Stromal accumulation of chondroitin sulphate in mammary tumours of dogs.

To contribute to the investigation of the composition of the extracellular matrix in epithelial tumours, mammary gland tissues of dogs (including tumours, hyperplasias and normal tissue as well as metastatic lesions in lymph nodes and lung) were studied histochemically and immunohistochemically for distribution of sulphated glycosaminoglycans (s-GAGs). The formaline-fixed tissue was stained by alcian blue at pH 5.8, using the 'critical electrolyte concentration' to study the degree of sulphation of s-GAGs. s-GAGs were characterized by degradation with enzymes and nitrous acid and by immunohistochemistry with two anti-chondroitin sulphate monoclonal antibodies. The light microscopic investigation of s-GAG deposits revealed a limited number of patterns of their distribution. The main s-GAGs found in the mammary gland tumours of dogs and in metastatic lesions were chondroitin sulphate (CS) and heparin/heparan sulphate (HEP/HS). CS accumulated in diffuse structures between epithelial cells as well as around clusters of tumour cells. The latter pattern, possibly representing a mesenchymal reaction to the tumour, was present in 74% of the tumours, and in 67% of these, highly sulphated CS was present. A diffuse accumulation of CS was present almost exclusively in complex and mixed tumours; because of the expression of the 3B3 epitope for CS in immature cartilage the spindle cells of complex tumours are argued to be the precursors of the cartilage in mixed tumours. HEP/HS was stored mainly in mast cells that were found in increased numbers in hyperplasias and tumours. By pretreatment of microscopic slides with chondroitinase AC or ABC immunostaining of fibronectin could be made possible in areas in which CS was abundantly present, suggesting that CS may mask fibronectin epitopes. It is concluded that CS with different degrees of sulphation is the most important s-GAG in the extracellular matrix of mammary tumours of dogs. CS and other s-GAGs accumulate at different sites and may have a different pathogenetic significance.

Necrotizing pneumonia in a cat caused by an orthopox virus.

A necrotizing pneumonia was observed in a domestic cat which had a clinical history of severe respiratory distress. Histology, immunohistology and electronmicroscopy revealed poxvirus as the causative agent. By the polymerase chain reaction and gene sequencing, an orthopox virus with 93% homology to cowpox virus was identified.

Lymphangiosarcomas in cats: a retrospective study of 12 cases.

Clinical, macroscopic, and histologic features of 12 lymphangiosarcomas in cats are described. Nine tumors were located in the subcutaneous tissue at the caudoventral abdominal wall (eight cats) or in the neck (one cat). The remaining three cats had lymphangiosarcomas around the cranial mesenteric artery (two cats) or precardial in the mediastinum (one cat). Macroscopically, the tumors were noncircumscribed, white, edematous, and intermixed with fat tissue. Histologic features varied from cleft-forming and cavernous growth to papilliform and solid patterns. Follow-up data were available for seven cats with subcutaneous lymphangiosarcomas. All these cats died or were euthanatized within 6 months after surgery because of poor wound healing, local recurrence, or distant metastases. The cats with abdominal or thoracic masses were either euthanatized at surgery or within 6 months after the first surgery because of recurrent chylothorax, chyloperitoneum, or distant metastases.

[A case of necrotizing meningoencephalitis in a pug dog (pug dog encephalitis--PDE)]. / Ein Fall von nekrotisierender Meningoenzephalitis beim Mops (pug dog encephalitis-PDE).

A three-year-old female pug dog was euthanized because of recurrent seizures. Pathological examination revealed severe multifocal necrosis confined to the cerebrum. Histologically, areas of malacia of different stages, with prominent gitter cell infiltration were observed. Furthermore, there was severe rarefication resulting in cavities separated by tissue bridges and blood vessels. In the adjacent tissue and in the meninges a moderate to severe non-purulent meningoencephalitis was evident. The lesions are consistent with those reported for pug dog encephalitis (PDE). In the present paper, the first case of PDE in Germany is described and an overview of the clinical symptoms, the neuropathological findings of this etiologically unknown disease, the differential diagnoses and the possible pathogenesis is given.

[Ascites as a result of peritoneal mesotheliomas in a horse]. / Aszites infolge eines peritonealen Mesothelioms bei einem Pferd.

An abdominal tumor was suspected after clinical evaluation in an eight-year-old, bay-coloured hannoveranian gelding. The diagnosis was based on the symptoms of ascites, on the results of the transcutaneous abdominal ultrasound examination and on the characteristic changes in the serum-electrophoresis. Postmortem a peritoneal mesothelioma was diagnosed. This primary tumor of the peritoneum is rarely described in horses.

[Leukoencephalomalacia in two horses--moldy corn poisoning in Germany?]. / Leukoenzephalomalazie bei zwei Pferden--Moldy Corn Poisoning in Deutschland?

Moldy corn poisoning is a mycotoxicosis of Fusarium sp. causing a disease termed equine leukoencephalomalacia (ELEM). This article reviews the literature on ELEM and describes two cases with clinical signs and morphological findings comparable with fusariotoxicosis. Since in both cases neither a fungus nor a toxin proof were performed, the different causes of leukoencephalomalacia in horses are discussed.

[Tumor cells on the inside and tumor cells on the outside. A contribution to the diagnosis and elucidation of the pathogenesis of mammary tumors in dogs]. / Tumorcellen van binnen en tumorcellen van buiten. Een bijdrage tot de diagnostiek en de opheldering van de pathogenese van mammatumoren van de hond.

This review discusses some recent experience with intra- and extracellular components of tumours as markers for tumour diagnosis. Intermediate filaments are cytoskeletal proteins of either epithelial or mesenchymal cells. Antibodies raised against human intermediate filament proteins cross-react with their canine counterparts. A study of the presence and distribution of intermediate filaments in normal mamma and mammary tumours of dogs showed that they do not contribute to a more adequate classification of the tumours. However, the presence of vimentin in epithelial tumour cells may be a marker of malignancy. Proteoglycans are extracellular matrix proteins, containing long chains of glycosaminoglycans (GAGs). Analysis of the presence of GAGs in canine mammary tumours showed that accumulation of chondroitin sulfate, frequently of an abnormal type, was a predominant finding in all tumour types, but not in normal mamma, and was not related to biological behaviour. Although demonstration of the before-mentioned tumour components does not help tumour diagnosis, it may contribute to the elucidation of the development of such tumours as the complex and mixed adenomas of the mamma of which cartilage and bone are important constituents of unknown origin.

Metabolism of ganglioside-amides in cultured human fibroblasts.

Metabolism of [3H]ganglioside derivatives GM3-amide and GM2-amide has been investigated in normal human skin fibroblasts. In a cell-free system the ganglioside analogues have been shown to enter biosynthetic pathways, their degradation, however, was curtailed at an early stage, as GM3-amide could not be hydrolysed by sialidase action. GM2-amide was susceptible to beta-hexosaminidase degradation yielding GM3-amide. When incorporated into fibroblasts [3H]GM2-amide was degraded to [3H]GM3-amide presumably in the lysosomes, and at the same time glycosylation to [3H]GD1a-monoamide took place most likely in the Golgi apparatus. [3H]GM3-amide, however, did not seem to reach the glycosylation sites in the Golgi apparatus. It could be detected in the lysosomes, where it was not degraded due to its sialidase resistance. From these results we conclude that in cells exogenously administered [3H]GM3-amide and [3H]GM2-amide both are directed to the lysosomes and that [3H]GM2-amide also reaches the Golgi apparatus. The synthesis of higher [3H]ganglioside-amides from incorporated [3H]GM2-amide can occur by direct glycosylation. [3H]GM3-amide, however, even if it reaches the Golgi compartment, does not enter the biosynthetic pathway.

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects.

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.

[Adrenoleukodystrophy in a pair of siblings]. / Adrenoleukodystrophie bei einem Geschwisterpaar.

The diagnosis of adrenoleukodystrophy (ALD) in two brothers was confirmed by the analysis of long-chain fatty acids in cultured skin fibroblasts. The 23 year old brother was treated for Addison's disease at the age of 7 years. His first symptoms of ALD developed at the age of 22. These were lack of concentration and compulsive disorder. The younger brother was noted to show behavioural changes and a decreased performance at school at age 9. The disease then progressed rapidly. Variability and diagnostic procedures are discussed in this report.

Diagnosis of infantile and juvenile forms of GM2 gangliosidosis variant 0. Residual activities toward natural and different synthetic substrates.

p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside, which is known to be a specific substrate for human hexosaminidase A, has recently been used successfully for diagnosis of variants B and B1 of GM2-gangliosidosis (Fuchs et al. 1983; Kytzia et al. 1983; Li et al. 1983). However, it is hydrolyzed by hexosaminidase S as well and is therefore not suitable for detection of patients with variant 0, who reach the normal range of activity toward this substrate. Assay of ganglioside GM2 cleaving activity in fibroblast extracts in the presence of the natural GM2 activator protein reveals residual hexosaminidase A activities of less than 2% of normal controls in two infantile and up to 7.5% in two juvenile patients with variant 0.

Release of sphingomyelin phosphodiesterase (acid sphingomyelinase) by ammonium chloride from CL 1D mouse L-cells and human fibroblasts. Partial purification and characterization of the exported enzymes.

In cultured human fibroblasts and mouse L-cells the lysosomotropic agent, ammonium chloride, caused release of acid sphingomyelinase into the culture medium. The water-soluble enzymes were partially purified by sequential chromatography on ConA-Sepharose, octyl-Sepharose and Sepharose CL-4B. Mouse sphingomyelinase was purified up to 64-fold and human sphingomyelinase 134-fold from the culture medium. Specific activities were 925 nmol/(h X mg) and 1 434 nmol/(h X mg), respectively. The final enzyme preparations obtained were free of other lysosomal enzyme activities tested and had very similar properties: optimal activity at pH 4.8 (mouse enzyme) and pH 4.4 (human enzyme), Km values of 6.2 X 10(-5)M and 2.4 X 10(-5)M, respectively, and an apparent molecular mass of 68 kDa. In isoelectric focusing the enzymes peaked at pH 4.78 (mouse enzyme) and pH 4.75 (human enzyme).

Variant of GM2-gangliosidosis with hexosaminidase A having a severely changed substrate specificity.

The levels of hexosaminidase A activity in cultivated fibroblasts of two patients with GM2-gangliosidosis were close to the normal range with 4-methylumbelliferyl-beta-D-2-acetamido-2-deoxyglucopyranoside and 4-methylumbelliferyl-beta-D-2-acetamido-2-deoxygalactopyranoside as substrates, and the enzymes were normal in most parameters analyzed. However, the enzymes of both patients were almost completely inactive against two specific substrates for hexosaminidase A, rho-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside, and ganglioside GM2 in the presence of GM2-activator. Fibroblast extracts of both patients showed normal hexosaminidase B and GM2-activator activity, the latter was strongly decreased in two cases with variant AB. It is suggested that human hexosaminidase A may contain two different active sites which might be inactivated separately by different mutations.

[Collagen metabolism of fetal bone cultured in vitro]. / Untersuchungen über den Kollagenstoffwechsel in vitro gezüchteter fetaler Knochenanlagen.

Cultures of murine fetal bone explants were used as experimental model for the investigation of drug effects on mesenchymal metabolic processes, particularly the collagen metabolism. As parameters for growth or metabolic processes, resp., the increase in size, the DNA-, uronic acid- and hydroxyproline content of the explants were determined under various modifications of the culture conditions. Addition of ascorbic acid to the culture medium caused in concentrations from 2.5 micrograms/ml an increase of the hydroxyproline content and of the size and in concentrations from 20 or 40 micrograms/ml, resp., also an increase of the DNA- and uronic acid content of the explants. Proline exerted in concentrations up to 100 micrograms/ml no significant influences on the determined parameters and showed in concentrations from 150 micrograms/ml inhibitory effects. Substitution of the horse serum of the culture medium by chick embryonic extract resulted in a decrease of growth and metabolic activity of the explants. Under the influence of an oxygen content of 10% in the gas phase of the culture system all determined parameters with exception of the DNA-content were significantly increased as compared with 20% oxygen content, whereas concentrations of 30% oxygen or more led to reduction of growth and uronic acid content of the explants.