Treatment with methylnaltrexone is associated with increased survival in patients with advanced cancer.

BACKGROUND: Methylnaltrexone (MNTX), a peripherally acting µ-opioid receptor (MOR) antagonist, is FDA-approved for treatment of opioid-induced constipation (OIC). Preclinical data suggest that MOR activation can play a role in cancer progression and can be a target for anticancer therapy. PATIENTS AND METHODS: Pooled data from advanced end-stage cancer patients with OIC, despite laxatives, treated in two randomized (phase III and IV), placebo-controlled trials with MNTX were analyzed for overall survival (OS) in an unplanned post hoc analysis. MNTX or placebo was given subcutaneously during the double-blinded phase, which was followed by the open-label phase, allowing MNTX treatment irrespective of initial randomization. RESULTS: In two randomized, controlled trials, 229 cancer patients were randomized to MNTX (117, 51%) or placebo (112, 49%). Distribution of patients' characteristics and major tumor types did not significantly differ between arms. Treatment with MNTX compared with placebo [76 days, 95% confidence interval (CI) 43-109 versus 56 days, 95% CI 43-69; P = 0.033] and response (laxation) to treatment compared with no response (118 days, 95% CI 59-177 versus 55 days, 95% CI 40-70; P < 0.001) had a longer median OS, despite 56 (50%) of 112 patients ultimately crossing over from placebo to MNTX. Multivariable analysis demonstrated that response to therapy [hazard ratio (HR) 0.47, 95% CI 0.29-0.76; P = 0.002) and albumin ≥3.5 (HR 0.46, 95% CI 0.30-0.69; P < 0.001) were independent prognostic factors for increased OS. Of interest, there was no difference in OS between MNTX and placebo in 134 patients with advanced illness other than cancer treated in these randomized studies (P = 0.88). CONCLUSION: This unplanned post hoc analysis of two randomized trials demonstrates that treatment with MNTX and, even more so, response to MNTX are associated with increased OS, which supports the preclinical hypothesis that MOR can play a role in cancer progression. Targeting MOR with MNTX warrants further investigation in cancer therapy. CLINICAL TRIALS NUMBER: NCT00401362, NCT00672477.

Increased µ-opioid receptor expression in metastatic lung cancer.

BACKGROUND: We and others have previously demonstrated that the µ-opioid receptor (MOR) is overexpressed in several human malignancies. There is a seven-fold increase in MOR in cell lines of human lung cancer. In animal models, overexpression of MOR promotes tumour growth and metastasis. We, therefore, examined whether MOR expression is increased in metastatic lung cancer. METHODS: In this study, we examined the association between MOR expression and metastasis in archived biopsy samples from patients with lung cancer. Paraffin-embedded patient material was stained using MOR antibody and scored qualitatively by two independent pathologists using a four-point scale. RESULTS: In human lung cancer and normal adjacent lung samples obtained from 34 lung cancer patients, MOR expression was increased significantly in cancer samples from patients with lung cancer compared with adjacent control tissue (P=0.0242). When the samples from patients with metastatic lung cancer were separated from the cohort of the total number of patients with lung cancer, we observed an approximately two-fold increase in MOR expression (P=0.0013). CONCLUSIONS: The association between the expression of MOR and the progression of the tumour is consistent with the hypothesis of a direct effect of MOR on cancer progression.

Electronic health records: new opportunities for clinical research.

Clinical research is on the threshold of a new era in which electronic health records (EHRs) are gaining an important novel supporting role. Whilst EHRs used for routine clinical care have some limitations at present, as discussed in this review, new improved systems and emerging research infrastructures are being developed to ensure that EHRs can be used for secondary purposes such as clinical research, including the design and execution of clinical trials for new medicines. EHR systems should be able to exchange information through the use of recently published international standards for their interoperability and clinically validated information structures (such as archetypes and international health terminologies), to ensure consistent and more complete recording and sharing of data for various patient groups. Such systems will counteract the obstacles of differing clinical languages and styles of documentation as well as the recognized incompleteness of routine records. Here, we discuss some of the legal and ethical concerns of clinical research data reuse and technical security measures that can enable such research while protecting privacy. In the emerging research landscape, cooperation infrastructures are being built where research projects can utilize the availability of patient data from federated EHR systems from many different sites, as well as in international multilingual settings. Amongst several initiatives described, the EHR4CR project offers a promising method for clinical research. One of the first achievements of this project was the development of a protocol feasibility prototype which is used for finding patients eligible for clinical trials from multiple sources.

Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor.

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.

Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: role of receptor transactivation.

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, agents that inhibit angiogenesis have important therapeutic implications in numerous malignancies. We examined the effects of methylnaltrexone (MNTX), a peripheral mu opioid receptor antagonist, on agonist-induced human EC proliferation and migration, two key components in angiogenesis. Using human dermal microvascular EC, we observed that morphine sulfate (MS), the active metabolite, morphine-6-glucuronide (M6G), DAMGO ([d-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkaphalin) and VEGF induced migration which were inhibited by pretreatment with MNTX at therapeutically relevant concentration (0.1 microM). The biologically inactive metabolite morphine-3-glucuronide (M3G) did not affect EC migration. We next examined the mechanism(s) by which MNTX inhibits opioid and VEGF-induced angiogenesis using human pulmonary microvascular EC. MS and DAMGO induced Src activation which was required for VEGF receptor transactivation and opioid-induced EC proliferation and migration. MNTX inhibited MS, DAMGO and VEGF induced tyrosine phosphorylation (transactivation) of VEGF receptors 1 and 2. Furthermore, MS, DAMGO and VEGF induced RhoA activation which was inhibited by MNTX or VEGF receptor tyrosine kinase inhibition. Finally, MNTX or silencing RhoA expression (siRNA) blocked MS, DAMGO and VEGF-induced EC proliferation and migration. Taken together, these results indicate that MNTX inhibits opioid-induced EC proliferation and migration via inhibition of VEGF receptor phosphorylation/transactivation with subsequent inhibition of RhoA activation. These results suggest that MNTX inhibition of angiogenesis can be a useful therapeutic intervention for cancer treatment.

Security and confidentiality approach for the Clinical E-Science Framework (CLEF).

OBJECTIVES: CLEF is an MRC sponsored project in the E-Science programme that aims to establish methodologies and a technical infrastructure for the next generation of integrated clinical and bioscience research. METHODS: The heart of the CLEF approach to this challenge is to design and develop a pseudonymised repository of histories of cancer patients that can be accessed by researchers. Robust mechanisms and policies have been developed to ensure that patient privacy and confidentiality are preserved while delivering a repository of such medically rich information for the purposes of scientific research. RESULTS: This paper summarises the overall approach adopted by CLEF to meet data protection requirements, including the data flows, pseudonymisation measures and additional monitoring policies that are currently being developed. CONCLUSION: Once evaluated, it is hoped that the CLEF approach can serve as a model for other distributed electronic health record repositories to be accessed for research.

Social stress differentially regulates neuroendocrine responses in laying hens: I. Genetic basis of dopamine responses under three different social conditions.

Effects of genetic-environmental interactions on plasma dopamine (DA) concentrations were studied in White Leghorn chickens selected for both high (HGPS) or low (LGPS) group productivity and survivability resulting from cannibalism and flightiness. Plasma DA levels were measured from chickens in three social treatments: single-, two-, or ten-hen cages. The two-hen treatment consisted of paired chickens from three genetic lines: HGPS, LGPS and a commercial strain, Dekalb XL (DXL). In HGPS/DXL and LGPS/DXL pairs, the DXL hen was used as a standardized genetic competitor. The ten-hen treatment contained only hens from the same line, which is similar to the original selection condition. After 7 weeks housing in the social environments, LGPS hens in the ten-hen treatment had greater plasma DA concentrations than HGPS hens (P<0.05). Compared to levels in the ten-hen treatment from the same line, plasma DA concentrations in both HGPS and LGPS hens were significantly lower in the two-hen treatment (average mean, 0.09 vs. 0.15 ng/ml and 0.22 vs. 0.44 ng/ml, P<0.05, respectively), but significantly higher in the single-hen treatment (average mean, 0.44 vs. 0.15 ng/ml and 1.78 vs. 0.44 ng/ml, P<0.05 and P<0.01, respectively). In the single-hen treatment, LGPS hens had greater plasma DA levels than HGPS hens (P<0.05). The results provide evidence of genetically related differences in the regulation of chickens' plasma DA concentrations in response to social stress. These differences may magnify the behavioral and physiological differences observed in the lines under basal and challenged conditions. These results suggest that these chicken lines may provide a new model for investigating effects of DA on the control of behavioral, neural and endocrine responses to stress.

Social stress in laying hens: differential effect of stress on plasma dopamine concentrations and adrenal function in genetically selected chickens.

Genetic selection for high or low group productivity and survivability (HGPS, LGPS) has created two phenotypically distinct chicken lines. Each line has unique characteristics in behavioral and physiological adaptability to multiple-bird cage system. The present study was designed to examine whether these differences reflect genetic variation in the control of plasma dopamine (DA) concentrations and adrenal function in response to social stress. Chickens from the HGPS and LGPS lines were randomly assigned to single- or 10-bird cages at 17 wk of age. The 10-bird cages were the same as those used in the development of the two lines. Differences in regulation of DA concentrations and adrenal function in response to different social environments were measured between the two lines when the study was conducted at 24 wk of age. In the 10-bird cages, the HGPS line had lower levels of DA (P < 0.05) and heavier adrenal glands (AG, P < 0.05) than those of the LGPS line, but concentrations of corticosterone (CORT) from the two lines were not significantly different. In the single-bird cages, DA levels in both lines were greater than in that of their siblings in the 10-bird cages, but a greater increase was found in the LGPS line (P < 0.01 and P < 0.05, 405% vs. 293%). Likewise, both lines had lower concentrations of CORT (P < 0.05) in the single- vs. 10-bird cages, but the AG were less heavy in the LGPS line but not in HGPS line in the single-bird cages (P < 0.05). The results indicated that the two strains reacted differently in terms of their stress hormone levels in the two different environments. These differences could contribute to the behavioral and physiological differences existing between the two lines.

Social stress in laying hens: differential dopamine and corticosterone responses after intermingling different genetic strains of chickens.

White Leghorn chickens were genetically selected for high (HGPS) or low (LGPS) group productivity and survivability. The selection resulted in two genetic lines with marked opposite changes in cannibalism and flightiness when housed in multiple-colony battery cages without beak trimming. The objective of the study was to examine whether the genetic selection differentially affected the neuroendocrine system of chickens from different strains in response to social stress. Based on the previous studies, social stress was induced by randomly pairing 17-wk-old hens from three genetic lines, i.e., HGPS, LGPS, and Dekalb XL (DXL), to form three mixed-line combinations. At 24 wk of age, the concentrations of plasma dopamine (DA) and corticosterone (CORT) showed no differences in DXL hens housed with HGPS or LGPS hens (P > 0.05). However, different regulations of DA and adrenal function were found between HGPS and LGPS hens when paired with DXL hens. Compared to HGPS hens, LGPS hens had greater levels of DA and CORT (P < 0.01 and P < 0.05, respectively). In addition, under the HGPS-LGPS social treatment, the concentrations of DA but not CORT were greater in LGPS hens than in HGPS hens (P < 0.05 and P > 0.05, respectively). The results indicated genetic selection for production and survivability differentially altered DA and CORT systems in response to social stress. The data suggested, compared to LGPS hens, HGPS hens had a better coping capability to social stress, which might have been responsible for their higher productivity and survivability.

Comparison of molecular and antibiotic resistance profile methods for the population analysis of Bradyrhizobium spp. (TGx) isolates that nodulate the new TGx soybean cultivars in Africa.

AIMS: Comparison of molecular and antibiotic resistance profile methods to identify an easy method that can differentiate between strains of introduced Bradyrhizobium japonicum and the indigenous Bradyrhizobium spp. (TGx) isolates which nodulate the newly developed TGx soybean cultivars in Africa. METHODS AND RESULTS: Restriction fragment length polymorphism (RFLP) of 16S rDNA generated by five restriction enzymes, banding patterns in Southern hybridization using nod and nif genes as probes, and resistance patterns of the isolates to nine antibiotics, were used to group 26 Bradyrhizobium spp. (TGx) isolates and four other Bradyrhizobium strains. The clusters of isolates obtained from the four grouping methods were all different, although all methods revealed large genetic diversity among the isolates. CONCLUSIONS: Results indicate that the antibiotic resistance profile method is as good as the three molecular methods used in this study for phylogenetic grouping of the Bradyrhizobium spp. (TGx) isolates, which may serve as a basis for further characterization of selected isolates from each group. SIGNIFICANCE AND IMPACT OF THE STUDY: The antibiotic resistance profile method can be used as a simple means of assessing genetic variability and grouping of a large number of Bradyrhizobium spp. (TGx) isolates. Representative isolates from each group can then be selected for further characterization.

Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth.

In this study we initially examined the interaction between CD44v3 (a hyaluronan (HA) receptor) and Vav2 (a guanine nucleotide exchange factor) in human ovarian tumor cells (SK-OV-3.ipl cell line). Immunological data indicate that both CD44v3 and Vav2 are expressed in SK-OV-3.ipl cells and that these two proteins are physically linked as a complex in vivo. By using recombinant fragments of Vav2 and in vitro binding assays, we have detected a specific binding interaction between the SH3-SH2-SH3 domain of Vav2 and the cytoplasmic domain of CD44. In addition, we have observed that the binding of HA to CD44v3 activates Vav2-mediated Rac1 signaling leading to ovarian tumor cell migration. Further analyses indicate that the adaptor molecule, growth factor receptor-bound protein 2 (Grb2) that is bound to p185(HER2) (an oncogene product), is also associated with the CD44v3-Vav2 complex. HA binding to SK-OV-3.ipl cells promotes recruitment of both Grb2 and p185(HER2) to the CD44v3-Vav2 complex leading to Ras activation and ovarian tumor cell growth. In order to determine the role of Grb2 in CD44v3 signaling, we have transfected SK-OV-3.ipl cells with Grb2 mutant cDNAs (e.g. Delta N-Grb2 that has a deletion in the amino-terminal SH3 domain or Delta C-Grb2 that has a deletion in the carboxyl-terminal SH3 domain). Our results clearly indicate that the SH3 domain deletion mutants of Grb2 (i.e. the Delta N-Grb2 (and to a lesser extent the Delta C-Grb2) mutant) not only block their association with p185(HER2) but also significantly impair their binding to the CD44v3-Vav2 complex and inhibit HA/CD44v3-induced ovarian tumor cell behaviors. Taken together, these findings strongly suggest that the interaction of CD44v3-Vav2 with Grb2-p185(HER2) plays an important role in the co-activation of both Rac1 and Ras signaling that is required for HA-mediated human ovarian tumor progression.

Effects of group selection for productivity and longevity on blood concentrations of serotonin, catecholamines, and corticosterone of laying hens.

Selection of a line of White Leghorn chickens for high group productivity and longevity resulted in reducing cannibalism and flightiness in multiple-hen cages. Improvements in survival might have been due to changes of physiological homeostasis. The objective of the present study was to test the hypothesis that genetic selection for high (HGPS) and low (LGPS) group productivity and survivability also altered regulation of neuroendocrine homeostasis. Hens were randomly assigned to individual cages at 17 wk of age. At 21 wk of age, blood concentrations of dopamine, epinephrine, norepinephrine, and serotonin were measured using HPLC assay. Blood concentrations of corticosterone were measured using radioimmunoassay. The LGPS hens had greater blood concentrations of dopamine and epinephrine than the HGPS hens (P < 0.01). The blood concentration of norepinephrine was not significantly different between the lines, but the ratio of epinephrine to norepinephrine was greater in the LGPS hens (P < 0.01). The blood concentrations of serotonin were also higher in the LGPS hens compared to those in the HGPS hens (P < 0.01). Although the HGPS hens tended to have a higher level of blood corticosterone, the difference was not significant (1.87 +/- 0.19 vs. 1.49 +/- 0.21 ng/mL; P = 0.08). The results suggest that selection for group productivity and survivability alters the chickens' neuroendocrine homeostasis, and these changes may correlate with its line-unique coping ability to domestic environments and survivability.

Effect of genetic selection for group productivity and longevity on immunological and hematological parameters of chickens.

A line of White Leghorn chickens was selected for high group productivity and longevity resulting in improved survival and feather score as well as reduced cannibalism and flightiness. Improvements in survival might have also been due to improved immunity. The present study was designed to test the hypothesis that selection for high (HGPS) and low (LGPS) group productivity and survivability also altered immune and hematological parameters. The LGPS line was an intense reverse selected line of the HGPS line at the eighth generation of development. Hens were randomly assigned to individual cages at 17 wk of age. Blood samples were collected from the hens at 21 wk of age. Subsets of T lymphocytes (CD4+, CD8+, and gammadelta cells) were measured using flow cytometry. Concentrations of plasma IgG were quantified with western blot analysis and immunoprecipitation assay. Hematological parameters were collected from blood smears. The HGPS hens had significantly higher percentages of blood lymphocytes and CD4+:CD8+ ratios of circulating T cells (P < 0.01) and tended to have more, but not significantly, gammadelta T cells (P = 0.07) than the LGPS hens. In contrast, the LGPS hens exhibited eosinophilia and heterophilia and greater heterophil:lymphocyte ratios (P < 0.01). The concentrations of plasma IgG were also significantly higher in the LGPS hens (P < 0.01). These results suggest that genetic selection for group productivity and longevity also alters the immunological and hematological systems of hens. The line difference in regulation of T cells, leukocytes, and production of IgG may suggest that different genes or modes of gene action are involved.

Bradyrhizobium spp. (TGx) isolates nodulating the new soybean cultivars in Africa are diverse and distinct from bradyrhizobia that nodulate North American soybeans.

The newly developed cultivars of soybean in Africa, known as Tropical Glycine cross (TGx), are nodulated by bradyrhizobia indigenous to African soils, here designated Bradyrhizobium spp. (TGx). Isolates of Bradyrhizobium spp. (TGx) obtained from nodules of TGx soybeans that were inoculated with soils from 65 locations in six African countries were characterized and grouped into 11 phylogenetic clusters on the basis of RFLP of the 16S rRNA gene. Five restriction enzymes (RsaI, HinfI, MspI, CfoI and HaeIII) established RFLP groups within these Bradyrhizobium spp. (TGx) isolates, which were used to construct a phylogenetic tree showing their genetic relationship with other Bradyrhizobium species. RFLP analysis indicated that Bradyrhizobium spp. (TGx) is a heterogeneous group with some isolates related to Bradyrhizobium japonicum and Bradyrhizobium elkanii strains and some to Bradyrhizobium spp. (misc.) reference strains isolated from a variety of tropical legumes. The heterogeneity within the large phylogenetic clusters was further examined through analysis of randomly amplified polymorphic DNA (RAPD) using GC-rich PCR primers. The RAPD analysis showed additional heterogeneity in the Bradyrhizobium spp. (TGx) phylogenetic clusters, which was not revealed by separations based on RFLP analysis. The Bradyrhizobium spp. (TGx) isolates were classified into effective and ineffective types based on their symbiotic performance on TGx soybean. The isolates were randomly distributed throughout the phylogenetic clusters regardless of their symbiotic effectiveness on TGx soybean.

Differential expression of tenascin by astrocytes associated with the supraoptic nucleus (SON) of hydrated and dehydrated adult rats.

The present study evaluated the expression of tenascin by astrocytes in the supraoptic nucleus and associated ventral glial limitans (SON-VGL) under conditions that induce reversible changes in neuronal organization (dehydration and rehydration). Immunostaining of astroglia cultured from rat neonatal SON-VGL confirmed that these cells are capable of both expressing and secreting tenascin. Observations of immunostained tissue sections from adult rats revealed tenascin immunoreactivity primarily in the VGL and dendritic zone, subjacent to SON neuronal somata. Comparison of immunostained tissues from hydrated and dehydrated animals showed an apparent decrease in the intensity of immunostaining with dehydration. Subsequent Western blots of similar tissues confirmed the presence of the 210-220-kDa tenascin protein in the SON-VGL. SON-VGL tissues from control, dehydrated, and rehydrated rats were then studied by using SDS-PAGE and quantitative gel densitometry. A consistent decrease in tenascin concentration was observed by 6 days of dehydration that, with rehydration, reversed back toward or beyond control levels. Together, these observations indicate that SON-VGL astrocytes variably express tenascin and that this protein may play a role in adult SON plasticity.

Detergent solubilization of membrane-bound methane monooxygenase requires plastoquinol analogs as electron donors.

Quinols can provide reducing equivalents for the membrane-bound form of methane monooxygenase (pMMO), substituting for NADH in whole cells and membranes. Furthermore, quinols are effective reductants for the detergent-solubilized enzyme, whereas NADH is ineffective. The decyl analog of plastoquinol and duroquinol (2,3,5,6-tetramethylbenzoquinol) provide the greatest methane monooxygenase activity in whole cells and membrane suspensions, as well as detergent-solubilized samples. Lauryl maltoside is by far the best detergent for solubilization of catalytically active methane monooxygenase. Optimal pMMO activity in the detergent-solubilized fraction is obtained with a ratio of approximately 1.7 mg of detergent per milligram of membrane protein, independent of protein concentration. The detergent-solubilized pMMO retains its sensitivity to inhibition by cyanide, acetylene, and EDTA. It is also stimulated by exogenous copper, as in isolated membrane fractions. Reaction of the detergent-solubilized enzyme with [14C]acetylene results in labeling of a 26-kDa peptide, analogous to the behavior observed for isolated membrane suspensions. The selectivity of pMMO for duroquinol and decyl-plastoquinol, relative to other structurally similar quinols, suggests that the enzyme obtains reducing equivalents directly from a quinol (probably plastoquinol) in vivo.