Analysis of Swine Leukocyte Antigen Haplotypes in Yucatan Miniature Pigs Used as Biomedical Model Animal.

The porcine major histocompatibility complex (MHC) is called swine leukocyte antigen (SLA), which controls immune responses and transplantation reactions. The SLA is mapped on pig chromosome 7 (SSC7) near the centromere. In this study, 3 class I (SLA-1, SLA-3, and SLA-2) and 3 class II (DRB1, DQB1, and DQA) genes were used for investigation of SLA haplotypes in Yucatan miniature pigs in Korea. This pig breed is a well-known model organism for biomedical research worldwide. The current study indicated that Korean Yucatan pig population had 3 Class I haplotypes (Lr-4.0, Lr-6.0, and Lr-25.0) and 3 class II haplotypes (Lr-0.5, Lr-0.7, and Lr-0.25). The combinations of SLA class I and II haplotype together, 2 homozygous (Lr-4.5/4.5 and Lr-6.7/6.7) and 3 heterozygous (Lr-4.5/6.7, Lr-4.5/25.25, and Lr-6.7/25.25) haplotypes were identified, including previously unidentified new heterozygous haplotypes (Lr-4.5/4.7). In addition, a new SLA allele typing method using Agilent 2100 bioanalyzer was developed that permitted more rapid identification of SLA haplotypes. These results will facilitate the breeding of SLA homozygous Yucatan pigs and will expedite the possible use of these pigs for the biomedical research, especially xenotransplantation research.

A Split-face Study on Rejuvenation Efficacy According to Monopolar Radiofrequency Tip Size.

Monopolar radiofrequency (RF) non-invasively tightens and rejuvenates the skin by stimulating collagen fiber production. Since the introduction of the monopolar RF device in the early 2000's, RF devices have advanced and they can rejuvenate of periorbital and forehead wrinkles, as well as skin laxity of the lower face and neck. We compared the differences in the treatment effects based on the tip size. This randomized split-face study comprised 31 participants aged 29 to 75 years old (three males and 28 females) who underwent one session of monopolar RF; one side of the face was treated with a 3cm2 tip and the other with a 4cm2 tip. Facial wrinkle scores were measured on the upper face and the lower face before and after treatment for up to three months. Significant improvement was observed in the periorbital area (p<0.001), forehead (p=0.72), and glabellar (p=0.63) treated with a smaller tip. However, nasolabial folds (p=0.8) and marionette lines (p=0.13) showed better improvement when treated with a larger tip.

Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation.

OBJECTIVE: To determine whether the level of lysophosphatidylcholine (lysoPC) generated by lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with severity of inflammation in human atherosclerotic plaques. Elevated plasma Lp-PLA2 is associated with increased cardiovascular risk. Lp-PLA2 inhibition reduces atherosclerosis. Lp-PLA2 hydrolyzes low-density lipoprotein-oxidized phospholipids generating lysoPCs. According to in vitro studies, lysoPCs are proinflammatory but the association between their generation and plaque inflammation remains unknown. METHODS AND RESULTS: Inflammatory activity in carotid plaques (162 patients) was determined immunohistochemically and by analyzing cytokines in homogenates (multiplex immunoassay). LysoPCs were quantified using mass spectrometry and Lp-PLA2 and the lysoPC metabolite lysophosphatidic acid (LPA) by ELISA. There was a strong correlation among lysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 in plaques. LysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 correlated with interleukin-1ß, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, regulated on activation normal T-cell expressed and secreted, and tumor necrosis factor-α in plaques. High lysoPC and Lp-PLA2 correlated with increased plaque macrophages and lipids and with low content of smooth muscle cells, whereas LPA only correlated with plaque macrophages. Lp-PLA2, lysoPC 16:0, 18:0, and 18:1, but not LPA were higher in symptomatic than in asymptomatic plaques. CONCLUSIONS: The associations among Lp-PLA2, lysoPCs, LPA, and proinflammatory cytokines in human plaques suggest that lysoPCs play a key role in plaque inflammation and vulnerability. Our findings support Lp-PLA2 inhibition as a possible strategy for the prevention of cardiovascular disease.

4-Chlorotetrazolo[1,5-a]quinoxaline inhibits activation of Syk kinase to suppress mast cells in vitro and mast cell-mediated passive cutaneous anaphylaxis in mice.

4-Chlorotetrazolo[1,5-a]quinoxaline is a quinoxaline derivative. We aimed to study the effects of 4-chlorotetrazolo[1,5-a]quinoxaline on activation of mast cells in vitro and in mice. 4-Chlorotetrazolo[1,5-a]quinoxaline reversibly inhibited degranulation of mast cells in a dose-dependent manner, and also suppressed the expression and secretion of TNF-α and IL-4 in mast cells. Mechanistically, 4-chlorotetrazolo[1,5-a]quinoxaline inhibited activating phosphorylation of Syk and LAT, which are crucial for early FcεRI-mediated signaling events, as well as Akt and MAP kinases, which play essential roles in the production of various pro-inflammatory cytokines in mast cells. Notably, although 4-chlorotetrazolo[1,5-a]quinoxaline inhibited the activation of Fyn and Syk, minimal inhibition was observed in mast cells in the case of Lyn. Furthermore, consistent with its in vitro activity, 4-chlorotetrazolo[1,5-a]quinoxaline significantly suppressed mast cell-mediated passive cutaneous anaphylaxis in mice. In summary, the results from this study demonstrate that 4-chlorotetrazolo[1,5-a]quinoxaline shows an inhibitory effect on mast cells in vitro and in vivo, and that this is mediated by inhibiting the activation of Syk in mast cells. Therefore, 4-chlorotetrazolo[1,5-a]quinoxaline could be useful in the treatment of mast cell-mediated allergic diseases.

Interleukin-33 stimulates formation of functional osteoclasts from human CD14(+) monocytes.

Interleukin (IL)-33 is a recently described pro-inflammatory cytokine. Here we demonstrate IL-33 as a regulator of functional osteoclasts (OCs) from human CD14(+) monocytes. IL-33 stimulates formation of tartrate-resistant acid phosphatase (TRAP)(+) multinuclear OCs from monocytes. This action was suppressed by anti-ST2 antibody, suggesting that IL-33 acts through its receptor ST2, but not by the receptor activator of NF-κB ligand (RANKL) decoy, osteoprotegerin, or anti-RANKL antibody. IL-33 stimulated activating phosphorylations of signaling molecules in monocytes that are critical for OC development. These included Syk, phospholipase Cγ2, Gab2, MAP kinases, TAK-1, and NF-κB. IL-33 also enhanced expression of OC differentiation factors including TNF-α receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells cytoplasmic 1, c-Fos, c-Src, cathepsin K, and calcitonin receptor. IL-33 eventually induced bone resorption. This study suggests that the osteoclastogenic property of IL-33 is mediated through TRAF6 as well as the immunoreceptor tyrosine-based activation motif-dependent Syk/PLCγ pathway in human CD14(+) monocytes.

Deficiency of peroxiredoxin 2 exacerbates angiotensin II-induced abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease characterized by structural deterioration of the aorta caused by inflammation and oxidative stress, leading to aortic dilatation and rupture. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, has been reported as a potential negative regulator of inflammatory vascular diseases, and it has been identified as a protein that is increased in patients with ruptured AAA compared to patients with nonruptured AAA. In this study, we demonstrated that PRDX2 was a pivotal factor involved in the inhibition of AAA progression. PRDX2 levels were increased in AAA compared with those in normal aortas in both humans and mice. Ultrasound imaging revealed that the loss of PRDX2 accelerated the development of AAA in the early stages and increased AAA incidence in mice infused with angiotensin II (Ang II). Prdx2-/- mice infused with Ang II exhibited increased aortic dilatation and maximal aortic diameter without a change in blood pressure. Structural deterioration of the aortas from Prdx2-/- mice infused with Ang II was associated with increases in the degradation of elastin, oxidative stress, and intramural thrombi caused by microhemorrhages, immature neovessels, and the activation of matrix metalloproteinases compared to that observed in controls. Moreover, an increase in inflammatory responses, including the production of cell adhesion molecules and the accumulation of inflammatory cells and proinflammatory cytokines due to PRDX2 deficiency, accelerated Ang II-induced AAA progression. Our data confirm that PRDX2 plays a role as a negative regulator of the pathological process of AAA and suggest that increasing PRDX2 activity may be a novel strategy for the prevention and treatment of AAA.

Anti-inflammatory effects of Artemisia princeps in antigen-stimulated T cells and regulatory T cells.

OBJECTIVES: The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells and antigen-expanded regulatory T cells. METHODS: CD4(+)CD25(-) T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [(3)H]thymidine and, after harvesting the cells, [(3)H]thymidine incorporation was measured. For analysis of interleukin-2 and interferon-gamma secreted from CD4(+)CD25(-) T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4(+)CD25(-) T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography-mass spectrometry. KEY FINDINGS: A. princeps (30 microg/ml) effectively suppressed proliferation of CD4(+)CD25(-) T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-gamma in anti-CD3/CD28-stimulated CD4(+)CD25(-) T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. CONCLUSIONS: The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4(+)CD25(-) T cells and increasing activity of expanded regulatory T cells.

Curcumin, a constituent of curry, suppresses IgE-mediated allergic response and mast cell activation at the level of Syk.

BACKGROUND: Activation of mast cells through the high-affinity receptor for IgE (FcepsilonRI) underlies atopic allergic reactions. Curcumin can block this activation, but the mechanism and the effects of curcumin on IgE-mediated allergic reactions are unknown. OBJECTIVES: We sought to determine the antiallergic activity of curcumin in vivo and its mechanism of action in mast cells. METHODS: The antiallergic activity of curcumin was evaluated in mast cell cultures and the passive cutaneous anaphylaxis model. The effects of curcumin on mast cell signaling events were examined by using immunoblotting, immunoprecipitation, RT-PCR, and other molecular biologic approaches. RESULTS: Curcumin inhibited antigen-mediated activation of mast cells and passive cutaneous anaphylaxis in mice. Suppression of degranulation and secretion of TNF-alpha and IL-4 was apparent at concentrations as low as 3 micromol/L curcumin in activated mast cells. Similar concentrations of curcumin suppressed Syk-dependent phosphorylations of the adaptor proteins linker of activated T cells and Grb2-associated binder 2, which are critical for mast cell activation. Although curcumin did not inhibit the phosphorylation of Syk itself, it directly inhibited Syk kinase activity in vitro. Further downstream, activating phosphorylations of Akt and the mitogen-activated protein kinases p38, p44/42 (extracellular signal-regulated kinase 1/2), and c-Jun N-terminal kinase, which are critical for the production of inflammatory cytokines, were also inhibited. CONCLUSIONS: Curcumin inhibits Syk kinase-dependent signaling events in mast cells and might thus contribute to its antiallergic activity. Therefore curcumin might be useful for the treatment of mast cell-related immediate and delayed allergic diseases.

Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line.

Interleukin (IL)-32 was recently identified as a new cytokine which induces various proinflammatory cytokines in human monocytes and macrophages. Therefore, IL-32 has been primarily studied in inflammatory models such as rheumatoid arthritis and inflammatory bowel diseases. The regulation of endogenous IL-32 in other immune cells remains unknown. In the present study, we stimulated Jurkat T cells with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and examined IL-32 expression at both the mRNA and protein levels. All mRNAs of the four IL-32 isoforms and the 12-15 kDa IL-32 protein were independent of PHA and PMA stimulation, however a 9 kDa molecular weight IL-32 protein in the cell culture supernatant was induced by PHA and PMA after 16 h of stimulation. Compared to other human cell lines, the Jurkat cell line constitutively expressed a 12-15 kDa molecule of IL-32, which is smaller than the known IL-32 isoforms. We used IL-32 shRNA to examine the specificity of the 12-15 kDa molecule. Upon IL-32 shRNA transfection, the 12-15 kDa band was decreased specifically as compared to the control scrambled clone. Thus, the constitutive expression of IL-32 mRNA as well as the predominant production of a smaller sized IL-32 isoform in Jurkat cells may implicate a role for IL-32 in human T cell leukemia.

Mast cell-mediated allergic response is suppressed by Sophorae flos: inhibition of SRC-family kinase.

Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC(50) values of approximately 31.6 microg/mL (RBL-2H3 mast cells) and approximately 47.8 microg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-alpha and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.

Polygoni cuspidati radix inhibits the activation of Syk kinase in mast cells for antiallergic activity.

The antiallergic activity of Polygoni cuspidati radix (PR) and the mechanism of action by which it functions were investigated in this study. The extract of PR exhibited potent inhibitory activity in mast cells; its IC50 values were 62 +/- 2.1 microg/ml for RBL-2H3 mast cells and 46 +/- 3.2 microg/m for bone marrow-derived mast cells by antigen stimulation, and it also suppressed the expression of tumor necrosis factor-alpha and interleukin-4 in RBL-2H3 cells. According to the in vivo animal allergy model, it inhibited a local allergic reaction, passive cutaneous anaphylaxis, in a dose-dependent manner. With regard to its mechanism of action, PR inhibited the activating phosphorylation of Syk, a key signaling protein for the activation of mast cells. It also suppressed Akt and the mitogen-activated protein kinases ERK1/2, p38, and JNK, which are critical for the production of various inflammatory cytokines in mast cells. The results of the study indicate that the antiallergic activity of PR is mediated through the inhibition of histamine release and allergic cytokine production by the inhibition of Syk activating phosphorylation in mast cells.

In-vitro and in-vivo anti-inflammatory action of the ethanol extract of Trachelospermi caulis.

In this study, we aimed to investigate the anti-inflammatory activity, antinociceptive activity and the action mechanism of Trachelospermi caulis extract. The anti-inflammatory effects were investigated using arachidonic acid, 12-O-tetradecanoylphorbol 13-acetate or carrageenan-induced oedema assays. Antinociceptive activity, using the acetic acid-induced writhing model, was also tested in mice. The extract exhibited dose-dependent and significant (P<0.05 at 100-400 mg kg-1) anti-inflammatory and antinociceptive activity in the animals. To further understand the mechanism of activity, we investigated whether the extract inhibited the expression of inducible nitric oxide synthase (iNOS), the production of nitric oxide (NO) and the expression of TNF-alpha from murine macrophage RAW 264.7 cells. Similar to the in-vivo activity, the iNOS expression, NO production and TNF-alpha expression were found to be dose dependent and significantly suppressed by the extract through the inhibition of the p38 MAP kinase/NF-kappaB pathway. Taken together, the results presented here suggest that T. caulis extract may be useful for the treatment of various inflammatory diseases.

Analysis of erythema after Er:YAG laser skin resurfacing.

BACKGROUND: Postoperative erythema can be expected to occur in every patient after laser resurfacing, and pigmentary disturbances may be related to the intensity and the duration of erythema. OBJECTIVE: This study was undertaken to assess the clinical features of erythema, the factors that influence its duration, and the relation between the duration of erythema and the incidence of hyperpigmentation and hypopigmentation in skin of Asian persons after Er:YAG laser resurfacing. METHODS: A total of 218 patients (skin phototypes III to V) were recruited and treated with a short-pulsed Er:YAG laser, a variable-pulsed Er:YAG laser, or a dual-mode Er:YAG laser for skin resurfacing. Clinical assessments were performed retrospectively using medical charts and serial photographs. RESULTS: Postoperative erythema was observed in all patients after Er:YAG laser resurfacing with a mean duration of 4.72 months. In 98.2% of patients, erythema faded completely within 12 months. Postinflammatory hyperpigmentation was observed in 38.1% of patients after Er:YAG laser resurfacing. CONCLUSIONS: Skin phototype, level of ablation, and depth of thermal damage caused by a long-pulsed laser appear to be important factors that affect the duration of erythema. Moreover, prolonged erythema was related to the risk of postinflammatory hyperpigmentation.

The anti-inflammatory effects of Pyrolae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO production.

The extract of Pyrolae herba (PH), which has been used as an anti-inflammatory folk remedy in Korea and China, was investigated for its anti-inflammatory action using arachidonic acid, 12-O-tetradecanoylphorbol 13-acetate or carrageenan-induced edema assays. The anti-nociceptive activity of PH was also tested in mice using the acetic acid-induced writhing model. PH showed dose-dependent and significant (P<0.05 at 100-400mg/kg) anti-inflammatory and anti-nociceptive activities in the animal assays. The mechanism of the activities of PH was examined by testing the extract to determine if it inhibits the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) from the murine macrophages, RAW 264.7 cells. Similar to the in vivo activities, both the iNOS expression and NO production were significantly suppressed by PH in a dose-dependent manner. PH also inhibited the activating phosphorylation of p38 MAP kinase and NF-kappaB in these cells. These results provide a scientific basis to explain the effects of PH as an anti-inflammatory folk remedy in Asian countries.

The synergistic effect of phytohemagglutinin and interferon-gamma on the expression of tumor necrosis factor-alpha from RAW 264.7 cells.

Tumor necrosis factor-alpha (TNF-alpha) is a major cytokine of host immune reaction by foreign agents. Phytohemagglutinin (PHA) is a dynamic contributor to mitogenic stimulation and augmentation of host immune defense. Interferon-gamma (IFN-gamma) induces induction of cytokines in macrophages and lymphocytes. The aim of this study was to examine the synergistic effects of PHA plus low dose IFN-gamma on TNF-alpha mRNA production, cytosolic levels, and secretion in RAW 264.7 cells. The cells were stimulated with PHA or IFN-gamma using various concentrations for various times. The effects of PHA on TNF-alpha expression appeared in dose- and time-dependent manners. The maximum doses of PHA and IFN-gamma to produce them were 300 microg/ml PHA and 10 ng/ml IFN-gamma. The optimum time of PHA for the TNF-alpha mRNA production and release were 6 and 7 h after stimulation, respectively, whereas the time of IFN-gamma on them was achieved at 3 and 8 h. Although the TNF-alpha mRNA production, cytosolic levels, and secretion from the cells were slightly detected under 10 microg/ml PHA and 1 ng/ml IFN-gamma, the combination of PHA (10 microg/ml) and IFN-gamma (1 ng/ml) greatly increased them, indicating the synergistic effect of PHA plus low dose IFN-gamma on TNF-alpha expression.

Effect of a fermented ginseng extract, BST204, on the expression of cyclooxygenase-2 in murine macrophages.

This paper investigates how BST204, a fermented ginseng extract, affects the expression and mechanism of cyclooxygenase-2 (COX-2). BST204 was prepared by incubating crude ginseng extract with ginsenoside-beta-glucosidase. Unexpectedly, BST204 had no effect on the level of COX-2 protein in unstimulated RAW 264.7 cells, and it suppressed the level of COX-2 protein and PGE(2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. It did not show any suppressive effect, though, on the COX-2 mRNA level. To investigate the suppressive mechanism of COX-2 protein, the activating phosphorylation of p70 S6 kinase and 4E-BP1, which are important for translation, were measured. The phosphorylation of p70 S6 kinase, not 4E-BP1, was increased by LPS in a time-dependent manner, and was inhibited by BST204 in a dose-dependent manner. The expression of COX-2 protein, however, was partially suppressed by rapamycin, an upstream inhibitor of p70 S6 kinase. Therefore, this paper suggests that the suppression of COX-2 protein by BST204 was partially correlated with the inhibition of p70 S6 kinase activation.

Inhibitory effects of a fermented ginseng extract, BST204, on the expression of inducible nitric oxide synthase and nitric oxide production in lipopolysaccharide-activated murine macrophages.

In this study, the effects of BST204, a fermented ginseng extract, on the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production are looked into. Crude ginseng extract was incubated with ginsenoside-beta-glucosidase to prepare BST204. BST204, unlike lipopolysaccharide (LPS) and crude ginseng extract, did not affect the level of iNOS protein and NO production in unstimulated RAW 264.7 cells. However, it suppressed the level of iNOS protein and NO production in LPS-stimulated RAW 264.7 cells but did not manifest the same effect on the iNOS mRNA level. An investigation of the activating phosphorylation of p70 S6 kinase and 4E-BP1, which are important for translation, was conducted to investigate the suppressive mechanism of iNOS protein. LPS increased the phosphorylation of p70 S6 kinase, but not 4E-BP1, in a time-dependent manner, and BST204 inhibited it in a dose-dependent manner. The expression of iNOS protein, however, was partially suppressed by rapamycin, an upstream inhibitor of p70 S6 kinase. Therefore, this paper suggests that the suppression of iNOS protein by BST204 was partially correlated with the inhibition of p70 S6 kinase activation.

Inhibitory activity of Chrysanthemi sibirici herba extract on RBL-2H3 mast cells and compound 48/80-induced anaphylaxis.

The effects of extracts from various oriental medicinal herbs on mast cell-mediated allergic reaction were investigated. Among them, Chrysanthemi sibirici herba ethanol extract exerted the potent inhibitory activity on antigen-induced degranulation in RBL-2H3 mast cells. Chrysanthemi sibirici herba dose-dependently inhibited DNP-BSA or compound 48/80-induced degranulation in RBL-2H3 mast cells, with IC(50) values of approximately 49 microg/ml and 76 microg/ml, respectively. This extract strongly suppressed compound 48/80-induced systemic anaphylaxis by 48.7% at a dose of 300 mg/kg in mice. Chrysanthemi sibirici herba also inhibited the expression of TNF-alpha and the activation of the MAP kinase, ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of activating phosphorylation of ERK1/2. These results lead us to conclude that Chrysanthemi sibirici herba may be used clinically to treat various allergic diseases.