Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Animals ; Disease Models, Animal ; Electromyography ; Humans ; Irritable Bowel Syndrome ; diagnosis ; physiopathology ; Reflex

Objective To explore the influence of inhaled glucocorticosteroid on ?-glutamylcysteine synthetase(?-GCS) in inflammatory cell of sputum in children with asthma.Methods Twenty-two asthmatic children were divided into 2 groups according to treatment.The children who were treated by inhaled budesonide combined with salbutamol were due to group A and the others inhaling salbutamol only were due to group B,the healthy children were acted as healthy control group(group C).The glutation(GSH),total GSH and the activity of ?-GCS in sputum were measured respectively;Expression of ?-GCS in inflammatory cell of sputum were detected by immunohistochemistry;the expression of ?-GCS heavy chain(?-GCS-h) mRNA were detected by reverse transcriptase-polymerase chain reaction(RT-PCR).Results 1.The total GSH[(1.08?0.14) ?mol/L] and oxidized glutathione(GSSG)[(0.37?0.09) ?mol/L] were decreased in sputum of group A of post-treatment compared with pre-treatment(Pa

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

OBJECTIVETo investigate the value of the noninvasive examinations for localization of insulinoma.

METHODSThe clinical materials of 88 cases of insulinoma treated in Peking Union Medical College Hospital between January 2005 and November 2008 were analyzed retrospectively. There were 40 males and 48 females aged from 15 to 74 years old (averaged, 46.5 years old).

RESULTSThe positive rates for localization were 19.3% (17/88), 52.4% (11/21), 95.5% (64/67), 1/6, 30.0% (6/20), 83.9% (26/31), 8/8 and 5/5 respectively in transabdominal ultrasound, enhanced CT, multislice spiral CT pancreatic perfusion, MRI, somatostatin receptor scintigraphy, endoscopic ultrasound, laparoscopic ultrasound and intraoperative ultrasound. Thirty-one multiple tumors in 8 patients were resected. The positive rates of locating the multiple insulinomas of multislice spiral CT pancreatic perfusion and Intraoperative Ultrasound were 48.4% (15/31) and 100% (14/14) respectively.

CONCLUSIONSThe locating diagnosis of insulinoma has came to noninvasive examination era. Multislice spiral CT pancreatic perfusion is the first choice for localization. Intraoperative ultrasound might play an important role in locating tumors for patients with multiple insulinomas.

Adolescent ; Adult ; Aged ; Female ; Humans ; Insulinoma ; diagnosis ; Male ; Middle Aged ; Pancreatic Neoplasms ; diagnosis ; Retrospective Studies ; Young Adult

Objective To summarize the diagnostic and therapeutic experiences for multiple insulinoma. Methods Clinical data of 34 cKsefl of multiple insulinoma treated in Peking Union Medical College Hospital between 1984 and 2007 were analyzed retrospectively. Results Multiple insulinoma was identified in these 34 cases for 37 instances.Malignant insulinoma was found in 2 cases.Three cases suffered from postoperative recurrent multipie tumors.35.3% cases belonged to MEN1;13.5% cases were of insulinoma combined with islet hyperplasia;43.2% cases had 3 or more than 3 insulinomas;Fifteen cases (40.5%)had had a misdiagnosis.45.2%tumors were smaller than 1 cm in diameter:88.9%multiple insulinonla located at the body and tail of the pancreas.Enucleation of multiple tumors was performed for 48.7 percent of cases. Conclusions Most multiple insulinomas were small,it was difficult for preoperative examination to locate all the tumors therefore.Being on the alert against multiple insulinoma and such measures as careful exploration,intraoperative blood glucose determination.fine needle aspiration biopsy,frozen sections helps to avoid missing multiple imuhnoma.

Objective:To investigate whether the nutrition support team (NST) benefits esophageal carcinoma (EC) patients who are concurrently undergoing chemoradiotherapy. Methods: Between June 2012 and December 2013, 40 EC patients undergoing chemoradiotherapy were divided into the NST group and routine treatment (RT) group, with 20 patients in each group. At the end of chemoradiotherapy, the nutritional status, incidence of complications, and completion rates of radiotherapy were evaluated. The length of hospital stay (LOS) and cost were also compared between the two groups. Results:The nutrition and blood parameter values of the NST group were better (P<0.05) than those of the RT group. The incidence of complications was lower in the NST group (P<0.05) than that in the RT group. In addition, all patients in the NST group achieved the treatment plan, whereas five of the patients in the RT group interrupted or delayed the plan (P<0.05). The average LOS decreased by 3.8 d (P<0.05), and the hospitalization costs were reduced to 6300 RMB person-times (P>0.05) for the patients of the NST group. Conclusion: NST could maintain the nutritional status and improve the treatment compliance and tolerance of EC patients undergoing chemoradiotherapy, thereby shortening the LOS time and reducing the costs.

Objective To determine the urinary iodine level of people in Yunyang and Bishan County of Chongqing and explore into its influencing factors. Methods Using multistage cluster stratified simple random sample method, Yunyang and Bishan County were chosen as research spots, then thirty children aged 8-10 in each 3 primary school of the 2 counties were selected using stratified randomization sampling method to inspected their urine and household salt for iodine and the iodine content in drinking water. Results Five hundred and seventy-one urine samples were inspected and the urinary iodine median was 261.47 μg/L. 5.78% (33/571) and 37.48%(214/571) of samples had an urinary iodine median less than 100 μg/L and more than 300 μg/L. The urinary iodine median of Yunyang County was higher than that of Bishan (H = 7.42, P < 0.01). The iodine salt coverage rate, the qualified rate and edible qualified iodine salt rate respectively were 99.64%(554/556), 94.22% (522/554) and 93.88% (522/556) in 556 samples of family table salt. Eighty-seven samples of drinking water were inspected, resulting an averaged iodine content of 8.81 and 2.97 μg/L, respectively in the 2 counties. Conclusions The 2 counties are all the area of iodine deficiency. The urinary iodine level, although meeting the demand of eliminating iodine deficiency diseases, is a little bit higher given that iodized salt of present doage has been taken for a long time. The content of iodized salt should be adjusted accordingly.

OBJECTIVETo study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs.

METHODSDCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis.

RESULTSThe proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05).

CONCLUSIONSTwo kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

Carcinoma, Hepatocellular ; pathology ; ultrastructure ; Cell Line, Tumor ; Cell Shape ; Dendritic Cells ; drug effects ; immunology ; Glucans ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; ultrastructure ; Lymphocyte Culture Test, Mixed ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; Transcription Factor RelA ; metabolism

Objective To study the effect of electroacupuncture on repair of spinal cord injury and its effect on somatosensory evoked potential ( SEP) in dog models of intervertebral disc prolapse. Methods Nine Beagle dogs were randomly divided into three groups. In the model group and electroacupuncture group, the dog disc prolapse models were made by balloon compression, and in the electroacupuncture group, electroacupuncture was used every day for 14 days after operation. The model group was not treated after surgery. Sham operation was performed in the control group. Each dog was scored according to the Texas Spinal Cord Injury Scale for Dogs (TSCIS) scores before surgery (day 0) and on days 1, 4, 7, 14 after surgery. At the same time, SEP wave was measured using an EMG Evoked Potential Measuring Systerm and its latency and amplitude were analyzed. Results There was a significant difference in TSCIS scores between the model group, electroacupuncture group and control group at 1 day after operation. There was a significant difference between the electroacupuncture and model groups at 14 days after surgery. The amplitude of SEP in the model and electroacupuncture groups was significantly different from that in the control group at 1 day after operation, and there was a significant differ-ence between the electroacupuncture and model groups at 14 days after operation. There was a significant difference in the latency of SEP between the model and electroacupuncture groups at 4 days after operation, and between the electroacupunc-ture and model groups after at 14 days after operation. Conclusions Electroacupuncture can effectively promote healing of spinal cord injury in dogs with intervertebral disc prolapse, improve the TSCIS scores, restore SEP waveform, shorten the latency and enhance the amplitude. SEP can reflect the degree of spinal cord injury to a certain extent, and can be used to evaluate the effect of electroacupuncture treatment in these dogs.