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Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p

Endogenous production of carbon monoxide (CO) is affected by inflammatory phenomena and ischemia-reperfusion injury. Precise measurement of exhaled endogenous CO (eCO) is possible thanks to a laser spectrometer (ProCeas® from AP2E company). We assessed eCO levels of human lung grafts during the normothermic Ex-Vivo Lung Perfusion (EVLP). ProCeas® was connected in bypass to the ventilation circuit. The surgical team took the decision to transplant the lungs without knowing eCO values. We compared eCO between accepted and rejected grafts. EVLP parameters and recipient outcomes were also compared with eCO values. Over 7 months, eCO was analyzed in 21 consecutive EVLP grafts. Two pairs of lungs were rejected by the surgical team. In these two cases, there was a tendency for higher eCO values (0.358 ± 0.52 ppm) compared to transplanted lungs (0.240 ± 0.76 ppm). During the EVLP procedure, eCO was correlated with glucose consumption and lactate production. However, there was no association of eCO neither with edema formation nor with the PO 2 /FiO 2 ratio per EVLP. Regarding post-operative data, every patient transplanted with grafts exhaling high eCO levels (>0.235 ppm) during EVLP presented a Primary Graft Dysfunction score of 3 within the 72 h post-transplantation. There was also a tendency for a longer stay in ICU for recipients with grafts exhaling high eCO levels during EVLP. eCO can be continuously monitored during EVLP. It could serve as an additional and early marker in the evaluation of the lung grafts providing relevant information for post-operative resuscitation care., Competing Interests: Author KJ was employed by the company AP2E Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brenckmann, Briot, Ventrillard, Romanini, Barbado, Jaulin, Trocme, De Wolf, Glorion and Sage.)

Preventing the cytokine storm observed in COVID-19 is a crucial goal for reducing the occurrence of severe acute respiratory failure and improving outcomes. Here, we identify Aldo-Keto Reductase 1B10 (AKR1B10) as a key enzyme involved in the expression of pro-inflammatory cytokines. The analysis of transcriptomic data from lung samples of patients who died from COVID-19 demonstrates an increased expression of the gene encoding AKR1B10. Measurements of the AKR1B10 protein in sera from hospitalised COVID-19 patients suggests a significant link between AKR1B10 levels and the severity of the disease. In macrophages and lung cells, the over-expression of AKR1B10 induces the expression of the pro-inflammatory cytokines Interleukin-6 ( IL-6) , Interleukin-1β ( IL-1β ) and Tumor Necrosis Factor a ( TNF α ), supporting the biological plausibility of an AKR1B10 involvement in the COVID-19-related cytokine storm. When macrophages were stressed by lipopolysaccharides (LPS) exposure and treated by Zopolrestat, an AKR1B10 inhibitor, the LPS-induced production of IL-6 , IL-1β , and TNFα is significantly reduced, reinforcing the hypothesis that the pro-inflammatory expression of cytokines is AKR1B10-dependant. Finally, we also show that AKR1B10 can be secreted and transferred via extracellular vesicles between different cell types, suggesting that this protein may also contribute to the multi-organ systemic impact of COVID-19. These experiments highlight a relationship between AKR1B10 production and severe forms of COVID-19. Our data indicate that AKR1B10 participates in the activation of cytokines production and suggest that modulation of AKR1B10 activity might be an actionable pharmacological target in COVID-19 management.

Background and Aims: Markers predicting complications of post-hepatitis C cirrhosis are needed. We asked whether changes in noninvasive markers of fibrosis can predict liver-related complications., Methods: This was a case-controlled study using a prospective national cohort (ANRS-CO12-CIRVIR) of 1323 HCV-infected patients with compensated cirrhosis: 97 patients who developed liver-related complications such as hepatocellular carcinoma or hepatic decompensation (cases) matched in age, sex and follow-up duration were compared with 257 patients without complications (controls). Actitest/Fibrotest™, Inflameter/Fibrometer™, ELF™ and Fibroscan™ were performed at baseline and yearly. Samples based on Propensity score matching were built and mixed linear models performed. Outcomes in a sustained virological response (SVR) negative population and a SVR-positive population were also described., Results: At baseline, all characteristics of patients were similar between the groups. All fibrosis tests were statistically higher for cases compared to controls, Fibroscan™ excepted: Fibrotest™: 0.83±0.13 vs. 0.77±0.16; Fibrometer™: 0.93±0.07 vs. 0.90±0.11; ELF™: 11.4±1.0 vs. 11.0±1.2 (P<0.02). The mean follow-up was 5.7±1.9 years. Over a 3-year period, the significant difference in fibrosis marker values between cases and controls remained constant; with a trend toward a decrease in inflammation markers in controls, independent of SVR status., Conclusions: Baseline noninvasive serum fibrosis and inflammation markers were significantly higher in patients developing a complication than in controls. During the follow-up only inflammatory markers decreased in controls, but not in cases, and thus could potentially be used to predict the occurrence of complications in cirrhotic patients., (Copyright © 2019. Published by Elsevier Masson SAS.)

Background: This study aimed to identify transplantation characteristics and biomarkers that predict outcomes for kidney transplant (KT) patients from donors after circulatory death (DCDs)., Methods: Consecutive patients receiving a KT from a DCD in our center between 2014 and 2016 were included; the reference population was recipients with a living donor KT. The urinary tubular injury biomarker-to-creatinine ratio and serum lactate dehydrogenase (LDH) were measured at post-transplant days 1 and 3. The primary outcome was the occurrence of delayed graft function (DGF). Descriptive and receiver operating characteristic analyses were performed., Results: Forty-one patients were included in the analysis: 15 (36.59%) DCD KTs (9 of which suffered from DGF) and 26 (63.41%) living donor KTs. For the primary endpoint, neutrophil gelatinase-associated lipocalin, N-acetyl-beta-D-glucosaminidase, urinary tubular injury biomarker-to-creatinine ratio, and LDH areas under the curve were 1 and 0.96 (95% confidence interval: 0.84-1.0), 1 and 0.92 (95% confidence interval: 0.73-1.0), respectively. Among the transplant characteristics, only the 30-minute resistive index on the perfusion machine was significantly higher in DCD KTs with DGF vs those without DGF (0.26 mm Hg/mL/min [0.20; 0.32] vs 0.14 mm Hg/mL/min [0.12; 0.16], P = .05). Median 3-month creatinine clearance among DGF DCD KTs was 49 mL/min/1.73 m 2 [IQR: 42; 65] and 65 mL/min/1.73 m 2 [IQR: 62; 66] among DCD KTs without DGF (P = .22)., Conclusion: In the DCD KT population, clinical and biological markers were identified that provided predictive tools for DGF. Thus, systematic measurement of these biomarkers, particularly LDH, could improve the management of kidney graft recipients' immunosuppressive therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Calprotectin is a calcium binding protein produced by neutrophils and monocytes locally at the site of inflammation in order to trigger the innate immunity receptors. This unique characteristic makes it a good proxy for evaluation of local inflammation in chronic inflammatory rheumatic diseases. Complete data suggest, in inflammatory rheumatic diseases, a relevant role of calprotectin in the inflammatory process. The interest of serum or plasma calprotectin dosage has been studied intensively, in the current years, especially in rheumatoid arthritis, spondyloarthritis, juvenile idiopathic arthritis and ANCA associated vasculitis. Calprotectin seems to be a great candidate as biomarker to assess and monitor disease activity, to predict structural progression or response to the treatment. Calprotectin showed its ability to predict radiological progression in rheumatoid arthritis and ankylosing spondylitis. Serum calprotectin can predict the risk of relapse in ANCA associated vasculitis and the risk of inflammatory bowel disease in spondyloarthritis. Nevertheless, studies report controversial result requiring replication in other large cohort. The lack of assay standardization between studies is a problem to replicate and compare studies. In this review, we discuss on the interest of systemic calprotectin in chronic inflammatory rheumatic disease as a diagnostic, activity or prognostic biomarker., (Copyright © 2019 Société française de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.)

Background: This study aimed to assess cardiac and pulmonary pathophysiological responses during cooling and extracorporeal life support (ECLS) rewarming in a porcine model of deep hypothermic cardiac arrest (DHCA). In addition, we evaluated whether providing a lower flow rate of ECLS during the rewarming phase might attenuate cardiopulmonary injuries., Methods: Twenty pigs were cannulated for ECLS, cooled until DHCA occurred and subjected to 30 min of cardiac arrest. In order to assess the physiological impact of ECLS on cardiac output we measured flow in the pulmonary artery using Doppler echocardiography as well as a modified thermodilution technique using the Swan-Ganz catheter (injection site in the right ventricle). The animals were randomized into two groups during rewarming: a group with a low blood flow rate of 1.5 L/min (LF group) and a group with a normal flow rate of 3.0 L/min (NF group). The ECLS temperature was adjusted to 5 °C above the central core. Cardiac output, hemodynamics and pulmonary function parameters were evaluated., Results: During the cooling phase, cardiac output, heart rhythm and blood pressure decreased continuously. Pulmonary artery pressure tended to increase at 32 °C compared to the initial value (20.2 ± 1.7 mmHg vs. 29.1 ± 5.6 mmHg, p = 0.09). During rewarming, arterial blood pressure was higher in the NF than in the LF group at 20° and 25 °C (p = 0.003 and 0.05, respectively). After rewarming to 35 °C, cardiac output was 3.9 ± 0.5 L/min in the NF group vs. 2.7 ± 0.5 L/min in LF group (p = 0.06). At the end of rewarming under ECLS cardiac output was inversely proportional to the ECLS flow rate. Moreover, the ECLS flow rate did not significantly change pulmonary vascular resistance., Discussion: Using a newly developed experimental model of DHCA treated by ECLS, we assessed the cardiac and pulmonary pathophysiological response during the cooling phase and the ECLS rewarming phase. Despite lower metabolic need during hypothermia, a low ECLS blood flow rate during rewarming did not improved cardiopulmonary injuries after rewarming., Conclusion: A low ECLS flow rate during the rewarming phase did not attenuate pulmonary lesions, increased blood lactate level and tended to decrease cardiac output after rewarming. A normal ECLS flow rate did not increase pulmonary vascular resistance compared to a low flow rate. This experimental model on pigs contributes a number of pathophysiological findings relevant to the rewarming strategy for patients who have undergone accidental DHCA.

Objectives: Extracorporeal life support (ECLS) is the reference rewarming technique of accidental deep hypothermic cardiac arrest (DHCA). This study was designed to examine the impact of different rewarming blood flow rates and temperature setting of ECLS on cardiopulmonary lesions after DHCA in a porcine model of accidental hypothermia., Methods: Twenty-four pigs were cannulated for ECLS, cooled until DHCA occurred, and subjected to 30 minutes of cardiac arrest. During the rewarming phase, we compared a low blood flow rate of 1.5 L/min versus a high flow rate of 3.0 L/min as well as two-temperature-setting rewarming strategies: a temperature during ECLS adjusted to 5°C above the central core temperature versus 38°C maintained throughout the rewarming phase. Cardiac output, hemodynamics and pulmonary function parameters were evaluated. Biologic markers of ischemia-reperfusion injuries were analyzed at baseline and at the end of the experiment., Results: DHCA occurred at 21.2 ± 2°C. There was a trend for better cardiac output in groups with high blood flow (p = 0.053), with no interaction between ECLS flow and temperature (p = 0.63), a trend toward lower pulmonary vascular resistance (PVR; p = 0.075) and a significant decrease in arterial PVR in groups with high blood flow (p = 0.013) with no interaction (p = 0.47 and p = 0.60 for PVR and arterial PVR, respectively). Serum interleukin-6, tumor necrosis factor-α, receptor for advanced glycation end products (RAGE), and neuron-specific enolase were significantly increased between baseline and endpoint. The increase in the serum RAGE concentration was higher in the 38°C rewarming temperature groups compared to 5°C above adjusted temperature. There were no other significant differences in biomarkers., Conclusions: We developed a porcine model of DHCA treated by ECLS. Our data suggest that cardiac output tended to improve with a high-flow-rate rewarming strategy while a high-temperature delta between core temperature and ECLS increased the RAGE markers of lung injury., (© 2016 by the Society for Academic Emergency Medicine.)

Background and Objective: Obstructive sleep apnoea (OSA) could be an independent risk factor for non-alcoholic fatty liver disease (NAFLD) occurrence and progression. The impact of continuous positive airway pressure (CPAP) treatment on non-invasive markers of NAFLD has not been studied. The aim of this study was to evaluate the effect of 6-12 weeks of effective CPAP on the FibroMax test (comprising components including the SteatoTest, NashTest and FibroTest) through three randomized sham controlled studies., Methods: The FibroMax test was performed in 103 obstructive sleep apnoea patients (apnoea + hypopnoea index > 15/h) enrolled in a randomized study comparing sham versus effective CPAP., Results: At baseline, 40.4% of patients in the sham CPAP group and 45.5% in the CPAP group exhibited liver steatosis. Furthermore, 39.6% of patients in the sham CPAP group and 58.4% in the CPAP group displayed borderline or possible non-alcoholic steatohepatitis (NASH). Six to twelve weeks of effective CPAP did not demonstrate any impact on reducing steatosis, NASH or liver fibrosis even after adjustment for gender, BMI, baseline apnoea + hypopnoea index and severity of liver injury., Conclusion: A number of non-invasive markers of liver damage are increased in untreated obstructive sleep apnoea patients, potentially contributing to cardiometabolic risk, but they do not improve after 6-12 weeks of effective CPAP treatment., Clinical Trial Registration: NCT01196845 (ADISAS), NCT00464659 (MneSAS) and NCT00669695 (StatinflaSAS) at ClinicalTrials.gov., (© 2015 Asian Pacific Society of Respirology.)

The NADPH oxidases, Nox, are transmembrane hemoproteins, whose exclusive function is to reduce molecular oxygen to produce superoxide anion O2°- and consequently highly reactive oxidant and toxic oxygen species, ROS. Among the 7 NADPH oxidases expressed in humans, Nox4 is the sole Nox isoform present in human primary chondrocytes. Nox4 was suggested as one of the main actors involved in cartilage degradation in osteoarthritis. The stimulation of chondrocytes, the only cell present in cartilage, by IL-1β results in the activation of Nox4. This leads to an increase of ROS production which in turn could regulate signaling pathways sensitive to oxidative stress such as gene-encoding matrix metalloproteases MMP1, MMP13 and Adamalysin ADAMTS4. A deep understanding of Nox4 structure/function and mechanisms of regulation could lead both to the identification of new therapeutic targets and to the development of innovative strategies for appropriate osteoarthritis treatment.

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS-producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase-induced emphysema using NOX2- and NOX1-deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2-positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase-induced alveolar airspace enlargement and elastin degradation were prevented in NOX2-deficient mice, but not in NOX1-deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase-9 (MMP-9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage-restricted functional NOX2 in elastase-induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68-positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase-induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage-specific NOX2 participates in elastase-induced emphysema through the involvement of SIRT1/MMP-9 pathways in mice., (© 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Crohn's disease (CD) and irritable bowel syndrome (IBS) involve brain-gut dysfunctions where vagus nerve is an important component. The aim of this work was to study the association between vagal tone and markers of stress and inflammation in patients with CD or IBS compared to healthy subjects (controls). The study was performed in 73 subjects (26 controls, 21 CD in remission and 26 IBS patients). The day prior to the experiment, salivary cortisol was measured at 8:00 AM and 10:00 PM. The day of the experiment, subjects completed questionnaires for anxiety (STAI) and depressive symptoms (CES-D). After 30 min of rest, ECG was recorded for heart rate variability (HRV) analysis. Plasma cortisol, epinephrine, norepinephrine, TNF-alpha and IL-6 were measured in blood samples taken at the end of ECG recording. Compared with controls, CD and IBS patients had higher scores of state-anxiety and depressive symptomatology. A subgroup classification based on HRV-normalized high frequency band (HFnu) as a marker of vagal tone, showed that control subjects with high vagal tone had significantly lower evening salivary cortisol levels than subjects with low vagal tone. Such an effect was not observed in CD and IBS patients. Moreover, an inverse association (r =  -0.48; p<0.05) was observed between the vagal tone and TNF-alpha level in CD patients exclusively. In contrast, in IBS patients, vagal tone was inversely correlated with plasma epinephrine (r =  -0.39; p<0.05). No relationship was observed between vagal tone and IL-6, norepinephrine or negative affects (anxiety and depressive symptomatology) in any group. In conclusion, these data argue for an imbalance between the hypothalamus-pituitary-adrenal axis and the vagal tone in CD and IBS patients. Furthermore, they highlight the specific homeostatic link between vagal tone and TNF-alpha in CD and epinephrine in IBS and argue for the relevance of vagus nerve reinforcement interventions in those diseases.

Background & Aims: Fibrosis blood tests have been validated in chronic hepatitis C. Their diagnostic accuracy is less documented in hepatitis B. The aim of this study was to describe the diagnostic performance of FibroTest®, FibroMeter®, and HepaScore® for liver fibrosis in hepatitis B compared to hepatitis C., Methods: 510 patients mono-infected with hepatitis B or C and matched on fibrosis stage were included. Blood tests were performed the day of the liver biopsy. Histological lesions were staged according to METAVIR., Results: Fibrosis stages were distributed as followed: F0 n=76, F1 n=192, F2 n=132, F3 n=54, F4 n=56. Overall diagnostic performance of blood tests were similar between hepatitis B and C with AUROC ranging from 0.75 to 0.84 for significant fibrosis, 0.82 to 0.85 for extensive fibrosis and 0.84 to 0.87 for cirrhosis. Optimal cut-offs were consistently lower in hepatitis B compared to hepatitis C, especially for the diagnosis of extensive fibrosis and cirrhosis, with decreased sensitivity and negative predictive values. More hepatitis B than C patients with F ⩾3 were underestimated: FibroTest®: 47% vs. 26%, FibroMeter®: 24% vs. 6%, HepaScore®: 41% vs. 24%, p<0.01. Multivariate analysis showed that hepatitis B (0R 3.4, 95% CI 1.2-19.2, p<0.02) and low γGT (OR 7.3, 95% CI 2.0-27.0, p<0.003) were associated with fibrosis underestimation., Conclusion: Overall the diagnostic performance of blood tests is similar in hepatitis B and C. The risk of underestimating significant fibrosis and cirrhosis is however greater in hepatitis B and cannot be entirely corrected by the use of more stringent cut-offs., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Studies have shown that the clinical impact of Janus kinase 2 (JAK2) inhibitors in primary myelofibrosis patients is due to the regulation of cytokine levels, suggesting that cytokine profiles might play a critical role in myeloproliferative neoplasms (MPNs) physiopathology. In this study, we compared the plasma cytokine profiles of polycythemia vera (PV) patients and essential thrombocythemia (ET) patients as a function of their JAK2 V617F status and the presence of thrombohemorrhagic complications. Using a multiplex cytokine assay, cytokine measurements were taken of the plasma of 17 PV patients and 21 ET patients. Twenty-two of these patients (10 PV and 12 ET) experienced at least one thrombohemorrhagic manifestation before diagnosis. We showed that cytokine levels were significantly increased in PV and ET patients compared with normal values and that several positive correlations existed between the cytokine concentrations and the biological parameters in each MPN. The comparison between the cytokine profiles of ET and PV patients showed a statistically significant increase of interleukin (IL)-4, IL-8, granulocyte macrophage-colony stimulating factor, interferon -γ, monocyte chemotactic protein -1, platelet derived growth factor-BB, and vascular endothelial growth factor in the ET group. Only tumor necrosis factor-α and platelet derived growth factor-BB were specifically impacted by the JAK2 V617F status of the PV and ET patients, respectively, suggesting that there are both JAK2 V617F-driven and JAK2 V617F-independent inflammatory responses in MPNs. We also showed that the subgroup of PV patients with vascular complications displayed significantly different concentrations of IL-12(p70) and granulocyte macrophage-colony stimulating factor compared with patients without vascular complications. Altogether, these data suggest that cytokine measurement might be useful for the clinical and therapeutic stratification of PV and ET patients., (Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Nocturnal hypoxia, the hallmark of OSA, is a potential contributing factor for nonalcoholic fatty liver disease (NAFLD). NAFLD severity and its implication in OSA-related endothelial dysfunction have not been investigated in a large, unselected OSA population, including nonobese subjects., Methods: Noninvasive blood tests (SteatoTest, NashTest, and FibroTest) were used to evaluate steatosis, nonalcoholic steatohepatitis (NASH), and fibrosis in a large cohort of patients with OSA. In the same group, endothelial function and its links with NAFLD severity were assessed., Results: Of the 226 subjects included who were referred for suspicion of OSA (men, 55%; median age, 56 years; median BMI, 34.2 kg/m2 [33% with BMI<30 kg/m2]), 61.5% exhibited moderate or severe steatosis. By multivariate analysis, independent factors for liver steatosis were, as expected, triglyceride levels (P<.0001) and insulin resistance (P=.0004) as well as nocturnal cumulative time spent<90% of oxygen saturation (CT90) (P=.01). Thirty-eight percent had borderline or possible NASH (N1 or N2 with NashTest). CT90 was significantly associated with borderline or possible NASH (P=.035) in univariate but not in multivariate analysis. The dose-response relationship between the severity of nocturnal hypoxia and liver injury was established only in morbid obesity and not in lean. Multivariate models showed that steatosis was independently associated with endothelial dysfunction after adjustment for confounders., Conclusions: In a large, unselected OSA population, the severity of nocturnal hypoxia was independently associated with steatosis. Preexisting obesity exacerbated the effects of nocturnal hypoxemia. NAFLD is a potential mechanism of endothelial dysfunction in OSA.

The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the "killed but metabolically active" (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery.

Background & Aims: Blood tests and transient elastography (Fibroscan™) have been developed as alternatives to liver biopsy. This ANRS HCEP-23 study compared the diagnostic accuracy of nine blood tests and transient elastography (Fibroscan™) to assess liver fibrosis, vs. liver biopsy, in untreated patients with chronic hepatitis C (CHC)., Methods: This was a multicentre prospective independent study in 19 French University hospitals of consecutive adult patients having simultaneous liver biopsy, biochemical blood tests (performed in a centralized laboratory) and Fibroscan™. Two experienced pathologists independently reviewed the liver biopsies (mean length=25±8.4 mm). Performance was assessed using ROC curves corrected by Obuchowski's method., Results: Fibroscan™ was not interpretable in 113 (22%) patients. In the 382 patients having both blood tests and interpretable Fibroscan™, Fibroscan™ performed similarly to the best blood tests for the diagnosis of significant fibrosis and cirrhosis. Obuchowski's measure showed Fibrometer® (0.86), Fibrotest® (0.84), Hepascore® (0.84), and interpretable Fibroscan™ (0.84) to be the most accurate tests. The combination of Fibrotest®, Fibrometer®, or Hepascore® with Fibroscan™ or Apri increases the percentage of well classified patients from 70-73% to 80-83% for significant fibrosis, but for cirrhosis a combination offers no improvement. For the 436 patients having all the blood tests, AUROC's ranged from 0.82 (Fibrometer®) to 0.75 (Hyaluronate) for significant fibrosis, and from 0.89 (Fibrometer® and Hepascore®) to 0.83 (FIB-4) for cirrhosis., Conclusions: Contrarily to blood tests, performance of Fibroscan™ was reduced due to uninterpretable results. Fibrotest®, interpretable Fibroscan™, Fibrometer®, and Hepascore® perform best and similarly for diagnosis of significant fibrosis and cirrhosis., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is characterized by synaptic dysfunction and cardinal neuropathological features including amyloid plaques and neurofibrillary tangles. Soluble amyloid-beta (Abeta) can suppress synaptic activities by interacting with alpha7 nicotinic acetylcholine receptors (alpha7nAChRs). Here, we show that alpha7nAChR and NMDA glutamatergic receptor (NMDAR) activities are severely compromised in synaptosomes prepared from AD and Abeta(1-42) (Abeta42)-exposed control frontal cortex slices from postmortem tissue. Whereas Abeta(12-28) prevents Abeta42 from binding to alpha7nAChRs, 2-[2-(4-bromophenyl)-2-oxoethyl]-1-methyl pyridinium (S 24795), a novel alpha7nAChR partial agonist, facilitates release of Abeta42 from Abeta42-alpha7nAChR and -Abeta42 complexes. S 24795 interacts with alpha7nAChR and Abeta(15-20) region of the Abeta42 to enable partial recovery of the alpha7nAChR and NMDAR channel function. These findings suggest that the Abeta-alpha7nAChR interaction may be an upstream pathogenic event in AD and demonstrate that some recovery of neuronal channel activities may be achieved in AD brains by removing Abeta from alpha7nAChRs.

Objective: Systemic sclerosis (SSc) is a multisystem disease characterized by microvascular dysfunction and excessive fibrosis. However, the relationship between these 2 features remains unclear. Endothelial dysfunction can be assessed by quantifying plasma asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase. Matrix remodeling can be assessed by quantifying serum tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Both biomarkers are elevated in patients with SSc. Our objective was to test whether plasma ADMA is correlated with serum TIMP-1., Methods: We enrolled 91 subjects, 39 patients with SSc, 28 patients with primary Raynaud's phenomenon (RP), and 24 healthy volunteers. Plasma ADMA concentrations were measured by liquid chromatography-tandem mass spectrometry. Serum TIMP-1 concentrations were determined by ELISA., Results: Mean ADMA concentrations were higher in patients with SSc (0.68 microM +/- 0.12) than in patients with primary RP or healthy volunteers (respectively, 0.56 microM +/- 0.14 and 0.62 microM +/- 0.12; p = 0.002). Median serum TIMP-1 concentrations were increased in patients with SSc compared to primary RP and healthy volunteers [12 (9-15), 11 (8-13), and 10 (7-13) nM, respectively; p = 0.05]. In the SSc group, we observed a statistically significant correlation between plasma ADMA and serum TIMP-1 (r = 0.34, p = 0.035)., Conclusion: These data are consistent with our hypothesis of an association of endothelial dysfunction and matrix remodeling in scleroderma spectrum disorders.

One approach for the identification of therapeutic agents for Alzheimer's disease has focused on the research of alpha7 nAChR-selective agonists such as the partial agonists 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) and, more recently, 2-[2-(4-bromophenyl)-2-oxoethyl]-1-methyl pyridinium (S 24795). An alternative approach for targeting alpha7 nAChR has been the development of positive modulators for this receptor. In this study we examined the interactions between full or partial agonists and positive modulators of alpha7 nAChRs in situ in brain tissue. Three positive modulators were used, 5-hydroxyindole (5-HI), 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), and genistein. Whole-cell recordings were performed in stratum radiatum interneurons from rat brain slices. Hippocampal interneurons were stimulated by ACh, choline, S 24795, or 4OH-GTS-21, before and after bath perfusion with the positive modulators. 5-HI was not effective at potentiating 200 microM 4OH-GTS-21-evoked responses, however 5-HI induced a sustained potentiation of responses evoked by 30 microM 4OH-GTS-21. When 1 mM ACh and 200 microM 4OH-GTS-21 were applied alternately alpha7-mediated responses to both agonists were reduced, suggesting that high concentration of 4OH-GTS-21 produces residual inhibition or desensitization and that 5-HI is not effective at overcoming receptor desensitization. Similar results were obtained with alpha7 receptors expressed in Xenopus oocytes. Interestingly, responses evoked by S 24795 were potentiated by 5-HI but not by genistein. Additionally, PNU-120596 was able to potentiate alpha7-mediated responses, regardless of the nature of the agonist. We demonstrated that the potentiation of alpha7 nAChR response would depend on the nature and the effective concentration of the agonist involved and its particular interaction with the positive modulator.

Objectives: To evaluate the diagnostic accuracy of liver fibrosis tests and its influencing factors in a meta-analysis with individual data., Design and Methods: Four independent centers provided four blood tests and Metavir staging from 825 patients with chronic hepatitis C., Results: FibroMeter AUROC (0.840) for significant fibrosis was superior to those of Fibrotest (0.803, p=0.049), APRI (0.789, p=0.001) and Hepascore (0.781, p<0.001). The misclassification rate was lower for FibroMeter (23%) than for Fibrotest and Hepascore (both 28%, p<0.001). The variation in the diagnostic cut-offs of tests among centers, reflecting the overall reproducibility, was: FibroMeter: 4.2%, APRI: 24.0%, Fibrotest: 24.2%, Hepascore: 35.0%. Accordingly, the proportion of patients diagnosed with significant fibrosis changed: FibroMeter: 0.8%, Hepascore: 2.4% (p=0.02 vs FibroMeter), Fibrotest: 5.8% (p<10(-3)), APRI: 18.2% (p<10(-3))., Conclusions: This study on clinical applicability shows significant differences in diagnostic accuracy, inter-center reproducibility, and robustness of biomarkers to changes in population characteristics between blood tests.

Aims: Matrix metalloproteinases (MMPs) play a major role in wound healing: they can degrade all components of the extracellular matrix. In diabetic foot ulcers there is an excess of MMPs and a decrease of the tissue inhibitors of MMPs (TIMPs). This imbalance is probably one cause of impaired healing. However, little is known about changes in MMPs during wound healing., Methods: Sixteen patients with neuropathic diabetic foot ulcers participated. Wound fluid was collected regularly during the 12-week follow-up period, for measurement of MMP-1, MMP-2, MMP-8, MMP-9 and TIMP-1. Results were analysed by the degree of wound healing: good healers (defined by a reduction of at least 82% in initial wound surface at 4 weeks) and poor healers (reduction of less than 82% in wound surface at 4 weeks)., Results: In good healers, levels of MMP-8 and -9 secreted by inflammatory cells decreased earlier. The initial levels of MMP-1 were similar in good and poor healers (P = 0.1) but rose significantly at week 2 in good healers (P = 0.039). There was a significant correlation between a high ratio of MMP-1/TIMP-1 and good healing (r = 0.65, P = 0.008). Receiver Operator Curve (ROC) analysis showed that an MMP-1/TIMP-1 ratio of 0.39 best predicted wound healing (sensitivity = 71%, specificity = 87.5%)., Conclusions: A high level of MMP-1 seems essential to wound healing, while an excess of MMP-8 and -9 is deleterious, and could be a target for new topical treatments. The MMP-1/TIMP-1 ratio is a predictor of wound healing in diabetic foot ulcers.

Results: In comparison with 5-month-old mice, 18- to 19-month-old mice exhibited a severe and specific memory impairment in a contextual serial discrimination (CSD) task involving the learning and remembering of two successive spatial discriminations carried out on two distinct floors. This impairment was specific, as spatial memory, simultaneously tested on a simple discrimination (SD) task, was not affected in these aged mice. This deficit was completely reversed by 9-day per os administration of S 24795, a partial agonist of alpha 7 nicotinic receptors, at either 0.3 or 1.0 mg/kg. Memantine, an NMDA receptor antagonist, also had a memory-enhancing effect at a dose of 3.0 mg/kg, but not at 0.3 mg/kg., Conclusions: The memory-enhancing effect of S 24795 was due to a strong enhancement of contextual memory as indicated by a decrease in interference rate, whereas memantine enhanced spatial/semantic memory. S 24795 was more effective than memantine and also appears to be more specific to flexible forms of memory, one of the first cognitive domains (i.e. episodic memory) affected in Alzheimer's disease.

The effects of S 24795, a newly developed partial agonist at alpha7 nAChRs, were tested on synaptic transmission and plasticity using extracellular field excitatory postsynaptic potentials (fEPSPs) evoked in the CA1 region by Schaffer collateral stimulation in hippocampal slices obtained from adult mice. S 24795 reduced the amplitude of the fEPSPs in a concentration-dependent manner with an IC(50) of 127 microM and a Hill coefficient of 1.1. The reduction in amplitude of the fEPSPs started at S 24795 concentrations higher than 3muM and reached 71% of controls at 300 microM. This effect was mediated by alpha7 nAChRs since it was blocked by nAChR antagonists and was not observed in alpha7 -/- mice. This effect was probably due to a reduction in glutamate release from presynaptic terminals since it was associated with a significant increase in the paired pulse ratio. In addition, S 24795 (100 microM) significantly reduced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents, recorded in the whole cell configuration of the patch clamp technique (in voltage clamp mode), further supporting a presynaptic site of action of S 24795. In addition, S 24795 at 3 microM, a concentration that did not affect basic synaptic transmission, potentiated LTP. This effect was mediated by alpha7 nAChRs since it was prevented by MLA (10 nM) and was absent in alpha7 -/- mice. Galantamine an allosteric modulator of nAChRs, at the concentrations of 0.3-3 microM, failed to potentiate LTP. In view of its powerful effect on LTP and on cognitive function, S 24795 can be considered a novel useful tool for the treatment of AD patients and other senile forms of dementia.

S 24795 evoked methyllycaconitine-sensitive inward currents in voltage-clamped hippocampal interneurons with maximum amplitude about 14% that of ACh-evoked responses. Experiments with rat alpha7 receptors expressed in Xenopus oocytes confirmed that S 24795 is a partial agonist of alpha7 nAChR with an EC(50) of 34+/-11 microM and I(max) of approximately 10% relative to ACh. When 60 microM ACh was co-applied to alpha7-expressing oocytes along with increasing concentrations of S 24795, there was a progressive decrease in response compared to the responses to 60 microM ACh alone (IC(50) 45+/-9 microM). The positive allosteric modulator 5-hydroxyindole potentiated ACh- and S 24795-evoked responses of alpha7 receptors in both oocytes and hippocampal interneurons. In hippocampal slice experiments, depending on the ACh concentrations in the application pipette and the ratio of ACh to S 24795, co-application of S 24795 with ACh variously increased, decreased, or had no effect on responses, compared to ACh alone. In order to estimate the effective dilution factor for the pressure application experiments, we tested alpha7 receptors in oocytes with ACh alone and in co-application with S 24795 at the same ratios as in the slice experiments, but at varying dilution factors. The pattern of interaction seen in the slice experiments was most closely matched under the conditions of a 3:100 dilution, suggesting that the pipette solution was diluted approximately 30-fold at the site of action. This dilution factor was consistent with the potency of ACh and S 24795 in the oocyte expression system (EC(50)s approximately 30 microM).

Oxidative stress leads to vascular damage and participates in the pathomechanisms of aortic dissection and aneurysm formation. Here we study aortic dissection in mice deficient in the superoxide-generating reduced nicotinamide-adenine dinucleotide phosphate oxidase NOX1. Seven days of treatment with the hypertensive agent angiotensin II (3 mg/kg per day) led to aortic dissection in 23% of wild-type C57BL/6J mice but in only 4% of NOX1-deficient mice (P=0.05). In contrast, treatment of wild-type C57BL/6J mice with the hypertensive agent norepinephrine (12 mg/kg per day), did not lead to aortic dissection or sudden death, suggesting that hypertension is not sufficient to cause aortic dissection. Interestingly, norepinephrine-dependent blood pressure elevations were conserved in NOX1-deficient mice, demonstrating that, different from angiotensin II, it acts through NOX1-independent hypertensive mechanisms. The resistance of NOX1-deficient mice to angiotensin II-induced aortic dissection suggests a role for NOX1-dependent alterations of the vascular wall. We, therefore, studied gene expression and protease/inhibitor equilibrium. cDNA array analysis demonstrated differential effects of angiotensin II on gene expression in wild-type and NOX1-deficient mice. Tissue inhibitor of metalloproteinase 1 was increased both on the mRNA and the protein level in aortas from NOX1-deficient mice. Thus, our results demonstrate that NOX1 is involved in the mechanisms of angiotensin II-dependent aortic dissection. As one underlying mechanism, we have identified NOX1-dependent suppression of tissue inhibitor of metalloproteinase 1 expression, which could lead to tissue damage through an altered protease/inhibitor balance.

Background/aims: Non-invasive markers of liver fibrosis have recently been developed as an alternative to liver biopsy. The aim of this study was to compare the diagnostic performance of 6 scores (MP3, Fibrotest, Fibrometer, Hepascore, Forns' score and APRI)., Methods: We studied 180 chronic hepatitis C patients. Liver fibrosis was staged according to the METAVIR scoring system., Results: Overall diagnostic performance of scores determined by AUROCs ranged from 0.86 for Fibrometer to 0.78 for Forns' score (NS) for discriminating F0F1 versus F2F3F4. For discriminating F0F1F2 versus F3F4, AUROCs ranged from 0.91 for Fibrometer to 0.78 for Forns' score (p<0.02). Significant or extensive fibrosis was predicted in 10-86% of patients with positive predictive value (PPV) ranging from 55% to 94%. Using logistic regression, statistical independence was demonstrated for MP3, Fibrotest and APRI. Diagnostic performance of paired-combination scores was then evaluated. The best combinations could select one-third of patients for whom either absence of significant fibrosis or presence of extensive fibrosis could be predicted with more than 90% of certainty., Conclusions: Current non-invasive scores give reliable information on liver fibrosis in one-third of chronic hepatitis C patients, especially when used in combination.

We have recently described a fibrosis index combining serum procollagen type III N-terminal peptide (PIIINP) and matrix metalloproteinase 1 (MMP-1) concentrations for evaluating the amount of liver fibrosis in chronic hepatitis C patients. The aims of the present study were to validate this score in another cohort of patients and to assess its variations along those of TIMP-1, hyaluronic acid (HA) and MMP-9 during antiviral treatment. Seventy-nine patients treated by interferon-alpha and ribavirin for 24 or 48 weeks were included. A liver biopsy was performed within the 6 months before the start of treatment. Serum markers were measured in serum collected the day of the liver biopsy, at start of treatment, and every 3 months during treatment and a 6-month follow-up period. The PIIINP/MMP-1 index was significantly correlated to the METAVIR fibrosis (r = 0.68, P < 0.001). Its overall diagnostic value defined by the area under the receiver operating characteristics curves was 0.77 for discriminating F1 vs F2F3F4, and 0.81 for discriminating F1F2 vs F3F4, and was better than that observed for HA and TIMP-1. At the end of follow-up, the PIIINP/MMP-1 index significantly decreased in responders and remained stable in nonresponder patients. This decrease occurred early and continued regularly during the treatment period. This variation was because of both a decrease of PIIINP and an increase of MMP-1 concentrations. HA and TIMP-1 serum concentrations were also significantly lower at the end of follow-up in responder patients, but early changes were minimal and not influenced by the response to treatment. Our study shows that a noninvasive index combining PIIINP and MMP-1 is a useful tool to follow-up fibrosis change during and after antiviral therapy chronic hepatitis C patients.

Reactive oxygen species (ROS) are regulators of redox-sensitive cell signaling pathways. In osteoarthritis, human interleukin-1beta is implicated in cartilage destruction through an ROS-dependent matrix metalloproteinase production. To determine the molecular source of ROS production in the human IL-1beta (hIL-1beta)-sensitive chondrocyte immortalized cell line C-20/A4, transfected cells were constructed that overexpress NAD(P)H oxidases. First, RT-PCR analysis showed that the C-20/A4 cell line expressed Nox2, Nox4, p22( phox ), and p67( phox ), but not p47( phox ). It was found that ROS production by C-20/A4 chondrocytes does not depend on PMA and ionomycin activation. This indicates that Nox2 was not involved in the production of ROS. In C- 20/A4 cells that overexpress Nox4, hIL-1beta stimulated ROS production three times more than the normal production of C-20/A4 cells. Moreover, there was a fourfold increase in the production of collagenase (MMP-1) by chondrocytes that overexpress Nox4. Interestingly, MMP-1 production in cells that overexpress Nox2 was not sensitive to hIL-1beta. These data suggest that under hIL-1beta stimulation, C-20/A4 chondrocytes produce MMP-1 through a Nox4-mediated, ROS-dependent pathway.

The major difficulty for high-throughput screening of therapeutic protein candidates in experimental animal models of pathologies or for structural studies is their fast and efficient production. The tissue inhibitors of metalloproteinases (TIMPs) considered to play a role in many physiological and pathological processes, such as arthritis or cancer, by inhibiting matrix metalloproteinases or acting as signalling molecules, have always been produced with huge difficulties. We hereby propose a new method to overproduce human recombinant TIMP-1 by transient expression in HEK293E cells, followed by a one-step chromatography purification, yielding in only 2 weeks, dozens of milligrams of pure, stable, glycosylated and active protein for in vitro and in vivo studies. This easy to set up, rapid, and efficient method could be applied for any naturally secreted mammalian protein.

Objectives: Histological examination of liver biopsy is currently required in the management of patients with chronic hepatitis C. Our aim was to evaluate the diagnostic utility of a panel of circulating markers in detecting the stage of fibrosis., Methods: One hundred and ninety four-patients who had undergone a percutaneous liver biopsy before antiviral treatment, and 194 age- and sex-matched healthy subjects were studied. Serum levels of hyaluronate, procollagen type III N-terminal peptide (PIIINP), matrix metalloproteinases (MMP)-1, MMP-2, MMP-9 and their tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 were determined by RIA and ELISA. Histological lesions were staged according to the METAVIR score., Results: Hyaluronate, PIIINP, TIMP-1, and TIMP-2 serum levels were significantly higher in patients than in controls. Six markers were significantly correlated with fibrosis: MMP-2 (r = 0.28; p < 0.01), TIMP-1 (r = 0.42; p < 0.001), HA (r = 0.50; p < 0.001), PIIINP (r = 0.62; p < 0.0001), MMP-1 (r = -0.32; p < 0.01), and MMP-9 (r = -0.22; p < 0.05). By multivariate analysis, only PIIINP and MMP-1 were independently associated with fibrosis, and were combined using the equation of the logistic regression model. Using receiver-operating characteristics analysis, the area under the curve of the score to discriminate mild (FO/F1) from significant fibrosis (F2/F3/F4) was 0.82, with a sensitivity of 60% for a specificity of 92%., Conclusion: Our results suggest that combining two serum markers reflecting fibrogenesis (PIIINP) and fibrolysis (MMP-1) may provide a useful tool for evaluating liver fibrosis.