Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides

PURPOSE: G protein-coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). EXPERIMENTAL DESIGN: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. RESULTS: The IC50 value of [(nat)In]-RM1 was 14 +/- 3.4 nmol/L. [(nat/111)In]-RM1 was found to bind to the GRPR with a Kd of 8.5 +/- 2.7 nmol/L compared with a Kd of 0.6 +/- 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 +/- 0.2 nmol/L) compared with [111In]-AMBA (0.7 +/- 0.1 nmol/L). [(nat)Lu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [(nat)In]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 +/- 0.8% IA/g versus 3.69 +/- 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. CONCLUSION: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor-targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors.

Baylis-Hillman reaction assisted synthesis of substituted 5,8-dihydro-isoxazolo[4,5-c]azepin-4-one: A novel isoxazole-annulated heterocycle

The novel synthesis of a new isoxazole-annulated heterocycle namely 5,8-dihydro-isoxazolo[4,5-c]azepin-4-one described herein is based on the reaction of benzyl amine with acetates of Baylis-Hillman adducts generated from 3-aryl-5-formyl-isoxazole-4-carboxylate

Studies toward the construction of substituted piperidine-2-ones and pyridine-2-ones from Baylis-Hillman adducts: Discovery of a facile synthesis of 5-methyl-4-oxo-6-aryl-3-aza-bicyclo[3.1.0]hexane-1-carboxylates

Studies toward the construction of functionalised piperidone derivatives from derivatives of Baylis-Hillman adducts are described. Interestingly the 6-oxo-4-aryl-piperidine-3-carboxylates generated during the study serve as precursor for the facile synthesis of 4-oxo-6-aryl-3-aza-bicyclo[3.1.0]hexane-1-carboxylates

Impact of the CTLA-4/CD28 axis on the processes of joint inflammation in rheumatoid arthritis

OBJECTIVE: The importance of the costimulatory molecules CD28 and CTLA-4 in the pathologic mechanism of rheumatoid arthritis (RA) has been demonstrated by genetic associations and the successful clinical application of CTLA-4Ig for the treatment of RA. This study was undertaken to investigate the role of the CTLA-4/CD28 axis in the local application of CTLA-4Ig in the synovial fluid (SF) of RA patients. METHODS: Quantitative polymerase chain reaction was used to analyze the expression of proinflammatory and antiinflammatory cytokines in ex vivo fluorescence-activated cell sorted CTLA-4+ and CTLA-4- T helper cells from the peripheral blood and SF of RA patients. T helper cells were also analyzed for cytokine expression in vitro after the blockade of CTLA-4 by anti-CTLA-4 Fab fragments or of B7 (CD80/CD86) molecules by CTLA-4Ig. RESULTS: CTLA-4+ T helper cells were unambiguously present in the SF of all RA patients examined, and they expressed increased amounts of interferon-γ (IFNγ), interleukin-17 (IL-17), and IL-10 as compared to CTLA-4- T helper cells. The selective blockade of CTLA-4 in T helper cells from the SF in vitro led to increased levels of IFNγ, IL-2, and IL-17. The concomitant blockade of CD28 and CTLA-4 in T helper cells from RA SF by CTLA-4Ig in vitro resulted in reduced levels of the proinflammatory cytokines IFNγ and IL-2 and increased levels of the antiinflammatory cytokines IL-10 and transforming growth factor β. CONCLUSION: Our ex vivo and in vitro results demonstrate that the CTLA-4/CD28 axis constitutes a drug target for not only the systemic, but potentially also the local, application of the costimulation blocking agent CTLA-4Ig for the treatment of RA.

Chlamydia prevalence in young attenders of rural and regional primary care services in Australia: a cross-sectional survey.

OBJECTIVE To estimate chlamydia prevalence among 16-29-year-olds attending general practice clinics in Australia. DESIGN, PARTICIPANTS AND SETTING A cross-sectional survey was conducted from May 2010 to December 2012. Sexually experienced 16-29-year-olds were recruited from 134 general practice clinics in 54 rural and regional towns in four states and in nine metropolitan clinics (consecutive patients were invited to participate). Participants completed a questionnaire and were tested for chlamydia. MAIN OUTCOME MEASURE Chlamydia prevalence. RESULTS Of 4284 participants, 197 tested positive for chlamydia (4.6%; 95% CI, 3.9%-5.3%). Prevalence was similar in men (5.2% [65/1257]; 95% CI, 3.9%-6.4%) and women (4.4% [132/3027]; 95% CI, 3.5%-5.2%) (P = 0.25) and high in those reporting genital symptoms or a partner with a sexually transmissible infection (STI) - 17.0% in men (8/47; 95% CI, 2.8%-31.2%); 9.5% in women (16/169; 95% CI, 5.1%-13.8%). Nearly three-quarters of cases (73.4% [130/177]) were diagnosed in asymptomatic patients attending for non-sexual health reasons, and 83.8% of all participants (3258/3890) had attended for non-sexual health reasons. Prevalence was slightly higher in participants from rural and regional areas (4.8% [179/3724]; 95% CI, 4.0%-5.6%) than those from metropolitan areas (3.1% [17/548]; 95% CI, 1.5%-4.7%) (P = 0.08). In multivariable analysis, increasing partner numbers in previous 12 months (adjusted odds ratio [AOR] for three or more partners, 5.11 [95% CI, 2.35-11.08]), chlamydia diagnosis in previous 12 months (AOR, 4.35 [95% CI, 1.52-12.41]) and inconsistent condom use with most recent partner (AOR, 2.90 [95% CI, 1.31-6.40]) were significantly associated with chlamydia in men. In women, increasing partner numbers in previous 12 months (AOR for two partners, 2.59 [95% CI, 1.59-4.23]; AOR for three or more partners, 3.58 [95% CI, 2.26-5.68]), chlamydia diagnosis in previous 12 months (AOR, 3.13 [95% CI, 1.62-6.06]) and age (AOR for 25-29-year-olds, 0.23 [95% CI, 0.12-0.44]) were associated with chlamydia. CONCLUSIONS Chlamydia prevalence is similar in young men and women attending general practice. Testing only those with genital symptoms or a partner with an STI would have missed three-quarters of cases. Most men and women are amenable to being tested in general practice, even in rural and regional areas.

Asymmetric cyclopropanation of olefins catalysed by Cu(I) complexes of chiral pyridine-containing macrocyclic ligands (Pc-L*)

The synthesis and characterisation of copper(I) complexes of chiral pyridine-containing macrocyclic ligands (Pc-L*) and their use as catalysts in asymmetric cyclopropanation reactions are reported. All ligands and metal complexes were fully characterised, including crystal structures of some species determined by X-ray diffraction on single crystals. This allowed characterising the very different conformations of the macrocycles which could be induced by different substituents or by metal complexation. The strategy adopted for the ligand synthesis is very flexible allowing several structural modifications. A small library of macrocyclic ligands possessing the same donor properties but with either C-1 or C-2 symmetry was synthesized. Cyclopropane products with both aromatic and aliphatic olefins were obtained in good yields and enantiomeric excesses up to 99%.

The structure of the asteroid 4 Vesta as revealed by models of planet-scale collisions

Asteroid 4Vesta seems to be a major intact protoplanet, with a surface composition similar to that of the HED (howardite-eucrite-diogenite) meteorites. The southern hemisphere is dominated by a giant impact scar, but previous impact models have failed to reproduce the observed topography. The recent discovery that Vesta's southern hemisphere is dominated by two overlapping basins provides an opportunity to model Vesta's topography more accurately. Here we report three-dimensional simulations of Vesta's global evolution under two overlapping planet-scale collisions. We closely reproduce its observed shape, and provide maps of impact excavation and ejecta deposition. Spiral patterns observed in the younger basin Rheasilvia, about one billion years old, are attributed to Coriolis forces during crater collapse. Surface materials exposed in the north come from a depth of about 20kilometres, according to our models, whereas materials exposed inside the southern double-excavation come from depths of about 60-100kilometres. If Vesta began as a layered, completely differentiated protoplanet, then our model predicts large areas of pure diogenites and olivine-rich rocks. These are not seen, possibly implying that the outer 100kilometres or so of Vesta is composed mainly of a basaltic crust (eucrites) with ultramafic intrusions (diogenites).

Molecular characterisation and expression of Atlantic cod (Gadus morhua) myoglobin from two populations held at two different acclimation temperatures.

Much previous research has demonstrated the plasticity of myoglobin concentrations in both cardiac and skeletal myocytes in response to hypoxia and training. No study has yet looked at the effect of thermal acclimation on myoglobin in fish. Atlantic cod (Gadus morhua) from two different populations, i.e. the North Sea and the North East Arctic, were acclimated to 10 and 4 degrees C. Both the myoglobin mRNA and myoglobin protein in cod hearts increased significantly by up to 3.7 and 2.3 fold respectively as a result of acclimation to 4 degrees C. These increments were largest in the Arctic population, which in earlier studies have been shown to possess cold compensated metabolic demands at low temperatures. These metabolic demands associated with higher mitochondrial capacities may have driven the increase in cardiac myoglobin concentrations, in order to support diffusive oxygen supply. At the same time the increase in myoglobin levels may serve further functions during cold acclimation, for example, protection of the cell against reactive oxygen species, and scavenging nitric oxide, thereby contributing to the regulation of mitochondrial volume density.

Function and formation of $\beta$1,4-galactosyltransferase-specific adhesions during growth and differentiation of F9 embryonal carcinoma cells

Galactosyltransferase (GalTase) is localized in the Golgi, where it functions in oligosaccharide synthesis, as well as on the cell surface where it serves as a cell adhesion molecule. GalTase-specific adhesions are functional in a number of important biological events, including F9 embryonal carcinoma (EC) cell adhesions. GalTase-based adhesions are formed by recognition and binding to terminal N-acetylglucosamine (GlcNAc) residues on its glycoprotein counterpart on adjacent cell surfaces. The object of this work has been to investigate the formation and function of GalTase-specific adhesions during F9 cell growth and differentiation. We initially investigated GalTase synthesis during differentiation and found that the increase in GalTase activity was specific for the Golgi compartment; surface GalTase levels remained constant during differentiation. These data indicated that the increase in cell adhesions expected with increased cell-matrix interaction in differentiated F9 cells is not the consequence of increased surface GalTase expression and, more interestingly, that the two pools of GalTase are under differential regulation. Synthesis and recognition of the consociate glycoprotein component was next investigated. Surface GalTase recognized several surface glycoproteins in a pattern that changes with differentiation. Uvomorulin, lysosome-associated membrane protein-1 (LAMP-1), and laminin were recognized by surface GalTase and are, therefore, potential components in GalTase-specific adhesions. Furthermore, these interactions were aberrant in an adhesion-defective F9 cell line that results, at least in part, from abnormal oligosaccharide synthesis. The function played by surface GalTase in growth and induction of differentiation was examined. Inhibition of surface GalTase function by a panel of reagents inhibited F9 cell growth. GalTase expression at both the transcription and protein levels were differentially regulated during the cell cycle, with surface expression greatest in the G1 phase. Disruption of GalTase adhesion by exposure to anti-GalTase antibodies during this period resulted in extension of the G2 phase, a result similar to that seen with agents known to inhibit growth and induce differentiation. Finally, other studies have suggested that a subset of cell adhesion molecules have the capability to induce differentiation in EC cells systems. We have determined in F9 cells that dissociating GalTase adhesion by galactosylation of and release of the consociate glycoproteins induces differentiation, as defined by increased laminin synthesis. The ability to induce differentiation by surface galactosylation was greatest in cells grown in cultures promoting cell-cell adhesions, relative to cultures with minimal cell-cell interactions. ^