Mechanistic insights into cytosolic molecular chaperones in protein unfolding and disaggregation

Under optimal non-physiological conditions of low concentrations and low temperatures, proteins may spontaneously fold to the native state, as all the information for folding lies in the amino acid sequence of the polypeptide. However, under conditions of stress or high protein crowding as inside cells, a polypeptide may misfold and enter an aggregation pathway resulting in the formation of misfolded conformers and fibrils, which can be toxic and lead to neurodegenerative illnesses, such as Alzheimer's, Parkinson's or Huntington's diseases and aging in general. To avert and revert protein misfolding and aggregation, cells have evolved a set of proteins called molecular chaperones. Here, I focussed on the human cytosolic chaperones Hsp70 (DnaK) and HspllO, and co-chaperone Hsp40 (DnaJ), and the chaperonin CCT (GroEL). The cytosolic molecular chaperones Hsp70s/Hspll0s and the chaperonins are highly upregulated in bacterial and human cells under different stresses and are involved both in the prevention and the reversion of protein misfolding and aggregation. Hsp70 works in collaboration with Hsp40 to reactivate misfolded or aggregated proteins in a strict ATP dependent manner. Chaperonins (CCT and GroEL) also unfold and reactivate stably misfolded proteins but we found that it needed to use the energy of ATP hydrolysis in order to evict over- sticky misfolded intermediates that inhibited the unfoldase catalytic sites. Ill In this study, we initially characterized a particular type of inactive misfolded monomeric luciferase and rhodanese species that were obtained by repeated cycles of freeze-thawing (FT). These stable misfolded monomeric conformers (FT-luciferase and FT-rhodanese) had exposed hydrophobic residues and were enriched with wrong ß-sheet structures (Chapter 2). Using FT-luciferase as substrate, we found that the Hsp70 orthologs, called HspllO (Sse in yeast), acted similarly to Hsp70 as were bona fide ATP- fuelled polypeptide unfoldases and was much more than a mere nucleotide exchange factor, as generally thought. Moreover, we found that HspllO collaborated with Hsp70 in the disaggregation of stable protein aggregates in which Hsp70 and HspllO acted as equal partners that synergistically combined their individual ATP-consuming polypeptide unfoldase activities to reactivate the misfolded/aggregated proteins (Chapter 3). Using FT-rhodanese as substrate, we found that chaperonins (GroEL and CCT) could catalytically reactivate misfolded rhodanese monomers in the absence of ATP. Also, our results suggested that encaging of an unfolding polypeptide inside the GroEL cavity under a GroES cap was not an obligatory step as generally thought (Chapter 4). Further, we investigated the role of Hsp40, a J-protein co-chaperone of Hsp70, in targeting misfolded polypeptides substrates onto Hsp70 for unfolding. We found that even a large excess of monomeric unfolded a-synuclein did not inhibit DnaJ, whereas, in contrast, stable misfolded a-synuclein oligomers strongly inhibited the DnaK-mediated chaperone reaction by way of sequestering the DnaJ co-chaperone. This work revealed that DnaJ could specifically distinguish, and bind potentially toxic stably aggregated species, such as soluble a-synuclein oligomers involved in Parkinson's disease, and with the help of DnaK and ATP convert them into from harmless natively unfolded a-synuclein monomers (chapter 5). Finally, our meta-analysis of microarray data of plant and animal tissues treated with various chemicals and abiotic stresses, revealed possible co-expressions between core chaperone machineries and their co-chaperone regulators. It clearly showed that protein misfolding in the cytosol elicits a different response, consisting of upregulating the synthesis mainly of cytosolic chaperones, from protein misfolding in the endoplasmic reticulum (ER) that elicited a typical unfolded protein response (UPR), consisting of upregulating the synthesis mainly of ER chaperones. We proposed that drugs that best mimicked heat or UPR stress at increasing the chaperone load in the cytoplasm or ER respectively, may prove effective at combating protein misfolding diseases and aging (Chapter 6).  - Dans les conditions optimales de basse concentration et de basse température, les protéines vont spontanément adopter un repliement natif car toutes les informations nécessaires se trouvent dans la séquence des acides aminés du polypeptide. En revanche, dans des conditions de stress ou de forte concentration des protéines comme à l'intérieur d'une cellule, un polypeptide peu mal se replier et entrer dans un processus d'agrégation conduisant à la formation de conformères et de fibrilles qui peuvent être toxiques et causer des maladies neurodégénératives comme la maladie d'Alzheimer, la maladie de Parkinson ou la chorée de Huntington. Afin d'empêcher ou de rectifier le mauvais repliement des protéines, les cellules ont développé des protéines appelées chaperonnes. Dans ce travail, je me suis intéressé aux chaperonnes cytosoliques Hsp70 (DnaK) et HspllO, la co-chaperones Hsp40 (DnaJ), le complexe CCT/TRiC et GroEL. Chez les bactéries et les humains, les chaperonnes cytosoliques Hsp70s/Hspl 10s et les « chaperonines» sont fortement activées par différentes conditions de stress et sont toutes impliquées dans la prévention et la correction du mauvais repliement des protéines et de leur agrégation. Hsp70 collabore avec Hsp40 pour réactiver les protéines agrégées ou mal repliées et leur action nécessite de 1ATP. Les chaperonines (GroEL) déplient et réactivent aussi les protéines mal repliées de façon stable mais nous avons trouvé qu'elles utilisent l'ATP pour libérer les intermédiaires collant et mal repliés du site catalytique de dépliage. Nous avons initialement caractérisé un type particulier de formes stables de luciférase et de rhodanese monomériques mal repliées obtenues après plusieurs cycles de congélation / décongélation répétés (FT). Ces monomères exposaient des résidus hydrophobiques et étaient plus riches en feuillets ß anormaux. Ils pouvaient cependant être réactivés par les chaperonnes Hsp70+Hsp40 (DnaK+DnaJ) et de l'ATP, ou par Hsp60 (GroEL) sans ATP (Chapitre 2). En utilisant la FT-Luciferase comme substrat nous avons trouvé que HspllO (un orthologue de Hsp70) était une authentique dépliase, dépendante strictement de l'ATP. De plus, nous avons trouvé que HspllO collaborait avec Hsp70 dans la désagrégation d'agrégats stables de protéines en combinant leurs activités dépliase consommatrice d'ATP (Chapitre 3). En utilisant la FT-rhodanese, nous avons trouvé que les chaperonines (GroEL et CCT) pouvaient réactiver catalytiquement des monomères mal repliés en absence d'ATP. Nos résultats suggérèrent également que la capture d'un polypeptide en cours de dépliement dans la cavité de GroEL et sous un couvercle du complexe GroES ne serait pas une étape obligatoire du mécanisme, comme il est communément accepté dans la littérature (Chapitre 4). De plus, nous avons étudié le rôle de Hsp40, une co-chaperones de Hsp70, dans l'adressage de substrats polypeptidiques mal repliés vers Hsp70. Ce travail a révélé que DnaJ pouvait différencier et lier des polypeptide mal repliés (toxiques), comme des oligomères d'a-synucléine dans la maladie de Parkinson, et clairement les différencier des monomères inoffensifs d'a-synucléine (Chapitre 5). Finalement une méta-analyse de données de microarrays de tissus végétaux et animaux traités avec différents stress chimiques et abiotiques a révélé une possible co-expression de la machinerie des chaperonnes et des régulateurs de co- chaperonne. Cette meta-analyse montre aussi clairement que le mauvais repliement des protéines dans le cytosol entraîne la synthèse de chaperonnes principalement cytosoliques alors que le mauvais repliement de protéines dans le réticulum endoplasmique (ER) entraine une réponse typique de dépliement (UPR) qui consiste principalement en la synthèse de chaperonnes localisées dans l'ER. Nous émettons l'hypothèse que les drogues qui reproduisent le mieux les stress de chaleur ou les stress UPR pourraient se montrer efficaces dans la lutte contre le mauvais repliement des protéines et le vieillissement (Chapitre 6).

Phenotypic heterogeneity of antigen-specific CD4 cells under different conditions of antigen persistence and antigen load

RESUME Nous n'avons pas de connaissance précise des facteurs à l'origine de l'hétérogénéité phénotypique des cellules T CD4 mémoires. Une troisième population phénotypique des cellules T CD4 mémoires, caractérisée par les marqueurs CD45RA+CCR7- a été identifiée dans cette étude. Cette population présente un état de différentiation avancée, comme en témoigne son histoire de réplication, ainsi que sa capacité de prolifération homéostatique. Les réponses des cellules T CD4 mémoires à différentes conditions de persistance et charge antigénique ont trois patterns phénotypiques différents, caractérisés par les marqueurs CD45RA et CCR7. La réponse CD4 mono -phénotypique CD45RA-CCR7+ ou CD45RA- CCR7- est associée à des conditions d'élimination de l'antigène (telle la réponse CD4 tétanos spécifique) ou à des conditions de persistance antigénique et de virémie élevée (telle la réponse HIV chronique ou la primo-infection CMV) respectivement. D'autre part, les réponses T CD4 multi -phénotypiques CD45RA-CCR7+ sont associées à des conditions d'exposition antigénique prolongée et de faible virémie (telles les infections CMV, EBV et HSV ou les infections HIV chez les long term non progressons). La réponse mono -phénotypique CD45RA- CCR7+ est propre aux cellules T CD4 secrétant de IL2, définies également comme centrales mémoires, la réponse CD45RA- CCR7- aux cellules T CD4 secrétant de l'IFNγ et finalement la réponse mufti-phénotypique aux cellules T CD4 secrétant à la fois de l'IL2 et de l' IFNγ. En conclusion, ces résultats témoignent d'une régulation de l'hétérogénéité phénotypique par l'exposition et la charge antigénique. ABSTRACT The factors responsible for the phenotypic heterogeneity of memory CD4 T cells are unclear. In the present study, we have identified a third population of memory CD4 T cells characterized as CD45RA+CCRT that, based on its replication history and the homeostatic proliferative capacity, was at an advanced stage of differentiation. Three different phenotypic patterns of memory CD4 T cell responses were delineated under different conditions of antigen (Ag) persistence and load using CD45RA and CCR7 as markers of memory T cells. Mono-phenotypic CD45RA'CCR7+ or CD45RA'CCR7' CD4 T cell responses were associated with conditions of Ag clearance (tetanus toxoid-specific CD4 T cell response) or Ag persistence and high load (chronic HIV-1 and primary CMV infections), respectively. Multi-phenotypic CD45RA CCR7+, CD45RA'CCRT and CD45RA+CCRT CD4 T cell responses were associated with protracted Ag exposure and low load (chronic CMV, EBV and HSV infections and HIV-1 infection in long-term nonprogressors). The mono-phenotypic CD45RA'CCR7+ response was typical of central memory (TCM) IL-2-secreting CD4 T cells, the mono-phenotypic CD45RA CCRT response of effector memory (TEM) IFN-γ -secreting CD4 T cells and the multi-phenotypic response of both IL-2- and IFN-γ -secreting cells. The present results indicate that the heterogeneity of different Ag-specific CD4 T cell responses is regulated by Ag exposure and Ag load.

An objective anisotropic elastic plastic model and Algorithm applicable to bone mechanics

The implicit projection algorithm of isotropic plasticity is extended to an objective anisotropic elastic perfectly plastic model. The recursion formula developed to project the trial stress on the yield surface, is applicable to any non linear elastic law and any plastic yield function.A curvilinear transverse isotropic model based on a quadratic elastic potential and on Hill's quadratic yield criterion is then developed and implemented in a computer program for bone mechanics perspectives. The paper concludes with a numerical study of a schematic bone-prosthesis system to illustrate the potential of the model.

Interleukin-28B polymorphisms, IP-10, viral load and age predict the outcome of therapy in genotype 1 hepatitis C virus under real-life conditions

Background and Aims: IL28B polymorphisms, interferon (IFN)-gamma inducible protein-10 (IP-10) levels and the homeostasis model assessment of insulin resistance (HOMA-IR) score have been reported to predict rapid (RVR) and sustained (SVR) virological response in chronic hepatitis C (CHC), but it is not known whether these factors represent independent, clinically useful predictors. The aim of the study was to assess factors (including IL28B polymorphisms, IP-10 levels and HOMA-IR score) independently predicting response to therapy in CHC under real life conditions.Methods: Multivariate analysis of factors predicting RVR and SVR in 280 consecutive, treatment-naive CHC patients treated with pegylated IFN alpha and ribavirin in a prospective multicenter study.Results: Independent predictors of RVR were HCV RNA < 400,000 IU/ml (OR11.37; 95% CI 3.03-42.6), rs12980275 AA (vs. AG/GG) (OR 7.09; 1.97-25.56) and IP-10 (OR 0.04; 0.003-0.56) in HCV genotype 1 patients and lower baseline γ-glutamyl-transferase levels (OR = 0.02; 0.0009-0.31) in HCV genotype 3 patients. Independent predictors of SVR were rs12980275 AA (OR 9.68; 3.44-27.18), age < 40 yrs (OR = 4.79; 1.50-15.34) and HCV RNA < 400,000 IU/ml (OR 2.74; 1.03-7.27) in HCV genotype 1 patients and rs12980275 AA (OR = 6.26; 1.98-19.74) and age < 40 yrs (OR 5.37; 1.54-18.75) in the 88 HCV genotype 1 patients without a RVR. RVR was by itself predictive of SVR in HCV genotype 1 patients (32 of 33, 97%; OR 33.0; 4.06-268.32) and the only independent predictor of SVR in HCV genotype 2 (OR 9.0, 1.72-46.99; p=0.009) or 3 patients (OR 7.8, 1.43-42.67; p=0.01).Conclusions: In HCV genotype 1 patients, IL28B polymorphisms, HCV RNA load and IP-10 independently predict RVR. The combination of IL28B polymorphisms, HCV RNA level and age may yield more accurate pretreatment prediction of SVR. HOMA-IR score is not associated with viral response.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

Abrupt decrease in serum testosterone levels after an oral glucose load in men: implications for screening for hypogonadism.

OBJECTIVE: This study examines the physiological impact of a glucose load on serum testosterone (T) levels in men with varying glucose tolerance (GT). DESIGN: Cross-sectional study. PATIENTS AND METHODS: 74 men (19-74 years, mean 51·4 ± 1·4 years) underwent a standard 75-g oral glucose tolerance test with blood sampling at 0, 30, 60, 90 and 120 min. Fasting serum glucose, insulin, total T (and calculated free T), LH, SHBG, leptin and cortisol were measured. RESULTS: 57% of the men had normal GT, 30% had impaired GT and 13% had newly diagnosed type 2 diabetes. Glucose ingestion was associated with a 25% decrease in mean T levels (delta = -4·2 ± 0·3 nm, P < 0·0001). T levels remained suppressed at 120 min compared with baseline (13·7 ± 0·6 vs 16·5 ± 0·7 nm, P < 0·0001) and did not differ across GT or BMI. Of the 66 men with normal T levels at baseline, 10 (15%) had levels that decreased to the hypogonadal range (<9·7 nm) at one or more time points. SHBG, LH and cortisol levels were unchanged. Leptin levels decreased from baseline at all time points (P < 0·0001). CONCLUSIONS: Glucose ingestion induces a significant reduction in total and free T levels in men, which is similar across the spectrum of glucose tolerance. This decrease in T appears to be because of a direct testicular defect, but the absence of compensatory changes in LH suggests an additional central component. Men found to have low nonfasting T levels should be re-evaluated in the fasting state.

Effect of testosterone on immunocompetence, parasite load, and metabolism in the common wall lizard (Podarcis muralis)

Testosterone can benefit individual fitness by increasing ornament colour, aggressiveness, and sperm quality, but it can also impose both metabolic and immunological costs. However, evidence that testosterone causes immuno suppression in freely living populations is scant. We studied the effects of testosterone on one component of the immune system (i.e., the cell-mediated response to phytohaemagglutinin), parasite load, and metabolic rate in the common wall lizard, Podarcis muralis (Laurenti, 1768). For analyses of immunocompetence and parasitism, male lizards were implanted at the end of the breeding season with either empty or testosterone implants and were returned to their site of capture for 5-6 weeks before recapture. For analyses of the effects of testosterone on metabolic rate, male lizards were captured and implanted before hibernation and were held in the laboratory for 1 week prior to calorimetry. Experimental treatment with testosterone decreased the cell-mediated response to the T-cell mitogen phytohemagglutinin and increased mean metabolic rate. No effects of testosterone on the number of ectoparasites, hemoparasites, and resting metabolic rate could be detected. These results are discussed in the framework of the immunocompetence handicap hypothesis and the immuno-redistribution process hypothesis. [Authors]

A new electrophysiological index for working memory load in humans.

Working memory, the ability to store and simultaneously manipulate information, is affected in several neuropsychiatric disorders which lead to severe cognitive and functional deficits. An electrophysiological marker for this process could help identify early cerebral function abnormalities. In subjects performing working memory-specific n-back tasks, event-related potential analysis revealed a positive-negative waveform (PNwm) component modulated in amplitude by working memory load. It occurs in the expected time range for this process, 140-280 ms after stimulus onset, superimposed on the classical P200 and N200 components. Independent Component Analysis extracted two functional components with latencies and topographical scalp distributions similar to the PNwm. Our results imply that the PNwm represents a new electrophysiological index for working memory load in humans.

Capsaicin mimics mechanical load-induced intracellular signaling events: involvement of TRPV1-mediated calcium signaling in induction of skeletal muscle hypertrophy.

Mechanical load-induced intracellular signaling events are important for subsequent skeletal muscle hypertrophy. We previously showed that load-induced activation of the cation channel TRPV1 caused an increase in intracellular calcium concentrations ([Ca ( 2+) ]i) and that this activated mammalian target of rapamycin (mTOR) and promoted muscle hypertrophy. However, the link between mechanical load-induced intracellular signaling events, and the TRPV1-mediated increases in [Ca ( 2+) ]i are not fully understood. Here we show that administration of the TRPV1 agonist, capsaicin, induces phosphorylation of mTOR, p70S6K, S6, Erk1/2 and p38 MAPK, but not Akt, AMPK or GSK3β. Furthermore, the TRPV1-induced phosphorylation patterns resembled those induced by mechanical load. Our results continue to highlight the importance of TRPV1-mediated calcium signaling in load-induced intracellular signaling pathways.

Effect of inspiratory threshold loading on ventilatory kinetics during constant-load exercise.

Humoral factors play an important role in the control of exercise hyperpnea. The role of neuromechanical ventilatory factors, however, is still being investigated. We tested the hypothesis that the afferents of the thoracopulmonary system, and consequently of the neuromechanical ventilatory loop, have an influence on the kinetics of oxygen consumption (VO2), carbon dioxide output (VCO2), and ventilation (VE) during moderate intensity exercise. We did this by comparing the ventilatory time constants (tau) of exercise with and without an inspiratory load. Fourteen healthy, trained men (age 22.6 +/- 3.2 yr) performed a continuous incremental cycle exercise test to determine maximal oxygen uptake (VO2max = 55.2 +/- 5.8 ml x min(-1) x kg(-1)). On another day, after unloaded warm-up they performed randomized constant-load tests at 40% of their VO2max for 8 min, one with and the other without an inspiratory threshold load of 15 cmH2O. Ventilatory variables were obtained breath by breath. Phase 2 ventilatory kinetics (VO2, VCO2, and VE) could be described in all cases by a monoexponential function. The bootstrap method revealed small coefficients of variation for the model parameters, indicating an accurate determination for all parameters. Paired Student's t-tests showed that the addition of the inspiratory resistance significantly increased the tau during phase 2 of VO2 (43.1 +/- 8.6 vs. 60.9 +/- 14.1 s; P < 0.001), VCO2 (60.3 +/- 17.6 vs. 84.5 +/- 18.1 s; P < 0.001) and VE (59.4 +/- 16.1 vs. 85.9 +/- 17.1 s; P < 0.001). The average rise in tau was 41.3% for VO2, 40.1% for VCO2, and 44.6% for VE. The tau changes indicated that neuromechanical ventilatory factors play a role in the ventilatory response to moderate exercise.

An iterative Multiscale Finite-Volume algorithm converging to the exact solution

The multiscale finite volume (MsFV) method has been developed to efficiently solve large heterogeneous problems (elliptic or parabolic); it is usually employed for pressure equations and delivers conservative flux fields to be used in transport problems. The method essentially relies on the hypothesis that the (fine-scale) problem can be reasonably described by a set of local solutions coupled by a conservative global (coarse-scale) problem. In most cases, the boundary conditions assigned for the local problems are satisfactory and the approximate conservative fluxes provided by the method are accurate. In numerically challenging cases, however, a more accurate localization is required to obtain a good approximation of the fine-scale solution. In this paper we develop a procedure to iteratively improve the boundary conditions of the local problems. The algorithm relies on the data structure of the MsFV method and employs a Krylov-subspace projection method to obtain an unconditionally stable scheme and accelerate convergence. Two variants are considered: in the first, only the MsFV operator is used; in the second, the MsFV operator is combined in a two-step method with an operator derived from the problem solved to construct the conservative flux field. The resulting iterative MsFV algorithms allow arbitrary reduction of the solution error without compromising the construction of a conservative flux field, which is guaranteed at any iteration. Since it converges to the exact solution, the method can be regarded as a linear solver. In this context, the schemes proposed here can be viewed as preconditioned versions of the Generalized Minimal Residual method (GMRES), with a very peculiar characteristic that the residual on the coarse grid is zero at any iteration (thus conservative fluxes can be obtained).

Fatal and nonfatal AIDS and non-AIDS events in HIV-1-positive individuals with high CD4 cell counts according to viral load strata.

BACKGROUND: This study compared the incidence of fatal and nonfatal AIDS and non-AIDS events in HIV-positive individuals with a CD4 cell count more than 350  cells/μl among viral load strata: low (<500  copies/ml), intermediate (500-9999.9  copies/ml) and high (≥ 10000  copies/ml). METHODS: Individuals contributed person-years at risk if their most recent CD4 cell count was more than 350  cells/μl. Follow-up was censored if their CD4 cell count dropped below 350  cells/μl. Poisson regression analysis investigated the relationship between viraemia and the incidence of AIDS and non-AIDS events. RESULTS: Three hundred and fifty-four AIDS events occurred during 51 732  person-years of follow-up (PYFU), crude incidence rate of AIDS across the three strata was 0.53, 0.90 and 2.12 per 100 PYFU, respectively. After adjustment, a higher rate of AIDS was observed in individuals with moderate [incidence rate ratio (IRR) 1.44, 1.02-2.05, P = 0.03] and high viraemia had a higher rate (IRR 3.91, 2.89-5.89, P < 0.0001) compared with low viraemia. Five hundred and seventy-two non-AIDS events occurred during 43 784 PYFU, the crude incidence rates were 1.28, 1.52, and 1.38 per 100 PYFU, respectively. After adjustment, particularly for age, region of Europe and starting combination antiretroviral therapy, there was a 61% (IRR 1.61, 1.21-2.14, P = 0.001) and 66% (IRR 1.66, 1.17-2.32, P = 0.004) higher rate of non-AIDS in individuals with intermediate and high viraemia compared with low viraemia. CONCLUSION: In individuals with a CD4 cell count more than 350  cells/μl, an increased incidence of AIDS and a slightly increased incidence of non-AIDS was found in those with uncontrolled viral replication. The association with AIDS was clear and consistent. However, the association with non-AIDS was only apparent after adjustment and no differences were observed between intermediate and high viraemia.

Risk assessment of recurrence in sporadic retinoblastoma using a molecular-based algorithm

Le rétinoblastome (Rb) est une tumeur provenant des cellules rétiniennes progénitrices des photorécepteurs. C'est la tumeur pédiatrique maligne la plus fréquente avec une incidence par naissance évaluée entre 1/15Ό00 et 1/20Ό00. Les enfants atteints de Rb sont diagnostiqué dans leur grande majorité avant l'âge de 4 ans, soit le temps nécessaire à la différentiation et à la maturation des photorécepteurs et donc à la disparition de la cellule d'origine du Rb. La survie du patient, la sauvegarde oculaire et le pronostic visuel restent excellents pour autant que le traitement ne soit pas différé. Dans sa variante non héréditaire (60%) le Rb est toujours unilatéral et sporadique. Le Rb héréditaire de transmission dominante autosomique (40%), se décline sous toutes les formes, familiale (10%) ou sporadique (30%), que l'atteinte soit unilatérale ou bilatérale. La majorité des mutations causales sont uniques et distribuées de façon aléatoire sur la totalité du gène RB1 sans région prédisposante. La détection de ces mutations est couteuse et chronophage, tout en présentant un taux de détection relativement bas; surtout dans les cas de Rb sporadiques unilatéraux. Dans le but d'identifier les patients présentant un risque réel de développer un Rb, et de réduire le nombre d'examens sous narcose requis pour le dépistage de la maladie chez les sujets à risque, nous avons développé une stratégie sensible, rapide, efficace et peu couteuse basée sur une analyse de l'haplotype intragénique. Cet algorithme prend en compte a) la perte d'hétérozygotie intratumorale du gène RB1, b) l'origine paternelle préférentielle des nouvelles mutations germinales et c) un risque a priori dérivé des données empiriques de Vogel. Pendant la période allant de janvier 1994 à décembre 2006, nous avons comparé l'apparition de nouveau Rb parmi la fratrie et la descendance de patient atteints au nombre de nouveaux cas attendus calculé par notre algorithme. 134 familles ont été étudiées. L'analyse moléculaire a été effectuée chez 570 personnes dont 99 patients âgés de moins de 4 ans et donc à risque de développer un Rb. Parmi cette cohorte, nous avons observé l'apparition d'un cas de Rb, alors que les risques cumulés a posteriori calculé par notre algorithme prédisait l'apparition de 1.77 nouveau cas. Dans cette étude, nous avons pu valider notre algorithme prédisant la récurrence de Rb chez les parents de 1er degré de patients atteints. Cet outil devrait grandement faciliter le conseil génétique ainsi que le suivi des patients à risque de développer un Rb, surtout dans les cas ou le séquençage direct du gène RB1 n'est pas disponible ou est resté non informatif. - Purpose: Most RBI mutations are unique and distributed throughout the RBI gene. Their detection can be time-consuming and the yield especially low in cases of conservatively-treated sporadic unilateral retinoblas-toma (Rb) patients. In order to identify patients with true risk of developing Rb, and to reduce the number of unnecessary examinations under anesthesia in all other cases, we developed a universal sensitive, efficient and cost-effective strategy based on intragenic haplotype analysis. Methods: This algorithm allows the calculation of the a posteriori risk of developing Rb and takes into account (a) RBI loss of heterozygosity in tumors, (b) preferential paternal origin of new germline mutations, (c) a priori risk derived from empirical data by Vogel, and (d) disease penetrance of 90% in most cases. We report the occurrence of Rb in first degree relatives of patients with sporadic Rb who visited the Jules Gonin Eye Hospital, Lausanne, Switzerland, from January 1994 to December 2006 compared to expected new cases of Rb using our algorithm. Results: A total of 134 families with sporadic Rb were enrolled; testing was performed in 570 individuals and 99 patients younger than 4 years old were identified. We observed one new case of Rb. Using our algorithm, the cumulated total a posteriori risk of recurrence was 1.77. Conclusions: This is the first time that linkage analysis has been validated to monitor the risk of recurrence in sporadic Rb. This should be a useful tool in genetic counseling, especially when direct RBI screening for mutations leaves a negative result or is unavailable.

High bacterial load in negative pressure wound therapy (NPWT) foams used in the treatment of chronic wounds.

No earlier study has investigated the microbiology of negative pressure wound therapy (NPWT) foam using a standardized manner. The purpose of this study is to investigate the bacterial load and microbiological dynamics in NPWT foam removed from chronic wounds (>3 months). To determine the bacterial load, a standardized size of the removed NPWT foam was sonicated. The resulting sonication fluid was cultured, and the colony-forming units (CFU) of each species were enumerated. Sixty-eight foams from 17 patients (mean age 63 years, 71% males) were investigated. In 65 (97%) foams, â0/00¥âeuro0/001 and in 37 (54%) â0/00¥2 bacterial types were found. The bacterial load remained high during NPWT treatment, ranging from 10(4) to 10(6) CFU/ml. In three patients (27%), additional type of bacteria was found in subsequent foam cultures. The mean bacterial countâeuro0/00±âeuro0/00standard deviation was higher in polyvinyl alcohol foam (6.1âeuro0/00±âeuro0/000.5 CFU/ml) than in polyurethane (5.5âeuro0/00±âeuro0/000.8 CFU/ml) (pâeuro0/00=âeuro0/000.02). The mean of log of sum of CFU/ml in foam from 125âeuro0/00mmHg (5.5âeuro0/00±âeuro0/000.8) was lower than in foam from 100âeuro0/00mmHg pressure (5.9âeuro0/00±âeuro0/000.5) (pâeuro0/00=âeuro0/000.01). Concluding, bacterial load remains high in NPWT foam, and routine changing does not reduce the load.