Adding abiraterone or docetaxel to long-term hormone therapy for prostate cancer: directly randomised data from the STAMPEDE multi-arm, multi-stage platform protocol.

Background: Adding abiraterone acetate with prednisolone (AAP) or docetaxel with prednisolone (DocP) to standard-of-care (SOC) each improved survival in systemic therapy for advanced or metastatic prostate cancer: evaluation of drug efficacy: a multi-arm multi-stage platform randomised controlled protocol recruiting patients with high-risk locally advanced or metastatic PCa starting long-term androgen deprivation therapy (ADT). The protocol provides the only direct, randomised comparative data of SOC + AAP versus SOC + DocP. Method: Recruitment to SOC + DocP and SOC + AAP overlapped November 2011 to March 2013. SOC was long-term ADT or, for most non-metastatic cases, ADT for ≥2 years and RT to the primary tumour. Stratified randomisation allocated pts 2 : 1 : 2 to SOC; SOC + docetaxel 75 mg/m2 3-weekly×6 + prednisolone 10 mg daily; or SOC + abiraterone acetate 1000 mg + prednisolone 5 mg daily. AAP duration depended on stage and intent to give radical RT. The primary outcome measure was death from any cause. Analyses used Cox proportional hazards and flexible parametric models, adjusted for stratification factors. This was not a formally powered comparison. A hazard ratio (HR) <1 favours SOC + AAP, and HR > 1 favours SOC + DocP. Results: A total of 566 consenting patients were contemporaneously randomised: 189 SOC + DocP and 377 SOC + AAP. The patients, balanced by allocated treatment were: 342 (60%) M1; 429 (76%) Gleason 8-10; 449 (79%) WHO performance status 0; median age 66 years and median PSA 56 ng/ml. With median follow-up 4 years, 149 deaths were reported. For overall survival, HR = 1.16 (95% CI 0.82-1.65); failure-free survival HR = 0.51 (95% CI 0.39-0.67); progression-free survival HR = 0.65 (95% CI 0.48-0.88); metastasis-free survival HR = 0.77 (95% CI 0.57-1.03); prostate cancer-specific survival HR = 1.02 (0.70-1.49); and symptomatic skeletal events HR = 0.83 (95% CI 0.55-1.25). In the safety population, the proportion reporting ≥1 grade 3, 4 or 5 adverse events ever was 36%, 13% and 1% SOC + DocP, and 40%, 7% and 1% SOC + AAP; prevalence 11% at 1 and 2 years on both arms. Relapse treatment patterns varied by arm. Conclusions: This direct, randomised comparative analysis of two new treatment standards for hormone-naïve prostate cancer showed no evidence of a difference in overall or prostate cancer-specific survival, nor in other important outcomes such as symptomatic skeletal events. Worst toxicity grade over entire time on trial was similar but comprised different toxicities in line with the known properties of the drugs. Trial registration: Clinicaltrials.gov: NCT00268476.

Comparison of efficacy of letrozole and clomiphene citrate in ovulation induction in Indian women with polycystic ovarian syndrome.

OBJECTIVES: The objective of this study was to compare the efficacy of letrozole versus clomiphene citrate as an ovulation induction drug in polycystic ovarian syndrome (PCOS) patients of Indian origin. METHOD: One hundred and forty seven infertile PCOS patients were randomly given letrozole (2.5 mg) (n = 69) or clomiphene (100 mg) (n = 78) from day 3 to day 7 of menstrual cycle, followed-up with transvaginal serial folliculometry from day 9. 10,000 IU of human chorionic gonadotropin (hCG) was administered when at least 1 ovarian follicle was ≥ 18 mm in size. RESULTS: The pertinent results of the study are as follows: on the day of hCG injection, mean E2 level was significantly higher in the clomphene citrate group (817 ± 286.70 pg/ml) in comparison with letrozole group (444.03 ± 85.42 pg/ml). Mean endometrial development was 8.72 ± 1.41 mm in the letrozole and 8.78 ± 1.16 mm in the clomiphene group (P = 0.004). CONCLUSION: Letrozole has beneficial effect on endometrium, thereby potentially increasing pregnancy rates after successful ovulation induction in women with PCOS.

Changes in the allergenicity during different preparations of Pomfret, Hilsa, Bhetki and mackerel fish as illustrated by enzyme-linked immunosorbent assay and immunoblotting.

BACKGROUND: Although the identification and characterization of several fish allergens have already been reported, there is almost no data on Indian fish allergens and the effect of thermal processing on their allergenicity. This study aimed at the evaluation of the changes in the level of allergenicity of 4 highly consumed Indian fishes, i.e. pomfret, hilsa, bhetki and mackerel, that occurred after boiling and frying. METHODS: In this study 110 patients with fish hypersensitivity as evidenced by clinical history and symptoms were recruited based on their positive skin prick test results. The raw, boiled and fried muscle extracts of the 4 fishes were prepared, and each extract was tested by ELISA and immunoblotting with patients' sera. RESULTS: ELISA and immunoblotting studies demonstrated that the raw muscle extracts of pomfret, hilsa, bhetki and mackerel were allergenic. While the allergenicity of boiled and fried extracts of pomfret and hilsa was considerably reduced, maximum allergenicity of bhetki was demonstrated in the fried extract. The degree of allergenicity of bhetki was demonstrated in the order fried>boiled>raw while that of mackerel followed the order raw>boiled approximately fried. CONCLUSION: The specific IgE-binding activity and immunoblot profile clearly showed that pomfret and hilsa fish allergens are heat-labile, while allergens of bhetki and mackerel maintained strong reactivity even after thermal treatment.

Effect of thyroid hormone on the development and gene expression of hormone receptors in rat testes in vivo.

Thyroid hormone is known to play a pivotal role in the regulation of prepuberal rat testes development and function with specific influence on the differentiation of Sertoli cells, the only cell type that expresses thyroid hormone receptors in testes. To explore in vivo effects of thyroid hormone on testes development and the regulation of testicular gene expression, the hyper- and hypothyroid rat models were established by T3 injection to pups (ip 100 microg/kg bw) and by oral administration of 6-N-propyl-2-thiouracil (PTU) to the lactating mother from days 1 to 21 post-delivery. Half of the rats from each group were sacrificed at 21 days of age, and the other half were allowed to recover with discontinued treatments from day 22 to day 50. At 21 days of age, a significantly elevated serum T3 level was observed in hyperthyroid rats (179.5 ng/dl) vs controls (97.5 ng/dl), and in hypothyroid rats a significantly lower level of T3 was detected (26.1 ng/dl). However, serum T4 concentration was significantly lower in both hyper- (0.105 microg/dl) and hypothyroid (0.058 microg/dl) rats compared to the controls (2.48 microg/dl). In recovered rats in which the serum T3 and T4 were restored to normal, the serum T levels remained remarkably lower in both hyper- and hypothyroid rats. The significantly decreased body and testes weights observed in both hyper- and hypothyroid rats at 21 days of age were not restored by the time they were 50 days old. Histological analyses of testes of 21-day-old hypothyroid rats revealed smaller-sized seminiferous tubules, incomplete lumen formation and delayed germ cell differentiation and in hyperthyroid rats an increased number of early stage spermatocytes was found. Testicular mRNA levels of follicle-stimulating hormone receptor (FSH-R), luteinizing hormone receptor (LH-R) and androgen binding protein (ABP) were studied by Northern blot hybridization. At 21 days of age data showed that FSH-R mRNA levels were significantly higher in both hyper- and hypothyroid rat testes compared to controls, but no differences were detected in recovered 50-day-old rats. Significantly decreased ABP mRNA levels were detected only in hypothyroid rat testes compared to those in both the hyperthyroid and control groups at 21 days of age, but no significant change was observed in recovered 50-day-old rats. To further evaluate the effect of thyroid hormone on the Leydig cell function, the 2.3/2.6 kb specific LH-R hybridization bands were detected with rat LH-R cRNA probe. Significant suppression of LH-R mRNA levels was only observed in the hypothyroid rat testes at 50 days of age. The testicular thyroid hormone receptors (TRs) and the regulation of TR by thyroid hormone were investigated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Both TRalpha and TRbeta mRNAs were identified in the testes from 21- and/or 50-day-old rats. TRalpha mRNA levels were significantly increased in hypothyroid rat testes and were suppressed in hyperthyroid rats at 21 days of age and no changes of TRalpha mRNA were found in recovered animals. Our in vivo data strongly suggest that the thyroid hormone directly affects the development of prepuberal testes and the regulation of FSH-R and ABP gene expression in Sertoli cells, as well as the LH-R mRNA levels in Leydig cells, which may lead to further modulating the effect of gonadotropins on testes function.

In vitro testing of platinum-based drugs on a panel of human ovarian tumour cell lines.

Much improvement in the treatment of ovarian cancer has been achieved since the introduction of platinum compounds in the 1980s, with the result that single-agent platinum-based therapy following primary surgery is now the standard treatment for advanced ovarian cancer. The main therapeutic effect of chemotherapy is based on the sensitivity of the patient's tumour to the drug. However, testing a new chemical compound on humans requires much care, time and resources, whereas prior testing of drugs on cancer cell lines may indicate those drugs particularly suited to treatment of a specific disease. This study investigates the actions of two established platinum-based chemotherapeutic agents (cisplatin and carboplatin) on a panel of 10 human ovarian cancer cell lines. Each cell line was plated onto 96-well tissue culture plates, incubated for 72 hours with the drug, formalin-fixed and then assessed using the methylene blue colorimetric microassay to detect viable cells. The IC50 values for each cell line were calculated in order to assess the toxicity of each drug, and a wide range of responses were observed across the 10 cell lines investigated. This suggests that the panel reflected the heterogeneous nature of ovarian cancer, a malignancy in which a huge range of drug sensitivities can be seen even among tumours of the same histological type. The results indicate that the panel could be of use either as a primary screen to test new drugs against ovarian cancer or to investigate the drug resistance that is so common in this disease.

B-subunit of phosphate-specific transporter from Mycobacterium tuberculosis is a thermostable ATPase.

The B-subunit of phosphate-specific transporter (PstB) is an ABC protein. pstB was polymerase chain reaction-amplified from Mycobacterium tuberculosis and overexpressed in Escherichia coli. The overexpressed protein was found to be in inclusion bodies. The protein was solubilized using 1.5% N-lauroylsarcosine and was purified by gel permeation chromatography. The molecular mass of the protein was approximately 31 kDa. The eluted protein showed ATP-binding ability and exhibited ATPase activity. Among different nucleotide triphosphates, ATP was found to be the preferred substrate for M. tuberculosis PstB-ATPase. The study of the kinetics of ATP hydrolysis yielded K(m) of approximately 72 microm and V(max) of approximately 0.12 micromol/min/mg of protein. Divalent cation like manganese was inhibitory to the ATPase activity. Magnesium or calcium, on the other hand, had no influence on the functionality of the enzyme. The classical ATPase inhibitors like sodium azide, sodium vanadate, and N-ethylmaleimide were without any effect but an ATP analogue, 5'-p-fluorosulfonylbenzoyl adenosine, inhibited the ATPase function of the recombinant protein with a K(i) of approximately 0.40 mm. Furthermore, there was hardly any ATP hydrolyzing ability of the PstB as a result of mutation of the conserved aspartic acid residue to lysine in the Walker motif B, confirming the recombinant protein is an ATPase. Interestingly, analysis of the recombinant PstB revealed that it is a thermostable ATPase; thus, our results highlight for the first time the presence of such an enzyme in any mesophilic bacteria.

Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis.

We reported in an earlier study that active efflux of drug has a predominant role in conferring resistance in a laboratory-generated ciprofloxacin-resistant mutant of Mycobacterium smegmatis. This mutant exhibited mRNA level overexpression, as well as chromosomal amplification, of the gene pstB, encoding the putative ATPase subunit of phosphate specific transport (Pst) system. We demonstrate here that this mutant shows enhanced phosphate uptake and that inactivation of pstB in the parental strain results in loss of high affinity phosphate uptake and hypersensitivity to fluoroquinolones. These findings suggest a novel role of the Pst system in active efflux, in addition to its involvement in phosphate transport.

Involvement of a natural transport system in the process of efflux-mediated drug resistance in Mycobacterium smegmatis.

The phosphate-specific transporter (Pst) in bacteria is a multi-subunit system which belongs to the ABC family of transporters. The gene forms part of an operon and it is involved in phosphate uptake in prokaryotes. Its import function is known to be operative only under conditions of phosphate starvation. However, we found overexpression of this transporter in a Mycobacterium smegmatis strain selected for ciprofloxacin resistance (CIPr) which was grown under conditions in which the phosphate-scavenging function of this operon was inoperative. In CIPr cells, active efflux of the drug plays a predominant role in conferring high levels of fluoroquinolone resistance. We therefore investigated the role of this transporter in the process of efflux-mediated drug resistance by inactivating the pst operon in the CIPr strain. Phenotypic characterization of the resulting strain, CIPrd, showed a striking reduction in the minimal inhibitory concentration (MIC) of ciprofloxacin and in the drug extrusion profile as well. Genotype analysis, on the other hand, revealed partial disruption of the pst operon in CIPrd as a consequence of transporter gene amplification. Furthermore, disruption of this operon in wild-type cells resulted in hypersensitivity to ciprofloxacin and other xenobiotics to which CIPr cells exhibited cross-resistance. Thus our results provide strong evidence that Pst is a natural membrane transport system that has the ability to promote drug efflux in addition to its phosphate-scavenging function in the CIPr strain.

Mycoplasma detection in cell cultures: a comparison of four methods.

Mycoplasma is a common contaminant of tissue culture samples. Infection is persistent, difficult to detect and diagnose, and very difficult to cure. The concentration of mycoplasma in infected cultures can be as high as 10(7) colony-forming units per mL, and their presence can change many of the cell reactions, including altering cell growth rate, inducing morphological changes or cell transformation, and mimicking virus infection. Therefore, it should be assumed that a mycoplasma-contaminated cell line may be significantly influenced in every respect, and, thus, experimental data derived from such a cell line is likely to be invalid. Contamination is not obvious, either macroscopically or microscopically; thus, routine mycoplasma testing is essential for any cell culture laboratory. Many of the testing procedures developed so far are time-consuming, expensive, inconclusive and unsuitable for screening large numbers of test specimens. This study compares DNA staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and PCR ELISA, to determine which is the best procedure for routine assessment of cell cultures. All four methods gave reproducible results with both infected and non-infected cell lines. Both ELISA methods were easy to perform, reproducible and easily interpreted.

Role of an ABC importer in mycobacterial drug resistance.

Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIPr) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIPr.

Synergistic activation of yeast-expressed rat androgen receptor by modulators of protein kinase-A.

We have employed a yeast (Saccharomyces cerevisiae) based rat androgen receptor expression system to examine the cross-talk between different signalling pathways. We report here the synergistic modulation of androgen regulated transcriptional activation of beta-galactosidase reporter activity by the activators of protein kinase-A, like forskolin and 8-bromo-cyclic AMP. A similar ligand-dependent enhancement of reporter activity compared to a DHT treated control has been noticed with okadaic acid, which is a potent inhibitor of protein phosphatase. The activation could be blocked by protein kinase-A/C inhibitor, H7. Forskolin treatment neither altered levels of receptor mRNA nor [3H]R1881 binding to the receptor. Although it promotes binding of receptor to an androgen response element, forskolin was unable to activate subsequent interaction with the transcription machinery in the absence of androgen. Additionally, the synergistic actions of these activators were independent of the degree of androgen response element occupancy. Anti-androgens, cyproterone acetate and flutamide, which failed to exhibit antagonistic behaviour with yeast expressed receptor, were able to antagonize only the forskolin mediated augmentation of reporter activity. Finally, analyses of mutants established the role of DNA and steroid binding domains of receptor for this synergism.

Activation of rat androgen receptor by androgenic ligands is unaffected by antiandrogens in Saccharomyces cerevisiae.

The E. coli lacZ has been utilized as a reporter to evaluate ligand-mediated activation of the rat androgen receptor (AR) in Saccharomyces cerevisiae strain YCR1. beta-galactosidase activity was androgen-specific and was found to be inducible approximately 260-fold by dihydrotestosterone (DHT), testosterone and R1881. None of the antiandrogens tested was able to antagonize the DHT-dependent induction of beta-galactosidase activity. In the gel retardation assay, exposure of the receptor to DHT in vitro led to the formation of a protein-DNA complex that was not detected in yeast extracts unexposed to hormone. However, activation of AR by a steroidal (cyproterone acetate) and a non-steroidal antiandrogen (flutamide) either alone or in combination with DHT also results in a similar migration pattern. Additionally, LEM1, the ABC transporter that selectively modulates the biological potency of steroids in yeast, although operative in YCR1, was not responsible for antiandrogen resistance. These results thus indicate the involvement of other non-receptor factor(s) in mediating the effect of antiandrogens in yeast.

Identification of an ABC transporter gene that exhibits mRNA level overexpression in fluoroquinolone-resistant Mycobacterium smegmatis.

We describe here the PCR amplification of a DNA fragment (mtp1) from Mycobacterium smegmatis using primers derived from consensus sequences of the ABC family of transporters. The fragment encodes amino acid sequences that exhibited significant homology with different ABC transporters. Amino acid sequence alignment of the full length gene with other transporters identified the ABC protein as the B-subunit of the phosphate specific transporter. Strikingly, a M. smegmatis colony which exhibited a high level of ciprofloxacin resistance showed mRNA level overexpression of mtp1. Thus this is the first report in any prokaryote indicating differential expression of an ABC transporter in a fluoroquinolone resistant colony.

Stereotactic multiple are radiotherapy. IV--Haemangioblastoma.

Our initial experience in the treatment of haemangioblastoma using conventional external beam radiotherapy and stereotactic radiotherapy (radiosurgery), by the linear accelerator method, is reported. Six haemangioblastomas in five patients were treated with a mean follow-up of 40 months (range 14-60). Five haemangioblastomas in four patients were treated with stereotactic radiotherapy, where four showed complete radiological response and the fifth was static. Neurological symptoms and signs improved in those patients. The sixth haemangioblastoma was situated close to the pituitary and optic chiasm, and was treated with conventionally fractionated external beam radiotherapy. The lesion showed partial response. No complications were noted in this patient group. This series complements and extends the relatively sparse published literature demonstrating that radiotherapy is an effective option for treating haemangioblastomas. Radiosurgery often lends itself particularly well to these discrete lesions allowing highly focused treatment. For patients with multiple and metachronous cerebellar haemangioblastomas as part of the von Hipple-Lindau syndrome, the data support a policy of conventionally fractionated external beam radiotherapy to the whole cerebellum of 50-55 Gy followed, after a period of time, by radiosurgery to persisting lesions (patients 3 and 4).

Involvement of an efflux system in mediating high level of fluoroquinolone resistance in Mycobacterium smegmatis.

A wild type strain of Mycobacterium smegmatis mc2 155 was serially adapted to 64 fold of minimal inhibitory concentration of an antimycobacterial agent, ciprofloxacin. This clone (CIPr) exhibited cross resistance to ofloxacin and ethidium bromide. The rate of drug efflux was accelerated in CIPr compared to the wild type strain. Verapamil, a calcium channel blocker, enhanced the drug accumulation in CIPr by diminishing the efflux and thus reversed the resistant phenotype. Additionally, a missense mutation was detected in the quinolone resistance determining region of the DNA-gyrase A subunit of CIPr. Taken together, these results suggest that drug efflux plays a major role in conferring such a high level of resistance in CIPr, in addition to the mutation in the DNA-gyrase locus.

Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains.

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins.

Effects of prolactin and fish pituitary extract on plasma calcium levels in common carp, Cyprinus carpio.

The effects of prolactin (PRL) from both mammalian and piscine sources on plasma calcium levels in common carp, Cyprinus carpio, were investigated. Injections of ovine prolactin (oPRL) or homologous fish pituitary extract or partially purified prolactin of murrel, Channa punctatus pituitary (mPRL), caused significant increases in total and ultrafiltrable plasma calcium. Larger hypercalcemic responses were observed in fish kept in high-calcium fresh water and normal tap water than in fish in low-calcium fresh water. Injections of oPRL caused dose-dependent increases in plasma calcium level. The highest dose (1 mg/kg body wt) of oPRL had a reduced hypercalcemic effect. Administration of EGTA (200 microM/kg body wt) reduced the stimulatory effects of daily injections of oPRL or mPRL on plasma calcium in freshwater common carp under laboratory conditions. These results indicate that PRL may be involved in regulating plasma calcium levels in fish.

Metal oxyanion stabilization of the rat glucocorticoid receptor is independent of thiols.

The ability of sodium molybdate, both to stabilize the steroid binding activity of glucocorticoid receptors and to prevent the activation of receptor-steroid complexes to a DNA binding species, has long been thought to involve thiols. Two receptor thiols in particular, Cys-656 and Cys-661 of rat receptors, have been suspected. The requirements for the action of molybdate, as well as two other metal oxyanions (tungstate and vanadate) known to exert the same effects as molybdate, have now been examined with receptors in which these thiols, or a third cysteine in the steroid binding cavity (Cys-640), have been mutated to serine. No mutation prevented any metal oxyanion from either stabilizing steroid-free receptors or blocking the activation of complexes for binding to nonspecific or specific DNA sequences. Thus, Cys-640, Cys-656, and Cys-661 are not required for any of the effects of molybdate, tungstate, or vanadate with rat glucocorticoid receptors. Studies with hybrid receptors, and with a 16-kDa steroid binding core fragment containing only 3 cysteines at positions 640, 656, and 661, indicated that no cysteine of the rat receptor was needed to maintain responsiveness to molybdate. Even when all of the thiol groups in crude cytosol were blocked by reaction with excess methyl methanethiol-sulfonate, each metal oxyanion was still able to stabilize the steroid binding of receptors. These results argue that molybdate, tungstate, and vanadate each interact with the receptor or an associated nonreceptor protein(s) in a manner that does not require thiols. An indirect mechanism of molybdate action was evaluated in light of the recent report that the whole cell actions are mediated by increased levels of intracellular cGMP. Under cell-free conditions, however, the effects of molybdate could not be reproduced by cGMP derivatives. Evidence consistent with a direct effect was that molybdate, tungstate, or vanadate each modified the kinetics of proteolysis of wild type receptors at 0 degrees C by trypsin, presumably due to induced conformational changes of the receptor. This alteration of trypsin digestion constitutes yet another effect of metal oxyanions on the glucocorticoid receptor.