Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac239delta3-vaccinated rhesus macaques

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01- animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01- animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.     

Role of genes that modulate host immune responses in the immunogenicity and pathogenicity of vaccinia virus

Poxvirus vaccine vectors, although capable of eliciting potent immune responses, pose serious health risks in immunosuppressed individuals. We therefore constructed five novel recombinant vaccinia virus vectors which contained overlapping deletions of coding regions for the B5R, B8R, B12R, B13R, B14R, B16R, B18R, and B19R immunomodulatory gene products and assessed them for both immunogenicity and pathogenicity. All five of these novel vectors elicited both cellular and humoral immunity to the inserted HIV-BH10 env comparable to that induced by the parental Wyeth strain vaccinia virus. However, deletion of these immunomodulatory genes did not increase the immunogenicity of these vectors compared with the parental vaccinia virus. Furthermore, four of these vectors were slightly less virulent and one was slightly more virulent than the Wyeth strain virus in neonatal mice. Attenuated poxviruses have potential use as safer alternatives to current replication-competent vaccinia virus. Improved vaccinia virus vectors can be generated by deleting additional genes to achieve a more significant viral attenuation.     

Vacuolating cytotoxin A is associated with increased thrombin generation in gastric mucosa

BACKGROUND: Activation of the coagulation system is a critical response for both the repair of tissue injury and the host defense against microbial pathogens. Activation of the coagulation cascade culminates with the generation of thrombin. In vitro studies have shown that thrombin protects gastric epithelial cells from injury. The present study was undertaken to assess in vivo the relationship between gastric intramucosal generation of thrombin and Helicobacter pylori infection. MATERIALS AND METHODS: This study comprised 59 patients with gastroduodenal disorders. There were 27 patients with H. pylori infection (Hp+), 14 without it (Hp-), and 18 patients with cured H. pylori infection (Hp c). The gastric intramucosal concentrations of thrombin-antithrombin complex (TAT), epidermal growth factor (EGF), prostaglandin E2 (PGE2), and vacuolating cytotoxin A (VacA) were measured by specific immunoassays. RESULTS: The level of TAT was significantly increased in patients with Hp+ compared to Hp- and Hp c. The levels of TAT, EGF and PGE2 were higher in VacA (+) patients than in those with VacA (-). VacA induced significant expression of tissue factor in gastric epithelial cells in vitro. The gastric intramucosal level of VacA antigen was proportionally and significantly correlated with TAT, EGF and PGE2 in Hp+ patients. The level of TAT was proportionally and significantly correlated with EGF in Hp+ patients but not in Hp- and HP c patients. CONCLUSIONS: These results showed that VacA produced by H. pylori is associated with increased thrombin generation, and that thrombin may play a protective role in H. pylori-associated gastroduodenal disorders.     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

A dioxin sensitive gene, mammalian WAPL, is implicated in spermatogenesis

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disruptor that produces a variety of toxic effects. We have isolated a mouse homolog of the hWAPL gene, termed mouse WAPL (mWAPL), as a target of TCDD by cDNA representational difference analysis from mouse embryonic stem cells. A statistically significant increase in mWAPL expression was observed at 0.1 microM TCDD in AhR-/- mouse embryonic fibroblast cells. Interestingly, at 1 microM TCDD, mWAPL mRNA levels decreased in AhR+/+ cells, but further increased in AhR-/- cells. hWAPL and mWAPL were highly expressed only in testes among normal tissue samples, and we observed mWAPL localization in the synaptonemal complex of testicular chromosomes. In addition, mouse testes decreased the expression of mWAPL mRNA after a single intraperitoneal injection of TCDD. Thus, mammalian WAPL such as hWAPL and mWAPL may be involved in spermatogenesis and be target genes mediating the reproductive toxicity induced by TCDD.     

Cyclooxygenase-3 Gene Expression in Alzheimer Hippocampus and in Stressed Human Neural Cells

The cyclooxygenase (COX) superfamily of prostaglandin synthase genes encode a constitutively expressed COX-1, an inducible, highly regulated COX-2, and a COX-3 isoform whose RNA is derived through the retention of a highly structured, G + C-rich intron 1 of the COX-1 gene. As generators of oxygen radicals, lipid mediators, and the pharmacological targets of nonsteroidal anti-inflammatory drugs (NSAIDs), COX enzymes potentiate inflammatory neuropathology in Alzheimer's disease (AD) brain. Because COX-2 is elevated in AD and COX-3 is enriched in the mammalian CNS, these studies were undertaken to examine the expression of COX-3 in AD and in [IL-1beta + Abeta42]-triggered human neural (HN) cells in primary culture. The results indicate that while COX-2 remains a major player in propagating inflammmation in AD and in stressed HN cells, COX-3 may play ancillary roles in membrane-based COX signaling or when basal levels of COX-1 or COX-2 expression persist.     

Inhibitory effects of red wine extracts on endothelial-dependent adhesive interactions with monocytes induced by oxysterols

Red wine polyphenolic compounds have been demonstrated to possess antioxidant properties, and several studies have suggested that they might constitute a relevant dietary factor in the protection from coronary heart disease. The aim of the present study is to examine whether red wine extracts (RWE) can ameliorate oxysterol-induced endothelial response, and whether inhibition of adhesion molecule expression is involved in monocyte adhesion to endothelial cells. Surface expression and mRNA levels of adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1) were determined by ELISA and RT-PCR performed on human aortic endothelial cells (HAEC) monolayers stimulated with 7beta-hydroxycholesterol or 25-hydroxycholesterol. Incubation of HAEC with oxysterols (10 microM) increased expression of adhesion molecules in a time-dependent manner. Pretreatment of HAEC with RWE at final concentrations of 1, 10, and 100 ng/ml significantly inhibited the increase of surface protein expression and mRNA levels. Adherence of monocytes to oxysterol-stimulated HAEC was increased compared to that of unstimulated cells. Treatment of HAEC with RWE significantly inhibited adherence of monocytes. These results suggest that RWE works as an anti-atherogenic agent through the inhibition of endothelial-dependent adhesive interactions with monocytes induced by oxysterols.     

Effects of alpha-tocopherol on an animal model of tauopathies

We have reported that transgenic (Tg) mice overexpressing human tau protein develop filamentous tau aggregates in the CNS. We overexpressed the smallest human tau isoform (T44) in the mouse CNS to model tauopathies. These tau Tg mice acquire age-dependent CNS pathologies, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Therefore, these Tg mice are a model that can be exploited for drug discovery in studies that target amelioration of tau-induced neurodegeneration as well as for elucidating mechanisms of tau pathology in various neurodegenerative tauopathies. Oxidative stress has been implicated in the pathogenesis of various neurodegenerative diseases, including tauopathies, and many epidemiological, clinical, and basic studies have suggested the neuroprotective effects of vitamin E in neurodegenerative diseases. To elucidate the role of oxidative damage in the pathological mechanisms of these Tg mice, we fed them alpha-tocopherol, the major component of antioxidant vitamin E. Supplementation of alpha-tocopherol suppressed and/or delayed the development of tau pathology, which correlated with improvement in the health and attenuation of motor weakness in the Tg mice. These results suggest that oxidative damage is involved in the pathological mechanisms of the tau Tg mice and that treatment with antioxidative agents like alpha-tocopherol may prevent neurodegenerative tauopathies.     

The inhibitory effect of alacepril, an angiotensin-converting enzyme inhibitor, on endothelial inflammatory response induced by oxysterol and TNF-alpha

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-alpha. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-alpha-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-alpha. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-alpha.     

7-Ketocholesterol enhances the expression of adhesion molecules on human aortic endothelial cells by increasing the production of reactive oxygen species

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, alpha-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.