Interaction of volkensin with HeLa cells: binding, uptake, intracellular localization, degradation and exocytosis.

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10(-10) M) and had a similar number of binding sites (2 x 10(5)/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.

Ricin toxicity to microglial and monocytic cells.

Microglial cells, like macrophages, are very sensitive to ricin, a galactose-specific toxic lectin belonging to the family of ribosome-inactivating proteins. This toxin can be taken up by most cells through the binding of its B chain to galactose-containing molecules on the cell membrane. In macrophagic cell types it can be internalised also by mannose receptors which are present on the surface of these cells. Endocytosis of the toxin by either pathway was evaluated by ricin toxicity to primary cultures of rat microglial cells and to a microglial N11 cell line in the presence or absence of lactose and mannan, which compete for the endocytosis via the ricin lectin chain or cellular mannose receptors, respectively. Results were compared with those obtained in cultures of mouse macrophages, human monocytes, and a monocytic JM cell line. All cultures were protected from ricin toxicity more by lactose than by mannan, indicating that ricin endocytosis via its lectin B chain is prevalent over that mediated by cellular mannose receptors. However, a partial protection by mannan was observed in all cases but not-stimulated N11 cells, either in the form of direct protection or of significant additional protection over that afforded by lactose. Mannose receptor expression by N11 cells was negative before, and positive after, treatment with endotoxin, as assessed by the specific binding of 125I-mannose-bovine serum albumin. Moreover, a partial protection from ricin toxicity by mannan was induced in the N11 microglial line after stimulation, consistently with an inducible expression of the mannose receptor by activated cells switched towards a microglial phenotype.

Cytogenetic investigation of chordomas of the skull.

We report the first cytogenetic investigation of cranial chordoma. Three cranial chordomas were examined, two of which could be further histopathologically classified as chondroid chordomas. In addition, we have included a case of chordoma of a cervical vertebra to compare the cytogenetic abnormalities. Diagnosis was made at histological and immunohistochemical levels. The three cases of cranial chordoma showed a normal karyotype, while one vertebra showed 46,XY,t(6;11)(q12;q23). Chordomas, particularly those containing cartilage, have to be distinguished from chondrosarcomas of the skull base. Such a distinction is normally based on expression of epithelial markers which usually are lacking in chondrosarcoma. Cytogenetic investigation may eventually prove to be useful in the distinction of the two lesions, if chromosome anomalies are consistently absent in chordoma, although some chondrosarcomas may also present a normal karyotype. Such a distinction has clinical implications because chondroid chordomas show better survival, whereas chondrosarcomas show a propensity to infiltrate the surrounding tissues.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

[Cytogenetics of tumors of the central nervous system: a case study and review of the literature]. / Citogenetica di tumori del sistema nervoso centrale: studio casistico e revisione della letteratura.

The introduction of cytogenetic techniques, especially chromosome banding techniques, has facilitated a more detailed study of the chromosomal basis of hematological malignancies and solid tumours. With the advent of molecular cell biology many new insights have been gained into the pathogenetic mechanisms of cancer. With G banding technique we have studied chromosomal aberrations of 174 tumours of the Central Nervous System in adults and the results obtained have been compared with the literature data.

Intracranial germ cell tumour (embryonal carcinoma with teratoma) with complex karyotype including isochromosome 12p.

We report the chromosomal characteristics of a malignant teratoma with embryonal carcinoma component located in the pineal region of a 15-year-old boy. Chromosome analysis showed a near-triploid complex karyotype (62 chromosomes), including two copies of an isochromosome 12p, confirmed by fluorescence in situ hybridization analysis. The present findings indicate that isochromosome 12p formation is probably associated with the development of malignant germ cell tumours.

[Cytogenetic analysis of hypophyseal adenoma. Study of 9 cases and review of the literature]. / Analisi citogenetica degli adenomi ipofisari. Studio di 9 casi e revisione della letteratura.

INTRODUCTION: To the best of our knowledge, no specific chromosomal abnormalities have been found in the literature in pituitary adenomas. In the present study, we investigated 9 cases of pituitary adenoma and reviewed the current literature. MATERIALS AND METHODS: Nine cases of pituitary adenoma have been studied with immunohistochemistry and cytogenetic using short-term cultures. RESULTS: All tumors had a normal karyotype. Three cases were clinically non-secreting adenomas, three cases produced prolactin, two showed growth hormone production and one thyroid-stimulating hormone. One of the cases showed dural invasion. CONCLUSIONS: We compared our results with those published in the current literature. It appears that pituitary adenomas do not have specific numerical and structural abnormalities and mostly show a normal karyotype.

Mammary foam cells. Characterization by immunohistochemistry and in situ hybridization.

Cells showing abundant, finely vacuolized cytoplasm (foam cells) are found frequently in most benign lesions of the breast and in certain malignant breast tumours. The origin of mammary foam cells (FCs) has not been clarified, and we therefore studied the morphological features of mammary FCs in a series of 50 benign lesions. The FCs were subdivided, on the basis of their distribution into FCs lining the glandular lumina, intraluminal FCs, intraepithelial-pagetoid FCs, and stromal FCs. The lesions were tested with a panel of antibodies against macrophage (MAC 387, CD68) and epithelial (epithelial membrane antigen [EMA], gross cystic disease fluid protein 15 [GCDFP15] and cytokeratin) markers. The lesions were examined for the presence of PIP/GCDFP15-specific mRNA by an in situ hybridization technique. Three different types of FCs were identified. Type A FCs are epithelial cells (positivity with EMA and cytokeratin) and show apocrine differentiation (positivity with GCDFP15 antiserum and expression of PIP/GCDFP15 mRNA). Type B FCs are of macrophage origin, as they are positive with the macrophage markers and lack cytokeratin and PIP/GCDFP15 mRNA. Finally, type C FCs show an intermediate profile between an epithelial cell and a macrophage: they are both CD68 and GCDFP15 positive and show a thin peripheral rim of positivity with anti-cytokeratin antibody. They lack PIP/GCDFP15 mRNA. Our results indicate the possibility of a spectrum of phenotypes in mammary FCs, from epithelial-apocrine cells to macrophage-derived phagocytic cells.

Simulated ischaemia-reperfusion conditions increase xanthine dehydrogenase and oxidase activities in rat brain slices.

Xanthine dehydrogenase and oxidase activities increased by 87% in rat brain slices after 30 min in vitro ischaemia. A further 41% increase was induced by 30 min simulated reperfusion of ischaemic slices. No conversion from the dehydrogenase to the oxidase activity was observed. The increment of enzyme activity was not due to neosynthesis of the enzyme, since it was not affected by the addition of cycloheximide during the ischaemic incubation. The increased oxygen-dependent form of the enzyme could aggravate the ischaemic brain injury by free radicals production, in particular after reperfusion.

Toxicity of ricin and volkensin, two ribosome-inactivating proteins, to microglia, astrocyte, and neuron cultures.

Ricin and volkensin, two potent toxins belonging to the family of ribosome-inactivating proteins (RIPs), have been largely exploited in recent years in in vivo experiments of neuronal degeneration consequent to suicide transport or immunolesioning. We have determined both the toxicity of, and the inhibition of, protein synthesis by ricin and volkensin in in vitro cultures enriched in microglial cells, astrocytes, or neurons. In microglial cultures, 50% of toxicity (estimated by LDH released from dead cells) after 24 h exposure to RIPs was obtained with volkensin at 2.2x10(-12) M concentration and 50% of protein synthesis inhibition at 2x10(-14) M concentration. Both values were higher by about one order of magnitude in astrocyte-enriched cultures. Toxicity of, and inhibition of, protein synthesis by, ricin were lower for both cell types by about 1 order of magnitude as compared to volkensin. Cerebellar granule neurons in culture survived remarkably well to 24 h exposure to ricin or volkensin, although their protein synthesis was effectively inhibited by the two toxins with a potency similar to that found for astrocytes. These results demonstrate that glial cells, in particular microglia, are very sensitive to RIPs toxicity and should, therefore, be a primary target of these toxins when injected in vivo. Thus, the damage observed after in vivo experiments could be partly related to diffusion of toxic substances from early-affected glial cells.

Ribosome-inactivating lectins with polynucleotide:adenosine glycosidase activity.

Lectins from Aegopodium podagraria (APA), Bryonia dioica (BDA), Galanthus nivalis (GNA), Iris hybrid (IRA) and Sambucus nigra (SNAI), and a new lectin-related protein from Sambucus nigra (SNLRP) were studied to ascertain whether they had the properties of ribosome-inactivating proteins (RIP). IRA and SNLRP inhibited protein synthesis by a cell-free system and, at much higher concentrations, by cells and had polynucleotide:adenosine glycosidase activity, thus behaving like non-toxic type 2 (two chain) RIP. APA and SNAI had much less activity, and BDA and GNA did not inhibit protein synthesis.

Toxicity and cytotoxicity of nigrin b, a two-chain ribosome-inactivating protein from Sambucus nigra: comparison with ricin.

Nigrin b, a lectin isolated from the bark of elderberry (Sambucus nigra L.), has structure and enzymatic activity similar to that of ricin and other type 2 ribosome inactivating proteins (RIPs), and yet is much less toxic to cells and animals. In an attempt to explain this difference, we studied (1) the cytotoxicity of both lectins at 18 and 37 degrees C, and in the presence of substances interfering with intracellular routing, and (2) the binding of nigrin b to, and its uptake and degradation by HeLa cells, in parallel with ricin. As compared with the latter, (1) less nigrin b was bound and more was degraded by cells, with a resulting lower concentration remaining inside the cells, and (2) there is evidence for a different intracellular routing followed by the two lectins. These results may explain at least partly the different cytotoxicity and consequently the lower toxicity to mice of nigrin b compared with ricin.

Hepatoxicity of ricin, saporin or a saporin immunotoxin: xanthine oxidase activity in rat liver and blood serum.

Male Wistar rats each received an i.p injection of the ribosome-inactivating proteins ricin or saporin, or a Ber-H2 (anti-CD30)-saporin immunotoxin at a dose corresponding to three times the LD50 calculated for mice. Animals were killed 24, 48 or 72 h after treatment. Histological examination showed hepatic necrosis in all treated animals, although the sinusoidal lining was affected only in ricin-poisoned rats. The activities of xanthine dehydrogenase (D-form) and oxidase (O-form) were determined spectrophotometrically in liver and serum samples. In ricin-treated animals the liver enzyme was progressively converted from the D- to the O-form, which accounted for more than 60% of total activity after 48 h of poisoning, whilst no change in the xanthine oxidase activity was found in the serum. In the liver of rats treated with free or Ber-H2-conjugated saporin, the D-form was more than 75%, as in normal animals. In the same animals the serum xanthine oxidase activity was up to three-fold control values. The determination of serum xanthine oxidase may prove helpful in the evaluation of liver damage in patients treated with immunotoxins. It may become a diagnostic tool for the differential diagnosis of liver diseases.

Excitotoxic increase of xanthine dehydrogenase and xanthine oxidase in the rat olfactory cortex.

Excitotoxic lesions induced by systemic injection of kainic acid, resulted in 2-3-fold increase of xanthine dehydrogenase and xanthine oxidase activities in the rat olfactory cortex 48-72 h after drug administration. A significant increase of the xanthine oxidase/dehydrogenase ratio was also observed at 4 and 48 h post-injection. No similar changes were noticed in the hippocampus. The enhancement of enzyme activity seems to be primarily a consequence of the altered cell composition in damaged area. Free radicals produced by the increased oxygen-dependent form of the enzyme could in turn aggravate the excitotoxic brain injury.

In vivo and in vitro uptake of an anti-CD30/saporin immunotoxin by rat liver parenchymal and nonparenchymal cells.

A Ber-H2/saporin immunotoxin, consisting of the single-chain ribosome-inactivating protein saporin-S6 and the anti-CD30 monoclonal antibody Ber-H2, gave encouraging results in the treatment of refractory Hodgkin's disease but caused a transient hepatotoxicity. The accumulation of Ber-H2/saporin conjugate and of its components by rat liver parenchymal and nonparenchymal cells was studied. The in vivo concentration of intravenously injected Ber-H2/saporin, saporin or Ber-H2 in nonparenchymal cells was 4-, 25- and 11-fold higher, respectively, than that in parenchymal cells. Adherent in vitro cultured nonparenchymal cells, mostly Kupffer cells, accumulated the proteins approximately 10 times more than parenchymal cells; traces of free saporin were taken up by both types of cells. In vitro protein synthesis by both cell types was inhibited by 50% at nanomolar concentrations of saporin. Nonparenchymal cells were sensitive to Ber-H2/saporin at picomolar concentrations, whereas parenchymal cells were unaffected by the immunotoxin up to 100 pmol/L. The results of the uptake of, and the sensitivity to, the immunotoxin suggest that the sensitivity of liver cells is proportional to the uptake and that the in vivo damage to parenchymal cells is at least in part mediated by the toxicity to nonparenchymal liver cells.

Serum pancreatic enzyme assays in acute abdomen: a comparative prospective study.

Serum amylase, pancreatic isoamylase, lipase, trypsinogen and elastase-1 were measured in 100 consecutive patients who were emergency admissions to a surgical department, and in 27 selected patients with proven acute pancreatitis who served as controls. The final diagnoses in the 100 patients of the study group were: acute pancreatitis in eight patients, other digestive diseases in 87, and urogenital tract diseases in five. In the control group, pancreas-specific enzymes were abnormally high in all patients and amylase in 26 out of 27. In the study group, all enzymes were markedly high in all eight patients with acute pancreatitis. In the remaining 92 patients, serum amylase was abnormally high in seven, and at least one pancreatic enzyme was elevated in 16. These elevations were generally mild. The diagnostic efficiency, i.e., the percentage of patients correctly classified, was 96% for pancreatic isoamylase and lipase, 93% for amylase, 91% for elastase-1, and 84% for trypsinogen. We conclude that serum lipase turbidimetric assay is the most suitable test for emergency diagnosis of acute pancreatitis, because it is highly sensitive and specific and simply and quickly performed.