Objective To investigate the levels of IL-33 in the lung induced by Aspergillus fumigatus (A.fumigatus) exposure in bronchial asthmatic rats and the effects of dexamethasone.Methods Twenty-four Wistar rats were randomly and equally divided into group A (normal control),group B (asthma group),group C (A.fumigatus-exposed group),and group D (dexamethasone-treated group).B,C and D group were sensitized and challenged with ovalbumin (OVA) to establish asthmatic models.Then,group C and D were given A.fumigatus spores intranasally.Group D was pre-treated with dexamethasone during ovalbumin challenge.Group A was sensitized and challenged intranasally with normal saline as control.Airway hyperresponsiveness,eosinophils percentage and serum IgE levels were measured to confirm the establishment of asthmatic model.Expression of IL-33 in the lung were quantified by ELISA and qRT-PCR.Lung tissues and blood were plated on the potato dextrose agar (PDA) medium and cultivated for 24 h to measure the number of colony.Results Eosinophils percentage in bronchoalveolar lavage fluid (BALF),IgE levels in serum and IL-33 levels in the lung in group B and C were much more higher than in group A (P＜0.05),and the increasing range in group C was more obvious than group B.However,the changes of sRaw values in these groups had no statistical significance.Esosinophils percentage in BALF and IL-33 levels in the lung were significantly decreased (P＜0.05),and IgE levels in serum was slightly decreased (P＞0.05) in group D when compared with group C,but the ratio of A.fumigatus colonization in the lung was higher than group C.Conclusions Dexamethasone can inhibit the expression of IL-33 in the lung induced by A.fumigatus exposure in an asthmatic rat,but it also increases the risk of fungal colonization in the airway.Anti-IL-33 antibody treatment needs further research.

Objective To investigate the effects of Gefarnate on expression of myeloperoxidase (MPO),cyelooxygenase-1 (COX-1) and COX-2 in trinitrobenzene sulphonic acid (TNBS) induced experimental colitis in rats and its therapeutic effects on ulcerative colitis. Methods Forty female Sprague-Dawley (SD) rats were randomly divided into 4 groups with 10 each. The rats in group A, B and C were infused with TNBS/alcohol by enema. After the production of colitis, the rats in group A or B were treated daily with 1 ml of normal saline or with 1 ml of 5-ASA (100 mg/kg) by enema,and those in group C were treated daily with 1 ml of Gefarnate by gavage. Group D was served as normal control. After the production of colitis,animals were sacrificed at day 7 and 14 with 5 in each group. The macroscopic changes of the colon were evaluated according to disease activity index (DAD scoring and histological change was assessed by HE staining. MPO activity of the mucosa was detected by biochemical methods. Expressions of COX-1 and COX-2 in tissues were detected by immunohistochemistry. Results Compared with group A, macroscopic and histological scores and MPO activity were significantly decreased in group B and C (P<0.05). The expressions of COX-1 at day 7 and 14 were 1.86±0.51 and 1.96±0.41 in group B, 1.73±0.68 and 1.79±0.6 in group C, 1.91±0.34 and 1.99±0.45 in group D, respectively, which were significantly higher than those in group A (0.87±0.18 and 0.93±0.15, P<0.05). Whereas the expressions of COX-2 at day 7 and 14 were 1.53±0.19 and 0.73±0.15 in group B, 1.73±0.94 and 0.86±0.29 in group C, 0.24±0.18 and 0.18±0. 16 in group D, respectivley, which were significantly lower that those in group A (3.50±0.2;3 and 3.06±0.27). There was a significant difference between group D and group B or C (P<0.05). Conclusions Gefarnate provides a therapeutic effect during TNBS-induced colitis in rats, which is similar to that of 5-ASA. The mechanisms are involved in decreasing the concentration of colonic MPO and regulating the expression of COX-1/COX-2.

Breast ; pathology ; Breast Neoplasms ; blood supply ; pathology ; Carcinoma, Ductal, Breast ; blood supply ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; blood supply ; parasitology ; Female ; Humans ; Hyperplasia ; Microcirculation ; pathology ; Neovascularization, Pathologic ; pathology

Objective Studies show that the abnormal expression of EphB4 plays an important role in the development and progression of colon cancer .The present study aims to provide some experimental evidence for the gene therapy of colon cancer by con -structing a lentiviral expression vector carrying the homo EphB4 gene and further establishing colon cancer cell lines with stable overex-pression of EphB4. Methods A series of oligonucleotides (oligo) encoding the homo EphB4 gene were ligated together by PCR and then cloned into a lentiviral expression vector pLenti 6.3-MCS-IRES2-EGFP.After confirmed by sequencing , the vector pLenti6.3-EphB4-IRES2-EGFP and its helper vectors were mixed and co-transfected into 293 T cells to obtain recombinant virus containing the EphB4 gene.The lentiviral titer was detected and the resulting recombinant lentiviruses carrying EphB4 or control viruses only carrying green fluorescence protein (GFP) were used to infect the human colon cancer cell lines .The expression of GFP was determined under the inverted fluorescence microscope and the level of EphB 4 mRNA in the infected cells detected by qPCR . Results The lentiviral expression vector pLenti6.3-EphB4-IRES2-EGFP carrying correct homo EphB4 gene sequence was successfully constructed .The titer of the recombinant EphB4 lentiviral supernatant Lenti6.3-EphB4 was 1 ×108 TU/mL.The expression of GFP was observed in the trans-duced cells under the fluorescence microscope , and that of EphB4 mRNA in the transfected SW480 and Coca-2 cells was significantly up-regulated as compared with the control and blank groups . Conclusion The homo EphB4 gene was successfully amplified and cloned.A lentiviral expression vector was successfully constructed , and so were colon cancer cell lines stably overexpressing EphB 4, which may shed light on the lentivirus-mediated genetic therapy for colon cancer .

Objective Studies show that the role of EphB 4 in the development and progression of cancer is correlated to its ligand EphrinB2.The present study was to observe the effect of the changes in EphB4 on the expression of EphrinB2 by constructing and identifying microRNA ( miRNA) interference vectors targeting the EphB4 gene in colon cancer cells . Mte hods According to the EphB4 gene sequence , 3 pairs of oligo DNA sequences of miRNA were designed .The single strand of oligo DNA was annealed to form double-strand DNA, and then connected with the plasmid pcDNA 6.2-GW/EmGFP-miRNA.The expression vector pcDNA6.2-GW/EmGFP-miR-EphB4 was linked to pDONR221 and pLenti6/V5-DEST to construct the lentiviral expression vector pLenti 6/V5-DEST-EphB4, which was cotransfected with packaging mix (pLP1, pLP2 and pLP/VSVG) into 293FT cells by lipofectamine 2000 transfec-tion to produce lentivirus , and the lentivirus titer was measured by infection of HEK 293 cells.The stable cell lines were selected and cultured.The expression levels of EphB4 and EphrinB2 were examined by qPCR. Results Three miRNA interference vectors SR-1, SR-2, and SR-3 targeting the EphB4 gene were successfully constructed , with SR-3 exhibiting the most significant interference efficien-cy.The constructed lentiviral vector pLenti 6/V5-DEST-EphB4 was successfully packaged in 293FT cells.The virus titer was 7 ×108 Caco-2 cells. Conclusion The exogenous EphB4 expression could be significantly inhibited by treatment with specific miRNA in co-lon cancer cells .The correlation of EphB4 and EphrinB2 may be effected by many factors and need further studies .

Objective To study the expression changes of IL-1β、FN-α6、IFN-γ and TNFR-m18 in peripheral blood within 2 hours in epileptic mice. Methods Semi quantitative real-time PCR was used to test the mRNA expres?sion level of IL-1β、FN-α6、IFN-γand TNFR-m18 in peripheral blood from normal and pilocarpine-induced epileptic mice at different time points (10 min, 30 min, 1 h and 2 h). Results The mRNA expression level of IL-1βincreased at 30 min(1.8±0.07), 1 h(2.9±0.98)and 2 h(1.45±0.11)after pilocarpine-induced status epilepticus comparing with that of control and SE 10 min(0.81±0.09)(P<0.05). The IFN-α6 mRNA expression level was lower at 10 min(0.59±0.05, P<0.05) than that of control. IFN-γmRNA expression level was higher at 10 min(2.85±0.11) than that of control and at oth?er time points during SE(P<0.01). TNFR-m18 mRNA expression level was higher at 1h(2.84±0.15) than that of control, and at other time points during SE(P<0.01). Conclusion The immune system of epileptic state is active, the imbalance of cytokine expression in peripheral blood may be related to the immune pathological process of acute stage of epilepsy.

To improve the solubility of quercetin ( QT) , one of flavonoids that can inhibit the proliferation of vari-ous types of cancer cells, the novel amphiphilic polymer N-( 4-methylimidazole)-hydroxyethyl-chitosan ( MHC) , synthetized by chemical derivatization from chitosan, was used as the self-assembly micelles of QT. The formed polymer was characterized by 1 H NMR, elemental analysis and pyrene fluorescence spectrometry. The formulation of MHC micelles loading quercetin was optimized through single factor experiment. Then the optimized formulation was obtained as follows:the concentration of MHC was 0. 67% and the ratio of drug and carrier was 1 ∶10. The micelles particle size was ( 99. 21 ± 1. 71) nm, Zeta potential was +( 20. 01 ± 0. 72) mV and drug loading was ( 5. 42 ± 0. 32 )%. The in vitro release curve was investigated and was found to conform to Higuchi equation of Q=0. 1101 t1/2 -0. 064. The results of in vivo experiment showed that the mean rentention time and bioavail-ability of the MHC-QT micelles were 21. 42 h and 57. 49 μg h/mL, respectively, compared to 0. 30 h and 2. 50 μg h/mL of the free QT solution. These indicated that the MHC micelles could significantly improve the solubility of QT, the drug sustained-release effect and bioavailability, which would used as carrier for the anti-tumor drugs.

Toxicology ; Translational Medical Research

Objective To establish the microtiter plate radiobinding assay (RBA) of IA-2A and to evaluate its clinical application. Methods The purified ~(35)S - IA-2 was incubated with sera for 24 hours on a 96-well V-shaped bottom plate, and then transferred to the Millipore plate coated with protein A, and counted with liquid scintillation and luminescence counters after washing. The IA-2A levels were detected in 162 patients with type 1 diabetes(T1DM),210 patients with newly diagnosed type 2 diabetes(T2DM) and 224 healthy controls to evaluate clinical application of IA-2A RBA. Results 1. The intra-coefficient of variation (CV) of the assay was 4.1%～10.0%, and the inter CV was 5.7%～12.8%. 2. The results from DASP 2005 showed that the sensitivity and specificity of the assay were 72% and 98%. The results of IA-2A from two methods of RBA and classical radioligand assay(RLA) were significantly correlated (r=0.962,P＜0.001) with a consistency of 96.5%. 3. When compared with the healthy controls, T1DM patients had higher positivity for IA-2A (22.8% vs 0.89%, χ~2=49.9,P＜0.001), but no significant difference was found in T2DM patients(2.4% vs 0.89%, χ~2=1.5,P＞0.05). 4. The consistency rate of IA-2A measurement was 100% between RBA using finger tip blood and RLA using venous blood (r=0.977,P＜0.001). Conclusions The microtiter plate RBA of IA-2A using finger tip blood has high sensitivity, specificity and reproducibility, and possesses a good clinical value.

Objective To explore the characteristics of proinsulin secretion in latent autoimmune diabetes in adults (LADA). Methods Fasting and 2 h sera in oral glucose tolerance test from 36 LADA patients, 37 type 2 diabetic patients and 43 healthy controls were collected to test glucose, proinsulin (PI) and C-peptide (CP) by radioimmune assay. Glutamie acid decarboxylase antibodies (GAD-Ab) were determined by radioligand assay.Results (1) Fasting proinsulin (FPI) and 2 h proinsulin (PPI) level in LADA patients were lower than those in type 2 diabetic patients (P<0.05), being both significantly inereasad compared with healthy controls (P<0.05 or P<0.01); The ratios of FPI/FCP and PPI/PCP (%) in LADA were beth significantly higer than those of type 2 diabetes mellitus and controls (P<0.05 or P<0.01); (2) LADA type-1 (GAD-Abe>0.3) patients showed lower PI levels(P<0.05 or P<0.01) and higher PI/CP ratio (all P<0.05) than LADA type-2 (0.05≤GAD-Ab<0.3); Meanwhile, there were no significant differences in above parameters between LADA type-2 and type 2 diabetes meUitus (P>0.05). (3) GAD-Ab index was negatively correlated with FPI and PPI in LADA group (r=-0.236 and-0.268, both P<0.05), and positively correlated with PPI/PCP (r=0.254, P=0.030).Meanwhile BMI was positively correlated with FPI, PPI and PI/CP in type 2 diabetes mellitus (all P<0.01). No factor entered the multiple regression analysis for predieting the hyperproinsulinemia and dispropriately elevated proinsulin levels in LADA patients. (4) According to the 99.5 th percentile of proinsulinemia in the healthy controls, which is defined as the cutoff point dispropriately elevated proinsulin levels, the proportion of subjects with fasting dispropriately elevated proinsulin levels (FPI/FCP) were 77.8%, 62.2% and 2.3% in LADA, type 2 diabetes meUitus and controls respectively, and PPI/PCP 83.3%, 51.4% and 2.3% respectively. Conclusion LADA patients, as well as type 2 diabetic patients, all showed hyperproinsulinemia and disproportionately elevated proiasulin levels that were one of characteristics of defective β-cell function. Moreover, disproportionately elevated nproinsulin level is more evident in LADA patients than that in type 2 diabetics and this may be related to humoral immunity.