Targeting MCL-1 sensitizes FLT3-ITD-positive leukemias to cytotoxic therapies.

Patients suffering from acute myeloid leukemias (AML) bearing FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) have poor outcomes following cytarabine- and anthracyclin-based induction therapy. To a major part this is attributed to drug resistance of FLT3-ITD-positive leukemic cells. Against this background, we have devised an antibody array approach to identify proteins, which are differentially expressed by hematopoietic cells in relation to activated FLT3 signaling. Selective upregulation of antiapoptotic myeloid cell leukemia-1 (MCL-1) was found in FLT3-ITD-positive cell lines and primary mononuclear cells from AML patients as compared with FLT3-wild-type controls. Upregulation of MCL-1 was dependent on FLT3 signaling as confirmed by its reversion upon pharmacological inhibition of FLT3 activity by the kinase inhibitor PKC412 as well as siRNA-mediated suppression of FLT3. Heterologously expressed MCL-1 substituted for FLT3 signaling by conferring resistance of hematopoietic cells to antileukemia drugs such as cytarabine and daunorubicin, and to the proapoptotic BH3 mimetic ABT-737. Conversely, suppression of endogenous MCL-1 by siRNA or by flavopiridol treatment sensitized FLT3-ITD-expressing hematopoietic cells to cytotoxic and targeted therapeutics. In conclusion, MCL-1 is an essential effector of FLT3-ITD-mediated drug resistance. Therapeutic targeting of MCL-1 is a promising strategy to overcome drug resistance in FLT3-ITD-positive AML.

[Strategies for hormonal ovarian stimulation in IVF, based on materials of the centre for assisted reproduction "varna"].

During a two years' period (2008-2009), 834 patients underwent IVF in the Centre. Long protocol was used where normal response was expected; for high risk patients was used protocol with antagonist and induction of ovulation with agonist or 5 000 IU HCG; and when poor response was expected, flare up protocol was applied. 363 clinical pregnancies were registered--50% for transfer and 43.4% for cycle. Experience shows that antagonist - agonist protocol reduces significantly but not completely the risk of OHSS. On poor responders, the standard short protocol shows better results than any of the recently suggested protocols. In future, the so called "controlled ovarian hyperstimulation" will be performed only in its soft version and when certain indications exist.

Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

Widespread detection of VEB-1-type extended-spectrum beta-lactamases among nosocomial ceftazidime-resistant Pseudomonas aeruginosa isolates in Sofia, Bulgaria.

A total of 132 ceftazidime-resistant clinical isolates of Pseudomonas aeruginosa were collected during 2001-2005 from 5 university hospitals in Sofia, Bulgaria to assess the current levels of antimicrobial susceptibility and to evaluate resistance mechanisms to beta-lactams. Antimicrobial susceptibilities were detected by a disk diffusion method and E-test. Polymerase chain reaction amplification and sequencing of bla(VEB-1 )and bla(PER-1 )were performed. The antibiotic resistance rates were: to piperacillin 90.2%, piperacillin/tazobactam 52.3%, ceftazidime 94.7%, cefepime 88.6%, cefpirome 98.5%, aztreonam 85.6%, imipenem 66.6%, meropenem 63.6%, amikacin 81.1%, gentamicin 84.8%, tobramycin 89.4%, netilmicin 57.6%, ciprofloxacin 83.4%. Structural genes for VEB-1 extended-spectrum beta -lactamases (ESBLs) were found in 75 (56.8%) of the isolates. PER-1 ESBLs were not detected. The VEB-1-producing strains were more resistant than VEB-1 non-producers to amikacin, gentamicin, tobramycin and ciprofloxacin ( P<0.001). VEB-1 appears to have a significant presence among ceftazidime-resistant P. aeruginosa isolates from Sofia.

[Uroinfections and the possibilities of therapy with levofloxacin]. / Uroinfektsii i vuzmozhnosti za terapevtichno povliiavane s levofloxacin.

UNLABELLED: The common occurrence, of inflammatory diseases in the urinary tract and the genitals in men, is quite a reason for identification of the agents and looking for some new chances of therapeutic treatment. MATERIALS AND METHODS: For a period of two years (2002-2003) we accomplished 4810 microbiological tests of urine and prostate secretion on patients with uroinfections. The cultures are completed on standard nutrition medium, as the biochemical identification of the strains is accomplished by the systems mini API and "bio Merieux" BBL Crystal, "Becton Dickinson". Meanwhile for a period of one year, 113 patients with different uroinfections are treated and studied. In 16 (14.1%) Levofloxacin (Tavanic-R) is applied i.v. 500mg/daily as in the rest cases 97 (85.9%) Tavanic are applied per oral in different therapeutic doses. RESULTS AND DISCUSSIONS: The most common agent in our study was the E. Coli, as for the year 2003 we read an increase of the infections caused by E.Coli with 3.4% comparing to the previous one (38.5% to 35.1%). The differentiation of patients treated with Levofloxacin in groups is completed as follows: with acute uncomplicated cystitis--pyelonephritis after ESWL--15.9%; with postoperative uroinfections--20.3%; with chronic post-tuberculosis pyelonephritis--4.4%; and with inflammatory diseases of the male genitals--37.2%.

Spontaneous fusion of 'os acetabuli' after triple pelvic osteotomy.

In acetabular dysplasia, an overloading of the acetabular rim can cause a stress fracture, creating an 'os acetabuli', or a lesion of the acetabular labrum. At puberty, the os acetabuli seems to be the epiphysis of the os pubis. We present the case of a 14-year-old girl with acetabular dysplasia and spontaneous fusion of an 'os acetabuli' after biomechanical correction by triple pelvic osteotomy. Our report supports the correctness of the biomechanical principle: reorientation of the acetabulum results in a better coverage of the femoral head, reduces the stress at the acetabular rim, shifts the os acetabuli out of the stress region, and may allow union of the bony fragment with the acetabulum.

[The results of using ICSI at the Center for Assisted Reproduction, Varna]. / Rezultati ot prilozhenieto na IKSI v Tsentura za asistirana reproduktsiia--Varna.

The authors present the results of 31 consecutive cycles of ICSI. Fertilization occurred in 251 /88%/ of 279 oocytes which resulted in 8 clinical pregnancies /27% per transfer/--3 singleton, 2 multiple uterine pregnancies, 2 abortions and 1 ectopic pregnancy.

Directed alterations of the X-chromosomal ribosomal gene repeats in mutant sc8 of Drosophila melanogaster.

The patterns of the ribosomal DNA (rDNA) repeat units in seven Drosophila melanogaster inversional mutants have been studied. Among them, only the In(1)sc8 and its deletional derivative Df(1)mal12 female rDNAs exhibited significant reduction in the size of nearly all units, compared to the wild-type females (Canton S, Oregon R). Further investigation shows that each kind of repeat (insertion-free, insertion-containing) in the Xsc8 rDNA array is highly enriched with short (reduced to 4 kilobases) intergenic spacers (IGSs). We revealed two main types of rearrangements. Only part of the 4 kb IGSs display variable length deletions (0.2-0.6 kb) at the 5' ends, within the so-called '1900' base pair (bp) region, recognizable by restriction endonuclease AluI. The presence of additional 100-150 bp DNA in the start portion of this region has also been demonstrated. In contrast, the 3' end spacer regions, corresponding to the external transcribed spacer, do not show any changes in size. These data indicate how reductions of approximately 1.1 kb DNAs in sc8 IGSs, carrying both the rearranged and non-rearranged '1900' sequences, are achieved: the fixed decrease of a number of 240 bp AluI subrepeats, clustered in the central IGS portion, also contribute. None of the other similar inversional mutants examined has so many IGS variants. Therefore, alterations in the Xsc8 rRNA gene cluster seem not to be dependent on its inversional status.

Complex alterations of the ribosomal gene spacers in mutant sc8 of Drosophila melanogaster.

Extreme changes in the proportions of the rDNA intergenic spacers (IGSs) have previously been demonstrated by us in the X-chromosomal rDNA array of the Drosophila melanogaster mutant sc8, where nearly all IGSs were found to be reduced in size. We have cloned different structural variants of the Xsc8 ribosomal units and mapped their spacers by restriction endonuclease analysis. Most of the IGSs exhibited complex rearrangements within the subrepeated region at their 5' terminus. The sequence data revealed the presence of an additional 100 bp subrepeat. In addition, extended deletions/substitutions down to the 18S gene were also found. Examination of the Ysc8 IGS polymorphism indicates that some of the X-chromosomal IGS variants are probably the result of X-Y interchanges. The rarity of alternative recombination products implies that the overabundant deleted spacers in sc8 are generated by nonreciprocal recombination. Our results demonstrate nonrandom distribution of deletional breakpoints within the "1900" region in sc8 and show that the deletions do not truncate the internal IGS subregions at either breakpoint but eliminate them as discrete blocks.

Pharmacokinetic and pharmacodynamic approach for comparing two therapeutic regimens using amikacin.

The efficiencies of two dosage schedules of amikacin (2 x 10 mg/kg of body weight per 24 h and 1 x 20 mg/kg/24 h intramuscularly for 5 days) against Pseudomonas aeruginosa sepsis in rabbits were compared. Blood samples were drawn at various times after the first application, and amikacin concentrations in serum were assayed microbiologically. The dynamics of the bactericidal effect of amikacin was simulated in vitro with the same strain of P. aeruginosa. No regrowth was found with the 20-mg/kg dose when the bacterial inoculum was in contact with experimental and theoretically predicted serum amikacin concentrations. The killing effect was present even when the drug levels decreased considerably below the MIC. The interrelationship between simulated amikacin concentrations in serum and the corresponding average killing rates was described appropriately by the standard Emax model. The higher amikacin dose performed its bacterial effect faster and the drug persisted longer in the blood. The two amikacin regimens were therapeutically equivalent, but the once-daily schedule had some advantages over the twice-daily drug administration which became evident when both the pharmacokinetic and the pharmacodynamic parameters of the drug were considered.

[Contamination during transurethral manipulations of the upper urinary tract]. / Kontaminatsiia pri transuretralni manipulatsii vurkhu gornite pikochni putishta.

A four-year observation was carried out on a series of 289 patients who had undergone miscellaneous transurethral operations and manipulations on the upper urinary tract. Clinical and microbiological studies were performed in an attempt to record the incidence of urinary tract infections as a consequence of contamination following transurethral manipulations. Random groups of in-lying patients at two urologic clinics served as controls. It was found that the hazard of contamination with pathogenic bacteria causative agents of urinary tract infections in the study group was smaller than usually believed. The mean infection rate was 17.3 per cent, vs. 35.6 and 45.5 per cent respectively in the control groups. Higher was the risk of contamination after manipulations terminating without temporary drainage of the urine from the upper urinary tract. In the contamination processes was involved a broader spectrum of microorganisms with more resistant strains. Conclusions are drawn on the possibilities to reduce the contamination levels and to prevent the consequences of infection. This should be achieved not by limiting the manipulations, but by mastering the endoscopic technique and nontraumatic work with this technique.

[Staphylococcal coagglutination test for detecting antibody-coated bacteria in the urine]. / Stafilokokov koaglutinatsionen test za otkrivane na bakterii, pokriti s antitela, v urinata.

An easy and rapid staphylococcal coagulation test is described for detection of bacteria, covered with antibodies in urine of patients with chronic pyelonephritis as an additional index of the etiological role of the isolated microorganisms and localization of the pathological process. Inactivated and stabilized protein A containing strain S. aureus Cowan's (SSM 2352) was used. The coagulation test is negative in infectious processes of the lower segments of urinary system and bacterial contamination of urine.

[Microbial spectrum in chronic pyelonephritis and the drug sensitivity of the microorganisms]. / Mikroben spektur pri khronichni pielonefriti i lekarstvena chuvstvitelnost na mikroorganizmite.

The authors studied the etiological spectrum of 226 patients with chronic pyelonephritis, with 926 microbic strains isolated from the urocultures. The most frequent microbic causative agents were E. coli (40,82%), Proteus (a total of 17,82%), Enterobacter (8,86%), Klebsiella (8,64%), Pseudomonas (6,16%), Enterococcus (5,94%). The drug susceptibility of the majority of the isolated gram-negative microorganisms versus ampicillin and chloramphenicol, widely used in out-patient department practice, is relatively low. An important condition for the effectiveness of the treatment is the consideration given to the susceptibility of the microbic strains, isolated from the uroculture.