Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

MMP-9 as predictive factor for response and progression free survival in breast cancer patients treated with bevacizumab and pegylated liposomal doxorubicin. A multicenter, single-arm phase II trial (SAKK24/06). On behalf of the Swiss Group for Clinical Cancer Research (SAKK).

The benefit of bevacizumab (Bv) has been shown in different tumors including colorectal cancer, renal cancer, pulmonary non-small cell cancer and also breast cancer. However to date, there is no established test evaluating the angiogenic status of a patient and monitoring the effects of anti-angiogenic treatments. Tumor angiogenesis is the result of a balance between multiple pro- and anti¬angiogenic molecules. There is very little published clinical data exploring the impact of the anti-angiogenic therapy on the different angiogenesis-related molecules and the potential role of these molecules as prognostic or predictive factors.

Pseudoprogression after high-dose busulfan-thiotepa with autologous stem cell transplantation and radiation therapy in children with brain tumor: impact on survival

Objective: To demonstrate the incidence, time course, predisposing factor and reversibility of neurotoxicity in children with brain tumors treated with high dose busulfan-thiotepa with autologous stem cell transplantation (ASCT) and radiation therapy in our institutional experience.Materials and Methods: We performed a retrospective analysis of prospectively collected data. Between May 1988 and May 2007, 110 patients, median age 3.6 years (range, 1 months-15.3 years), with brain tumors were treated with surgical intervention and conventional chemotherapy. All patients received one course of high-dose busulfan-thiotepa with stem cell rescue, followed or preceded by radiotherapy.Results: Twenty-three patients (21%) developed neuroradiological abnormalities on follow-up imaging studies at a median time of 9.2 months (range, 5.6-17.3 months) after day 0 of ASCT. All MRI-lesions appeared in patients receiving radiotherapy after ASCT and were localized inside the 50-55 Gy isodoses. They disappeared in 14 of 23 patients with a median time of 8 months (range, 3-17 months). The presence of MRI-abnormalities was a favorable prognostic factor for overall survival on univariate analysis (hazard ratio: 0.12, 95% confidence interval [0.04, 0.33]), with a 5-year overall survival in patients with MRI-abnormalities of 84% (95% CI, 62-94), comparedto 27% (95% CI, 19-37) in those without lesions. On multivariate analysis, the presence of MRI-abnormalities was an independent prognostic factor for overall survival.Conclusion: MRI-detectable brain abnormalities are common early findings in children treated with high-dose busulfan-thiotepa followed by radiation therapy, and may mimic early tumor recurrence. They are correlated with a better outcome.

TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak.

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway.

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

One-year survival and neurological outcome after pediatric cardiopulmonary resuscitation.

OBJECTIVE: Reported survival after cardiopulmonary resuscitation (CPR) in children varies considerably. We aimed to identify predictors of 1-year survival and to assess long-term neurological status after in- or outpatient CPR. DESIGN: Retrospective review of the medical records and prospective follow-up of CPR survivors. SETTING: Tertiary care pediatric university hospital. PATIENTS AND METHODS: During a 30-month period, 89 in- and outpatients received advanced CPR. Survivors of CPR were prospectively followed-up for 1 year. Neurological outcome was assessed by the Pediatric Cerebral Performance Category scale (PCPC). Variables predicting 1-year survival were identified by multivariable logistic regression analysis. INTERVENTIONS: None. RESULTS: Seventy-one of the 89 patients were successfully resuscitated. During subsequent hospitalization do-not-resuscitate orders were issued in 25 patients. At 1 year, 48 (54%) were alive, including two of the 25 patients with out-of-hospital CPR. All patients died, who required CPR after trauma or near drowning, when CPR began >10 min after arrest or with CPR duration >60 min. Prolonged CPR (21-60 min) was compatible with survival (five of 19). At 1 year, 77% of the survivors had the same PCPC score as prior to CPR. Predictors of survival were location of resuscitation, CPR during peri- or postoperative care, and duration of resuscitation. A clinical score (0-15 points) based on these three items yielded an area under the ROC of 0.93. CONCLUSIONS: Independent determinants of long-term survival of pediatric resuscitation are location of arrest, underlying cause, and duration of CPR. Long-term survivors have little or no change in neurological status.

IDH1/IDH2 mutations predict survival in glioma and AML

Mutations in the isocitrate dehydrogenase family genes 1 or 2 (IDH1/2) have been discovered by high through put sequencing approaches inglioma and acute myeloid leukemia (AML) and related myeloproliferativeneoplasms. In both diseases, the discovery of IDH mutations has identifieda prognostically new subtype with distinct pathogenetic evolution. Ingliomas mutations are mostly found in IDH1 (>90%). They are infrequent inprimary glioblastoma (GBM) (<10%), but common in secondary GBM thatevolve from lower grade glioma (60−90%). Mutations in IDH1 precede p53mutations or 1p/19q co-deletions in sporadic low grade glioma, hence arean early evant. Co-deletions of 1p/19q, characteristic for oligodenroglioma,are highly associated with IDH1/2 mutations, while they are mutuallyexclusive with EGFR amplifications, a hall mark of primary GBM. IDH1 or 2mutations are associated with younger patient age, but absent in childhoodgliomas, and have a better prognosis that seems to be consistent in gradeII through IV gliomas. In myeloid malignancies mutations are more likelyin IDH2 and are found in de novo and secondary AML (12−18%) andpre-leukemic clonal malignancies (5% chronic; 20% transformed). IDH1/2mutations are strongly associated with NPM1 mutations that are found in30% of novo cytogenetically normal AML. In CN-AML with mutated NPM1,without FLT3 internal tandem duplication (ITD) IDH mutations constitutean adverse prognostic factor. Mutations in the metabolic enzymes IDH1 or2 result in a neomorphic reaction, generating high levels of the metabolite2-hydroxyglutarate (2-HG). IDH mutations are mutually exclusive with TET2mutations in myeloid malignancies that led to the discovery that high levelsof 2-HG inhibit the a-KG dependent dioxygenase TET2. TET2 is involved inepigenetic regulation and mediates demethylation of DNA. This mechanismis in accordance with the association of a methylator phenotype with loss offunction of TET2 by mutation or indirectly by mutation of IDH1/2 in myeloidmalignancies and gliomas, respectively.Metabolism meets Epigenetics. These discoveries will have importantclinical implications: IDH1/2 mutants may serve as unique targets fortherapy. Further, the high concentrations of the onco-metabolite 2-HGgenerated by IDH1/2 mutants, may serve as biomarker in the serum ofpatients with myeloid malignancies and may be amenable by magneticresonance spectroscopy in glioma patients.

Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide.

PURPOSE: Temozolomide is a novel oral alkylating agent with demonstrated efficacy as second-line therapy for patients with recurrent anaplastic astrocytoma and glioblastoma multiforme (GBM). This phase II study was performed to determine the safety, tolerability, and efficacy of concomitant radiation plus temozolomide therapy followed by adjuvant temozolomide therapy in patients with newly diagnosed GBM. PATIENTS AND METHODS: Sixty-four patients were enrolled onto this open-label, phase II trial. Temozolomide (75 mg/m(2)/d x 7 d/wk for 6 weeks) was administered orally concomitant with fractionated radiotherapy (60 Gy total dose: 2 Gy x 5 d/wk for 6 weeks) followed by temozolomide monotherapy (200 mg/m(2)/d x 5 days, every 28 days for six cycles). The primary end points were safety and tolerability, and the secondary end point was overall survival. RESULTS: Concomitant radiation plus temozolomide therapy was safe and well tolerated. Nonhematologic toxicities were rare and mild to moderate in severity. During the concomitant treatment phase, grade 3 or 4 neutropenia, thrombocytopenia, or both were observed in 6% of patients, including two severe infections with Pneumocystis carinii. During adjuvant temozolomide, 2% and 6% of cycles were associated with grade 3 and 4 neutropenia or thrombocytopenia, respectively. Median survival was 16 months, and the 1- and 2-year survival rates were 58% and 31%, respectively. Patients younger than 50 years old and patients who underwent debulking surgery had the best survival outcome. CONCLUSION: Continuous daily temozolomide and concomitant radiation is safe. This regimen of concomitant chemoradiotherapy followed by adjuvant chemotherapy may prolong the survival of patients with glioblastoma. Further investigation is warranted, and a randomized trial is ongoing.

VEGF is required for growth and survival in neonatal mice.

We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development.

Role of disease activity for decision making ability in early multiple sclerosis

Background and purpose: Decision making (DM) has been defined as the process through which a person forms preferences, selects and executes actions, and evaluates the outcome related to a selected choice. This ability represents an important factor for adequate behaviour in everyday life. DM impairment in multiple sclerosis (MS) has been previously reported. The purpose of the present study was to assess DM in patients with MS at the earliest clinically detectable time point of the disease. Methods: Patients with definite (n=109) or possible (clinically isolated syndrome, CIS; n=56) MS, a short disease duration (mean 2.3 years) and a minor neurological disability (mean EDSS 1.8) were compared to 50 healthy controls aged 18 to 60 years (mean age 32.2) using the Iowa Gambling Task (IGT). Subjects had to select a card from any of 4 decks (A/B [disadvantageous]; C/D [advantageous]). The game consisted of 100 trials then grouped in blocks of 20 cards for data analysis. Skill in DM was assessed by means of a learning index (LI) defined as the difference between the averaged last three block indexes and first two block indexes (LI=[(BI-3+BI-4+BI-5)/3-(BI-1+B2)/2]). Non parametric tests were used for statistical analysis. Results: LI was higher in the control group (0.24, SD 0.44) than in the MS group (0.21, SD 0.38), however without reaching statistical significance (p=0.7). Interesting differences were detected when MS patients were grouped according to phenotype. A trend to a difference between MS subgroups and controls was observed for LI (p=0.06), which became significant between MS subgroups (p=0.03). CIS patients who confirmed MS diagnosis by presenting a second relapse after study entry showed a dysfunction in the IGT in comparison to the other CIS (p=0.01) and definite MS (p=0.04) patients. In the opposite, CIS patients characterised by not entirely fulfilled McDonald criteria at inclusion and absence of relapse during the study showed an normal learning pattern on the IGT. Finally, comparing MS patients who developed relapses after study entry, those who remained clinically stable and controls, we observed impaired performances only in relapsing patients in comparison to stable patients (p=0.008) and controls (p=0.03). Discussion: These results raise the assumption of a sustained role for both MS relapsing activity and disease heterogeneity (i.e. infra-clinical severity or activity of MS) in the impaired process of decision making.

The beta-catenin--TCF-1 pathway ensures CD4(+)CD8(+) thymocyte survival.

The association of trans-acting T cell factors (TCFs) or lymphoid enhancer factor 1 (LEF-1) with their coactivator beta-catenin mediates transient transcriptional responses to extracellular Wnt signals. We show here that T cell maturation depends on the presence of the beta-catenin--binding domain in TCF-1. This domain is necessary to mediate the survival of immature CD4(+)CD8(+) double-positive (DP) thymocytes. Accelerated spontaneous thymocyte death in the absence of TCF-1 correlates with aberrantly low expression of the anti-apoptotic protein Bcl-x(L). Increasing anti-apoptotic effectors in thymocytes by the use of a Bcl-2 transgene rescued TCF-1-deficient DP thymocytes from apoptosis. Thus, TCF-1, upon association with beta-catenin, transiently ensures the survival of immature T cells, which enables them to generate and edit T cell receptor (TCR) alpha chains and attempt TCR-mediated positive selection.

Alveolar echinococcosis: from a deadly disease to a well-controlled infection. Relative survival and economic analysis in Switzerland over the last 35 years.

BACKGROUND/AIMS: Alveolar echinococcosis (AE) is a serious liver disease. The aim of this study was to explore the long-term prognosis of AE patients, the burden of this disease in Switzerland and the cost-effectiveness of treatment. METHODS: Relative survival analysis was undertaken using a national database with 329 patient records. 155 representative cases had sufficient details regarding treatment costs and patient outcome to estimate the financial implications and treatment costs of AE. RESULTS: For an average 54-year-old patient diagnosed with AE in 1970 the life expectancy was estimated to be reduced by 18.2 and 21.3 years for men and women, respectively. By 2005 this was reduced to approximately 3.5 and 2.6 years, respectively. Patients undergoing radical surgery had a better outcome, whereas the older patients had a poorer prognosis than the younger patients. Costs amount to approximately Euro108,762 per patient. Assuming the improved life expectancy of AE patients is due to modern treatment the cost per disability-adjusted life years (DALY) saved is approximately Euro6,032. CONCLUSIONS: Current treatments have substantially improved the prognosis of AE patients compared to the 1970s. The cost per DALY saved is low compared to the average national annual income. Hence, AE treatment is highly cost-effective in Switzerland.

Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation.

Root diseases caused by fungal pathogens can be suppressed by certain rhizobacteria that effectively colonize the roots and produce extracellular antifungal compounds. To be effective, biocontrol bacteria need to be present at sufficiently high cell densities. These conditions favor the operation of positive feedback mechanisms that control the production of antifungal compounds in biocontrol strains of fluorescent pseudomonads, via both transcriptional and post-transcriptional mechanisms.