Inflammatory extracellular vesicles prompt heart dysfunction via TRL4-dependent NF-κB activation.

Background: After myocardial infarction, necrotic cardiomyocytes release damage-associated proteins that stimulate innate immune pathways and macrophage tissue infiltration, which drives inflammation and myocardial remodeling. Circulating inflammatory extracellular vesicles play a crucial role in the acute and chronic phases of ischemia, in terms of inflammatory progression. In this study, we hypothesize that the paracrine effect mediated by these vesicles induces direct cytotoxicity in cardiomyocytes. Thus, we examined whether reducing the generation of inflammatory vesicles within the first few hours after the ischemic event ameliorates cardiac outcome at short and long time points. Methods: Myocardial infarction was induced in rats that were previously injected intraperitoneally with a chemical inhibitor of extracellular-vesicle biogenesis. Heart global function was assessed by echocardiography performed at 7, 14 and 28 days after MI. Cardiac outcome was also evaluated by hemodynamic analysis at sacrifice. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langendorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory macrophages (M1) were studied in-vitro in primary rat neonatal cardiomyocytes. Results: Inflammatory response following myocardial infarction dramatically increased the number of circulating extracellular vesicles carrying alarmins such as IL-1α, IL-1β and Rantes. Reducing the boost in inflammatory vesicles during the acute phase of ischemia resulted in preserved left ventricular ejection fraction in vivo. Hemodynamic analysis confirmed functional recovery by displaying higher velocity of left ventricular relaxation and improved contractility. When added to the perfusate of isolated hearts, post-infarction circulating vesicles induced significantly more cell death in adult cardiomyocytes, as assessed by cTnI release, comparing to circulating vesicles isolated from healthy (non-infarcted) rats. In vitro inflammatory extracellular vesicles induce cell death by driving nuclear translocation of NF-κB into nuclei of cardiomyocytes. Conclusion: Our data suggest that targeting circulating extracellular vesicles during the acute phase of myocardial infarction may offer an effective therapeutic approach to preserve function of ischemic heart

Transduction of Brain Dopamine Neurons by Adenoviral Vectors Is Modulated by CAR Expression: Rationale for Tropism Modified Vectors in PD Gene Therapy

Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo.Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals.These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD

Rapid Accumulation of CD14+CD11c+ Dendritic Cells in Gut Mucosa of Celiac Disease after in vivo Gluten Challenge

Of antigen-presenting cells (APCs) expressing HLA-DQ molecules in the celiac disease (CD) lesion, CD11c(+) dendritic cells (DCs) co-expressing the monocyte marker CD14 are increased, whereas other DC subsets (CD1c(+) or CD103(+)) and CD163(+)CD11c(-) macrophages are all decreased. It is unclear whether these changes result from chronic inflammation or whether they represent early events in the gluten response. We have addressed this in a model of in vivo gluten challenge.Treated HLA-DQ2(+) CD patients (n = 12) and HLA-DQ2(+) gluten-sensitive control subjects (n = 12) on a gluten-free diet (GFD) were orally challenged with gluten for three days. Duodenal biopsies obtained before and after gluten challenge were subjected to immunohistochemistry. Single cell digests of duodenal biopsies from healthy controls (n = 4), treated CD (n = 3) and untreated CD (n = 3) patients were analyzed by flow cytometry.In treated CD patients, the gluten challenge increased the density of CD14(+)CD11c(+) DCs, whereas the density of CD103(+)CD11c(+) DCs and CD163(+)CD11c(-) macrophages decreased, and the density of CD1c(+)CD11c(+) DCs remained unchanged. Most CD14(+)CD11c(+) DCs co-expressed CCR2. The density of neutrophils also increased in the challenged mucosa, but in most patients no architectural changes or increase of CD3(+) intraepithelial lymphocytes (IELs) were found. In control tissue no significant changes were observed.Rapid accumulation of CD14(+)CD11c(+) DCs is specific to CD and precedes changes in mucosal architecture, indicating that this DC subset may be directly involved in the immunopathology of the disease. The expression of CCR2 and CD14 on the accumulating CD11c(+) DCs indicates that these cells are newly recruited monocytes

Derivation of a Myeloid Cell-Binding Adenovirus for Gene Therapy of Inflammation

The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5). Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR), with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP), with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease

Wolfram Syndrome: New Mutations, Different Phenotype

BACKGROUND: Wolfram Syndrome (WS) is an autosomal recessive neurodegenerative disorder characterized by Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, and Deafness identified by the acronym "DIDMOAD". The WS gene, WFS1, encodes a transmembrane protein called Wolframin, which recent evidence suggests may serve as a novel endoplasmic reticulum calcium channel in pancreatic β-cells and neurons. WS is a rare disease, with an estimated prevalence of 1/550.000 children, with a carrier frequency of 1/354. The aim of our study was to determine the genotype of WS patients in order to establish a genotype/phenotype correlation. METHODOLOGY/PRINCIPAL FINDINGS: We clinically evaluated 9 young patients from 9 unrelated families (6 males, 3 females). Basic criteria for WS clinical diagnosis were coexistence of insulin-treated diabetes mellitus and optic atrophy occurring before 15 years of age. Genetic analysis for WFS1 was performed by direct sequencing. Molecular sequencing revealed 5 heterozygous compound and 3 homozygous mutations. All of them were located in exon 8, except one in exon 4. In one proband only an heterozygous mutation (A684V) was found. Two new variants c.2663 C>A and c.1381 A>C were detected. CONCLUSIONS/SIGNIFICANCE: Our study increases the spectrum of WFS1 mutations with two novel variants. The male patient carrying the compound mutation [c.1060_1062delTTC]+[c.2663 C>A] showed the most severe phenotype: diabetes mellitus, optic atrophy (visual acuity 5/10), deafness with deep auditory bilaterally 8000 Hz, diabetes insipidus associated to reduced volume of posterior pituitary and pons. He died in bed at the age of 13 years. The other patient carrying the compound mutation [c.409_424dup16]+[c.1381 A>C] showed a less severe phenotype (DM, OA)

Heterogeneity of human monocytes: an optimized four-color flow cytometry protocol for analysis of monocyte subsets.

Monocytes are central mediators in the development of atherosclerotic plaques. They circulate in blood and eventually migrate into tissue including the vessel wall where they give rise to macrophages and dendritic cells. The existence of monocyte subsets with distinct roles in homeostasis and inflammation suggests specialization of function. These subsets are identified based on expression of the CD14 and CD16 markers. Routinely applicable protocols remain elusive, however. Here, we present an optimized four-color flow cytometry protocol for analysis of human blood monocyte subsets using a specific PE-Cy5-conjugated monoclonal antibody (mAb) to HLA-DR, a PE-Cy7-conjugated mAb to CD14, a FITC-conjugated mAb to CD16, and PE-conjugated mAbs to additional markers relevant to monocyte function. Classical CD14(+)CD16(-) monocytes (here termed "Mo1" subset) expressed high CCR2, CD36, CD64, and CD62L, but low CX(3)CR1, whereas "nonclassical" CD14(lo)CD16(+) monocytes (Mo3) essentially showed the inverse expression pattern. CD14(+)CD16(+) monocytes (Mo2) expressed high HLA-DR, CD36, and CD64. In patients with stable coronary artery disease (n = 13), classical monocytes were decreased, whereas "nonclassical" monocytes were increased 90% compared with healthy subjects with angiographically normal coronary arteries (n = 14). Classical monocytes from CAD patients expressed higher CX(3)CR1 and CCR2 than controls. Thus, stable CAD is associated with expansion of the nonclassical monocyte subset and increased expression of inflammatory markers on monocytes. Flow cytometric analysis of monocyte subsets and marker expression may provide valuable information on vascular inflammation. This may translate into the identification of monocyte subsets as selective therapeutic targets, thus avoiding adverse events associated with indiscriminate monocyte inhibition

Mitigation of Fire Damage and Escalation by Fireproofing: a Risk-Based Strategy

Passive fire protection by the application of fireproofing materials is a crucial safety barrier in the prevention of the escalation of fire scenarios. Fireproofing improves the capacity of process items and of support structures to maintain their structural integrity during a fire, preventing or at least delaying the collapse of structural elements. Maintenance and cost issues require however to apply such protection only where an actual risk of severe fire scenarios is present. Available methodologies for fireproofing application in on-shore installation do not consider the effect of jet-fires. In the present study, a risk-based methodology aimed at the protection from both pool fire and jet fire escalation was developed. The procedure addresses both the prevention of domino effect and the mitigation of asset damage due to the primary fire scenario. The method is mainly oriented to early design application, allowing the identification of fireproofing zones in the initial phases of lay-out definition

A risk-based methodology to identify fire protection zones

Fire is among the most dangerous accident scenarios that may affect the process and chemical industry. Beside the immediate and direct harms to operators and population, fire may also cause damages to plant structures, that may in turn trigger severe secondary scenarios (domino effect) affecting a greater number of people and/or causing relevant asset damages. As a matter of fact, fire may cause the collapse of structural elements due to the loss of mechanical properties at high temperatures. Fireproofing is usually applied to improve the capacity of support structures to maintain their structural integrity during a fire, and to prevent or at least to delay the collapse of structural elements. However, due to cost issues, fireproofing should be ap-plied only to elements present in areas where an actual risk of severe fire scenarios exist. The available inter-national standards for the application of fireproofing in onshore chemical and process plants do not provide appropriate criteria to identify the areas where the structural elements must be protected. In particular, avail-able methodologies do not consider the effect of jet-fires and are based on a deterministic approach. Account-ing for the effect of both pool fire and jet fire, the present contribution suggests a new risk-based methodol-ogy for the identification of fire protection zones where fireproofing coatings should be applied. The developed methodology is oriented to early design application, allowing the identification of fireproofing zones in the initial phases of lay-out definition. Thus, it is built up on simplified criteria based on radiant heat intensity to identify the extension of fire protection zones. The key point of the methodology is the choice of the fire scenarios to use in the consequence evaluation. These are selected applying a semi-quantitative risk analysis. The characteristics of the initial events are used to evaluate the characteristics of the final outcome scenarios. A risk matrix approach is used to assess the fire risk level of each zone. In order to demonstrate the features and the potentials of the proposed methodology, a case-study was carried out, based on the actual layout of an Oil&Gas facility. The results show that the proposed methodology provides a robust identifica-tion of fire protection zones, giving useful and timely indications for the application of fire protections to structural plant elements