The ro-vibrational ν2 mode spectrum of methane investigated by ultrabroadband coherent Raman spectroscopy.

We present the first experimental application of coherent Raman spectroscopy (CRS) on the ro-vibrational ν2 mode spectrum of methane (CH4). Ultrabroadband femtosecond/picosecond (fs/ps) CRS is performed in the molecular fingerprint region from 1100 to 2000 cm-1, employing fs laser-induced filamentation as the supercontinuum generation mechanism to provide the ultrabroadband excitation pulses. We introduce a time-domain model of the CH4 ν2 CRS spectrum, including all five ro-vibrational branches allowed by the selection rules Δv = 1, ΔJ = 0, ±1, ±2; the model includes collisional linewidths, computed according to a modified exponential gap scaling law and validated experimentally. The use of ultrabroadband CRS for in situ monitoring of the CH4 chemistry is demonstrated in a laboratory CH4/air diffusion flame: CRS measurements in the fingerprint region, performed across the laminar flame front, allow the simultaneous detection of molecular oxygen (O2), carbon dioxide (CO2), and molecular hydrogen (H2), along with CH4. Fundamental physicochemical processes, such as H2 production via CH4 pyrolysis, are observed through the Raman spectra of these chemical species. In addition, we demonstrate ro-vibrational CH4 v2 CRS thermometry, and we validate it against CO2 CRS measurements. The present technique offers an interesting diagnostics approach to in situ measurement of CH4-rich environments, e.g., in plasma reactors for CH4 pyrolysis and H2 production.

Coherent Raman spectroscopy on hydrogen with in-situ generation, in-situ use, and in-situ referencing of the ultrabroadband excitation.

Time-resolved spectroscopy can provide valuable insights in hydrogen chemistry, with applications ranging from fundamental physics to the use of hydrogen as a commercial fuel. This work represents the first-ever demonstration of in-situ femtosecond laser-induced filamentation to generate a compressed supercontinuum behind a thick optical window, and its in-situ use to perform femtosecond/picosecond coherent Raman spectroscopy (CRS) on molecular hydrogen (H2). The ultrabroadband coherent excitation of Raman active molecules in measurement scenarios within an enclosed space has been hindered thus far by the window material imparting temporal stretch to the pulse. We overcome this challenge and present the simultaneous single-shot detection of the rotational H2 and the non-resonant CRS spectra in a laminar H2/air diffusion flame. Implementing an in-situ referencing protocol, the non-resonant spectrum measures the spectral phase of the supercontinuum pulse and maps the efficiency of the ultrabroadband coherent excitation achieved behind the window. This approach provides a straightforward path for the implementation of ultrabroadband H2 CRS in enclosed environment such as next-generation hydrogen combustors and reforming reactors.

Left Ventricular Diastolic Dysfunction in ARDS Patients.

BACKGROUND: The aim of this study was to evaluate the possible presence of diastolic dysfunction and its possible effects in terms of respiratory mechanics, gas exchange and lung recruitability in mechanically ventilated ARDS. METHODS: Consecutive patients admitted in intensive care unit (ICU) with ARDS were enrolled. Echocardiographic evaluation was acquired at clinical PEEP level. Lung CT-scan was performed at 5 and 45 cmH2O. In the study, 2 levels of PEEP (5 and 15 cmH2O) were randomly applied. RESULTS: A total of 30 patients were enrolled with a mean PaO2/FiO2 and a median PEEP of 137 ± 52 and 10 [9-10] cmH2O, respectively. Of those, 9 patients (30%) had a diastolic dysfunction of grade 1, 2 and 3 in 33%, 45% and 22%, respectively, without any difference in gas exchange and respiratory mechanics. The total lung weight was significantly higher in patients with diastolic dysfunction (1669 [1354-1909] versus 1554 [1146-1942] g) but the lung recruitability was similar between groups (33.3 [27.3-41.4] versus 30.6 [20.0-38.8] %). Left ventricular ejection fraction (57 [39-62] versus 60 [57-60]%) and TAPSE (20.0 [17.0-24.0] versus 24.0 [20.0-27.0] mL) were similar between the two groups. The response to changes of PEEP from 5 to 15 cmH2O in terms of oxygenation and respiratory mechanics was not affected by the presence of diastolic dysfunction. CONCLUSIONS: ARDS patients with left ventricular diastolic dysfunction presented a higher amount of lung edema and worse outcome.

Coherent N2 + emission mediated by coherent Raman scattering for gas-phase thermometry.

We report on the generation of coherent emission from femtosecond (fs) laser-induced filaments mediated by ultrabroadband coherent Raman scattering (CRS), and we investigate its application for high-resolution gas-phase thermometry. Broadband 35-fs, 800-nm pump pulses generate the filament through photoionization of the N2 molecules, while narrowband picosecond (ps) pulses at 400 nm seed the fluorescent plasma medium via generation of an ultrabroadband CRS signal, resulting in a narrowband and highly spatiotemporally coherent emission at 428 nm. This emission satisfies the phase-matching for the crossed pump-probe beams geometry, and its polarization follows the CRS signal polarization. We perform spectroscopy on the coherent N2 + signal to investigate the rotational energy distribution of the N2 + ions in the excited B2Σu + electronic state and demonstrate that the ionization mechanism of the N2 molecules preserves the original Boltzmann distribution to within the experimental conditions tested.

Is it possible to conduct clinical trials during a pandemic? The example of a trial of hydroxychloroquine.

OBJECTIVES: to examine the factors that, in the context of the current pandemic, have influenced the conduct of a randomized clinical trial on hydroxychloroquine in Italy. DESIGN: the trend of enrolment in the PROTECT study, "A randomized study with Hydroxychloroquine versus observational support for prevention or early phase treatment of Coronavirus disease (COVID-19)" (Eudract number: 2020-001501-24, NCT04363827), conducted in the period from May to September 2020, was analysed to evaluate the possible association of the enrolment rate with the amount of information published in the national and local press on hydroxychloroquine. SETTING AND PARTICIPANTS: the PROTECT clinical study is an Italian interventional superiority study, open label, with cluster randomization, aimed at evaluating whether treatment with hydroxychloroquine can reduce the percentage of symptomatic subjects compared to observation only in a population of subjects exposed to SARS-CoV-2 virus consisting of cohabitants/contacts of COVID-19 patients and asymptomatic or paucisymptomatic subjects diagnosed with COVID-19. MAIN OUTCOME MEASURES: the number of asymptomatic or paucisymptomatic COVID-19 patients and the number of contacts/cohabitants of COVID-19 patients enrolled in the Protect study from May to September 2020. RESULTS: from May to September 2020, the number of patients diagnosed with COVID-19 enrolled in the PROTECT clinical trial showed a decrease consistent with the number of news on hydroxychloroquine appearing in the national and local press, starting from the time when the first criticisms of the efficacy of hydroxychloroquine were made known; the number of contacts/cohabitants of COVID-19 patients showed a more marked and more timely decrease. CONCLUSIONS: in the context determined by the current COVID-19 pandemic, conducting a controlled clinical trial is strongly influenced by public opinion on scientific issues. Adherence to a clinical study can become highly problematic and invalidate the possibility of answering a scientific question and the validity of a project. In the current pandemic situation, randomized controlled trials may not always be the optimal tool to reach the expected scientific evidence, due to a number of problems. It is preferable to use a sequential or adaptive design. Furthermore, study protocols should implement innovative approaches that also include the involvement of participants in the decision-making process. In any case, the influence of public information on scientific issues is an extremely important factor to consider in the design of clinical trials in exceptional situations such as a pandemic.

Oral CorticoSteroid sparing with biologics in severe asthma: A remark of the Severe Asthma Network in Italy (SANI).

According to the data derived from several national and international registries, including SANI (Severe Asthma Network Italy), and considering the strong impact that frequent or regular use of oral corticosteroid has on quality of life (QoL) of severe asthmatics, as well as on the costs for managing corticosteroid-related diseases, oral corticosteroid sparing up to withdrawal should be considered a primary outcome in the management of severe asthma. New biologics have clearly demonstrated that this effect is possible, with concomitant reduction in the rate of exacerbations and in symptom control. Then, there is no reason for using so frequently oral corticosteroid before having explored all alternatives currently available for a large part of severe asthmatics.

Pure-rotational 1D-CARS spatiotemporal thermometry with a single regenerative amplifier system.

We report spatiotemporal pure-rotational coherent anti-Stokes Raman spectroscopy (CARS) in a one-dimensional imaging arrangement obtained with a single ultrafast regenerative amplifier system. The femtosecond pump/Stokes photon pairs, used for impulsive excitation, are delivered by an external compressor operating on a ∼35% beam split of the uncompressed amplifier output (2.5 mJ/pulse). The picosecond 1.2 mJ probe pulse is produced via the second-harmonic bandwidth compression (SHBC) of the ∼65% remainder of the amplifier output (4.5 mJ/pulse), which originates from the internal compressor. The two pump/Stokes and probe pulses are spatially, temporally, and repetition-wise correlated at the measurement, and the signal generation plane is relayed by a wide-field coherent imaging spectrometer onto the detector plane, which is refreshed at the same repetition rate as the ultrafast regenerative amplifier system. We demonstrate 1 kHz cinematographic 1D-CARS gas-phase thermometry across an unstable premixed methane/air flame-front, achieved with a single-shot precision <1% and accuracy <3%, 1.4 mm field of view, and an excellent <20µm line-spread function.

Perceived Well-Being and Quality of Life in People with Typical and Atypical Development: The Role of Sports Practice.

Sports and physical activity are recognized as a source of psycho-physical well-being. Therefore, practicing sports can improve the perception of the quality of life (QoL). The study investigated in adults with atypical development (AD) and typical development (TD) if the perceptions of well-being and QoL may depend on the frequency of practicing sports. Participants were 51 AD (age M = 32.98, SD = 7.81; 45.1% female) and 270 TD adults (age M = 26.50, SD = 6.52; 79.3% female) subsequently divided into three groups: 1) people not practicing sports, or 2) practicing it occasionally, or 3) practicing it regularly. We adopted general self-efficacy, life satisfaction, positive well-being, and psychological distress related to physical exercise as measures of well-being, WHOQOL-BREF as QoL, and I-TIPI-R as indicators of Big Five personality factors. Questionnaires were completed online. Practicing sports influenced the perception of well-being and QoL. For the AD group, sporting practice seems to have assumed an equalization effect, eliminating the statistical differences between well-being and QoL measures of AD and TD groups. Associations emerged between Big Five and well-being and QoL indices with some differences as a function of group characteristics. In summary, results suggest that practicing sports is strongly associated with improvements in perceptions of well-being and QoL, especially in disabled persons.

Mepolizumab for severe eosinophilic asthma: a real-world snapshot on clinical markers and timing of response.

Background: Few studies have provided real-world evidence of mepolizumab efficacy and safety. We aimed to evaluate mepolizumab for severe eosinophilic asthma in daily clinical practice.Research design and methods: Patients included in the RINOVA (Interdisciplinary Network for the management of severe asthma in Veneto region, Italy) database were investigated. Blood eosinophil count, forced expiratory volume in 1 second, % of predicted (FEV1%), fractional exhaled nitric oxide (FeNO), asthma control test (ACT), oral steroid (OCS) intake, and exacerbation rate were evaluated during mepolizumab treatment.Results: 69 patients were enrolled (mean age: 55.1 years; 60.9% females). A significant improvement was detected at one month with respect to blood eosinophils (median level at baseline: 710/µl; -620/µl, p < 0,001), FEV1% (median value at baseline 87; range: 79-101; +4, p = 0.001) and ACT (median value at baseline 18; range: 14-20.5;+4, <0.001). A significant reduction of FeNO was observed six months after the treatment start, when the exacerbation rate and the mean OCS dose significantly decreased (respectively: Δ reduction -3; p < 0.001 and -5 mg; p < 0.001).Conclusions: Our study provides real-world evidence of mepolizumab safety and confirms its dramatic steroid sparing effect. The greatest clinical change (ACT and FEV1) was observed within the first month.

Asthma management in a specialist setting: Results of an Italian Respiratory Society survey.

BACKGROUND: Asthma considerably impairs patients' quality of life and increases healthcare costs. Severity, morbidity, and degree of disease control are the major drivers of its clinical and economic impact. National scientific societies are required to monitor the application of international guidelines and to adopt strategies to improve disease control and better allocate resources. AIM: to provide a detailed picture of the characteristics of asthma patients and modalities of asthma management by specialists in Italy and to develop recommendations for the daily management of asthma in a specialist setting. METHOD: A quantitative research program was implemented. Data were collected using an ad hoc questionnaire developed by a group of specialists selected by the Italian Pneumology Society/Italian Respiratory Society. RESULTS: The records of 557 patients were analyzed. In the next few years, specialists are expected to focus their activity patients with more severe disease and will be responsible for selection of patients for personalized biological therapy; however, only 20% of patients attending Italian specialist surgery can be considered severe. In 84.4% of cases, the visit was a follow-up visit requested in 82.2% of cases by the specialist him/herself. The Asthma Control Test is used only in 65% of patients. When available, a significant association has been observed between the test score and asthma control as judged by the physician, although concordance was only moderate (κ = 0.68). Asthma was considered uncontrolled by the specialist managing the case in 29.1% of patients; nevertheless, treatment was not stepped up in uncontrolled or partly controlled patients (modified in only 37.2% of patients). CONCLUSIONS: The results of this survey support re-evaluation of asthma management by Italian specialists. More resources should be made available for the initial visit and for more severely ill patients. In addition, more extensive use should be made of validated tools, and available drugs should be used more appropriately.

Short-term Survival of Patients with Lung Metastases from Colorectal and Non-colorectal Cancer Who Underwent Pulmonary Metastasectomy.

The lung is a common site of metastases, whose prevalence varies as a function of the primary tumor site, which is usually colorectal cancer (CRC), breast carcinoma, or genitourinary cancers, such as ovary, urinary bladder and renal cell carcinomas. The aim of the present study was to analyze whether the site of primitive tumor affects overall survival (OS) of patients with lung metastases (LMs) who underwent pulmonary metastasectomy. The data of 41 patients with surgically treated CRC (Group A=22 patients) and non-colorectal carcinomas (Group B=19 patients), who developed matachronous LMs and underwent pulmonary metastasectomy with curative intent, were analyzed. The origin of non-colorectal LMs was genitourinary cancer in nine and breast cancer in 10 patients. Overall, there were 22 men and 19 women, with a median age of 65 years (range=31-80); 18 patients had a solitary metastatic tumor, while 23 had two or more LMs. Twenty-nine patients underwent wedge resection, through thoracotomy or video-assisted thoracic surgery, while 12 underwent pulmonary lobectomy. Seventy-five LMs were resected with a 5-tear OS of 48.8%. No difference was found between elderly (≥65 year-old) and younger patients (p=0.26), and between those with solitary or multiple LMs (p=0.62) in terms of survival rate. The female patients had a worse OS (31.6% vs. 63.6%; odds ratio (OR)=3.79, 95% confidence interval (CI)=1.03-13.91, p=0.003) compared to males, independent of the origin of primary cancer. There was no difference in the cumulative survival rates (OR=1.65, 95%CI=0.48-5.69, p=0.42) between Groups and the log-rank test (p=0.75) was not significant. In conclusion, the main pathological characteristics of metastatic lesions and advanced age do not appear to be associated with a poor prognosis in patients with LMs, while the female gender is a negative prognostic factor. Thus, the primary tumor site should not be considered a major criterion in selecting patients for pulmonary metastasectomy.

Open source software in a practical approach for post processing of radiologic images.

PURPOSE: The purpose of this paper is to evaluate the use of open source software (OSS) to process DICOM images. MATERIALS AND METHODS: We selected 23 programs for Windows and 20 programs for Mac from 150 possible OSS programs including DICOM viewers and various tools (converters, DICOM header editors, etc.). The programs selected all meet the basic requirements such as free availability, stand-alone application, presence of graphical user interface, ease of installation and advanced features beyond simple display monitor. Capabilities of data import, data export, metadata, 2D viewer, 3D viewer, support platform and usability of each selected program were evaluated on a scale ranging from 1 to 10 points. RESULTS: Twelve programs received a score higher than or equal to eight. Among them, five obtained a score of 9: 3D Slicer, MedINRIA, MITK 3M3, VolView, VR Render; while OsiriX received 10. CONCLUSIONS: OsiriX appears to be the only program able to perform all the operations taken into consideration, similar to a workstation equipped with proprietary software, allowing the analysis and interpretation of images in a simple and intuitive way. OsiriX is a DICOM PACS workstation for medical imaging and software for image processing for medical research, functional imaging, 3D imaging, confocal microscopy and molecular imaging. This application is also a good tool for teaching activities because it facilitates the attainment of learning objectives among students and other specialists.

Carboxy-terminal telopeptide (CTX) and amino-terminal propeptide (PINP) of type I collagen as markers of bone metastases in patients with non-small cell lung cancer.

The early diagnosis of non-small cell lung carcinoma (NSCLC) is difficult, and 30-40% of patients with NSCLC develop bone metastases (BMs) during the course of their disease. Because the delayed demonstration of skeletal involvement may seriously affect survival, there is a need for early diagnosis of BMs. Unfortunately, the sensitivity of common serum tumor markers is low and they are used mainly for monitoring the efficacy of therapy and detection of recurrence. The aim of this study was to evaluate the utility of a panel of serum biomarkers in patients with NSCLC and BMs. Sixteen patients (11 males, 5 females; median age=64 years, range 54-68 years) with NSCLC and BMs (cases), and 18 age- and stage-matched patients without BMs (controls) underwent measurement of serum carboxy-terminal telopeptide of type I collagen (CTX), tartrate-resistant acid phosphatase isoform type 5b (TRAP5b) and amino-terminal propeptide of type I collagen (PINP), carcinoembryonic antigen (CEA) and fragments of cytokeratin 19 (CYFRA 21-1. CTX (443.7 ± 945.1 vs. 402.7 ± 28.4 pg/ml, p=0.003) and PINP (75.9 ± 11.4 vs. 64.1 ± 7.5 µg/l, p=0.001) were significantly higher in patients with BMs, while the mean value of the other markers did not differ (p=NS) between cases and controls. The sensitivity, specificity and accuracy were 73.3%, 86.7% and 79.4% for CTX; 55.5%, 62.5% and 58.8% for CEA; 65.0%, 78.6% and 70.6% for CYFRA; 30.4%, 76.2% and 67.6% for TRAP5b; and 72.2%, 81.2% and 76.5% for PINP, respectively. The area under the receiver operating characteristic curve (AUC) for CTX was 0.68. In conclusion, CTX and PINP measurement can be useful in monitoring patients with NSCLC during follow-up, with the aim of detecting BMs early.

Talc pleurodesis as surgical palliation of patients with malignant pleural effusion. Analysis of factors affecting survival.

Malignant pleural effusion (MPE) is common in most patients with advanced cancer, especially in those with lung cancer, metastatic breast carcinoma and lymphoma. This complication usually leads patients to suffer from significant dyspnea, which may impair their mobility and reduce their quality of life. In patients with MPE, several interventions have been shown to be useful for palliation of the symptoms, including talc pleurodesis. The aim of this study was to evaluate prognostic factors for survival of patients with symptomatic MPE who underwent palliative video-assisted thoracoscopic (VATS) talc pleurodesis. Thirty-five patients with MPE underwent VATS, evacuation of the pleural fluid and talc pleurodesis with large-particle talc. There were 22 (62.9%) males and 13 (37.1%) females, with an overall median age of 69 years (range 42-81 years). The main causes of MPE were non-small cell lung carcinoma, breast or ovarian cancer and malignant pleural mesothelioma. The age did not differ (p=0.88) between men (68.6±11.6 years) and women (68.0±8.7 years). The mean quantity of pleural effusion was 2005.7±1078.9 ml, while the overall survival was 11.2±8.9 months. We did not find any relationship between survival and gender (log-rank test, p=0.53) or underlying malignancy associated with MPE (p=0.89, 0.48 and 0.36 for secondary cancer, lung cancer and mesothelioma, respectively). Similarly, no correlation was found between survival and age of the patients (Cox's regression, p=0.44) or quantity of pleural effusion (p=0.88). Our results show that the prognosis of patients after talc pleurodesis is independent of age, gender, type of malignancy and amount of pleural effusion, thus, suggesting the utility of treating all patients with symptomatic MPE early.

Improved quality of life in patients with malignant pleural effusion following videoassisted thoracoscopic talc pleurodesis. Preliminary results.

Malignant pleural effusion (MPE) is a common, debilitating complication of several types of advanced malignancy, which may significantly reduce the quality of life of patients. There are several options to treat MPE, including thoracentesis, placement of a long-term indwelling pleural catheter and chemical pleurodesis. The best treatment is still debated, but talc remains the agent of choice to achieve pleurodesis. Forty-six patients (28 men and 18 women; median age 67 years, range 47-82 years) with MPE related to different malignancies underwent video-assisted thoracoscopy talc pleurodesis. There were 26 (56.5%) patients with non-small cell lung cancer, 8 (17.4%) with breast cancer, 7 (15.2%) with pleural mesothelioma and 5 (10.9%) with other malignancies. The average operative time was 28±8 minutes, and the duration of chest tube drainage was 9.4±4.1 days. Side-effects were mild (temporary pain, fever for 2-3 days), affecting only three (12%) patients. Two patients (8%) died during hospitalization, due to progression of disease. Overall, pre- and postoperative Karnofsky performance index (KI) and Medical Research Council (MRC) dyspnea score were 62.1±12.2 vs. 71.3±13.2 (p=0.014), and 4.2±0.8 vs. 2.7±1.0 (p<0.001), respectively. A significant relationship between total amount of preoperative pleural effusion and both KI (R=-0.54, p=0.002) and MRC (R=0.64, p=0.0001) was found. No correlation (p=NS, log-rank test) was found between preoperative KI or MRC and underlying malignancy related to MPE. In conclusion, thoracoscopic large-particle talc pleurodesis is a feasible and effective treatment for MPE, significantly improving quality of life of patients.

Hypoxemia and hypoventilation syndrome improvement after laparoscopic bariatric surgery in patients with morbid obesity.

BACKGROUND: The objective of this study was to evaluate the relationship between oxygen partial pressure (pO(2)), awake oxymetric saturation (SpO(2)), body mass index (BMI), and percentage of excess weight loss (EWL) in extremely severe obesity (BMI >50 kg m(-2)) and hypoxemia, before and after laparoscopic Roux-en-Y gastric bypass. PATIENTS AND METHODS: A group of 11 obese patients aged 41.2 + or - 10.2 years (4 men, 7 women, median BMI=52.3 kg/m(2), range 50.2-57.1) were prospectively enrolled in the study. BMI, arterial blood gas measurements, and spirometry were obtained before and after (6 and 12 months) surgery. RESULTS: The main preoperative parameters were SpO(2)=88.3 + or - 3.9%, predicted forced vital capacity (FVC)=84.5 + or - 8.3%, predicted forced expiratory volume exhaled in one second (FEV1)=79.9+/-10.1%. No relationship (p>0.01) was found between BMI, SpO(2), and FEV1. A significant correlation between SpO(2) and both paO(2) (R=0.74, p=0.009) and EWL (R=-0.75, p=0.008) was found. Three, 6, and 12 months after surgery EWL was 18.9%, 26.4%, and 39.6% (p<0.001), respectively. At one-year follow-up SpO(2), FVC, and FEV1 were 96.2 + or - 3.2% (p<0.001), 112.3 + or - 9.9% (p<0.001), and 101.6 + or - 18.8% (p=0.003), respectively. CONCLUSION: In patients with extremely severe obesity, bariatric surgery may improve significantly both SpO(2) and spirometric parameters, and EWL represents the factor that impacted the results.

Heme oxygenase induction by cyanidin-3-O-beta-glucoside in cultured human endothelial cells.

The aim of the present research was to investigate the effect of cyanidin-3-O-beta-glucoside (C3G) on heme oxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS) and dimethylarginine dimethylamino hydrolase-2 (DDAH-2) expression in cultured endothelial cells. Different concentrations (0.00625-250 microM) of C3G were tested in order to investigate possible beneficial and harmful effects of C3G. Our data demonstrated that C3G increased the induction of eNOS and HO-1 in a dose-dependent manner. Higher concentration (62.5-250 microM) also resulted in increase of isoprostane, cGMP and PGE2 levels and in induction of iNOS with consequent oxidative stress. In conclusion, our data evidence that C3G may exert various protective effects against endothelial dysfunction, whereas potentially harmful effects of C3G appear to be limited to concentrations very difficult to be reached in physiological conditions unless there is abundant oral supplementation.

High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells.

The mechanisms involved in endothelial dysfunction are multifactorial. A correlation between oxidative stress and derangements of nitric oxide synthase (NOS) pathways in altered endothelial homeostasis has been most studied and demonstrated in different pathophysiological conditions. NOS activities are regulated by endogenous inhibitors such as asymmetric dimethyl-L-arginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since recent data demonstrated that some endothelial dysfunction may be related to reduced expression and/or activity of DDAH, the aim of the present research was to investigate the expression of DDAH-2 and NOS isoforms in high glucose-mediated oxidative stress. Endothelial cells were incubated with normal (7 mM) and high concentrations (33 mM) of D-glucose for 5 days; mannose (26 mM) plus D-glucose (7 mM) was used as osmotic control. Data obtained in the present study show that the exposure for 5 days to high glucose increases oxidative stress, reduces DDAH-2 and eNOS expression and increases iNOS expression. These results indicate that DDAH-2 and iNOS/eNOS dysregulation may play a key role in high glucose-mediated oxidative stress, suggesting that selective modulation of DDAH isoforms may result in selective inhibition/activation of NOS isoforms, thereby providing a novel strategy of approach in vascular complications of several pathologies.

Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100microM). CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoes modification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Western blot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.