Gaucher's disease

Gaucher disease is an uncommon autosomic recessive disorder. The disease is caused by a deficiency in the activity of the lysosomal enzyme glucocerebrosidase which is responsible for the degradation of glucosylceramide, résulting from the breakdown of red and white cell-membranes. In the absence of enzyme glucosylceramide accumulates in the lysosomes of macrophages. This accumulation leads to hepatomegaly, splenomegaly with subsequent haematologic abnormalities (leucopenia, anemia, thrombopenia) and bone manifestations. Three types of Gaucher disease are described: type 1 is the most common, type 2 and 3 are associated with neurologic symptoms. Macrophages are the likely cellular source of biochemical abnormalities: elevated blood level of ferritin, angiotensin converting enzyme, immunoglobulins and haemostasis abnormalities. Lysosomal perturbations lead to increased blood level of tartrate resistant acid phosphatase and chitotriosidase. Enzyme replacement therapy is available in France since 1991. In 2002, 136 patients are treated. The efficacy is overt on the asthenia, organomegaly and haematological manifestations. Bone pains disappear or decrease in intensity, however bone complications may be irreversible justifying treatment initiations before the appearance of lesions that may lead to serious functional impairment.     

Splenic lipids in Gaucher's disease

Column chromatography (on cellulose, silicic acid, and Florisil) and thin-layer chromatography were employed for the separation and purification of lipid fractions from normal and Gaucher spleens. A new hydrolysis procedure, followed by paper chromatography, was used for identification of sugar moieties. A nonhydrolytic combined colorimetric procedure, with anthrone and orcinol, was used for the estimation of glucose and galactose separately in glycolipids. The limitations of this method were examined. Spleens from two control subjects and three patients with Gaucher's disease have been examined in detail. In all Gaucher spleens, the predominant feature was the massive acccumulation of glucocerebroside; neutral ceramide oligohexoside levels were probably within the normal range, as were other neutral lipids and phospholipids. In one case examined for gangliosides, these were increased twentyfold. One Gaucher spleen, in which others had reported that the stored "cerebroside" contained predominantly lactose as the saccharide moiety, has been examined in detail and it has been established that the stored material was, in fact, glucocerebroside, ceramide lactoside levels not being significantly elevated. In a further nine cases glucose was the major sugar detected in the splenic lipids.     

Characterization of lysosomal hydrolases that are elevated in Gaucher's disease.

Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase⁵ activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [³H]galactose labeled galactocerebrosides as substrates and with [³H]galactose labeled lactocerebroside under the “lactosylceramidase I”⁵ assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of G_mi-ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and G_mi-ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. G_mi-ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β-d-galactopyranoside and [³H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.     

Cytokines in health and disease

Cytokines are cellular regulators of non-immunoglobulin character. The studies of interferon, a representative cytokine, support the view that cytokines are information molecules forming a network in the animal organism. Their main task is to protect the homeostasis of the organism. This may be disturbed both by external and internal causes. The results of the studies of interferon appearing in patients with systems lupus erythematosus do not support the assumption that interferons of this type may play a role in aetiology of autoimmune diseases.     

Cytokines in inflammatory bowel disease

Over the past decade, much has been learned regarding the role of various cytokines in the pathogenesis of inflammatory bowel disease. Several cytokine 'knockout' models in mice have been shown to develop colitis, while alterations in the production of various cytokines has been documented in human Crohn's disease and ulcerative colitis. In recent years, attempts have been made to treat these diseases through modulation of cytokine production or action. This review focuses on the cytokines that have been implicated in the pathogenesis of inflammatory bowel disease. The evidence for and against a role for particular cytokines in intestinal inflammation is reviewed, as is the experimental and clinical data suggesting that cytokines are rational targets for the development of new therapies.     

The distribution of glucocerebroside in the liver of patients with Gaucher's disease.

Two Gaucher livers obtained at autopsy were examined for uniformity of glucocerebroside content. Six needle biopsies were obtained from one liver and twenty from the other, Samples were analyzed for glucocerebroside content by thin-layer and gas-liquid chromatography. One of the livers was partitioned into four sections with five biopsies taken at random from each section. This method supposes that if differences in glycolipid concentrations exist, then these differences will be exhibited among the liver sections. Our findings indicate that stored glucocerebroside is uniformly distributed throughout the liver since all the observed glycolipid levels were within ± 1.95 sample standard deviations of the sample mean.     

The bilayer nature of deposits occurring in Gaucher's disease

Evidence is presented for the structure of the Gaucher cell deposit existing as a series of bilayers that are each 60 Å thick and are gradually twisted along their length. This evidence was obtained by freeze-etching studies and by X-ray diffraction studies that were used to calculate possible electron density profiles for each bilayer. A model is presented which shows the probable arrangement of the aggregated glucocerebroside molecules.     

Composition of the membranous deposits occurring in Gaucher's disease

We have isolated and purified low-density (1.08 g/ml) glucocerebroside-rich deposits from the spleen of a patient with Gaucher's disease. The organic phase obtained by chloroform-methanol (2:1) extraction of the deposits contains glucocerebroside, phospholipid, and free cholesterol in an approximate molar ratio of 12:3:2. The lipid-free residue accounting for approximately 12% of the dry weight of the deposits, contained protein and glycoprotein and accounts for the observation that the low-density deposits are significantly more dense than pure β-glucocerebroside (1.06 g/ml). The nonlipid residue contains galactose, hexosamine, and sialic acid and a PAS-staining glycoprotein with an approximate molecular weight of 95,000. This glycoprotein is extensively degraded by Pronase, but only when the structure of the deposits has been destroyed by organic solvent extraction. The nonlipid residue also contains several polypeptides with subunit molecular weights of 26,000–38,000.The deposits appear as a series of twisted bilayers, each 60 Å thick when prepared by freeze-etching methods. These bilayers are considered to be densely packed since staining methods visualize only the external surface.     

Impaired trafficking of mutants of lysosomal glucocerebrosidase in Gaucher's disease

Gaucher's disease is the most inherited lysosomal storage disorder. Except for a few cases, the broad phenotypic heterogeneity of Gaucher's disease can be neither predicted from defined mutations nor from differences in residual enzyme activity. Here, we analyse the intracellular trafficking of glucocerebrosidase as an underlying mechanism for the expression of the clinical phenotype. Biosynthetic labeling studies combined with immunofluorescence analyses with fibroblasts from patients with the defined mutations N370S, L444P, D409H and G202R unequivocally demonstrate a retarded transport of glucocerebrosidase carrying the mutation N370S and a transport block in the ER of the enzyme with the mutations G202R, L444P and D409H. We asked whether cellular components in the patients' fibroblasts other than glucocerebrosidase are implicated in the onset of the disease. For this, mutant cDNA's corresponding to the phenotypes N370S, G202R and L444P were expressed in the mouse fibroblasts NIH3T3. Essentially similar biochemical and cellular features were revealed as compared to the patients' fibroblasts strongly suggesting that these mutations are exclusively responsible for the characterized phenotypes. Interestingly, the immunoglobulin binding protein (BiP) binds wild type and the mutant N370S but not the G202R and L444P variants suggesting a discriminatory role played by this chaperone associated with the severity of the disease.     

Cytokines and chronic liver disease

From an immunological point of view, the healthy liver has been usually associated with the phenomenon of tolerance. A microenvironment of regulatory cytokines produced by liver Kuppfer cells and liver sinusoidal endothelial cells has contributed, together with resident dendritic cells, to generate a tolerogenic environment in this tissue. In this review we discussed the intrahepatic responses to different sorts of liver injury, such as hepatotrophic viruses, alcohol or putative self-antigens. In each case we analyzed the impact of different cytokines in the clinical outcome of the different pathological situations.