A 20-Year Longitudinal Study of Plasma Chitotriosidase Activity in Treated Gaucher Disease Type 1 and 3 Patients-A Qualitative and Quantitative Approach.

Chitotriosidase is an enzyme produced and secreted in large amounts by activated macrophages, especially macrophages loaded with phagocytozed glycosphingolipid in Gaucher disease. Macrophages phagocytose decayed blood cells that contain a lot of sphingolipid-rich cell membranes. In Gaucher disease, due to a deficit in beta-glucocerebrosidase activity, the phagocytozed substrate glucocerebroside cannot undergo further catabolism. In such a situation, macrophages secrete chitotriosidase in proportion to the degree of overload. Gaucher disease (GD) is a recessively inherited disorder resulting in storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. It is directly caused by the deficiency of beta-glucocerebrosidase (GBA) activity. Chitotriosidase has been measured systematically each year in the same group of 49 patients with type 1 and 3 GD for over 20 years. Our analysis showed that chitotriosidase is very sensitive biomarker to enzyme replacement therapy (ERT). The response to treatment introduction is of an almost immediate nature, lowering pathologically high chitotriosidase levels by a factor of 2 in a time scale of 8 months, on average. Long term enzyme replacement therapy (ERT) brings chitotriosidase activity close to reference values. Finally, reducing the dose of ERT quickly boosts chitotriosidase activity, but restoring the initial dose of treatment brings chitotriosidase level of activity back onto the decreasing time trajectory.

Alternative approaches to antifungal therapies.

The expansive use of immunosuppressive medications in fields such as transplantational medicine and oncology, the higher frequency of invasive procedures in an ageing population and the HIV/AIDS pandemic have increased the frequency of systemic fungal infections. At the same time, increased resistance of pathogenic fungi to classical antifungal agents has led to sustained research efforts targeting alternative antifungal strategies. In this review, we focus on two promising approaches: cationic peptides and the targeting of fungal virulence factors. Cationic peptides are small, predominantly positively charged protein fragments that exert direct and indirect antifungal activities, one mechanism of action being the permeabilization of the fungal membrane. They include lysozyme, defensins and cathelicidins as well as novel synthetic peptides. Among fungal virulence factors, the targeting of candidal secreted aspartic proteinases seems to be a particularly promising approach.

Correlation of bone marrow response with hematological, biochemical, and visceral responses to enzyme replacement therapy of nonneuronopathic (type 1) Gaucher disease in 30 adult patients.

PURPOSE: This investigation correlates the marrow response with the degree of increase in hemoglobin (Hb) and platelet count (Plt); decrease in concentrations of plasma tartrate-resistant acid phosphatase, serum angiotensin-converting enzyme (ACE), serum ferritin, and plasma chitotriosidase; and decrease in liver and spleen size, measured by ultrasonography. METHODS: Thirty adult patients with nonneuronopathic (type 1) Gaucher disease (23% splenectomized) were followed for mean periods of 12 and 36 months. RESULTS: The series achieved highly significant mean responses in all hematological, biochemical, and visceral parameters over both observation periods; over a mean 36 months, 19 (63%) had a marrow response, 11 (37%) did not. Six of 7 splenectomized patients attained a marrow response. There were no significant differences between the marrow responders and nonresponders in age, sex, spleen status, Gaucher genotype distribution, mean baseline hematological or biochemical values or liver size, or mean follow-up. At a mean follow-up of 12 months, no significant differences yet were evident between marrow responders and nonresponders in seven of eight response parameters, but marrow responders had a significantly greater reduction in plasma chitotriosidase and a trend toward significantly greater reduction in serum ACE. At a mean follow-up of 36 months, marrow responders had a significantly greater mean increase in Plt and mean decrease in serum ferritin concentration and liver size than did marrow nonresponders; the trend toward greater ACE reduction in marrow responders deepened. However, chitotriosidase response now was equivalent in both subgroups. Marrow nonresponders had a significantly greater mean diminution in spleen size than did marrow responders, but the marrow nonresponders had significantly larger mean spleen size at baseline. Marrow nonresponders also had a greater mean increase in Hb than did marrow responders, but the difference was not significant. The two subgroups had equivalent mean reductions of plasma TRAP. CONCLUSIONS: The clinical relevance of qualitative MRI of the bone marrow in monitoring patients receiving ERT for nonneuronopathic Gaucher disease show that marrow nonresponders can derive considerable benefit from ERT across a variety of other response parameters and suggest an influence of spleen size on marrow response to ERT.

[The effect of the treatment of skin wounds in rats with hydrolytic enzymes on the synthetic activity of the epidermocytes]. / Vliianie obrabotki kozhnykh ran u krys gidroliticheskimi fermentami na sinteticheskuiu aktivnost' épidermotsitov.

Epidermis cells synthetic activity was studied by a luminescent microscopy method. Different conditions of skin wound healing in intrascapular region were investigated on 84 rats-males. Daily effect of 0.2% trypsin or 0.1% ronidase solution on the healing surface for 20 min has been shown to increase basal layer cells synthetic activity of forming epidermis in comparison with control. However, maximum raise of epidermis cells functional activity after ronidase influence was observed already on the 4th day, while after trypsin action--only on the 7th. Epithelial cells synthetic activity decreases several days before total skin defect epithelialization. In all experiments their activity stabilizes not less than one month after the operation. We have come to a conclusion that the mechanism of healing acceleration due to proteolytic enzymes depends on basal layer cells synthetic activity of forming epithelium.

Beta-hexosaminidase isozymes and replacement therapy in Gm2 gangliosidosis.

The problem of cell targeting of lysosomal enzymes is a critical one in the development of strategies for therapeutic enzyme replacement in lysosomal storage diseases. In principle, posttranscriptional isozymes with different carbohydrate-chain composition may be helpful in exploiting existing glycosyl-specific receptors on target cells, if the receptor specificities are known and match the glycosyl composition of available isozymes. In practice, however, the choice is limited to isozymes that can be obtained from tissues available in abundance, such as placenta or blood plasma. Our early experiments show that one can interfere with the interaction between hepatic (RES) receptor and enzyme glycosyl chain, to obtain extrahepatic targeting of beta-hexosaminidase, with catabolic effects. This approach, of course, does not have an immediate therapeutic application, as it involves injection of large amounts of foreign material in order to inhibit hepatic uptake. Modification of the glycosyl chain may be the method of choice in selected instances [Furbish et al. 1981], but is applicability again depends on the knowledge of receptor specificity on target cells and on composition of the glycosyl chain of the enzyme in question. Our recent experiments are a first step toward obtaining enzyme forms that can be endocytosed efficiently by mechanisms that are independent of glycosyl-specific receptors. Charge-mediated, absorptive endocytosis can be obtained by covalent coupling of cationic PLL to beta-hexosaminidase. Given the abundance of negative surface charges on most cell types [Weiss, 1969], this approach may be applicable to different target cells and organs, and possibly also to lysosomal enzymes other than beta-hexosaminidase. The existence of glycosyl recognition signals on beta-hexosaminidase can be obviated by simple chemical manipulations, such as Na-metaperiodate oxidation, which effectively prevents hepatic RES uptake [Rattazzi et al, 1982]. In combination with PLL conjugation, this may ultimately result in an enzyme form that escapes the undesired, preferential RES uptake and is efficiently endocytosed by most cells. It will remain to be seen if this artificially created isozyme (for which we propose the name "ersatzyme") is catabolically effective. This can easily be verified in our animal model, along the lines followed to demonstrate the catabolic effects of native Hex A. Finally, the recent developments in molecular genetics, which allows production of human proteins in bacterial systems by recombinant DNA techniques, make it very likely that abundant beta-hexosaminidase may be similarly obtained for therapeutic applications.(ABSTRACT TRUNCATED AT 400 WORDS)

Towards enzyme replacement in GM2 gangliosidosis: organ disposition and induced central nervous system uptake of human beta-hexosaminidase in the cat.

The rapid plasma clearance of human placental beta-hexosaminidase in the cat is due mainly to a receptor-mediated mechanism recognizing terminal N-acetyl glucosaminyl and mannosyl residues on glycoproteins. Using a sensitive single radial immunodiffusion assay, specific for human beta-hexosaminidase, we have shown that, in normal cats, the liver is responsible for most of the clearance of human beta-hexosaminidase. Two hours after injection of approximately 6 X 10(6) U beta-hexosaminidase/kg bw, 70-90% of the enzyme was recovered in the liver. Spleen, kidney, lung, bone, bone, pancreas, adrenals, testes and ovaries, cardiac and skeletal muscle, lymph nodes, and placenta, however, also participated in the clearance, although specific uptake in most organs was < 5% of that of liver. Exogenous beta-hexosaminidase was also present in bile, indicating that the hepatocytes are involved in clearance. Injection of terminal mannose-rich S. cerevisiae mannans (50-150 mg/kg bw), prolonged the plasma half-life of the enzyme (t 1/2 up to 290 min). In these animals, beta-hexosaminidase uptake by liver was reduced to < 10% of controls but uptake by other organs was not proportionally or uniformly reduced, suggesting the existence of different uptake mechanisms in different tissues. Permeability of the blood-brain barrier was induced by exposing cats to 100% O2 at 2.5 ATA for 90 min. Injection of 6 X 10(6) U beta-hexosaminidase/kg bw during or immediately after exposure resulted in apparent uptake of enzyme by nervous tissue, qualitatively detectable by immunologic methods, but below the limits of sensitivity of the radial immunoassay(ie < 150 U/gr). When enzyme uptake by liver was inhibited by injection of ovomucoid or mannans, however, the hyperbaric oxygen-induced apparent uptake of beta-hexosaminidase by brain, cerebellum, and spinal cord was 200-500 U/gr of blood-free tissue, suggesting that the transport mechanism involved (presumably at the level of the nervous system vascular endothelium) is different from the carbohydrate-dependent hepatic uptake. The mechanism by which hyperbaric oxygenation induces permeability of the blood-brain barrier is not clear. The combination of this procedure (routinely used in human therapy) with specific inhibition of hepatic uptake, however, appears to be a promising approach for lysosomal enzyme targeting to the central nervous system.

Enzyme replacement in Tay-Sachs disease.

Enzyme replacement therapy was attempted with two Tay-Sachs-diseased individuals--a 14-month-old child and a 7-week-old infant. Treatment consisted of repeated weekly intrathecal injections of pure hexosaminidase A. Injection of this enzyme resulted in almost complete disappearance of GM2 from the serum, but did not bring about dissolution of the GM2 membranous cytoplasmic bodies in the brain, as detected by electronmicroscopy. Both patients tolerated the treatment without apparent clinical complications, but no clear-cut improvement was noted as a result of prolonged injections of hexosaminidase A. Since this treatment was initiated in both an advanced stage and a very early stage of the disease, we conclude that enzyme replacement treatment by this route is not beneficial for patients with Tay-Sachs disease.

Investigations in enzyme replacement therapy in lipid storage diseases.

Enzyme replacement appears to offer much promise as specific therapeutic procedures for patients with Fabry's disease and Gaucher's disease. However, enzyme replacement in patients with Tay-Sachs disease and other heritable metabolic disorders where the central nervous system is affected will require first the development of effective methods for the delivery of exogenous enzymes to the brain; such methods are not yet available.

The lipid storage diseases: new concepts and control.

The nature of the enzymatic defect is now well established in ten inherited disorders of lipid metabolism. This information has provided for the development of facile, sensitive tests using readily available materials such as washed leukocytes or cultured skin fibroblasts for the diagnosis of these disorders; the detection of heterozygous carriers of these traits; and the monitoring of pregnancies at risk for any of these conditions. Recent investigations of enzyme replacement therapy have shown great promise for the treatment of patients with Fabry's disease and those with the adult form of Gaucher's disease. Additional procedures must be developed for successful enzyme replacement in patients where the central nervous system is damaged by the accumulation of lipids.

Sanfilippo disease type B: enzyme replacement and metabolic correction in cultured fibroblasts.

alpha-Acetylglucosaminidase, purified from human placent, corrected the defect in mucopolysaccharide degradation when added to culture fibroblasts from patients with Sanfilippo disease type B. A small cellular concentration of enzyme gave a large corrective effect. The half-life of disappearance of enzyme activity was 4 to 7 days.