CNTF up-regulation of TIMP-2 in neuroblastoma cells.

The ciliary neurotrophic factor (CNTF) can regulate survival and differentiation of many types of developing and adult neurons; in metastatic SK-N-BE neuroblastoma cells, it promotes differentiation and neurite outgrowth. The expression of Gelatinase A (MMP-2) and its specific tissue inhibitor (TIMP-2), a degradative system whose balance is involved in matrix invasion and metastasis, was investigated in SK-N-BE cells cultured with and without CNTF or NGF. Zymographic analysis of conditioned media revealed that the cells constitutively secrete two gelatinases, mainly pro-MMP-2 but also traces of pro-MMP-9. In a time-course experiment in the presence of 25 ng/ml of CNTF, the MMP-2 mRNA expression showed no significant modulation, while TIMP-2 mRNA up-regulated to > 2-fold after 48 h and then fell dramatically. At the same concentrations, NGF showed no effect. TIMP-2 mRNA expression showed a dose-dependent increase of up to 8-fold from 1 to 250 ng/ml of CNTF and increased secretion of TIMP-2 was confirmed by Western blotting. MMP-2 was only slightly over-expressed under the same conditions, at either mRNA or protein level, with no correlation with neurocytokine concentration. These results suggest that boosting the expression of TIMP-2 by CNTF could restrain both matrix degradation following nervous system injury and neuroblastoma aggressiveness.

Identification of the 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein in human mononuclear leukocytes.

The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation.

Main clinical manifestations of a bleeding diathesis: an often disregarded aspect of medical and surgical history taking.

A suitable clinical evaluation of a bleeding diathesis is often forgone. The young doctor is often unprepared to describe in an accurate way the different types of bleeding. An adequate classification and adequate clinical information about a bleeding diathesis are instead of paramount importance. Bleeding may be cutaneous, mucous, articular, muscular, parenchymal, intracavitary, orificial. Each of these sites and forms may have diagnostic implications. An accurate description of the several forms of cutaneous bleeding (petechiae, purpuric spots, ecchymosis, haematomas, etc.) is needed for referrals and for controls. The correct evaluation of cutaneous bleeding manifestations of children (battered child syndrome) is absolutely important for clinical and medico-legal purposes. The same is true for the battering syndrome seen in women abused by their spouses. The grading of haemarthrosis in haemophilia patients is important for the follow-up. A proper description of haematuria is essential in suggesting the probable site of bleeding (kidney or bladder or urethra). A proper evaluation of bleeding may give also useful information on the general health status of the patients (presence of anaemia, poor nutrition, renal insufficiency, etc.). The combination of bleeding and thrombosis in the same patient is also a clinical challenge. The relationship between haemorrhage and thrombosis may be sequential or concomitant. Sequential thrombosis may occur in a patient confined in bed for a brain haemorrhage. Concomitant thrombosis and bleeding occur in DIC and in patients with thrombosis being treated with anticoagulants. Finally, it should be kept in mind that a proper evaluation of the bleeding diathesis of a given patient may help the caring doctor in ordering appropriate laboratory tests (e.g. a platelet count for petechiae, a PTT for a patient with haemarthrosis, etc.).

Expression study on D123 gene product: evidence for high positivity in testis.

A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared with D123, three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in alpha-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.