Profiling of 19-norandrosterone sulfate and glucuronide in human urine: implications in athlete's drug testing.

19-Norandrosterone (19-NA) as its glucuronide derivative is the target metabolite in anti-doping testing to reveal an abuse of nandrolone or nandrolone prohormone. To provide further evidence of a doping with these steroids, the sulfoconjugate form of 19-norandrosterone in human urine might be monitored as well. In the present study, the profiling of sulfate and glucuronide derivatives of 19-norandrosterone together with 19-noretiocholanolone (19-NE) were assessed in the spot urines of 8 male subjects, collected after administration of 19-nor-4-androstenedione (100mg). An LC/MS/MS assay was employed for the direct quantification of sulfoconjugates, whereas a standard GC/MS method was applied for the assessment of glucuroconjugates in urine specimens. Although the 19-NA glucuronide derivative was always the most prominent at the excretion peak, inter-individual variability of the excretion patterns was observed for both conjugate forms of 19-NA and 19-NE. The ratio between the glucuro- and sulfoconjugate derivatives of 19-NA and 19-NE could not discriminate the endogenous versus the exogenous origin of the parent compound. However, after ingestion of 100mg 19-nor-4-androstenedione, it was observed in the urine specimens that the sulfate conjugates of 19-NA was detectable over a longer period of time with respect to the other metabolites. These findings indicate that more interest shall be given to this type of conjugation to deter a potential doping with norsteroids.

Que faire des nouveaux anti-épileptiques [Clinical utilization of new anti-epileptic agents].

Since the beginning of the 1990's, a dozen of new anti-epileptic drugs have been on the market or will be soon. This article reviews the daily clinical utilisation of new anti-epileptic drugs. It considers, without being complete, the current opinions and tendencies. The new anti-epileptic substances are generally as efficient as conventional medications. However, they are better tolerated and are more easily used in combination with conventional anti-epileptic drugs. Polytherapy is certainly the form of treatment, which is used in the most cases of resistant epilepsies. The surgical treatment can be used in only a very limited number of cases. The objective of treatment is the complete control of seizures, with minimum secondary effects. Though this objective is rarely reached, the NAE significantly improves the quality of life of patients suffering from severe epilepsy. The utilisation of NAE is not without risk. Increase in the frequency and severity of seizures may occur; we should remember that severe adverse effects appeared in the post-marketing period of the use of Vigabatrine and Felbamate. Therefore, we must remain vigilant in the clinical use of the anti-epileptic drugs.

PGE2-induced colon cancer growth is mediated by mTORC1.

The inflammatory prostaglandin E2 (PGE2) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE2 directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE2-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE2 increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE2 EP4 receptor was responsible for transducing the signal to mTORC1. Moreover, PGE2 increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE2-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE2 increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE2 mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

Appropriateness of therapy for active Crohn's disease: results of a multidisciplinary international Expert Panel (EPACT II)

INTRODUCTION: The development of novel therapies and the increasing number of trials testing management strategies for luminal Crohn's disease (CD) have not filled all the gaps in our knowledge. Thus, in clinical practice, many decisions for CD patients need to be taken without high quality evidence. For this reason, a multidisciplinary European expert panel followed the RAND method to develop explicit criteria for the management of individual patients with active, steroid-dependent (ST-D) and steroid-refractory (ST-R) CD. AIMS & METHODS: Twelve international experts convened in Geneva, Switzerland in December 2007, to rate explicit clinical scenarios, corresponding to real daily practice, on a 9-point scale according to the literature evidence and their own expertise. Median ratings were stratified into three categories: appropriate (7-9), uncertain (4-6) and inappropriate (1-3). RESULTS: Overall, panelists rated 296 indications pertaining to mild-to-moderate, severe, ST-D, and ST-R CD. In anti-TNF naïve patients, budesonide and prednisone were found appropriate for mild-moderate CD, and infliximab (IFX) when those had previously failed or had not been tolerated. In patients with prior success with IFX, this drug with or without co-administration of a thiopurine analog was favored. Other anti-TNFs were appropriate in case of intolerance or resistance to IFX. High doses steroids, IFX or adalimumab were appropriate in severe active CD. Among 105 indications for ST-D or ST-R disease, the panel considered appropriate the thiopurine analogs, methotrexate, IFX, adalimumab and surgery for limited resection, depending on the outcome of prior therapies. Anti-TNFs were generally considered appropriate in ST-R. CONCLUSION: Steroids, including budesonide for mild-to-moderate CD, remain first-line therapies in active luminal CD. Anti-TNFs, in particular IFX with respect to the amount of available evidence, remain second-line for most indications. Thiopurine analogs are preferred to anti-TNFs when steroids are not appropriate, except when anti-TNFs were previously successful. These recommendations are available online (www.epact.ch). A prospective evaluation of these criteria in a large database in Switzerland in underway to validate these criteria.

Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH.

Alpha-ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and alpha-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His6-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the alpha-ketoglutarate-dependent dioxygenases and share the specific motif HXDX(24)TX(131)HX(10)R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent Km values were 99 microM for (R)-mecoprop, 164 microM for (R)-dichlorprop, and 3 microM for alpha-ketoglutarate for RdpA and 132 microM for (S)-mecoprop, 495 microM for (S)-dichlorprop, and 20 microM for alpha-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 microM for SdpA and >230 microM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace alpha-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAalpha-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.

Optimal management of elderly patients with glioblastoma.

Median age at diagnosis in patients with glioblastoma (GB) is slowly increasing with an aging population in Western countries, and was 64years in 2006. The number of patients age 65 and older with GB will double in 2030 compared with 2000. Survival in this older cohort of patients is significantly less than seen in younger patients. This may in part be related to more aggressive biology of tumor, reduced use of standard management approaches, increased toxicity of available therapies, and increased presence of comorbidities in this older patient population. Limited data do support the use of more extensive resection in these patients. Randomized data support the use of post-operative radiotherapy (RT) versus supportive care, but do not demonstrate a benefit for the use of the standard 6weeks course of RT over hypofractionated RT given over 3weeks. Preliminary data of randomized studies raise the possibility of temozolomide alone as an option for these patients. The use of 6weeks of RT with concurrent and adjuvant temozolomide has been associated with reasonably good survival in several uncontrolled small series of selected older patients; however, this better outcome may be related to the selection of better prognosis patients rather than the specific therapy utilized. The current National Cancer Institute of Canada (NCIC) and European Organization for Research and Treatment of Cancer (EORTC) CE.6/26062/22061 randomized study of short course RT with or without concurrent and adjuvant temozolomide will help determine the optimal therapy for this older cohort with currently available therapies.

Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target?

Tumor angiogenesis is an essential step in tumor progression and metastasis formation. Suppression of tumor angiogenesis results in the inhibition of tumor growth. Recent evidence indicates that vascular integrins, in particular alpha V beta 3, are important regulators of angiogenesis, including tumor angiogenesis. Integrin alpha V beta 3 antagonists, such as blocking antibodies or peptides, suppress tumor angiogenesis and tumor progression in many preclinical tumor models. The potential therapeutic efficacy of extracellular integrin antagonists in human cancer is currently being tested in clinical trials. Selective disruption of the tumor vasculature by high doses of tumor necrosis factor (TNF) and interferon gamma (IFN-gamma), and the antiangiogenic activity of nonsteroidal anti-inflammatory drugs are associated with the suppression of integrin alpha V beta 3 function and signaling in endothelial cells. Furthermore, expression of isolated integrin cytoplasmic domains disrupts integrin-dependent adhesion, resulting in endothelial cell detachment and apoptosis. These results confirm the critical role of vascular integrins in promoting endothelial cell survival and angiogenesis and suggest that intracellular targeting of integrin function and signaling may be an alternative strategy to extracellular integrin antagonists for the therapeutic inhibition of tumor angiogenesis.

Dose-dependent influence of didanosine on immune recovery in HIV-infected patients treated with tenofovir.

BACKGROUND: Antiretroviral therapy (ART) containing tenofovir disoproxil fumarate (TDF) and didanosine (ddI) has been associated with poor immune recovery despite virologic success. This effect might be related to ddI toxicity since ddI exposure is substantially increased by TDF. OBJECTIVE: To analyze whether immune recovery during ART with TDF and ddI is ddI-dose dependent. DESIGN AND METHODS: A retrospective longitudinal analysis of immune recovery measured by the CD4 T-cell slope in 614 patients treated with ART containing TDF with or without ddI. Patients were stratified according to the tertiles of their weight-adjusted ddI dose: low dose (< 3.3 mg/kg), intermediate dose (3.3-4.1 mg/kg) and high dose (> 4.1 mg/kg). Cofactors modifying the degree of immune recovery after starting TDF-containing ART were identified by univariable and multivariable linear regression analyses. RESULTS: CD4 T-cell slopes were comparable between patients treated with TDF and a weight-adjusted ddI-dose of < 4.1 mg/kg per day (n = 143) versus TDF-without-ddI (n = 393). In the multivariable model the slopes differed by -13 CD4 T cells/mul per year [95% confidence interval (CI), -42 to 17; P = 0.40]. In contrast, patients treated with TDF and a higher ddI dose (> 4.1 mg/kg per day, n = 78) experienced a significantly impaired immune recovery (-47 CD4 T cells/microl per year; 95% CI, -82 to -12; P = 0.009). The virologic response was comparable between the different treatment groups. CONCLUSIONS: Immune recovery is impaired, when high doses of ddI (> 4.1 mg/kg) are given in combination with TDF. If the dose of ddI is adjusted to less than 4.1 mg/kg per day, immune recovery is similar to other TDF-containing ART regimen.

Immunocytochemical and pharmacological characterization of metabotropic glutamate receptors of the vestibular end organs in the frog.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1alpha) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10-100 microM) enhanced the resting discharge frequency. MCPG (5-100 microM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by L-glutamate (L-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The L-Glu agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Reduction of plasma alanine aminotransferase during vigabatrin treatment.

In 9 drug-resistant patients with partial seizures treated with vigabatrin, gamma-vinyl GABA (VGB), alanine aminotransaminase (ALAT) activity in plasma was significantly reduced. Comparison of in vitro with in vivo measurements led us to conclude that this reduction is mainly an in vivo phenomenon, perhaps due to cross-enzyme inhibition. The assessment of two biological variables linked with ALAT, glucose and alanine levels under fasting conditions, failed to show any significant metabolic alterations. VGB is an effective drug for partial epilepsy. Our observations do not suggest that reduced ALAT activity is of clinical concern.

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

mTORC2 regulates PGE2-mediated endothelial cell survival and migration.

Prostaglandin E(2) (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE(2)-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE(2)-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE(2)-mediated endothelial cell responses.

Several neuronal and axonal types form long intrinsic connections in the cat primary auditory cortical field (AI).

Intrinsic connections in the cat primary auditory field (AI) as revealed by injections of Phaseolus vulgaris leucoagglutinin (PHA-L) or biocytin, had an anisotropic and patchy distribution. Neurons, labelled retrogradely with PHA-L were concentrated along a dorsoventral stripe through the injection site and rostral to it; the spread of rostrally located neurons was greater after injections into regions of low rather than high characteristic frequencies. The intensity of retrograde labelling varied from weak and granular to very strong and Golgi-like. Out of 313 Golgi like retrogradely labelled neurons 79.6% were pyramidal, 17.2% multipolar, 2.6% bipolar, and 0.6% bitufted; 13.4% were putatively inhibitory, i.e. aspiny or sparsely spiny multipolar, or bitufted. Individual anterogradely labelled intrinsic axons were reconstructed for distances of 2 to 7 mm. Five main types were distinguished on the basis of the branching pattern and the location of synaptic specialisations. Type 1 axons travelled horizontally within layers II to VI and sent collaterals at regular intervals; boutons were only present in the terminal arborizations of these collaterals. Type 2 axons also travelled horizontally within layers II to VI and had rather short and thin collateral branches; boutons or spine-like protrusions occurred in most parts of the axon. Type 3 axons travelled obliquely through the cortex and formed a single terminal arborization, the only site where boutons were found. Type 4 axons travelled for some distance in layer I; they formed a heterogeneous group as to their collaterals and synaptic specializations. Type 5 axons travelled at the interface between layer VI and the white matter; boutons en passant, spine-like protrusions, and thin short branches with boutons en passant were frequent all along their trajectory. Thus, only some axonal types sustain the patchy pattern of intrinsic connectivity, whereas others are involved in a more diffuse connectivity.