Ora Medalia

Anti-neoplastic activity of paclitaxel on experimental superficial bladder cancer: In vivo and in vitro studies

The effects of intravesical administration of paclitaxel (taxol) in a bladder tumor model in mice, as well as the drugs in vitro activity on the same tumor cells, have been studied. Two cell lines, derived from MBT‐2 cells, were employed in these experiments. The T50 line (obtained by many passages in mice) was much more aggressive in vivo than the T5 line. In vivo paclitaxel treatment for 3 days after T5 implantation resulted in a considerable retardation of tumor growth, whereas under the same conditions the T50 line was much less, although still significantly, affected. When treatment was started 1 day after tumor implantation, both tumor variants were affected by paclitaxel to the same extent. The in vitro experiments utilized the MiCK assay, which allows continuous recording of the kinetics of cell growth. These studies revealed a 39.8% inhibition of cell growth by 2.10−8M paclitaxel in the T50 line and a 30‐fold increase in concentration had only a small additional effect on the degree of inhibition. At 2.10−8M paclitaxel, growth of T5 was inhibited by 21.7%, which increased to 35.2% at 6.10−7M. The treated cells displayed bundles of microtubuli, as described for other paclitaxel‐treated cells. Int. J. Cancer, 70:297–301, 1997.

Prevention of Ascending Pyelonephritis in Mice by Urease Inhibitors

The effect of 2 urease inhibitors, hydroxyurea and thiourea, on experimental ascending pyelonephritis by P. mirabilis was studied in mice undergoing water diuresis. It was found tha

Basic secretagogues activate protein tyrosine phosphorylation and release of arachidonic acid in mast cells via a novel protein kinase C and phosphatidylinositol 3‐kinase‐dependent mechanism

Mast cells play a central role in inflammatory and immediate‐type allergic reactions. These granulated cells release by a process of regulated exocytosis a variety of biologically active substances which are either preformed (e.  g. histamine), or synthesized de novo following activation [e.  g. metabolites of arachidonic acid (AA) and multifunctional cytokines]. Exocytosis in mast cells is activated either in response to aggregation of the receptors for immunoglobulin E (FcϵRI) or by the direct activation of pertussis toxin‐sensitive G‐proteins by a class of receptor mimetic agents, collectively known as basic secretagogues of mast cells. In the present study we show that compound 48/80 (c48/80), a synthetic member of the class of basic secretagogues, stimulates protein tyrosine phosphorylation of a number of as yet unidentified cellular substrates.These phosphorylations were inhibited by the tyrphostin AG‐18, by the phosphatidylinositol 3‐kinase inhibitor wortmannin and by the protein kinase C inhibitors K252a and GF109203X. These inhibitors also inhibited the release of AA induced by c48/80 but had no effect on exocytosis. Taken together, our findings suggest that basic secretagogues induce protein tyrosine phosphorylation as part of their parallel multiple signaling pathways which are presumably mediated by more than one G‐protein. Both protein kinase C and phosphatidylinositol 3‐kinase serve as intermediates in this signaling pathway. The protein tyrosine kinase signaling pathway, which mediates the activation of AA release, does not contribute to secretion of the preformed mediators such as histamine, but it might largely contribute to the de novo production of inflammatory mediators like leukotrienes and prostaglandins.

O-glycosylation is essential for intracellular targeting of synaptotagmins I and II in non-neuronal specialized secretory cells

We have examined the trafficking of synaptotagmin (Syt) I and II in the mast cell line rat basophilic leukemia (RBL-2H3). We demonstrate that both Syt I and Syt II travel through the plasma membrane and require endocytosis to reach their final intracellular localization. However, N- or C-terminal tagging of Syt II, but not of Syt I, prevents its internalization, trapping the tagged protein at the plasma membrane. Furthermore, a chimeric protein comprising a tagged luminal domain of Syt II fused with the remaining domains of Syt I also localizes to the plasma membrane, whereas a chimera consisting of tagged luminal domain of Syt I fused with Syt II colocalizes with Syt I on secretory granules. We also show that endocytosis of both Syt I and Syt II is strictly dependent on O-glycosylation processing, whereby O-glycosylation mutants of either protein fail to internalize and remain at the plasma membrane. Our results indicate that the luminal domains of Syt I and Syt II govern their internalization capacity from the plasma membrane and identify O-glycosylation as playing a crucial role in Syt trafficking in non-neuronal secretory cells.

Synaptotagmin II negatively regulates MHC class II presentation by mast cells

Synaptotagmins (Syts), comprise a gene family of proteins, implicated in the control of protein traffic. Rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells (MMC), express at least four distinct Syt homologues, including Syt II, Syt III, Syt V and Syt IX. Synaptotagmin II is located at the late/endosomal/lysosomal compartment, where it negatively regulates lysosomal exocytosis. Mast cells may contribute to immune defense mechanisms by presenting MHC class II/antigen complexes and triggering T cell-dependent immune responses. We now demonstrate that RBL-2H3 mast cells, which express reduced levels of Syt II (<5%) by transfection with Syt II antisense cDNA, are able to release MHC class II molecules. We further show that release of both MHC class II molecules and of the lysosomal enzyme cathepsin D is stimulated by lipopolysaccharide (LPS, 1 microg/ml, 48h). We show further that LPS reduces by >40% the level of Syt II expression in both RBL-2H3 and bone marrow-derived mast cells (BMMC). This effect is both dose and time-dependent. These results indicate that Syt II can be down-regulated by external inflammatory signals, resulting in the amplification of mast cell function. Finally, our results implicate Syt II as an important and novel regulator of MHC class II presentation.

Comparison of Nuclear DNA Content in Locally Invasive and Noninvasive Papillary Carcinoma of the Thyroid Gland

Extrathyroidal invasion of papillary carcinoma of the thyroid gland has a very bad prognosis. A retrospective study was performed on 40 specimens from patients with papillary carcinoma of the thyroid gland to find out whether DNA ploidy correlated with aggressive tumor behavior. The nuclear DNA content of 20 locally aggressive papillary thyroid carcinomas was studied by flow cytometry. The results were compared with those of a matched control group of 20 patients with noninvasive papillary tumors. Forty percent of the tumors with spread to extrathyroid tissue were aneuploid, whereas all the tumors without such extension were diploid. This difference was statistically significant (p < 0.003). The data suggest that the differentiation of locally noninvasive and invasive papillary thyroid carcinomas may be potentially possible by nuclear DNA determination.

Spontaneous Bacterial Clearance in Experimental Urinary Tract Infections in Mice: Cellular Aspects of the Process

The morphological aspects of Proteus mirabilis clearance from the urinary tract of experimentally infected mice were investigated. For this purpose, two groups of mice were compared: a group infected with the bacterium and a group that had just recovered from the infection. Differences between the two groups were found in extent of bladder and kidney inflammation, depth of infiltration into the bladder wall, and in the increment of bladder volume. There was no clear-cut difference between the two groups in number of lymphocytes and monocytes, but the number of granulocytes was significantly lower in the bladder and kidneys of the recently healed animals. Likewise, the plasma cells were fewer in healed animals than in infected ones, the difference being statistically significant for bladders and on the verge of significance for kidneys.

DNA ploidy in papillary carcinoma of the thyroid gland in children and adolescents.

OBJECTIVE To evaluate DNA ploidy in papillary thyroid carcinoma in children in correlation to the clinical course of the disease. METHODS Flow cytometric DNA ploidy measurements were performed on formalin-fixed, paraffin-embedded tumor specimens from 14 children and 14 adult patients with papillary carcinoma of the thyroid gland. Analysis of DNA content was performed blind to patients age and clinical presentation. RESULTS Seven patients presented with cervical metastasis, one patient had distal metastasis and four patients had local invasion. All patients underwent total thyroidectomy. Seven children underwent bilateral modified neck dissection. Twenty-five tumors expressed diploid DNA content. No statistically significant difference in DNA content was observed between the tumors from child and adult patients. No correlation was found between DNA content and aggressive presentation in the pediatric group. CONCLUSION Our primary results indicate that diploid DNA content is common in papillary thyroid carcinoma in children and aggressive clinical presentation is not associated with DNA aneuploidy. Larger prospective studies and long-term clinical follow-up is warranted to document the clinical significance of these observations.

Enhanced cytologic detection of early stage mouse bladder tumor following induction of uroepithelial cell shedding.

Previous studies from our laboratory have shown that some Escherichia coli endotoxins are capable of inducing massive normal urothelial cell shedding. In the present study we investigated whether endotoxin-induced shedding improves cytologic detection of early stage mouse bladder cancer. Mouse bladder tumor (MBT-2) cells were implanted intravesically in the submucosa of C3H female mice. Ten to 21 days later the bladders were irrigated with saline followed by instillation of endotoxin. The bladder contents of each mouse were aspirated and examined cytologically together with the bladder wash specimen. Shedding of epithelial cells was observed in only 32% of the saline irrigated specimens compared with 93% after endotoxin instillation (p < 0.00001). Analysis showed an overall accuracy rate of 39% after saline barbotage versus 78% following endotoxin administration (p < 0.00001). These results indicate that intravesical instillation of specific bacterial endotoxin significantly increases the extent of cytologic detection of early stage superficial bladder cancer.