Advanced ovarian cancer is the leading non-breast gynaecologic cause of malignant pleural effusion. Aim of this study was to assess the efficacy of mitoxantrone sclerotherapy as a palliative treatment of malignant pleural effusions due to ovarian cancer.Sixty women with known ovarian cancer and malignant recurrent symptomatic pleural effusion were treated with chest tube drainage followed by intrapleural mitoxantrone sclerotherapy. Survival, complications and response to pleurodesis were recorded. The data are expressed as the mean ± SEM and the median.The mean age of the entire group was 64 ± 11,24 years. The mean interval between diagnosis of ovarian cancer and presentation of the effusion was 10 ± 2,1 months. Eighteen patients (30%) had pleural effusion as the first evidence of recurrence. The mean volume of effusion drained was 1050 ± 105 ml and chest tube was removed within 4 days in 75% of patients. There were no deaths related to the procedure. Side effects of chemical pleurodesis included fever (37–38,5°C) chest pain, nausea and vomiting. At 30 days among 60 treated effusions, there was an 88% overall response rate, including 41 complete responses and 12 partial responses. At 60 days the overall response was 80% . The mean survival of the entire population was 7,5 ± 1,2 months.Mitoxantrone is effective in the treatment of malignant pleural effusion secondary to ovarian cancer without causing significant local or systemic toxicity. Cancer accounts for 40% of all pleural effusions, especially in patients over 50 years old . BronchoApproximately two thirds of malignant pleural effusions occur in women because of the strong association with breast and ovarian cancer . AdvanceThe general approach to managing malignant effusions is determined by symptoms, performance status of the patient, expected survival and response of the known primary tumor to systemic treatment. Intervention options range from observation in the case of asymptomatic effusions through simple thoracentesis to more invasive methods such as thoracoscopy, pleuroperitoneal shunting and pleurectomy. In patients with reasonable survival expectancy and good performance status every attempt should be made to prevent recurrence of the effusion. Intercostal tube drainage with instillation of a sclerosing agent, resulting in the obliteration of the pleural space, is the most widely used method to control recurrent symptomatic malignant pleural effusions.Aim of this study was to study 60 patients with ovarian cancer who had a pleural effusion as a direct consequence of metastatic disease and to assess the efficacy of mitoxantrone as a sclerosing agent.Over an 8-year period (1996–2003), all patients with known ovarian malignancy and recurrent symptomatic malignant pleural effusion referred to Thoracic Surgery Department of Theagenio Cancer Hospital for drainage and sclerotherapy, were eligible to participate in this study. This study was approved by the Theagenio Cancer Research Ethics Committee and patients were included after giving their informed consent.All patients satisfied the following eligibility criteria:1. Known ovarian malignancy.2. Recurrent symptomatic malignant pleural effusion. The diagnosis established by positive pleural fluid cytology on thoracentesis or positive pleural biopsy.3. Evidence of expansion of the lung after fluid drainage and abscence of bronchial obstruction and/or fibrosis preventing lung expansion.4. No previous intrapleural therapy.5. Predicted survival of >1 month.Patients were ineligible if they had a history of cardiac disease, obstructive jaundice or surgery within the previous month. No patient had systemic chemotherapy immediately prior to or during the first 30-day interval following sclerotherapy. Sixty women fulfilled the above eligibility criteria.Pretreatment assessment was performed during admission and included history and physical examination, full blood count, liver biochemistry, electrocardiogram, a pre-drainage base line posteroanterior and lateral chest radiograph and other imaging as clinically indicated.th or 6th intercostal space under local anesthesia and in some case additional intravenous benzodiazepines and/or narcotics. The pleural effusion was drained to dryness initially by gravity and followed if necessary by suction from a wall-mounted suction pump using a pressure of 20 cm H2O usually for 12–24 hours to achieve complete drainage of the effusion and lung re-expansion. Daily tube outputs were recorded and when drainage fell below 100 ml in a 24 h period, posteroanterior and lateral chest radiographs were obtained to assure that the fluid had been sufficiently evacuated, there were no loculated collections and the lung had fully re-expanded. Then the patients were eligible for pleurodesis.A chest tube (28–32 F) was inserted into the midaxillary line through the 5Fifty ml of normal saline solution containing 2 mg/kg lidocaine were infused through the chest tube. After 15 minutes, a pleurodesis solution containing a mixture of 40 mg mitoxantrone and 20 ml normal saline was infused into the pleural cavity, after which the tube was clamped for 2 hours, while the patients changed position (rotated 90°) every 15 minutes. The tube then was re-opened. If the post-sclerotherapy drainage was <100 ml per day thetube was removed.Complications related to the procedure were recorded. Post-sclerotherapy posteroanterior and lateral chest radiographs were obtained immediately after tube removal in order to be compared with others obtained 30 and 60 days later.The radiographic response was determined on posteroanterior and lateral chest radiographs by observing the level of fluid meniscus overlying the costophrenic or vertebrophrenic angles and was determined as follows: complete response(CR) – no re-accumulation of pleural fluid, partial response (PR) – fluid recurrence less than 50% of the original level without symptoms or not requiring repeat drainage, progressive disease (PD) – re-accumulation to or above the original level with symptoms and requiring repeat drainage.Survival was calculated from the day of diagnosis of pleural effusion to the day of death or to the last day of follow up if alive.The data are expressed as the mean ± SEM and the median.Sixty women were included in this study. The mean age of the entire group was 64 ± 11,24 years. The interval between diagnosis of ovarian cancer and the development of a subsequent malignant pleural effusion ranged from 1 to 36 months (mean: 10 ± 2,1 months). Fifty one patients 85%) had unilateral effusion and 9 (15%) bilateral. Histology, degree of differentiation and TNM stage [% had uniEigtheen patients (30%) had pleural effusion as the first manifestation of recurrent disease, whereas 42 patients (70%) were already diagnosed as having local or distant spread before the onset of pleural effusion. These 42 patients with preexisting metastases showed a variable pattern of secondary spread. Eighteen patients had parenchymal liver metastases and intraabdominal lymph nodes, 11 patients had liver metastases only, 8 had synchronous lung and liver metastases, 2 had lung metastases only, 1 patient had umbilical nodule, 1 had anterior wall abdominal wall infiltration and 1 had brain metastases.The mean volume of effusion drained was 1050 ± 105 ml (range: 450–1500 ml). Chest tube was removed within 4 days in 75% of patients (range: 3 – 10 days).There were no deaths related to the thoracostomy procedure. One patient experienced vasovagal reflex during the procedure with systemic hypotension and intense pleuritic pain. Hypotension was treated with intravenous fluids and the pain was controlled with narcotics. This episode lasted 20 minutes. The patient recovered without incident.The most frequent complications related to pleurodesis were fever (temperature > 37°C), chest pain, nausea and vomiting Table .Three patients died within 1 month of pleurodesis due to rapid progression metastatic disease. At 30 days, 57 patients were alive and 41 out of them had a complete response and 12 had a partial response. The overall response to chemical pleurodesis with mitoxantrone was 88% (53/57 patients). Four patients had progressive disease and revealed reaccumulation of fluid to or above the original level.At 60 days 52 patients were alive and 38 out of them had a complete response and 10 had a partial response. The overall response was 80% . Follow up ranged from 10 days to 38 months with a mean of 10 ± 1,36 months. Eight patients out of the 41, who initially had complete response developed later recurrent pleural effusion and needed again tube thoracostomy and a second attempt of chemical pleurodesis.The mean survival of the entire study population was 7,5 ± 1,2 months (median: 5,4 months).Management of malignant pleural effusions depends on the underlying malignancy, extent of disease, potential effectiveness of treatment and performance status. In patients with lymphoma, small cell lung cancer or germ cell neoplasms, pleural effusions may be controlled initially by systemic therapy alone. In patients with metastatic breast or non small cell lung carcinoma, local palliative treatment is often required. Since malignant pleural effusions are frequently a preterminal event with a 30-day mortality rate of 29 to 50%, treatment is directed toward symptomatic relief with minimal discomfort, inconvenience and cost -8.Local treatment options include repeated thoracenteses, chest tube drainage with sclerotherapy, pleuroperitoneal shunt or pleurectomy. Repeated thoracentesis is usually a temporizing measure and carries the risk for pneumothorax and pleural infection . InpatiePleural effusion due to metastatic ovarian cancer is a frequent phenomenon and as shown in our study it can occur as early as one month or as late as 36 months with a median of 8,5 months. This is in complete accord with the study of Cheng et al who found a median interval of 9 months . When efNumerous sclerosing agents have been used to treat malignant pleural effusions. Until recently, tetracycline was the most commonly used sclerosing agent with response rates ranging from 25 to 100% ,15. BecaBleomycin has been studied extensively as a sclerosing agent ,17. GoffTalc has proved to be one of the most effective sclerosing agents for treating malignant pleural effusions. Talc causes severe pleuritis resulting in effective pleurodesis but can worsen dyspnea and can result in respiratory failure . Other cMany other chemotherapeutic agents such as doxorubicin, cisplatin and cytarabine combination, etoposide, fluorouracil and mitomycin have been used for sclerotherapy. In addition radioactive isotopes, corynebacterium parvum, interferon and recombinant interleukin-2 have been instilled in the pleural space for treatment of malignant pleural disease. Response rate have been variable and less than optimal. Side effects are not inconsequential and thus none of these agents have gained widespread use .Mitoxantrone is a synthetic anthracenedione which has been demonstrated to be effective in the treatment of peritoneal and pleural effusion. From a pharmacological point of view, mitoxantrone may be an especially appropriate choice due to its higher molecular weight and polarity since this may be factor important in prolonging contact with the pleura. The mechanism of intrapleural action of mitoxantrone has not yet been established. Both the inflammatory and antineoplastic activity of mitoxantrone intrapleurally have been described ,24.Our findings are consistent with the findings of others. In a prospective study in 18 patients, Musch et al  reportedThere is only one study which proved mitoxantrone ineffective. Groth et al.  presenteOur study confirmed the majority of previous reports that mitoxantrone is an effective agent in controlling recurrent malignant pleural effusions. The overall 30-day response rate was 88%. Side effects were mild and rare.To develop new treatment plans for the management of pleural effusions, one must consider several requirements. First, no treatment regimen should exacerbate patients' symptoms, since palliation is the main aim. Second, seriously ill patients should not be subjected to procedures associated with high mortality and morbidity. Third, since about half the patients with pleural effusion will have no other clinically apparent metastases, treatment should be local rather than systemic. To be successful, the local treatment has to be effective and given at the first sign of the effusion, because inadequate or delayed treatment may eliminate the possibility of any subsequent therapy being effective, by producing loculation of the effusion.Pleural effusion often occurs during the course of ovarian cancer. Chemical pleurodesis via bedside thoracostomy has been shown to be effective and has become a common therapeutic approach. Using this approach we found mitoxantrone to be highly effective at controlling malignant pleural effusions and decreasing the associated symptoms of dyspnea and pain. Our data justify further studies in a controlled setting to elucidate the biological action and prognostic relevance of mitoxantrone in the treatment of malignant pleural effusions and to compare this agent with other treatment procedures.None declared.NB conceived the study.and performed thestatistical analysis. MV and RV participated in the design of the study. KK and CT conceived of the study and participated in its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Animals in the wild are constantly confronted with decisions: Where to nest? Who to mate? Where's the best forage? To explore the mechanisms underlying such decisions, animal behavior studies often incorporate concepts from economic theory. Mainstream models of choice in both economics and biology predict that preferences will be rational, or consistent across contexts, as a result of being motivated by self interest or, in the case of animals, reproductive success. Yet many studies report that when making decisions people often take shortcuts, using cognitive heuristics that may lead to economically irrational decisions, with similar claims now showing up in animal behavior studies.In a new study, Cynthia Schuck-Paim, Lorena Pompilio, and Alex Kacelnik ask whether studies applying economic rationality to animal behavior are controlling for potentially confounding effects inherent in such approaches. The authors suggest that observed “breaches of rationality” may stem from differences in the physiological state of animals “unwittingly imposed” by experimental design rather than from real irrational decisions.Choice studies typically offer subjects a range of choices that include clearly superior and inferior alternatives. While humans can simply hear about the various alternatives and their respective properties, animals must be trained to learn about the different choices. This difference is far from trivial, Schuck-Paim et al. argue, and could well require different interpretations of results in animal and human studies. In fact, economic theory states that optimal choices depend on both the properties of the option and the chooser's state. Training animals to learn of different choices typically involves giving them food rewards, which means that an animal's energetic state—that is, hungry versus sated—will change over a day of training. A bird that's eaten an ounce of birdseed is more likely to opt for an “irrational” option—say, a choice that dispenses little food—than one that's hungry.To examine this theoretical constraint under experimental conditions, Schuck-Paim et al. trained European starlings to choose between two rich food sources  and one of two poorer “decoys” in different contexts. One of the focal options offered more food while the other offered a shorter delay between pecking a key and receiving the food, but their amount/delay ratios were equal. The decoys were considered less preferable because their ratio of amount to delay was lower than that of the focal options. But the decoys could potentially confound the results because repeated training to each decoy could sate a bird's appetite to different degrees: although amount/delay was equal among the decoys, their long term energetic consequences differed.The authors tested for preference between the focal options under three experimental conditions: altering the birds' food intake/energetic state with no decoys; changing the decoy and not controlling for its corresponding energetic contributions; and changing the decoy but controlling for its energetic consequence .Schuck-Paim and collaborators show that the birds' preferences between the focal options differed significantly between treatments, in apparent breach of economic rationality; the preference for the larger reward option over the shorter delay option was much stronger when the trial involved lower accumulated intake than when the accumulated intake was high. Introducing the decoys resulted in an “irrational” preference only when the decoys were allowed to have an effect on food intake, suggesting that the choice resulted from the birds' energetic state rather than from cognitive mechanisms of choice similar to those used to explain irrationality in human subjects.The authors offer an evolutionary and mechanistic explanation for why animal preference might be governed by energetic state, including the possibility that animals are less motivated to focus exclusively on the richest option when they are well fed. But they are careful to disabuse the notion that “state-dependence accounts for all reported inconsistencies in animal choice” or that animals do not employ cognitive mechanisms of choice similar to those of humans. Altogether, Schuck-Paim and co-authors argue, these results warn that studies appropriating ideas from other disciplines can introduce confounding effects. And that researchers would do well to carefully examine the underlying causes of observed animal behaviors when testing ideas formulated in a nonbiological framework.


RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system.Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein.Here we demonstrate that both transient and constitutive expression of the Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.These results indicate that at least part of the process of RNAi in  Neurospora crassa [Caenorhabditis elegans [Arabidopsis thaliana [Drosophila cells [RNA interference (RNAi) is a process in which the introduction of dsRNAs into cells leads to inactivation of expression of genes containing homologous sequences by sequence-specific degradation of mRNA (for reviews see ,2). RNAia crassa -12, Caen elegans -15 and Athaliana -18. dsRNla cells -22 and bla cells , and an Tobacco etch virus (TEV), was initially characterised as a determinant of virus movement in plants [Cucumber mosaic virus (CMV), a cucumovirus, interferes with the systemic spread of the silencing signal, preventing initiation of silencing in newly emerging tissues [Cowpea mosaic virus, the geminivirus African cassava mosaic virus, the potexvirus Narcissus mosaic virus, the tobamovirus TMV, the sobemovirus Rice yellow mottle virus (RYMV), the tombusvirus Tomato bushy stunt virus (TBSV) and the tobravirus Tobacco rattle virus (TRV) also were able to suppress GFP silencing [Drosophila cells of expression of some silencing suppressors from plant viruses.PTGS in plants can operate as a defence mechanism against virus infection (for reviews see ,23) and n plants  and subsn plants . HC-Pro n plants  and appen plants . In cont tissues . A surveilencing . Althougilencing . Thus, iilencing . We haveDrosophila DS2 cells and their effect on dsRNA-mediated silencing of a lacZ gene examined using a previously described system [The TEV HC-Pro or CMV 2b proteins were transiently expressed in d system . Expressd system  as a negd system . NortherDrosophila cells when they were transfected with a lacZ expression plasmid (pMT/V5-His/lacZ) (Table lacZ gene demonstrating induction of RNAi. No significant decrease in the number of cells stained was observed when cells were transfected with pMT/V5-His/lacZ and dsRNA derived from the green fluorescent protein gene indicating that the silencing was specific. Co-transfection of Drosophila cells with pMT/V5-His/lacZ, lacZ-specific dsRNA and an HC-Pro expression vector (pMT-HC-Pro) resulted in staining of ~46% of cells, indicating that RNAi was being suppressed. Transfection of cells with the mutant pMT-HC-Pro-K along with pMT/V5-His/lacZ and dsRNA resulted in no suppression of RNAi. This indicates that a mutation affecting the ability of HC-Pro to suppress PTGS in plants also affects suppression of RNAi in Drosophila cells. Transfection of cells with pMT-2b along with pMT/V5-His/lacZ and dsRNA resulted in no increase in the percentage of transfected cells staining for β-galactosidase activity compared to cells transfected only with pMT/V5-His/lacZ and dsRNA.β-galactosidase activity could be detected by staining in ~80% of Z) Table . ~17% ofet al., 2000) was used as a control in order to eliminate the possibility that stable transformation of the cells could interfere with the RNAi mechanism. The transfection efficiency of all transformed lines with pMT/V5-His/lacZ was lower than that of the control DS2 cells (Table Stable cell lines expressing HC-Pro (DS2-HC-Pro), or CMV 2b (DS2-2b) were produced in order to improve the efficiency of the RNAi suppression assay by reducing the number of different nucleic acid molecules needed for co-transfection. Immunoblotting of lysates of DS2-2b cells confirmed that the 2b protein accumulated in these cells Fig , lane 8 ls Table ; possiblls Table . TransfeDrosophila cells.Similar results were obtained when the DS2-2b cells were grown in the presence of CuSO4 to induce expression of the virus protein before transfection with pMT/V5-His/lacZ and dsRNA, indicating that the CMV 2b protein could suppress neither the initiation nor the maintenance of the dsRNA-induced silencing in the Drosophila cell system could be used as a screen for RNAi suppression effects caused by other virus proteins. We chose to examine the potential RNAi suppression activity of a protein from the tobravirus TRV. This virus is able to suppress transgene silencing in plants [We were interested to determine if the n plants  but the n plants , howeverNicotiana benthamiana plants with PVX alone initially induced vein chlorosis and systemic leaf curling, although the plants continued to grow. Inoculation with PVX carrying the 16K gene caused similar initial symptoms but led to tip necrosis and death of the plants [One characteristic of plant virus silencing suppressor proteins is often significant enhancement of disease symptoms when they are over-expressed from a viral vector . Similare plants .Drosophila cells after transfection of cells with the expression plasmid pMT-16K, and the 16K protein could be detected by western blotting when the cells were induced with CuSO4  that is defective in suppression of gene silencing [Drosophila cells. This strongly supports the idea that the TEV HC-Pro protein targets the same component of the plant PTGS system and the Drosophila RNAi system. Our demonstration that the TRV 16K protein also is able to interfere with RNAi in Drosophila cells, leads us to suggest that many other plant virus silencing suppressor proteins are likely to be functional in this system.Suppression of RNAi in  ability . These p ability ,27. Thesilencing  but is eilencing  was alsolacZ gene alone that there is considerable variation in the amount of staining of individual cells indicating differences in the amount of gene expression in individual cells. There is no reason to suppose that this variability in gene expression is limited to the lacZ gene and it is possible that low levels of the suppressors may fail to inhibit silencing in some of the cells within a culture. Secondly, the experimental protocol requires a time delay between transfection of the cells with the lacZ plasmid and dsRNA before induction of expression of the suppressor proteins in order to allow the cells to recover from the transfection procedure. Some production of siRNAs from the dsRNA may possibly occur in cells during this lag period before the suppressors are induced. For these reasons the usefulness of the Drosophila RNAi system for studying the mechanisms of action of plant virus suppressor proteins may be limited.SiRNAs were detected in our cell lines expressing HC-Pro and the TRV 16K protein even though suppression of silencing was observed. This is not totally unexpected for a number of possible reasons. Firstly, suppression of RNAi is partial in our cultures indicating that some RNAi, and presumably siRNA production, does occur in some of the cells. We have observed with cells expressing the Arabidopsis that are impaired in PTGS have an increased susceptibility to CMV, showing that PTGS can operate as a defensive system that targets viruses [Drosophila and possibly other organisms as well as allowing detailed characterisation of the mode of action of the virus suppressor proteins. Furthermore, it is becoming apparent that RNAi or PTGS may play a role in processes other than defence against foreign RNAs. Transformation of plants with TEV HC-Pro or rgs-CaM, a plant-encoded PTGS-suppressor protein related to calmodulin, interrupted normal development and led to the formation of differentiated tumours at the stem/root junction [Arabidopsis gene CARPEL FACTORY is related to Drosophila Dicer and is involved in plant development and fertility [C. elegans also appears to have a role in both RNAi and germ-line development 15]. Components of the RNAi mechanism including a homolog of Dicer are also involved in synthesis of short temporal RNAs that regulate developmental timing in C. elegans [Mutants of  viruses . Plant v viruses . The supjunction . The Araertility , and the elegans -42. InteDrosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi.These results indicate that at least part of the process of RNAi in KpnI-XhoI fragment into pMT/V5-HisA (Invitrogen) to produce plasmid pMT-HC-Pro. The same primers were also used to clone a mutant HC-Pro gene  of the TEV genome containing the HC-Pro sequence was amplified by reverse transcription – polymerase chain reaction (RT-PCR) using as a template RNA from an infected plant, and primers HC-Pro-1 (5'-CCGGTACCATGAGCGACAAATCAATCTCTGAGGC-3') and HC-Pro-2 (5'-GGCTCGAGCTACACATCTCGGTTCATCCCTCC-3'). The primers add an ATG codon to the start of the open reading frame and the HC-Pro gene was cloned as a   from trlacZ gene was amplified using pcDNA3.1/HisB/lacZ (Invitrogen) as a template and primers lacZ-1 (5'- TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC-3') and lacZ-2 (5'- TAATACGACTCACTATAGGGCAAACGGCGGATTGACCG-3'). Both primers contain T7 RNA polymerase promoter sequences (shown underlined). The PCR product was used to direct synthesis of dsRNA using T7 RNA polymerase (Invitrogen) after which the DNA template was removed by DNase digestion.cDNA corresponding to ~500 bp of the 5' end of the lacZ expression plasmid pMT/V5-His/lacZ were supplied as part of the Drosophila Expression System (Invitrogen) and cells were grown according to the manufacturer's instructions. Cells were grown in 60 mm dishes and transfected by calcium phosphate co-precipitation with various mixtures of 10 μg each of pMT/V5-His/lacZ and the suppressor expression plasmid DNAs and 5 μg dsRNA. In control transfections, 10 μg of an empty expression plasmid (pMT/V5-HisB) replaced the suppressor expression plasmid. After transfection the cells were washed twice in DES medium and grown for eight hours before expression of proteins was induced by addition of CuSO4 to a final concentration of 500 μM. Stably transformed lines expressing the HC-Pro, 2b or a6k genes were established by co-transfection of cells with pMT-HC-Pro or pMT-2b and pCo-Hygro (Invitrogen) followed by selection of transformed cells in medium containing hygromycin. Cells were stained 48 hrs after transfection to detect lacZ gene expression using a β-Gal Staining Kit (Invitrogen). Four randomly selected fields of view each containing ~100 cells were selected in each of duplicate plates and the number of cells staining blue was counted.DS2 cells, DES expression medium and the Cell lysates were analysed by polyacrylamide gel electrophoresis and separated proteins were transferred to nitocellulose using a carbonate buffer . The bloDrosophila cells using TriPure Isolation Reagent (Boehringer). Samples of RNA were separated by electrophoresis on formaldehyde/agarose gels, transferred to nylon membrane and probed with digoxigenin-labelled DNA probes corresponding to ~500nts at the 5' end of the lacZ gene or to the HC-Pro cDNA described above, as appropriate and essentially as described [lacZ probe, and HC-Pro-1 and HC-Pro-2 for the HC-Pro probe. Digoxigenin-labeled RNA Molecular Weight Marker II  was run as a size marker.RNA was extracted from escribed . The proDrosophila cell cultures were fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad), following the manufacturers instructions. ~20 μg of the short RNA species were separated by electrophoresis in a 15% polyacrylamide gel containing 7 M Urea and electroblotted as described by Llave et al. [lacZ gene as described above was synthesised from a PCR fragment containing the gene fragment downstream of a T7 RNA polymerase promoter, and hybridised to the blots according to the manufacturer instructions (Roche Diagnostics). Induction and detection of siRNAs to GFP or to 35S promoter sequences in agroinfiltrated plant tissue was performed as described by Canto et al. [For detection of short (~23nt) RNA species total RNA preparations from e et al. . After eo et al. .None declared.Drosophila expression experiments, northern blots and drafted the manuscript. SD carried out the immunoassays. TC carried out some of the siRNA assays. SM participated in the design of the study, carried out some of the siRNA assays and contributed to the manuscript. All authors read and approved the final manuscript.BR conceived of the study, and participated in its design, carried out the


In recent years out-of-hours primary care in the Netherlands has changed from practice-based to large-scale cooperatives. The purpose of this study is to determine patient satisfaction with current out-of-hours care organised in general practitioner (GP) cooperatives, and gain insight in factors associated with this satisfaction.From March to June 2003, 2805 questionnaires were sent to patients within three weeks after they had contacted the GP cooperative in their region. The study was conducted in the province of Limburg in the South of the Netherlands. One-third of these questionnaires was sent to patients who had only received telephone advice, one-third to patients who attended the GP cooperative for consultation, and one-third to patients who received a home visit. Four weeks after the first reminder, a non-respondents telephone interview was performed among a random sample of 100 patients. Analyses were performed with respect to the type of consultation.The total response was 42.4% (1160/2733). Sixty-seven percent of patients who received telephone advice only reported to be satisfied with out-of-hours care. About 80% of patients who went to the GP cooperative for consultation or those receiving a home visit, reported to be satisfied. Factors that were strongly associated with overall satisfaction included, the doctor's assistant's attitude on the phone, opinion on GP's treatment, and waiting time.Patients seem generally satisfied with out-of-hours primary care as organised in GP cooperatives. However, patients who received telephone advice only are less satisfied compared to those who attended the GP cooperative or those who received a home visit. In recent years, out-of-hours primary care in the Netherlands has been substantially reorganised. Formerly, general practitioners (GPs) used to perform these services in small locum groups (6 to 8 GPs) in which they joined a rota system. Nowadays, out-of-hours care is organised in large-scaled GP cooperatives 45 to 120 GPs) following examples in the UK and Denmark [20 GPs foThe initiative of reorganising out-of-hours care has come mainly from the profession itself, motivated by increased dissatisfaction with the organisation of former out-of-hours primary care services. This dissatisfaction was mainly due to the high perceived workload (after out-of-hours service a regular day of work followed), and poor separation between work and private life. The main advantage of the reorganisation was the substantial reduction of number of hours a GP has to be on call. Furthermore, the organisation of out-of-hours care became much more professional by installing management, employing doctor's assistants, and using chauffeured cars. Studies have indicated that GPs appear to be generally satisfied with out-of-hours care organised in cooperatives.Not only did things change for doctors, but also patients experienced some important changes in out-of-hours primary care. Generally, the reorganisation caused a shift from more personal care to more anonymous care, with increased distance to the GP. Formerly, when patients needed primary care outside office hours, the probability of being seen by their own or a local GP with whom they were familiar, was higher. In addition, when patients contacted the GP during out-of-hours in the past, they were most likely to speak to the GP himself on the phone. Nowadays, the phone is staffed by a doctor's assistant who decides what action should follow the patient's call. Moreover, out-of-hours care used to be delivered by local GPs, indicating short distances to the GP's practice. In large-scale GP cooperatives, the distance to a GP outside office hours will have increased substantially for most patients.We expected that patient satisfaction would have been reduced after the reorganisation, because factors that guaranteed personal out-of-hours care at a short distance, that may be important to patients, were changed substantially. Furthermore, in Denmark it has been shown that after the out-of-hours primary care reform patient satisfaction dropped significantly,5.Patient satisfaction with out-of-hours primary care has quite often been investigated, especially in the UK -11. MostInsight in patient satisfaction with out-of-hours care supplies the health care provider with important information on the patient's perception of the quality of that care. During the last years, Dutch GP cooperatives have often received negative publicity in newspapers. The reorganisation has had some important implications for patients, and therefore research on their opinions about current out-of-hours care is warranted. The purpose of this study is to determine patient satisfaction with current out-of-hours care, and to determine how satisfaction is related to different aspects of the patient's contact with a GP cooperative.The study was conducted in the province of Limburg in the South of the Netherlands. With respect to out-of-hours primary care, the province is organisationally divided in five regions. Two of these regions each have two GP cooperatives (NL and ML), one region (OZL) has one GP cooperative with two satellite centres, and in the other two regions (WM and MH) only one GP cooperative is operational. All cooperatives but one (MH) are organisationally separate from the emergency department of the local hospital, and are located nearby the hospital. This implies that patients may choose between attending the emergency department and the GP cooperative for medical problems during out-of-hours. The MH cooperative is located at the emergency department of the region's only hospital and sees all patients needing out-of-hours care, except for those having a referral for emergency care.st of September 2001.In total, these seven GP cooperatives cover a population of about 1.1 million people , and are fully operational since the 1To determine relevant issues for the questionnaire we interviewed GPs and managers involved with out-of-hours primary care. In addition, we analysed the process for a patient contacting the GP cooperative for all three loci of care (telephone advice, consultation at the cooperative, and home visits) separately to make sure that all facets of the GP cooperative a patient faces would be incorporated in the questionnaire. Moreover, we also analysed unpublished Dutch questionnaires in this field, and the patient satisfaction questionnaire developed by McKinley et al. . Based oFinally three questionnaires were constructed for each of the three types of consultations (telephone advice, consultation at the cooperative, and home visit). The three questionnaires differed on items related to the specific type of contact, but general items were the same for all three questionnaires. In this way it was possible to avoid complex skip sections which lengthen the questionnaire and can reduce the response rate. We used a balanced Likert five point scale  to record responses.The questionnaire related to telephone advice contained six initial scales measuring: accessibility of the cooperative by phone, doctor's assistant's attitude, questions asked by the assistant, advice given by the assistant, urgency of patient's complaint, and overall satisfaction.The questionnaire related to consultations at the cooperative contained ten initial scales: accessibility of the cooperative by phone, doctor's assistant's attitude, questions asked by the assistant, urgency of patient's complaint, waiting time at the cooperative, waiting room, distance to the cooperative, GP's attitude, treatment by GP, and overall satisfaction.The questionnaire related to home visits contained eight initial scales: accessibility of the cooperative by phone, doctor's assistant's attitude, questions asked by the assistant, urgency of patient's complaint, waiting time until GP arrives, GP's attitude, treatment by GP, and overall satisfaction.In addition, patient characteristics such as, age, gender, level of education, and health insurance  were recorded. Patients were also asked which type of consultation they expected prior to their contact with the GP cooperative, and whether they thought that the right diagnosis had been made.From March to June 2003 a sample of 2805 patients – who had contacted the GP cooperative in their region – received a questionnaire by mail. Patients received this questionnaire within three weeks after they had contacted the GP cooperative. Sampling was performed per GP cooperative within the four-month period. With respect to patients who received telephone advice only and those who attended the GP cooperative, a computer program randomly selected each fourth patient contact with the GP cooperative backwards from the moment of sampling. Since the number of home visits is limited, all 150 patients, who were visited by a GP from the cooperative, prior to the moment of sampling received a questionnaire. These procedures assured that the time between receiving the questionnaire and the contact with the GP cooperative was not more than three weeks.Per region 450 questionnaires were sent out; 150 to patients who received only telephone advice, 150 to patients who visited the GP cooperative, and 150 to patients who received a home visit. Because of parallel research, more questionnaires were sent out in one of the regions (WM): 1005 questionnaires equally distributed among the three types of patient contact with the GP cooperative. The study size was chosen based on previous research by McKinley et al,13, who The study was approved by the Institutional Medical Ethics Board.Three to four weeks after the questionnaire had been distributed, a reminder was sent to patients who had not returned the questionnaire, with the exception of the WM area. Four weeks after the last reminder, a random sample of 100 patients who had not responded, was contacted by phone. They were asked about their reasons not to return the questionnaire, and about their opinion on the contact they had with the GP cooperative. This interview was performed during office hours, during a three-week period.Principal components analysis with varimax rotation was used to test whether the items could be assumed to measure similar aspects or components of patients' opinions about their contact with the GP cooperative. Next, Cronbach's alpha coefficient was calculated to estimate the internal consistency as a measure for reliability for each component. Finally, scale scores were calculated per component by summing the scores per item and expressing the total result as a percentage of the maximum score for each scale,14. ScalThe relationship between individual variables and overall satisfaction was analysed using multiple regression analysis, with subscale satisfaction scores as covariates. Variables that did not significantly contribute to the regression model were excluded from the final model. In case of missing data, listwise deletion of missing cases was applied. All data were analysed using SPSS-pc, version 10.0.5.Seventy-two of the 2805 questionnaires were excluded, either because they could not be delivered (patient had moved or gave a wrong address), the patient had died, or the patient was sent a double questionnaire (multiple contacts). Eventually the response was 42.4% 1160/2733). Generally more women responded to the questionnaire, and about three-quarter of the respondents had public health insurance (table 160/2733.Forty percent (366/908) of the patients who had received telephone advice only, returned the questionnaire. 67% of these patients responded to be satisfied (44.3%) or very satisfied 22.3%) with their contact with the GP cooperative, and 57% thought that the current out-of-hours care was an improvement compared to the former situation. We identified the same six scales that were initially set to represent patients' opinions on aspects of primary out-of-hours care  of the patients who attended the GP cooperative returned the questionnaire. Approximately 80% of these patients reported to be satisfied (54.6%) or very satisfied (26.3%) with their contact with the GP cooperative, and 61% thought that the current out-of-hours care was an improvement compared to the former situation. We identified nine scales that represent patients' opinions on aspects of primary out-of-hours care  of the patients that received a home visit by a GP from the cooperative returned the questionnaire. About 81% of these patients reported to be satisfied (42.8%) or very satisfied (38.8%) with their contact with the GP cooperative, and 61% thought that the current out-of-hours care was an improvement compared to the former situation. We identified six multi-item scales that represented the patient's opinion on different aspects of out-of-hours primary care, with Cronbach's alpha coefficients between 0.73 and 0.96. Two initial scales have been merged into one scale; these were patient's opinion on the GP's attitude and the treatment by the GP. All other identified scales were the same as the initial scales. Detailed information on the scales and items can be found in table We found that five variables predicted overall satisfaction, with a variance explained of 51%  and those who received a home visit (72.5 ± 1.37) Table . HoweverOut of 100 randomly selected patients, who had not returned the questionnaire, we were able to reach 63 by phone. Of these 63 non-respondents 35 (55.6%) were male and 28 (44.4%) were female. Many of them reported that they had forgotten to return the questionnaire (40%). A minority said not to be interested (6.7%) or did not find it needful (6.7%). Most non-respondents (46.7%) gave other reasons like, no time, too difficult, or had lost the questionnaire.Of these patients, about 71% reported to be satisfied or very satisfied about their contact with the GP cooperative.The results of this study indicate that patients were generally satisfied about their contact with the GP cooperative. Patients who received telephone advice only, however, were less satisfied compared to those who attended the GP cooperative and those who received a home visit. A small majority believes that current out-of-hours care is an improvement compared to the former situation.The response rate in our study is not as high as presented previously by others who investigated patient satisfaction with out-of-hours primary care,7-9,11. In the process of determining relevant aspects of out-of-hours care to patients, we consulted the province patient organisation and studied discussions on out-of-hours care in newspapers. We have not used patient interviews, although this might have identified other relevant domains of out-of-hours care. However, we think that the current questionnaire captures many relevant domains of out-of-hours care to patients as well as to health professionals.Based on results of a Danish study ,5, we exWe have no reason to believe that the results of this study cannot be generalised to other regions in the Netherlands. Most GP cooperatives in the Netherlands are comparable, with respect to organisation and population size, to those in this study. In addition, the region in our study includes both rural and urban areas. Despite the similarities with out-of-hours primary care in other countries such as Ireland, the UK and Denmark, there are also differences with respect to the way these cooperatives are organised, and therefore care should be taken when generalising these results to other countries.We identified various factors that are closely related to overall satisfaction. These factors give important insight in aspects of the GP cooperative that really matter in the patient's opinion on out-of-hours care. The patient's opinion on the doctor's assistant's attitude on the phone proved to be the strongest predictor of overall satisfaction with respect to those having received telephone advice and those that received a home visit. Also for those attending the GP cooperative, this factor was a relatively strong predictor; in this group the patient's satisfaction with the GP's treatment was by far the strongest predictor of overall satisfaction. Thus, it appears that the patients' impression of the first contact they have with the cooperative, which is mostly through telephone, strongly influences overall satisfaction.In accordance with other studies we found that patients who received telephone advice only, are generally less satisfied with the out-of-hours service, compared to those attending the GP cooperative and those receiving a home visit,5,8,9,11It is questionable whether extra information to the public on the process of the telephone triage process will adjust patients' expectations. Similar to what Salisbury et al suggesteThis study has shown that patients are generally satisfied with out-of-hours care, but that patients with telephone advice only are less satisfied than those attending the cooperative or receiving a home visit. The patient's opinion on several aspects of out-of-hours care can predict overall satisfaction, with different predictors regarding the three types of consultations. However, the accessibility by phone and the doctor's assistant's attitude on the phone are always significantly related to overall satisfaction, regardless of the type of consultation. This implies that when trying to improve overall satisfaction one should always focus on at least these two factors. The questionnaire used in this study has potential for use as a standardised instrument for assessing satisfaction with out-of-hours care in The Netherlands for either research or service monitoring.The author(s) declare that they have no competing interests.CU participated in the design of the study, performed the statistical analysis, and drafted this manuscript. AA, SH, PZ, and HC participated in the design of the study, supervised the project, and provided critical edits to this manuscript.The pre-publication history for this paper can be accessed here:


Both Taxotere and Capecitabine have shown anti-cancer activity against various cancers including prostate cancer. In combination, Taxotere plus Capecitabine has demonstrated higher anti-cancer activity in advanced breast cancers. However, the molecular mechanisms of action of Taxotere and Capecitabine have not been fully elucidated in prostate cancer.The total RNA from PC3 and LNCaP prostate cells untreated and treated with 2 nM Taxotere, 110 μM Furtulon (active metabolite of Capecitabine), or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 hours, was subjected to Affymetrix Human Genome U133A Array analysis. Real-time PCR and Western Blot analysis were conducted to confirm microarray data.Taxotere and Furtulon down-regulated some genes critical for cell proliferation, cell cycle progression, transcription factor, cell signaling, and oncogenesis, and up-regulated some genes related to the induction of apoptosis, cell cycle arrest, and differentiation in both cell lines. Taxotere and Furtulon also up-regulated some genes responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents.Taxotere and Furtulon caused the alternation of a large number of genes, many of which may contribute to the molecular mechanisms by which Taxotere and Furtulon inhibit the growth of prostate cancer cells. This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against prostate cancer. Prostate cancer is the most common cancer and the second leading cause of cancer related deaths in men in the United States with an estimated 230,110 new cases and 29,500 deaths in 2004 [Taxus baccata. Its known basic mechanism of action is that it binds to tubulin and deranges the equilibrium between microtubule assembly and disassembly during mitosis [XL and bcl-2, and upregulation of p21WAF1 and p53 [Taxotere, a member of taxane family, is semi-synthesized from an inactive taxoid precursor extracted from the needles of the European yew,  mitosis . Stabili mitosis . Taxoter mitosis ,6. In me mitosis ,8. Thus, and p53 ,10. We h and p53 .in vivo and in vitro. After being converted to 5-FU, Capecitabine inhibits essential cellular biosynthetic processes and is incorporated into DNA to inhibit normal bioprocess function of the cell [Capecitabine is an orally administered systemic prodrug of 5'-deoxy-5-fluorouridine (5-DFUR or Furtulon) which is converted to 5-fluorourasil (5-FU) . Capecitthe cell . Capecitthe cell -16. 5-FUthe cell ,17.Clinical trials showed that chemotherapeutic combination with Taxotere and Capecitabine resulted in improved objective response rate and overall survival without a significant increase in the treatment related adverse effects in metastatic breast cancer and advanced non-small cell lung carcinoma ,19. Howe2atmosphere at 37°C. Taxotere  was dissolved in DMSO to make 4 μM stock solution. Furtulon  was dissolved in PBS to make 100 mM stock solution. For growth inhibition, PC3 and LNCaP cells were treated with Taxotere (1, 2, and 4 nM), Furtulon (50, 100, and 200 μM), or 1 nM Taxotere plus 50 μM Furtulon for one to three days. Control PC3 and LNCaP cells received 0.01% DMSO or 0.1% PBS for same time points. After treatment, PC3 and LNCaP cells were incubated with MTT (0.5 mg/ml, Sigma, St. Louis, MO) at 37°C for two hours and then with isopropyl alcohol at room temperature for one hour. The spectrophotometric absorbance of the samples was determined by using ULTRA Multifunctional Microplate Reader (TECAN, Durham, NC) at 595 nm. The concentrations of Taxotere and Furtulon used for our in vitro studies are easily achievable in humans, suggesting that our experimental results are relevant for human applications. The experiment was repeated three times and a t test was performed to verify the significance of cell growth inhibition after treatment.PC3 (ATCC, Manassas, VA) and LNCaP (ATCC) human prostate cancer cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO24 primer instead of the oligo(dT) provided in the kit. Then, the biotin-labeled cRNA was transcripted in vitro from cDNA by using BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY), and purified by RNeasy Mini Kit. The purified cRNA was fragmented by incubation in fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to Human Genome U133A Array (Affymetrix, Santa Clara, CA), which contains 22,215 human gene probes, and hybridized to the probes in the array. After washing and staining, the arrays were scanned. Two independent experiments were performed to verify the reproducibility of results.PC3 and LNCaP cells were treated with 2 nM Taxotere, 110 μM Furtulon, or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 h. Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA) and purified by RNeasy Mini Kit and RNase-free DNase Set  according to the manufacturer's protocols. cDNA for each sample was synthesized by Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) using the T7-(dT)t test between treated and untreated samples. Clustering and annotation of the gene expression were analyzed by using Cluster and TreeView [The gene expression levels of samples were normalized and analyzed by using Microarray Suite, MicroDB™, and Data Mining Tool software (Affymetrix, Santa Clara, CA). The absolute call  and average difference of 22,215 gene expressions in a sample, and the absolute call difference, fold change, average difference of gene expressions between two or several samples were normalized and identified using these software. Statistical analysis of the mean expression average difference of genes, which show >2 fold change, was performed using a TreeView , Onto-Ex2O) in SmartCycler II . The PCR program was initiated by 10 min at 95°C before 40 thermal cycles, each of 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative Ct method and were normalized by actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes Table  with varWAF1 (1:500, Upstate, Lake Placid, NY), anti-Bax (1:10000, Trevigen, Gaithersburg, MD), anti-survivin (1:200, Alpha Diagnostic, San Antonio, TX), anti-CDC2 (1:200, Santa Cruz, Santa Cruz, CA), anti-cyclin A (1:250, NeoMarkers, Union City, CA), anti-cyclin B (1:200, Santa Cruz, Santa Cruz, CA), anti-cyclin E (1:250, NeoMarkers), and anti-β-actin (1:10000, Sigma, MO) primary antibodies, and subsequently incubated with secondary antibody conjugated with fluorescence dye. The signal was then detected and quantified by using Odyssey infrared imaging system (LI-COR, Lincoln, NE).We also conducted Western Blot analysis to verify the alterations of genes at the level of translation for selected genes with varying expression profiles. The PC3 and LNCaP cells were treated with 1 and 2 nM Taxotere or 50 and 110 μM Furtulon for 24, 48, and 72 hours. After treatment, the cells were lysed in 62.5 mM Tris-HCl and 2% SDS, and protein concentration was measured using BCA protein assay (PIERCE, Rockford, IL). The proteins were subjected to 10% or 14% SDS-PAGE, and electrophoretically transferred to nitrocellulose membrane. The membranes were incubated with anti-cathepsin C (1:200, Santa Cruz, Santa Cruz, CA), anti-p16 (1:200, Santa Cruz, Santa Cruz, CA), anti-IKKα (1:100, Santa Cruz, Santa Cruz, CA), anti-p21MTT assay showed that the treatment of PC3 and LNCaP prostate cancer cells with Taxotere, Furtulon, or lower concentration of Taxotere plus Furtulon resulted in dose and time-dependent inhibition of cell proliferation Figure , demonstMicroarray analysis showed that the alterations of gene expression were occurred as early as 6 hours of Taxotere and/or Furtulon treatment, and were more evident with longer treatment Table  and 3.CIP1, VDUP1, BTG, etc), and tumor suppression , transcription factors (transcription factor A, ATF5, TAF1131L, FOXM1, etc), and oncogenesis (GRO oncogene, BRCA1 associated RING domain, tumor-associated nuclear protein p120, etc) in Taxotere and/or Furtulon treated prostate cancer cells  that showed no change in mono-treatment Table  and 5, sTaxotere and Furtulon also up-regulated some genes (S-100P, ALDH1A3, casein kinase, annexin, etc) responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents Table  and 3. TTo verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes with varying expression profiles for real-time RT-PCR and Western Blot analysis. The results of real-time RT-PCR for these selected genes were in direct agreement with the microarray data Figure . The samIt has been known that Taxotere binds to microtubules while Capecitabine is incorporated into DNA, inhibiting the bioprocess in cancer cells ,13. HoweWAF1, p16INK4A, and p18INK4C have been demonstrated to arrest the cell cycle and inhibit the growth of cancer cells [WAF1 and other growth inhibitor genes (BTG2, VDUP1, anti-proliferative B-cell translocation gene 1) [INK4A and p18INK4C were also observed in combination treatment but not in mono-treatment, suggesting the synergic effect of combination treatment. These observations are novel in Taxotere and/or Furtulon treated prostate cancer cells.It has been well known that CDCs regulate the molecules related to the cell cycle initiation and progression and that cyclins associate with cyclin-dependent protein kinases (CDKs) and CDCs to control the process of cell cycle ,24. The er cells ,24. Our er cells -27 were  gene 1) ,29 were Induction of apoptosis by chemotherapeutic agents also leads to the inhibition of cancer cell growth. It has been reported that Taxotere is able to induce apoptosis by caspase-3 dependent or independent cell death mechanism . CapecitWe also found that Taxotere and/or Furtulon inhibited the expression of transcription factors (FOXM1, ATF5, TFAM, TAFII31L), translation factors (EIF1A, EIF5A), oncogene (GRO1, GRO3, BRCA1-associated protein, tumor-associated nuclear protein p120), and heat shock protein, and up-regulated the genes for differentiation (prostate differentiation factor). These results are novel, and suggest the beneficial effects of Taxotere and/or Furtulon on the inhibition of cancer cell growth and oncogenesis.It is important to note that Taxotere and/or Furtulon also up-regulated the expression of some genes which are known to induce cell resistance to chemotherapeutic agents and to favor cell survival. Among these genes, calcium-binding protein S100P has been found to be highly expressed in cells which develop acquired resistance to anti-tumor agents . The oveTaxotere showed no effect on AR expression while Furtulon down-regulated AR expression in LNCaP cells, suggesting that the combination could be superior in AR-positive cells. The genes altered by Taxotere and/or Furtulon with respect to the control of cell growth, apoptosis, transcription, oncogenesis, and metastasis in androgen insensitive PC3 cells are different from that in androgen sensitive LNCaP cells, suggesting that the effects of Taxotere and Furtulon may be mediated by both AR-dependent and independent signaling pathways. We observed up-regulation of tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2, and protease inhibitor 3 in Taxotere and/or Furtulon treated PC3 cells, suggesting that Taxotere and/or Furtulon may exert anti-metastatic effect. However, we also observed increase in the expression of MMP1, MMP9, cathepsin B, uPA, and tPA in Taxotere and Furtulon treated PC3 cells, therefore, more experimental studies are needed to reveal the overall effect of Taxotere and Furtulon on metastatic processes. These results were not observed in androgen sensitive LNCaP cells, suggesting difference in effects that could be mediated through different cell signal transduction pathways.In conclusion, Taxotere and/or Furtulon directly and indirectly caused changes in the expression of many genes that are critically involved in the control of cell proliferation, apoptosis, transcription, translation, oncogenesis, angiogenesis, metastasis, and drug resistance Figure . These fThe author(s) declare that they have no competing interests.FHS designed the study and prepared the manuscript. YL carried out cell growth inhibition, microarray and Western Blot analysis and drafted the manuscript. MH participated in the design of the study. RL and SHS carried out real-time PCR. JE prepared Furtulon reagent. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


Growing evidence shows that interleukin 13 (IL-13) may play an essential role in the development of airway inflammation and bronchial hyper-responsiveness (BHR), two defining features of asthma. Although the underlying mechanisms remain unknown, a number of reports have shown that IL-13 may exert its deleterious effects in asthma by directly acting on airway resident cells, including epithelial cells and airway smooth muscle cells. In this report, we hypothesize that IL-13 may participate in the pathogenesis of asthma by activating a set of "pro-asthmatic" genes in airway smooth muscle (ASM) cells.Microarray technology was used to study the modulation of gene expression of airway smooth muscle by IL-13 and IL-13R130Q. TaqMan™ Real Time PCR and flow cytometry was used to validate the gene array data.IL-13 and the IL-13 polymorphism IL-13R130Q (Arg130Gln), recently associated with allergic asthma, seem to modulate the same set of genes, which encode many potentially interesting proteins including vascular cellular adhesion molecule (VCAM)-1, IL-13Rα2, Tenascin C and Histamine Receptor H1, that may be relevant for the pathogenesis of asthma.The data supports the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma. Recent reports using murine models of allergic asthma have shown that the Th2 type cytokine IL-13 may play a critical role in the pathogenesis of asthma, either by regulating airway inflammation, mucus hyper-secretion or airway hyper-responsiveness . EvidencUsing complementary molecular approaches, we investigated the effect of IL-13 on the transcription of "pro-asthmatic" genes in human airway smooth muscle cells (HASMC). The effect of IL-13 was compared to that of IL-13R130Q, a naturally occurring isoform resulting in a change from glutamine to arginine residues in the coding region that is associated with high serum IgE levels . Interes2, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F12 medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) 100 U/ml penicillin (Gibco), 0.1 mg/ml streptomycin (Gibco), and 2.5 μg/ml fungizone (Gibco). Aliquots of the cell suspension were plated at a density of 1.0 × 104 cells/cm2. The cells were cultured in Ham's F12 media supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and this was replaced every 72 h. Human ASM cells in subculture during the second through to fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin [Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the tracheal muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCld myosin . Unless Total cellular RNA was isolated from IL-13 (50 ng/ml), IL-13R130Q (50 ng/ml) or control treated HASMC using the RNeasy mini kit  as per manufacturer's instructions. The IL-13 was purchased from R&D Systems (Minneapolis, MN) and the IL-13R130Q was generated in house at Centocor Inc. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, New Jersey). Samples that demonstrated high quality (ratio of 28S rRNA and 18S rRNA is greater than 1.7) were submitted for microarray analysis.A complimentary DNA (cDNA) microarray, or DNA chip (Target B), containing a total of 8159 human gene cDNA clones from Research Genetics (IMAGE consortium, Huntsville, AL), Incyte Genomics (Santa Clara, CA) and internal sources was used in this study. All clones have been verified by DNA sequencing and are printed as 2 independent spots on a given chip. Duplicate chips were used for each RNA sample. Non-linear normalization between duplicate chips allowed each clone to be averaged to a single intensity value for each RNA sample.RNA amplification, probe synthesis and labeling, cDNA chip hybridization and washing were performed as described previously . AgilentIn this study, fifty one-color cDNA microarrays were used to profile gene expression in human airway smooth muscle cells from 3 donors stimulated with IL-13, or its variant IL-13R130Q at 2 time points (6 hr and 18 hr). Untreated samples from the same group of donors were used as control. The samples being analyzed are listed in Table th percentile of all measurements in that sample. The intensity of each clone was then normalized to the median intensity of that clone in the untreated control group. The normalized intensity was then log transformed.Purified cDNA probes were hybridized to two microarrays, each containing two spots for each cDNA. Raw intensity data from the cDNA arrays were first normalized within each sample. Linear normalization and then nonlinear normalization was performed within each sample. Outlier intensity data points (greater than 1.4 fold away from the median of replicate measurements) were identified and removed from the data sets. The average intensity was generated by calculating the arithmetic mean of nonoutlier intensity values. The averaged intensity for each clone was further normalized across all samples. Chip-to-chip normalization was performed by dividing the averaged intensity of each clone by the 50.0Using Partek Pro™ 5.1, sources of variance, such as batch effects, were identified by Principle Component Analysis (PCA) and appropriate factors were taken into account in the Analysis of Variance (ANOVA). ANOVA was performed to identify the genes that were differentially expressed by cytokine stimulation. Treatment (IL-13 and IL-13R130Q), time (6 hour and 18 hour), and donor (1, 2, and 3) were the three main effects considered in ANOVA. P-value cutoff was 0.05.Benjamini and Hochberg false discovery rate (FDR) was performed for multiple testing correction. After comparing the gene lists from IL-13 and IL-13R130Q treatments, it was clear that these two treatments resulted in the regulation of the same set of genes. Subsequently, samples from these two treatments were combined and regarded as replicates in ANOVA. Furthermore, outliner samples in the data set were detected by PCA and removed to improve the detection power.As an alternative approach, fold change comparisons (cutoff = 1.5 fold) between a treated condition and the control were carried out within each donor by using GeneSpring™ 6.2 . A gene ® RT reagents (Applied Biosystems) at 37°C for 120 min followed by 25°C for 10 min. Forty ng of cDNA per reaction were used in the Real Time PCR using the ABI Prism® 7900 sequence detection system . In the presence of AmpliTaq Gold DNA plolymerase , the reaction was incubated for 2 min at 50°C followed by 10 min at 95°C. Then the reaction was run for 40 cycles at 15 sec, 95°C and 1 min, 60°C per cycle. Assays-on-Demand™ primers and probes (Applied Biosystems) were used in the PCR. The Real Time PCR data was analyzed using the standard curve method.1 μg of total RNA from each of the IL-13 (50 ng/ml) or IL-13R130Q (50 ng/ml) or control treated HASMC were used for the reverse transcription (RT) reaction. The RT reaction was performed as per protocol using TaqManFlow cytometry was performed as described previously with slight modifications . Brieflyin vivo relevance of these particular concentrations remains unknown.IL-13 may exert its deleterious effects in asthma by directly altering gene expression in airway resident cells such as epithelial cells or ASM cells -7,20. InThree donors were used and two types of analyses were carried out . Both IL-13 and IL-13R130Q generated a similar expression profile i.e., genes regulated by IL-13 were the same as those regulated by IL-13R130Q at the 1.5 fold cutoff. Table TaqMan™ Real Time PCR was used to validate VCAM1, IL-13Rα2, Tenascin C and Histamine Receptor H1. As shown in Figure In order to validate the modulatory effect of IL-13 on VCAM-1 and IL-13Rα2 genes at their protein level, flow cytometry was performed to confirm the up regulation of VCAM-1 and IL-13Rα2 in HASMC by IL-13. As shown in Figure Recent evidence using various experimental approaches such as gene-deficient mice, soluble inhibitors or transgene overexpressing IL-13 in the airways have highlighted the critical role of IL-13 in the pathogenesis of allergic asthma, possibly due to its ability to regulate goblet cell metaplasia, mucus hypersecretion and airway hyper-responsiveness . Few repin vivo [in vitro in isolated airways preparations [Our present study further supports the concept that IL-13 regulates the expression of different "pro-asthmatic" genes that are potentially important in the regulation of all three key features of asthma, i.e., airway inflammation, airway remodeling and bronchial hyper-responsiveness (for details see Table in vivo  or in viarations ,29,30. AThe receptor complex by which IL-13 regulates cellular function comprises the IL-13Rα1, which binds IL-13 and forms a complex with the IL-4Rα to initiate signal transduction via the JAK/STAT6 pathway . IL-13RαThese data further support the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma. Additional studies are clearly needed to define the transcriptional regulation of the different "pro-asthmatic" genes by IL-13, which may lead to novel therapeutic approaches for the treatment of allergic asthma.FS, LL, YA and RAP participated in the conception and design of the study. FS coordinated the study and along with YA drafted the manuscript. OT performed the RNA isolation and flow cytometry experiments. CH and KL performed the microarray data analysis. MB performed the Real Time PCR experiments. DG and BA proof read the manuscript. All authors read and approved the final manuscript.


Clinical Research faces perhaps a certain grain of confusion. Often, it is mistakenly reduced to case report. Caveats of even greater risks may be hidden in the secrets of patents and interests of manufacturers covertly promoting their products. Thus, a book clearly stating what a clinical trial is, well located within the scientific frame, appears as a significant contribution enhancing the importance and complexity of XXIst Century human health.Clinical Research, a branch of Applied Research, finds in clinical trials the most definitive tool for evaluation of its actual applicability. The book is divided in 19 chapters, each setting up unequivocally the Fundamental Point to be treated, as for example the introductory Chapter 1: A properly planned and executed clinical trial is a powerful experimental technique for assessing the effectiveness of an intervention. A clinical trial compares the effect and value of intervention(s) against a control in human beings. Intervention refers either to a drug, a procedure or a technological device, thus implying that biomedical engineering scientists and professionals might be involved in it. Clinical Research always presents early stages where the laboratory work is mandatory. This book emphasizes the concept all along, so placing the clinical trial in its proper hierarchical perspective. There is laboratory work during drug, device and/or procedure development, animal studies and early tests in small number of human beings. Case reports, typical of the hospital, find their place as a warning of adverse effects or as a flag of an unexpected beneficial effect.The book is preceded by two editions (1981 and 1985) and well backed by the experience and prestige of its authors. In Chapter 2 the authors emphasize that a clinical trial must have a primary question. It should be carefully selected, as any subsidiary question must be, too. Otherwise, the investigators may lose the track. Chapter 3 deals with the study population, underlying the importance of unambiguous eligibility criteria, the latter having a direct influence on the recruitment of participants and, thus, on the final number involved in the project (which may end up with not enough data). Chapter 4 outlines the basic study design; it stresses the demand of a control group and the need of randomization for assigning participants to control and intervention groups.Sample Size, addresses the question of how many subjects is enough. The trial must have sufficient statistical power to detect differences between groups. Therefore, calculation of sample size with provision for adequate levels of significance is essential. Several situations with their respective examples are presented making of this pages an invaluable tool. Unfortunately, several equations have been printed in such a way that there is confusion on whether some parameters are factors or exponents.An extremely important subject is the randomization process, essential to avoid any possible bias (Chapter 5). Several forms are presented: simple, blocked and stratified. More elaborate procedures make use of adaptive methods. There are examples and an appendix with an algorithm of general applicability. Bias (a systematic error) is one of the main concerns; thus, a double-blind design is essential (Chapter 6). Chapter 7, Baseline refers to the status of participants before the start of the intervention (Chapter 8), while Chapter 9 deals with the recruitment of participants. The latter depends on developing a careful plan with multiple strategies. Chapter 10 enters into data collection and quality control, wisely underlining that no study can be better than the quality of its data. Monitoring and even auditing are thus necessary. The history of new medical interventions abounds in sad experiences. Hence, adverse effects, if any at all, must be searched and assessed. Chapter 11 clearly probes into this complex aspect. Sadly, perhaps due to sheer enthusiasm , often it has played a secondary role in clinical trials, as stated by the authors.Chapter 12, by Michelle J. Naughton and Sally A. Shumaker, catches the assessment of health-related quality of life, that is, trying to elucidate if the intervention improved, say, physical, psychological and social functioning, perception of well-being and health status, personal productivity, sexual life, sleep disturbances and other parameters. Not an easy task, indeed, although in certain extreme situations  may lead into deep controversies.The remaining seven chapters deal, respectively, with participant adherence to the program, survival analysis, monitoring response variables (37 pages), issues in data analysis , closeout (that is, how to properly terminate the study), reporting and interpreting of results (one of the most difficult steps because recommendations will be produced that may end up in massive use of the intervention), and finally a chapter devoted to multicenter trials.The book provides an exceedingly good collection of references so that the interested reader can deepen in a particular subject. All in all, I would highly recommend it, to the physician, basic investigator and biomedical engineer. For the beginner, instead, it may be a little hard for some parts require good knowledge of statistics. It is no doubt excellent material for a course.