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Biomedical subjects

Janusz Skierski

Publications and source records attributed to Janusz Skierski.

6 recordsLinked to original sources
PubMed2006

EGFP fluorescence as an indicator of cancer cells response to methotrexate.

Methotrexate action in viable cells was monitored by registering changes in EGFP (Enhanced Green Fluorescent Protein) fluorescence intensity. Treatment with 1 microM methotrexate for 48 h of human colorectal adenocarcinoma C85 cells, stably transfected to express EGFP, caused 5-fold increase in EGFP fluorescence assayed by flow cytometry with no distinct increase in EGFP protein level. This was correlated with morphological changes, including an increase of cell granularity and cell shape flattening, as well as cell cycle G1 phase arrest revealed by DNA content analysis. Methotrexate removal allowed the morphology of the cells in culture to revert in 10 days to normal. The cells that survived methotrexate exposure were propagated as C85r cell subline and displayed kinetics of methotrexate sensitivity parallel to that of the parental C85 line. As the increase in EGFP fluorescence could also be visualized by fluorescence microscopy, this reporter system may be employed to image methotrexate action in cancer cells in living models.

Adenocarcinoma↗
PubMed2006

High dose of intravenously given glucocorticosteroids decrease IL-8 production by monocytes in multiple sclerosis patients treated during relapse.

The aim of our study was to determine whether high doses of intravenous methylprednisolone have significant impact on immune parameters during the multiple sclerosis (MS) exacerbations. Peripheral blood of 32 MS patients was evaluated, using two-color flow cytometry before glucocorticosteroids and after 7 days from starting therapy. Significant increase of B cells, decrease of NK cells and monocytes producing IL-8 were observed after treatment. IL-8 is one of the cytokines responsible for blood-brain-barrier disruption and migration of immune cells to the central nervous system; in this aspect, explaining glucocorticosteroid effects during MS exacerbations.

Adult↗
PubMed2005

Tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) as selective inhibitors of protein kinase CK2: evaluation of their effects on cells and different molecular forms of human CK2.

The development of selective cell-permeable inhibitors of protein kinase CK2 has represented an important advance in the field. However, it is important to not overlook the existence of discrete molecular forms of CK2 that arise from the presence of distinct isozymic forms, and the existence of the catalytic CK2 subunits as free subunits and in complexes with the regulatory CK2beta subunits and, possibly, other proteins. This review examines two recently developed, and presently widely applied, CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBBt) and the related 4,5,6,7-tetrabromobenzimidazole (TBBz), the latter of which was previously shown to discriminate between different molecular forms of CK2 in yeast. We have shown, by spectrophotometric titration, that TBBt, with a pK(a) approximately 5, exists in solution at physiological pH almost exclusively (>99%) as the monoanion; whereas TBBz, with a pKa approximately 9, is predominantly (>95%) in the neutral form, both of obvious relevance to their modes of binding. In vitro, TBBt inhibits different forms of CK2 with Ki values ranging from 80 to 210 nM. TBBz better discriminates between CK2 forms, with Ki values ranging from 70 to 510 nM. Despite their general similar in vitro activities, TBBz is more effective than TBBt in inducing apoptosis and, to a lesser degree, necrosis, in transformed human cell lines. Finally, development of shRNA strategies for the selective knockdown of the CK2alpha and CK2alpha' isoforms reinforces the foregoing results, indicating that inhibition of CK2 leads to attenuation of proliferation.

Apoptosis↗
PubMed2003

Changes of percentages in immune cells phenotypes and cytokines production during two-year IFN-beta-1a treatment in multiple sclerosis patients.

OBJECTIVE: The aim of the study was to find out whether INF-beta-1a influences the immune profile of peripheral blood (PB) leukocytes in MS patients. METHOD: We have studied 20 patients with relapsing-remitting form of MS treated with INF-beta-1a using twocolor cytometry. We determined immune cells phenotypes and production of some cytokines: IL-4, IL-10, IL-12, IFN-gamma, before drug administration and after starting the treatment. RESULTS: In MS patients an increased percentage of CD14(+)CD86(+) cells and CD3(+)CD25(+) cells was noticed after 6, 9 and 12 months of INF-beta-1a therapy. Among cytokine-producing cells we noted an increased fraction of CD3(+)IL-4, CD14(+)IL-10 and CD14(+)IL-12 cells after 12 months, which decreased to the level observed before treatment after 24-month therapy. CONCLUSIONS: IFN-beta-1a treatment was associated with significant changes in immune response. This effect was mostly evident within the first year of treatment.

Adjuvants, Immunologic↗
PubMed2002

Apoptosis during ectromelia orthopoxvirus infection is DEVDase dependent: in vitro and in vivo studies.

Ectromelia virus (EV), which causes mousepox, is a member of the orthopoxviruses that are defined as being able to suppress apoptosis. Caspase-3 is one of the key effector proteases which regulates the apoptotic cascade and which is responsible for DNA fragmentation observed during apoptosis. It is well known that viruses, especially poxviruses, can inhibit caspase activity. Here, we report that EV can regulate apoptosis in vitro, suppressing the activity of caspases recognizing the DEVD (Asp-Glu-Val-Asp) motif (caspase-3 and -7) before successful virus replication is completed. Caspase-3 activity measurement showed that an increase in caspase-3 activity preceded the peak of DNA fragmentation demonstrated by TUNEL staining of L929 and RK-13 cells. By using specific caspase inhibitors (Ac-DEVD-CHO, Ac-IETD-CHO and zVAD-fmk), we showed that caspase-3 and -7 (DEVDases) are major effector caspases during EV-induced apoptosis in permissive L929 and RK-13 cell cultures. Apoptosis in vivo seems to play an important role during viraemia as well as during the clearance of EV from genetically susceptible BALB/c (H-2(d)) mice. However, as shown by measurement of caspase-3 activity, caspase-3 protein detection and M30-antibody staining, both DEVDases seem to play an important role during EV clearance from draining lymph nodes and conjunctivae at 15 days p.i. up to 20 days p.i., whereas in the liver and spleen DNA fragmentation coexisted with viral multiplication and secondary viraemia. Apoptosis was DEVDase dependent only in the liver, while spleen DNA fragmentation observed between 5 and 10 days p.i. was caspase independent. Therefore, we conclude that DEVDase- (caspase-3- and caspase-7-) dependent apoptosis is an important mechanism regulating the resolution of EV infection.

Animals↗