F. Šmíd

Cardiocyte storage and hypertrophy as a sole manifestation of Fabry's disease. Report on a case simulating hypertrophic non-obstructive cardiomyopathy.

Fabrys disease was diagnosed in an adult patient as a lipid storage-induced non-obstructive hypertrophic cardiomyopathy. Stable angina pectoris started 15 years before death, was followed by slowly progressive heart failure and repeated pulmonary thromboembolism with death at 63 years. Autopsy disclosed enormous cardiomegaly (1100 g), cardiac storage of ceramide trihexoside (CTH) of the same intensity as in classical cases of generalized Fabrys disease (11 mg lipid/g wet weight) restricted to cardiocytes. Other tissues (liver, kidney, brain, pancreas, pulmonary artery, coronary arteries) were free of storage. Using proton magnetic resonance analysis on formaldehyde-fixed tissue the stored CTH was identified as globotriaosylceramide. It was enzymatically degradable by control cell cultures but left uncleaved by mutant reference Fabry cells. Alpha — galactosidase activities in peripheral leucocytes of all four of the patients daughters were in the heterozygous range. The diagnostic difficulties in this monosymptomatic novel variant of Fabrys disease are stressed.

Niemann-Pick disease type C

SummaryA complex neuropathological study of two cases of Niemann-Pick disease (NPD) type C (NPDC) revealed some novel features in the chemical pathology of the neuronal storage. Lipid histochemistry showed the presence of a lipid which met the criteria of a neuronal glycosphingolipid. Sphingomyelin (SM) was not detected in the neurones in any of the regions examined. Lipid chemical analysis of total extracts and of partially purified lysosomal fraction of the brain cortex showed markedly increased levels of neutral ceramide hexosides especially of glucosylceramide and ceramide dihexoside (mostly of its slower band). Phospholipids were not significantly increased. Monosialogangliosides GM2 and GM3 were increased only slightly. The storage process displayed the well known fine structure and was accompanied by a marked secondary increase in some lysosomal enzyme activities. There was neuroaxonal dystrophy (NAD) of considerable intensity and extent. Many spheroids contianed masses of degenerated organelles and neurofilaments in various proportions and displayed variable activities of acid phosphatase, nonspecific esterase and dehydrogenases. There was marked brain atrophy accompanied in one case by severe demyelination. Enzyme studies revealed partial decrease of sphingomyelinase (SMase) and betaglucosidase activities in cultured fibroblasts, as well as lack of cathodic SMase activity on isoelectric focusing. No defects of these enzymes were found in the brain samples. The findings are regarded as significant since they indicate a biochemical defect in which SM is not primarily involved and which may thus be fundamentally different from that in type A of NPD.

Photodynamic Therapy of Nonmelanoma Skin Cancer with Topical Hypericum perforatum Extract : A Pilot Study

Hypericin, the photoactive compound of Hypericum perforatum, is probably the most powerful photosensitizer found in nature. This compound has shown high potency in the photodynamic treatment of tumor cells. However, there is only limited knowledge regarding the photodynamic effect of hypericin on nonmelanoma skin cancer cells. The aim of this prospective study was to investigate the efficacy of photodynamic therapy with topical application of an extract of H. perforatum in actinic keratosis, basal cell carcinoma (BCC) and morbus Bowen (carcinoma in situ). The study was carried out on 34 patients—eight with actinic keratoses (AKs), 21 with BCC and five with Bowen’s disease. The extract of H. perforatum was applied on the skin lesions under occlusion and that was followed by irradiation with 75 J cm−2 of red light 2 h later. The treatment was performed weekly for 6 weeks on average. The percentage of complete clinical response was 50% for AKs, 28% in patients with superficial BCC and 40% in patients with Bowen’s disease. There was only a partial remission seen in patients with nodular BCCs. A complete disappearance of tumor cells was found in the histologic preparation of 11% of patients with superficial BCCs and 80% in the patients with Bowen’s disease. All patients complained of burning and pain sensations during irradiation. Although the results of this first clinical trial could be regarded as disappointing, there are still possibilities for improvement. Better preparation of the lesions, enhancement of hypericin delivery and other types of light exposure procedures could significantly improve the clinical outcomes of this relatively inexpensive treatment modality.

Niemann-Pick disease type C with enhanced glycolipid storage

A case of infantile neurovisceral disease was classified according to the morphological and chemical analysis of fixed tissue as a chemically different type of Niemann-Pick disease (NPD) type C, with glycolipids dominating the storage process. The diagnosis was reached on the basis of massive accumulation of neutral glycolipids in visceral storage elements (hepatocytes and macrophages) as an outstanding feature of lipid histochemistry. Chemical lipid analysis corroborated the findings by detecting a manyfold increase of glucosyl ceramide, lactosyl ceramide, ceramide trihexoside and GM3 ganglioside. In addition, macrophages contained variable quantities of sphingomyelin. The brain showed slightly increased quantities of lactosylceramide (Slower fraction) and glucosyl ceramide. Apart from the classical neuronal storage changes there was also marked neuroaxonal dystrophy. In terms of quality, the glycolipid spectrum was comparable to that of NPD type C, in terms of quantity, the changes were consistent with those in so-called lactosyl-ceramidosis, which, however, was reclassified as NPD type C only recently. In our view, the present case is the second published observation of lactosylceramidosis classifiable as a glycolipid (GL) variety of NPD type C in which the normally considerable tendency to glycolipid storage is further enhanced while the storage of sphingomyelin is less expressed.

Exogenous Administration of Gangliosides Inhibits FcεRI-Mediated Mast Cell Degranulation by Decreasing the Activity of Phospholipase Cγ

Gangliosides released from tumor cells, as well as administered exogenously, suppress the immune responses by largely unknown mechanisms. We show here that a pretreatment of rat basophilic leukemia cells with isolated brain gangliosides inhibited the release of preformed secretory mediators from cells activated via FcεRI but not Thy-1 glycoprotein. Exogenously administered gangliosides also affected the cell-substrate adhesion and the levels of polymeric filamentous actin in Ag-activated cells. Although the production of phosphoinositides was also decreased, enzymatic activity of phosphatidylinositol 3-kinase was not inhibited. Gangliosides had no or only marginal effect on the association of aggregated FcεRI with glycosphingolipid-enriched membranes and on tyrosine phosphorylation of FcεRI and the linker for activation of T cells. Though pretreatment with gangliosides did not inhibit the association of linker for activation of T cells with phospholipase C (PLC)γ1 and PLCγ2, tyrosine phosphorylation of these enzymes, as well as their enzymatic activities and association with detergent-insoluble signaling assemblies were reduced. This resulted in a decreased production of inositol 1,4,5-trisphosphate and an inhibition of Ca2+ mobilization. The combined data support the concept that exogenously administered gangliosides interfere with those properties of glycosphingolipid-enriched membranes that are important for the formation of plasma membrane-associated signaling assemblies containing PLCγ but not for initial tyrosine phosphorylation of FcεRI subunits.

Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at −20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier.

Changes in GM1 ganglioside content and localization in cholestatic rat liver.

Abstract(Glyco)sphingolipids (GSL) are believed to protect the cell against harmful environmental factors by increasing the rigidity of plasma membrane. Marked decrease of membrane fluidity in cholestatic hepatocytes was described but the role of GSL therein has not been investigated so far. In this study, localization in hepatocytes of a representative of GSL, the GM1 ganglioside, was compared between of rats with cholestasis induced by 17α-ethinylestradiol (EE) and vehicle propanediol treated or untreated animals. GM1 was monitored by histochemical reaction employing cholera toxin B-subunit. Our findings in normal rat liver tissue showed that GM1 was localized in sinusoidal and canalicular hepatocyte membranes in both peripheral and intermediate zones of the hepatic lobules, and was nearly absent in central zones. On the contrary, in EE-treated animals GM1 was also expressed in central lobular zones. Moreover, detailed densitometry analysis at high magnification showed greater difference of GM1 expression between sinusoidal surface areas and areas of adjacent cytoplasm, caused as well by increased sinusoidal staining in central lobular zone as by decreased staining in cytoplasm in peripheral zone. These differences correlated with serum bile acids as documented by linear regression analyses. Both GM1 content and mRNA corresponding to GM1-synthase remained unchanged in livers; the enhanced expression of GM1 at sinusoidal membrane thus seems to be due to re-distribution of cellular GM1 at limited biosynthesis and could be responsible for protection of hepatocytes against harmful effects of bile acids accumulated during cholestasis.

Chromatography and spectrofluorometry of brain fluorophores in neuronal ceroid lipofuscinosis (NCL)

The aim of the present work was to develop a chromatographic system for the separation of individual fluorophores extracted from neuronal ceroid lipofuscinosis (NCL) brain and isolated storage bodies. Extracts from gray matter were best resolved on silica-gel HPTLC plates using a mixture of chloroform/methanol/water (55:45:10 by vol.). Two other chromatographic systems were tested which gave poorer separation. Corrected fluorescence spectra were obtained on the original extract and fluorescence intensity, especially at longer wavelengths was increased in both samples. Yellow and blue fluorophores were detected on HPTLC plates using a primary violet and secondary yellow filter with cut-off levels of 400 and 520 nm, respectively. Plates were photographed at 20 min, 2 h and 1 week after chromatography. With this filter system, up to 12 yellow bands of differing intensity were observed at 20 min but with time, some of these changed to blue as a result of autoxidation. NCL tissues emit yellow fluorescence when viewed under light microscopy, however extracted material did not demonstrate a distinct peak in this region of the spectrum which should be around 575 nm. HPTLC confirmed this observation and time studies revealed that autoxidation changes occur and must be carefully controlled to reduce artifacts. The discrepancy between extracted and non-extracted observations may be the result of superposition of multiple fluorophores with differing maxima and/or a self-absorption phenomenon. The combination of chromatographic separation and spectral analysis as described in this study, may be a valuable technique to further clarify the characteristics of compound fluorescent lipopigments. It is suggested that NCL fluorophores of human brain differ in their properties from other models.

An unusual case of phospholipidosis.

We present the results of a structural, histochemical and lipid-chromatographic study of tissues obtained at postmortem from an unusual case of phospholipidosis. A previous biopsy of the appendix and liver (Elleder et al., 1975a) had revealed a predominance of phosphoglyceride storage, principally of lysobisphosphatidic acid (LBPA) postmortem material showed that this lipid was stored exclusively in central neurons. In the spleen and the lymph node, however, sphingomyelin (SP) was shown, histochemically and chromatographically, to be the main lipid stored. Total sphingomyelinase (SPase) activity in the appendix was reduced to about 50% of normal. Neuroaxonal dystrophy (NAD) and a conspicuous discrepancy between the degree of distension of some neurons and their lipid content deserve special mention. The case is contrasted with classical sphingomyelinosis; the complexity of the Niemann-Pick group of diseases is discussed as an indication of the difficulties of classification of any atypical case.

Adrenal changes in Niemann-Pick disease: Differences between sphingomyelinase deficiency and type C

Structural, chemical, and histochemical analyses of adrenal tissue performed in 8 cases of Niemann-Pick disease (NPD) revealed stark differences of storage between spingomyelinase (SMase) deficiency (6 cases) and type C (2 cases). In all the full-blown cases of the SMase deficiency group, pronounced sphingomyelin (SM) storage was found in all the zones of the cortical epithelium with slightly increasing centripetal gradient. The storage resulted in the reduction or even disappearance of lipofuscinogenesis in the reticular zone, in the reduction of the physiological fat content, in the generalized foamy transformation of the epithelium, and in moderate organomegaly. The storage was expressed in both A and B types and was roughly proportional to the storage in other viscera. The stromal storage was confined to the vascular endothelium, and in particular, to the macrophages. One of the cases showed the presence of typical spirolactone bodies unmodified in fine structure by the lysosomal storage. Their most conspicuous enzymatic activity was that of non-specific esterase and NADH tetrazolium reductase. The adrenals in type C were macroscopically and histologically normal except for a variable population of stromal foam cells. Chemically, there was slight increase in all phospholipids with borderline or moderate percentual increase of SM. There was also slight increase in some of the lower neutral glycosphingolipids. Electron microscopy dislosed rudimentar storage in lower cortical layer epithelium which by its fine structure and according to results of lipid histochemistry was qualitatively different from that in SMase deficiency. The stromal storage was expressed mainly in macrophages in which there was histochemically detectable amount of SM. There was no storage detectable in medullary cells in neither group of NPD complex. The results point not only to striking quantitative differences in storage intensity between the 2 basic groups of NPD showing the cortical epithelium in type C as being remarkably resistant to the metabolic disorder, but also to difference in quality of the storage very much like that found in other tissues, too.