Induction of lytic Epstein-Barr virus (EBV) infection by synergistic action of rituximab and dexamethasone renders EBV-positive lymphoma cells more susceptible to ganciclovir cytotoxicity in vitro and in vivo

The purposeful induction of the lytic form of Epstein-Barr virus (EBV) infection combined with ganciclovir (GCV) treatment has been advocated as a novel strategy for EBV-positive B-cell lymphoma. We demonstrated that rituximab had a synergistic effect with dexamethasone on induction of the lytic EBV infection in CD20-positive lymphoma cells. Addition of GCV to the dexamethasone/rituximab-treated cells was more effective than dexamethasone/rituximab alone in killing EBV-positive lymphoma cells in vitro and in lymphoma-bearing nude mice but not in EBV-negative cells. These data suggest that induction of the lytic EBV infection with dexamethasone/rituximab in combination with GCV could be a potential virally targeted therapy for EBV-associated B-cell lymphoma.     

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.     

Low TLR4 expression by liver dendritic cells correlates with reduced capacity to activate allogeneic T cells in response to endotoxin

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8alpha- (myeloid) and CD11c+CD8alpha+ ("lymphoid-related") DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-gamma) and Th2 (IL-4) responses. When higher LPS concentrations (> or =100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.     

Rolling of Th1 cells via P-selectin glycoprotein ligand-1 stimulates LFA-1-mediated cell binding to ICAM-1

Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 or CCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.     

Expression of HSP47 in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia

Background

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and α-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP).

Methods

We reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and α-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining).

Results

The expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of α-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP.

Conclusion

Our results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen.     

Contribution of non-extensor muscles of the leg to maximal-effort countermovement jumping

Background  

The purpose of this study was to determine the effects of non-extensor muscles of the leg (i.e., muscles whose primary function is not leg extension) on the kinematics and kinetics of human maximal-effort countermovement jumping. Although it is difficult to address this type of question through experimental procedures, the methodology of computer simulation can be a powerful tool.     

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.