Imunosupresivní látky izolované z mikroorganizmů

Práce podává přehled imunosupresivních látek, soustřeďuje se na Cyklosporin A, FK 506 a Rapamycin, rozebírá jejich chemickou strukturu, mechanismus účinku, farmakokinetiku, stanovení hladiny a nežádoucí účinky. Zmiňuje také aplikaci v klinické praxi.Dokončená práce s úspěšnou obhajobo

Preparation of the polyclonal antibodies with anti-chymotrypsin specifity and determination of their functional affinity

Byly připraveny polyklonální specifické anti-chymotrypsinové protilátky imunizací tří jedinců druhu Oryctolagus cuniculus. Imunizační schémata se lišila dobou imunizace a množstvím chymotrypsinu v booster dávce. Průběh imunizace byl sledován stanovením titrů specifických protilátek metodou ELISA a stanovením aviditních indexů metodou eluce thiokyanátem amonným.Dokončená práce s úspěšnou obhajobo

Ionizing Radiation Sensitizes Leukemic MOLT-4 Cells to TRAIL-induced Apoptosis

One of perspective approaches in treatment of hematological malignancies is activation of death receptors for TRAIL. However, leukemia cells studied to date have shown variable susceptibility to TRAIL. Our study demonstrates that cells of acute T-lymphoblastic leukemia MOLT-4 are resistant to TRAIL and that ionizing radiation in the therapeutically achievable dose of 1 Gy sensitizes TRAIL-resistant cells MOLT-4 to the TRAIL-induced apoptosis by increase in death receptors for TRAIL DR5. When TRAIL is applied after the irradiation in the time of increased DR5 positivity more efficient cell killing is achieved

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation

Background: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. Material and methods: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. Results: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G2 phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 µg/l TRAIL for 16 h reduced D0 value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. Conclusion: TRAIL in concentration of 6 µg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction

Gamma-radiation-induced ATM-dependent signalling in human T-lymphocyte leukemic cells, MOLT-4

ATM kinase (ATM) is essential for activation of cell cycle check points and DNA repair in response to ionizing radiation (IR). In this work we studied the molecular mechanisms regulating DNA repair and cell death in human T-lymphocyte leukemic cells, MOLT-4. Apoptosis was evaluated by flow-cytometric detection of annexin V. Early apoptotic cells were determined as sub-G1 cells and late apoptotic cells were determined as APO2.7-positive ones. Proteins involved in ATM signalling pathway were analysed by Western-blotting. We observed a rapid (0.5 h) phosphorylation of ATM declining after 6 h after irradiation by all the doses studied (1.5, 3.0, and 7.5 Gy). Checkpoint kinase-2 (Chk-2) was also phosphorylated after 0.5 h but its phosphorylated form persisted 4, 2, and 1 h after the doses of 1.5, 3.0, and 7.5 Gy, respectively. The amount of p53 protein and its form phosphorylated on Ser-392 increased 1 h after irradiation (1-10 Gy). The lethal dose of 7.5 Gy caused an immediate induction and phosphorylation of p53 after 0.5 h post-irradiation. At the time of phosphorylation of p53, we found simultaneous phosphorylation of the oncoprotein Mdm2 on Ser-166. Neither ATM nor its downstream targets showed a dose-dependent response after 1 h when irradiated by the doses of 1-10 Gy. MOLT-4 cells were very sensitive to the effect of IR. Even low doses, such as 1.5 Gy, induced apoptosis 16 h after irradiation (evaluated according to the cleavage of nuclear lamin B to a 48-kDa fragment). IR-induced molecular signalling after exposure to all the tested doses was triggered by rapid phosphorylation of ATM and Chk-2. Subsequent induction of p53 protein and its phosphorylation was accompanied by concomitant phosphorylation of its negative regulator, oncoprotein Mdm2, and followed by induction of apoptosis

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to γ-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G0 phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing γH2A.X (1 h after γ-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3+ lymphocytes were A+. Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A+PI-) in comparison to non-stimulated PBMCs (38% A+ resp. 13.4%). After PHA-stimulation also the amount of γH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to γ-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3+ T lymphocytes by the dose of 4 Gy 65% of cells were A+