Seong-Kug Eo

Induction of CD8 T-cell-specific systemic and mucosal immunity against herpes simplex virus with CPG-peptide complexes

ABSTRACT Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs exert powerful adjuvant activity in vivo and in vitro. Administered with antigen they induce a population of antigen-specific CD8+ T cells. In this study we immunized C57BL/6 mice with bioactive CpG ODN combined with an immunodominant epitope derived from herpes simplex virus (HSV) glycoprotein B (amino acids 498 to 505; SSIEFARL) and analyzed the magnitude and durability of the peptide-specific response. The effectiveness of the CD8+ T-cell response as measured by peptide-specific tetramers, peptide-induced intracellular gamma interferon expression, and resistance to systemic and mucosal challenge during the acute and memory phases was compared with the response induced by immunization with recombinant vaccinia virus encoding SSIEFARL as a minigene (VvgB498-505). Confirming the reports of others, our results demonstrate that the CpG ODN-peptide approach generates an antigen-specific CD8+ T-cell population, but the frequency of CD8+ T cells is lower than that induced by VvgB498-505. Nevertheless, the protection level was comparable when mice were systemically and mucosally challenged during the acute phase. However, such responses by both groups waned with time and were functionally less effective. Still, our results indicate that the CpG ODN-peptide immunization system holds promise as a means of selectively inducing a CD8+ T-cell response against HSV.

Evaluation of the Methicillin-Resistant Staphylococcus aureus (MRSA)-Screen Latex Agglutination Test for Detection of MRSA of Animal Origin

ABSTRACT Methicillin (oxacillin)-resistant staphylococci (MRS) have emerged as major clinical and epidemiological pathogens, and there have been frequent reports of MRS infections in the veterinary field. The MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) was compared with an oxacillin agar screen test, MIC determination, and mecA PCR assay, the “gold standard.” In an analysis of 15 mecA-positive and 48 mecA-negative S. aureus animal isolates, as well as 9 mecA-positive and 147 mecA-negative, coagulase-negative staphylococcal animal isolates, the latex agglutination test surpassed the widely used oxacillin agar screen method and MIC determination, with a sensitivity and a specificity of 100%. The MRSA-Screen test is a reliable and rapid method of detecting MRS in the veterinary field.

7-Ketocholesterol Upregulates Interleukin-6 via Mechanisms That Are Distinct from Those of Tumor Necrosis Factor-α, in Vascular Smooth Muscle Cells

This study investigated the effects of 7-ketocholesterol on interleukin (IL)-6 expression in vascular smooth muscle cells (VSMC). Among the 7 IL examined, only IL-6 transcript was increased by 7-ketocholesterol treatment in human aorta smooth muscle cells. IL-6 transcripts increased up to 24 h after treatment with 7-ketocholesterol, and this effect was profoundly repressed by treatment with p38 MAPK inhibitors and to a lesser extent JNK inhibitors. 7α-Hydroxycholesterol, 27-hydroxycholesterol or cholesterol, however, did not induce IL-6 expression. Mechanisms of IL-6 induction by 7-ketocholesterol were investigated in comparison with tumor necrosis factor (TNF)-α. Whereas TNF-α activated IL-6 promoter, which was impaired by p38 MAPK inhibitors or by mutation in the NF-κB-binding site within the promoter region, 7-ketocholesterol did not affect IL-6 promoter activity. Instead, this oxysterol slowed degradation of IL-6 mRNA and increased the amount of cytoplasmic HuR. 7-ketocholesterol significantly increased the amount of intracellular IL-6 protein in the presence of brefeldin A. 7-Ketocholesterol also enhanced IL-6 release from VSMC. IL-6 release by 7-ketocholesterol, although significant, was not as remarkable as that induced by TNF-α. These data suggest that 7-ketocholesterol upregulates IL-6 via mechanisms distinct from TNF-α and contributes to the intra- and extracellular IL-6 deposits within the vasculature.

Extracellular heat shock protein 90 induces interleukin-8 in vascular smooth muscle cells

Oxidative stress results in sustained release of heat shock protein 90 (HSP90) from vascular smooth muscle cells (VSMCs). The aim of this article is to investigate whether extracellular HSP90 predisposes VSMCs to pro-inflammatory phenotype. Exposure of aortic smooth muscle cells to HSP90 elevated IL-8 release and IL-8 transcript via promoter activation. HSP90-induced IL-8 promoter activation was suppressed by dominant-negative forms of Toll-like receptor (TLR)-4 and MyD88, but not by dominant-negative-forms of TLR-3, TLR-2, and TRIF. IL-8 up-regulation in response to HSP90 was also attenuated by IkappaB, rasveratrol, curcumin, diphenyleneiodium, N-acetylcystein, U0126, and SB202190. Mutation at the NF-kappaB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed the promoter activation by HSP90. This study proposes that extracellular HSP90 would contribute to IL-8 elevation in the stressed vasculature, and that TLR-4, mitogen-activated protein kinases, NF-kappaB, and reactive oxygen species are involved in that process.

Thrombin promotes proinflammatory phenotype in human vascular smooth muscle cell

Expression of PAR, the thrombin receptor, is elevated in smooth muscle cell-rich areas in atherosclerotic plaques, where the cells change to proinflammatory or synthetic phenotype. In this study we investigated whether thrombin promotes a proinflammatory phenotype in vascular smooth muscle cell (VSMC), characterized by increased cytokine and chemokine synthesis. Thrombin not only elevated transcripts for IL-6, CXCL8, and CCL11 genes but also enhanced release of IL-6 and CXCL8 protein from human aortic smooth muscle cell (HAoSMC). Thrombin activated Akt, PKC and MAPK in HAoSMC, and thrombin-mediated expression of IL-6 and CXCL8 was significantly inhibited by LY294002, AKT IV, RO318220, and GF109203X as well as by diphenyleneiodium at the messenger RNA and the protein levels. SB202129 and U0126 also significantly attenuated thrombin-mediated release of IL-6 and CXCL8 proteins from HAoSMC. These results indicate that thrombin promotes proinflammatory phenotype in human VSMC and that PI3K, Akt, PKC, NADPH oxidase, and MAPK are involved in that process. We propose that activation VSMC in response to thrombin after endothelial injury and/or thrombus formation will enhance inflammation in vasculature.

27-hydroxycholesterol induces production of tumor necrosis factor-alpha from macrophages.

Enhanced production of TNF-α from macrophages promotes development and instability of atherosclerotic plaques, but involvement of lipid component in TNF-α production has not been clarified in atherosclerosis. We attempted to determine whether cholesterol oxidation products (oxysterols) could modify TNF-α production. Treatment of THP-1 cells with 27-hydroxycholesterol (27OHChol) or 7α-hydroxycholesterol (7αOHChol) resulted in a profound increase in TNF-α transcription, while treatment with an identical concentration of cholesterol and 7-ketochoelsterol did not lead to any change in TNF-α expression. Treatment with 27OHChol resulted in increased synthesis, as well as secretion, of TNF-α, while 7αOHChol led to increased synthesis of TNF-α without affecting secretion of the cytokine. Co-treatment with 7αOHChol or 27OHChol and LPS resulted in synergistically enhanced or augmented secretion of TNF-α. Treatment with TO-901317, pertussis toxin, PP2, and LY294002 resulted not only in attenuated transcription of TNF-α induced by 27OHChol and 7αOHChol, but also secretion of TNF-α enhanced by 27OHChol. This is the first report demonstrating enhanced production of TNF-α in macrophages by treatment with oxysterols which are detected in abundance in atheromatous lesions; in addition, results of the current study provide evidence indicating that certain types of oxysterols contribute to development of atherosclerosis by promoting production of proinflammatory cytokines.

An Allele-Specific PCR Assay for the Rapid and Serotype-Specific Detection of Salmonella Pullorum

Abstract Salmonella serovar Pullorum is a causative agent of pullorum disease (PD) in poultry and is responsible for severe economic losses to the poultry industry in many parts of the world. A definitive detection of Pullorum requires culture followed by serotyping and biochemical identification, a process that is tedious and takes several weeks to accomplish. We have developed a rapid allele-specific polymerase chain reaction (PCR) method based on the nucleotide polymorphism in rfbS gene sequence for the serotype-specific detection of Pullorum and its differentiation from the closely related Gallinarum. The specificity of this PCR assay was tested using DNA samples from Pullorum (n = 13), Salmonella serotypes other than Pullorum (n = 19), and closely related non-Salmonella organisms (n = 5). The PCR assay was highly serotype-specific as the PCR amplicon of 147 base pairs was observed only in the case of Pullorum, while all the other DNA samples tested PCR negative. A definitive identification of Pullorum cultures was possible in less than 3 hr. As little as 100 pg of SP DNA was detected. This allele-specific PCR method is highly specific as well as sensitive and may be an effective molecular tool in the rapid and serotype-specific detection of Pullorum and differentiation from other Salmonella species.

27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes

Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE(-/-) mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process.

Peptidoglycan enhances secretion of monocyte chemoattractants via multiple signaling pathways.

Peptidoglycan (PG) is detected in a high proportion in inflammatory cell-rich regions of human atheromatous plaques. In the present study, we determined the cellular factors involved in PG-mediated chemokine expression in mononuclear cells in order to understand the molecular mechanisms of inflammatory responses to bacterial pathogen-associated molecular patterns in the diseased artery. Exposure of human monocytic leukemia THP-1 cells to PG resulted in not only enhanced secretion of CCL2 and CCL4 but also profound induction of their gene transcripts, which were abrogated by oxidized 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine, an inhibitor of Toll-like receptors (TLRs)-2/4, but not by polymyxin B. PG enhanced phosphorylation of Akt and mitogen-activated protein kinases and activated protein kinase C. Pharmacological inhibitors such as SB202190, SP6001250, U0126, Akt inhibitor IV, rapamycin, and RO318220 significantly attenuated PG-mediated up-regulation of CCL2 and CCL4. We propose that PG contributes to vascular inflammation in atherosclerotic plaques by upregulating expression of mononuclear cell chemoattractants via TLR-2, protein kinase C, Akt, mTOR, and mitogen-activated protein kinases.

27-Hydroxycholesterol induces recruitment of monocytic cells by enhancing CCL2 production.

Deposition of lipids in the intima is followed by infiltration of inflammatory cells, like monocytic cells and T lymphocytes, in atherosclerosis. However, roles of lipids in the infiltration of the inflammatory cells are not clearly defined. We investigated the possible involvement of cholesterol or its catabolites in recruitment of monocytic cells. Consumption of a high cholesterol-diet resulted in enhanced expression of CCL2 in arteries of ApoE(-/-) mice. 27-Hydroxycholesterol, the most abundant cholesterol oxide in atherosclerotic lesions, significantly induced the transcription of CCL2 and enhanced secretion of corresponding protein by THP-1 monocytic cells. However, cholesterol and 7-ketocholesterol did not influence expression of CCL2. Conditioned media containing CCL2 induced migration of monocytic cells, and migration was abrogated in the presence of CCL2-neutralizing antibody. TO-901317, a synthetic LXR agonist, inhibited both production of CCL2 and migration of monocytic cells induced by 27-hydroxycholesterol. Expression of CCL2 induced by 27-hydroxycholesterol was blocked when Akt inhibitor IV was added and when Akt1 was knocked down. We propose that 27-hydroxycholesterol will trigger a sequence of events leading to recruitment of monocytes into atherosclerotic lesions.