Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

The effects of chemokine, adhesion and extracellular matrix molecules on binding of mesenchymal stromal cells to poly(l-lactic acid)

Abstract Background aims. Mesenchymal stromal cells (MSC) are pluripotent adult stem cells capable of osteogenesis and chondrogenesis to form bone and cartilage. This characteristic gives them the potential for bone and cartilage regeneration. Synthetic polymers have been studied to examine whether they could be used as a scaffold for tissue engineering. In the current study a two-dimensional (2-D) poly(l-lactic acid) (PLLA) scaffold was treated with chemokine, adhesion and extracellular matrix molecules with the aim of using biologic molecules to improve the attachment of human MSC. Methods. MSC were isolated from human bone marrow and applied to a 2-D PLLA scaffold. Chemokines ligand (CXCL12 and CXCL13), adhesion molecules [P-selectin, vascular cell adhesion molecule (VCAM)-1 and heparin] and extracellular matrix molecules (fibronectin and type IV collagen) were coated on the scaffold and their effects on the number of MSC that adhered were recorded. Results. When used alone CXCL12 and CXCL13 enhanced MSC adhesion, as did VCAM-1, P-selectin, fibronectin and collagen, but not heparin. The effects of VCAM-1, P-selectin and heparin were enhanced by the addition of CXCL12. Incubation of MSC with antibodies to integrins α4 and α5β1 inhibited their adhesion to VCAM-1 and fibronectin-treated PLLA respectively, suggesting that these integrins were involved in the MSC interactions. Conclusions. The use of certain chemokines and adhesion and extracellular matrix molecules, alone or in combination, is beneficial for the attachment of MSC to PLLA, and may be helpful as natural molecules in scaffolds for regenerative medicine.     

Dietary supplementation with vitamin E and C attenuates dexamethasone-induced glucose intolerance in rats

Glucocorticoid excess induces marked insulin resistance and glucose intolerance. A recent study has shown that antioxidants prevent dexamethasone (DEX)-induced insulin resistance in cultured adipocytes. The purpose of this investigation was to examine the effects of dietary vitamin E and C (Vit E/C) supplementation on DEX-induced glucose intolerance in rats. We hypothesized that feeding rats a diet supplemented with Vit E/C would improve glucose tolerance and restore insulin signaling in skeletal muscle, adipose, and liver and prevent alterations in AMPK signaling in these tissues. Male Wistar rats received either a control or Vit E/C-supplemented diet (0.5 g/kg diet each of L-ascorbate and DL-all rac-alpha-tocopherol) for 9 days prior to, and during, 5 days of daily DEX treatment (subcutaneous injections 0.8 mg/g body wt). DEX treatment resulted in increases in the glucose and insulin area under the curve (AUC) during an intraperitoneal glucose tolerance test. The glucose, but not insulin, AUC was lowered with Vit E/C supplementation. Improvements in glucose tolerance occurred independent of a restoration of PKB phosphorylation in tissues of rats stimulated with an intraperitoneal injection of insulin but were associated with increases in AMPK signaling in muscle and reductions in AMPK signaling and the expression of fatty acid oxidation enzymes in liver. There were no differences in mitochondrial enzymes in triceps muscles between groups. This study is the first to report that dietary Vit E/C supplementation can partially prevent DEX-induced glucose intolerance in rats.     

Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3(rd) instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these 'larva chips' to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila.     

Outcome of the first electron microscopy validation task force meeting

This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine.     

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.     

Vitamin D receptor signaling inhibits atherosclerosis in mice

Although vitamin D has been implicated in cardiovascular protection, few studies have addressed the role of vitamin D receptor (VDR) in atherosclerosis. Here we investigate the effect of inactivation of the VDR signaling on atherogenesis and the antiatherosclerotic mechanism of vitamin D. Low density lipoprotein receptor (LDLR)(-/-)/VDR(-/-) mice exhibited site-specific accelerated atherogenesis, accompanied by increases in adhesion molecules and proinflammatory cytokines in the aorta and cholesterol influx in macrophages. Macrophages showed marked renin up-regulation in the absence of VDR, and inhibition of renin by aliskiren reduced atherosclerosis in LDLR(-/-)/VDR(-/-) mice, suggesting that the renin-angiotensin system (RAS) promotes atherosclerosis in the absence of VDR. LDLR(-/-) mice receiving LDLR(-/-)/VDR(-/-) BMT developed larger lesions than LDLR(-/-) BMT controls. Moreover, LDLR(-/-) mice receiving Rag-1(-/-)/VDR(-/-) BMT, which were unable to generate functional T and B lymphocytes, still had more severe atherosclerosis than Rag-1(-/-) BMT controls, suggesting a critical role of macrophage VDR signaling in atherosclerotic suppression. Aliskiren treatment eliminated the difference in lesions between Rag-1(-/-)/VDR(-/-) BMT and Rag-1(-/-) BMT recipients, indicating that local RAS activation in macrophages contributes to the enhanced atherogenesis seen in Rag-1(-/-)/VDR(-/-) BMT mice. Taken together, these observations provide evidence that macrophage VDR signaling, in part by suppressing the local RAS, inhibits atherosclerosis in mice.     

Backseat drivers: the hidden influence of microbial viruses on disease

Because viral replication depends on the vigour of its host, many viruses have evolved incentives of fitness to pay their keep. When the viral host is a human pathogen, these fitness factors can surface as virulence: creating a Russian doll of pathogenesis where pathogens within pathogens complicate the disease process. Microbial viruses can even be independently immunogenic, as we recently reported for leishmania-virus. Thus, the incidence of this 'hyperpathogenism' is becoming an important clinical consideration and by appreciating the microbial-virus as a backseat driver of human disease, we could exploit its presence as a diagnostic biomarker and molecular target for therapeutic intervention. Here we discuss the prevalence of clinically relevant hyperpathogenism as well as the environmental sanctuaries that breed it.     

Anti-hypertensive effects of Rosuvastatin are associated with decreased inflammation and oxidative stress markers in hypertensive rats

Among their pleiotropic effects, statins exert antioxidant and anti-inflammatory properties. The aim of this study was to evaluate in normotensive (WKY) and in spontaneously hypertensive rats (SHR) the effect of rosuvastatin (ROSU) treatment on (1) plasma inflammation markers and endogenous NO synthase inhibitor (ADMA) levels, (2) reactive oxygen species (ROS) generated by circulating leukocytes and (3) vascular oxidative stress and tissue inflammation markers. Plasma cytokines were higher in SHR than in WKY, except for IL-4, which was lower in SHR than in WKY. SHR monocytes exhibited higher production of ROS than did WKY monocytes. In the experimental conditions, ROSU did not modify plasma cholesterol levels in SHR but attenuated the increase in systolic blood pressure. In SHR only, ROSU lessened pro-inflammatory cytokines and ADMA levels, increased IL-4 and reduced ROS production in circulating monocytes. These results demonstrate the beneficial effects of ROSU in SHR, independently of any lowering of cholesterol levels.     

The activating immunoreceptor NKG2D and its ligands are involved in allograft transplant rejection

Although the linkage between innate and adaptive immunity in transplantation has been recognized, the mechanisms underlying this cooperation remain to be fully elucidated. In this study, we show that early "danger" signals associated with transplantation lead to rapid up-regulation of NKG2D ligands. A second wave of NKG2D ligand up-regulation is mediated by the adaptive immune response to allografts. Treatment with an Ab to NKG2D was highly effective in preventing CD28-independent rejection of cardiac allografts. Notably, NKG2D blockade did not deplete CD8(+) T cells or NK1.1(+) cells nor affect their migration to the allografts. These results establish a functional role of NKG2D and its ligands in the rejection of solid organ transplants.