[Guidance to the Interpretation of Cardiopulmonary Exercise Testing]. / Spiroergometrie kompakt ­ Physiologie, Durchführung und Auswertung.

Cardiorespiratory fitness has been established as an independent overall predictor of morbidity and mortality. However, patients' symptoms or stated levels of exercise intolerance correlate only poorly with resting functional and imaging tests. Cardiopulmonary exercise testing (CPET) is the gold standard for the integrative assessment of the cardiocirculatory, pulmonary and metabolic response to exercise and can help identify the source of exercise limitation, monitor disease progression, evaluate treatment responsiveness and inform about prognosis. Though CPET offers more valuable and pertinent information with slightly more expenditure of time compared to other methods even at submaximal exercise levels, it remains underutilized for various reasons such as costs, reimbursement and expertise. CPET can be seen as a complex, but not necessarily difficult tool. The objective of this review was to provide a description of the underlying principles of physiology, and an easy-to-follow guidance to indications, methodology, and interpretative strategies of CPET.

Immature platelets do not reliably predict platelet recovery in patients with intensive chemotherapy or stem cell transplantation.

BACKGROUND AND OBJECTIVES: It has been reported that immature platelets may indicate imminent platelet (PLT) recovery following intensive chemotherapy and/or haematopoietic stem cell transplantation with the perspective to withhold unnecessary transfusions. The objective of our prospective study was to test immature PLTs as measured by two haematology analysers to predict PLT recovery. MATERIAL AND METHODS: This study monitored 51 courses of severe thrombocytopenia after chemotherapy in 35 adult haematology/oncology patients starting at a PLT count <70 × 109 /l until PLT recovery (>20 × 109 /l) or discharge from hospital. Immature platelets as measured by immature platelet fraction (IPF) (Sysmex XE-5000) and retPLT (Abbott CD-Sapphire) as well as reticulocyte and leucocyte count were evaluated regarding their usefulness to predict PLT recovery. Furthermore, the influence of platelet concentrate (PC) transfusions upon IPF and retPLT was assessed. RESULTS: With beginning of thrombocytopenia, most patients demonstrated elevated IPF% (median 8·6%) and retPLT% (median 6·8%). These elevated values significantly dropped after transfusions of PCs. IPF and retPLT values during nadir and at imminent PLT recovery were largely overlapping and receiver operating characteristics analysis demonstrated poor separation between both clinical situations. A reasonable cut-off could not be established to prospectively predict imminent PLT recovery for either of the parameters investigated. CONCLUSION: Measurements of IPF and retPLT lack predictive power regarding imminent PLT recovery in patients with severe thrombocytopenia following intensive chemotherapy. Decisions regarding prophylactic PC transfusions should not be based on these parameters.

Concerning emergence of a new vancomycin-resistant Enterococcus faecium strain ST1299/CT1903/vanA at a tertiary university centre in South Germany.

BACKGROUND: vanB-carrying vancomycin-resistant Enterococcus faecium (VREfm) of the sequence types 80 (ST80) and ST117 have dominated Germany in the past. In 2020, our hospital witnessed a sharp increase in the proportion of vanA-positive VREfm. AIM: To attempt to understand these dynamics through whole-genome sequencing (WGS) and analysis of nosocomial transmissions. METHODS: At our hospital, the first VREfm isolate per patient, treated during 2020, was analysed retrospectively using specific vanA/vanB PCR, WGS, multi-locus sequence typing (MLST), and core-genome (cg) MLST. Epidemiologic links between VRE-positive patients were assessed using hospital occupancy data. FINDINGS: Isolates from 319 out of 356 VREfm patients were available for WGS, of which 181 (56.7%) fulfilled the ECDC definition for nosocomial transmission. The high load of nosocomial cases is reflected in the overall high clonality rate with only three dominating sequence (ST) and complex types (CT), respectively: the new emerging strain ST1299 (100% vanA, 77.4% CT1903), and the well-known ST80 (90.0% vanB, 81.0% CT1065) and ST117 (78.0% vanB, 65.0% CT71). The ST1299 isolates overall, and the subtype CT1903 in particular, showed high isolate clonality, which demonstrates impressively high spreading potential. Overall, 152 out of 319 isolates had an allelic cgMLST difference of ≤3 to another, including 91 (59.6%) ST1299. Occupancy data identified shared rooms (3.7%), shared departments (6.2%), and VRE-colonized prior room occupants (0.6%) within 30 days before diagnosis as solid epidemiological links. CONCLUSION: A new emerging VREfm clone, ST1299/CT1903/vanA, dominated our institution in 2020 and has been an important driver of the increasing VREfm rates.

Whole-genome sequencing reveals two prolonged simultaneous outbreaks involving Pseudomonas aeruginosa high-risk strains ST111 and ST235 with resistance to quaternary ammonium compounds.

OBJECTIVE: Water-bearing systems are known as frequent Pseudomonas aeruginosa (PA) outbreak sources. However, many older buildings continue to have sanitary facilities in high-risk departments such as the ICU. We present two simultaneous prolonged multi-drug-resistant (MDR) PA outbreaks detected at the ICU of a pulmonology hospital, which were resolved by whole-genome sequencing (WGS). METHODS: Outbreak management and investigations were initiated in August 2019 after detecting two patients with nosocomial VIM-2-positive MDR PA. The investigations involved weekly patient screenings for four months and extensive environmental sampling for 15 months. All patient and environmental isolates were collected and analysed by WGS. RESULTS: From April to September 2019, we identified 10 patients with nosocomial MDR PA, including five VIM-2-positive strains. VIM-2-positive strains were also detected in nine sink drains, two toilets, and a cleaning bucket. WGS revealed that of 16 VIM-2-positive isolates, 14 were ST111 that carried qacE, or qacEΔ1 genes, whereas 13 isolates clustered (difference of ≤11 alleles by cgMLST). OXA-2 (two toilets), and OXA-2, OXA-74, PER-1 (two patients, three toilets) qacEΔ1-positive ST235 isolates dominated among VIM-2-negative isolates. The remaining seven PA strains were ST17, ST233, ST273, ST309 and ST446. Outbreak containment was achieved by replacing U-bends, and cleaning buckets, and switching from quaternary ammonium compounds (QUATs) to oxygen-releasing disinfectant products. CONCLUSION: Comprehension and management of two simultaneous MDR PA outbreaks involving the high-risk strains ST111 and ST235 were facilitated by precise control due to identification of different outbreak sources per strain, and by the in-silico detection of high-level QUATs resistance in all isolates.

Retrospective genome-oriented analysis reveals low transmission rate of multidrug-resistant Pseudomonas aeruginosa from contaminated toilets at a bone marrow transplant unit.

BACKGROUND: Prevention of toilet-to-patient transmission of multidrug-resistant Pseudomonas aeruginosa (MDR PA) poses management-related challenges at many bone marrow transplant units (BMTUs). AIM: To conduct a longitudinal retrospective analysis of the toilet-to-patient transmission rate for MDR PA under existing infection control (IC) measures at a BMTU with persistent MDR PA toilet colonization. METHODS: The local IC bundle comprised: (1) patient education regarding IC; (2) routine patient screening; (3) toilet flushing volume of 9 L; (4) bromination of toilet water tanks, and (5) toilet decontamination using hydrogen peroxide. Toilet water was sampled periodically between 2016 and 2021 (minimum every three months: 26 intervals). Upon MDR PA detection, disinfection and re-sampling were repeated until ≤3 cfu/100 mL was reached. Whole-genome sequencing (WGS) was performed retrospectively on all available MDR PA isolates (90 out of 117 positive environmental samples, 10 out of 14 patients, including nine nosocomial). FINDINGS: WGS of patient isolates identified six sequence types (STs), with ST235/CT1352/FIM-1 and ST309/CT3049/no-carbapenemase being predominant (three isolates each). Environmental sampling consistently identified MDR PA ST235 (65.5% ST235/CT1352/FIM-1), showing low genetic diversity (difference of ≤29 alleles by core-genome multi-locus sequence typing (cgMLST)). This indicates that direct toilet-to-patient transmission was infrequent although MDR PA was widespread (detection on 79 occasions, detection in every toilet). Only three MDR PA patient isolates can be attributed to the ST235/CT1352/FIM-1 toilet MRD PA population over six years. CONCLUSION: Stringent targeted toilet disinfection can reduce the potential risk for MDR PA acquisition by patients.

Oxidative stress induces unfolding protein response and inflammation in nasal polyposis.

BACKGROUND: Nasal polyposis, a chronic inflammatory disease affecting the upper airways, is a valuable and accessible model to investigate the mechanisms underlying chronic inflammation. The main objective of this study was to investigate a potential involvement of the unfolded protein response (UPR) in the context of oxidative stress and inflammation in nasal epithelial cells from nasal polyps (NP). METHODS: Epithelial cells from NP (n = 20) and normal mucosa (Controls, n = 15) in primary culture were analyzed by global proteomic approach and cell biology techniques for the glucose-regulated protein 78 (GRP78), the spliced X-box-binding protein 1 (sXBP-1), the glucose-regulated protein 94 (GRP94), and the calreticulin (immunoblot, mass spectrometry, immunocytochemistry). RESULTS: Proteomics analysis of human nasal epithelial cells in culture revealed the activation of the unfolded protein response in NP. Systematic cell biology and biochemical analysis of two markers (GRP78, sXBP-1) in the presence and absence of oxidative stress in NP showed a susceptibility of the unfolded protein response to oxidative stress compared to controls at least partially linked to an abnormal redox state of the protein disulfide-isomerase 4. This unfolded protein response was correlated with mitochondrial depolarization and secretion of interleukin 8 (IL-8) and leukotriene B4 (LTB4) and was prevented by mitochondrial antioxidant. CONCLUSIONS: We show the existence of UPR in nasal epithelial cells that is linked to oxidative stress leading to IL-8 and LTB4 secretions. These mechanisms may participate in chronic inflammation in nasal polyposis.

Long-term follow-up after biliary stent placement for postcholecystectomy bile duct strictures: a multicenter study.

BACKGROUND AND STUDY AIMS: Endoscopic stenting is a recognized treatment of postcholecystectomy biliary strictures. Large multicenter reports of its long-term efficacy are lacking. Our aim was to analyze the long-term outcomes after stenting in this patient population, based on a large experience from several centers in France. METHODS: Members of the French Society of Digestive Endoscopy were asked to identify patients treated for a common bile duct postcholecystectomy stricture. Patients with successful stenting and follow-up after removal of stent(s) were subsequently included and analyzed. Main outcome measures were long-term success of endoscopic stenting and related predictors for recurrence (after one stenting period) or failure (at the end of follow-up). RESULTS: A total of 96 patients were eligible for inclusion. The mean number of stents inserted at the same time was 1.9±0.89 (range 1-4). Stent-related morbidity was 22.9% (n=22). The median duration of stenting was 12 months (range 2-96 months). After a mean follow-up of 6.4±3.8 years (range 0-20.3 years) the overall success rate was 66.7% (n=64) after one period of stenting and 82.3% (n=79) after additional treatments. The mean time to recurrence was 19.7±36.6 months. The most significant independent predictor of both recurrence and failure was a pathological cholangiography at the time of stent removal. CONCLUSION: Endoscopic stenting helps to avoid surgery in more than 80% of patients bearing postcholecystectomy common bile duct strictures. However, a persistent anomaly on cholangiography at the time of stent removal is a strong predictor of recurrence and may lead to consideration of surgery.

Selective HSP90ß inhibition results in TNF and TRAIL mediated HIF1α degradation.

Signaling via TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Previous reports demonstrated that pro-survival signaling emanates from membrane resident TNF-R1 complexes (complex I) while only internalized TNF-R1 complexes are capable for DISC formation (complex II) and thus, apoptosis induction. Internalized TNF-R1 containing endosomes undergo intracellular maturation towards lysosomes, resulting in activation and release of Cathepsin D (CtsD) into the cytoplasm. We recently revealed HSP90 as target for proteolytic cleavage by CtsD, resulting in cell death amplification. In this study, we show that extrinsic cell death activation via TNF or TRAIL results in HSP90ß degradation. Co-incubation of cells with either TNF or TRAIL in combination with the HSP90ß inhibitor KUNB105 but not HSP90α selective inhibition promotes apoptosis induction. In an attempt to reveal further downstream targets of combined TNF-R1 or TRAIL-R1/-R2 activation with HSP90ß inhibition, we identify HIF1α and validate its ligand:inhibitor triggered degradation. Together, these findings suggest that selective inhibition of HSP90 isoforms together with death ligand stimulation may provide novel strategies for therapy of inflammatory diseases or cancer, in future.

Development and optimization of a carbon dioxide-aided cold microfiltration process for the physical removal of microorganisms and somatic cells from skim milk.

Physical removal of microorganisms from skim milk by microfiltration (MF) is becoming increasingly attractive to the dairy industry. Typically, this process is performed at temperatures of approximately 50 degrees C. Additional shelf-life and quality benefits might be gained by conducting the MF process at low temperatures. Cold MF could also minimize microbial fouling of the membrane and prevent the germination of thermophilic spores. The objective of this study was to optimize a cold MF process for the effective removal of microbial and somatic cells from skim milk. An experimental MF setup containing a tubular Tami ceramic membrane with a nominal pore size of 1.4 microm was used for MF of raw skim milk at a temperature of 6 +/- 1 degrees C. The processing conditions used were cross-flow velocities of 5 to 7 m/s, and transmembrane pressures of 52 to 131 kPa. All MF experiments were performed in triplicate. The permeate flux was determined gravimetrically. Microbiological, chemical, and somatic cell analyses were performed to evaluate the effect of MF on the composition of skim milk. The permeate flux increased drastically when velocity was increased from 5 to 7 m/s. The critical transmembrane pressure range conducive to maximum fluxes was 60 to 85 kPa. When MF was conducted under optimal conditions, very efficient removal of vegetative bacteria, spores, and somatic cells, as well as near complete transmission of proteins into the MF milk, was achieved. To further enhance the flux, a CO(2) backpulsing system was developed. This technique is able both to increase the flux and to maintain it steadily for an extended period of time. The CO(2)-aided cold MF process has the potential to become economically attractive to the dairy industry, with direct benefits for the quality and shelf life of dairy products.

[Recommendations for the management of bone demineralization in cystic fibrosis]. / Recommandations pour la prise en charge de la déminéralisation osseuse dans la mucoviscidose.

A high prevalence of low bone mineralization is documented in adult patients with cystic fibrosis (CF). Osteopenia is present in as much as 85% of adult patients and osteoporosis in 13 to 57% of them. In children, studies are discordant probably because of different control database. Denutrition, inflammation, vitamin D and vitamin K deficiency, altered sex hormone production, glucocorticoid therapy, and physical inactivity are well known risk factors for poor bone health. Puberty is a critical period and requires a careful follow-up for an optimal bone peak mass. This review is a consensus statement established by the national working group of the French Federation of CF Centers to develop practice guidelines for optimizing bone health in patients with CF. Recommendations for screening and for calcium, vitamin D and K supplementation are given. Further work is needed to define indications for treatment with biphosphonates and anabolic agents.

Baroreflex control of plasma norepinephrine and heart period in healthy subjects and diabetic patients.

Resting diabetic patients may have excessively rapid heart rates, reduced heart rate variability, and subnormal plasma catecholamine levels. Although all of these abnormalities may relate in some way to baroreceptor reflex function, there have been surprisingly few attempts to evaluate systematically baroreflex mechanisms in diabetic patients. Accordingly, we studied autonomic responses over a range of pharmacologically induced arterial pressure changes in 10 unselected young adult insulin-dependent diabetic patients who had no symptoms of autonomic neuropathy, and 12 age-matched nondiabetic subjects. Sympathetic responses were estimated from antecubital vein plasma norepinephrine levels, and parasympathetic responses were estimated from electrocardiographic R-R intervals and their variability (standard deviation). Both were correlated with other noninvasive indexes of peripheral and central nervous system function. Multiple derangements of baroreflex function were found in the diabetic patients studied. Sympathetic abnormalities included subnormal baseline norepinephrine levels, virtual absence of changes of norepinephrine levels during changes of arterial pressure, and supranormal pressor responses to phenylephrine infusions. Parasympathetic abnormalities included subnormal baseline standard deviations of R-R intervals, and R-R interval prolongations during elevations of arterial pressure which were unmistakably present, but subnormal. Our data suggest that in diabetic patients, subnormal baseline plasma norepinephrine levels may signify profound, possibly structural defects of sympathetic pathways. Subnormal resting levels of respiratory sinus arrhythmia may have different implications, however, since vagal, unlike sympathetic reflex abnormalities, can be reversed partly by arterial pressure elevations.

Autonomic pathophysiology in heart failure patients. Sympathetic-cholinergic interrelations.

We conducted this study in an effort to characterize and understand vagal abnormalities in heart failure patients whose sympathetic activity is known. We measured sympathetic (peroneal nerve muscle sympathetic recordings and antecubital vein plasma norepinephrine levels) and vagal (R-R intervals and their standard deviations) activities in eight heart failure patients and eight age-matched healthy volunteers, before and after parasympathomimetic and parasympatholytic intravenous doses of atropine sulfate. At rest, sympathetic and parasympathetic outflows were related reciprocally: heart failure patients had high sympathetic and low parasympathetic outflows, and healthy subjects had low sympathetic and high parasympathetic outflows. Low dose atropine, which is known to increase the activity of central vagal-cardiac motoneurons, significantly increased R-R intervals in healthy subjects, but did not alter R-R intervals in heart failure patients. Thus, our data document reciprocal supranormal sympathetic and subnormal parasympathetic outflows in heart failure patients and suggest that these abnormalities result in part from abnormalities within the central nervous system.

Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells.

UNLABELLED: We have previously shown (i) that the cystic fibrosis transmembrane regulator (CFTR) locates to lipid raft-like microdomains of epithelial cells upon TNF-α proinflammatory stimulation; and (ii) that TNF-α increases the membrane localization and the channel function of F508del-mutated CFTR. In the present work, we hypothesized that CFTR mutations modify the proteome of lipid rafts in the same proinflammatory conditions. We prepared lipid rafts from HeLa cells transfected with either wild-type or F508del-CFTR and incubated for 10min with 100U/mL of TNF-α. Proteins were extracted, trypsin digested, and peptides analyzed by high resolution MS. Proteins were quantified by a stable isotope labeling with amino acids in cell culture approach. Out of the 22 proteins differentially recruited in lipid rafts after proinflammatory exposure, 17 were increased in F508del cells with respect to wild-type, including two G-protein coupled receptors, three anion transporters, and one cell surface mucin. In both HeLa and bronchial epithelial cells we confirmed that G-protein coupled receptor 5A relocates to lipid rafts along with F508del-CFTR after TNF-α treatment. These results could enlighten the cross-talk between CFTR and TNF-α and its impact on the cell response to proinflammatory challenge. BIOLOGICAL SIGNIFICANCE: CFTR mutations are at the origin of cystic fibrosis. The latter disease is characterized, among other symptoms, by a defective management of infection and inflammation in the airways. Short exposure to the proinflammatory cytokine TNF-α targets mutated CFTR to the plasma membrane and increases its chloride channel activity. The results hereby presented show a substantial modification of the lipid raft proteome in the same conditions, and may enlighten the effect of this cytokine and the role of CFTR in the cell response to inflammation.

Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation.

Cystic fibrosis (CF) is a genetic disease due to mutations in the cystic fibrosis transmembrane regulator (CFTR), F508del-CFTR being the most frequent. Lipid raft-like microdomains (LRM) are regions of the plasma membrane that present a high cholesterol content and are insoluble to non-ionic detergents. LRM are essential functional and structural platforms that play an important role in the inflammatory response. CFTR is a known modulator of inflammation in LRM. Here we provide mass spectrometry data on the global impact of CFTR mutation and TNF-a stimulation on the LRM proteome. We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article "Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells" (Chhuon et al., in press [1]).

Potassium currents and effects of vitamin D-3 metabolites and cyclic GMP in rat osteoblastic cells.

A K+ current (IK1), activated by depolarization above -20 mV, showing voltage-dependent inactivation within a few seconds and reduced by 40% by 1 mM TEA, was observed in all cells. In a few cells, we also observed a progressive K(+)-current increase during cell dialysis. The developing current (IK2) was not sensitive to 1 mM TEA and did not inactivate. It was detectable over the whole voltage range and slowly increased during 10 s depolarizations. 1,25-(OH)2D3 and 24,25-(OH)2D3 did not affect IK1, but induced a small K(+)-current increase in some cells showing no IK2. This effect was not mimicked by cyclic GMP analogs which, on the contrary, induced a K(+)-current decrease. 24,25-(OH)2D3 (even at 10(-11)M, but not 1,25-(OH)2D3, strongly reduced IK2. The results further document the diversity of voltage-gated currents of osteoblastic cells, confirm the existence of immediate effects of vitamin D-3 metabolites, which are independent of classical 1,25-(OH)2D3 receptors.

Dual modulation of the L-type calcium current of rat osteoblastic cells by parathyroid hormone: opposite effects of protein kinase C and cyclic nucleotides.

Using whole-cell voltage-clamp recording of rat osteoblastic cells, we show that PTH-(1-34), known to stimulate protein kinase C (PKC) and adenylate cyclase, has a dual effect on the L-type calcium current. It induces a long-lasting increase and a superimposed reversible decrease, which can be separated by repeating hormone applications. The stimulatory effect is the only effect induced by the (3-34) fragment, able to stimulate PKC but unable to stimulate adenylate cyclase. The L current is stimulated by an active phorbol ester and is reduced by permeable analogues of cyclic AMP. Thus, the effect of PTH-(1-34) can be explained by the opposite effects of PKC and cyclic AMP. Dibutyryl cyclic GMP reduces the L current even more potently than dibutyryl cyclic AMP. The above modulations are all voltage-insensitive. These results led us to reinvestigate the effects of some vitamin D3 metabolites known to stimulate PKC and/or guanylate cyclase, and previously reported to affect the voltage-sensitivity of the L current. We only detected voltage-insensitive effects.

Diagnosis and prognosis of AIDS-related cholangitis.

OBJECTIVE: To determine more precisely the clinical and biological characteristics of AIDS-related cholangitis, and to investigate prognostic variables of this disease. DESIGN: Retrospective clinical and prognostic study. SETTING: Biliary unit, Bicêtre Hospital, France. PATIENTS: HIV-positive patients (n = 52) referred to the unit between December 1986 and June 1993 for biliary symptoms leading to the suspicion of AIDS-related cholangitis, (42 men; 10 women; mean age, 37 +/- 8 years). INTERVENTION: Endoscopic retrograde cholangiopancreatography (ERCP) was performed in order to determine the cause of the biliary symptoms. MAIN OUTCOME MEASURE: Clinical features and evolution of the cholangitis. RESULTS: Among the 52 patients, 45 met the ERCP criteria of AIDS-related cholangitis (36 men; nine women). The diagnosis of cholangitis was strongly suggested by abdominal ultrasonography in 47% of the cases. ERCP showed papillary stenosis, diffuse cholangitis, extrahepatic cholangitis alone, and intrahepatic cholangitis alone in 60, 67, 7 and 27%, respectively. Endoscopic sphincterotomy was performed in 28 patients. Pain was relieved by sphincterotomy in nine patients, but the other clinical or biological features were not influenced. One-year and 2-year survival rates were 41 +/- 7% and 8 +/- 4%, respectively. Multidimensional analysis using a Cox model showed that a lymphocyte count > 500 x 10(6)/l was the only independent predictive factor of better survival. CONCLUSION: AIDS-related cholangitis is a disease which leads preferentially to papillary stenosis or diffuse abnormalities of the biliary tract. Prognostic factors depend on the stage of the HIV infection. Another diagnosis of cholestasis was found in approximately 15% of the patients who showed biliary symptoms.