Complete genome sequencing of a genotype 3 hepatitis E virus strain identified in a swine farm in Italy

In this study, we investigated hepatitis E virus (HEV) infection in piglets sampled in two farms in southern Italy. The virus was detected in 11 out of 15 animals tested. Based on sequence analysis, the 6 Italian strains examined belonged to two clusters containing both swine and human strains of either genotype 3 subtype e or f from Europe and Japan. The two Italian strain clusters shared nucleotide identity of 81.8% and 87.5% in the ORF2 (capsid protein) and ORF1 (RdRp) diagnostic fragments, respectively, confirming the heterogeneity of genotype 3 viruses circulating in pigs in Italy. The complete genome of one genotype 3 subtype e strain and the full ORF2 and ORF3 coding regions of one of the genotype 3f strains, obtained in this study, were compared to other HEV sequences available on line (NCBI database). The results of analysis showed that porcine strains clustered together with human and swine strains detected in Europe. Most changes in the coding region corresponded to synonymous mutations, and only the ORF3 showed a positive selection. Further analyses are needed to understand the clinical significance of HEV genotypes and subtypes

Assessment of left ventricular diastolic dysfunction by tissue Doppler imaging in patients with sclerodermia.

Background. Myocardial involvement in sclerodermia is related to patchy myocardial fibrosis attributed to intermittent and intense ischemia produced by microvascular occlusion. Although cardiac dysfunction can lead to heart failure or sudden cardiac death (particularly in patients with skeletal myopathy), it may also remain clinically silent despite extensive involvement. The purpose of our study was the evaluation of patients with sclerodermia by conventional Doppler echocardiography and tissue Doppler imaging (TDI) in order to detect early patterns of myocardial dysfunction. Methods. Twenty-six patients (age 54 +/- 13 years) with sclerodermia were studied. Exclusion criteria were non sinusal rhythm, hypertensive, ischemic, or valvular heart disease. Left ventricular ejection fraction (LVEF), fractional shortening (LVFS), and mitral flow filling parameters (E/A ratio, DT) were determined. Offline analysis of the myocardial velocity data sets was performed using dedicated software (Aplio, Toshiba Corp.). Velocity traces from the left ventricular free wall at 3 levels (basal, mid cavity, and apical) were processed in different cineloops in the apical views. Systolic (Sw) and diastolic (Ew, Aw) wall velocities were determined. Normal patterns (type 1) and relaxation or restrictive abnormalities (type 2 and 3) were examined for both standard and TDI indices. Results. All patients were asymptomatic and had normal LV dimensions, fractional shortening and segmental wall motion. 12 patients (group I) had septal hypertrophy and/or abnormal mitral inflow pattern (type 2 or 3). 14 patients (group II) had none of these abnormalities. 425 segments were analysed by TDI and a type 2 or 3 pattern was found in 139 (33%), from 20 patients. Five patients had pulmonary artery pressure >35mmHg. In six patients there was heterogeneous pattern in different segments and in thirteen a type 1 pattern was found in all segments, which was concordant with mitral flow Doppler pattern. Group I patients had an abnormal TDI pattern in all but one (95%). Nine Group II patients (64%) showed an abnormal TDI pattern. A significant difference was found between Ew and Sw in Groups I and II (p = 0.003 and 0.005, respectively). Conclusion. Thus in patients with sclerodermia and no cardiac involvement as assessed by conventional echocardiography, TDI showed abnormalities of longitudinal systolic and diastolic left ventricular functio

The Cannabinoid Receptor <i>Type 2</i> as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

<div><p>Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.</p><p>Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor <i>subtype 2</i>, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.</p><p>In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.</p><p>We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.</p></div

Stimulation of CB2 directly stimulates the ERK2 pathway.

<p>(<b>A</b>) Western Blot experiment and the relative quantification for p-ERK2 expression normalized with respect to β-tubulin in total MSC lysates from different passages and (<b>B</b>) after CB2 selective stimulation at multiple time-points. Data show the maximum increase of the protein levels at P1 and after 20-40 minutes and 24 hours from CB2 selective stimulation with the synthetic agonist JWH-133. The minimal p-ERK2 expression is revealed at P6. The CB2 antagonist AM630 significantly counteracts the JWH-induced increase of p-ERK2. Data are represented as a mean ± SD from three different assays. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant.</p

2-AG is Chemoattractant for hMSCs through CB2 receptor.

<p>(A) Image-based detection of 2-AG chemoattractive effect on cultured hMSCs. Representative morphological data of hMSCs time-lapse migration recorded at starting point (0 min), 90 min and 120 min from the 2-AG (filled gray arrows) or vehicle (white arrows) exposure. The cells were maintained in the relative medium or pretreated with the CB2 antagonist AM630 (lower panel) 15 min before the exposure to 2-AG during the chemotaxic assay. The arrowheads indicate static reference points in the cell port chamber; the black arrows point to soma or cellular processes without noticeable movements. The numbers indicate the hMSCs or their processes affected by 2-AG chemoattractive movements. Scale bar = 30 microns. A source of HGF has been used as positive control. (B) Quantative data of percentage of hMSCs effected by chemotaxic movements. n = 300 cells per each treatment. *** <i>P</i><0.0001 of hMSCs migration toward 2-AG at 90 min and 120 min <i>versus</i> hMSCs migration at each different treatment and time course.</p

Endocannabinoid levels.

<p>Human mesenchymal stem cells (MSCs) contained measurable amounts of AEA, 2-AG (A), PEA and OEA (B). A significantly decrease of all the endocannabinoids was detected during expansion passages from P1 to P9 with respect to P0. The minimum amount of AEA, 2-AG and PEA was detected at P6 (AEA 1.26±0.27 pmol/mg extract; 2-AG 4.79±0.81 pmol/mg extract; PEA 20.10±4.76 pmol/mg extract). The minimum amount of OEA was detected at P5 (AEA 6.13±1.11 pmol/mg extract). Data are represented as a mean ± SD from N = 6 experiments. A t-test has been used for statistical analysis. <i>p</i><0.05 has been considered statistically significant.</p

CB2 selective stimulation is associated with mTOR pathway activation.

<p>(<b>A</b>) Western Blot experiment and the relative quantification for pS6K1 expression normalized with respect to β-tubulin in total MSC lysates from different passages and (<b>B</b>) after CB2 selective stimulation at multiple time-points. Data show the maximum increase of the protein levels at P1 and P9 and after 24 hours from CB2 selective stimulation with the synthetic agonist JWH-133. The minimal expression is revealed at P6. The CB2 antagonist AM630 reduces the JWH-induced increase of p-AKT. Data are represented as a mean ± SD from three different assays. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant.</p

CB2 expression level in hMSCs increases with their maturation.

<p>(<b>A</b>) Western Blot experiment and (<b>B</b>) the relative quantification for CB2 receptor expression in total MSC lysates from different passages normalized with respect to β-tubulin reveal a significant increase of the protein levels at P5 with a maximal expression at P6. Data are represented as a mean ± SD from three different assays. A t-test has been used for statistical analysis. <i>p</i><0.05 was considered statistically significant. (<b>C</b>) A positive staining for the specific mesenchymal CD105 marker (in red) and an increased CB2 receptor expression from passage P1 to P7 (in green) is highlighted by immunocytochemistry analysis. Scale bar 50 = 50 microns.</p