Analysis of susceptibility to human immunodeficiency virus 1 (HIV-1) by comparative genetics

Summary [résumé français voir ci-dessous] From the beginning of the 20th century the world population has been confronted with the human immune deficiency virus 1 (HIV-1). This virus has the particularity to mutate fast, and could thus evade and adapt to the human host. Our closest evolutionary related organisms, the non-human primates, are less susceptible to HIV-1. In a broader sense, primates are differentially susceptible to various retrovirus. Species specificity may be due to genetic differences among primates. In the present study we applied evolutionary and comparative genetic techniques to characterize the evolutionary pattern of host cellular determinants of HIV-1 pathogenesis. The study of the evolution of genes coding for proteins participating to the restriction or pathogenesis of HIV-1 may help understanding the genetic basis of modern human susceptibility to infection. To perform comparative genetics analysis, we constituted a collection of primate DNA and RNA to allow generation of de novo sequence of gene orthologs. More recently, release to the public domain of two new primate complete genomes (bornean orang-utan and common marmoset) in addition of the three previously available genomes (human, chimpanzee and Rhesus monkey) help scaling up the evolutionary and comparative genome analysis. Sequence analysis used phylogenetic and statistical methods for detecting molecular adaptation. We identified different selective pressures acting on host proteins involved in HIV-1 pathogenesis. Proteins with HIV-1 restriction properties in non-human primates were under strong positive selection, in particular in regions of interaction with viral proteins. These regions carried key residues for the antiviral activity. Proteins of the innate immunity presented an evolutionary pattern of conservation (purifying selection) but with signals of relaxed constrain if we compared them to the average profile of purifying selection of the primate genomes. Large scale analysis resulted in patterns of evolutionary pressures according to molecular function, biological process and cellular distribution. The data generated by various analyses served to guide the ancestral reconstruction of TRIM5a a potent antiviral host factor. The resurrected TRIM5a from the common ancestor of Old world monkeys was effective against HIV-1 and the recent resurrected hominoid variants were more effective against other retrovirus. Thus, as the result of trade-offs in the ability to restrict different retrovirus, human might have been exposed to HIV-1 at a time when TRIM5a lacked the appropriate specific restriction activity. The application of evolutionary and comparative genetic tools should be considered for the systematical assessment of host proteins relevant in viral pathogenesis, and to guide biological and functional studies. Résumé La population mondiale est confrontée depuis le début du vingtième siècle au virus de l'immunodéficience humaine 1 (VIH-1). Ce virus a un taux de mutation particulièrement élevé, il peut donc s'évader et s'adapter très efficacement à son hôte. Les organismes évolutivement le plus proches de l'homme les primates nonhumains sont moins susceptibles au VIH-1. De façon générale, les primates répondent différemment aux rétrovirus. Cette spécificité entre espèces doit résider dans les différences génétiques entre primates. Dans cette étude nous avons appliqué des techniques d'évolution et de génétique comparative pour caractériser le modèle évolutif des déterminants cellulaires impliqués dans la pathogenèse du VIH- 1. L'étude de l'évolution des gènes, codant pour des protéines impliquées dans la restriction ou la pathogenèse du VIH-1, aidera à la compréhension des bases génétiques ayant récemment rendu l'homme susceptible. Pour les analyses de génétique comparative, nous avons constitué une collection d'ADN et d'ARN de primates dans le but d'obtenir des nouvelles séquences de gènes orthologues. Récemment deux nouveaux génomes complets ont été publiés (l'orang-outan du Bornéo et Marmoset commun) en plus des trois génomes déjà disponibles (humain, chimpanzé, macaque rhésus). Ceci a permis d'améliorer considérablement l'étendue de l'analyse. Pour détecter l'adaptation moléculaire nous avons analysé les séquences à l'aide de méthodes phylogénétiques et statistiques. Nous avons identifié différentes pressions de sélection agissant sur les protéines impliquées dans la pathogenèse du VIH-1. Des protéines avec des propriétés de restriction du VIH-1 dans les primates non-humains présentent un taux particulièrement haut de remplacement d'acides aminés (sélection positive). En particulier dans les régions d'interaction avec les protéines virales. Ces régions incluent des acides aminés clé pour l'activité de restriction. Les protéines appartenant à l'immunité inné présentent un modèle d'évolution de conservation (sélection purifiante) mais avec des traces de "relaxation" comparé au profil général de sélection purifiante du génome des primates. Une analyse à grande échelle a permis de classifier les modèles de pression évolutive selon leur fonction moléculaire, processus biologique et distribution cellulaire. Les données générées par les différentes analyses ont permis la reconstruction ancestrale de TRIM5a, un puissant facteur antiretroviral. Le TRIM5a ressuscité, correspondant à l'ancêtre commun entre les grands singes et les groupe des catarrhiniens, est efficace contre le VIH-1 moderne. Les TRIM5a ressuscités plus récents, correspondant aux ancêtres des grands singes, sont plus efficaces contre d'autres rétrovirus. Ainsi, trouver un compromis dans la capacité de restreindre différents rétrovirus, l'homme aurait été exposé au VIH-1 à une période où TRIM5a manquait d'activité de restriction spécifique contre celui-ci. L'application de techniques d'évolution et de génétique comparative devraient être considérées pour l'évaluation systématique de protéines impliquées dans la pathogenèse virale, ainsi que pour guider des études biologiques et fonctionnelles

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results.

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.

OBJECTIVE: The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. DESIGN: We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. METHODS: Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. RESULTS: Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. CONCLUSION: As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

Les endocardites dues aux germes du groupe HACEK. A propos d'un cas d'endocardite native a Kingella kingae. [Endocarditis due to HACEK bacteria. A case report of endocarditis due to Kingella kingae]

Endocarditis is a common disease in hospital practice. Identification of the microorganism responsible for the valvular damage is essential to establish the prognosis and to determine the optimal antibiotic treatment. In some cases of endocarditis the diagnosis is laborious, especially when the responsible microorganism is difficult to detect using standard culture techniques. Here we report a case of native aortic valve endocarditis due to Kingella kingae, a Gram negative organism of the HACEK group. In addition we review 6 other cases of endocarditis caused by organism belonging to this group, treated in our hospital between 1983 and 1999. Epidemiological studies show that less than 5% of all cases of endocarditis are caused by organisms of the HACEK group. The diagnosis is often delayed because their slow growth on a standard culture medium. We describe clinical and microbiological characteristics of this group of endocarditis.

Addition of aripiprazole to the clozapine may be useful in reducing anxiety in treatment-resistant schizophrenia.

There exist many case reports and studies on the antipsychotic augmentation by aripirazole in partial responders to clozapine, the most seem to be finding a slight difference in the PANSS and CGI scores after the aripirazole addition. The results of our report are compatible with those of other studies but, we have found a considerable antianxiety action in both of the cases. The 5HT1A agonism of aripirazole could be hypothesized as mechanism contributing to this effect.

Appropriateness of respiratory care: evidence-based guidelines.

PRINCIPLES: Respiratory care is universally recognised as useful, but its indications and practice vary markedly. In order to improve the appropriateness of respiratory care in our hospital, we developed evidence-based local guidelines in a collaborative effort involving physiotherapists, physicians and health service researchers. METHODS: Recommendations were developed using the standardised RAND appropriateness method. A literature search was conducted based on terms associated with guidelines and with respiratory care. A working group prepared proposals for recommendations which were then independently rated by a multidisciplinary expert panel. All recommendations were then discussed in common and indications for procedures were rated confidentially a second time by the experts. The recommendations were then formulated on the basis of the level of evidence in the literature and on the consensus among these experts. RESULTS: Recommendations were formulated for the following procedures: non-invasive ventilation, continuous positive airway pressure, intermittent positive pressure breathing, intrapulmonary percussive ventilation, mechanical insufflation-exsufflation, incentive spirometry, positive expiratory pressure, nasotracheal suctioning and non-instrumental airway clearance techniques. Each recommendation referred to a particular medical condition and was assigned to a hierarchical category based on the quality of the evidence from the literature supporting the recommendation and on the consensus among the experts. CONCLUSION: Despite a marked heterogeneity of scientific evidence, the method used allowed us to develop commonly agreed local guidelines for respiratory care. In addition, this work fostered a closer relationship between physiotherapists and physicians in our institution.

Current approaches and challenges for the metabolite profiling of complex natural extracts.

Metabolite profiling is critical in many aspects of the life sciences, particularly natural product research. Obtaining precise information on the chemical composition of complex natural extracts (metabolomes) that are primarily obtained from plants or microorganisms is a challenging task that requires sophisticated, advanced analytical methods. In this respect, significant advances in hyphenated chromatographic techniques (LC-MS, GC-MS and LC-NMR in particular), as well as data mining and processing methods, have occurred over the last decade. Together, these tools, in combination with bioassay profiling methods, serve an important role in metabolomics for the purposes of both peak annotation and dereplication in natural product research. In this review, a survey of the techniques that are used for generic and comprehensive profiling of secondary metabolites in natural extracts is provided. The various approaches (chromatographic methods: LC-MS, GC-MS, and LC-NMR and direct spectroscopic methods: NMR and DIMS) are discussed with respect to their resolution and sensitivity for extract profiling. In addition the structural information that can be generated through these techniques or in combination, is compared in relation to the identification of metabolites in complex mixtures. Analytical strategies with applications to natural extracts and novel methods that have strong potential, regardless of how often they are used, are discussed with respect to their potential applications and future trends.

Insulin-like growth factor I (IGF I) stimulates DNA synthesis in fetal rat brain cell cultures.

Addition of insulin, IGF I or IGF II to serum-free cultures of fetal rat brain cells (gestation day 15/16) significantly stimulates DNA synthesis. The dose-response curves show that IGF I is more potent than insulin; half maximal stimulation of [3H]thymidine incorporation is obtained at about 0.4 nM IGF I and 14 nM insulin, respectively. Cultures initiated 2 days later (gestation day 17/18) showed a decreased responsiveness to both peptides. No additive effect was observed after combined addition of both peptides at near-maximal doses. Both peptides show a latency of action of about 12-18 h. In the presence of either IGF or insulin, neuronal as well as glial enzymes are increased, suggesting that neuronal and glial precursor cell division is influenced. IGF I and IGF II interact with a specific binding site for which insulin competes very weakly; however IGF I and IGF II bind with relatively high affinity to the insulin specific binding site. The present results support the hypothesis that both insulin and IGF stimulate mitotic activity by interacting with specific somatomedin receptors and suggest a physiological role of IGF in the developing brain.

Nouvelles techniques radiologiques pour les patients insuffisants rénaux [New radiological techniques to investigate patients suffering from chronic kidney disease].

Radiological investigations using gadolinium or intravenous iodinated contrast products are used cautiously in patients suffering from chronic kidney disease because of their risk of acute kidney injury and systemic nephrogenic fibrosis. In this article, we review several radiological alternatives that can be useful to obtain renal anatomical and/or functional information in this patient population. The basic principles, indications, and advantages and limitations of Doppler ultrasound with measurement of the resistance index, contrast-enhanced ultrasound, and a technique called BOLD-MRI (blood-oxygenation level dependent-MRI) are discussed.

Variations in IB1/JIP1 expression regulate susceptibility of beta-cells to cytokine-induced apoptosis irrespective of C-Jun NH2-terminal kinase signaling.

We previously reported that interleukin-1beta (IL-1beta) alone does not cause apoptosis of beta-cells, whereas when combined with gamma-interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), it exerts a distinct apoptotic effect. Studies in beta-cell lines indicated that IL-1beta reduced expression of islet brain (IB)-1/JNK interacting protein (JIP)-1, a JNK scaffold protein with antiapoptotic action. We examined whether variations in IB1/JIP-1 expression in purified primary beta-cells affect their susceptibility to cytokine-induced apoptosis. Exposure to IL-1beta for 24 h decreased cellular IB1/JIP-1 content by 66 +/- 17%; this IL-1beta effect was maintained in the presence of TNF-alpha + IFN-gamma, which did not influence IB1/JIP-1 levels by themselves. Addition of IL-1beta to TNF-alpha + IFN-gamma increased apoptosis from 20 +/- 2% to 59 +/- 5%. A similar increase in TNF-alpha + IFN-gamma-induced apoptosis was produced by adenoviral expression of antisense IB1/JIP-1 and was not further enhanced by addition of IL-1beta, indicating that IL-1beta-mediated suppression of IB1/JIP-1 in beta-cells increases their susceptibility to cytokine-induced apoptosis. However, adenovirally mediated overexpression of IB1/JIP-1 also potentiated TNF-alpha + IFN-gamma-induced apoptosis, suggesting that the antiapoptotic effect of IB1/JIP-1 depends on well-defined cellular levels. We conclude that the IB1/JIP-1 level in beta-cells can control their susceptibility to apoptosis independent of JNK signaling.

Transcatheter aortic valve implantation (TAVI): state of the art techniques and future perspectives.

Transcatheter aortic valve therapies are the newest established techniques for the treatment of high risk patients affected by severe symptomatic aortic valve stenosis. The transapical approach requires a left anterolateral mini-thoracotomy, whereas the transfemoral method requires an adequate peripheral vascular access and can be performed fully percutaneously. Alternatively, the trans-subclavian access has been recently proposed as a third promising approach. Depending on the technique, the fine stent-valve positioning can be performed with or without contrast injections. The transapical echo-guided stent-valve implantation without angiography (the Lausanne technique) relies entirely on transoesophageal echocardiogramme imaging for the fine stent-valve positioning and it has been proved that this technique prevents the onset of postoperative contrast-related acute kidney failure. Recent published reports have shown good hospital outcomes and short-term results after transcatheter aortic valve implantation, but there are no proven advantages in using the transfemoral or the transapical technique. In particular, the transapical series have a higher mean logistic Euroscore of 27-35%, a procedural success rate above 95% and a mean 30-day mortality between 7.5 and 17.5%, whereas the transfemoral results show a lower logistic Euroscore of 23-25.5%, a procedural success rate above 90% and a 30-day mortality of 7-10.8%. Nevertheless, further clinical trials and long-term results are mandatory to confirm this positive trend. Future perspectives in transcatheter aortic valve therapies would be the development of intravascular devices for the ablation of the diseased valve leaflets and the launch of new stent-valves with improved haemodynamic, different sizes and smaller delivery systems.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.