Search

Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) differ for triggers, mode of start, associated symptoms, evolution, and biochemical traits. Therefore, serious attempts are underway to partition them into subgroups useful for a personalized medicine approach to the disease. Here, we investigated clinical and biochemical traits in 40 ME/CFS patients and 40 sex- and age-matched healthy controls. Particularly, we analyzed serum levels of some cytokines, Fatty Acid Binding Protein 2 (FAPB-2), tryptophan, and some of its metabolites via serotonin and kynurenine. ME/CFS patients were heterogeneous for genetic background, trigger, start mode, symptoms, and evolution. ME/CFS patients had higher levels of IL-17A (p = 0.018), FABP-2 (p = 0.002), and 3-hydroxykynurenine (p = 0.037) and lower levels of kynurenine (p = 0.012) and serotonin (p = 0.045) than controls. Changes in kynurenine and 3-hydroxykynurenine were associated with increased kynurenic acid/kynurenine and 3-hydroxykynurenine/kynurenine ratios, indirect measures of kynurenine aminotransferases and kynurenine 3-monooxygenase enzymatic activities, respectively. No correlation was found among cytokines, FABP-2, and tryptophan metabolites, suggesting that inflammation, anomalies of the intestinal barrier, and changes of tryptophan metabolism may be independently associated with the pathogenesis of the disease. Interestingly, patients with the start of the disease after infection showed lower levels of kynurenine (p = 0.034) than those not starting after an infection. Changes in tryptophan metabolites and increased IL-17A levels in ME/CFS could both be compatible with anomalies in the sphere of energy metabolism. Overall, clinical traits together with serum biomarkers related to inflammation, intestine function, and tryptophan metabolism deserve to be further considered for the development of personalized medicine strategies for ME/CFS.

Funding Acknowledgements Type of funding sources: None. Background Left ventricular thrombosis (LVT) is a possible complication following myocardial infarction (MI). Besides infarct size, risk factors for LVT include ST-elevated MI (STEMI), anterior and apical location, reduced left ventricular ejection fraction (LVEF) and the presence of microvascular obstruction. Echocardiography quantified myocardial strain has been associated with LVT following MI. Recently, global longitudinal strain, calculated with feature tracking (FT) - CMR, emerged as an independent predictor of major cardiovascular events following MI. Anyway, the relationship between abnormalities on FT-CMR and LVT following MI is still unexplored. Aim of our study is to investigate the possible association between abnormal strain on FT-CMR and LVT following apical STEMI. Methods We performed a retrospective analysis including all patients with a previous apical STEMI, who underwent CMR at our Institute between August 2013 and October 2020. Patients with ongoing anticoagulant therapy were excluded. Differences in global and segmental strain on CMR between patients with and without LVT were tested in a propensity-matched sample, using LVEF, age, gender, time from MI diagnosis and number of LV segments with transmural late gadolinium enhancement (LGE) as covariates to assign propensity score. Furthermore, difference in terms of apical to global radial strain percentual deviation (AGD), calculated as [(Global Radial Strain – Apical Radial Strain)/Global Radial Strain] * 100, was tested. Results Of 356 patients with apical STEMI undergoing CMR at our center, 37 (10.4%) were diagnosed with LVT. After performing a propensity score matching, we obtained a sample of 36 pairs, with a mean age of 65 (SD 11) years, and a median EF of 35% (IQR 27-42); 59 (82%) of them were male. A significant difference in terms of apical radial strain was found between the two groups, with a median strain of 10.75 (IQR 6.8–16.5) in patients without LVT compared to a value of 5.25 (IQR 2.7–9-6) in patients with LVT (p = 0.007). No differences were found in terms of global longitudinal, radial and circumferential strain (p = 0.19, p= 0.2 and p= 0.49 respectively) and segmental circumferential and longitudinal strain. When considering the AGD parameter, a significant difference was found between the two groups, with a median deviation of 12% (IQR -20; +48) in patients without LVT and 51% (IQR +47; +75) in patients with LVT (p= 0.0003). Furthermore, an AGD value of 26% was found to be the most accurate in terms of sensitivity and specificity applying a Receiver Operating Characteristic (ROC) curve analysis (AUC 0.74; CI 0.62-0.85). Conclusions Among patients with transmural MI involving LV apex, reduced apical radial strain on FT-CMR is associated with the presence of LV thrombosis. Furthermore, among patients developing LV thrombi, a greater apical radial strain deviation from the global one was found, with a threshold value of 26% at ROC curve analysis.

Coronavirus disease 2019 poses a serious threat to public health. The protocol developed at the Azienda Sanitaria Universitaria Friuli Centrale (Italy) is based on clinical data, laboratory tests, chest echography and HRCT. Several therapeutic options are considered, since patients vary in disease severity, evolution and co-morbidities and because so far there are no clear indications about therapeutic strategy based on randomized clinical trial. In this protocol chest echography has a central role in categorizing patient status, follow-up and decision-making.

Funding Acknowledgements Type of funding sources: None. BACKGROUND. Stress echocardiography (SE) is widely used for the assessment of left ventricular (LV) function, diagnostic and prognostic stratification of patients with coronary artery disease and for assessment of mitral and aortic valve disease. However, the assessment of the right ventricle (RV) in general, and in particular in regard to the contractile reserve of the RV in patients with tricuspid valve (TV) disease is an area that has not been previously explored in adult patients. The physiology and function of the RV is different than that of the LV and the use of SE provides the possibility to test both systolic and diastolic function of the RV in response to increased loading conditions. This can potentially be used to assess the RV function prior to surgery and to predict which subset of patients may benefit from intervention on the TV before the RV displays signs of failure PURPOSE. We therefore propose a study to investigate the potential use of SE for the assessment of RV function in adult patients. The aim is to evaluate the feasibility of RV SE in any patients with more than trivial tricuspid regurgitation (TR) and to assess the presence and degree of RV contractile reserve. METHODS. We enrolled 81 patients undergoing a phisical or dobutamine SE for CV risk stratification or chest pain. Inclusion criteria were age≥ 18 years, normal baseline RV function (FAC> 35%, TAPSE> 16 mm). Exclusion criteria were presence of RV dysfunction, pulmonary stress hypertension, positive stress test for left myocardial ischemia, presence of moderate or severe valvular disease, grade III or higher diastolic dysfunction at baseline, severe respiratory, renal or hepatic dysfunction. We evaluated the average values of TAPSE, fractional area change (FAC), S wave, sPAP (pulmonary systolic blood pressure), RV strain during baseline and at the peak of the effort. We also assessed the reproducibility of these measurement between two different expert operators (blind analysis). RESULTS. We were able to measure the RV parameters both during baseline and at the peak of the effort in all patients, demonstrating an excellent feasibility. Differences in parameters collected at baseline and at peak were assessed using paired Wilcoxon signed rank test. All variables showed a statistical significant increase (p CONCLUSIONS. RV SE proved to be feasible and showed little inter-operator variability in patients with at least trivial TR. It provided valuable informations about RV contractile reserve that may help stratifying the risk of RV failure in patients undergoing TV surgery. Abstract Figure

Background The best policy to follow when nursing homes are massively hit by SARS-CoV2 is unclear. Aim To describe COVID-19 containment in a nursing home transformed into a caring center. Methods Physicians and nurses were recruited. The facility was reorganized and connected with the laboratory of the reference hospital. Ultrasound was used to diagnose pneumonia. Patients needing intensive care were transferred to the reference hospital. Hydroxychloroquine/azithromycin/enoxaparin were used initially, while amiodarone/enoxaparin were used at a later phase. Under both regimens, methylprednisolone was added for severe cases. Prophylaxis was done with hydroxychloroquine initially and then with amiodarone. Period covered: March 22–July 31, 2020. Results The facility was reorganized in two days. Ninety-two guests of the 121 (76%) and 25 personnel of 118 (21.1%) became swab test positive. Seven swab test negative patients who developed symptoms were considered to have COVID-19. Twenty-seven patients died, 23 swab test positive, 5 of whom after full recovery. Four patients needing intensive care were transferred (3 died). Mortality, peaking in April 2020, was correlated with symptoms, comorbidities, dyspnea, fatigue, stupor/coma, high neutrophil to lymphocyte ratio, C-reactive protein, interleukin-6, pro-calcitonin, and high oxygen need (p ≤ 0.001 for all). Among swab-positive staff, 3 had pneumonia and recovered. Although no comparison could be made between different treatment and prophylaxis strategies, potentially useful suggestions emerged. Mortality compared well with that of nursing homes of the same area not transformed into care centers. Conclusion Nursing homes massively hit by SARS-CoV-2 can become caring centers for patients not needing intensive care. Supplementary Information The online version contains supplementary material available at 10.1007/s40520-020-01784-w.

Background Clinically manifest cardiac sarcoidosis (CS) has a prevalence of 5%, but is more frequent in autoptic series (25%). Diagnosis is multiparametric and relies on clinical criteria and imaging findings, although a certain diagnosis, especially in the case of isolated CS (ICS), can only be based on endomyocardial biopsy. Cardiovascular magnetic resonance (CMR) has a comprehensive role in the assessment of CS: left ventricular (LV) dysfunction and extent of late gadolinium enhancement (LGE)are important predictors of prognosis, T2 mapping provides information on disease activity and global longitudinal strain (GLS) analysis can uncover subclinical LV impairment. Purpose To assess the prevalence of CS by CMR in patients with biopsy-proven extracardiacsarcoidosis (ECS); to describe the imaging characteristics of patients with ECS and those with high clinical suspicionof ICS; to investigate the contribution of more recent techniques to the diagnosis of CS alongside traditional LGE assessment. Methods We retrospectively enrolled 84 patients (66% males, mean age 59±13 years) referred to our centreforsuspected CS (biopsy-proven ECS, n=61; clinical presentation suggestive of CS,, n=23). CMR was performed on a 1.5T scanner, with a protocol comprehensive of biventricular functional assessment and post-contrast images; T2-STIR images (n=30), native myocardial T1 mapping (n=24) and T2 mapping (n=19) were also performed in selected patients. Tissue tracking analysis was perfomed in all patients using a dedicated software. Results Based on CMR findings, 35 patients (42%) with ECS did not show cardiac involvement (SS), 26 (31%) showed both cardiac and systemic involvement (CS-SS) and 23 (27%) had evidence of ICS (ICS). 43% of patients had history of arrhythmias, but life-threatening tachyarrhythmiaswere more frequent in patients with CS (p=0.02).Patients with CS had significantly lower LVEF (p Conclusions CMR findings consistent with CS were found in 49 patients referred for suspected CS. Patients with cardiac involvement, particularly if isolated, had significantly lower LVEF, greater LV volumes and more impaired GLS. Patients with SS, despite a normal LV function, showed mildly impaired GLS, subtending subclinical cardiac involvement. Funding Acknowledgement Type of funding source: None

Background Myocarditis is an infectious or autoimmune inflammatory disease of the myocardium; diagnosis relies on the exclusion of an acute coronary syndrome, and is confirmed by endomyocardial biopsy (EMB). Prognosis is highly variable, outcome predictors are not well defined. Purpose To identify clinical, imaging and immunological predictors of death, heart transplantation (HTx) and relapse in patients with myocarditis in the pre-immunosuppression era. Methods From 1993 to 2012 we consecutively enrolled 466 patients (68% male, mean age 37±17 years), 216 with clinically suspected and 250 with EMB-proven myocarditis. All patients underwent coronary angiogram and transthoracic echocardiogram, 44% of patients underwent cardiac magnetic resonance (CMR). Circulating auto-antibodies were measured in patients' sera by indirect immunofluorescence. All patients were prospectively followed-up at the local Cardio-immunology outpatient clinic. Results After a median follow-up of 50 months (IQR 25–75), 366 patients (79%) were alive, while 42 (9%) were dead or underwent HTx; 58 were lost to follow-up. Ten-year survival free from death or HTx was overall 83%, but was lower in patients with EMB-proven myocarditis (76% vs 94% in patients with clinically suspected myocarditis, p Conclusions In the pre-immunosuppressive era, young age and a previous episode of myocarditis were independent predictors of relapse, female gender, left ventricular dysfunction at presentation and high-titre organ-specific AHA and ANA were independent predictors of death and HTx, suggesting that autoimmune features in myocarditis predict worse prognosis. Funding Acknowledgement Type of funding source: None

Background Risk stratification for death and heart transplantation (HTx) in myocarditis is complex. A random forest (RF) is a tree-based machine learning technique (MLT) which is being increasingly used for clinical data analysis; it allows the detection of complex relationships between the outcome of interest and the covariates, overcoming the limits of traditional statistical analysis (i.e. regression approaches). Purpose To assess the potential role of clinical and diagnostic features at presentation as predictors of death and HTx in biopsy (Bx)-proven myocarditis using RF. Methods From January 1993 to August 2019, we consecutively enrolled 357 patients with Bx-proven myocarditis (65% male, median age 39 years, interquartile range (IQR) 26–51). An RF approach for survival data was used. Variables included in the analysis were: histology type by Bx, NYHA, type of presentation (infarct-like, arrhythmia, heart failure), viral genome detection on Bx, serum antiheart (AHA), antiintercalated disk (AIDA), anticardiac endothelial cells (AECA), antinuclear (ANA) autoantibodies, immunosuppressive therapy, cardiac catheterisation (left ventricular enddiastolic volume (LVEDV), mean capillary wedge pressure, right and left ventricular enddiastolic pressure) and 2-D echocardiographic measures (LVEDV, left ventricular ejection fraction (LVEF) at presentation and at follow-up, right ventricular fractional area change (FAC%), right ventricular diastolic area). Results The median follow-up time was of 1352 days (IQR 423.25–2535.75). At the end of follow-up, 42 patients were dead or transplanted. The 1-year, 5-year, and 10-year survival probabilities were of 0.928, 0.854, and 0.817, respectively. The most relevant predictors of death or HTx identified by the RF algorithm (according to the variable importance measure) were histological type, NYHA, clinical presentation, LVEF, and FAC%. Among the circulating auto-antibodies AECA were found to be the most important. Histological type was the strongest predictor of death/HT (100% relative importance, (RI)), giant cell myocarditis having a lower survival probability compared to other types. The next stronger predictors were advanced (III-IV) NYHA and heart failure presentation with lower survival probabilities (90% and 84% RI respectively). AECA-positive patients had lower survival probability compared to AECA negative ones (20% RI). The RF algorithm revealed an excellent predictive performance in the correct identification of all alive patients, with only 5 dead patients being misclassified (balanced accuracy 94%). Conclusions Autoimmune features, i.e Giant cell myocarditis and AECA, as well as severity of heart failure and of left ventricular disfunction at presentation were the strongest predictors of dismal prognosis. Our RF approach provides a new automated powerful tool for accurate risk stratification for death/HTx in Bx-proven myocarditis. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Budget Integrato per la Ricerca dei Dipartimenti (BIRD, year 2019), Padova University, Padova, Italy (project Title: Myocarditis: genetic background, predictors of dismal prognosis and of response to immunosuppressive therapy.)

Background Right ventricle (RV), tricuspid anulus (TA) and right atrium (RA) dilatation, are listed among the supportive signs to grade severe tricuspid regurgitation (TR) according to current EACVI and ESC guidelines. However, at present, there is no cut-off value to define RV, RA and TA dilatation associated to severe TR. Purpose Accordingly, we sought to identify the threshold values of RV, RA and TA size associated to severe TR. Methods 302 patients (59 ± 13 years, 54 % women) with functional TR underwent three- (3D) and two-dimensional (2D) echocardiography to obtain: 3D RV end diastolic volume (RVEDVi) indexed for body surface area (BSA), 3D RV end systolic volume indexed for BSA (RVESVi), 3D RA max volume indexed for BSA (3DRAi), 2D RA systolic volume indexed for BSA (3DRAi), 2D RV basal diameter (2DRVd), 2D RV basal diameter indexed for BSA (2DRVdi), 2D TA measured in the apical 4-chamber view and 2D TA measured in the apical 4-chamber view indexed for BSA. To identify the threshold values of the parameters that discriminate patients with right chamber enlargement associated to severe TR, we selected the probability which returns the best sum of sensitivity and specificity on the ROC curve of the model. Results According to EACVI multiparametric approach, 50/302 pts (17%) were found to have severe TR. As shown in Figure, 3DRAi > 45 ml/m2 and 2DRAi > 45 ml/m2 identified patients with RA enlargement associated to severe TR. RVEDVi and RVESVi did not show any predictive value for severe TR. Conversely, 2DRVd > 52 mm (or >30 mm/m2) was associated to severe TR. 2DTA > 42 mm ( or >24 mm/m2) was the selected threshold value for TA dilatation. Conclusions Our study provided the threshold values to define the right chamber and TA dilatation associated to severe TR. Implementation of those values in current guidelines can help clinicians to improve their accuracy to identify patients with severe TR. Abstract 47 Figure.

Background The right atrium (RA) is a highly dynamic chamber with 3 mechanical functions (reservoir, conduit, booster pump) and prognostic implications in heart failure (HF) and pulmonary hypertension (PH). However, RA function and its interplay with the right ventricular (RV) performance in patients (pts) with reduced left ventricular ejection fraction (LVEF) and without PH remain to be clarified. Methods We used three-dimensional echocardiography to study 55 pts (61 ± 14 years, 43 men) with LVEF Results Mean LVEF was 30 ± 7%. Mean echo-derived pulmonary vascular resistance was 1.64 ± 0.54 Wood units. 28 pts (51%) had reduced RA reservoir function (total EF = 34 ± 9%), 34 pts (62%) had reduced RA conduit function (passive EF = 15 ± 4%), and 10 pts (18%) had reduced RA pump function (active EF = 11 ± 3%). Pts with reduced RA reservoir function showed larger RV end-systolic volume (RVESV 124 ± 48ml vs. 90 ± 32ml; p = 0.004) and lower RVEF (38 ± 8% vs. 46 ± 6%; p RVESV was positively correlated with total (r2 = 0.47, p Conclusions RA dysfunction is not uncommon in pts with reduced LVEF, even in the absence of PH. In these pts, RA function is associated with significant changes in RV function. The RA acts as a dynamic modulator of RV pump function by redistributing RV filling and ejection force among reservoir, conduit and pump functions in the setting of altered hemodynamics. The clinical and prognostic significance of RA function in pts with reduced LVEF warrant further studies.

Background According to current EACVI guidelines, right ventricle (RV), tricuspid anulus (TA) and right atrium (RA) dilatation are supportive signs to identify severe functional tricuspid regurgitation (TR) by echocardiography. However, the ranking by which those parameters should be considered to identify severe TR remains to be clarified. Purpose Accordingly, the aim of this study is to compare RV, RA and TA association with severe TR and to rank them in order of importance to predict severe TR. Methods 302 patients (59 ± 13 years, 54 % women) with functional TR underwent two- and three-dimensional echocardiography. Using the nonparameteric Variable Importance (VIMP) software package, we assessed the relative importance of 6 differerent parameters (indexed by body surface area) to identify severe TR: 3D RV end diastolic volume (RVEDVi), 3D RV end systolic volume (RVESVi), 3D RA max volume (3DRAi), 2D RA systolic volume (3DRAi), 2D RV basal diameter (2DRVdi) and 2D TAi measured in the apical 4-chamber view. Results According to EACVI multiparametric approach, 50/302 pts (17%) were found to have severe TR. 3DRAi (VIMP = 0.075) was the most important predictor of severe TR. 2DRVdi (VIMP= 0.005) was the second most important parameter and was the only parameter of RV dilation (RVEDVi= -0.0011 and RVESVi= -0.0012) associated to severe TR. Also, 2DRAi (VIMP= 0.023), and 2D TAi (VIMP= 0.004) showed good predictive ability. Conclusions Among the various right heart structures undergoing remodeling in patients with functional TR, RA dilation was the most important predictor of severe TR. Also the RV basal diameter, but not the volumes, was a predictor of severe TR. This underlines the importance of the shape, more than the volume of the RV as a predictor of severe TR.

Background Morbidity and mortality associated with severe tricuspid regurgitation (TR) have prompted interest in new corrective transcatheter procedures. However, to properly select patients for interventional procedures, and to assess their effectiveness, a reliable and reproducible grading system of TR severity is mandatory. However, the cut-off values used by current guidelines to differentiate among mild, moderate and severe TR lack clinical validation. Purpose We aimed to obtain the threshold values of the currently recommended quantitative echocardiographic parameters used to grade TR severity using pts’ outcome as a reference. Methods 296 pts, with at least mild TR and complete 2D, 3D and Doppler echocardiographic study, were enrolled and assessed for potential confounders: age, NYHA class, left ventricular ejection fraction, coexistent valvular heart disease and right ventricular (RV) systolic pressure. Average diameter of the vena contracta (VCavg), effective regurgitant orifice area (EROA), regurgitant volume (RVol) and regurgitant fraction (RF) were obtained to grade TR severity. Median follow-up was 47 (17-80) months. The primary composite endpoint was the occurrence of death of any cause or hospitalization for right heart failure (RHF). Survival curves for the composite endpoint were divided in quartiles at median follow-up. Cut-off values for the echo parameters were derived to grade mild (below the 1st quartile), moderate (between 1st and 3rd quartiles), and severe (above the 3r quartile) TR. Results 33 deaths and 72 hospitalizations for RHF occurred. Event-free rate from death or RHF at the end of follow-up was 14%, 46% and 93% in pts with severe, moderate, and mild TR, respectively. Differences reached statistical significance early (at 1 month), and lasted during the whole follow-up period (Figure). The new threshold values for mild, moderate and severe TR are summarized in Table. Conclusions Partition values of quantitative echo-Doppler parameters used to grade mild, moderate and severe TR according to pts’ clinical outcome are significantly lower than those currently reported in guidelines. Further studies are needed to test if these new threshold values for severe TR will translate in earlier referral of pts to valve repair and improved prognosis. Mild Moderate Severe VCavg 6 mm EROA 0.30 cm² R Vol 30 ml RF 45% Abstract 38 Figure.

Background Cardiovascular magnetic resonance (CMR) is an accurate diagnostic tool providing detailed myocardial tissue characterization. Correlates of late gadolinium enhancement (LGE) with clinical and diagnostic features are poorly defined in endomyocardial biopsy (EMB)-proven myocarditis. Purpose We sought to identify clinical, laboratory and imaging correlates of LGE in patients with EMB-proven myocarditis. Methods We analyzed our prospective cohort of patients with EMB-proven myocarditis (n=366) to identify patients who underwent CMR (n=154, aged 39±13, 108 males). Presence of LGE was qualitatively assessed. CMR and EMB were performed in all cases at the time of hospitalization. Clinical, laboratory, and imaging features at diagnosis were analyzed to identify correlates of LGE presence. Results Demographic characteristics (age, gender), cardiovascular risk factors (hypertension, diabetes) and cardiovascular therapy did not differ between patients with and without LGE. Patients with history of myocarditis tended to have a higher prevalence of LGE (25 vs 4 patients, p=0.07). There was a trend towards a higher prevalence of LGE in patients presenting with heart failure or acute coronary syndrome (p=0.07) as opposed to those presenting with arrhythmias. Patients with LGE were more symptomatic in the 6 months preceding diagnosis (palpitations, p=0.03, chest pain p=0.02). Clinical left and right ventricular systolic dysfunction at presentation were more common in patients with LGE (p=0.02, p=0.02 respectively). LGE presence failed to distinguish patients according to EMB findings (active vs. borderline myocarditis) (p=0.66) or to the histological type of myocarditis. Troponin I levels were higher in patients with LGE (p=0.03). There was no difference in bi-ventricular volumes and function, as assessed both by echocardiography and heart catheterization. There was no correlation between anti-heart antibodies and LGE presence, nor were patients with LGE more likely to receive immunosuppressive therapy; however, response to immunosuppression tended to be more common in patients without LGE (13/13 vs 29/38 patients, p=0.09). The rate of heart transplant, death and myocarditis relapse did not differ between patients with and without LGE. Conclusions Presence of LGE on CMR was associated with longer symptom duration before diagnosis, presentation with heart failure and higher troponin release, but failed to correlate with specific EMB features or myocarditis aetiopathogenetic markers. Acknowledgement/Funding None

Background Myocarditis is an insidious and potentially fatal illness with different clinical presentations and an unpredictable course. Prompt recognition of high risk patients is of paramount importance in preventing major adverse events. Purpose To identify predictors of dismal prognosis in a large cohort of patients with biopsy proven myocarditis. Methods Univariate analysis was used to identify predictors of death and heart transplant in a prospective cohort of 366 patients with biopsy proven myocarditis (aged 38±17, male 66%) using student's test and contingency tables as appropriate. Results At the time of follow up 46 patients (13%) were dead or received heart transplant (DHTX), 283 (77%) were alive (A) and 37 (10%) lost at follow up. Age at presentation was 33±20 y in DHTX v.s 39±15 in A cohort (p=0.057). Clinical features predicting adverse prognosis included female gender (p=0.002), heart failure at presentation (p=0.000), NYHA class II to IV (p=0.000). Clinical and radiographic signs of both left and right heart failure suggested worse outcome (p=0.000) as well as ongoing anticoagulation therapy (p=0.009). On ECG right (R) or left (L) axis deviation was a strong predictor of events (p=0.000). From an echocardiography perspective the presence of mild to severe mitral regurgitation (p=0.03), reduced left ventricular systolic function (FE) (p=0.000), reduced right ventricular fractional area change (FAC) (p=0.035) was strongly correlated to death or heart transplant. On cardiac catheterization the variables predicting unfavourable outcome included reduced left ventricular systolic pressure (LVSP) (p=0.000), reduced mean aortic pressure (mAP) (p=0.002), increased mean right atrial pressure (RAP) (p=0.001), FE on angiography (p=0.000). On cardiac biopsy (Bx) negative predictors were giant cell histology type (p=0.000) and PCR positive for viral genome (p=0.02) particularly for parvovirus B19 (p=0.04), adenovirus (p=0.04), and Epstein Barr virus (EBV) (p=0.03). See Tab 1 Table 1 Conclusion Female gender, HF like presentation, reduced LV and RV systolic function, R or L axis deviation on ECG, presence of viral PCR or giant cell histology on Bx, reduced LVSP and mAP; increased RAP may be useful parameters to identify high risk patients on presentation. This may increase clinical efforts and surveillance in this subgroup in order to reduce the incidence of major adverse events.

Background Sarcoidosis is an immune-mediated disease; cardiac involvement, a granulomatous form of myocarditis, is under-recognised and prognostically relevant, as it can present with significant morbidity and mortality. Anti-heart autoantibodies (AHA) and anti-intercalated disk autoantibodies (AIDA) are reliable autoimmune markers in non-sarcoidosis myocarditis forms. Purpose The aim of this study was to assess the potential role of serum AHA and AIDA in cardiac sarcoidosis. Methods This is a cross-sectional study on a series of 29 patients with biopsy proven extra-cardiac sarcoidosis and with biopsy-proven or clinically suspected cardiac involvement, who were tested for AHA and AIDA. Patients were recruited in two recruiting tertiary centres, in USA and Italy. AHA and AIDA were detected by indirect immunofluorescence on human myocardium and skeletal muscle. Controls included sera from patients with non-inflammatory cardiac disease (NICD) (n=160), with ischemic heart failure (IHF) (n=141) and normal blood donors (NBD) (n=270). Results The frequencies of AHA and of AIDA were higher in sarcoidosis (86%; 62%) than in NICD (8%; 4%), IHF (7%; 2%), NBD (9%; 0%) (p=0.0001; p=0.0001 respectively). Sensitivity and specificity were: 86% and 92% for positive AHA and 62% and 98% for positive AIDA, respectively (see figure). Figure 1 Conclusions The detection of serum AHA and AIDA in biopsy-proven or clinically suspected cardiac sarcoidosis supports the involvement of heart-specific autoimmunity in the majority of our cases and may provide a novel non invasive diagnostic marker.

Background Biopsy-proven myocarditis may be infectious or autoimmune. Risk stratification in biopsy-proven myocarditis and the role of immunosuppressive therapy in autoimmune forms have not been completely defined. Purpose To identify clinical, instrumental and immunological predictors of death, cardiac transplantation and relapse in a prospective cohort of 314 biopsy-proven myocarditis patients, and describe the effect of immunosuppressive treatment on secondary outcome measures, e.g. left ventricular ejection fraction (LVEF), in a subgroup of 45 consecutive patients with biopsy-proven autoimmune myocarditis diagnosed in our Cardiology Clinic. Methods Univariate and multivariate Cox regression analysis were used to identify predictors of death, heart transplant, and relapse in a cohort of 314 patients with biopsy-proven myocarditis (male 75%, median age 37). Actuarial survival free from death or transplant was calculated by the Kaplan-Meier method. Results Actuarial survival free from death or heart transplantation was 83% at 5 years. Among the clinical, instrumental and immunological features at diagnosis, independent predictors of death or heart transplantation by multivariable analysis were a lower transthoracic echocardiographic biplane LVEF% (p=0.001) and high serum titre for anti-nucler (ANA) and anti-cardiac endothelial cell autoantibodies (AECA). The only independent predictor of relapse was previous history of myocarditis. Immunosuppressive therapy was associated with a significantly favorable effect on LVEF (LVEF pre-therapy 37% (26; 50 interquartile range) vs. LVEF post-therapy 59% (48; 65 interquartile range), respectively, p=0.000). Conclusions In biopsy-proven myocarditis left ventricular dysfunction at diagnosis and autoimmune pathogenesis are associated with dismal prognosis, immunosuppressive therapy with improved LVEF in autoimmune patients.

In patients with type 1 diabetes (T1D), the prevalence of non-alcoholic fatty liver disease (NAFLD) ranges from 10 to 53% and contrasting evidence suggests that vitamin D deficiency may favor liver fat accumulation. Here, we investigated the association between vitamin D status and NAFLD in adults with T1D. 220 consecutive adult T1D patients on multiple daily injections or continuous subcutaneous insulin infusion and not taking calcium or vitamin D supplements were included. Patient characteristics, 25(OH)D serum levels, and metabolic parameters were analyzed. Vitamin D status was defined as sufficiency ( ≥ 75 nmol/L; 30 ng/ml), insufficiency (50–75 nmol/L; 20–30 ng/ml), or deficiency (

In resource-limited countries, the diagnosis of pulmonary tuberculosis (TB) is based on clinical findings, chest radiography and the demonstration of acid-fast bacilli in sputum. Few data are available on the use of ultrasound (US) to diagnose pulmonary TB. Chest US was performed in patients with lung TB from a rural African setting, to look for signs of the disease and to clarify the role US may have in the diagnosis of pulmonary TB. Sixty adult patients diagnosed with lung TB underwent chest US. All patients had abnormal findings. The most frequent was a subpleural nodule (SUN), which was mostly multiple and also found in radiologically normal areas. Other findings were lung consolidations, cavitations, miliary patterns made of miniature SUNs, and pleural and pericardial effusions. Chest US is a complementary tool in evaluating patients with suspected lung TB in resource-limited settings where the disease has high prevalence.

Introduction: Emerging and reemerging viral infections represent a major concern for human and veterinary public health and there is an urgent need for the development of broad-spectrum antivirals. Areas covered: A recent strategy in antiviral research is based on the identification of molecules targeting host functions required for infection of multiple viruses. A number of FDA-approved drugs used to treat several human diseases are cationic amphiphilic drugs (CADs) that have the ability to accumulate inside cells affecting several structures/functions hijacked by viruses during infection. In this review we summarized the CADs’ chemical properties and effects on the cells and reported the main FDA-approved CADs that have been identified so far as potential antivirals in drug repurposing studies. Expert commentary: Although there have been concerns regarding the efficacy and the possible side effects of the off-label use of CADs as antivirals, they seem to represent a promising starting point for the development of broad-spectrum antiviral strategies. Further knowledge about their mechanism of action is required to improve their antiviral activity and to reduce the risk of side effects.

Surfactant protein B (SP-B) plays a key role in surfactant homeostasis affecting its biophysical properties and physiological function. Recently, a method to measure SP-B amount and kinetics from tracheal aspirates (TAs) became available. The main objective of this study was to improve the critical steps of the procedure to obtain a better SP-B sensitivity. We administered a 24 h continuous infusion of 1 mg/kg/h of 113 C-leucine to ten newborn infants. SP-B was isolated from serial TAs and its fractional synthesis rate, secretion time, peak time and half life were derived from 13 C enrichment curves obtained by gas chromatography mass spectrometry. SP-B amount in TAs was also assessed. During the extraction step, acidification and organic solvent ratio optimization doubled the recovery of SP-B from TAs, so did the elongation of the propylation time (from 20 min to 1 h) with enhanced leucine derivatization yield. Measurement of 13 C leucine enrichments, and therefore all SP-B kinetics parameters, were successfully calculated in all TAs samples due to the increase of SP-B yield. SP-B amount was 0.29 (0.16–0.41) % of total phospholipids with a minimum value of 0.08% belonging to one of the respiratory distress syndrome (RDS) patients. In conclusion, this new procedure enables accurate determination of SP-B kinetics even in the presence of low protein amount like in preterm RDS patients. Copyright © 2012 John Wiley & Sons, Ltd.

Pulmonary hypoplasia and hypertension account for significant morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH). Whether CDH is associated with surfactant dysfunction remains controversial. Therefore, we measured disaturated phosphatidylcholine (DSPC) and surfactant protein (SP)-B concentration in tracheal aspirates and their synthesis rate in infants with CDH compared to infants without lung disease. (2)H2O as a precursor of DSPC and 1-(13)C-leucine as a precursor of SP-B were administered to 13 infants with CDH and eight controls matched for gestational age. DSPC and SP-B were isolated from tracheal aspirates, and their fractional synthesis rate was derived from (2)H and (13)C enrichment curves obtained by mass spectrometry. DSPC and SP-B amounts in tracheal aspirates were also measured. In infants with CDH, SP-B fractional synthesis rate and amount were 62±27% and 57±22% lower, respectively, than the value found in infants without lung disease (p

Disaturated-phosphatidylcholine (DSPC) and phosphatidylglycerol (PG) are respectively the first and the third most abundant phospholipid in human alveolar surfactant. Their concentration decreases in airway surfactant of adults and infants with respiratory distress syndrome and cystic fibrosis. In this study, we used mass spectrometry (IRMS) to investigate the turnover of DSPC and PG in tracheal aspirates (TA) obtained from infants with normal or diseased lungs. We studied eight infants requiring mechanical ventilation: two with no lung disease, four with diaphragmatic hernia, one with ATP-binding cassette sub-family A member 3 heterozygote mutation and one with sepsis. Patients received deuterated water for 48 h as metabolic precursors of palmitate-DSPC and palmitate-PG. Serial TAs were obtained every 6 h for five days or until extubation. DSPC and PG were isolated from TA by column and high-performance thin layer chromatography. Deuterium enrichments of palmitate-DSPC and PG residues were measured by IRMS coupled with a gas chromatographer. Median secretion time (ST), peak time (PT) and fractional synthesis rate (FSR) were 3.7 [0.9– 13.4] h, 71.0 [52.2 – 85.2] h and 6.6 [6.3 – 11.1] %/day for DSPC and 19.3 [6.4 – 22.8] h, 49.0 [33.0 – 52.5] h and 5.8 [4.8 – 10.9] %/day for PG. This study shows that it is feasible to use deuterium derived from body water to trace simultaneously airway surfactant DSPC and PG in humans. When compared within the same patient, DSPC and PG had similar fractional synthesis rates, but PG had a shorter PT, suggesting differences in the life cycle of these essential surfactant components. Copyright © 2011 John Wiley & Sons, Ltd.

Thirty years after its introduction, the use of continuous subcutaneous insulin infusion (CSII) keeps increasing, especially among children and adolescents. The technique, when used properly, is safe and effective.Compared with traditional NPH-based multiple daily injections (MDI), CSII provides a small but clinically important reduction of HbA(1c) levels, diminishes blood glucose variability, decreases severe hypoglycaemic episodes and offers a better way to cope with the dawn phenomenon.Insulin analogues have improved the treatment of diabetes, eroding part of the place previously occupied by CSII, but CSII still remains the first option for patients experiencing severe hypoglycaemic episodes, high HbA(1c) values or marked glucose variability while being treated with optimized MDI. Furthermore CSII is better than MDI considering the effects on quality of life and the possibility to adjust insulin administration according to physical activity or food intake.CSII may be limited by cost. Present estimates suggest that CSII may be cost-effective just for patients experiencing a marked improvement in HbA(1c) or a decrease in severe hypoglycaemic episodes, but the effects on quality of life are difficult to measure.CSII does not merely imply wearing an external device; it requires a multidisciplinary team, intensive patient education and continuous follow up.

Amiodarone (AMI) is a potent antiarrhythmic agent; however, its clinical use is limited due to numerous side effects. In order to investigate the structure--cytotoxicity relationship, AMI analogues were synthesized, and then, using rabbit alveolar macrophages, were tested for viability and for the ability to interfere with the degradation of surfactant protein A (SP-A) and with the accumulation of an acidotropic dye. Our data revealed that modification of the diethylamino-beta-ethoxy group of the AMI molecule may affect viability, the ability to degrade SP-A and vacuolation differently. In particular, PIPAM (2d), an analogue with a piperidyl moiety, acts toward the cells in a similar manner to AMI, but is less toxic. Thus, it would be possible to reduce the cytotoxicity of AMI by modifying its chemical structure.

Summary The Ebola outbreak that has devastated parts of west Africa represents an unprecedented challenge for research and ethics. Estimates from the past three decades emphasise that the present effort to contain the epidemic in the three most affected countries (Guinea, Liberia, and Sierra Leone) has been insufficient, with more than 24 900 cases and about 10 300 deaths, as of March 25, 2015. Faced with such an exceptional event and the urgent response it demands, the use of randomised controlled trials (RCT) for Ebola-related research might be both unethical and infeasible and that potential interventions should be assessed in non-randomised studies on the basis of compassionate use. However, non-randomised studies might not yield valid conclusions, leading to large residual uncertainty about how to interpret the results, and can also waste scarce intervention-related resources, making them profoundly unethical. Scientifically sound and rigorous study designs, such as adaptive RCTs, could provide the best way to reduce the time needed to develop new interventions and to obtain valid results on their efficacy and safety while preserving the application of ethical precepts. We present an overview of clinical studies registered at present at the four main international trial registries and provide a simulation on how adaptive RCTs can behave in this context, when mortality varies simultaneously in either the control or the experimental group.

Ebola virus disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work, we investigated how amiodarone interferes with Ebola virus infection. Wild-type Sudan ebolavirus and recombinant vesicular stomatitis virus, pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate that the antiviral activity is correlated with drug ability to accumulate into and interfere with the endocytic pathway. Considering that it is well tolerated, especially in the acute setting, amiodarone appears to deserve consideration for clinical use in EVD., The anti-arrhythmic drug amiodarone, and one of its active metabolites interfere with the early steps of Ebola virus life cycle by blocking the fusion step between the viral envelope and the endosomal membrane.

In patients with chronic diseases education should improve knowledge about the disease and increase certainty in knowledge. We present here a technique to measure changes in certainty after an educational intervention. For this purpose, before and after a course, patients answer a questionnaire in which answers are accompanied by an estimate of the degree of certainty. Answers are then assigned to areas of knowledge defined a priori: mastered (certaintyor = 90%, correctnessor = 90%), hazardous (certaintyor = 90%, correctnessor = 50%), uncertain (certaintyor = 50%, correctnessor = 90%) and residual. Finally differences in the distribution of answers among different areas are analysed statistically. Using this technique in a group of patients with type I diabetes who followed a course on insulin use, we found significant changes in the distribution of answers among different areas of knowledge. Thus changes in certainty can be analysed quantitatively and used to evaluate better the effect of therapeutic education.

Previous studies have shown that surfactant apoprotein A (SP-A) and natural or synthetic surfactant can modulate the release of pro-inflammatory cytokines from alveolar mononuclear phagocytes. The aim of this study was to assess whether SP-A or Surfactant (Surf) from patients with pulmonary alveolar proteinosis (PAP) can affect the release of two chemokines (interleukin (IL)-8 and monocyte chemtactic peptide (MCP)-1) from human monocytes and rat lung type-II cells. In addition IL-8 and MCP-1 levels were assessed in the brochoalveolar lavage fluid (BALF) of seven patients with PAP and compared with those in a group of control subjects (n=5). SP-A, tested over a wide range of concentrations, significantly increased IL-8 and MCP-1 release from monocytes. SP-A retained its activity after collagenase digestion, but was not active after heat treatment. The release of IL-8 by monocytes was also stimulated by Surf. Finally, median BALF IL-8 and MCP-1 levels in PAP patients were significantly higher than in controls (9.50 and 9.51 pg x mL(-1) in controls versus 151.95 and 563.70 pg x mL(-1) in PAP, respectively) and significantly correlated with SP-A concentrations in BALF. Overall the results of this study support the view that the high content of alveolar surfactant apoprotein A may contribute to the upregulation of chemokine release in pulmonary alveolar proteinosis, thus contributing to airway inflammation.

Background: Term newborns with pneumonia show a reduced pulmonary compliance due to multiple and ill-defined factors. Surfactant proteins’ (SPs) changes could have a role in the reduced compliance but the matter is still unsettled. The aim of this study was to clarify the meaning of SPs changes during pneumonia in term newborns. Methods: In 28 term ventilated newborns, 13 with pneumonia and 15 with no lung disease, we measured SP-B, SP-A, disaturated-phosphatidylcholine (DSPC), and total phospholipids (PL) concentrations in tracheal aspirates at intubation and close to extubation. We also measured DSPC kinetics using (U-13C-PA)dipalmitoyl-phosphatidylcholine. Results: At baseline, SP-B, expressed as % of PL, was significantly different between the groups, being 3.5-fold higher in pneumonia than controls. Conversely, SP-A did not vary between the groups. At extubation, SP-B and SP-A concentrations had decreased significantly in newborns with pneumonia, while there was no significant change in controls. DSPC t1/2 was significantly shorter in the pneumonia group (11.8 (5.5–19.8) h vs. 26.6 (19.3–63.6) h, P = 0.011). Conclusion: In term newborns with pneumonia, SP-B increases with respect to PL, and DSPC is turned over at a faster rate. Disease’s resolution is associated with the restoration of the normal ratio between SP-B and PL. Supplementary information The online version of this article (doi:10.1038/pr.2015.123) contains supplementary material, which is available to authorized users.

Surfactant protein C (SP-C) is deemed as the surfactant protein most specifically expressed in type II alveolar epithelial cells and plays an important role in surfactant function. SP-C turnover in humans and its meaning in the clinical context have never been approached. In this study, we used mass spectrometry to investigate SP-C turnover in humans. We studied four infants and eight adults requiring mechanical ventilation. All patients had no lung disease. Patients received a 24-h continuous infusion of (13)C-leucine as precursor of SP-C, and serial tracheal aspirates and plasma samples were obtained every 6 h till 48 h. SP-C was isolated from tracheal aspirates by sorbent-phase chromatography. (13)C-leucine SP-C enrichment could be successfully measured in three infant and in four adult samples by using mass spectrometry coupled with a gas chromatographer. Median SP-C fractional synthesis rate, secretion time, and peak time were 15.7 (14.1-27.5)%/day, 6.0 (4.7-11.5) h, and 24 (20-27) h. In conclusion, this study shows that it is feasible to accurately determine SP-C turnover in humans by stable isotopes.

Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.

Graphical abstract Amiodarone, having basic pKa and high water solubility at acidic pH, accumulates within late endocytic compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking and inducing a Niemann-Pick C-like phenotype., Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control.

Background Patients with Adult Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover. Objectives To analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls). Methods 2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured. Results 1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p < 0.01), while the fractional synthesis rate of surfactant protein-B was not different. 3) In ARDS/ALI patients the concentrations of disaturated-phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively). Conclusions 1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

Background: While surfactant protein composition of bronchoalveolar lavage (BAL) has been described in lung diseases of adults and premature infants, scanty data are available on surfactant protein composition beyond the neonatal period.

Aims The superiority of continuous subcutaneous insulin infusion (CSII) over multiple daily injections (MDI) with glargine is uncertain. In this randomized cross-over study, we compared CSII and MDI with glargine in patients with Type 1 diabetes well controlled with CSII. The primary end-point was glucose variability. Methods Thirty-nine patients [38.1 ± 9.3 years old (mean ± sd), diabetes duration 16.6 ± 8.2 years, glycated haemoglobin (HbA1c) 7.6 ± 0.8%], already on CSII for at least 6 months, were randomly assigned to CSII with lispro or MDI with lispro and glargine. After 4 months they were switched to the alternative treatment. During the last month of each treatment blood glucose variability was analysed using glucose standard deviation, mean amplitude of glycaemic excursions (MAGE), lability index and average daily risk range (ADRR). As secondary end-points we analysed blood glucose profile, HbA1c, number of episodes of hypo- and hyperglycaemia, lipid profile, free fatty acids (FFA), growth hormone and treatment satisfaction. Results During CSII, glucose variability was 5–12% lower than during MDI with glargine. The difference was significant only before breakfast considering glucose standard deviation (P = 0.011), significant overall using MAGE (P = 0.016) and lability index (P = 0.005) and not significant using ADRR. Although HbA1c was similar during both treatments, during CSII blood glucose levels were significantly lower, hyperglycaemic episodes were fewer, daily insulin dose was less, FFA were lower and treatment satisfaction was greater than during MDI with glargine. The frequency of hypoglycaemic episodes was similar during both treatments. Conclusions During CSII, glucose variability is lower, glycaemic control better and treatment satisfaction higher than during MDI with glargine.

Amiodarone interferes with the endocytic pathway, inhibits proteolysis, and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles, and functional consequences of amiodarone accumulation remain unclear. Our objective was to study amiodarone uptake, clarify the origin of vacuoles, and investigate the effect of amiodarone on the life cycle of the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Using alveolar macrophages, we studied uptake of (125)I-amiodarone and (125)I-B2, an analog lacking the lateral group diethylamino-beta-ethoxy, and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. We found that (1) amiodarone associates with different cell membranes and accumulates in acidic organelles; (2) the diethylamino-beta-ethoxy group is an important determinant of uptake; (3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; (4) amiodarone inhibits the spreading in vitro of SARS coronavirus; and (5) trypsin cleavage of the viral spike protein before infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. We conclude that amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.

Background Pulmonary alveolar proteinosis (PAP) is a rare disease characterised by accumulation of lipoproteinaceous material within alveoli, occurring in three clinically distinct forms: congenital, acquired and secondary. Among the latter, lysinuric protein intolerance (LPI) is a rare genetic disorder caused by defective transport of cationic amino acids. Whole Lung Lavage (WLL) is currently the gold standard therapy for severe cases of PAP. Case presentation We describe the case of an Italian boy affected by LPI who, by the age of 10, developed digital clubbing and, by the age of 16, a mild restrictive functional impairment associated with a high-resolution computed tomography (HRCT) pattern consistent with pulmonary alveolar proteinosis. After careful assessment, he underwent WLL. Conclusion Two years after WLL, the patient has no clinical, radiological or functional evidence of pulmonary disease recurrence, thus suggesting that WLL may be helpful in the treatment of PAP secondary to LPI.

Background In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes. Methods We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of 13C-dipalmitoyl-phosphatidylcholine, we measured the 13C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds. Results In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08). Conclusion In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.

Diabetic patients should understand their disease correctly and be sure of what they know, but certainty is rarely considered by educators. Furthermore little is known about how certainty changes with time after an educational intervention. To clarify this, in 38 patients with type 1 diabetes (0.3-36 years duration) we analysed the effect of a course on insulin use by administering a questionnaire before the course, after the course and 1 and 3 years later.Answers, accompanied by a subjective estimate of the degree of certainty, were assigned to mastered knowledge (certaintyor=90%, correctnessor=90%), hazardous knowledge (certaintyor=90%, correctnessor=50%), uncertain knowledge (certaintyor=50%, correctnessor=90%) and residual knowledge (total-[mastered+hazardous+uncertain]). Answers were then counted and changes in distribution among areas were analysed by the chi2 test. We also followed the fate of wrong answers.The course increased mastered knowledge, while other types of knowledge decreased. With time mastered knowledge decreased, patients losing both correctness and certainty. The loss affected declarative knowledge, based purely on theory, more than procedural knowledge, which concerns the way things are done. Wrong answers, mostly given with high degree of certainty, were heterogeneous since some became correct after the course, some remained wrong, some became wrong after the course, some became mistaken after having been corrected earlier.The analysis of certainty helps in evaluating patient's knowledge; programmes tending to improve procedural knowledge are more likely to have long lasting effects; wrong answers need to be considered on a individual basis.

Backgroundα1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series ofin vitroexperiments aimed at investigating the nature and consequences of such an interaction.Methods and resultsAt an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.ConclusionWe conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.

Human surfactant protein C (hSP-C(1-197)) is synthesized as a 197 amino acid proprotein and cleaved to a mature 3.7 kD form. Although interstitial lung disease in patients with mutations of the hSP-C gene is becoming increasingly recognized, the mechanisms linking molecular events with clinical pathogenesis are not fully defined. We describe a full-term infant with respiratory insufficiency associated with a spontaneous heterozygous mutation resulting in a substitution of lysine for glutamic acid at position 66 (= E66K) of the proximal hSP-C COOH flanking propeptide. Lung histology and biochemical studies of the index patient (hSP-C(E66K)) revealed nonspecific interstitial pneumonia, increased alveolar total phospholipid lacking phosphatidylglycerol, and increased surfactant protein A. Localization of proSP-C from lung sections prepared from this patient using immunofluorescence and immunogold electron microscopy revealed abnormal proSP-C staining in endosomal-like vesicles of type II cells distinct from SP-B. To evaluate the effect of the E66K substitution on intracellular trafficking of proSP-C, fusion proteins consisting of enhanced green fluorescent protein (EGFP) and hSP-C(1-197) (wild type) or mutant hSP-C(E66K) were generated and transfected into A549 cells. EGFP/hSP-C(1-197) was expressed within CD-63-positive, EEA-1-negative vesicles, whereas EGFP/hSP-C(E66K) localized to EEA-1 positive vesicles. The E66K substitution is representative of a new class of SP-C mutation associated with interstitial lung disease that is diverted from the normal biosynthetic pathway. We propose that, similar to other storage disorders, lung injury results from induction of a toxic gain of function induced by the mutant product that is subject to genetic modifiers and environmental influences.

Amiodarone, an antiarrhythmic drug toxic toward the lung, is metabolized through sequential modifications of the diethylaminoethoxy group to mono- N-desethylamiodarone (MDEA), di- N-desethylamiodarone (DDEA), and amiodarone-EtOH (B2-O-EtOH), whose effects on lung cells are unclear. To clarify this, we exposed rabbit alveolar macrophages to analogs with different modifications of the diethylaminoethoxy group and then searched for biochemical signs of cell damage, formation of vacuoles and inclusion bodies, and interference with the degradation of surfactant protein A, used as a tracer of the endocytic pathway. The substances studied included MDEA, DDEA, and B2-O-EtOH, analogs with different modifications of the diethylaminoethoxy group, fragments of the amiodarone molecule, and the antiarrhythmic agents dronedarone (SR-33589) and KB-130015. We found the following: 1) MDEA, DDEA, and B2-O-EtOH rank in order of decreasing toxicity toward alveolar macrophages, indicating that dealkylation and deamination of the diethylaminoethoxy group represent important mechanisms of detoxification; 2) dronedarone has greater, and KB-130015 has smaller, toxicity than amiodarone toward alveolar macrophages; and 3) the benzofuran moiety, which is toxic to liver cells, is not directly toxic toward alveolar macrophages.