Objective:To clone and express panallergen profilin from the pollen of coco(Cocos nucifera Linnaeus).Methods:RT-PCR and RACE methods were applied to clone the full-length panallergen genes from coco pollen and the sequence was analyzed.The specific primers were designed.The ORF of profilin of coco pollen was amplified with RT-PCR and cloned into the expression vector pET 28a.Expression of the recombinant coco pollen profilin was carried out in E.coli BL21(DE3) and the purification of the recombinant protein was performed via affinity chromatography with Ni2+ coupled to sepharose.IgE reactivity to recombinant coco pollen profilin was investigated by immunoblot.Results:The complete sequence of coco pollen profilin was cloned.The sequence was 608 bp and included an open reading frame(396 bp) coding for 131 amino acids.Sequence analysis showed that the deduced protein was an acidic protein with an estimated molecular mass of 14.19 kD and a pI of 4.61.The GeneBank accession number of the clones was EF173598.After overexpressed in E.coli BL21(DE3),the recombinant protein was purified through affinity chromatography with Ni2+ coupled to sepharose.Immunoassay showed that the recombinant allergen has good IgE binding capacity.Conclusion:The profilin of coco pollen is expressed successfully in BL21(DE3),which will be used as a base for further study on coco pollen related allergy.

OBJECTIVE: Exploring the relationship between total and specific IgE in serum and allergen skin test of carvota mitis pollen-induced allergic rhinitis patients. METHOD: Four hundred and-twenty-nine carvota mitis pollen-induced allergic rhinitis patients and 243 healthy control subjects were recruited. The experimental group carried out skin tests. and pollen-specific IgE were also examined by BSA-ELISA method. Total IgE in serum of all of the subjects were determined by ELISA. RESULT: The positive rate of the total IgE level of the patients were much higher than those of the controls (66.2% vs. 15.6%, P < 0.01). No statistically significance was found between the positivity of skin test and serum specific IgE of the experimental group (chi2 = 0.758 8, P > 0.05). The difference between serum-specific IgE and total IgE was statistically significant (chi2 = 50.639, P<0.01). There was no statistical significance of specific IgE and the total IgE in serum between long term residents in Haikou and Hainan Tourisms (P > 0.05). CONCLUSION Allergen skin test and carvota mitis pollen-specific IgE are two effective methods for the diagnosis of carvota mitis pollen-induced allergic rhinitis. The detection of total IgE in serum of carvota mitis pollen-induced allergic rhinitis provides a reference value for diagnosis. The relationship between concentration of IgE in serum of the carvota mitis pollen-induced allergic rhinitis and allergen contact duration is waiting for further study.
Adolescent ; Adult ; Aged ; Allergens ; immunology ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Humans ; Immunoglobulin E ; blood ; immunology ; Male ; Middle Aged ; Pollen ; immunology ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; blood ; immunology ; Skin Tests ; Young Adult

Objective To explore the effect of Melatonin(MT) on CD4+CD25+ regulatory T cell (CD4+CD25+Tr)and airway inflammation in asthmatic rat.Methods Thirty-two SD rats were randomly divided into 4 groups,8 rats in each group.Asthmatic group:rats were immunized on day 1 and 7 by intraperitoneal inject of mixture of ovalbumin(OVA) and aluminumhydroxide.From day 14,the animals were allenged with aerosolized OVA for 20 min per day for 7 consecutive days.MT group:OVA-sensitized rats were injected intraperitoneally with 0.1 mg/kg MT 30 min before each OVA challenge.Dexamethasone group:OVA-sensitized rats were injected intraperitoneally with 0.5 mg/kg Dexamethasone 30 min before each OVA challenge.Control group:OVA for inhalation and MT for intraperitoneal injection was replaced with saline.After the last challenge,peripheral blood was stained to count the percentage of eosinophil(EOS).Then the rats were lavaged and total leukocytes counts in bronchoalveolar lavage fluid(BALF) were performed after staining with Wright-Giemsa staining.The EOS counts around the airway was counted after the histological section of lung staining with hematoxylin and eosin staining.The serum level of immunoglobulin E(IgE) was detected by immunoenhancement.The change of CD4+CD25+Tr was assessed with flow cytometry.SPSS 10.0 software was applied to analyze data. Results In asthmatic rats,the CD4+CD25+ Tr/ CD4+T cells ratio had significant negative relationship with the EOS counts around the airway and the total leukocytes counts in BALF (r=-0.73 P0.05).There was a significant decrease in the percentage of the eosinophils in peripheral blood,the eosinophil counts around the airway,the total leukocytes counts in BALF and the serum level of IgE in MT group compared with asthmatic group (Pa

cDNA for Insulin-like growth factor binding protein 3 was cloned and constructed a prokaryotic expression vector--pET-DsBA-IGFBP3. The construct was transformed into E. coli BL21 (DE3)plysS. The induced fusion protein (D-IGFBP3) was expressed successfully in soluble form. We obtained D-IGFBP3 the purify of which is over 95% after purification by His affinity chromatography. The product was identified by Western-blot. The cell assay showed that the obtained fusion protein can inhibit the growth of MCF-7 and bind with IGF-I in vitro.
Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, Affinity ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; metabolism ; pharmacology ; Insulin-Like Growth Factor I ; metabolism ; Protein Binding ; Recombinant Proteins ; isolation & purification ; metabolism ; pharmacology ; Solubility

UNLABELLEDProminent eosinophil airway inflammation is important in the pathogenesis of asthma. There is increasing evidence that the disorder of eosinophil apoptosis contributes to the mechanism. But most of the studies have been done in vitro or on animal models, very few were done among the adult asthmatics in vivo.

OBJECTIVEThe aim of this study was to elucidate the relationship between the apoptotic eosinophils and Bcl-2 in asthmatic children in vivo.

METHODSEleven mild to moderate asthmatic patients were recruited and the range of age was 7 - 14 years (9 males, 2 females), meanwhile 7 patients with lower respiratory infection were recruited as control and the range of age was 9 - 14 years (5 males, 2 females). Before and after inhaled glucocorticoid (GC) induced sputum, bronchoalveolar lavage (BAL), bronchial mucosa specimens and peripheral blood were obtained for measuring and comparing the changes of apoptotic EG(2)(+) cell by combining the techniques of TUNEL and immunohistochemistry, meanwhile the expression of Bcl-2 in bronchial mucosa specimens was measured by using the immunohistochemical assay.

RESULTSBefore the inhalation of GC, the apoptotic EG(2)(+) cells in asthmatics were significantly lower than that in control group (P < 0.01), and the numbers of EG(2)(+) cell in asthmatics group were significantly higher than that in control group (P < 0.001). After the treatment apoptotic EG(2)(+) cells in asthmatics were increased (P < 0.01), and the numbers of EG(2)(+)cell were decreased (P < 0.01, P < 0.05 and P < 0.05, respectively), FEV(1)% was increased (P < 0.05). Before the inhalation of GC, the numbers of Bcl-2(+) cell in asthmatic airway submucosa were higher than that in control group (P < 0.05) but after the treatment the number of Bcl-2(+) cell did not change significantly. (4) Before and after GC treatment the percentages of apoptotic eosinophils of peripheral blood in vivo had no significant changes compared with those of control subjects (P > 0.05). There was a positive correlation between apoptosis of EG(2)(+) cell in sputum, BAL, airway submucosa and FEV(1)% (P < 0.05).

CONCLUSIONApoptosis of EG(2)(+) cell decreased in the airway of asthmatic children and inducing EOS apoptosis is one of the important mechanism of inhaled GC therapy for asthma.

Adolescent ; Apoptosis ; Asthma ; blood ; drug therapy ; pathology ; Bronchoalveolar Lavage Fluid ; cytology ; Child ; Eosinophils ; cytology ; Female ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Respiratory Mucosa ; chemistry ; cytology

OBJECTIVETo prepare a transgenic tumor cell vaccine transfected the fusion gene of murine granulocyte-monocyte colony stimulating factor (mGM-CSF) and human interleukin-2 (hIL-2) using H22 cells, and explore its specific antitumor immunity against hepatocellular carcinoma.

METHODSThe eukaryotic vector expressing the fusion gene mGM-CSF/hIL-2 was transfected into H22 cells followed by radiation exposure to construct the tumor cell vaccine, which was used to immunize Balb/c mice by subcutaneous inoculation. The mice inoculated subcutaneously with H22 cells, cells transfected with the empty vector pcDNA(+), or with PBS served as the controls. A week later, H22 cells were injected peritoneally into Balb/c mice for establishing the tumor-bearing model, and their serum levels of interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) were detected using enzyme-linked immunosorbent assay (ELISA) with the survival of the mice recorded. The spleen cells were obtained from the mice immunized with the tumor cell vaccine, the tumor-bearing mice and the normal control mice to assess their cytotoxicity against the parental H22 cells in vitro using (51)C(r)-release assay.

RESULTSThe transgenic H22 cell vaccine transfected with mGM-CSF/hIL-2 fusion gene was successfully constructed. The killing rate of H22 cells by the spleen cells from the mice immunized with the transgenic cell vaccine was significantly higher than those by the spleen cells from the tumor-bearing mice or normal control mice (38.3% vs 13.6% and 7.5%, P<0.05). Serum IFN-gamma in the tumor-bearing mice immunized with the transgenic cell vaccine was significantly higher, and serum IL-10 significantly lower than those of the control groups (P<0.01). The survival time of the tumor-bearing mice injected with the transgenic cell vaccine was also significantly prolonged.

CONCLUSIONSyngeneic tumor cell vaccine genetically modified by mGM-CSF/hIL-2 fusion gene transfection can elicit specific cellular immune response and enhance the host antitumor immune response to extend the survival time of tumor-bearing mice.

Animals ; Cancer Vaccines ; genetics ; immunology ; Carcinoma, Hepatocellular ; immunology ; Cell Line, Tumor ; Genetic Vectors ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Immunotherapy ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-2 ; genetics ; immunology ; Liver Neoplasms ; immunology ; Mice ; Mice, Inbred BALB C ; Spleen ; cytology ; immunology ; Transfection

OBJECTIVETo study the effect of lycopene on red blood cell and the level of blood lipid.

METHODSAccording to the level of serum total cholesterol and weight, forty-eight adult male SD rats were divided randomly into six groups: normal control (group A), fed by normal feed; hyperlipidemia group (group B): fed by high fat diet; positive control group (group C): fed by high fat diet plus 10 mg * kg(-1) * d(-1) fluvastatin sodium; lycopene groups: fed by high fat diet plus 11 (group D), 22 (group E), 44 mg * kg(-1) * d(-1) (group F) lycopene through gavage, respectively. For all six groups, the level of serum total cholesterol (TC) and total triglyceride (TG) were measured at the end of 0, 1, 3 weeks of the study by taking samples from tail vein. At the end of the experiment, RBC and HGB were measured.

RESULTSAfter the rats were fed with high-fat feed for a week, models of hyperlipidemia rats were established. At the end of 3 weeks, TC of group A, B, C, D, E and F were (1.31 +/- 0.05), (19.40 +/- 0.54), (4.66 +/- 0.07), (7.18 +/- 0.06), (5.30 +/- 0.28), (4.49 +/- 0.23) mmol/L (F = 4395.72, P = 0.00), respectively;and TG were (0.42 +/- 0.01), (2.29 +/- 0.42), (0.69 +/- 0.03), (1.10 +/- 0.05), (0.63 +/- 0.02), (0.62 +/- 0.04) mmol/L (F = 127.26, P = 0.00), respectively; HGB were (143.13 +/- 6.33), (112.63 +/- 2.56), (124.75 +/- 3.62), (124.63 +/- 7.78), (132.38 +/- 6.41), (142.13 +/- 5.54) g/L (F = 34.14, P = 0.00), respectively; RBC were (6.75 +/- 0.60) x 10(12)/L, (5.08 +/- 0.75) x 10(12)/L, (7.14 +/- 0.82) x 10(12)/L, (5.94 +/- 1.09) x 10(12)/L, (6.18 +/- 0.36) x 10(12)/L and (7.31 +/- 0.58) x 10(12)/L (F = 10.35, P = 0.00), respectively.

CONCLUSIONLycopene have some protective effects on red blood cells of the hyperlipidemic rats by regulating the blood lipid and antioxidant.

Animals ; Carotenoids ; pharmacology ; Cholesterol ; blood ; Cholesterol, LDL ; blood ; Erythrocytes ; drug effects ; Hypercholesterolemia ; blood ; Lipids ; blood ; Male ; Rats ; Rats, Sprague-Dawley ; Triglycerides ; blood

The aim of the study is to investigate chemical constituents of the leaves of Pieris japonica. The isolation and purification of the constituents were performed by various chromatography and spectral analysis. Three new phenolic glucosides, erythro-syringoylglycerol 4-O-beta-D-glucoside (1), 1-(2-beta-D-glucopyranoxyl-4-methoxyl-6-hydroxyphenyl)-3-hydroxyl-l-propanone (3), erythro-l-(4-hydroxyl-3-methoxyphenyl)-2-[4-(3-beta-D-glucopyranoxypropyl)-2 ,6-dimethoxyphenoxy]-1, 3-propanediol (4), along with five known phenolic glucosides, syringoylglycerol 8-O-beta-D-glucoside (2), magnolenin C (5), syringaresinol mono-beta-D-glucoside (6), 3-(4-hydroxyl-3-methyphenyl)-1 -propanol-l-O-beta-D-glucoside (7) and 3, 5-dimethoxyl-4-hydroxybenzyl alcohol 4-O-beta-D-glucoside (8) were isolated and identified from the plant leaves. Compounds 1 and 2 inhibited significantly (P <0.01) the proliferation of murine T and B cells at concentration of 1 x 10(-6) mol L(-1), in vitro.
Ericaceae ; chemistry ; Glucosides ; chemistry ; isolation & purification ; Immunosuppressive Agents ; chemistry ; isolation & purification ; Lignans ; chemistry ; isolation & purification ; Phenols ; chemistry ; isolation & purification ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry

OBJECTIVETo compare the expression of CCL28 in minor and major salivary glands and clarify the role it plays in IgA secreting by minor salivary glands in oral cavity.

METHODSLabial gland and parotid samples were analyzed with real-time fluorescent quantitative PCR assay for CCL28 mRNA. Rank-sum test was used for data analysis using SPSS 10.0 software package.

RESULTSCCL28 mRNA was abundantly expressed in labial glands of healthy adults. Its expression was higher than that in parotids (P<0.01).

CONCLUSIONThe results of this article suggest that the expression level of CCL28 in labial glands is remarkably higher than that in parotids, which reminds us that the high concentration of IgA in minor salivary glands may be associated with their high expression of CCL28.

Adult ; Humans ; Lip ; Salivary Glands, Minor

OBJECTIVETo study early change features of microRNA (miRNA) in the peripheral blood of Sophorae Tonkinensis Radix et Rhizoma induced liver injury rats, and to look for the miRNA biomarkers in the peripheral blood of early liver injury.

METHODSSixty Wistar rats were randomly divided into the control group and the Sophorae Tonkinensis Radix et Rhizoma (abbreviated as STRR) group, 30 in each group. Rats in the STRR group was administered with STRR decoction at 12 g/kg (2 mL/100 g), while equal volume of the distilled water was given to those in the control group. Rats were anesthetized on day 3, 7, 14, and 28, and 28 days after withdrawal. The serum samples were withdrawn. The alanine aminotransferase (ALT), aspartate transaminase (AST), total bile (TBIL), alkaline phosphatase (ALP), total protein (TP), and albumin (ALB) were detected. The globulin (GLO) level was calculated. HE staining was performed on the liver tissue to observe the pathomorphological changes. The whole blood was collected on day 7, 14, and 28 to perform the microarray test. The differentially expressed miRNAs were screened and verified by RT-PCR.

RESULTSThe ALT activity obviously increased on day 7 - 28 in the STRR group (P <0.05). The histopathological results showed the degeneration and swelling of the liver cells on day 28. In the microarray test, there were 11, 22, and 13 up regulated expressed miRNAs on day 7, 14, and 28, respectively. There were 1, 13, 2 down regulated expressed miRNAs on day 7, 14, and 28, respectively. By target gene prediction and pathway analysis of differentially expressed miRNA on day 7, 14, and 28, they involved in regulating and controlling signal transduction, cellular interaction, cytoskeleton. Differentially expressed miRNA might possibly participate in the process of liver injury. The RT-PCR result of the expression of miR-291a-5p with the peak time efficiency on day 7 showed that the expressions of miR-291a-5p in the peripheral blood and the liver tissue were basically identical.

CONCLUSIONmiR-291a-5p could early indicate the liver injury, which could be taken as one of an early marker in STRR induced liver injury.

Animals ; Chemical and Drug Induced Liver Injury ; metabolism ; pathology ; Drugs, Chinese Herbal ; adverse effects ; Female ; Liver ; metabolism ; pathology ; Male ; MicroRNAs ; blood ; metabolism ; Rats ; Rats, Wistar