Activity of enzalutamide in men with metastatic castration-resistant prostate cancer is affected by prior treatment with abiraterone and/or docetaxel.

BACKGROUND: Enzalutamide and abiraterone are new androgen-axis disrupting treatments for metastatic castration-resistant prostate cancer (mCRPC). We examined the response and outcomes of enzalutamide-treated mCRPC patients in the real-world context of prior treatments of abiraterone and/or docetaxel. METHODS: We conducted a seven-institution retrospective study of mCRPC patients treated with enzalutamide between January 2009 and February 2014. We compared the baseline characteristics, PSA declines, PSA progression-free survival (PSA-PFS), duration on enzalutamide and overall survival (OS) across subgroups defined by prior abiraterone and/or docetaxel. RESULTS: Of 310 patients who received enzalutamide, 36 (12%) received neither prior abiraterone nor prior docetaxel, 79 (25%) received prior abiraterone, 30 (10%) received prior docetaxel and 165 (53%) received both prior abiraterone and prior docetaxel. Within these groups, respectively, ⩾30% PSA decline was achieved among 67, 28, 43 and 24% of patients; PSA-PFS was 5.5 (95% CI 4.2-9.1), 4.0 (3.2-4.8), 4.1 (2.9-5.4) and 2.8 (2.5-3.2) months; median duration of enzalutamide was 9.1 (7.3-not reached), 4.7 (3.7-7.7), 5.4 (3.8-8.4) and 3.9 (3.0-4.6) months. Median OS was reached only for the patients who received both prior abiraterone and docetaxel and was 12.2 months (95% CI 10.7-16.5). 12-month OS was 78% (59-100%), 64% (45-90%), 77% (61-97%) and 51% (41-62%). Of 70 patients who failed to achieve any PSA decline on prior abiraterone, 19 (27%) achieved ⩾30% PSA decline with subsequent enzalutamide. CONCLUSIONS: The activity of enzalutamide is blunted after abiraterone, after docetaxel, and still more after both, suggesting subsets of overlapping and distinct mechanisms of resistance.

The influence of coach reputation on the behavioral responses of male soccer players.

The present study examined the impact of reputation information on athletes' behavioral responses to coaches within a naturalistic, field-based setting. Using a between-group design, male soccer players (n = 35) were assigned to one of three experimental conditions (i.e., experienced reputation, inexperienced reputation, no reputation) prior to taking part in a coaching session delivered by an unknown coach. Participants' behaviors indicative of attention to coach instruction, effort and persistence, and willingness to participate in demonstrations were video recorded throughout the coaching session. Multivariate analyses of variance revealed that participants in the experienced reputation condition exhibited significantly greater attention to coach instruction, and greater effort and persistence during free practice than participants in the inexperienced reputation condition. Results related to participants' willingness to participate in demonstrations failed to yield any significant differences. The results provide further evidence to support the contention that athletes use reputation information as a basis for their initial expectancies of coaches, and such expectancies have the potential to influence athletes' behavior during coach-athlete interactions. The findings also indicate that expectancies based on positive information may be more powerful than negatively framed expectancies, and can be harnessed by coaches as a means of developing effective relationships with their athletes.

Hepatic and mesenteric nitric oxide synthase expression in a rat model of CCl(4)-induced cirrhosis.

BACKGROUND: Cirrhosis and portal hypertension are frequently linked with changes in expression of nitric oxide synthase (NOS) and/or endotoxaemia. AIMS: This study tested the following hypothesis: that inducible (i)NOS activity is increased within the visceral circulation concurrently with decreased constitutive (c)NOS activity in the hepatic sinusoids and that the concentration of NO metabolites in portal blood is consequent on endotoxin concentration. MATERIALS AND METHODS: Plasma concentrations of (nitrite + nitrate) and endotoxin, together with hepatic and mesenteric NOS activity (arginine/citrulline method) and protein expression (histochemistry) plus portal and arterial blood pressure, were determined in rats made severely cirrhotic by intragastric CCl(4) over 14 weeks (n = 6) compared with age-matched controls (n = 5). The concentrations of [nitrite + nitrate] and endotoxin in portal plasma were also directly compared in rats made cirrhotic for a period of 8-14 weeks (n = 10). RESULTS: In rats with advanced cirrhosis, arterial [nitrite + nitrate] was 93.1 (22.4) micromol/L (mean, SEM) compared with 29.1 (6.1) micromol/L in controls (P < 0.05); portal plasma [NO(2)(-) + NO3(-)] was 127.1 (27.2) compared with 24.7 (4.7) micromol/L in controls (P < 0.05). Cirrhotic rats had higher endotoxin concentration in plasma compared with controls (systemic: 85.0 (24.5) versus 1.7 (0.2) EU/ml, P < 0.05; portal: 180.3 (47.9) versus 1.7 (0.2) EU/ml, P < 0.05). The same severely cirrhotic rats possessed decreased cNOS activity in liver (2.95 [0.40] versus 5.29 [0.85] pmol/min/g; P < 0.05) and increased iNOS activity in mesentery (4.83 [1.23] versus 1.47 [0.15] pmol/min/g; P < 0.05) compared with controls. Histochemical observations confirmed these findings. Rats given CCl(4) for a period of 8-14 weeks possessed high endotoxin concentration in portal plasma, with correspondingly high [nitrite + nitrate] (r(2) = 0.954; P < 0.001). CONCLUSIONS: An endotoxin-induced increase in mesenteric iNOS activity and a decrease in hepatic cNOS activity may account for, respectively, the hyperdynamic visceral circulation and the increased intrahepatic resistance of cirrhosis.

Antibiotic prophylaxis in surgery of the intervertebral disc. A comparison between gentamicin and cefuroxime.

Antibiotics are often administrated prophylactically in spinal procedures to reduce the risk of infection of the disc space. It is still not known which antibiotics are able to penetrate the intervertebral disc effectively. In a prospective, randomised, double-blind clinical study, we examined the penetration of the intervertebral discs of two commonly used antibiotics, cefuroxime and gentamicin. The patients, randomised into two groups, received either 1.5 g of cefuroxime or 5 mg/kg of gentamicin prophylactically two hours before their intervertebral discs were removed. A specimen of blood, from which serum antibiotic levels were determined, was obtained at the time of discectomy. Therapeutic levels of antibiotic were detectable in the intervertebral discs of the ten patients who received gentamicin. Only two of the ten patients (20%) who received cefuroxime had a quantifiable level of antibiotic in their discs although therapeutic serum levels of cefuroxime were found in all ten patients. Our results show that cefuroxime does not diffuse into human intervertebral discs as readily as gentamicin. It is possible that the charge due to ionisable groups on the antibiotics can influence the penetration of the antibiotics. We therefore recommend the use of gentamicin in a single prophylactic dose for all spinal procedures in order to reduce the risk of discitis.

The effect of N(G)-monomethyl-L-arginine and tamoxifen on nitric oxide production in breast cancer cells stimulated by oestrogen and progesterone.

OBJECTIVE: To examine the capacity of oestrogen, or progesterone, or both to elicit the release of nitric oxide (NO) from T47D breast cancer cells in vitro. DESIGN: Prospective, longitudinal, controlled in vitro experiment. SETTING: University Medical School, United Kingdom. MATERIAL AND INTERVENTIONS: T47D breast cancer cells were stimulated by micromolar to picomolar doses of 17beta-oestradiol, or progesterone, or both, with or without inhibition of NO or tamoxifen at 24 and 48 hours. MAIN OUTCOME MEASURES: Concentration of NO metabolites (nitrite + nitrate) in the culture medium measured by chemiluminescence. RESULTS: Both hormones dose-dependently increased the proliferation of T47D without toxic effects over the range 10(-12)-10(-6) M. Both stimulated NO production at 24 hours, micomolar doses producing a pronounced (2-4 fold) increase in the concentration of NO metabolites in culture medium (p = 0.002 and p < 0.001 for oestradiol and progesterone, respectively). By contrast, incubation with hormones for 48 hours had little effect on the concentrations of NO metabolites. NO production induced by hormones was completely inhibited by the NO synthesis inhibitor N(G)-monomethyl-L-arginine (10(-5)-10(-3) M) and by tamoxifen (10(-8)-10(-4) M) (p < 0.001 in each case). CONCLUSIONS: Oestrogen and progesterone have a role in stimulating NO production in T47D breast cancer cells. Inhibition of NO synthesis might be a novel therapeutic approach for reducing hormone-associated angiogenesis in breast cancer.

Complete nucleotide sequence and genome organization of Beet soilborne mosaic virus, a proposed member of the genus Benyvirus.

The complete nucleotide sequences of RNAs 1 to 4 of Beet soilborne mosaic virus (BSBMV) were determined. The genomic organization of BSBMV is identical to Beet necrotic yellow vein virus (BNYVV), the type species of the genus Benyvirus. BSBMV RNA1 encodes a single large open reading frame (ORF) with similar replicase-associated motifs identified for BNYVV. BSBMV RNA2 has six potential ORFs with an organization resembling BNYVV RNA2. RNA3 and RNA4 resemble the analogous BNYVV RNAs, which encode proteins associated with symptom development and fungal transmission, respectively. The predicted ORFs on BNYVV and BSBMV reveal 23% to 83% amino acid identity and the overall nucleotide sequences are 35% to 77% identical. Based on sequence analyses, BSBMV is a new benyvirus that can be distinguished from BNYVV.

Bioglass 45S5 stimulates osteoblast turnover and enhances bone formation In vitro: implications and applications for bone tissue engineering.

We investigated the concept of using bioactive substrates as templates for in vitro synthesis of bone tissue for transplantation by assessing the osteogenic potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro. Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric analysis of the cell cycle suggested that the bioactive glass-ceramic substrate induced osteoblast proliferation, as indicated by increased cell populations in both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical analysis of the osteoblast differentiation markers alkaline phosphatase (ALP) and osteocalcin indicated that the bioactive glass-ceramic substrate augmented osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning electron microscopic observations of discrete bone nodules over the surface of the bioactive material, from day 6 onward, further supported this notion. A combination of fluorescence, confocal, transmission electron microscopy, and X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of cell aggregates which produced mineralized collagenous matrix. Control substrates did not exhibit mineralized nodule formation at any point studied up to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability to stimulate the growth and osteogenic differentiation of human primary osteoblasts. These findings have potential applications for tissue engineering where this bioactive glass substrate could be used as a template for the formation of bioengineered bone tissue.

cDNA probes for detection of specific dsRNAs from the fungal pathogen, Monosporascus cannonballus.

Monosporascus cannonballus is an ascomycete fungus that is the causative agent of Monosporascus root rot/vine decline, a serious disease of muskmelon and watermelon. Double-stranded RNA (dsRNA) was identified in approximately 60% of M. cannonballus isolates recovered from infected muskmelon plants in 1993. After repeated laboratory transfer on culture media, the majority of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to muskmelon. Initially, dsRNA purification and cDNA synthesis were attempted in three M. cannonballus isolates harboring dsRNAs. However, numerous difficulties were encountered due to the stable, double-stranded nature of the dsRNAs and contamination of the preparations by fungal rRNA. Several purification and cDNA protocols were evaluated and eventually modified into methods that were ultimately highly effective for cloning dsRNAs from M. cannonballus. The cDNAs derived from purified dsRNA preparations were cloned into a pUC119 plasmid vector and amplified in Escherichia coli. Nine cDNA clones were identified that are specific for medium-sized (ca. 3 kbp) dsRNAs associated with M. cannonballus isolate Ca91-17(96+). The methods used to make the cDNA clones of the dsRNAs in M. cannonballus may be useful for those working on fungal dsRNAs. In addition, these cDNAs may be useful for identifying dsRNAs associated with the hypovirulence phenotype.

Hepatic and splanchnic nitric oxide activity in patients with cirrhosis.

BACKGROUND: In animal models of cirrhosis, altered activity of nitric oxide (NO) has been implicated in the pathogenesis of increased intrahepatic portal vascular resistance and abnormal mesenteric vasodilatation. AIMS: To investigate NO activity in the liver and splanchnic vascular bed of patients with cirrhosis. METHODS: Activity of the calcium dependent constitutive and calcium independent inducible isoforms of NO synthase (cNOS and iNOS, respectively) was assayed biochemically in biopsy specimens of liver and a vascular portion of the greater omentum (representative of mesenteric vasculature) obtained from patients with cirrhosis undergoing liver transplantation (n=14) and non-cirrhotic control patients undergoing liver resection for metastases (n=9). The concentration of NO metabolites (NO2 + NO3) in portal and peripheral venous plasma was measured. RESULTS: The activity of cNOS was lower in cirrhotic compared with non-cirrhotic subjects for both liver and omentum. Hepatic and omental iNOS activities did not differ significantly between the two groups. Portal (NO2 + NO3) was threefold higher in cirrhotic than non-cirrhotic patients, but no differences were observed in systemic venous samples from the two groups. CONCLUSIONS: The activity of cNOS is diminished in the cirrhotic human liver. The resultant decrease in constitutive NO release may promote an increase in the intrahepatic portal vascular resistance. Elevated portal venous (NO2 + NO3) indicates enhanced splanchnic vascular release of NO in cirrhotic patients, but the absence of increased NOS activity in the mesenteric vasculature suggests differential regulation of NO synthesis within the splanchnic vascular bed.

Nitric oxide in fracture repair. Differential localisation, expression and activity of nitric oxide synthases.

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.

Nitric oxide mediated vasoreactivity during fracture repair.

An experimental model of fracture healing has been used to investigate whether nitric oxide mediated vascular reactivity, determined using laser Doppler flowmetry, is present in bone after a fracture. Times corresponding to Days 0, 1, 3, 7, 14, and 28 after fracture were used to study the injured and contralateral limbs in response to bolus intravenous administration of nitric oxide inhibitor, N-nitro-L-arginine methyl ester, and nitric oxide stimulator, acetylcholine. N-nitro-L-arginine methyl ester administration (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) caused a dose dependent increase in systemic blood pressure in each of the assessment groups; however, there was no statistical difference between the groups. Doppler flow readings at the fracture site showed measurable changes in local vascular reactivity after drug administration. At Day 1 after fracture, the magnitude of unit change in vascular reactivity in response to N-nitro-L-arginine methyl ester (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) was significantly higher in the fractured limb compared with the contralateral limb and also when compared with other points of assessment. These results show that nitric oxide mediated vasoreactivity is present about a fracture site and is maximal in the early healing phase, before returning to basal levels as healing progresses. This is compatible with an initial restoration of blood flow at a fracture site by nitric oxide dependent vasodilation of preexisting blood vessels, followed by ingrowth of less nitric oxide dependent angiogenic vessels during the later phase of repair.

A New Virus on Maize in Nigeria: Maize Mild Mottle Virus.

Maize (Zea mays) and itch grass (Rottboellia cochinchinensis) plants exhibiting a mild mosaic or mottle were collected from a farmer's field near Mokwa, Nigeria, in 1993. Icosahedral virions (approximately 28 to 30 nm) were purified from symptomatic tissue by differential centrifugation in 0.1 M phosphate buffer, pH 7.0. The virions are composed of one single-stranded positive-sense RNA of approximately 4,000 nucleotides and, as estimated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a capsid protein of approximately 28 kDa. The virus was readily detected in infected plants by enzyme-linked immunosorbent assay and immunoblot assays with a polyclonal rabbit antibody derived from purified virions. A partial cDNA library was generated with random primers. Sequence analyses of a cDNA clone representing a portion of the putative replicase gene aligned most closely with related sequences of viruses within the Tombusviridae. In particular, a region of 78 predicted amino acids surrounding the "GDD" replicase motif shares 73% identity with panicum mosaic virus and 61% identity with maize chlorotic mottle virus. The virus is readily transmitted by mechanical inoculation to sweet and dent corn, millet, and wheat. Currently it is not considered of economic importance in Nigeria. The data suggest that "maize mild mottle virus" is a newly identified virus infecting maize.

Endothelium-dependent vasodilatation produced by the L-arginine/nitric oxide pathway in normal and ischemic bone.

We used an experimental model of the perfused isolated rabbit tibia to investigate the vasodilatation produced by nitric oxide in the circulation of bone. Tibiae were perfused at a constant flow rate while the perfusion pressure was monitored continuously. Perfusion pressure was raised by the addition of noradrenaline to the perfusate, and dose responses were measured for bolus doses of acetylcholine and sodium nitroprusside. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis, was then added to the perfusate at a concentration of 10(-4) M, and the dose responses to acetylcholine and sodium nitroprusside were repeated. Measurements were performed on groups of bones after 0, 6, 12, and 24 hours of normothermic ischemia (n 5, 4, 6, and 9, respectively). Both acetylcholine and sodium nitroprusside produced significant vasodilatation after 0 and 6 hours' ischemia, but no significant response was observed after 12 or 24 hours of ischemia. The vasodilatation produced by acetylcholine was significantly attenuated when L-NAME was added to the perfusate, but the vasodilatation produced by sodium nitroprusside remained unchanged. These findings confirm endothelial production of NO by stimulation of muscarinic receptors on the endothelial cells in bone and indicate that vasodilatation via the L-arginine/NO pathway remains viable for 6 hours after normothermic ischemia.

Aseptic loosening of total hip replacement. Macrophage expression of inducible nitric oxide synthase and cyclo-oxygenase-2, together with peroxynitrite formation, as a possible mechanism for early prosthesis failure.

Aseptic loosening is a major cause of failure of total hip arthroplasty. The adverse tissue response to prosthetic wear particles, with activation of cytokine and prostanoid production, contributes to bone loss around the implants. We have investigated the possibility that inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) are expressed in macrophages in the pseudomembrane at the bone-implant interface, thereby contributing to the periprosthetic bone resorption. We also assessed whether peroxynitrite, a nitric oxide (NO)-derived oxidant associated with cellular injury, is generated in the membrane. Enzymatic activity of iNOS was measured using the arginine-citrulline assay technique and prostaglandin E2 (PGE2), as an indicator of COX-2 activity, was measured using an enzyme immunoassay. Cellular immunoreactivity for iNOS, nitrotyrosine (a marker of peroxynitrite-induced cellular injury) and COX-2 was assessed by quantitative peroxidase immunocytochemistry while immunofluorescence methods were used for subsequent co-localisation studies with CD68+ macrophages. The presence of calcium-independent iNOS activity and PGE2 production was confirmed in the homogenised interface membrane. Immunocytochemistry showed that periprosthetic CD68+ wear-debris-laden macrophages were the most prominent cell type immunoreactive for iNOS, nitrotyrosine and COX-2. Other periprosthetic inflammatory and resident cell types were also found to immunolocalise nitrotyrosine thereby suggesting peroxynitrite-induced protein nitrosylation and cellular damage not only in NO-producing CD68+ macrophages, but also in their neighbouring cells. These data indicate that both iNOS and COX-2 are expressed by CD68+ macrophages in the interface membrane and peroxynitrite-induced cellular damage is evident in such tissue. If high-output NO and peroxynitrite generation were to cause macrophage cell death, this would result in the release of phagocytosed wear debris into the extracellular matrix. A detrimental cycle of events would then be established with further phagocytosis by newly-recruited inflammatory cells and subsequent NO, peroxynitrite and prostanoid synthesis. Since both NO and PGE2 have been implicated in the induction and maintenance of chronic inflammation with resulting loss of bone, and peroxynitrite in the pathogenesis of disease states, they may be central to the pathogenesis of aseptic loosening.

Sensory and sympathetic innervation of the vertebral endplate in patients with degenerative disc disease.

We obtained intervertebral discs with cartilage endplates and underlying cancellous bone at operation from patients with degenerative disc disease and then used immunohistochemical techniques to localise the nerves and nerve endings in the specimens. We used antibodies for the ubiquitous neuronal protein gene product 9.5 (PGP 9.5). Immunoreactivity to neuropeptide Y was used to identify autonomic nerves and calcitonin gene-related peptide (CGRP) and substance P to identify sensory nerves. Blood vessels were identified by immunoreactivity with platelet-endothelial cell-adhesion molecule (CD31; PECAM). In a control group with no known history of chronic back pain, nerve fibres immunoreactive to PGP 9.5 and neuropeptide Y were most closely related to blood vessels, with occasional substance P and CGRP immunoreactivity. In patients with severe back pain and markedly reduced disc height, proliferation of blood vessels and accompanying nerve fibres was observed in the endplate region and underlying vertebral bodies. Many of these nerves were immunoreactive to substance P or CGRP, and in addition, substance P- and CGRP-immunoreactive nociceptors were seen unrelated to blood vessels. Quantification by image analysis showed a marked increase in CGRP-containing sensory nerve fibres compared with normal control subjects. We speculate that a chemotactic response to products of disc breakdown is responsible for the proliferation of vascularity and CGRP-containing sensory nerves found in the endplate region and vertebral body adjacent to degenerate discs. The neuropeptides substance P and CGRP have potent vasodilatory as well as pain-transmitting effects. The increase in sensory nerve endings suggests increase in blood flow, perhaps as an attempt to augment the nutrition of the degenerate disc. The increase in the density of sensory nerves, and the presence of endplate cartilage defects, strongly suggest that the endplates and vertebral bodies are sources of pain; this may explain the severe pain on movement experienced by some patients with degenerative disc disease.

Natural variation within the principal arginine-specific protease gene, prpR1, of Porphyromonas gingivalis.

RI, one of the major extracellular arginine-specific proteases of Porphyromonas gingivalis is a heterodimer composed of catalytic (alpha) and adhesin (beta) chains, encoded by the gene prpR1. The distribution of prpR1 and its variation within 43 isolates of P. gingivalis was determined. Chromosomal DNA was digested with Sma I and probed with a 32P-labeled DNA fragment from within the coding region for the alpha component of P. gingivalis W50. All isolates gave the expected 3.2 kb band, corresponding to the coding region for the alpha and beta components. The presence of a second locus (prR2) homologous to the alpha region of prpR1 was also detected. The 1.7-kb alpha coding region of prpR1 was amplified for subsequent restriction analysis. Following Taq I restriction all isolates gave identical patterns. With Rsa I, the majority of isolates (77%) could be placed into a single group. In conclusion, the prpR1 and prR2 loci are maintained in natural populations of P. gingivalis, and only minor polymorphism is detectable within the catalytic domain.