Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states.

The binding free energy for the interaction between serines 204 and 207 of the fifth transmembrane helix of the beta(2)-adrenergic receptor (beta(2)-AR) and catecholic hydroxyl (OH) groups of adrenergic agonists was analyzed using double mutant cycles. Binding affinities for catecholic and noncatecholic agonists were measured in wild-type and mutant receptors, carrying alanine replacement of the two serines (S204A, S207A beta(2)-AR), a constitutive activating mutation, or both. The free energy coupling between the losses of binding energy attributable to OH deletion from the ligand and from the receptor indicates a strong interaction (nonadditivity) as expected for a direct binding between the two sets of groups. However, we also measured a significant interaction between the deletion of OH groups from the receptor and the constitutive activating mutation. This suggests that a fraction of the decrease in agonist affinity caused by serine mutagenesis may involve a shift in the conformational equilibrium of the receptor toward the inactive state. Direct measurements using a transient transfection assay confirm this prediction. The constitutive activity of the (S204A, S207A) beta(2)-AR mutant is 50 to 60% lower than that of the wild-type beta(2)-AR. We conclude that S204 and S207 do not only provide a docking site for the agonist, but also control the equilibrium of the receptor between active (R*) and inactive (R) forms.

Model-based Segmentation and Image Fusion of 3D Computed Tomography and 3D Ultrasound of the Eye for Radiotherapy Planning

For radiotherapy treatment planning of retinoblastoma inchildhood, Computed Tomography (CT) represents thestandard method for tumor volume delineation, despitesome inherent limitations. CT scan is very useful inproviding information on physical density for dosecalculation and morphological volumetric information butpresents a low sensitivity in assessing the tumorviability. On the other hand, 3D ultrasound (US) allows ahigh accurate definition of the tumor volume thanks toits high spatial resolution but it is not currentlyintegrated in the treatment planning but used only fordiagnosis and follow-up. Our ultimate goal is anautomatic segmentation of gross tumor volume (GTV) in the3D US, the segmentation of the organs at risk (OAR) inthe CT and the registration of both. In this paper, wepresent some preliminary results in this direction. Wepresent 3D active contour-based segmentation of the eyeball and the lens in CT images; the presented approachincorporates the prior knowledge of the anatomy by usinga 3D geometrical eye model. The automated segmentationresults are validated by comparing with manualsegmentations. Then, for the fusion of 3D CT and USimages, we present two approaches: (i) landmark-basedtransformation, and (ii) object-based transformation thatmakes use of eye ball contour information on CT and USimages.

Dominant TNF-α(+) Mycobacterium tuberculosis-specific CD4(+) T cell responses discriminate between latent infection and active disease.

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4(+) T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4(+) T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry-based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4(+) T cells is a new tool for the rapid diagnosis of active tuberculosis disease.

From breathing to respiration.

The purpose of breathing remained an enigma for a long time. The Hippocratic school described breathing patterns but did not associate breathing with the lungs. Empedocles and Plato postulated that breathing was linked to the passage of air through pores of the skin. This was refuted by Aristotle who believed that the role of breathing was to cool the heart. In Alexandria, breakthroughs were accomplished in the anatomy and physiology of the respiratory system. Later, Galen proposed an accurate description of the respiratory muscles and the mechanics of breathing. However, his heart-lung model was hampered by the traditional view of two non-communicating vascular systems - veins and arteries. After a period of stagnation in the Middle Ages, knowledge progressed with the discovery of pulmonary circulation. The comprehension of the purpose of breathing progressed by steps thanks to Boyle and Mayow among others, and culminated with the contribution of Priestley and the discovery of oxygen by Lavoisier. Only then was breathing recognized as fulfilling the purpose of respiration, or gas exchange. A century later, a controversy emerged concerning the active or passive transfer of oxygen from alveoli to the blood. August and Marie Krogh settled the dispute, showing that passive diffusion was sufficient to meet the oxygen needs. © 2014 S. Karger AG, Basel.

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. 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 mildmoderate 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.