Characterization,expression, and evolutionary analysis of new TLR3 and TLR5M genes cloned from the spiny eel Mastacembelus armatus

Toll-like receptors (TLRs) play an important role in innate and adaptive immunity. Here, we identify two new TLRs from the spiny eel Mastacembelus armatus (TLR3 and membrane TLR5M). Both MaTLR3 and MaTLR5M were expressed in all tested tissues; expression was highest in liver and spleen, respectively. After infection with Vibrio parahaemolyticus, expression of both TLRs fluctuated and differed significantly from controls at several time points. The predicted three-dimensional model of the MaTLR3 and MaTLR5M proteins indicates that most sites under positive selection were located in the extracellular domains of TLRs. Evolutionary analysis detected positively selected sites in the ancestral lineages of vertebrates, amphibians and reptiles. Multiple ML methods recovered 10 positively selected sites in teleost TLR3 and 24 in TLR5M, and most sites were located in leucine-rich repeat domain, possibly related to an “arms-race” co-evolution with pathogens.     

Protein-bound polysaccharide activates dendritic cells and enhances OVA-specific T cell response as vaccine adjuvant

Protein-bound polysaccharide-K (PSK) is a hot water extract from Trametes versicolor mushroom. It has been used traditionally in Asian countries for its immune stimulating and anti-cancer effects. We have recently found that PSK can activate Toll-like receptor 2 (TLR2). TLR2 is highly expressed on dendritic cells (DC), so the current study was undertaken to evaluate the effect of PSK on DC activation and the potential of using PSK as a vaccine adjuvant. In vitro experiments using mouse bone marrow-derived DC (BMDC) demonstrated that PSK induces DC maturation as shown by dose-dependent increase in the expression of CD80, CD86, MHCII, and CD40. PSK also induces the production of multiple inflammatory cytokines by DC, including IL-12, TNF-α, and IL-6, at both mRNA and protein levels. In vivo experiments using PSK as an adjuvant to OVAp323–339 vaccine showed that PSK as adjuvant leads to enlarged draining lymph nodes with higher number of activated DC. PSK also stimulates proliferation of OVA-specific T cells, and induces T cells that produce multiple cytokines, IFN-γ, IL-2, and TNF-α. Altogether, these results demonstrate the ability of PSK to activate DC in vitro and in vivo and the potential of using PSK as a novel vaccine adjuvant.     

Total expression of HLA-G and TLR-9 in chronic lymphocytic leukemia patients

Suppressed immune status facilitates immune escape mechanisms that allow chronic lymphocytic leukemia cells to proliferate and expand. The expression of HLA-G could effectively inhibit the immune response. In immune response inhibitory signals follow activation of immune system which might be occur during bacterial or viral infection in CLL patients. In the current study we characterized two components of immune system, inhibitory molecule HLA-G with its receptor – CD85j and Toll-like receptor 9.     

Protective effects of melatonin against spinal cord injury induced oxidative damage in rat kidney: A morphological and biochemical study

Spinal cord injury (SCI) induced oxidative stress affects multiple organ systems including the kidney. We studied the possible protective effects of melatonin on SCI-induced oxidative damage in renal tissues of rats. Wistar albino rats (n = 24) were exposed to SCI and divided into vehicle- or melatonin-treated SCI groups. Melatonin was administred intraperitoneally at a dose of 10 mg/kg for seven days. Renal tissues were investigated by light and electron microscopy. Furthermore, tissue malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) and superoxide dismutase (SOD) activities were also determined. In the vehicle-treated SCI group, the renal histology was disturbed compared to controls, whereas the melatonin-treated SCI group showed significantly reduced degeneration of renal tissue as seen by both light and electron microscopy. MDA levels, MPO and SOD activities were increased and GSH levels were decreased in the vehicle-treated SCI group compared to controls. On the other hand, decreased MDA levels and MPO activities and increased GSH levels were observed in the melatonin-treated SCI group compared to vehicle-treated SCI group. These results showed that experimentally induced SCI caused oxidative stress in the rat kidney, whereas melatonin treatment reduced oxidative stress, suggesting that it may be used as a complementary therapy of renal problems occurring following SCI.     

Coupled experiment/finite element analysis on the mechanical response of porcine brain under high strain rates

This paper presents a coupled experimental/modeling study of the mechanical response of porcine brain under high strain rate loading conditions. Essentially, the stress wave propagation through the brain tissue is quantified. A Split-Hopkinson Pressure Bar (SPHB) apparatus, using a polycarbonate (viscoelastic) striker bar was employed for inducing compression waves for strain rates ranging from 50 to 750 s⁻¹. The experimental responses along with high speed video showed that the brain tissue’s response was nonlinear and inelastic. Also, Finite Element Analysis (FEA) of the SHPB tests revealed that the tissue underwent a non-uniform stress state during testing when glue is used to secure the specimen with the test fixture. This result renders erroneous the assumption of uniaxial loading. In this study, the uniaxial volume averaged stress–strain behavior was extracted from the FEA to help calibrate inelastic constitutive equations.     

Flow cytometric detection of endothelial microparticles (EMP): Effects of centrifugation and storage alter with the phenotype studied

Introduction

Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification.

Materials and Methods

Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1 550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 °C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 °C for 7 and 28 days. A final aliquot was further centrifuged at 10 000 g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 µm.

Results

High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p = 0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b = 0.611, p = 0.025).Storage at 4 °C did not affect EMP quantification. However, freezing at -80 °C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p < 0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance > 0.487; p < 0.05).

Conclusion

The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols.     

Replicating and identifying large cell neuroblastoma using high-dose intra-tumoral chemotherapy and automated digital analysis

PurposeLarge cell neuroblastomas (LCN) are frequently seen in recurrent, high-risk neuroblastoma but are rare in primary tumors. LCN, characterized by large nuclei with prominent nucleoli, predict a poor prognosis. We hypothesize that LCN can be created with high-dose intra-tumoral chemotherapy and identified by a digital analysis system.MethodsOrthotopic mouse xenografts were created using human neuroblastoma and treated with high-dose chemotherapy delivered locally via sustained-release silk platforms, inducing tumor remission. After recurrence, LCN populations were identified on H&E sections manually. Clusters of typical LCN and non-LCN cells were divided equally into training and test sets for digital analysis. Marker-controlled watershed segmentation was used to identify nuclei and characterize their features. Logistic regression was developed to distinguish LCN from non-LCN.ResultsImage analysis identified 15,000 nuclei and characterized 70 nuclear features. A 19-feature model provided AUC > 0.90 and 100% accuracy when > 30% nuclei/cluster were predicted as LCN. Overall accuracy was 87%.ConclusionsWe recreated LCN using high-dose chemotherapy and developed an automated method for defining LCN histologically. Features in the model provide insight into LCN nuclear phenotypic changes that may be related to increased activity. This model could be adapted to identify LCN in human tumors and correlated with clinical outcomes.     

The shift of macrophages toward M1 phenotype promotes aortic valvular calcification

Objective

The purpose of the present study was to comprehensively compare the phenotype profile of infiltrated macrophages in human noncalcified and calcific aortic valves, and to determine whether the shift of macrophage polarization modulates valvular calcification in vitro.