Cryptoporus volvatus polysaccharides attenuate LPS-induced expression of pro-inflammatory factors via the TLR2 signaling pathway in human alveolar epithelial cells.

CONTEXT: Cryptoporus volvatus (Peck) Hubb grows wild in China, and its fruiting bodies have been used traditionally to treat asthma and bronchitis. OBJECTIVES: This study evaluates the anti-inflammatory effect of Cryptoporus polysaccharides (CP) extracted from fruiting bodies of C. volvatus on lipopolysaccharide (LPS)-induced pro-inflammatory factors and the signaling pathways involved in human alveolar epithelial cells. MATERIALS AND METHODS: To evaluate the effects of CP on LPS-induced pro-inflammatory factors, A549 cells were pre-incubated with CP 1, 10, and 100 µg/ml for 1 h and then stimulated with LPS 10 µg/ml for 24 h. The expression of pro-inflammatory factors monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), Toll-like receptor 2 (TLR2), and phosphorylation of ERK1/2, p38, and NF-κB p65 were measured by q-PCR, ELISA, and western blotting. RESULTS: CP decreased LPS-induced mRNA expression of MCP-1, TNF-α, and IL-1ß (IC50 = 83.3, 85.2, and 91.6 µg/ml, respectively) and their correspondent protein expression (IC50 = 88.6, 76.4, and 81.6 µg/ml, respectively). Investigation of potential mechanisms indicated that CP 100 µg/ml reduced LPS-induced expression of TLR2 mRNA (66.9%, p < 0.01) and protein (63.2%, p < 0.01) that was a result of the decreased pro-inflammatory factors. LPS induction increased the expression of TLR2 and the phosphorylation of p38 and ERK1/2, NF-kB p65 concomitantly. CP 100 µg/ml inhibited the LPS-induced phosphorylation of the signaling proteins (p < 0.05). CONCLUSIONS: This suggests that CP pretreatment down-regulates LPS-mediated inflammation in lung epithelial cells. This study further confirmed that CP is a potential anti-inflammatory drug for the treatment of airway inflammatory diseases.

Epoxyeicosatrienoic acids attenuate cigarette smoke extract-induced interleukin-8 production in bronchial epithelial cells.

In response to endothelial cell activation, arachidonic acid can be converted by cytochrome P450 (CYP) epoxygenases to epoxyeicosatrienoic acids (EETs), which have potent vasodilator and anti-inflammatory properties. In this study, we investigated the effects of exogenous EETs on cigarette smoke extract (CSE)-induced inflammation in human bronchial epithelial cells (NCI-H292). We found that CSE inhibited the expression of CYP2C8 and mildly stimulated the expression of epoxide hydrolase 2 (EPHX2) but did not change the expression of CYP2J2. Treatment with 11,12-EET or 14,15-EET attenuated the CSE-induced release of interleukin (IL)-8 by inhibiting the phosphorylation of p38 mitogen-activated protein kinases (MAPKs). Our results demonstrated that CSE may reduce the anti-inflammatory ability of epithelial cells themselves by lowering the EET level. EETs from pulmonary epithelial cells may play a critical protective role on epithelial cell injury.

Effect of low-level laser therapy on allergic asthma in rats.

Asthma is a complex chronic inflammatory disease of the airways that involves the activation of many inflammatory and other types of cells. We investigated the effect of low-level laser therapy (LLLT) on allergic asthma in rats and compared its effect with that of the glucocorticoid budesonide. Asthma was induced by challenge and repeated exposure to ovalbumin. Asthmatic rats were then treated with LLLT or budesonide suspension. LLLT at 8 J/cm(2) once daily for 21 days could relieve pathological damage and airway inflammation in asthmatic rats. LLLT could decrease the total numbers of cells and eosinophils in bronchoalveolar lavage fluid. LLLT could reduce levels of IL-4 and increase IFN-γ levels in bronchoalveolar lavage fluid and serum, meanwhile reduce serum IgE levels. Flow cytometry assay showed that LLLT can regulate the Th1/Th2 imbalance of asthmatic rats. LLLT had a similar effect to that of budesonide. These findings suggest that the mechanism of LLLT treatment of asthma is by adjustment of Th1/Th2 imbalance. Thus, LLLT could take over some of the effects of budesonide for the treatment of asthma, thereby reducing some of the side effects of budesonide.

Apple polyphenol protects against cigarette smoke-induced acute lung injury.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease involving oxidative stress as well as a wide variety of cells activated from smoking cigarettes. There have been disappointingly few therapeutic advances in drug therapy for COPD. Plant polyphenols have been the topic of much research regarding their antioxidant activities and antiinflammatory and immunomodulatory effects. In the present study, we ask whether apple polyphenol provides protection against cigarette smoke (CS)-induced acute lung injury. METHODS: ICR mice were exposed to CS for 4 d with increasing exposure time for up to 6 h per day to elicit epithelial cells injury. One hour before smoke exposure, mice were treated with apple polyphenol (APP) by gavage; all examinations were performed 18 h after the last CS exposure. RESULTS: APP at 30, 100, or 300 mg not only significantly dose-dependently reduced the CS-induced accumulation of inflammatory cells and gene/protein expression of proinflammatory factors both in the lung and in bronchoalveolar lavage fluid, but also significantly reversed oxidative stress in the lungs. Additionally, treatment with APP also significantly regulated the CS-induced imbalance of matrix metalloproteinases-9/tissue inhibitor of metalloproteinase-1 expression in the lungs. To investigate further the possible signaling pathway of APP effects, we examined protein expression of p-P38 MAPK by immunohistochemistry that found treatment with APP significantly decreased the CS-induced increases of p-P38 expression in the lungs. CONCLUSION: Taken together, APP may be a potential dietary nutrient supplement agent to improve quality of life of COPD patients by inhibiting CS-exposed acute lung injury via P38 MAPK signaling pathway.

Shp2 plays an important role in acute cigarette smoke-mediated lung inflammation.

Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease, contains a variety of oxidative components that were implicated in the regulation of Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) activity. However, the contribution of Shp2 enzyme to chronic obstructive pulmonary disease pathogenesis remains unclear. We investigated the role of Shp2 enzyme in blockading CS-induced pulmonary inflammation. Shp2 levels were assessed in vivo and in vitro. Mice (C57BL/6) or pulmonary epithelial cells (NCI-H292) were exposed to CS or cigarette smoke extract (CSE) to induce acute injury and inflammation. Lungs of smoking mice showed increased levels of Shp2, compared with those of controls. Treatment of lung epithelial cells with CSE showed elevated levels of Shp2 associated with the increased release of IL-8. Selective inhibition or knockdown of Shp2 resulted in decreased IL-8 release in response to CSE treatment in pulmonary epithelial cells. In comparison with CS-exposed wild-type mice, selective inhibition or conditional knockout of Shp2 in lung epithelia reduced IL-8 release and pulmonary inflammation in CS-exposed mice. In vitro biochemical data correlate CSE-mediated IL-8 release with Shp2-regulated epidermal growth factor receptor/Grb-2-associated binders/MAPK signaling. Our data suggest an important role for Shp2 in the pathological alteration associated with CS-mediated inflammation. Shp2 may be a potential target for therapeutic intervention for inflammation in CS-induced pulmonary diseases.

Oral administration of allergen extracts from mugwort pollen desensitizes specific allergen-induced allergy in mice.

Clinically, sublingual immunotherapy (SLIT) using allergen extracts effectively alleviates the symptoms of allergic rhinitis and asthma. We hypothesized that oral administration of a high-dose of allergen extracts imitates SLIT, which may prevent IgE-related responses in allergic diseases. In the present study, we investigated the effects of oral administration of allergen extracts from mugwort pollen (MP) on allergen-induced inflammation and airway hyperresponsiveness (AHR) in an allergic mouse model. After administration of MPdrop containing Art v 1 and Art v 4 extracts derived from MP specifically in MP-sensitized mice, the effects of MPdrop on AHR, inflammatory cell accumulation, cytokine production in the bronchoalveolar lavage fluid and lung tissue, and serum IgE and IgG levels were investigated. The results indicated that MPdrop not only prevented the AHR in response to methacholine in a dose-dependent manner but also significantly reduced the total serum and allergen-specific IgE levels. All of the maximal effects were achieved at a dose of 100µg/(kgd) and were comparable to the effects of dexamethasone at a dose of 0.5mg/(kgd). Furthermore, oral administration of MPdrop dose-dependently elevated allergen-specific serum IgG2a levels, reduced total and allergen-specific IgE levels and normalized the imbalance between the Th1 cytokine IL-12 and Th2 cytokines IL-4 and IL-5. Finally, oral administration of MPdrop significantly reduced goblet cell hyperplasia and eosinophilia in the MP-sensitized allergic mouse model. These data suggest that MPdrop effectively improves specific allergen-induced inflammation and AHR in MP-sensitized and -challenged mice and provides the rationale for clinical use of MPdrop in the specific allergen-induced asthma.

Antiinflammatory effect of low-level laser therapy on Staphylococcus epidermidis endophthalmitis in rabbits.

A rabbit model of endophthalmitis was established to evaluate the antiinflammatory effect of low-level laser therapy (LLLT) as an adjunct to treatment for Staphylococcus epidermidis endophthalmitis. Rabbits were randomly divided into three groups to receive intravitreal injections into their left eye: group A received 0.5 mg vancomycin (100 µl), group B received 0.5 mg vancomycin + 0.2 mg dexamethasone (100 µl), and group C received 0.5 mg vancomycin (100 µl) and continuous wave semiconductor laser irradiation (10 mW, λ = 632 nm) focused on the pupil. Slit lamp examination and B-mode ultrasonography were conducted to evaluate the symptoms of endophthalmitis. Polymorphonuclear cells and tumour necrosis factor alpha (TNF-α) in aqueous fluid were measured at 0 h, and 1, 2, 3, 7 and 15 days. A histology test was conducted at 15 days. B-mode ultrasonography and histology revealed that groups B and C had less inflammation than group A at 15 days. Groups B and C had fewer polymorphonuclear cells and lower levels of TNF-α in aqueous fluid than group A at 2, 3 and 7 days (P < 0.05). There was no significant difference between groups B and C (P > 0.05). There was no significant difference between groups A, B and C at 15 days (P > 0.05). As an adjunct to vancomycin therapy to treat S. epidermidis endophthalmitis, LLLT has an antiinflammatory effect similar to that of dexamethasone.

Laboratory diagnosis of infectious endophthalmitis.

AIM: To analyze the lab diagnosis and etiology of infectious endophthalmitis. METHODS: The medical and microbial records of 36 patients diagnosed with infectious endophthalmitis and 8 patients diagnosed with intraocular lens (IOL)-related inflammation between Nov. 1999 and Dec. 2009 were retrospectively reviewed for lab diagnosis and etiology. RESULTS: The inflammatory cell counts in all aqueous humor specimens from infectious endophthalmitis patients were more than in all aqueous humor specimens from patients with IOL-related inflammation. Sixteen of the 36 aqueous humor samples (44.4%) and 11 of the 24 vitreous humor samples (45.8%) from infectious endophthalmitis patients showed positive results in smears; while 17 aqueous humor samples (47.2%) and 15 vitreous humor samples (62.5%) from infectious endophthalmitis patients showed positive results in culture. CONCLUSION: The inflammatory cell count may be an important index for infectious endophthalmitis; while, smears can show etiological information earlier.

Application of Lutein and Zeaxanthin in nonproliferative diabetic retinopathy.

AIM: To compare serum Lutein and Zeaxanthin (L/Z) concentrations between patients with nonproliferative diabetic retinopathy (NDR) and normal subjects, and to explore the effect of L/Z supplementation on serum L/Z level and visual function in NDR patients METHODS: SUBJECTS WERE DIVIDED INTO THREE GROUPS: 30 NDR patients supplied with Lutein 6mg/d and Zeaxanthin 0.5mg/d for three months (DR Group), 30 NDR patients without L/Z supplementation (DR Control Group) and 30 normal subjects (Control Group). Serum L/Z concentrations were measured by liquid high-resolution chromatography (HPLC). Visual acuity was recorded at baseline, 1 month, 2 months and 3 months post initial supplementation. Serum L/Z concentration were measured at baseline, 1 month and 2 months post initial supplementation. Contrast sensitivity (CS) and fovea thickness were recorded at baseline and 3 months post initial supplementation. RESULTS: Mean serum lutein concentrations in DR group were 0.0686±0.0296µg/mL and zeaxanthin concentration was 0.0137±0.0059µg/mL. The L/Z level of DR group was significantly lower compared to the control group (lutein: 0.2302±0.1308µg/mL, zeaxanthin: 0.0456±0.0266µg/m, P=0.000). The concentration of lutein and zeaxanthin in the DR control group at base line was 0.0714±0.0357µg/mL and 0.0119±0.0072µg/mL, respectively. There was no significant change of L/Z concentration in the DR control group during the study. Serum L/Z concentrations of DR group increased significantly after supplementation (F=109.124, P=0.000; F=219.207, P=0.000). Visual acuity improved significantly after medication. Compared with pre-medication, the average CS values of 1.5cpd, 3cpd and 6cpd after three months increased significantly (P=0.030,0.013,0.008) and the foveal thickness decreased. (P=0.05) CONCLUSION: Serum L/Z concentrations in DR patients are significantly lower than those in normal subjects, and L/Z intake can improve the visual acuity, CS and macular edema in DR patients, suggesting that L/Z supplementation might be targeted as potential potential therapeutic agents in treating NDR.

[Normal conjunctival flora in healthy infants aged from 1 to 4 months].

OBJECTIVE: To investigate the normal conjunctival bacterial flora isolated from healthy infants aged from 1 to 4 months and to analyze its correlated factors. METHODS: This was a cross-sectional study. From January to March 2009, 109 cases of healthy babies aged from 1 to 4 months were selected through simple random sampling when they accepted health examination provided by Tianjin Medical University Eye Center and Tianjin Women and Children's Health Center. Conjunctival swabs were taken from both eyes and inoculated onto blood agar plates. The isolated bacteria were classified professionally. The distribution of different bacteria was investigated as well as the possible correlated factors by using t-test, χ(2) test and binary logistic regression of SPSS 11.5. RESULTS: 26 cases of healthy babies (23.9%) got positive culture results which consisted 44 strains of ocular bacterial isolates. 25 strains were gram-positive cocci, while 18 strains gram-positive bacilli, and 1 strain gram-negative bacillus. The first three isolated bacteria were Diphthamide bacillus (17/44, 38.6%), Staphylococcus epidermidis (16/44, 36.4%), and Staphylococcus aureus (6/44, 13.6%). The two subtypes of Staphylococci coexisted with Diphthamide bacillus in 8 eyes of 36 eyes from which bacteria were isolated. The bacterial isolation rate through every month age group had no significant difference (χ(2) = 0.351, P > 0.05). There was no significant difference in the group of positive isolation (26 cases, 36 eyes) or negative isolation (83 cases, 166 eyes) through breast, formula or mixed feeding (χ(2) = 1.182, P > 0.05). The gender had no influence on the detection of bacteria (χ(2) = 0.001, P > 0.05). The birth weight, gestational age or examined age between the two groups had no significant difference (t = 0.078, t = 0.940, t = 0.686, P > 0.05). The gender, age, birth weight, gestational age and the way of feeding had no correlation with existence of conjunctival flora in these healthy infants (Wald χ(2) = 0.001, Wald χ(2) = 0.003, Wald χ(2) = 0.117, Wald χ(2) = 1.307, Wald χ(2) = 1.490, P > 0.05). CONCLUSIONS: The normal conjunctival flora might establish during the initial months of little babies. Staphylococcus epidermidis and Diphthamide bacillus are the most common bacteria. Future studies should be conducted to make sure whether there would be some factors correlated with the culture results.

Dynamic analysis of CD127 expression on memory CD8 T cells from patients with chronic hepatitis B during telbivudine treatment.

BACKGROUND: Accumulating evidence supports the theory that expression of CD127 on CD8 T cells during the process of antiviral immune response indicates a subset of effect CD8 T cells that successfully develop into fully protective memory. CD8 T cells expression of CD127 may be used as a predictor to evaluate disease status in chronic viral infection. The aim of this study was to investigate the CD127 expression level on different subsets of CD8 T cell and explore the relationship between CD127 expression on CD8 memory T cells and serum hepatitis B virus (HBV) DNA and hepatitis B e antigen (HBeAg) levels in patients with chronic hepatitis B (CHB). We also aimed to investigate the CD127 expression pattern on CD8 memory T cells of CHB patients who were treated with Telbivudine. METHODS/RESULTS: Twenty HBeAg-positive CHB patients were selected and treated with telbivudine 600 mg/day for 48 weeks. The memory CD8 T cells were characterized by expression of CD45RA and CD27 markers. CD127 expression on the CD8 T-cell surface was measured by four-colour flow cytometry. Our results showed that CD127 expression on memory CD8 T cells was reduced in CHB patients. There was a strong negative correlation between CD127 expression on memory CD8 T cells and serum HBV DNA and HBeAg levels in CHB patients. Moreover, successful antiviral therapy increased CD127 expression on CD8 memory T cells as well as on HBV-specific CD8 T cells in CHB patients. CONCLUSION: These results suggest that diminished CD127 expression on CD8 memory T cells of CHB patients is a potential mechanism explaining cellular immune function impairment in CHB infection, and that CD127 expression on CD8 memory T cells is a useful indicator for evaluating the effects of anti-HBV therapy.

Efficacy of telbivudine in HBeAg-positive chronic hepatitis B patients with high baseline ALT levels.

AIM: To evaluate the efficacy and safety of telbivudine (LDT) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients who have high baseline alanine aminotransferase (ALT) levels between 10 and 20 times the upper limit of normal. METHODS: Forty HBeAg-positive CHB patients with high baseline ALT levels between 10 and 20 times the upper limit of normal were enrolled and received LDT monotherapy for 52 wk. Another forty patients with baseline ALT levels between 2 and 10 times the upper limit of normal were included as controls. We compared the virological, biochemical, serological and side effect profiles between the two groups at 52 wk. RESULTS: By week 52, the mean decrease in hepatitis B virus (HBV) DNA level compared with baseline was 7.03 log(10) copies/mL in the high baseline ALT group and 6.17 log(10) copies/mL in the control group, respectively (P < 0.05). The proportion of patients in whom serum HBV DNA levels were undetectable by polymerase chain reaction assay was 72.5% in the high baseline ALT group and 60% in the control group, respectively (P < 0.05). In addition, 45.0% of patients in the high baseline ALT group and 27.5% of controls became HBeAg-negative, and 37.5% of those in the high baseline group and 22.5% of controls, respectively, had HBeAg seroconversion (P < 0.05) at week 52. Moreover, in the high baseline group, 4 out of 40 patients (10%) became hepatitis B surface antigen (HBsAg)-negative and 3 (7.5%) of them seroconverted (became HBsAg-positive). Only 1 patient in the control group became HBsAg-negative, but had no seroconversion. The ALT normalization rate, viral breakthrough, genotypic resistance to LDT, and elevations in creatine kinase levels were similar in the two groups over the 52 wk. CONCLUSION: High baseline ALT level is a strong predictor for optimal results during LDT treatment.

[hVEGF siRNA inhibits initial growth of A549 tumor cells in nude mice and angiogenesis on CAM assay].

OBJECTIVE: To investigate the effect of small interfering RNA (siRNA) targeting human vascular endothelial growth factor (hVEGF) on A549 cell growth in nude mice and angiogenesis on chorioallantoic membrane (CAM) assay. METHODS: Three pairs of hVEGF siRNA-plasmid and non-silencing-plasmid were constructed, and transfected into A549 cells through lipofectamine 2000, respectively. The most effective pair of hVEGF siRNA-plasmid was selected by ELISA and real-time RT-PCR. A549 cells transfected with selected hVEGF siRNA- plasmid, A549 cells transfected with non-silencing-plasmid and A549 cells without transfection were inoculated into nude mice, respectively. Chick embryos were randomly divided into four groups and CAM was treated by different solutions for 48 h: culture media DMEM as negative control group,un-transfected A549 cell culture supernatants as positive control group, hVEGF siRNA A549 cell culture supernatants as hVEGF siRNA group and nonsilencing siRNA A549 cell culture supernatants as non-silencing siRNA group. The CAMs were harvested on d12 for microscopic assays. RESULT: Compared with control group, hVEGF siRNA-plasmid induced 48% reduction in hVEGF secretion by A549 cells accompanied by 70% reduction in hVEGF mRNA. Compared with non-silencing siRNA group, the mean tumor volume of murine xenograft was reduced by 58% in hVEGF siRNA group; time for xenografts growing to 50 mm(3)was delayed by 5.4 d. hVEGF contents in xenograft were reduced by 54%; but mean doubling time of tumors and the growth rate of tumors were not significantly reduced. In CAM assays, hVEGF content was zero in negative group, and in hVEGF siRNA group that was 40%-44% of non-silencing siRNA group or positive group; vessels branch points of CAM in hVEGF siRNA group or non-silencing siRNA group or positive group were increased by 45%-55% compared with negative group; total vessel length of CAM in hVEGF siRNA group was increased by 53% compared with negative group, while in non-silencing siRNA group or positive group that was increased by 97% or 99%. Compared with negative control group, the proliferation of microvessels was increased when cell culture supernatant with hVEGF was added in hVEGF siRNA group, significant proliferated vessels were observed in non-silencing siRNA group or positive group. CONCLUSION: A plasmid-mediated hVEGF siRNA has been constructed and verified, which can effectively downregulate the expression of hVEGF in human A549 cells, resulting in the inhibition of angiogenesis. hVEGF siRNA can delay initial growth of A549 tumor xenograft but not reduce the growth rate.

Sex hormones and their receptors in patients with age-related cataract.

PURPOSE: To determine the sex hormone levels in the serum and aqueous humor in patients with age-related cataract and the corresponding hormone receptors in cataract lens epithelial cells (LECs). SETTING: Research Laboratory, International Intraocular Implant Training Center, Tianjin Medical University, Tianjin, China. METHODS: Serum and aqueous humor were drawn from patients with cataract and a control group. Enzyme-linked immunosorbent assay was used to determine the levels of estradiol, progesterone, and testosterone in the serum and the aqueous. The anterior lens capsules with attached LECs were obtained during cataract surgery, and the expressions of estrogen receptor, progesterone receptor, or androgen receptor were examined by immunohistochemistry. RESULTS: The testosterone level in the serum was significantly higher in the control group than in the cataract group. In the cataract group, there was no difference between sexes in the serum levels of estradiol or progesterone; however, the testosterone level in men was significantly higher than in women. The aqueous level of each hormone was lower than in serum; however, there was no difference between sexes and no association with corresponding serum levels. No estrogen, progesterone, or androgen receptor was detected in the LECs of patients with age-related cataract. CONCLUSIONS: There was no statistical difference between men and women or between the cataract and control groups in the levels of estradiol or progesterone in serum. There was no between-sex difference in the aqueous levels. It appears that sex hormone levels can be regarded only as a risk factor for cataractogenesis, not as a key factor.