Effect of DiscoGel treatment of the intervertebral disc at MRI.

AIM: To determine the impact of ethanol gel chemonucleolysis (EGCh) on the radiological picture of the treated intervertebral disc, the relationship between the initial radiological status and the clinical status of the patient after EGCh treatment, and the optimal radiographic criteria for qualifying a patient for EGCh treatment. MATERIALS AND METHODS: The study involved a group of 45 patients (25 men and 20 women) aged 23-68 years (46 ± 11) who underwent an EGCh procedure after qualification, radiography, and clinical questionnaire evaluation. RESULTS: The results showed a decrease in the size of the protrusion and Gadolinium-Enhanced (GI) zone in the treated intervertebral disc. The presence of a high-intensity zone (HIZ) on baseline magnetic resonance imaging was found to be a good predictor of the timing and outcome of treatment, and an increase in disc height was observed in adjacent segments. CONCLUSION: These findings suggest that EGCh is a promising treatment for spine diseases, and the HIZ on baseline magnetic resonance imaging can be used as a qualification criterion for this procedure.

Myokine irisin-induced protection against oxidative stress in vitro. Involvement of heme oxygenase-1 and antioxidazing enzymes superoxide dismutase-2 and glutathione peroxidase.

Irisin is a recently discovered myokine reported as protective protein released from exercising skeletal muscles. Although myokines were recently reported to possess the antioxidizing properties, the impact of irisin on the functions of macrophages with respect to its anti-inflammatory potential has not been fully elucidated. Here, we determined the ability of irisin to interact with reactive oxygen species (ROS) in RAW 264.7 murine macrophages. The macrophages were pre-incubated with irisin (0 - 50 nM), some of which had undergone additional co-incubation with bacterial lipopolysaccharide (LPS) (100 ng/ml). Cell viability, the reactive oxygen species scavenging potential as well as the mRNA and protein expression of key oxidative stress factors such as superoxide dismutase 1 (SOD-1), superoxide dismutase 2 (SOD-2), glutathione peroxidase (GSH-Px), catalase 9 (Cat-9), nuclear factor (erythroid-derived 2)-like 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) were evaluated. We found that irisin applied in a concentration of 50 nM significantly attenuated the production of harmful H2O2 and this effect appears to be mediated by a significant increase in the expression of key enzymes involved with antioxidative stress pathways including SOD, GSH-Px and Cat-9 predominantly observed after stimulation of these cells with LPS. We conclude that 1) irisin exhibits a potent antioxidant and anti-inflammatory activities in non-stimulated and LPS-stimulated isolated murine macrophages in vitro, and 2) this protective and antioxidative activity of irisin in vitro might be considered as an important component of protective action of this peptide in vivo, especially under condition of exercise.

Mitochondrial transmembrane potential in spontaneous and camptothecin-induced apoptosis of melanotic and amelanotic melanoma cells.

In this work we tried to estimate the role of mitochondria in the ability of cells of two: melanotic and amelanotic transplantable melanoma lines to undergo spontaneous and camptothecin-induced apoptosis. We measured mitochondrial transmembrane potential (DeltaPsi) changes during culture without (spontaneous apoptosis) and with camptothecin (induced apoptosis) by using JC-1 staining and flow cytometry analysis. Cytochrome c release and PARP cleavage as the biological effects of DeltaPsim changes belonging to the phenomena observed during apoptosis were estimated by Western blotting. The results of our investigations showed in both transplantable melanoma cells the features indicating apoptosis: DeltaPsi changes, cytochrome c release and PARP cleavage, but the degree of observed changes depended on the phenotype of melanoma cells examined. After camptothecin treatment the changes were more pronounced in the amelanotic melanoma cells- the more aggressive line.

Heterogeneity of the surface material in isolated cells of transplantable hamster melanomas.

The heterogeneity of the surface material released by trypsin in isolated cells of melanotic and amelanotic melanomas was studied by the method of separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoretic patterns of the surface material derived from two kinds of melanoma showed some differences. The differences in the surface proteins and glycoproteins seem to be related to the biological properties of both melanomas.

Persistence of non-typeable Haemophilus Influenzae in the pharynx of children with adenotonsillar hypertrophy after treatment with azithromycin.

This study was performed in children with adenotonsillar hypertrophy to evaluate the effect of azithromycin (AZT) on the presence of NTHi in monocyte/macrophages (CD14(+) cells) of adenoids/tonsils and the persistence of NTHi after adenotonsillectomy. A total of 36 pediatric patients participated in the study: 20 children were treated with AZT before adenotonsillectomy, and 16 children did not receive the antibiotic prior to surgery. NTHi were identified by culture and PCR in swabs and tissue samples. NTHi was detected in the lysates of CD14(+) cells by fluorescence in situ hybridization (FISH) and by culture. The molecular typing was used to cluster NTHi isolates from each child. The intracellular NTHi was found in 10 (62.5%) untreated patients and was identified in three (15%) azithromycin-treated patients (P = 0.003). The proportion of the persistent NTHi strains was similar in both groups. AZT treatment followed by adenotonsillectomy did not completely eliminate NTHi from pharynges; however, it significantly reduced the risk of carriage of Haemophilus influenzae inside the CD14(+) cells.

The DNA ploidy and proliferative activity of transplantable melanoma cells in regard to their secretory function.

The study concerns DNA ploidy and proliferative activity of the cells of two hamster transplantable melanoma lines - differing in many biological features - in the light of possible changes in their secretory activity. DNA ploidy was determined from the index of propidium iodide (PI) stained DNA content (DI), while the proliferative activity was defined as the percentage of cells in S and G2/M cell cycle phases. The secretory activity was described by determining total protein, interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), oncostatin M (OSM), interleukin 10 (IL-10) and nitric oxide (NO) content in the supernatants after 1, 6, 24 and 48 hours in cell culture. Our results indicate that melanotic melanoma cells (Ma) have a near-tetraploid DNAcontent and about 18% of proliferating cells, while amelanotic melanoma cells (Ab) - have a near-triploid DNA content and almost twice as many proliferating cells. The Ab cells, in comparison with Ma cells, secreted in vitro less total protein and most of the cytokines examined except OSM, but a spontaneous alteration of transplantable melanoma was accompanied by an increase of the quantity and dynamics of NO secretion. So, the cells of two melanoma lines have their own characteristic pattern of secretory function. But, the aneuploidy which accompanied the changes in cell differentiation of the studied melanoma lines, although seemed to reflect their changes in the proliferative activity, nevertheless did not reflect, in a direct way, differences in the secretion of the substances studied.

Characterization of NO and cytokine release by transplantable melanoma cell lines in relationship to their differentiation.

Changes in the secretory activity of two transplantable melanoma lines (differing in many biological features) as regards nitric oxide (NO) release and interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and oncostatin M (OSM) secretion were the subject of the present study. The results obtained show that a spontaneous alteration of the hamster native melanoma line into an amelanotic one is accompanied by a global change of the secretory activity, but there was not a distinct correlation between the endogenous cytokine secretion and NO secretion by cells of both melanoma lines.

Reactivity of peripheral blood lymphocytes in hamsters with transplanted melanomas in the rosette test.

Capability of peripheral blood lymphocytes of control and immunized hamsters of spontaneously binding sheep erythrocytes was studied at two different temperatures: at 29 degrees C to detect "Fc-negative" subpopulations of T lymphocytes, and at 4 degrees C to detect "Fc-positive" subpopulations. The percentage of rosettes in melanoma-immunized hamsters decreased at 29 degrees C and increased at 4 degrees C. More pronounced changes were observed after immunization with amelanotic melanoma.

Studies on antigenicity and immunogenicity of transplantable melanoma in hamster by using migration inhibition test.

The migration of spleen cells taken from hamster bearing transplantable melanoma was measured in the presence of melanoma cells isolated from appropriate tumors. The inhibition of macrophage migration in the presence of both amelanotic and melanotic tumor cells was observed, however higher response in the case of amelanotic tumor cells was detected.

Comparison of the antigenicity of melanotic and amelanotic melanoma cells after the release of surface glycoproteins by trypsin.

The antigenicity of isolated cells of malanotic and amelanotic melanomas after the release of surface glycoproteins by trypsin was studied by the migration inhibition test. It has been found that after treatment with trypsin the antgenicity of melanotic melanoma cells increased, while the amelanotic melanoma cells lost their ability to inhibit macrophage migration.

Study on the complement-dependent cytotoxic effect of the sera of hamsters bearing transplanted melanoma.

Complement-dependent cytotoxic effect of the sera of hamsters bearing transplanted melanomas: melanotic and amelanotic, was examined in relation to isolated melanocytes of these melanomas. A specific, cytotoxic effect, ranging 34-47% of the sera of hamsters with both types of melanomas in relation to the cells of these tumors was found. No relationship between the biological properties of melanoma and cytotoxic activity of serum was noted.

Comparative analysis of the surface antigens of transplantable melanomas in hamsters. II. Examination of serologic reactivity of sera with isolated melanocytes by the cross-reaction and absorption test.

The reactivity of sera of hamsters bearing melanotic and amelanotic melanoma with isolated melanocytes was evaluated using immunoadherence test and cross-reactions. Besides, the ability of neoplastic melanocytes to absorption of antibodies from these sera, was tested. The reactivity of cells of both melanomas with serum of hamsters bearing the original type--melanotic melanoma--appeared the same and twice as high in amelanotic cells with serum of animals bearing amelanotic melanoma. This suggests that in the course of spontaneous alteration, the new form--amelanotic, does not lose the original antigens but acquires new ones, not present on the melanotic cells. The above findings were confirmed by the absorption test.

Surface glycoprotein components in isolated melanotic melanoma cells in the golden hamster.

A study of glycoprotein components in trypsin-digested material from the surface of isolated melanotic melanoma cells showed presence of amino sugars, protein-bound hexoses, fucose and sialic acids. The high content of fucose and low content of sialic acids in the surface material from the cells were striking.

Characterization of surface antigens of transplantable melanomas in hamster on the basis of the macrophage migration inhibition test.

Migration of peritoneal macrophages and macrophages isolated from spleen of hamsters with transplanted melanotic melanoma in presence of amelanotic melanoma cells as an antigen was studied. Furthermore, migration of macrophages in hamsters grafted with amelanotic melanoma in presence of melanotic melanoma cells as an antigen was determined. In both cases, inhibition of migration of macrophages was not observed which suggests that both melanoma lines do not have common antigens but individually specific ones.

Concanavalin A induced agglutinability of the isolated cells of hamster melanotic and amelanotic melanomas.

The agglutination of cells isolated from solid melanotic and amelanotic malanomas in hamsters by Concanavalin A was studied. It has been found that the agglutination intensity of the two kinds of melanoma cells was different and depended on the concentration of the lectin. It has been suggested that the different susceptibility of the cells to Concanavalin A is related to changes of surface glycoproteins, resulting from a spontaneous alteration of the melanotic melanoma into amelanotic one.

Comparative analysis of surface antigens of transplantable melanoma in hamsters. I. Studies on serological reactivity of sera with isolated melanocytes by using the immunoadherence test.

Immunoadherence test was used to study the reactivity of sera of hamsters immunized with transplantable melanotic and amelanotic melanomas. In the sera of immunized animals the presence of antibodies, specifically reacting with melanocytes isolated from these tumors, was confirmed.

Expression and activity of P-glycoprotein in transplantable hamster melanomas.

In the study described here we investigated the possibility of an association between the aggressiveness of melanoma and multidrug resistance phenotype by analyzing the expression and activity of P-glycoprotein (Pgp) in two genetically related transplantable hamster melanomas--a melanotic (Ma) and an amelanotic (Ab) form --which differed in aggressiveness and metastatic potential. Flow cytometric analysis of Pgp activity (using a verapamil-sensitive rhodamine R123 exclusion test) as well as Western blotting of cellular lysates showed its preferential (although not very marked) expression in the Ab melanoma cells. The Ab melanoma cells also exhibited a higher proportion of tumor-infiltrating lymphocytes (TIL), mostly of T cell phenotype, that may have reflected a higher immunogenicity of the tumor. In conclusion, Pgp activity appeared to be associated with less-differentiated more aggressively metastasizing melanoma (the Ab variant) although its role in maintaining this phenotype remains to be established.

Heterogeneity of peritoneal macrophages in hamsters-bearing transplantable melanomas in relation to their transglutaminase.

The level of transglutaminase activity has been investigated by the fluorescence method in peritoneal macrophages of control and transplantable melanomas bearing hamsters. If the transglutaminase activity is concerned the macrophages in no one of the groups examined were a homogenous population. Differences in macrophage heterogeneity in hamsters bearing two melanoma lines of the same origin but showing changed biological properties were observed.

Tumoricidal effect of macrophages on transplantable melanoma cells with regard to their sensitivity to exogenous cytokines.

We studied the correlation between the cytotoxic effect of hamster peritoneal macrophages from animals bearing transplantable melanomas of common origin but differing in many biological features on the cells of those melanomas and the sensitivity of the melanoma cells to exogenous OSM, TNF-alpha and IL6. Our results did not show such a correlation.

Changes of expression of peritoneal macrophages Fc and C3 receptors induced by transplantable melanomas.

Expression of Fc and C3 receptors was studied in the rosette tests on isolated peritoneal macrophages of control and melanoma-bearing hamsters. In hamsters with transplanted melanomas an increase of the percentage of macrophages with Fc and C3 receptor expression was observed. The increase was prominent among macrophages from animals with transplanted amelanotic melanoma, a tumor line with greater malignancy and changed antigenicity and immunogenicity.