Venous thromboembolism in Cushing syndrome: results from an EuRRECa and Endo-ERN survey.

Background: Patients with Cushing syndrome (CS) are at increased risk of venous thromboembolism (VTE). Objective: The aim was to evaluate the current management of new cases of CS with a focus on VTE and thromboprophylaxis. Design and methods: A survey was conducted within those that report in the electronic reporting tool (e-REC) of the European Registries for Rare Endocrine Conditions (EuRRECa) and the involved main thematic groups (MTG's) of the European Reference Networks for Rare Endocrine Disorders (Endo-ERN) on new patients with CS from January 2021 to July 2022. Results: Of 222 patients (mean age 44 years, 165 females), 141 patients had Cushing disease (64%), 69 adrenal CS (31%), and 12 patients with ectopic CS (5.4%). The mean follow-up period post-CS diagnosis was 15 months (range 3-30). Cortisol-lowering medications were initiated in 38% of patients. One hundred fifty-four patients (69%) received thromboprophylaxis (including patients on chronic anticoagulant treatment), of which low-molecular-weight heparins were used in 96% of cases. VTE was reported in six patients (2.7%), of which one was fatal: two long before CS diagnosis, two between diagnosis and surgery, and two postoperatively. Three patients were using thromboprophylaxis at time of the VTE diagnosis. The incidence rate of VTE in patients after Cushing syndrome diagnosis in our study cohort was 14.6 (95% CI 5.5; 38.6) per 1000 person-years. Conclusion: Thirty percent of patients with CS did not receive preoperative thromboprophylaxis during their active disease stage, and half of the VTE cases even occurred during this stage despite thromboprophylaxis. Prospective trials to establish the optimal thromboprophylaxis strategy in CS patients are highly needed. Significance statement: The incidence rate of venous thromboembolism in our study cohort was 14.6 (95% CI 5.5; 38.6) per 1000 person-years. Notably, this survey showed that there is great heterogeneity regarding time of initiation and duration of thromboprophylaxis in expert centers throughout Europe.

Rationale and design of the multicentric, double-blind, double-placebo, randomized trial APrepitant versus HYdroxyzine in association with cytoreductive treatments for patients with myeloproliferative neoplasia suffering from Persistent Aquagenic Pruritus. Trial acronym: APHYPAP.

BACKGROUND: Aquagenic pruritus (AP), an intense sensation of scratching induced after water contact, is the most troublesome aspect of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). Mostly described in polycythemia vera (PV, ~ 40%), it is also present in essential thrombocythemia (ET) and primary myelofibrosis (PMF) (10%). Even if this symptom can decrease or disappear under cytoreductive treatments, 30% of treated MPN patients still persist with a real impact on the quality of life (QoL). Because its pathophysiology is poorly understood, efficient symptomatic treatments of AP are missing. The neuropeptide substance P (SP) plays a crucial role in the induction of pruritus. Several studies showed the efficacy of aprepitant, an antagonist of SP receptor (NK-1R), in the treatment of chronic pruritus but never evaluated in AP. The objectives of APHYPAP are twofold: a clinical aim with the evaluation of the efficacy of two drugs in the treatment of a persistent AP for MPN patients and a biological aim to find clues to elucidate AP pathophysiology. METHODS/DESIGN: A multicentric, double-blind, double-placebo, randomized study will include 80 patients with MPN (PV or ET or PMF) treated since at least 6 months for their hemopathy but suffering from a persistent AP (VAS intensity ≥6/10). Patients will be randomized between aprepitant (80 mg daily) + placebo to match to hydroxyzine OR hydroxyzine (25 mg daily) + placebo to match to aprepitant for 14 days. At D0, baseline information will be collected and drugs dispense. Outcome measures will be assessed at D15, D30, D45, and D60. The primary study endpoint will be the reduction of pruritus intensity below (or equal) at 3/10 on VAS at D15. Secondary outcome measures will include the number of patients with a reduction or cessation of AP at D15 or D60; evaluation of QoL and AP characteristics at D0, D15, D30, D45, and D60 with MPN-SAF and AP questionnaires, respectively; modification of plasmatic concentrations of cytokines and neuropeptides at D0, D15, D30, and D60; and modification of epidermal innervation density and pruriceptor expression at D0 and D15. DISCUSSION: The APHYPAP trial will examine the efficacy of aprepitant vs hydroxyzine (reference treatment for AP) to treat persistent AP in MPN patients. The primary objective is to demonstrate the superiority of aprepitant vs hydroxyzine to treat persistent AP of MPN patients. The treatment received will be considered efficient if the AP intensity will be reduced at 3/10 or below on VAS after 14 days of treatment. The results of this study may provide a new treatment option for this troublesome symptom and also give us more insights in the pathophysiology understanding of AP. TRIAL REGISTRATION: APHYPAP. NCT03808805 , first posted: January 18, 2019; last update posted: June 10, 2021. EudraCT 2018-090426-66.

Haematological parameters associated with postpartum haemorrhage after vaginal delivery: Results from a French cohort study.

BACKGROUND: Immediate postpartum haemorrhage (PPH) is a major, feared and often unpredictable issue. Besides many clinical risk factors, some biological parameters could also be predictive of PPH. OBJECTIVE: To study simple and easily accessible haematological parameters as potential risk factors for PPH after vaginal delivery. METHODS: All women who had a vaginal delivery between April 1, 2013 and May 29, 2015 in the maternity ward of Brest University Hospital (France) were included, after oral informed consent obtained. Clinical data were collected by obstetricians or midwives during antenatal care visits, labour and delivery, and recorded by trained research assistants. Haematological variables, including immature platelet fraction, were measured from a blood sample systematically collected at the entrance in the delivery room. PPH, measured with a graduated collector bag, was defined as blood loss of at least 500 ml. RESULTS: 2742 women were included. PPH occurred in 141 (5%) women. Seven clinical factors were independently associated with PPH: pre-eclampsia (OR 5.85, 95%CI 2.02, 16.90), multiple pregnancy (OR 3.28, 95%CI 1.21, 8.91), assisted reproduction (OR 2.75, 95%CI 1.45, 5.20), antepartum bleeding (OR 2.15, 95%CI 1.24,3.73), post-term delivery (OR 1.93, 95%CI 1.17, 3.17), obesity (OR 2.95, 95%CI 1.76, 4.93) and episiotomy (OR 2.51, 95%CI 1.63, 3.74). Three haematological factors were additionally identified as independent risk factors for PPH: platelets < 150 Giga/L (OR 2.98, 95%CI 1.63, 5.46), fibrinogen < 4.5 g/l (OR 1.86, 95%CI 1.21, 2.87) and APTT ratio ≥ 1.1 (OR 2.16, 95%CI 1.31, 3.57). Immature platelet fraction was not associated with PPH. CONCLUSION: Besides classical clinical risk factors, this study identifies simple haematological parameters as risk factors for PPH.

Weight of salivary gland ultrasonography compared to other items of the 2016 ACR/EULAR classification criteria for Primary Sjögren's syndrome.

OBJECTIVE: Major salivary gland ultrasonography (SGUS) is widely used for the diagnosis of primary Sjögren's syndrome (pSS). Our objective was to assess the contribution of SGUS compared to other items of the 2016 ACR/EULAR pSS classification criteria, based on expert opinion. METHODS: A secure web-based relational database was used by 24 experts from 14 countries to assess 512 realistic vignettes developed from data of patients with suspected pSS. Each vignette provided classification criteria items and information on history, clinical symptoms and SGUS findings. Each expert assessed 64 vignettes, and each vignette was assessed by 3 experts. A diagnosis of pSS was defined according to at least 2 of 3 experts. Validation was performed in the independent French DiapSS cohort of patients with suspected pSS. RESULTS: A criteria-based pSS diagnosis and SGUS findings were independently associated with an expert diagnosis of pSS (P < 0.001). The derived diagnostic weights of individual items in the 2016 ACR/EULAR criteria including SGUS were as follows: anti-SSA, 3; focus score ≥ 1, 3; SGUS score ≥ 2, 1; positive Schirmer's test, 1; dry mouth, 1; and salivary flow rate < 0.1 mL/min, 1. The corrected C statistic area under the curve for the new weighted score was 0.96. Adding SGUS improves the sensitivity from 90.2 % to 95.6% with a quite similar specificity 84.1% versus 82.6%. Results were similar in the DiapSS cohort: adding SGUS improves the sensitivity from 87% to 93%. CONCLUSION: SGUS had similar weight compared to minor items, and its addition improves the performance of the 2016 ACR/EULAR classification criteria.

Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.

OBJECTIVES: Early detection of Pseudomonas aeruginosa lung positivity is a key element in cystic fibrosis (CF) management. PCR has increased the accuracy of detection of many microorganisms. Clinical relevance of P. aeruginosa quantitative PCR (qPCR) in this context is unclear. Our aim was to determine P. aeruginosa qPCR sensitivity and specificity, and to assess the possible time saved by qPCR in comparison with standard practice (culture). METHODS: A multicentre cohort study was conducted over a 3-year period in 96 patients with CF without chronic P. aeruginosa colonization. Sputum samples were collected at each visit. Conventional culture and two-step qPCR (oprL qPCR and gyrB/ecfX qPCR) were performed for 707 samples. The positivity criteria were based on the qPCR results, defined in a previous study as follow: oprL qPCR positivity alone if bacterial density was <730 CFU/mL or oprL qPCR combined with gyrB/ecfX qPCR if bacterial density was ≥730 CFU/mL. RESULTS: During follow up, 36 of the 96 patients with CF were diagnosed on culture as colonized with P. aeruginosa. This two-step qPCR displayed a sensitivity of 94.3% (95% CI 79.7%-98.6%), and a specificity of 86.3% (95% CI 83.4%-88.7%). It enabled P. aeruginosa acquisition to be diagnosed earlier in 20 patients, providing a median detection time gain of 8 months (interquartile range 3.7-17.6) for them. CONCLUSIONS: Implementing oprL and gyrB/ecfX qPCR in the management of patients with CF allowed earlier detection of first P. aeruginosa lung positivity than culture alone.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism.

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.

Extrahepatic biliary tract in chinchilla (Chinchilla laniger, Molina).

The aim of the study was the macromorphological analysis of extrahepatic biliary tract in chinchilla (Chinchilla laniger Molina). Bile ducts, the gall bladder and portal vein were injected with coloured latex. Using the technique of dissection, bile ducts were isolated from the liver lobes. It was found that the cystic duct in this species is rarely single. Hepatic ducts form a system of multiple anastomosing structures running in the hepatoduodenal ligament. Many bile duct openings were observed in the duodenal papilla. The results confirm wide variations of the biliary tract in mammals and may be important for comparative analysis of the morphological differentiation of these structures in small mammals.

Fibroblast growth factor 23 and Klotho as cardiovascular risk factors in heart transplant recipients.

BACKGROUND: Fibroblast growth factor (FGF) 23 is one of the most recently discovered FGFs. This phosphaturic hormone produced in bones is a risk factor for cardiovascular diseases and thus mortality. Klotho is an essential coreceptor for FGF23 and at the same time it is known as a "longevity" hormone. There are no data considering FGF23 and Klotho roles in heart transplant (HT) recipients. The aim of this study was to assess Klotho and FGF23 serum concentration in heart transplant recipients depending on immunosuppressive therapy regimen and comorbidities. METHODS: Eighty-four stable heart transplant recipients were enrolled in the study; 22 healthy volunteers served as control subjects. FGF23 and Klotho protein concentration, markers of renal function, such as cystatin C and neutrophil gelatinase-associated lipocalin (NGAL), and heart failure markers, such as copeptine and N-termiinal pro-B-type natriuretic peptide (NT-proBNP), were evaluated. RESULTS: FGF23 concentration was significantly higher in the HT group whereas Klotho protein was significantly lower. FGF23 correlated with creatinine level (r = 0.72; P < .001), estimated glomerular filtration rate (eGFR; r = -0.32; P < .01), cystatin C (r = 0.36; P < .01), NGAL (r = 0.51; P < .001), hemoglobin (r = -0.39; P < .001), NT-proBNP (r = 0.51; P < .001), high-density lipoprotein (HDL; r = 0.27; P < .05), intraventricular septum thickness (r = 0.42; P < .01) and right ventricular systolic pressure (r = 0.34; P < .05). Klotho protein correlated only with age (r = -0.21; P < .05), creatinine (r = -0.21; P < .05), and eGFR (r = -0.31; P < .01). FGF23 concentration was significantly higher in patients with eGFR <60 mL/min whereas Klotho protein was significantly lower. FGF23 predictors were renal function (creatinine concentration; ß = 0.45; P = .0001), HDL (ß = 0.33; P = .003), intraventricular septum thickness (ß = 0.38; P = .0003), and right ventricular systolic pressure (ß = 0.34; P = .003), explaining 70% of FGF23 variability. CONCLUSIONS: FGF23/Klotho system disorders in HT recipients are related to cardiovascular system function and kidney failure and could cause increased risk of cardiovascular disease.

Zonulin and iron metabolism in heart transplant recipients.

BACKGROUND: In patients after heart transplantation, anemia is relatively common and is associated with impaired kidney function, subclinical inflammatory state, and immunosuppressive treatment. Zonulin-prehaptoglibin-2 is newly discovered protein with poorly defined function. Hemoglobin binds haptoglobin, and this stable complex prevents oxidative stress caused by hemoglobin. Zonulin is necessary for integrity of intracellular tight junction in the gut. Taking into consideration iron metabolism, including its absorption in the gut, the aim of this study was to assess zonulin levels in heart transplant recipients and their possible correlations with iron status, immunosuppressive therapy, and kidney function. METHODS: The study was performed with 80 stable heart transplant recipients and 22 healthy volunteers. Zonulin, iron status, and inflammatory markers were assessed with the use of commercially available kits. RESULTS: Zonulin correlated with intraventricular diameter (r = 0.30; P < .05), right ventricle systemic pressure (r = 0.27; P < .05), and hemoglobin (r = 0.21; P < .05). There were no correlations between zonulin and iron status. Zonulin was significantly lower in heart transplant recipients than in healthy volunteers (P < .001). Kidney function, immunosuppressive regimen, New York Heart Association functional class, sex, and presence of anemia did not affect zonulin level. CONCLUSIONS: Zonulin, despite its effect on the absorption of different nutrients and other substances and hypothethic role in oxidative stress, seems not to play a role in the pathogenesis of anemia in heart transplant recipients. Its physiologic role remains obscure.

YKL-40, a novel marker of cardiovascular complications, is related to kidney function in heart transplant recipients.

BACKGROUND: YKL-40 is an inflammatory glycoprotein involved in endothelial dysfunction and expressed in macrophages in the earliest lesions of atherosclerosis. Elevated serum YKL-40 levels are independently associated with the presence and extent of coronary artery disease and cardiovascular mortality. Because there are no data on heart transplant recipients and because they are prone to cardiovascular complications, the aim of this study was to assess YKL-40 in this population with particular attention to its relationship with endothelial damage. We studied 84 patients after heart transplantation. Healthy volunteers served as control subjects. METHODS: Complete blood count, urea, creatinine, lipids, fasting glucose, N-terminal pro-B-type natriuretic peptide (NT-proBNP), and iron status were studied with the use of standard laboratory methods. We assessed YKL-40, copeptin, markers of inflammation high sensitivity C-reactive protein (hsCRP) and interleukin (IL) 6, and markers of endothelial cell injury von Willebrand factor (vWF) and midkine with the use of commercially available assays. RESULTS: Mean levels of YKL-40, IL-6, vWF, and hsCRP were significantly higher in heart allograft recipients than in the control group (P < .001). In univariate analysis, YKL-40 was related to kidney function (creatinine, r = 0.63 [P < .001]; estimated glomerular filtration rate, r = -0.44 [P < .001]), NT-proBNP (r = 0.45; P < .001), age (r = 0.33; P < .01), time after transplantation (r = 0.23; P < .05), copeptin (r = -0.42; P < .001), soluble transferrin receptor (r = 0.24; P < .05), hemoglobin (r = -0.42; P < .001), transferrin (r = -0.31; P < .01), haptoglobin (r = 0.39; P < .001), cystatin C (r = 0.55; P < .001), ejection fraction (r = -0.28; P < .05), New York Heart Association functional class (r = -0.41; P < .01), hsCRP (r = 0.26; P < .05), IL-6 (r = 0.23; P < .05), vWF (r = -0.40; P < .001), and midkine (r = 0.33; P < .01). In multivariate analysis, only creatinine was found to be a predictor of YKL-40 (ß = 0.59; P = .02), explaining 56% of the variation in YKL-40 levels in heart allograft recipients. CONCLUSIONS: YKL-40 may contribute to the enhanced risk of cardiovascular complications mainly owing to impaired renal function in patients after heart transplantation.

Occurrence of waterborne pathogens and Escherichia coli at offshore drinking water intakes in lake Ontario.

The occurrence of waterborne pathogens was investigated at three drinking water intakes located about 2 km offshore in Lake Ontario. Water sampling was conducted over 3 years for Campylobacter spp., Cryptosporidium spp., Giardia spp., cultivable enteric viruses, and water quality parameters. All pathogens were detected in the offshore source water for each water treatment plant (WTP1 to WTP3), although at relatively low frequencies and concentrations. Giardia was the most common pathogen, occurring in 36% of water samples from the influent of WTP1 (n = 46), and with a maximum concentration of 0.70 cysts/liter in this influent. Cryptosporidium occurred as frequently as 15% in the WTP2 influent (n = 35), with a maximum concentration of 0.40 oocysts/liter in the WTP1 influent. The human Bacteroidales HF183 DNA marker was most common in the WTP1 influent (19%), and this was the only WTP where the Cryptosporidium hominis genotype was detected. No water quality parameter was predictive of pathogen occurrence across all three WTP influents. Escherichia coli was often below detection when pathogens were detected, and spikes in E. coli concentrations often did not coincide with pathogen occurrence. After summer rain events, river plumes had E. coli concentrations as high as 222 CFU/100 ml in surface waters 2 km offshore, without impacting drinking water intakes below the thermocline on the lake bottom. At times, prechlorination to control mussels at offshore intake cribs compromised the use of E. coli for "raw" water quality assessment, particularly for chlorine-resistant Cryptosporidium. E. coli measured by standard methods did not reliably predict pathogen occurrence at drinking water intakes in offshore ecosystems.

[Evaluation of preoperative non-invasive ventilation in thoracic surgery for lung cancer: the preOVNI study GFPC 12-01]. / Évaluation de la ventilation non invasive préopératoire avant chirurgie de résection pulmonaire. Étude préOVNI GFPC 12-01.

BACKGROUND: Surgical resection is the best treatment for stage I and II non-small cell lung cancer. Despite an improvement in the perioperative management of cancer patients and specialization of surgical teams, morbidity and mortality remains significant. Non-invasive ventilation (NIV) is an effective therapeutic option in hypercapnic respiratory failure. It also improves functional and gasometric parameters when undertaken before surgery. The objective of the preOVNI study is to demonstrate that preoperative non-invasive ventilation for 7 days, at home, reduces the postoperative respiratory and cardiovascular complications of lung resection surgery, in a high-risk population. METHODS: A prospective, randomized, controlled open-labelled multicentric French study, under the supervision of the Groupe Français de Pneumocancérologie (GFPC), comparing 7 days of preoperative non-invasive ventilation with standard treatment. Inclusion criteria are: patients suitable for lobectomy or segmentectomy for primary bronchial carcinoma and presenting with obstructive or restrictive lung disease, obesity or chronic cardiac insufficiency. The primary criterion is a composite one, including all respiratory and cardiac complications. The number of patients is 150 in each treatment arm, 300 in total. EXPECTED RESULTS: We think that preoperative NIV will be able to reduce the rate of postoperative complications. If this objective is achieved, the management of these patients could be changed.

[Comparison between ETCO2 values measured by the Smart Capnoline™ and the PACO2 in intubated then extubated postoperative cardiac surgery patients]. / Comparaison de la mesure de la PETCO(2) obtenue avec le Smart Capnoline™ avec la PaCO(2) en postopératoire de chirurgie cardiaque chez des patients intubés puis extubés.

OBJECTIVE: To compare the PaCO(2) with the ETCO(2) obtained with the Smart Capnoline™ in the postoperative setting of cardiac surgery during ventilation and after extubation TYPE OF STUDY: Prospective, observational. PATIENTS: Twenty patients after cardiac surgery. METHODS: In the intensive care unit, arterial blood gases were measured concomitantly with ETCO(2), and difference between PaCO(2) and ETCO(2) were calculated. Three CO(2) sensors were utilized: Filterline H set for intubated patients, Smart Capnoline HO(2) (nasal version) and Smart Capnoline O(2) (bucconasal version) after extubation. Data were compared with Wilconson test and the intraclass correlation coefficient was calculated. RESULTS: The difference PaCO(2) - ETCO(2) was significantly larger in extubated patients compared to intubated patients, which is also confirmed for the bucconasal sensor (intubated patients: 6.6 ± 4.3 mmHg, nasal sensor: 9.3 ± 3.5 mmHg, bucconasal sensor: 15,4 ± 12.9 mmHg). CONCLUSION: In the postoperative setting of cardiac surgery, ETCO(2) measurements allow a reliable estimation of PaCO(2) in intubated patients in contrast to measurements in extubated patients. The bucconasal CO(2) sensor does not show more reliable measurements compared to nasal sensors in the postoperative setting of cardiac surgery.

Association of HLA haplotypes with paroxysmal nocturnal hemoglobinuria.

The pathologies of paroxysmal nocturnal hemoglobinuria (PNH) are primarily caused by somatic mutation in the PIG-A gene in hematopoietic stem cells resulting in glycosyl phosphatidylinositol deficiency and accumulation of phosphatidylinositol (PI) in plasma membranes. The mechanism of pathologic clone domination over normal hematopoietic clones in PNH patients is not yet understood. Forty-four PNH patients, including 9 with aplastic anemia traits (AA/PNH), 31 without full aplasia in bone marrow (de novo PNH, or dn/PNH), and 4 with unclassified PNH, and 200 ethnically matched controls were tested for the HLA A, B, C, DRB1, and DQB1 alleles and haplotype associations. The top block association analysis showed the primary association of PNH with 3 haplotype fragments: the class I fragment A*2501-Cw*1203-B*1801 (risk ratio [RR], 6.64; P=.00012), and 2 class II fragments: DRB1*1501-DQB1*0602 (RR, 7.09; P=.0000015) and DRB1*0401-DQB1*0301 (RR, 6.76, P=.0093). The stratified analysis revealed that the A*2501-Cw*1203-B*1801 haplotype associated preferentially with AA/PNH, and its component HLA molecule showed immunodominant antiapoptotic peptides derived from PI-activated phospholipase D; whereas the DRB1*1501-DQB1*0602 haplotype was associated strongly with dn/PNH and presented immunodominant class II-derived autopeptides. We concluded that certain HLA haplotypes were associated with PNH much more strongly than their allelic components. At least 3 HLA haplotype blocks (A*2501-Cw*1203-B*1801, DRB1*1501-DQB1*0602, and DRB1*0401-DQB1*0301) were primarily associated with PNH. Our results supported the hypothesis of the roles in AA/PNH of antiapoptotic and in dn/PNH of autoimmune mechanisms.

Distribution of double-strand breaks induced by ionizing radiation at the level of single DNA molecules examined by atomic force microscopy.

Double-strand breaks (DSBs) are the most critical radiation-induced lesions, because they result in the fragmentation of the DNA molecule and because a single unrepaired DSB may lead to cell death. We present the results of radiation-induced fragmentation of plasmid DNA analyzed by atomic force microscopy (AFM) to allow the visualization of individual DNA molecules. Linear PhiX174 plasmid DNA was exposed to a wide range of doses of low-LET X rays and high-LET carbon, nickel and uranium ions. The induced DNA fragments were detected and measured based on the recorded AFM images and fragment length distributions were derived for each radiation type and dose. The results show a dose- and radiation type-dependent DNA fragmentation with a significantly larger fraction of short fragments produced by high-LET radiation compared to X rays. This can be considered as experimental evidence of DSB clustering due to inhomogeneous energy deposition at the level of the plasmid DNA molecule. Additionally, the experimentally derived fragment profiles were compared and found to be in agreement with the prediction of a model simulating the fragmentation of DNA molecules induced by radiation.

Cell cycle arrest and aberration yield in normal human fibroblasts. II: Effects of 11 MeV u-1 C ions and 9.9 MeV u-1 Ni ions.

PURPOSE: To investigate further the relationship between high linear energy transfer (LET) induced cell cycle arrests and the yield of chromosome aberrations observable in normal human fibroblasts at the first post-irradiation mitosis. MATERIALS AND METHODS: Normal human fibroblasts (AG01,522C) were exposed in G0/G1 to either 11 MeV u(-1) C ions (LET = 153.5 keV microm(-1)) or 9.9 MeV u(-1) Ni ions (LET = 2,455 keV microm(-1)), subcultured in medium containing 5-Bromo-2'-deoxyuridine (BrdU) and at multiple time-points post-irradiation the yield of chromosomal damage, the mitotic index and the cumulative BrdU-labelling index were determined. Furthermore, a mathematical approach was used to analyse the entire cell population. RESULTS: Following high LET exposure normal fibroblasts suffer a transient delay into S-phase and into mitosis as well as a prolonged, probably permanent cell cycle arrest in the initial G0/G1-phase. Cells that reach the first mitosis at early times carried less aberrations than those collected at later times indicating a relationship between cell cycle delay and the number of aberrations. However, with respect to the whole cell population, only a few aberrant fibroblasts are able to progress to the first mitosis. For all endpoints studied the relative biological effectiveness (RBE) of C ions is in the range of 2 - 4, while for Ni ions RBE < 1 is estimated. In contrast, when compared on a per particle basis Ni ions with the higher ionization density were found to be more effective. CONCLUSIONS: Detailed analysis of the data demonstrates that the number of fibroblasts at risk for neoplastic transformation is significantly reduced by a chronic cell cycle arrest in the initial G0/G1-phase and, for the first time, the LET-dependence of this effect has been shown.

Alveolar response to Pseudomonas aeruginosa: role of the type III secretion system.

The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoU-independent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains of Pseudomonas aeruginosa in a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivated exsA gene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1beta, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role in P. aeruginosa pathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSS-related virulence factors in addition to ExoU.