[The role of autophagy in PM2.5-induced inflammation in human nasal epithelial cells].

Objective: To explore the role of autophagy in PM2.5-induced inflammation in human nasal epithelial cells and related mechanism. Methods: Human nasal epithelial cells were exposed to different concentration of PM2.5 for different times, and the expression levels of microtubule-associated protein-1 light chain-3 Ⅱ (LC3 Ⅱ) and Beclin1 proteins were measured by Western blot. The typical autophagosome and autolysosome were observed by using transmission electron microscopy (TEM). To observe autophagic flux, mRFP-GFP-LC3 plasmid was transfected to nasal epithelial cells and the punctate staining of mRFP-GFP-LC3 were determined by confocal laser scanning microscope. The expression of inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatant were assessed by enzyme-linked immunosorbent assay (ELISA). To assess the role of autophagy in PM2.5-mediated inflammation, autophagy related gene Atg5 and Beclin-1 were silenced by siRNA knockdown, and inflammatory cytokines were analyzed.GraphPad Prism 6.0 was used for statistical analysis. Results: PM2.5 exposure increased the expression of LC3 Ⅱ and Beclin-1 proteins in a dose- (in PM2.5 group with concentration of 0, 15, 30, 60, 120 µg/ml, the expression of LC3 Ⅱ was 0.021±0.001(x±s), 0.037±0.002, 0.058±0.005, 0.075±0.006, 0.085±0.004, respectively, F=126.8, P<0.05; the expression of Beclin-1 was 0.002±0.000, 0.003±0.000, 0.005±0.000, 0.007±0.001, 0.008±0.001, respectively, F=137.3, P<0.05) and time-dependent manner (in PM2.5 group with exposure time of 0, 3, 6, 12, 24 h, the expression of LC3Ⅱ was 0.160±0.007, 0.222±0.003, 0.251±0.015, 0.483±0.029, 0.585±0.035, respectively, F=215.3, P<0.05; the expression of Beclin-1 was 0.059±0.002, 0.080±0.002, 0.087±0.002, 0.183±0.007, 0.228±0.005, respectively, F=137.3, P<0.05) in human nasal epithelial cells. TEM analysis showed typical autophagosome and autolysosome in cells after PM2.5 exposure for 24 h. PM2.5 significantly increased the number of yellow and red dots representing autophagosomes and autolysosomes respectively, indicating autophagic flux was elevated. Moreover, PM2.5 enhanced the secretion of inflammatory cytokines such as IL-6 and TNF-α, which was dramatically prevented by Atg5-siRNA and Beclin-1-siRNA. Conclusion: Autophagy plays an important role in PM2.5-caused inflammation response in nasal epithelial cells, which can induce release of inflammatory factors such as IL-6 and TNF-α and advance the inflammatory reaction.

[Effect of PM2.5 on inflammatory factors and pathology of nasal mucosa in a rat model of allergic rhinitis].

Objective: To investigate the effect of PM2.5 exposure on nasal inflammatory cytokines and nasal mucosal pathology in a rat model of allergic rhinitis (AR). Methods: Twenty-four healthy female SD rats were randomly divided into 3 groups by random number table method, with 8 rats in each group: normal control group (NC group), ovalbumin (OVA) induced AR model (AR group), and AR model group inhaled to PM2.5 at 200 µg/m(3), 3 h/d, for 30 d (ARE group). Nasal symptoms including sneezing, nasal rubs and nasal secretion were recorded. Levels of OVA specific IgE in serum, interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-ɑ) in nasal irrigating solution were measured by enzyme-linked immunosorbent assay (ELISA). The histopathological changes of nasal mucosa were observed by HE staining. SPSS 17.0 software was used to analyze the data. Results: The number of sneezing, nasal rubs and the amount of nasal secretion in the ARE group were significantly higher than that in the AR group and the NC group (number of sneezing (15.38±1.68) times/15 min vs (11.63±1.13) times/15 min vs (1.75±0.71) times/15 min, number of nasal rubs (27.75±2.12) times/15 min vs (21.25±2.96) times/15 min vs (5.25±1.04) times/15 min, amount of nasal secretion (18.90±2.07) mg vs (13.83±1.81) mg vs (3.78±0.41) mg, F values was 236.089, 224.139, 183.971, respectively, all P<0.001). Statistically significant differences in OVA specific IgE, IL-6 and TNF-ɑ levels were observed in ARE group exceeded AR group and NC group (OVA specific IgE (25.42±2.51) ng/ml vs (18.07±1.07) ng/ml vs (1.47±0.26) ng/ml, IL-6 (123.30±18.86) pg/ml vs (63.49±11.29) pg/ml vs (16.87±3.29) pg/ml, TNF-ɑ (162.50±38.15) pg/ml vs (72.96±11.28) pg/ml vs (27.52±4.15) pg/ml, F values was 481.604, 138.277, 63.938, respectively, all P<0.001). HE staining showed that the nasal epithelial cells of NC group were intact and neatly arranged. Nasal mucosa epithelial cells were arranged in disorder in AR group, with tissue structure swelling. Partial shedding of nasal epithelial cells, mucosal basement membrane thickening, submucosal tissue interstitial edema, vasodilation and gland hyperplasia were found in ARE group. Conclusion: An increase inflammatory factors level such as IL-6 and TNF-ɑ aggravates pathological damage of nasal mucosa in a rat model of AR by exposure to PM2.5.

[Application of image-guided system in endoscopic sinus and skull base surgery].

Objective:To evaluate the applicative value of image-guided system in endoscopic sinus and skull base surgeries. Method:A total of 103 endoscopic surgical procedures were performed.All these procedures were conducted with the utilization of image-guided system, among which there were 92 cases of sinonasal-skull base surgery(including nasal sinuses resection of benign and malignant tumors involving skull base lesions, the cumulative orbital lesion resection of nasal sinus lesions, etc. ), 6 repair of cerebrospinal fluid leak, 3 pituitary adenoma resection, 2 traumatic neuropathy optic nerve decompression. Result:With the utilization of image-guided system, all patients had successful surgery without major and minor complications. The image-guided system provided high precision with short registration time. Conclusion:Image-guided system can help the surgeon to identify accurately the vital anatomic landmarks of sinus and skull base, improving surgical accuracy and safety as well as reducing or avoiding the intraoperative and postoperative complications.

[The effect of PM2.5 on mucus secretion and ultrastructureof nasal mucosa in allergic rhinitis model].

Objective:The aim of this study is to the roles of nasal lavage fluid levels of MUC5AC, goblet cell hyperplasia and ultrastructure of nasal mucosa following ambient PM2.5 exposure in a rat model of allergic rhinitis(AR).Method:Female Sprague-Dawley rats were assigned randomly into 3 groups: a negative control group(NC group),an ovalbumin(OVA)-induced AR model group(AR group), and AR model group(ARE group) inhaled to PM2.5 at 200 µg/m³, 3 h/d, for 30 days. Nasal symptoms, levels of MUC5AC in nasal lavage fluid(NLF), were measured in each individual rat.Goblet cell hyperplasia were examined histologically with PAS-stained. Nasal mucosa tissue ultrastructure were observed by scanning electron microscope.Result:PM2.5 significantly increased the number of sneezes, nasal rubs and the amount of nasal secretion in rats with AR.Statistically significant differences in MUC5AC levels and goblet cell hyperplasia were observed between the AR model exposure to PM2.5 and the AR model group.The nasal mucosa of AR model exposure to PM2.5 was disordered,lodged,assembled and twisted.Conclusion:Our data indicate that an increase MUC5AC level in NLF and the development of nasal goblet cell hyperplasia may provide some clues for determining the pathogenic mechanisms of AR by exposure to PM2.5.

Identification and characterization of the antigen presenting cell in rat autoimmune myocarditis: evidence of bone marrow derivation and non-requirement for MHC class I compatibility with pathogenic T cells.

In the rat, autoimmune myocarditis can be produced by the infusion of activated myosin peptide specific, CD4(+), class II restricted, effector T cells. Whether antigen presenting cells (APCs), which interact with these effector T cells in the heart, are a fixed population of cells (resident dendritic, macrophage, or endothelial cells), or a dynamic bone marrow derived population has not yet been demonstrated in vivo. To study this question, bone marrow chimeras were generated using inbred Brown Norway (BN) rats, which are resistant to autoimmune myocarditis, and transplanting them after lethal irradiation with (LewisxBN) F1 bone marrow. BN rats differ at both MHC loci from the susceptible inbred Lewis rats. Two months after bone marrow transplantation, chimeric animals received Lewis T cells specific for a myocarditogenic peptide antigen. To characterize the cardiac APCs, immunohistochemistry using a battery of antibodies including Lewis-specific and broadly reactive antibodies for both MHC class I and class II, was performed on chimeric hearts, with and without infused Lewis T cells, and non-transplanted BN control hearts.All chimeric rats infused with allogeneic (Lewis), anti-cardiac myosin peptide effector T cells displayed the lesions of myocarditis. Myocarditis was not present in non-transplanted BN controls given either Lewis or F1 derived myocarditogenic T cells, nor in chimeric animals which did not receive myocarditogenic T cells, thus excluding graft vs host disease as the explanation for the inflammation in chimeric hearts with myocarditis. Marrow derived cells expressing both Lewis class I and class II MHC molecules were demonstrated on perivascular cells in the myocardium of all chimeric animals, and on infiltrating cells in chimeric animals with myocarditis. Cells expressing Lewis-specific MHC antigens were not detected in the non-transplanted BN controls. Furthermore, immunohistochemistry using broadly reactive antibodies demonstrated MHC class II on perivascular cells with a dendritic morphology in all hearts but not on endothelial cells or cardiac myocytes. These results support the hypothesis that in vivo, cardiac APCs which result in MHC class II restricted, T cell induced myocarditis are a dynamic bone marrow derived population and not a fixed population. In order to address the potential requirement of MHC class I for the initiation of autoimmune myocarditis, myocarditogenic T cells derived from either Lewis or DA(RP) rats were infused into a member of the other strain. These strains share common MHC class II genes but differ at the MHC class I loci. Myocarditis identical to that produced in the syngeneic animal was successfully transferred by the MHC class I mismatched T cells, but only after the recipient animal's native immune system was mildly suppressed. These results further support the primary role for professional antigen presentation via MHC class II restriction to the effector T cells at the initiation of autoimmune myocarditis in the heart.Together, these experiments confirm that activated effector T cells, in order to produce myocarditis, require MHC class II compatible APCs in the heart, that these APCs are bone marrow derived, and will endogenously take up and present local antigens in the target organ after bone marrow reconstitution.

PECAM-1 (CD31) expression in the central nervous system and its role in experimental allergic encephalomyelitis in the rat.

The role of T cell activation associated adhesion molecules on lymphocyte traffic and the initiation of inflammation has received considerable attention. This study, using a new monoclonal antibody (mAb) TLD-3A12, describes the distribution of PECAM-1 (CD31), an Ig supergene family adhesion molecule thought to be important in leukocyte transmigration during inflammation, in rat lymphoid organs and spinal cord. PECAM expression within the CNS is confined to endothelial cells of the blood brain barrier (BBB). Induction of inflammation within the CNS using the adoptive transfer of myelin reactive CD4+ T cells results in the de novo expression of immune adhesion and accessory molecules in the spinal cord, while the level of PECAM appeared only mildly increased. The distribution of PECAM on CNS endothelial cells became more diffuse during EAE induction, possibly the result of endothelial cell activation. In vitro studies demonstrate a partial inhibition of antigen-specific CD4+ T cell proliferation following anti-PECAM mAb treatment. Treatment of Lewis rats with TLD-3A12 antibody prior to T cell injection and throughout EAE induction does not result in a delay in the onset of clinical signs or weight loss, nor does it decrease the incidence and severity of disease. These data suggest that the expression of PECAM by CNS endothelial cells is not a requirement for the initiation of inflammation and clinical signs of EAE following the adoptive transfer of encephalitogenic lymphocytes. Thus, cells requiring PECAM-1 to migrate and perform their pathogenic functions are not critical to the development of rat EAE.