasma alpha-galactosidase A (alpha-D-galactosidase; alpha-D-galactoside galactohydrolase, EC 3.2.1.22) isozymes was undertaken in two brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Six unentrapped doses (2000 units/kg) of each isozyme were administered intravenously to the respective recipients during a 117-day period. The circulating half-life of the splenic isozyme was about 10 min, whereas that for the plasma isozyme was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either isozyme after successive administrations. After each dose of the splenic isozyme, the concentration of the accumulated circulating substrate, trihexosylceramide (globotriaosylceramide), decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma isozyme decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations gradually returned to preinfusion values by 36-72 hr.

Enzyme replacement therapy in Gaucher's and Fabry's disease.

Glucocerebrosidase and ceramidetrihexoside-alpha-galactosidase were obtained in a high degree of purity from human placental tissue. The enzymes were infused in Gaucher and Fabry patients, respectively. Following the administration of the proteins to supplement the genetically determined deficiencies, there resulted a specific reduction in the accumulated hlycolipids in the circulation and liver. These results indicate that enzyme replacement may provide hope for the clinical treatment of these disorders.