In this study membrane oestradiol (E) binding sites in the medial preoptic area-anterior hypothalamus (MPOA-AH) of ovariectomized (OVX) rats were characterized using standard radioligand binding techniques employing E conjugated to bovine serum albumin (BSA) at position 6 and radiolabeled with 125I (E-6-[125I-BSA]). In previous studies binding of a radioactive conjugate of progesterone (P) and BSA (P-3-[125I-BSA]) was examined using the same membrane preparation. E-6-[125I-BSA] binding was linear across a tissue concentration range of 0.005-0.02 mg protein/0.1 ml of membrane suspension. An association T1/2 of 9.5 min and a dissociation T1/2 of 52.1 min for E-6-[125I-BSA] were derived from kinetic experiments. Competition binding experiments revealed high (Ki=0.63+/-(0.50 nM) and low (Ki=161.5(96.5 nM) affinity binding sites for E-6-[125I-BSA], demonstrating different binding parameters than shown in our previous work for P-3-[125I-BSA] binding. Further studies on MPOA-AH membranes treated with cholera toxin (CTX) and GTPgammaS suggested that E-6-BSA binding sites are associated with G proteins. E-6-[125I-BSA] binding demonstrated both high-and low-affinity sites. GTPgammaS added to the assay reduced both E-6-[125I-BSA] and P-3-[125I-BSA] binding suggesting that G proteins are associated with both binding sites. Extensive analysis of both E-6-[125I-BSA] and P-3-[125I-BSA] binding sites demonstrated a reciprocal relationship such that high-affinity E-6-[125I-BSA] binding sites exhibit low affinity for P-3-[125I-BSA] and low-affinity E-6-[125I-BSA] binding sites exhibit high affinity for P-3-[125I-BSA]. Preincubating membranes with CTX or GTPgammaS reduced high-affinity E-6-[125I-BSA] binding and enhanced high-affinity P-3-[125I-BSA] binding. These results suggest that, in the MPOA-AH, membrane steroid binding sites exist in two interconvertible conformations that preferentially bind either E-6-BSA or P-3-BSA, depending on their association with a G protein. Additional studies with free steroids revealed that: (1) oestrogens (17beta-oestradiol, diethylstilbestrol) as well as synthetic oestrogen antagonists tamoxifen and ICI 182 780 displaced P-3-[125I-BSA] further suggesting a relationship between membrane binding sites for E and P-3-[125I-BSA] binding sites; and (2) treatment of OVX rats with E decreased displacement by P-3-BSA and increased displacement by ICI 182,780 and tamoxifen suggesting these antagonists affect membrane P-3-[125I-BSA] binding sites after in-vivo E treatment. The membrane binding sites for E and P demonstrate interrelationships not demonstrated by their nuclear receptors.

Background: Imatinib is tyrosine kinase inhibitor (TKI) and as a targeted anti-cancer agent has significantly changed chronic myeloid leukemia (CML) prognosis and patient survival. Currently TKI is the main therapy in CML Philadelphia chromosome-positive (Ph-positive) cases. When generics of imatinib appeared in the pharmaceuticals market, reimbursement policies in many countries switched to using generics or encouraged use of generic imatinib to lower the expenses. Cost savings were substantial; however, for doctors and CML patients the efficacy, safety and quality of generic imatinib were an issue of concern. Objective: Since the global number of CML patients, who in the future will have to switch from original imatinib to generic imatinib, is high, the aim of study was to monitor, whether during 24 months of generic imatinib usage patients maintain the achieved major molecular response (MMR) or whether the treatment results are inferior. Methods: We conducted a retrospective study, which included CML patients, who were above 18 years of age and who until May 2013 had used at least for 2 years (24 months) the original imatinib, and following that used at least for 24 months one of the generic imatinib medicines. In 2013, before switching to generic imatinib, all patients had reached MMR in accordance with European LeukemiaNet (ELN) Guidelines. Every three months blood count, BCR-ABL fusion gene (BCR-ABL), biochemical analysis and side effect were monitored. Results: Our study proved that CML patients, who had achieved MMR by original imatinib therapy, retained MMR during 24 months of generic imatinib therapy. Nobody was switched to second line generation TKI. During observation period neither haematological, nor non-hematological toxicity was found. Conclusion: Our study proved that CML patients, who had achieved MMR by original imatinib therapy, retained MMR during 24 months of generic imatinib therapy. This demonstrates that generic imatinib is not inferior to original imatinib. As to expenses, the annual costs of generic imatinib are lower by 96%, which is a significant benefit to health-care financing.

Background: Up to now, the immune status of chronic lymphocytic leukemia (CLL) patients in association with the expression of zeta-chain-associated protein kinase 70 (ZAP-70) in leukemic cells has not been evaluated. Aim: The aim of this work was the study of the peripheral blood (PB) T-lymphocyte phenotypes in ZAP-70-positive (ZAP-70+) and ZAP-70-negative (ZAP-70−) untreated patients with CLL. Materials and Methods: ZAP-70-, CD25-, CD3-, CD4-, and CD8-positive lymphocytes were enumerated by flow cytometry in PB of 120 untreated CLL patients. CD8+, CD3+CD4+ and CD3+CD25+ cells were counted for the non-leukemic lymphocytes. Results: The patients were distributed into two groups: the ZAP-70+ group of high CLL progression (n = 61), and the ZAP-70− group of low CLL progression (n = 59). In the ZAP-70+ group, the ratio CD4/CD8 (0.33 ± 0.62; p = 0.001) and the numbers of the CD3+ (34.8 ± 8.1%; p = 0.01), CD3+CD4+ (24.4% ± 4.8; p = 0.001), and CD3+CD25+ (6.2 ± 0.91%; p = 0.001) lymphocytes were reduced and the percentage of the CD8+ cells (73.1 ± 4.6%; p = 0.0001) was above the norm. In the ZAP-70− group, the number of the CD3+CD4+ cells (36.9 ± 6.1%; p = 0.001) was within the norm, but the numbers of the CD8+ (11.3 ± 1.1%; p = 0.0001) and CD3+ (41.2 ± 5.3%; p = 0.05) lymphocytes were reduced; the ratio CD4/ CD8 (3.26 ± 0.88; p = 0.001) and the percentage of the CD3+CD25+ cells (27.1 ± 3.4%; p = 0.0001) were above the norm. Conclusions: Our data show that the increased CD4/CD8 ratio, caused by the reduced number of the CD8+ lymphocytes, and the increased number of CD3+CD25+ cells are characteristic for the ZAP-70− group (slow progressing) of untreated CLL patients. In ZAP-70+ patients, the CD4/CD8 ratio was significantly below the norm indicating an active disease process. Results of our study contribute to identification of CLL patients with different prognosis in routine diagnostic/prognostic procedures.

Chemokines and their receptors direct migration and infiltration of immune cells. CCR1 and CCR2 maintain sequence similarity and respond to a number of the same chemokines secreted in lymphoid organs. Expression of CD38 on leukemic cells has been associated with poor clinical outcomes in patients with chronic lymphocytic leukemia (CLL) and is considered as the negative predictor of progression. In our study of newly diagnosed CLL patients, which included 39 CD38-positive and 22 CD38-negative patients, CCR1 and/or CCR2 were always detected, using flow cytometry, on the peripheral blood (PB) CD19+CD5+ lymphocytes in patients with >30% of the CD38+ CD19+CD5+ lymphocytes (n = 16). Spearman’s rank correlation analysis determined correlations between the frequency of the CCR1- and CCR2-expressing PB CD19+CD5+ lymphocytes and the frequency of the CD38-positive CD19+CD5+ lymphocytes (rs = 0.50 and rs = 0.38, respectively). No significant correlations were observed between ZAP70 mRNA expression levels in PB mononuclear cells and the frequency of the circulating CCR1+ or CCR2+ CD19+CD5+ lymphocytes. Further association studies are needed to verify prognostic relevance of the CCR1/CCR2 expression on leukemic cells in CLL patients at diagnosis. We suggest that CCR1/CCR2 signaling pathways could represent attractive targets for development of CLL anti-progression therapeutics.