Real-time and non-invasive acute lung rejection diagnosis using confocal LASER Endomicroscopy in lung transplant recipients: Results from the CELTICS study.

BACKGROUND AND OBJECTIVE: Traditionally, the diagnosis of acute rejection (AR) relies on invasive transbronchial biopsies (TBBs) to obtain histopathological samples. We aimed to evaluate the diagnostic yield of probe-based confocal laser endomicroscopy (pCLE) as a complementary and non-invasive tool for ACR screening, comparing its results with those obtained from TBBs. METHODS: Between January 2015 and April 2022, we conducted a retrospective study of all lung transplant recipients aged over 18 years at Toulouse University Hospital (France). All patients who underwent bronchoscopies with both TBBs and pCLE imaging were included. Two experienced interpreters (TV and MS) reviewed the pCLE images independently, blinded to all clinical information and pathology results. RESULTS: From 120 procedures in 85 patients, 34 abnormal histological samples were identified. Probe-based confocal laser endomicroscopy revealed significant associations between both alveolar (ALC) and perivascular (PVC) cellularities and abnormal histological samples (p<0.0001 and 0.003 respectively). Alveolar cellularity demonstrated a sensitivity (Se) of 85.3 %, specificity (Spe) of 43 %, positive predictive value (PPV) of 37.2 % and negative predictive value (NPV) of 88.1 %. For PVC, Se was 70.6 %, Spe 80.2 %, PPV 58.5 % and NPV 87.3 %. Intra-interpreter correlation (TV) was 88.3 % for the number of vessels (+/-1), 98.3 % for ALC and 90 % for PVC. Inter-interpreter correlation (TV and MS) was 80 % for vessels (+/-1), 97.5 % for ALC and 83.3 % for PVC. CONCLUSION: Our study demonstrates the feasibility of incorporating pCLE into clinical practice, demonstrating good diagnostic yield and reproducible outcomes in the screening of AR in lung transplant recipients.

[Epidemiology of fibrosing interstitial lung diseases in the department of Haute Garonne]. / EPIDemio : épidémiologie des pneumopathies interstitielles diffuses (PID) fibrosantes en Haute-Garonne.

EPIDemio study is a multicenter, prospective and observational study. The objective is to estimate the prevalence and incidence of fibrosing interstitial lung diseases (ILDs) in the department of Haute Garonne (31) in France. Fifty-five pulmonologists from the Toulouse university hospital and 8 private establishments participated in this study. Two hundred and fifty-six cases of fibrosing ILDs were reported (gross overall prevalence: 22.8/100,000 and estimated 30.1/100,000. Idiopathic ILDs represent 55.8% of fibrosing ILDs ahead of systemic disease-related ILDs (24.6%) and ILDs associated with environmental exposure (13.3%). Idiopathic pulmonary fibrosis (IPF) represents 35.9% of fibrosing ILDs, which corresponds to a minimal prevalence of 8.2/100,000 and an estimated prevalence of 11.2/100,000. This study confirms epidemiological data collected in France and Europe.

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment.

We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.

[Prevalence of smoking cessation during pregnancy according to trimester]. / Taux de sevrage tabagique chez la femme enceinte en fonction des trimestres.

OBJECTIVE: To investigate the smoking cessation period during pregnancy. PATIENTS AND METHODS: Questionnaire-based, descriptive study of 979 pregnant women in four regions of France. The variables analysed included the characteristics of the mother and neonate at delivery, the smoking habits of the mother before and during pregnancy, the perception of risk linked to smoking, and the reasons for giving up smoking. RESULTS: Eighteen percent of women smoked until delivery. Forty-five percent of women gave up smoking during pregnancy, usually in the first trimester. More precisely, about one woman who smoked out of 50 gives up in order to prepare pregnancy. The proportion of women who stop smoking in each of the three trimesters of pregnancy is 84,1, 8,8 and 7,1% respectively. DISCUSSION AND CONCLUSION: Most women appear to stop smoking before any intervention therapy is possible. The first contact with a midwife or an obstetrician takes place whereas smoking cessation is already successful.

[Adolescents with cystic fibrosis: the approach to transition from paediatric to adult care]. / Transition enfant-adulte au cours de la mucoviscidose.

Because of early and effective therapies, an increasing numbers of young people with cystic fibrosis (CF) reach adulthood. Preparing for and maintaining high quality CF care in the adult healthcare is critical for prolonged survival. Because adverse health consequences occur when inadequate transition arrangements are in place, safely transferring patients from pediatric to adult care is a priority. Key features include an early preparation, planning and self-management skills, a coordinated approach and a detailed communication between patients, families, pediatric and adult teams. Formal transition protocols and audits can support the process and be helpful for multidisciplinary teams.

Recruitment of cdc2 kinase by DNA topoisomerase II is coupled to chromatin remodeling.

Although initiation of chromosome condensation during early prophase is linked temporally to the appearance of the mitotic cdc2 kinase in the nucleus, it is not known what targets the kinase to the nucleus and how this is coupled to chromatin remodeling. We now report that cdc2 kinase forms stable molecular complexes with the nuclear enzyme DNA topoisomerase II, which is associated with marked stimulation of both DNA binding and catalytic activity of topoisomerase II, albeit in a phosphorylation-independent manner. The molecular interaction is required for recruitment of cdc2 kinase, as shown by incubation of purified enzymes with chicken erythrocyte nuclei, which have neither endogenous topoisomerase II nor cdc2 kinase. The physical association between the two enzymes alters the DNA/topoisomerase II interaction as shown by pulse-field electrophoresis after incubation of intact nuclei with the specific topoisomerase II inhibitor VM-26. Furthermore, the presence of both enzymes, but not either enzyme alone, is accompanied by extensive chromatin remodeling converting the interphase nuclei into precondensation chromosomes with striking resemblance to early prophase structures. Our results reveal a novel property of cyclin-dependent kinases and demonstrate that the recruitment of cdc2 kinase by topoisomerase II is coupled to chromatin remodeling.

[Obstetrical ultrasound screening and motivation to stop smoking]. / Echographies obstétricales et motivation à l'arrêt du tabac.

OBJECTIVES: To evaluate whether ultrasound screening during pregnancy increases the ability to stop or reduce smoking. MATERIALS AND METHODS: This multicenter questionnaire study was performed to describe smoking characteristics among pregnant women and evaluate the factors that influence the ability to reduce smoking during pregnancy. Questionnaire-based, descriptive study of 979 pregnant women in four regions of France. The variables analyzed included the characteristics of the mother and neonate at delivery, the smoking habits of the mother before and during pregnancy, and the reasons for giving up smoking. RESULTS: We report only results about ultrasound scan. One third of women, who smoked at the start of pregnancy, reported that ultrasound was a positive tool to enhance motivation to reduce smoking. CONCLUSION: Ultrasound screening may be a positive factor to reduce smoking. Our study focused on motivation could give a tool in this way. However most women who stop smoking during pregnancy do so in the first trimester (84%) mainly before the first ultrasound exam. Thus, the routine ultrasound scan itself does not seem to influence attitudes to stop smoking any further. But it may be included in comprehensive and individualised anti-smoking support.

[Smoking cessation: practice guidelines]. / Sevrage tabagique: conduite pratique.

Smoking is a habit sustained and amplified by dependency on nicotine. Despite knowing the risks to their health, smokers have great difficulty in stopping. The syndrome of nicotine withdrawal and the related complications when stopping smoking: depression, weight gain, are adequate justifications of the many failures to stop smoking. However, we have now come out of the empiricism, effective treatment is available and scientifically validated international recommendations have been established. They involve: the practice of minimal advice which consists of questioning every patient about smoking habits and encouraging them to stop; the treatments of nicotine substitution, patch, chewing gum, tablets or inhaler, used at effective dosage and sometimes in association with each other; more recently, Bupropion (Zyban, LP), a psychotropic inhibitor of Dobutamine and Noradrenalin recapture; behavioural and cognitive therapies, alone or in association with pharmacological therapy. The measurement of the score of tobacco dependency with the Fagerström test enables definition of a therapeutic strategy. Of course, these treatments are only effective in smokers motivated to stop smoking. The decision to stop smoking should only be taken after a period of reflection during which the role of information and advice given by all health professionals is primordial. Also, the long-term follow-up and counsel are essential to prevent relapse, especially during the first year.

[Ultrasonic features of congenital anomalies of the gallbladder]. / Aspects échographiques des anomalies congénitales de la vésicule biliaire.

Congenital anomalies of the gallbladder (GB) correspond to the ultimate manifestation of the wide range of anatomical variations that may affect the GB. Anomalies may be numerical: duplication, accessory GB, and, rarely, agenesis. Anomalies of position are more common: ectopic GB, floating or wandering GB. Morphological anomalies (septations) are the most common GB anomalies. Most of these anomalies may result in pitfalls at US imaging.

[How can we help pregnant women stop smoking?]. / Peut-on aider les femmes enceintes qui fument?

Smoking is a behavior maintained and enhanced by nicotine-induced dependence. Despite awareness and knowledge of the associated health risks many smokers find it considerably difficult to quit. Nicotine withdrawal symptoms and undesirable effects such as depression and weight gain serve as justification for the numerous unsuccessful attempts in smoking cessation. Yet, we have now come to the end of empiricism. Treatments that have been shown to work exist and international evidence-based recommendations for cessation interventions have been established: brief advice, nicotine replacement therapy and behavioral and cognitive therapies. Measuring nicotine dependence using the Fagerström test helps to define the therapeutic strategy. Blood cotinine level can be measured during pregnancy. It is obvious that therapies can only work for smokers who are motivated to stop smoking. Before reaching the decision to quit, the smoker goes through a process during the course of which the role of information and advice from health professionals are paramount. When they became pregnant, young women are not necessary ready to quit. For many of them, pregnancy is sufficient motivation enabling them to stop smoking, but for thirty percent of them, probably the most dependent, it would be very difficult to stop without a specific program of help.

Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition.

The G2-M transition of the cell cycle is triggered by the p34(cdc2)/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34(cdc2) (TP-YP) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-YP), which can be obtained in vitro by treatment of TP-YP by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-YP may be involved in the autocatalytic amplification of the p34(cdc2)/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.

The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast.

Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe. Spd1p overexpression blocks the onset of both S phase and mitosis. In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset. High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G(2) requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes. We propose that high levels of Spd1p in G(2) cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis. The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G(2 )rad/hus checkpoint pathway to block mitosis.

Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition.

Activation of Cdc2-cyclin B (or M phase-promoting factor (MPF)) at the prophase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B. We here investigated the regulation of cyclin B phosphorylation using the starfish oocyte model. Cyclin B phosphorylation is not required for Cdc2 kinase activity; both the prophase complex dephosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylated on cyclin B with protein phosphatase 2A display high kinase activities. An in vitro assay of cyclin B kinase activity closely mimics in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.

Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.

Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

Purification of GSK-3 by affinity chromatography on immobilized axin.

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.