Soo-Ki Kim

Dependence on p38 MAPK signalling in the up-regulation of TLR2, TLR4 and TLR9 gene expression in Trichomonas vaginalis-treated HeLa cells.

Toll‐like receptors (TLRs) are pattern recognition receptors (PRRs) that recognize conserved pathogen‐associated molecular patterns (PAMPs) synthesized by micro‐organisms. Despite the essential requirement for TLRs in prokaryotic infection, the pattern and regulation of TLR gene expression by Trichomonas vaginalis in the mucocutaneous barrier are still unknown. Our hypothesis is that T.u2003vaginalis‐infected epithelial cells are major effector cells in the skin barrier. These cells function as a central regulator of TLR gene expression, thus accelerating the process of barrier dysfunction via increased release of chemokines and proinflammatory cytokines. To test this hypothesis, RT‐PCR was performed on TLRs, interleukin (IL)‐8 and tumour necrosis factor (TNF)‐α. Stimulation of HeLa cells by T.u2003vaginalis was observed to up‐regulate TLR2, 4 and 9 mRNA expression as well as that of IL‐8 and TNF‐α. To further clarify the molecular mechanism of barrier devastation triggered by these up‐regulatory stimuli, we examined the profiles of extracellular signal‐regulated kinase (ERK), p38 mitogen‐activated protein kinase (MAPK) and nuclear factor (NF)‐κB activation in HeLa cells using specific inhibitors. Interestingly, pretreatment of HeLa cells with the p38 MAPK inhibitor SB203580 demonstrated inhibition of T.u2003vaginalis‐induced up‐regulation of TLR2, 4, and 9 mRNA expression. By contrast, inhibition of ERK or NF‐κB activation failed to block T.u2003vaginalis‐induced up‐regulation of TLR9 mRNA expression or TLR2 and TLR4 mRNA expression, respectively. In addition, pretreatment with SB203580 reduced epithelium‐derived IL‐8 and TNF‐α release evoked by T.u2003vaginalis. Our results show that T.u2003vaginalis infection of the mucocutaneous barrier could up‐regulate TLR2, 4 and 9 gene expression via the p38 MAPK signalling pathway in epithelial cells; this process then leads to modulation of p38 MAPK‐dependent IL‐8 and TNF‐α release from the epithelium.

Involvement of oxidative stress in simvastatin-induced apoptosis of murine CT26 colon carcinoma cells.

Recent studies have suggested that oxidative stress may play a role in the cytotoxic activity of statins against cancer cells. The objective of this study was to elucidate the role of oxidative stress in the cytotoxicity of simvastatin in murine CT26 colon carcinoma cells and B16BL6 melanoma cells. We found that CT26 cells were more sensitive to simvastatin than B16BL16 cells. Interestingly, exposure to simvastatin causes significant apoptotic cell death and perturbations in parameters indicative of oxidative stress in CT26 cells. Moreover, the increase in oxidative stress parameters and cell death were suppressed by isoprenoids including mevalonolactone, farnesyl pyrophosphate ammonium salt, geranylgeranyl pyrophosphate ammonium salt, and coenzyme Q10, and by antioxidants including N-acetyl cysteine, reduced glutathione, superoxide dismutases (SOD), and catalase (CAT) alone or in combination, but were promoted by an inhibitor of glutathione synthesis, L-buthionine-sulfoximine. The signaling pathway induced by simvastatin breaks down the antioxidant defense system by suppressing the expression of reactive oxygen species scavengers, particularly Mn-SOD, CAT, GPx1, and SESN 3, thereby inducing oxidative stress and apoptotic cell death. Collectively, our results demonstrate that simvastatin induces colon cancer cell death at least in part by increasing intracellular oxidative stress and inducing apoptosis.

Fluvastatin inhibits expression of the chemokine MDC/CCL22 induced by interferon-γ in HaCaT cells, a human keratinocyte cell line

Background and purpose:u2002 The macrophage‐derived chemokine (MDC/CCL22) is a prototypic Th2‐type chemokine intimately involved in Th2‐skewed allergic diseases, such as atopic dermatitis and asthma. The statins (3‐hydroxy‐3‐methyl glutaryl coenzyme A reductase inhibitors) have been demonstrated to relieve allergic inflammation. However, the immunological effects and mechanisms of statins against atopic dermatitis remain unknown, at least in vitro. This study aimed to define how different statins affect MDC expression in HaCaT cells, a human keratinocyte cell line.

Differential expression, shedding, cytokine regulation and function of TNFR1 and TNFR2 in human fetal astrocytes.

Tumor necrosis factor (TNF)-α induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF-α receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-α receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-α receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-α, IL-1, and IFN-γ, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-κB activation and TNF-α induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kB activation and TNF-α induction are blocked by TNFR1 neutralizing antibody treatments.

FoxO3a suppresses the senescence of cardiac microvascular endothelial cells by regulating the ROS-mediated cell cycle

FoxO3a plays an important role in the aging process and decreases with age. However, the potential regulatory roles of FoxO3a in processes involved in cardiac microvascular endothelial cell (CMEC) senescence, and its underlying molecular mechanisms have not been elucidated. This study demonstrates that FoxO3a is deactivated in senescent CMECs together with the inhibition of proliferation and tube formation. Furthermore, the activation of the antioxidant enzymes catalase and SOD, downstream FoxO3a targets, was significantly decreased, thereby leading to cell cycle arrest in G1-phase by increased ROS generation and subsequently the activation of the p27(Kip1) pathway. However, FoxO3a overexpression in primary low-passage CMECs not only significantly suppressed the senescence process by increasing the activation of catalase and SOD but also markedly inhibited ROS generation and p27(Kip1) activation, although it failed to reverse cellular senescence. Moreover, both cell viability and tube formation were greatly increased by FoxO3a overexpression in primary CMECs during continuous passage. In addition, FoxO3a, deficiency in low-passage CMECs, accelerated the senescence process. Collectively, our data suggest that FoxO3a suppresses the senescence process in CMECs by regulating the antioxidant/ROS/p27(Kip1) pathways, although it fails to reverse the cellular senescent phenotype.

CpG ODN, Toll Like Receptor (TLR)-9 Agonist, Inhibits Metastatic Colon Adenocarcinoma in a Murine Hepatic Tumor Model

BACKGROUNDnColorectal liver metastases (mets) are often refractory to conventional therapies. CpG oligodeoxynucleotide 1826 (CpG), a Toll like receptor (TLR)-9 agonist, inhibits murine tumor growth by augmenting Th1 immunity. The impact of CpG on metastatic colon tumors is unknown. The purpose of this study was to determine the effect of CpG on the growth of hepatic colon cancer mets.nnnMETHODSnTwo studies with separate control groups were performed using 40 Balb/C mice (study A, CpG 50 μg/dose; study B, 100 μg/dose; n = 9-11/subgroup). Tumors were induced via portal vein injection of 2 × 10(4) CT26 colon tumor cells. After surgery, the mice were randomized; test groups were given 14 daily intraperitoneal (i.p.) CpG injections (50 or 100 μg/dose) while the control group received i.p. saline. On d 21 mice were sacrificed, the livers and spleens excised and weighed and the mets counted (reported as median ± 95% confidence interval [CI]) and histologically assessed.nnnRESULTSnThe CpG mice had significantly fewer hepatic mets/mouse (study A, median two nodules, 95% CI, 0-3; study B, 0 nodules, 95% CI 0-0) than the control mice (study A, 6 nodules, 95% CI, 3-9, P = 0.002; Study B, 6 nodules, 95% CI, 3-9, P < 0.001). In study B, there were no mets in 9/11 CpG mice (versus 2/10 for CpG 50 μg and 0/19 for control mice). The mean liver/spleen weights of the CpG mice in both studies were significantly greater than in control mice. Histologically, high mitotic rates were noted in control mets while fewer tumor cells and histiocytic and lymphocytic infiltrates were found in CpG livers.nnnCONCLUSIONSnCpG inhibited liver tumor growth in this model (100 μg/dose more than 50 μg/dose). CpG was associated with increased liver and spleen weights not related to tumor burden. Increased lymphocytic and histiocytic infiltrates were noted in CpG-treated tumor nodules.

Immunohistochemical distribution of toll-like receptor 4 in preterm human fetal membrane.

Aim:u2002 Toll‐like receptor 4 (TLR‐4) is the major receptor used for recognition of specific gram negative bacteria by the host immune system. The role of TLR‐4 has been revealed in preterm parturition. This study aims to demonstrate the immunohistochemical expression of TLR‐4 with regard to histological layers and anatomical regions of the human fetal membranes.

Tenofovir versus tenofovir plus entecavir for chronic hepatitis B with lamivudine resistance and entecavir resistance

We compared the viral suppressive efficacy of tenofovir disoproxil fumarate (TDF) mono‐rescue therapy (TDF group) and TDF plus entecavir (ETV) combination‐rescue therapy (TDF + ETV group) in chronic hepatitis B (CHB) patients with lamivudine resistance and entecavir resistance. One hundred and thirty‐three CHB patients with lamivudine and entecavir resistance were investigated. Ninety‐six patients were treated with TDF and 37 with TDF + ETV for at least 6 months. We compared the virologic response rate (HBV DNA level <20 IU/mL) between the two groups and identified the predictive factors of treatment outcome. There were no significant differences between the two groups in demographic characteristics. Up to 24 months [median: 18 (range 6‐24) months], 85.4% and 89.2% of the TDF group and TDF + ETV group, respectively, achieved a virologic response (P=.068). Only the HBV DNA level at baseline was significantly associated with a virologic response in the multivariate analysis. In a subanalysis of patients with HBV DNA levels ≥4 log (IU/mL) at baseline, a higher proportion of patients in the TDF + ETV group than the TDF group achieved a virologic response (92.9% vs 68.3%; P<.001), while 90% of patients with HBV DNA (IU/mL) levels <4 log in all both TDF and TDF + ETV groups achieved a virologic response. TDF mono‐rescue therapy is a reasonable option in patients with lamivudine resistance and entecavir resistance. However, the combination strategy should be considered in patients with high baseline HBV DNA levels.

Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation

Recognition of oligodeoxynucleotides containing CpG motifs (CpG-ODNs) by toll-like receptor 9 (TLR9) inhibits RANKL-induced osteoclastogenesis from precursors. This inhibitory effect suggests the possibility of using this strategy to block pathological bone loss. However, the enhancing effect of CpG-ODNs on OC formation from RANKL-primed pre-osteoclasts (pOCs) has hampered their clinical use. In this report, we developed a CpG-KSK13 oligonucleotide with an alternative CpG motif, and tested its effect on osteoclastogenesis in comparison with previously used murine CpG motif (CpG-1826) or human CpG motif (CpG-2006) oligonucleotides. Murine CpG-1826 inhibited RANKL-induced OC formation from BMMs but not from RANKL-primed pOCs, while CpG-KSK13 treatment strongly inhibited OC formation from both BMM and primed pOC cells. CpG-KSK13 also showed a potent inhibitory effect on human OC differentiation using peripheral blood mononuclear cells (PBMCs), which was in contrast to the species-specific response of murine CpG-1826 or human CpG-2006. Moreover, CpG-KSK13 effectively inhibited NFATc1 activity, but not NF-kappaB or AP-1 activity, and decreased TREM-2 promoter activity and subsequent surface expression of the TREM-2 protein induced by M-CSF and RANKL. These results demonstrate that the recognition of CpG-KSK13 via TLR9 inhibits osteoclastogenesis by down-regulating TREM-2 expression. Thus, our findings provide evidence for the potential use of CpG-KSK13 as an anti-osteoclastogenic agent for human and for pre-clinical animals.

Cancer immunotherapeutic effects of novel CpG ODN in murine tumor model

While CpG oligodeoxynucleotides (ODN) are excellent candidates for cancer immunotherapeutics, the numbers of usable CpG ODNs are limited in current clinical settings. To resolve this, we investigated whether novel CpG ODN (KSK-CpG) would be an effective immunotherapeutic in a murine tumor model by affecting in vivo and in vitro parameters, such as survival span, the number of tumor nodules, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activity and interleukin (IL)-6 or IL-12 cytokine release in splenocytes. We found that KSK-CpG was effective in the murine cancer model by way of prolonging survival span, reducing the number of tumor nodules, augmenting NK cell and CTL cytotoxicity, as well as evoking IL-6 and IL-12 cytokine release in splenocytes. Collectively, these data demonstrate that KSK-CpG is active against the highly malignant B16BL6 and EL4 tumor mouse model via innate immune augmentation.