Production Flow Analysis - cases from manufacturing and service industry

Production flow analysis (PFA) is a well-established methodology used for transforming traditional functional layout into product-oriented layout. The method uses part routings to find natural clusters of workstations forming production cells able to complete parts and components swiftly with simplified material flow. Once implemented, the scheduling system is based on period batch control aiming to establish fixed planning, production and delivery cycles for the whole production unit. PFA is traditionally applied to job-shops with functional layouts, and after reorganization within groups lead times reduce, quality improves and motivation among personnel improves. Several papers have documented this, yet no research has studied its application to service operations management. This paper aims to show that PFA can well be applied not only to job-shop and assembly operations, but also to back-office and service processes with real cases. The cases clearly show that PFA reduces non-value adding operations, introduces flow by evening out bottlenecks and diminishes process variability, all of which contribute to efficient operations management.

Vascular dysfunction in offspring of preeclampsia is related to preeclampsia per se rather than genetically determined

Objectives: Epidemiological studies suggest that adverse events in utero may predispose to premature cardiovascular disease in adulthood, but the mechanisms are not known. Recently, we found that young apparently healthy offspring of mothers with preeclampsia (PE) display systemic endothelial dysfunction. This problem could be related to PE per se or to a genetic abnormality that predisposes the mother to PE and the offspring to vascular dysfunction. To distinguish between these two possibilities, we assessed vascular function in offspring of PE, their siblings who were born after a normal pregnancy, and in control subjects.Methods: We measured endothelium-dependent vasodilation (flow-mediated vasodilation, FMD), in 10 pairs of healthy normotensive siblings, one born after PE (age 15±6 y; mean±SD), the other after normal pregnancy (17±6y) and in 17 (16±7y) controls. All subjects were born at term.Results: The vascular function in siblings of PE who were born after normal pregnancy was normal and comparable to the one in controls (8.6±1.5% vs. 8.1±1.3%, P=0.32), whereas offspring of PE displayed a roughly 30% smaller FMD than the two other groups (5.9±1.6%, P<0.005 vs. both siblings and controls, Figure). The endothelial dysfunction in the offspring of PE was not related to a difference in the central arterial blood pressure or arterial oxygen saturation, because they were comparable in the 3 groups. Figure 1. FMD in the three groups.Conclusions: These findings provide the first evidence that vascular dysfunction in offspring of PE is caused by PE itself, rather than by a genetic abnormality that predisposes the mother to PE and the offspring to a vascular defect. Prevention of PE and/or its successful treatment is expected to prevent vascular dysfunction and premature cardiovascular morbidity and mortality in the offspring.

Nuclear receptor PPARS function in stem cell differentiation and bone physiology

In adult, bone remodeling is a permanent process, reaching an annual turnover of about 10% of the skeleton. Bone remodeling requires the sequential and coordinated actions of the hematopoietic origin osteoclasts, to remove bone and the mesenchymal origin osteoblasts to replace it. An increased level of bone resorption is the primary cause of age-related bone loss often resulting in osteopenia, and is the major cause of osteoporosis.¦Peroxisome proliferator-activated receptors (PPARs), which are expressed in three isotypes, PPARa, PPARp and PPARy, are ligand-activated transcription factors that control many cellular and metabolic processes, more particularly linked to lipid metabolism. In bone, previous works has shown that PPARy inhibits osteogenesis by favoring adipogenesis from common mesenchymal progenitors. In addition, the pro-osteoclastogenesis activity of PPARy results in an increased bone resorption. Accordingly, treatment with PPARy agonist such as the anti-diabetic drug TZD causes bone loss and accumulation of marrow adiposity in mice as well as in postmenopausal women. The aim of the present thesis work was to elucidate the PPARs functions in bone physiology.¦The initial characterization of the PPARP" bone phenotype mainly revealed a decreased BMD. In vitro studies exploring the potency of mesenchymal stem cells to differentiate in osteoblast showed no differences depending on the genotype. However, we could demonstrate an effect of PPARp in partially inhibiting osteoclastogenesis. These results are further sustained by a study made in collaboration with the group of Dr Kronke, which showed an impressive protection against ovariectomy-generated bone loss when the females are treated with a PPARp agonist.¦Observations in PPARy null mice are more complex. The lab has recently been able to generate mice carrying a total deletion of PPARy. Intriguingly, the exploration of the bone phenotype of these mice revealed paradoxical findings. Whereas short bones such as vertebrae exhibit an elevated BMD as expected, long bones (tibia and femur) are clearly osteoporotic. According to their activity when set in culture, osteoblast differentiation normally occurs. Indeed the phenotype can be mainly attributed to a high density of osteoclasts in the cortical bone of PPARy null mice, associated to large bone resorption areas.¦Our explorations suggest a mechanism that involves regulatory processes linking osteoclastogenesis to adipogenesis, the latter being totally absent in PPARy null mice. Indeed, the lack of adipose tissue creates a favorable niche for osteoclastogenesis since conditioned medium made from differentiated adipocyte 3T3L1 inhibited osteoclastogenesis from both PPARy-/- and WT cells. Thus, adipokines deficiency in PPARy-/- mice contributes to de- repress osteoclastogenesis. Using specific blocking antibody, we further identified adiponectin as the major player among dozens of adipokines. Using flow cytometry assay, we explored the levels at which the osteoclastic commitment was perturbed in the bone marrow of PPARy-/- mice. Intriguingly, we observe a general decrease for hematopoietic stem cell and lineage progenitors but increased proportion of osteoclast progenitor in PPARy-/- bone marrow. The general decrease of HSC in the bone marrow is however largely compensated by an important extra-medullary hematopoeisis, taking place in the liver and in the spleen.¦These specific characteristics emphasize the key role of PPARy on a cross road of osteogenesis, adipogenesis and hematopoiesis/osteoclastogenesis. They underline the complexity of the bone marrow niche, and demonstrate the inter-dependance of different cell types in defining bone homeostasis, that may be overseen when experimental design single out pure cell populations.¦Chez l'adulte, même après la fin de la croissance, le renouvellement des os se poursuit et porte sur environ 10% de l'ensemble du squelette adulte, par année. Ce renouvellement implique à la fois des mécanismes séquentiels et coordonnés des ostéoclastes d'origine hématopoïetique, qui dégradent l'os, et des ostéoblastes d'origine mésenchymale, qui permettent la régénération de l'os. La perte en densité osseuse due à l'âge entraîne un fort niveau de résorption, conduisant souvent à une ostéopénie, elle-même cause de l'ostéoporose.¦Les trois isotypes PPAR (Peroxisome proliferator-activated receptor, PPARa, PPARp, et PPARy) sont des récepteurs nucléaires qui contrôlent de nombreux mécanismes cellulaires et métaboliques, plus particulièrement liés au métabolisme lipidique. Au niveau osseux, des travaux précédents ont montré que PPARy inhibe l'ostéoblastogenèse en favorisant la formation d'adipocytes à partir de la cellule progénitrice commune. De plus, l'activité pro- ostéoclastogénique de PPARy induit une résorption osseuse accrue. Condormément à ces observations, les patients diabétiques traités par les thiazolidinediones qui agissent sur PPARy, ont un risque accrue d'ostéoporose liée à une perte osseuse accrue et un accroissement de l'adiposité au niveau de la moelle osseuse. Dans ce contexte, l'objectif de mon travail de thèse a été d'élucider le rôle des PPAR dans la physiologie osseuse, en s'appuyant sur le phénotype des souris porteuses de mutation pour PPAR.¦La caractérisation initiale des os des souris porteuses d'une délétion de ΡΡΑΕφ a principalement révélé une diminution de la densité minérale osseuse (DMO). Alors que l'ostéogenèse n'est pas significativement altérée chez ces souris, l'ostéoclastogenèse est elle augmentée, suggérant un rôle modérateur de ce processus par ΡΡΑΕΙβ. Ces résultats sont par ailleurs soutenus par une étude menée par le groupe du Dr Krônke en collaboration avec notre groupe, et qui monte une protection très importante des souris traitées par un activateur de PPARP contre l'ostéoporose provoquée par l'ovariectomie.¦Les observations concernant PPARy donnent des résultats plus complexes. Le laboratoire a en effet été capable récemment de générer des souris portant une délétion totale de PPARy. Alors que les os courts chez ces souris présentent une augmentation de la DMO, comme attendu, les os longs sont clairement ostéoporotiques. Ce phénotype corrèle avec une densité élevée d'ostéoclastes dans l'os cortical de ces os longs. Deux processus semblent contribuer à ce phénotype. En premier lieu, nous démontrons qu'un milieu conditionné provenant de cultures de cellules 3T3-L1 différenciées en adipocytes contiennent une forte activité inhibitrice d'osteoclastogenesis. L'utilisation d'anticorps neutralisant permet d'identifier l'adiponectine comme l'un des facteurs principaux de cette inhibition. Les souris PPARy étant totalement dépourvues d'adipocytes et donc de tissu adipeux, la sécrétion locale d'adiponectine dans la moelle osseuse est donc également absente, entraînant une désinhibition de l'ostéoclastogenèse. En second lieu, des analyses par FACS révèle une proportion accrue des cellules progénitrices d'ostéoclastes dans la moelle osseuse. Cela s'accompagne par une diminution globale des cellules souches hématopoïétiques, qui est cependant largement compensée par une importante hématopoëise extra-médullaire, dans le foie comme dans la rate.¦L'ensemble de notre travail montre toute l'importance de PPARy au carrefour de l'ostéogenèse, adipogenèse, et hématopoëise/osteoclastogenèse. Il souligne la complexité de la niche que représente la moelle osseuse et démontre l'inter-dépendance des différents types cellulaires définissant l'homéostasie osseuse, complexité qui peut facilement être masqué lorsque le travail expérimental se concentre sur le comportement d'un type cellulaire donné.

Foot skin blood flow following infrainguinal revascularization for critical lower limb ischemia.

INTRODUCTION: The aim of this study was to assess the blood flow in the feet before and after lower limb revascularization using laser Doppler imaging (LDI). METHODS: Ten patients with critical lower limb ischemia were prospectively enrolled from June to October 2004. All patients underwent successful unilateral surgical interventions including above-knee bypass, distal bypass and endarterectomy. Skin blood flow (SBF) over the plantar surface of both forefeet and heels was measured by LDI 24h before and 10 days after revascularization, expressed in perfusion units (PU), and reported as mean+/-SD. RESULTS: Measurements in the forefoot and heel were similar. Before revascularization mean SBF was significantly lower in the ischemic foot (130+/-71 PU) compared to the contralateral foot (212+/-68 PU), p<0.05. After revascularization a significant increase of the SBF in the forefoot (from 135+/-67 to 202+/-86 PU, p=0.001) and hindfoot (from 148+/-58 to 203+/-83, p=0.001) was observed on the treatment side. However, a large decrease of the SBF was seen in forefoot and hindfoot on the untreated side (from 250+/-123 PU to 176+/-83 and from 208+/-116 to 133+/-40, p=0.001, respectively). CONCLUSION: This study confirms the benefits of revascularization in patients with nonhealing foot lesions due to critical limb ischemia. A significant increase of the SBF was observed on the treatment side. However, an unexpected decrease was observed on the untreated side.

Immediate improvement in coronary flow reserve after alcohol septal ablation in patients with hypertrophic obstructive cardiomyopathy.

OBJECTIVES: To examine whether percutaneous alcohol septal ablation affects coronary flow reserve (CFR) in patients with hypertrophic cardiomyopathy (HCM). METHODS: CFR was measured immediately before and after septal ablation in patients with symptomatic obstructive HCM. CFR was also obtained in normal subjects (NL) for comparison. RESULTS: Patients with HCM (n = 11), compared with NL (n = 22), had a lower mean (SD) baseline CFR (1.96 (0.5) vs 3.0 (0.7), p<0.001), a lower coronary resistance (1.04 (0.45) vs 3.0 (2.6), p = 0.002), a higher coronary diastolic/systolic velocity ratio (DSVR; 5.1 (3.0) vs 1.8 (0.5), p = 0.04) and a lower hyperaemic coronary flow per left ventricular (LV) mass (0.73 (0.4) vs 1.1 (0.6) ml/min/g, p = 0.007). Septal ablation in the HCM group (n = 7) reduced the outflow tract gradient but not the left atrial or LV diastolic pressures. Ablation resulted in immediate normalisation of CFR (to 3.1 (1), p = 0.01) and DSVR (to 1.9 (0.8), p = 0.09) and an increase in coronary resistance (to 1.91 (0.6), p = 0.02). This was probably related to an improvement in the systolic coronary flow. CONCLUSIONS: This study demonstrates that successful septal ablation in patients with symptomatic HCM results in immediate improvement in CFR, which is reduced in HCM partly because of the increased systolic contraction load.

Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase.

BACKGROUND: Insulin resistance and arterial hypertension are related, but the underlying mechanism is unknown. Endothelial nitric oxide synthase (eNOS) is expressed in skeletal muscle, where it may govern metabolic processes, and in the vascular endothelium, where it regulates arterial pressure. METHODS AND RESULTS: To study the role of eNOS in the control of the metabolic action of insulin, we assessed insulin sensitivity in conscious mice with disruption of the gene encoding for eNOS. eNOS(-/-) mice were hypertensive and had fasting hyperinsulinemia, hyperlipidemia, and a 40% lower insulin-stimulated glucose uptake than control mice. Insulin resistance in eNOS(-/-) mice was related specifically to impaired NO synthesis, because in equally hypertensive 1-kidney/1-clip mice (a model of renovascular hypertension), insulin-stimulated glucose uptake was normal. CONCLUSIONS: These results indicate that eNOS is important for the control not only of arterial pressure but also of glucose and lipid homeostasis. A single gene defect, eNOS deficiency, may represent the link between metabolic and cardiovascular disease.

Alterations in Postural Control during the World's Most Challenging Mountain Ultra-Marathon

We investigated postural control (PC) effects of a mountain ultra-marathon (MUM): a 330-km trail run with 24000 m of positive and negative change in elevation. PC was assessed prior to (PRE), during (MID) and after (POST) the MUM in experienced ultra-marathon runners (n = 18; finish time = 126+/-16 h) and in a control group (n = 8) with a similar level of sleep deprivation. Subjects were instructed to stand upright on a posturographic platform over a period of 51.2 seconds using a double-leg stance under two test conditions: eyes open (EO) and eyes closed (EC). Traditional measures of postural stability (center of pressure trajectory analysis) and stabilogram-diffusion analysis (SDA) parameters were analysed. For the SDA, a significantly greater short-term effective diffusion was found at POST compared with PRE in the medio-lateral (ML; Dxs) and antero-posterior (AP) directions (Dys) in runners (p<0.05) The critical time interval (Ctx) in the ML direction was significantly higher at MID (p<0.001) and POST (p<0.05) than at PRE in runners. At MID (p<0.001) and POST (p<0.05), there was a significant difference between the two groups. The critical displacement (Cdx) in the ML was significantly higher at MID and at POST (p<0.001) compared with PRE for runners. A significant difference in Cdx was observed between groups in EO at MID (p<0.05) and POST (p<0.005) in the ML direction and in EC at POST in the ML and AP directions (p<0.05). Our findings revealed significant effects of fatigue on PC in runners, including, a significant increase in Ctx (critical time in ML plan) in EO and EC conditions. Thus, runners take longer to stabilise their body at POST than at MID. It is likely that the mountainous characteristics of MUM (unstable ground, primarily uphill/downhill running, and altitude) increase this fatigue, leading to difficulty in maintaining balance.

Impact of tight glycemic control on cerebral glucose metabolism after severe brain injury: a microdialysis study.

OBJECTIVES: To analyze the effect of tight glycemic control with the use of intensive insulin therapy on cerebral glucose metabolism in patients with severe brain injury. DESIGN: Retrospective analysis of a prospective observational cohort. SETTING: University hospital neurologic intensive care unit. PATIENTS: Twenty patients (median age 59 yrs) monitored with cerebral microdialysis as part of their clinical care. INTERVENTIONS: Intensive insulin therapy (systemic glucose target: 4.4-6.7 mmol/L [80-120 mg/dL]). MEASUREMENTS AND MAIN RESULTS: Brain tissue markers of glucose metabolism (cerebral microdialysis glucose and lactate/pyruvate ratio) and systemic glucose were collected hourly. Systemic glucose levels were categorized as within the target "tight" (4.4-6.7 mmol/L [80-120 mg/dL]) vs. "intermediate" (6.8-10.0 mmol/L [121-180 mg/dL]) range. Brain energy crisis was defined as a cerebral microdialysis glucose <0.7 mmol/L with a lactate/pyruvate ratio >40. We analyzed 2131 cerebral microdialysis samples: tight systemic glucose levels were associated with a greater prevalence of low cerebral microdialysis glucose (65% vs. 36%, p < 0.01) and brain energy crisis (25% vs.17%, p < 0.01) than intermediate levels. Using multivariable analysis, and adjusting for intracranial pressure and cerebral perfusion pressure, systemic glucose concentration (adjusted odds ratio 1.23, 95% confidence interval [CI] 1.10-1.37, for each 1 mmol/L decrease, p < 0.001) and insulin dose (adjusted odds ratio 1.10, 95% CI 1.04-1.17, for each 1 U/hr increase, p = 0.02) independently predicted brain energy crisis. Cerebral microdialysis glucose was lower in nonsurvivors than in survivors (0.46 +/- 0.23 vs. 1.04 +/- 0.56 mmol/L, p < 0.05). Brain energy crisis was associated with increased mortality at hospital discharge (adjusted odds ratio 7.36, 95% CI 1.37-39.51, p = 0.02). CONCLUSIONS: In patients with severe brain injury, tight systemic glucose control is associated with reduced cerebral extracellular glucose availability and increased prevalence of brain energy crisis, which in turn correlates with increased mortality. Intensive insulin therapy may impair cerebral glucose metabolism after severe brain injury.

Selectome update: quality control and computational improvements to a database of positive selection.

Selectome (http://selectome.unil.ch/) is a database of positive selection, based on a branch-site likelihood test. This model estimates the number of nonsynonymous substitutions (dN) and synonymous substitutions (dS) to evaluate the variation in selective pressure (dN/dS ratio) over branches and over sites. Since the original release of Selectome, we have benchmarked and implemented a thorough quality control procedure on multiple sequence alignments, aiming to provide minimum false-positive results. We have also improved the computational efficiency of the branch-site test implementation, allowing larger data sets and more frequent updates. Release 6 of Selectome includes all gene trees from Ensembl for Primates and Glires, as well as a large set of vertebrate gene trees. A total of 6810 gene trees have some evidence of positive selection. Finally, the web interface has been improved to be more responsive and to facilitate searches and browsing.

Discordant secular trends in elevated blood pressure and obesity in children and adolescents in a rapidly developing country

BACKGROUND: The effect of the increasing prevalence of obesity on blood pressure (BP) secular trends is unclear. We analyzed BP and body mass index secular trends between 1998 and 2006 in children and adolescents of the Seychelles, a rapidly developing island state in the African region. METHODS AND RESULTS: School-based surveys were conducted annually between 1998 and 2006 among all students in 4 school grades (kindergarten and 4th, 7th, and 10th years of compulsory school). We used the Centers for Disease Control and Prevention criteria to define obesity and elevated BP. The same methods and instruments were used in all surveys. Some 25 586 children and adolescents 4 to 18 years of age contributed 43 867 observations. Although the prevalence of obesity in boys and girls increased from 5.1% and 6.0%, respectively, in 1998 to 2000 to 8.0% and 8.7% in 2004 to 2006, the prevalence of elevated BP decreased from 8.4% and 9.8% to 6.9% and 7.8%. During the interval, mean age-adjusted body mass index increased by 0.57 kg/m(2) in boys and 0.58 kg/m(2) in girls. Mean age- and height-adjusted systolic BP decreased by -3.0 mm Hg in boys and -2.8 mm Hg in girls, whereas mean diastolic BP did not change substantially in boys (-0.2 mm Hg) and increased slightly in girls (0.4 mm Hg). CONCLUSIONS: At a population level, the marked increase in the prevalence of obesity in children and adolescents in the Seychelles was not associated with a commensurate secular rise in mean BP.

Genome-wide association study identifies eight loci associated with blood pressure.

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N ≤ 71,225 European ancestry, N ≤ 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10(-24)), CYP1A2 (P = 1 × 10(-23)), FGF5 (P = 1 × 10(-21)), SH2B3 (P = 3 × 10(-18)), MTHFR (P = 2 × 10(-13)), c10orf107 (P = 1 × 10(-9)), ZNF652 (P = 5 × 10(-9)) and PLCD3 (P = 1 × 10(-8)) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

Changes in cerebral compartmental compliances during mild hypocapnia in patients with traumatic brain injury.

The benefit of induced hyperventilation for intracranial pressure (ICP) control after severe traumatic brain injury (TBI) is controversial. In this study, we investigated the impact of early and sustained hyperventilation on compliances of the cerebral arteries and of the cerebrospinal (CSF) compartment during mild hyperventilation in severe TBI patients. We included 27 severe TBI patients (mean 39.5 ± 3.4 years, 6 women) in whom an increase in ventilation (20% increase in respiratory minute volume) was performed during 50 min as part of a standard clinical CO(2) reactivity test. Using a new mathematical model, cerebral arterial compliance (Ca) and CSF compartment compliance (Ci) were calculated based on the analysis of ICP, arterial blood pressure, and cerebral blood flow velocity waveforms. Hyperventilation initially induced a reduction in ICP (17.5 ± 6.6 vs. 13.9 ± 6.2 mmHg; p < 0.001), which correlated with an increase in Ci (r(2) = 0.213; p = 0.015). Concomitantly, the reduction in cerebral blood flow velocities (CBFV, 74.6 ± 27.0 vs. 62.9 ± 22.9 cm/sec; p < 0.001) marginally correlated with the reduction in Ca (r(2) = 0.209; p = 0.017). During sustained hyperventilation, ICP increased (13.9 ± 6.2 vs. 15.3 ± 6.4 mmHg; p < 0.001), which correlated with a reduction in Ci (r(2) = 0.297; p = 0.003), but no significant changes in Ca were found during that period. The early reduction in Ca persisted irrespective of the duration of hyperventilation, which may contribute to the lack of clinical benefit of hyperventilation after TBI. Further studies are needed to determine whether monitoring of arterial and CSF compartment compliances may detect and prevent an adverse ischemic event during hyperventilation.

Development of Elispot and CFSE flow cytometric assays for detecting beryllium sensitivity

Background: Beryllium sensitization (BeS) is caused by exposure to beryllium in the workplace and may progress to chronic beryllium disease (CBD). This granulomatous lung disorder mimicks sarcoidosis clinically, but is characterized by beryllium specific CD4+ T-cells immune response. BeS is classically detected by beryllium lymphocyte proliferation test (BeLPT), but this assay requires radioactivity and is not very sensitive. In the context of a study aiming to evaluate if CBD patients are misdiagnosed as sarcoidosis patients in Switzerland, we developed EliSpot and CFSE beryllium flow cytometric test. Methods: 23 patients considered as having sarcoidosis (n = 21), CBD (n = 1) and possible CBD (n = 1) were enrolled. Elispot was performed using plate covered with gamma-IFN mAb. Cells were added to wells and incubated overnight at 37 °C with medium (neg ctrl), SEB (pos ctrl) or BeSO4 at 1, 10 and 100 microM. Anti-IFN-gamma biotinylated mAb were added and spots were visualized using streptavidinhorseradish peroxidase and AEC substrate reagent. Results were reported as spot forming unit (SFU). For Beryllium specific CFSE flow cytometry analysis, CFSE labelled cells were cultured in the presence of SEB and 1, 10 or 100 microM BeSO4. Unstimulated CFSE labeled cells were defined as controls. The cells were incubated for 6 days at 37 °C and 5% CO2. Surface labelling of T-lymphocytes and vivid as control of cells viability was performed at the time of harvest. Results: Using EliSpot technology, we were able to detect a BeS in 1/23 enrolled patients with a mean of 780 SFU (cut off value at 50 SFU). This positive result was confirmed using different concentration of BeSO4. Among the 23 patients tested, 22 showed negative results with EliSpot. Using CFSE flow cytometry, 1/7 tested patients showed a positive result with a beryllium specific CD4+ count around 30% versus 45% for SEB stimulation as positif control and 0.6 % for negative control. This patient was the one with a positive EliSpot assay. Conclusions: The preliminary data demonstrated the feasibility of Elispot and CFSE flow cytometry to detect BeS. The patient with a beryllium specific positive EliSpot and CFSE flow cytometry result had been exposed to beryllium at her workplace 20 years ago and is still regularly controlled for her pulmonary status. A positive BeLPT had already been described in 2001 in France for this patient. Further validation of these techniques are in progress.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Calcium entry blockade attenuates the acute blood pressure rise induced by cigarette smoking.

The purpose of this study was to assess whether the administration of a calcium entry blocker can prevent the acute blood pressure rise induced by cigarette smoking. Seven male habitual smokers were included. After 45 min of equilibration, they took in randomized single-blind fashion at a 1 week interval either a placebo or nifedipine, 10 mg p.o. Thirty minutes thereafter, the subjects smoked within 10 min two cigarettes containing 1.4 mg of nicotine each. In addition to heart rate and skin blood flow (laser Doppler method), blood pressure of the median left finger was monitored continuously for 100 min using a noninvasive device (Finapres). Nifedipine induced an increase in skin blood flow that was not influenced by smoking. This skin blood flow response was observed although nifedipine had by itself no effect on systemic blood pressure. The calcium antagonist markedly attenuated the blood pressure rise induced by cigarette smoking. However, it tended to accentuate the heart rate acceleration resulting from inhalation of nicotine-containing smoke.