Regulation of heparanase expression in coronary artery disease in diabetic, hyperlipidemic swine

Objective Enzymatic degradation of the extracellular matrix is known to be powerful regulator of atherosclerosis. However, little is known about the enzymatic regulation of heparan sulfate proteoglycans (HSPGs) during the formation and progression of atherosclerotic plaques. Methods and results Swine were rendered diabetic through streptozotocin injection and hyperlipidemic through a high fat diet. Arterial remodeling and local endothelial shear stress (ESS) were assessed using intravascular ultrasound, coronary angiography and computational fluid dynamics at weeks 23 and 30. Coronary arteries were harvested and 142 arterial subsegments were analyzed using histomorphologic staining, immunostaining and real time PCR. Heparanase staining and activity was increased in arterial segments with low ESS, in lesions with thin cap fibroatheroma (TCFA) morphology and in lesions with severely degraded internal elastic laminae. In addition, heparanase staining co-localized with staining for CD45 and MMP-2 within atherosclerotic plaques. Dual staining with gelatinase zymography and heparanase immunohistochemical staining demonstrated co-localization of matrix metalloprotease activity with heparanase staining. A heparanase enzymatic activity assay demonstrated increased activity in TCFA lesions, subsegments with low ESS and in macrophages treated with oxidized LDL or angiotensin II. Conclusions Taken together, our results support a critical role for heparanase in the development of vulnerable plaques and suggest a novel therapeutic target for the treatment of atherosclerosis.Novartis (Firm)Boston Scientific CorporationNational Institutes of Health (U.S.) (Grant R01 GM49039

Influenza sequence and epitope database

Influenza epidemics arise through the acquisition of viral genetic changes to overcome immunity from previous infections. An increasing number of complete genomes of influenza viruses have been sequenced in Asia in recent years. Knowledge about the genomes of the seasonal influenza viruses from different countries in Asia is valuable for monitoring and understanding of the emergence, migration and evolution of strains. In order to make full use of the wealth of information from such data, we have developed an integrated user friendly relational database, Influenza Sequence and Epitope Database (ISED), that catalogs the influenza sequence and epitope information obtained in Asia. ISED currently hosts a total of 13 020 influenza A and 2984 influenza B virus sequence data collected in 17 countries including 9 Asian countries, and a total of approximately 545 amantadine-resistant influenza virus sequences collected in Korea. ISED provides users with prebuilt application tools to analyze sequence alignment and different patterns and allows users to visualize epitope-matching structures, which is freely accessible at http://influenza.korea.ac.kr and http://influenza.cdc.go.kr

Isoperimetric Inequalities in Simplicial Complexes

In graph theory there are intimate connections between the expansion properties of a graph and the spectrum of its Laplacian. In this paper we define a notion of combinatorial expansion for simplicial complexes of general dimension, and prove that similar connections exist between the combinatorial expansion of a complex, and the spectrum of the high dimensional Laplacian defined by Eckmann. In particular, we present a Cheeger-type inequality, and a high-dimensional Expander Mixing Lemma. As a corollary, using the work of Pach, we obtain a connection between spectral properties of complexes and Gromov's notion of geometric overlap. Using the work of Gunder and Wagner, we give an estimate for the combinatorial expansion and geometric overlap of random Linial-Meshulam complexes

Augmented Expression and Activity of Extracellular Matrix-Degrading Enzymes in Regions of Low Endothelial Shear Stress Colocalize With Coronary Atheromata With Thin Fibrous Caps in Pigs

Background—The molecular mechanisms that determine the localized formation of thin-capped atheromata in the coronary arteries remain unknown. This study tested the hypothesis that low endothelial shear stress augments the expression of matrix-degrading proteases and thereby promotes the formation of thin-capped atheromata. : Methods and Results—Intravascular ultrasound–based, geometrically correct 3-dimensional reconstruction of the coronary arteries of 12 swine was performed in vivo 23 weeks after initiation of diabetes mellitus and a hyperlipidemic diet. Local endothelial shear stress was calculated in plaque-free subsegments of interest (n=142) with computational fluid dynamics. At week 30, the coronary arteries (n=31) were harvested and the same subsegments were identified. The messenger RNA and protein expression and elastolytic activity of selected elastases and their endogenous inhibitors were assessed. Subsegments with low preceding endothelial shear stress at week 23 showed reduced endothelial coverage, enhanced lipid accumulation, and intense infiltration of activated inflammatory cells at week 30. These lesions showed increased expression of messenger RNAs encoding matrix metalloproteinase-2, -9, and -12, and cathepsins K and S relative to their endogenous inhibitors and increased elastolytic activity. Expression of these enzymes correlated positively with the severity of internal elastic lamina fragmentation. Thin-capped atheromata developed in regions with lower preceding endothelial shear stress and had reduced endothelial coverage, intense lipid and inflammatory cell accumulation, enhanced messenger RNA expression and elastolytic activity of MMPs and cathepsins, and severe internal elastic lamina fragmentation. : Conclusions—Low endothelial shear stress induces endothelial discontinuity and accumulation of activated inflammatory cells, thereby augmenting the expression and activity of elastases in the intima and shifting the balance with their inhibitors toward matrix breakdown. Our results provide new insight into the mechanisms of regional formation of plaques with thin fibrous caps.Novartis Pharmaceuticals CorporationBoston Scientific CorporationHellenic Heart FoundationHellenic Atherosclerosis SocietyAlexander S. Onassis Public Benefit FoundationPropondis FoundationHellenic Harvard FoundationA.G. Leventis FoundationPhilip Morris International. External Research ProgramAmerican Heart Association (Scientist Development Grant)National Institutes of Health (U.S.) (Grant NIHR01 GM49039

Safety, Humoral and Cell Mediated Immune Responses to Two Formulations of an Inactivated, Split-Virion Influenza A/H5N1 Vaccine in Children

BACKGROUND:Highly pathogenic influenza A/H5N1 has caused outbreaks in wild birds and poultry in Asia, Africa and Europe. It has also infected people, especially children, causing severe illness and death. Although the virus shows limited ability to transmit between humans, A/H5N1 represents a potential source of the next influenza pandemic. This study assesses the safety and immunogenicity of aluminium hydroxide adjuvanted (Al) and non adjuvanted influenza A/Vietnam/1194/2004 NIBRG-14 (H5N1) vaccine in children. METHODS AND FINDINGS:In a Phase II, open, randomised, multicentre trial 180 children aged 6 months to 17 years received two injections, 21 days apart, of vaccine containing either: 30 microg haemagglutinin (HA) with adjuvant (30 microg+Al) or 7.5 microg HA without adjuvant. An additional 60 children aged 6-35 months received two "half dose" injections (ie 15 microg+Al or 3.8 microg). Safety was followed for 21 days after vaccination. Antibody responses were assessed 21 days after each injection and cellular immune responses were explored. Vaccination appeared well tolerated in all age groups. The 30 microg+Al formulation was more immunogenic than 7.5 microg in all age groups: in these two groups 79% and 46% had haemagglutinination inhibition antibody titres > or =32 (1/dil). Among 6-35 month-olds, the full doses were more immunogenic than their half dose equivalents. Vaccination induced a predominantly Th2 response against H5 HA. CONCLUSIONS:This influenza A(H5N1) vaccine was well tolerated and immunogenic in children and infants, with Al adjuvant providing a clear immunogenic advantage. These results demonstrate that an H5N1 Al-adjuvanted vaccine, previously shown to be immunogenic and safe in adults, can also be used in children, the group most at risk for pandemic influenza

Rapid semi-automated quantitative multiplex tandem PCR (MT-PCR) assays for the differential diagnosis of influenza-like illness

<p>Abstract</p> <p>Background</p> <p>Influenza A, including avian influenza, is a major public health threat in developed and developing countries. Rapid and accurate detection is a key component of strategies to contain spread of infection, and the efficient diagnosis of influenza-like-illness is essential to protect health infrastructure in the event of a major influenza outbreak.</p> <p>Methods</p> <p>We developed a multiplexed PCR (MT-PCR) assay for the simultaneous diagnosis of respiratory viruses causing influenza-like illness, including the specific recognition of influenza A haemagglutinin subtypes H1, H3, and H5. We tested several hundred clinical specimens in two diagnostic reference laboratories and compared the results with standard techniques.</p> <p>Results</p> <p>The sensitivity and specificity of these assays was higher than individual assays based on direct antigen detection and standard PCR against a range of control templates and in several hundred clinical specimens. The MT-PCR assays provided differential diagnoses as well as potentially useful quantitation of virus in clinical samples.</p> <p>Conclusions</p> <p>MT-PCR is a potentially powerful tool for the differential diagnosis of influenza-like illness in the clinical diagnostic laboratory.</p

Spatially controlled cell adhesion on three-dimensional substrates

The microenvironment of cells in vivo is defined by spatiotemporal patterns of chemical and biophysical cues. Therefore, one important goal of tissue engineering is the generation of scaffolds with defined biofunctionalization in order to control processes like cell adhesion and differentiation. Mimicking extrinsic factors like integrin ligands presented by the extracellular matrix is one of the key elements to study cellular adhesion on biocompatible scaffolds. By using special thermoformable polymer films with anchored biomolecules micro structured scaffolds, e.g. curved and µ-patterned substrates, can be fabricated. Here, we present a novel strategy for the fabrication of µ-patterned scaffolds based on the “Substrate Modification and Replication by Thermoforming” (SMART) technology: The surface of a poly lactic acid membrane, having a low forming temperature of 60°C and being initially very cell attractive, was coated with a photopatterned layer of poly(L-lysine) (PLL) and hyaluronic acid (VAHyal) to gain spatial control over cell adhesion. Subsequently, this modified polymer membrane was thermoformed to create an array of spherical microcavities with diameters of 300 µm for 3D cell culture. Human hepatoma cells (HepG2) and mouse fibroblasts (L929) were used to demonstrate guided cell adhesion. HepG2 cells adhered and aggregated exclusively within these cavities without attaching to the passivated surfaces between the cavities. Also L929 cells adhering very strongly on the pristine substrate polymer were effectively patterned by the cell repellent properties of the hyaluronic acid based hydrogel. This is the first time cell adhesion was controlled by patterned functionalization of a polymeric substrate with UV curable PLL-VAHyal in thermoformed 3D microstructures

Hemagglutinin from the H5N1 Virus Activates Janus Kinase 3 to Dysregulate Innate Immunity

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in humans. There are no effective vaccines or antiviral therapies currently available to control fatal outbreaks due in part to the lack of understanding of virus-mediated immunopathology. In our study, we used hemagglutinin (HA) of H5N1 virus to investigate the related signaling pathways and their relationship to dysregulated innate immune reaction. We found the HA of H5N1 avian influenza triggered an abnormal innate immune signalling in the pulmonary epithelial cells, through an unusual process involving activation of Janus kinase 3 (JAK3) that is exclusively associated with γc chain and is essential for signaling via all γc cytokine receptors. By using a selective JAK3 inhibitor and JAK3 knockout mice, we have, for the first time, demonstrated the ability to target active JAK3 to counteract injury to the lungs and protect immunocytes from acute hypercytokinemia -induced destruction following the challenge of H5N1 HA in vitro and in vivo. On the basis of the present data, it appears that the efficacy of selective JAK3 inhibition is likely based on its ability to block multiple cytokines and protect against a superinflammatory response to pathogen-associated molecular patterns (PAMPs) attack. Our findings highlight the potential value of selective JAK3 inhibitor in treating the fatal immunopathology caused by H5N1 challenge

Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication

RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection

Increased indoleamine-2,3-dioxygenase activity is associated with poor clinical outcome in adults hospitalized with influenza in the INSIGHT FLU003Plus study

BACKGROUND: Indoleamine-2,3-dioxygenase (IDO) mediated tryptophan (TRP) depletion has antimicrobial and immuno-regulatory effects. Increased kynurenine (KYN)-to-TRP (KT) ratios, reflecting increased IDO activity, have been associated with poorer outcomes from several infections. METHODS: We performed a case-control (1:2; age and sex matched) analysis of adults hospitalized with influenza A(H1N1)pdm09 with protocol-defined disease progression (died/transferred to ICU/mechanical ventilation) after enrollment (cases) or survived without progression (controls) over 60 days of follow-up. Conditional logistic regression was used to analyze the relationship between baseline KT ratio and other metabolites and disease progression. RESULTS: We included 32 cases and 64 controls with a median age of 52 years; 41% were female, and the median durations of influenza symptoms prior to hospitalization were 8 and 6 days for cases and controls, respectively (P = .04). Median baseline KT ratios were 2-fold higher in cases (0.24 mM/M; IQR, 0.13-0.40) than controls (0.12; IQR, 0.09-0.17; P ≤ .001). When divided into tertiles, 59% of cases vs 20% of controls had KT ratios in the highest tertile (0.21-0.84 mM/M). When adjusted for symptom duration, the odds ratio for disease progression for those in the highest vs lowest tertiles of KT ratio was 9.94 (95% CI, 2.25-43.90). CONCLUSIONS: High KT ratio was associated with poor outcome in adults hospitalized with influenza A(H1N1)pdm09. The clinical utility of this biomarker in this setting merits further exploration. CLINICALTRIALSGOV IDENTIFIER: NCT01056185