Introducing isotonic fluids into pediatric oncology.

Objective: Hypotonic fluids are commonly used in pediatric oncology despite evidence that these fluids can lead to hospital-acquired hyponatremia. This practice is most likely due to lack of data evaluating risks and benefits of isotonic fluids in pediatric oncology. To address this issue, our study investigates the effects of exchanging hypotonic fluids with isotonic fluids in a large pediatric oncology unit. Study Design: Prevalence of laboratory disorders before and after the change to balanced, isotonic fluids for all patients are compared in this retrospective analysis. Disturbances in electrolyte levels, fluid-, acid-base balance and kidney function were examined. Results: The rate of hyponatremia was reduced using isotonic fluids. There were no hypernatremic events. Volume overload might increase the use of furosemide when using isotonic fluids. Potassium and bivalent cation levels increased. The risk of acidosis is greatly reduced, whereas alkalosis was more frequent due to furosemide use. The rate of acute kidney injury did not increase. Conclusion: Using isotonic fluids for hyper-hydration in pediatric oncology lead to a modest reduction of hospital-acquired hyponatremia without causing hypernatremia, but the effects on fluid balance need further investigation. The additional intake of bivalent cations and buffering anions in balanced fluids has measurable effects.

Blinatumomab in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

OBJECTIVE: Pediatric patients with relapsed or refractory acute lymphoblastic leukemia have a poor prognosis. We here assess the response rates, adverse events, and long-term follow-up of pediatric patients with relapsed/refractory acute lymphoblastic leukemia receiving blinatumomab. METHODS: Retrospective analysis of a single-center experience with blinatumomab in 38 patients over a period of 10 years. RESULTS: The median age at onset of therapy was 10 years (1-21 years). Seventy-one percent of patients had undergone at least one hematopoietic stem cell transplantation (HSCT) prior to treatment with blinatumomab. We observed a response to blinatumomab in 13/38 patients (34%). The predominant side effect was febrile reactions, nearly half of the patients developed a cytokine release syndrome. Eight events of neurotoxicity were registered over the 78 cycles (15%). To date, nine patients (24%) are alive and in complete molecular remission. All survivors underwent haploidentical HSCT after treatment with blinatumomab. CONCLUSIONS: Despite heavy pretreatment of most of our patients, severe adverse events were rare and response rates encouraging. Blinatumomab is a valuable bridging salvage therapy for relapsed or refractory patients to a second or even third HSCT.

Arsenic trioxide in pediatric cancer - a case series and review of literature.

Arsenic trioxide (ATO) has become an established component of treatment protocols for acute promyelocytic leukemia (APL) with excellent efficacy and no relevant sustained toxicity. Part of its action has been attributed to the inhibition of Hedgehog signaling (Hh) which enables a possible therapeutic approach as many pediatric tumor entities have been associated with increased Hh activity. We retrospectively analyzed 31 patients with refractory and relapsed pediatric cancer who were treated with ATO at the University Children's Hospital of Tuebingen. Additionally a literature review on the clinical and preclinical use of ATO in pediatric cancer treatment was performed.ATO alone as well as combinations with other drugs have proven effective in vitro and in mouse models of various pediatric malignancies. However, only few data on the clinical use of ATO in pediatric patients besides APL exist. In our patient sample, ATO was overall well tolerated in the treatment of various pediatric cancers, even in combination with other cytostatic drugs. Due to distinct tumor entities, differently progressed disease stages and varying co-medication, no clear statement can be made regarding the efficacy of ATO treatment. However, patients with proven Hh activation in molecular tumor profiling surpassed all other patients, who received ATO in an experimental treatment setting, in terms of survival. As molecular profiling of tumors increases and enhanced Hh activity can be detected at an early stage, ATO might expand its clinical use to other pediatric malignancies beyond APL depending on further clinical studies.

Posaconazole plasma concentration in pediatric patients receiving antifungal prophylaxis after allogeneic hematopoietic stem cell transplantation.

Posaconazole has been proven to be effective for antifungal prophylaxis in adults after hematopoietic stem cell transplantation (HSCT). Due to low gastrointestinal resorption of posaconazole suspension, bioavailability is impaired. Fatty food improves the uptake of posaconazole, but insufficient data on the pharmacokinetics of posaconazole in pediatric patients are available so far. The single-center analysis investigated 161 posaconazole serum concentrations in 27 pediatric patients after HSCT receiving 12 mg·kg BW(-1)·d(-1) posaconazole suspension depending on age, gender, and intestinal graft-versus-host (iGvHD) disease, and the influence of posaconazole on cyclosporine A plasma concentrations. To improve the uptake of posaconazole, one patient cohort received higher fat nutrition with the drug administration. A comparison of the regular nutrition and higher-fat nutrition groups revealed the following values: 31 (27.4%) versus 8 (16.7%) < 500 ng/ml; 12 (10.6%) versus 7 (14.6%) 500-700 ng/ml; 8 (7.1%) versus 6 (12.5%) 700-1000 ng/ml; 51 (45.1%) versus 21 (43.8%) 1000-2000 ng/ml; and 11 (9.7%) versus 6 (12.5%) > 2000 ng/ml. The mean posaconazole concentrations in patients with regular nutrition was 1123 ± 811 ng/ml and with higher-fat nutrition was 1191 ± 673 ng/ml. Posaconazole levels in patients with iGvHD were significantly lower (P = 0.0003) than in patients without GvHD. The majority of samples showed a sufficient posaconazole concentration above 700 ng/ml. Posaconazole levels were slightly higher in patients with higher-fat nutrition and significantly lower in patients with iGvHD. Cyclosporine A levels were not significantly higher during posaconazole administration.

Teachers' proactive behaviour: Interactions with job characteristics and professional competence in a longitudinal study.

BACKGROUND: In times of accelerating changes, teachers who proactively engage in activities towards school improvement and innovation are increasingly needed. Still, studies on factors that affect teachers' proactive behaviour are rare. AIMS: Integrating previous research on proactive behaviour within the Job Demand-Resources (JD-R) Model, this paper investigates how job characteristics (time pressure, bureaucratic structures, participative climate, personal initiative of the team) and aspects of teachers' professional competence (self-efficacy, self-regulation skills and knowledge) contribute to and interact with their proactive behaviour. SAMPLE: A total of 130 German secondary school teachers (M(SD)age = 44.05 (11.36), 65% female) participated in this study. METHODS: We employed a full two-wave panel design, with measurement points 5 months apart. The data were analysed with (moderated) single indicator modelling and a cross-lagged panel model. RESULTS: While teachers' self-efficacy in implementing change and self-regulation skills predicted their concurrent proactive behaviour, job characteristics and teachers' knowledge had no such cross-sectional effects. In addition, we found an interaction effect of time pressure and teachers' self-efficacy on proactive behaviour. Including the second measurement point, data indicated no cross-lagged effects of the job and personal factors on proactive behaviour. However, cross-lagged analysis revealed that teachers' proactive behaviour predicted their later self-efficacy in implementing change and the time pressure they perceive. CONCLUSIONS: Examining both cross-sectional and longitudinal effects, this study highlights the importance of measurements over time when analysing factors that influence teachers' proactive behaviour: While aspects of professional competence appear to be trainable concurrent resources, time pressure can limit their effect. Finally, cross-lagged effects of teachers' proactive behaviour on their later self-efficacy and time pressure appear as influential in the long run.

Caspofungin as antifungal prophylaxis in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective analysis.

BACKGROUND: Pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) often receive intravenous liposomal amphotericin B (L-AmB) as antifungal prophylaxis. There are no guidelines for antifungal prophylaxis in children in this situation. Caspofungin (CAS), a broad-spectrum echinocandin, could be an effective alternative with lower nephrotoxicity than L-AmB. METHODS: We retrospectively analyzed the safety, feasibility, and efficacy of CAS in our center, and compared the results with L-AmB as antifungal monoprophylaxis in pediatric patients undergoing HSCT. 60 pediatric patients received L-AmB (1 or 3 mg/kg bw/day) and another 60 patients received CAS (50 mg/m2/day) as antifungal monoprophylaxis starting on day one after HSCT. The median ages of patients receiving L-AmB and CAS were 7.5 years and 9.5 years, respectively. RESULTS: No proven breakthrough fungal infection occurred in either group during the median treatment period of 23 days in the L-AmB group and 24 days in the CAS group. One patient receiving CAS developed probable invasive aspergillosis. During L-AmB treatment, potassium levels significantly decreased below normal values. Patients treated with L-AmB had more drug-related side effects and an increased need for oral supplementation with potassium, sodium bicarbonate and calcium upon discharge as compared with the CAS group. CAS was well-tolerated and safe in this cohort of immunocompromised pediatric patients, who underwent high-dose chemotherapy and HSCT. CONCLUSION: Prophylactic CAS and L-AmB showed similar efficacy in this biggest cohort of pediatric patients after allogeneic HSCT reported, so far. A prospective randomized trial in children is warranted to allow for standardized guidelines.

Role of Catalyst in Optimizing Fluid Catalytic Cracking Performance During Cracking of H-Oil-Derived Gas Oils.

Three H-Oil gas oils, heavy atmospheric gas oil (HAGO), light vacuum gas oil (LVGO), heavy vacuum gas oil (HVGO), and two their blends with hydrotreated straight run vacuum gas oils (HTSRVGOs) were cracked on two high unit cell size (UCS) lower porosity commercial catalysts and two low UCS higher porosity commercial catalysts. The cracking experiments were performed in an advanced cracking evaluation fluid catalytic cracking (FCC) laboratory unit at 527 °C, 30 s catalyst time on stream, and catalyst-to-oil (CTO) variation between 3.5 and 7.5 wt/wt The two high UCS lower porosity catalysts were more active and more coke selective. However, the difference between conversion of the more active high UCS lower porosity and low UCS higher porosity catalysts at 7.5 wt/wt CTO decreased in the order 10% (HAGO) > 9% (LVGO) > 6% (HVGO) > 4% (80% HTSRVGO/20% H-Oil VGO). Therefore, the catalyst performance is feedstock-dependent. The four studied catalysts along with a blend of one of them with 2% ZSM-5 were examined in a commercially revamped UOP FCC VSS unit. The lower UCS higher porosity catalysts exhibited operation at a higher CTO ratio achieving a similar conversion level with more active higher UCS lower porosity catalysts. However, the higher UCS lower porosity catalysts made 0.67% Δ coke that was higher than the maximum acceptable limit of 0.64% for this particular commercial FCC unit (FCCU), which required excluding the HVGO from the FCC feed blend. The catalyst system containing ZSM-5 increased the LPG yield but did not have an impact on gasoline octane. It was found that the predominant factor that controls refinery profitability related to the FCCU performance is the FCC slurry oil (bottoms) yield.

Efficacy, Safety And Feasibility Of Antiemetic Prophylaxis With Fosaprepitant, Granisetron And Dexamethasone In Pediatric Patients With Hemato-Oncological Malignancies.

BACKGROUND: Chemotherapy-induced nausea and vomiting (CINV) are a major burden for patients undergoing emetogenic chemotherapy. International guidelines recommend an antiemetic prophylaxis with corticosteroids, 5-HT3R-antagonists and NK1R-antagonists. The NK1R-antagonist fosaprepitant has shown favorable results in pediatric and adult patients. There is little pediatric experience with fosaprepitant. METHODS: This non-interventional observation study analyzed 303 chemotherapy courses administered to 83 pediatric patients with a median age of 9 years (2-17 years), who received antiemetic prophylaxis either with fosaprepitant and granisetron with or without dexamethasone (fosaprepitant group/FG; n=41), or granisetron with or without dexamethasone (control group/CG; n=42), during moderately (CINV risk 30-90%) or highly (CINV risk>90%) emetogenic chemotherapy. The two groups' results were compared with respect to the safety and efficacy of the antiemetic prophylaxis during the acute (0-24hrs after chemotherapy), delayed (>24-120hrs after chemotherapy) and both CINV phases. Laboratory and clinical adverse events were compared between the two cohorts. RESULTS: Adverse events were not significantly different in the two groups (p>0.05). Significantly fewer vomiting events occurred during antiemetic prophylaxis with fosaprepitant in the acute (23 vs 142 events; p<0.0001) and the delayed (71 vs 255 events; p<0.0001) CINV phase. In the control group, the percentage of chemotherapy courses with vomiting was significantly higher during the acute (24%/FG vs 45%/CG; p<0.0001) and delayed CINV phase (28%/FG vs 47%/CG; p=0.0004). Dimenhydrinate (rescue medication) was administered significantly more often in the CG, compared to the FG (114/FG vs 320/CG doses; p<0.0001). Likewise, in the control group, dimenhydrinate was administered in significantly more (p<0.0001) chemotherapy courses during the acute and delayed CINV phases (79 of 150; 52.7%), compared to the fosaprepitant group (45 of 153; 29.4%). CONCLUSION: Antiemetic prophylaxis with fosaprepitant and granisetron with or without dexamethasone was well tolerated, safe and effective in pediatric patients. However, larger prospective trials are needed to evaluate these findings.

Local catheter-based delivery of antithrombotic or antiproliferative drugs: a new concept for prevention of restenosis.

BACKGROUND: Drug eluting stents have reduced the incidence of restenosis after percutaneous coronary interventions significantly, but cause concern about long term safety. Local drug delivery using special application catheters is an alternative approach for intracoronary pharmacotherapy. Besides the fact, that no problematic coating as drug carrier has to be used, a local delivery independent of the stent itself by using catheter techniques offers further advantages - such as the possibility to treat the whole vessel wall, stent edges and adjacent vessel segments and not only the area close to the stent struts. METHODS AND RESULTS: We have developed a new local catheter-based delivery system for local intracoronary pharmacotherapy. An antithrombotic as well as an antiproliferative therapy concept for prevention of restenosis are presented in the manuscript. Our data show that local drug delivery of platelet glycoprotein VI and paclitaxel were effective in the reduction of thrombus formation and neointima formation in experimental animal models. CONCLUSIONS: A combination of early antithrombotic and antiatherosclerotic mechanisms may be a realistic and effective approach to minimize postinterventional thromboischemic events and neointima formation. These results may contribute to an advanced and even combined local intracoronary pharmacotherapy in near future, independent of stent coatings.

Urea, Uric Acid, Prolactin and fT4 Concentrations in Aqueous Humor of Keratoconus Patients.

PURPOSE: Keratoconus is a noninflammatory disease of the cornea associated with progressive thinning and conical shape. Metabolic alterations in the urea cycle, with changes in collagen fibril stability, oxidative stress, thyroid hormones and prolactin with regulatory effect on biosynthesis and biomechanical stability of corneal stroma, may all play a role in keratoconus etiology. Our purpose was to determine urea, uric acid, prolactin and free thyroxin (fT4) concentrations in human aqueous humor (hAH) of keratoconus and cataract patients. METHODS: hAH was collected from 100 keratoconus (penetrating keratoplasty) (41.9 ± 14.9 years, 69 males) and 100 cataract patients (cataract surgery) (71.2 ± 12.4 years, 58 males). Urea, uric acid, prolactin and fT4 concentrations were measured by Siemens clinical chemistry or immunoassay system. For statistical analysis, a generalized linear model (GLM) was used. RESULTS: Urea concentration was 11.88 ± 3.03 mg/dl in keratoconus and 16.44 ± 6.40 mg/dl in cataract patients, uric acid 2.04 ± 0.59 mg/dl in keratoconus and 2.18 ± 0.73 mg/dl in cataract groups. Prolactin concentration was 3.18 ± 0.34 ng/ml in keratoconus and 3.33 ± 0.32 ng/ml in cataract patients, fT4 20.57 ± 4.76 pmol/l in KC and 19.06 ± 3.86 pmol/l in cataract group. Urea concentration was effected through gender (p = 0.039), age (p = 0.001) and diagnosis (p = 0.025). Uric acid concentration was not effected through any of the analyzed parameters (p > 0.056). Prolactin and fT4 concentration were effected only through diagnosis (p = 0.009 and p = 0.006). CONCLUSIONS: Urea and prolactin concentrations are decreased, fT4 concentration is increased in aqueous humor of keratoconus patients, and uric acid concentration remains unchanged. Urea concentration in aqueous humor is also increased in older and male patients. Therefore, metabolic disorder and hormonal balance may both have an impact on keratoconus development. Further studies are necessary to assess the specific impact.

Telomere length and telomerase activity in the BCR-ABL-transformed murine Pro-B cell line BaF3 is unaffected by treatment with imatinib.

OBJECTIVE: Imatinib mesylate is a novel tyrosine kinase inhibitor used for the treatment of Philadelphia chromosome positive (Ph+) leukemia and other malignancies. In previous studies, we found significant telomere shortening in Ph+ cells from patients with chronic myeloid leukemia (CML). Interestingly, imatinib treatment was found to lead to a normalization of previously shortened telomere length in CML patients. Based on recent reports demonstrating that c-ABL phosphorylates hTERT and thereby inhibits hTERT activity, a direct effect of imatinib on hTERT activity leading to telomere elongation in BCR-ABL-positive cells has been proposed by others. Such an effect could be of potential importance for telomere maintenance in Ph+ cells by facilitating clonal selection and progression of the disease to blast crisis. METHODS: We investigated the impact of imatinib on telomere length and telomerase activity of the interleukin-3 (IL-3)-dependent murine pro-B cell line BaF3 and the BCR-ABL-positive, IL-3-independent transfectant BaF3p185 in vitro. RESULTS: When BaF3 and BaF3p185 cells were treated with imatinib (the latter being rescued with IL-3), no effect on either telomerase activity or telomere length was observed. These findings can be explained by the cytoplasmatic localization of BCR-ABL found in BaF3p185 as compared to the nuclear localization of telomerase (and c-ABL). CONCLUSION: As opposed to recent reports for c-ABL, we do not see evidence for a functional interaction between BCR-ABL and hTERT in this model system arguing against imatinib-mediated upregulation of hTERT as a crucial factor for clonal selection and disease progression of CML.

Telomere length and hTERT expression in patients with acute myeloid leukemia correlates with chromosomal abnormalities.

BACKGROUND AND OBJECTIVES: Acute myeloid leukemia (AML) is a malignant, genetically heterogenous disorder characterized by uncontrolled growth of immature myeloid cells. The aim of this study was to analyze whether telomere length and/or hTERT expression are correlated with clonal chromosomal aberrations in AML. DESIGN AND METHODS: Telomere length in mononuclear cells derived from 137 previously untreated patients with >or= 80% blasts was analyzed by flow fluorescent in situ hybridization. Results were expressed in telomere fluorescence Units (1 TFU=1 kb). The expression of hTERT, including its different splice variants was studied by reverse transcription-polymerase chain reaction. RESULTS: Age-adjusted telomere length in AML patients was significantly reduced as compared to in matched controls, consisting of peripheral blood granulocytes from healthy individuals (0-90 years) (median: -2.5 TFU; p<0.001). Patients with an aberrant karyotype had significantly shorter telomeres than patients with a normal karyotype (median -3.0 vs. -2.3 TFU; p=0.03). The shortest telomeres were found in patients with multiple aberrations (median -3.7 TFU; p=0.03). hTERT expression was found to be correlated with chromosomal abnormalities as well as with the detection of functional hTERT splicing variants. INTERPRETATION AND CONCLUSIONS: These findings suggest an important role of intense telomere loss in the development of genetic instability during the pathogenesis of AML. It is assumed that critical telomere shortening in AML blasts could lead to telomerase activation and therefore prevent blasts from replicative senescence, one possible mechanism for clonal selection and disease progression. Therefore, telomere length might serve as a prognostic marker for AML patients. These findings would have to be confirmed in large, prospective studies.

Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites.

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

The Apis mellifera Filamentous Virus Genome.

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

Normalization of previously shortened telomere length under treatment with imatinib argues against a preexisting telomere length deficit in normal hematopoietic stem cells from patients with chronic myeloid leukemia.

Telomeres are composed of TTAGGG repeats and associated proteins. In somatic cells, telomere repeats are lost with each cell division, eventually leading to genetic instability and cellular senescence. In previous studies, we described substantial and disease stage-specific telomere shortening in peripheral blood (PB) leukocytes from patients with chronic myeloid leukemia (CML). Here, we sought to determine whether age-adjusted telomere length in PB granulocytes (deltaTEL(gran)) is associated with response to treatment with the selective tyrosine kinase inhibitor imatinib. A total of 517 samples from 206 patients in chronic phase (CP), accelerated phase (AP), and blast crisis (BC) before and up to 706 days after initiation of imatinib therapy (median: 144 days) were analyzed by quantitative fluorescence in situ hybridization of interphase cells in suspension (Flow-FISH); telomere fluorescence was expressed in molecular equivalents of soluble fluorochrome units (MESF). Telomere length in samples from start of treatment up to day 144 was significantly shorter (mean +/- SE; -1.5 +/- 0.3 kMESF) compared to samples from patients treated for more than 144 days (-0.8 +/- 0.3 kMESF, p = 0.035). In patients with repeated measurements, a significant increase in telomere length under treatment was observed. Median telomere length in major remission was found to be significantly longer compared to patients without response to treatment measured either by cytogenetics (n = 246, p < 0.05), interphase FISH (n = 204, p = 0.002), or quantitative RT-PCR (n = 371, p < 0.05). In conclusion, the increase in telomere length under treatment with imatinib reflects a shift from Ph+ to Ph- cells in the PB of patients with CML.

Functional p53 is required for effective execution of telomerase inhibition in BCR-ABL-positive CML cells.

OBJECTIVE: In chronic myeloid leukemia (CML), increased cellular turnover of hematopoietic cells driven by the oncogene BCR-ABL leads to accelerated telomere shortening despite increased telomerase activity. It has been postulated that shortened telomeres, particularly in the context of increased telomerase activity, might facilitate accumulation of genetic aberrations and, consequently, disease progression from chronic phase to accelerated phase and blast crisis. Therefore, inhibition of telomerase might be a promising approach in CML therapy. MATERIAL AND METHODS: To investigate the therapeutic potential of telomerase inhibition in this model disorder, we used a small molecule telomerase inhibitor, BIBR1532 as well as expression of a dominant-negative mutant of hTERT (DNhTERT-IRES-GFP) in the p53-negative CML blast crisis cell line K562 and characterized the effects in long-term culture. Furthermore, we expressed an inducible p53 construct (vector pBabe-p53ER(tam)) via retroviral transduction in cells with critically short telomeres and in cells with a normal telomere length to explain the role of the tumor suppressor in response to critical telomere shortening in BCR-ABL-positive cells. RESULTS: BIBR1532-treated bulk cultures did not show altered growth kinetics despite significant telomere shortening to a critical length of approximately 5 kb. In comparison, DNhTERT-expressing clones either lost telomere length, leading to a significant but transient slow down in proliferation but eventually all escaped senescence/crisis (group I) or, alternatively, remained virtually unaffected despite measurable telomerase inhibition (group II). Further analyses of group I clones revealed impaired DNA damage response and an accumulation of dicentric chromosomes. However, upon restoration of p53 in telomerase-negative K562 clones with critically short telomeres, immediate reinduction of apoptosis and complete eradication of cells was observed, whereas vector control cells continued to escape from crisis. CONCLUSIONS: These results suggest that the success of strategies aimed at telomerase inhibition in CML is highly dependent on the presence of functional p53 and should be explored preferentially in chronic phase CML.

Catheter-based delivery of fluid paclitaxel for prevention of restenosis in native coronary artery lesions after stent implantation.

BACKGROUND: Stents eluting antiproliferative drugs reduce the incidence of restenosis but delay healing of the vascular wall. We assessed the safety and efficacy of catheter-based local delivery of fluid paclitaxel in patients with coronary de novo stenoses after implantation of a bare metal stent. METHODS AND RESULTS: We conducted a prospective, randomized trial comparing the local delivery of fluid paclitaxel after bare metal stent implantation (group I) with the implantation of a bare metal stent (group II) and the implantation of a paclitaxel-eluting stent (group III) in 204 patients. The primary end point was in-stent late lumen loss. Secondary end points included binary restenosis rate >50%, minimal lumen diameter, diameter stenosis, and a composite clinical end point (major adverse cardiac events and revascularization of the target lesion) 6 months after intervention. At 6 months, angiography showed an in-stent late lumen loss of 0.61+/-0.44 mm in group I versus 0.99+/-0.72 mm in group II (I versus II, P=0.0006) and 0.44+/-0.48 mm in group III (noninferiority of I versus III, P=0.023). The 1-sided 95% CI for the true difference of the means of in-stent late lumen loss in groups I and III was -infinity to 0.3174188. The cumulative overall rate of major cardiac events was 13.4% in group I, 26.8% in group II, and 14.9% in group III. Target lesion revascularization rate was 13.4% (group I), 22.1% (group II), and 13.4% (group III). CONCLUSIONS: Additional antiproliferative treatment of de novo lesions in native coronary arteries with catheter-based delivery of fluid paclitaxel after bare metal stenting was safe and significantly reduced neointimal proliferation, restenosis, and clinical events compared with bare metal stent implantation alone.

Catheter-based local antiproliferative therapy in kissing balloon technique for in-stent stenosis of coronary artery bifurcation lesions.

There is currently no promising interventional solution for in-stent stenosis in previously stented bifurcation lesions, even with drug-eluting stents. Rather than being restricted to stent struts, catheter-based local antiproliferative therapy offers the advantage of homogenous drug transfer to the whole vessel wall, and thereby allows for intracoronary pharmacotherapy without adding additional layers of metal into an already stented lesion. The newly developed GENIE catheter (Acrostak Corp, Switzerland), applied in the kissing balloon technique, allows for delivery of liquid paclitaxel into whole bifurcation lesions without repeat stent implantation. After conventional percutaneous transluminal coronary angioplasty, local delivery of paclitaxel using two GENIE catheters in the kissing balloon technique was performed in three patients (left anterior descending, left circumflex and right coronary arteries) with highly symptomatic in-stent bifurcation stenoses. The intervention was feasible and safe in all coronary arteries. Final angiography and control angiography after six months showed good results. No major adverse cardiac events occurred 30 days and six months after intervention. The patients, who represent a group at high risk of recurrent instent restenosis, remained asymptomatic since the local drug delivery. They did not require insertion of a drug-eluting stent or crossover to coronary artery bypass surgery. In conclusion, this new treatment strategy proved to be safe and effective in this first human experience and offers a promising alternative to surgery or implantation of additional stents in these patients.

Targeting telomerase activity by BIBR1532 as a therapeutic approach in germ cell tumors.

Germ cell tumors (GCT) possess a high activity of telomerase, a ribonucleoprotein complex compensating the erosion of telomeres during cell division by adding TTAGGG-repeats to the telomeric ends of chromosomes. Cisplatin, the most important drug in the treatment of GCT, preferentially acts on G-rich regions like telomeres. Inhibiting telomerase in tumors can result in telomere shortening and senescence and could increase the efficacy of chemotherapy in refractory patients. The study evaluated the promise of the small molecule telomerase inhibitor BIBR1532 as single agent and assessed a possible synergism with cisplatin in a preclinical model of GCT.GCT-derived cell line 2102EP was cultured with or without 10 microM of BIBR1532. Cell expansion was quantified in population doublings (PD). Telomere length was analyzed by fluorescence in situ hybridization and flow cytometry (flow-FISH). The sensitivity of the cells towards cisplatin was determined by MTT-assay. Telomerase activity was assessed by TRAP assay. After 300 PD, telomere length diminished from 18.5 kb +/- 0.59 kb to 8.9 +/- 0.1 kb in BIBR1532 treated 2102 EP cells as compared to 14.5 +/- 0.0 kb in untreated control cells. Treated cells did not show altered growth kinetics compared to untreated counterparts. Despite effective shortening of telomeres, the sensitivity of the treated cells towards cisplatin did not increase. Concomitant treatment with BIBR1532 and cisplatin did not result in accelerated telomere shortening. Telomere length can be shortened significantly by telomerase inhibition in GCT cell line models. However, possibly in view of their extensive telomere "reserve," telomerase inhibition did neither result in increased sensitivity of 2102 EP cells to cisplatin nor did co-treated cells show accelerated telomere shortening.

Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel therapeutic target in BCR-ABL-positive leukemias identified by a proteomics approach.

Inhibition of BCR-ABL tyrosine kinase with imatinib represents a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular BCR-ABL downstream targets, we analyzed differences in global protein expression in BCR-ABL-positive K562 cells treated with or without imatinib in vitro. Among the 19 proteins found to be differentially expressed, we detected the down-regulation of eukaryotic initiation factor 5A (eIF5A), a protein essential for cell proliferation. eIF5A represents the only known eukaryotic protein activated by posttranslational hypusination. Hypusination inhibitors (HIs) alone exerted an antiproliferative effect on BCR-ABL-positive and -negative leukemia cell lines in vitro. However, the synergistic dose-response relationship found for the combination of imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell-cycle analysis, and CFSE labeling of primary CD34+ CML cells. Specificity of this effect could be demonstrated by cotreatment of K562 cells with imatinib and siRNA against eIF5. In conclusion, through a comparative proteomics approach and further functional analysis, we identified the inhibition of eIF5A hypusination as a promising new approach for combination therapy in BCR-ABL-positive leukemias.