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 explore the characteristics of changes in neutrophil gelatinase-associated lipocalcin (NGAL), kidney injury molecule-1 (KIM-1) and interleukin-18 (IL-18) in Phytolaccae Radix-induced kidney injury in rats and the significance of the combined detection.

METHODWistar rats were divided into three groups: high and low dose (crude drug 40, 20 g x kg(-1) x d(-1)) Phytolaccae Radix decoction groups and the control group, and orally administrated with distilled water or equal volume of Phytolaccae Radix decoction for 35 consecutive days. Their blood and urine samples were collected on day 7, 14, 21, 28, 35,42. The anatomical analysis was conducted for each group. The contents of serum total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CR) and urinary TP and ALB were detected-by means of biochemical analyzer. The concentrations of urinary NGAL, KIM-1 and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA). The morphological changes of renal pathology were observed by light or electron microscopy. The curve areas of various serum or urine indexes and the combined detection were compared by receiver operating characteristic curve (ROC curve).

RESULTRats were given Phytolaccae Radix decoction at the doses of 40, 20 g crude drug/kg daily for 35 consecutive days to induce kidney injury characterized by the degeneration of renal tubular epithelial cell and protein cast. The injury was partially reversible during the recovery period. Compared with the control group, the content of serum BUN, CR and urinary TP in each dose group mostly showed a downward trend. On day 21, the content of urinary ALB obviously increased till the end of administration. The contents of urinary NGAL, KIM-1 and IL-18 began increasing on day 7. Since day 14, high and low dose groups showed significant difference (P<0.01). The high dose group even showed notable changes during the recovery period. According to ROC analysis, the curve areas of NGAL, KIM-1 and IL-18 were 0.846, 0.837 and 0.863 (P <0.01), respectively, much higher than that of BUN and CR. The area of the combined detection was up to 0.947.

CONCLUSIONUrinary NGAL, IL-18 and KIM-1 could forecast and indicate the occurrence and development of renal injury to some degree, and show higher sensitivity and site specificity. The combined detection could further improve the test efficiency.

Acute-Phase Proteins ; genetics ; metabolism ; Animals ; Cell Adhesion Molecules ; genetics ; metabolism ; Drugs, Chinese Herbal ; adverse effects ; Female ; Humans ; Interleukin-18 ; genetics ; metabolism ; Kidney ; drug effects ; injuries ; metabolism ; Kidney Diseases ; etiology ; genetics ; metabolism ; Lipocalins ; genetics ; metabolism ; Male ; Proto-Oncogene Proteins ; genetics ; metabolism ; Rats ; Rats, Wistar

Objective To observe the dynamic changes of prevalence of Keshan disease (KD) in Hebei province from 1990 to 2007, to provide scientific basis for its prevention and treatment. Methods The surveillance data of KD was analyzed according to "the National Scheme of KD Surveillance and the Surveillance of KD" (W/T 78-1996) in Hebei province from 1990 to 2007 by the Institute for Prevention of Endemic Disease in Hebei Province Center for Disease Control and Prevention. The data included physical examination, electrocardiogram (ECG), the chest X-ray film of KD patients and the suspected patients, as well as selenium contents of hair collected in 1990, 1992 and 1999. Results No new cases of acute and subacute types of KD patients were found at the surveillance sites from 1990 to 2007. Thirty-five cases of new latent KD and one case of spontaneous chronic KD were identified respectively. Prevalence of chronic and latent KD ranged from 1.12% (8/713) to 8.18% (27/330) and 2.29% (19/831) to 8.20% (45/549) in Hebei province from 1990 to 2007, respectively. The prevalence of KD in children aged 3 - 14 years old and childbearing woman aged 20 - 45 years old decreased year by year, however population over 45 years old were more likely suffering from KD. The major abnormal changes of ECG in KD) patients were complete fight bundle branch block, ST-T change frequent premature ventricular contraction, and left anterior faseicular block. The prevalence of the heart enlargement in KD patients was 47.00% (211/449) averagely, and the prevalence of heart enlargement of medium grade increased remarkably after 2005 [28.57%(8/28) - 48.39%(15/31)]. The average mortality in chronic KD patients was 18.0%(18/100) from 1990 to 2007. Conclusions The prevalence of KD decreased slowly in Hebei province. Hebei province is still the region with higher prevalence of KD around the country, and the tasks of prevention and treatment of KD is still urgent. Enhancing the surveillance of of KD and carrying out management and treatment of KD patients should be emphasized in the future.

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 express soluble HA of A/H1N1 influenza virus in drosophila S2 cell line and identify its bio-activity.

METHODSHA gene was amplified from A/Shenzhen/71/09 virus strain using RT-PCR, then we constructed pAC5.1-HA expression vector, which was co-transfected into S2 cell with pCoblast vector. After transfection, stable S2 cell was selected through Blasticindin. HA in the supernatant was identified with Western Blot assay and purified with Ni-column. Recombinant HA was immunized into BALB/c mice 3 times, and the Abs titers were evaluated with ELISA.

RESULTSWe successfully cloned HA gene with 1.7 x 10(3) bp of A/Shenzhen/71/09 virus strain and got recombinant pAC5. 1-HA expression vector. Stable S2 cell line was established after transfection and selection, which continuously expressed HA with molecular weight 75 x 10(3) D. After immunization with HA, the Abs titers were 1:1280 and 1: 5120 respectively on 10 d, 30 d.

CONCLUSIONWe expressed soluble HA with good bio-activity, which contributed to research on immune diagnosis, subunit vaccine, and monoclonal Abs for influenza.

Animals ; Blotting, Western ; Cell Line ; Drosophila ; Female ; Gene Expression ; Hemagglutinin Glycoproteins, Influenza Virus ; analysis ; chemistry ; genetics ; metabolism ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; metabolism ; Influenza, Human ; virology ; Mice ; Mice, Inbred BALB C ; Solubility

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