An expression plasmid carrying anthrax protective antigen (PA) gene was constructed, which has an OmpA signal sequence attached to the 5' end of PA gene. The plasmid was transformed into E. coli and induced to express recombinant PA (rPA) . The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange, hydrophobic interaction chromatography, and gel filtration, about 15 mg of 95 % pure rPA was obtained from 1-liter culture. The bioactivity of rPA was proved by in vitro cytotoxicity assay. The polyclonal antiserum from rabbits immunized with rPA could inhibit the action of anthrax lethal toxin in vitro, which suggests that antibodies against rPA can provide high passive protection against anthrax. The results reported here may be helpful to develop a safe and efficacious recombinant PA vaccine against anthrax.
Amino Acid Sequence ; Animals ; Anthrax Vaccines ; immunology ; Antigens, Bacterial ; chemistry ; genetics ; immunology ; toxicity ; Bacterial Toxins ; chemistry ; genetics ; immunology ; toxicity ; Base Sequence ; Mice ; Molecular Sequence Data ; Plasmids ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.

METHODSTwo DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.

RESULTSThe standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.

CONCLUSIONThe Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.

DNA, Viral ; blood ; Humans ; Parvovirus B19, Human ; genetics ; Polymerase Chain Reaction ; methods

The aim of this study was to investigate the curative effect of amifostine (AMF) combined with recombinant human erythropoietin (rhEPO) on the aged patients with myelodysplastic syndrome. Two aged MDS patients (one aged 91; another 86) were treated with amifostine and rhEPO over a period of 4 weeks. The results showed that a short-term curative effect was observed and transfusion interval was prolonged in both patients after 4 week treatment with 5 x 0.4 g AMF plus 3 x 6,000 U rhEPO per week. The reticulocyte count in MDS-RA patient returned to normal at first week of treatment and still remained in normal level for 4 weeks; leukocyte, hemoglobin and platelet values in peripheral blood of MDS-RCMD patient obviousby increased, the abnormally increased reticulocyte value displayed a decrease trend after amifostine plus rh-EPO treatment. In conclusion, amifostine plus rhEPO may have a good therapeutic effect for aged MDS patients, and its clinical long-term curative effect still needs further evaluation.
Aged ; Aged, 80 and over ; Amifostine ; therapeutic use ; Drug Therapy, Combination ; Erythropoietin ; therapeutic use ; Humans ; Male ; Myelodysplastic Syndromes ; drug therapy ; Recombinant Proteins ; Time Factors ; Treatment Outcome

The present study was aimed to investigate the damage effect of hepatocyte growrh promoting substance (HGS) on the HL-60 cell DNA in vitro and to explore the possible mechanism underlying the effect. Experiment was divided into 3 groups: one was control group, in which 0.9% NaCl solution was added, and other two were experimental group 1 and group 2, where 22.5 microg/ml and 40 microg/ml HGS were added, respectively. HL-60 cell growths were compared between groups with and without HGS. Single cell gel electrophoresis (SCGE) was used to detect DNA damage of HL-60 cell. DNA electrophoresis was used to detect the apoptosis of HL-60 cells caused by HGS. The results showed that the inhibitory effects of HGS on growth of HL-60 cells were observed in group with 22.5 microg/ml and group with 40 microg/ml after culture for 2 days, the DNA ladder and the apoptosis of HL-60 cells occurred in these 2 groups on day 2 after addition of HGS, the counts of HL-60 cells with comet tail in these experimental groups were found to be more increased in comparison with control group. In conclusion, the HGS can inhibit the growth of HL-60 cell and the apoptosis of HL-60 cells should be induced through pathway of DNA damage caused by HGS.
Animals ; Animals, Newborn ; Apoptosis ; drug effects ; genetics ; Cattle ; Cell Proliferation ; drug effects ; Comet Assay ; DNA Damage ; DNA, Neoplasm ; analysis ; genetics ; HL-60 Cells ; Humans ; Peptides ; pharmacology

The purpose of this study was to explore the effect of amifostine in treating aged patients with idiopathic thrombocytopenic purpura (ITP). Three aged ITP patients (one aged 88, one aged 75, another aged 65) were treated with amifostine over a period of 4 weeks for each. The results showed that the early positive responses were observed in all 3 aged patients after treatment with amifostine 5 x 400 mg per week for 4 weeks, the recovery of platelet counts to normal level were found in 2 cases after 1 week and in 1 case after 2 weeks. Following 4 months after stop of treatment with amifostine, the normal platelet counts still remains in all patients, no more blood transfusions, hormonal preparation and gamma-globulin were given. Amifostine has never been reported elewhere as to treat of aged ITP. In conclusion, amifostine has a good early therapeutic effect on aged ITP patients, but its clinical outcome of long-term still needs further evaluation.
Aged ; Aged, 80 and over ; Amifostine ; therapeutic use ; Drug Administration Schedule ; Humans ; Male ; Platelet Count ; Purpura, Thrombocytopenic, Idiopathic ; drug therapy ; Treatment Outcome

OBJECTIVETo look for the active fraction of ethanol extract of Genkwa Flos (EGF) induced hepatotoxicity and develop an UPLC fingerprint of the active fraction.

METHODTarget fraction of EGF induced hepatotoxicity was guided by the serum biochemical and histopathology methods. The UPLC method was applied to establish the chromatographic fingerprint. The separation was achieved on a BEH C18 column (2.1 mm x 50 mm, 1.7 microm) with a mobile phase consisting of acetonitrile and water containing 0.05% phosphate acid running gradient elution. The detection was carried out at 210 nm and the analysis was finished within 10 min.

RESULTThe chloroform phase of EGF could be responsible for the hepatotoxicity of this herb. The common mode of the UPLC fingerprint was set up under the established condition. There were 17 common peaks in fourteen batches of herbs, eight of which were identified, and the similar degrees of the fourteen batches to the common mode were between 0.890-0.999.

CONCLUSIONIt is easy to locate the chloroform extraction of EGF with hepatotoxicity. And the UPLC fingerprint was developed for the above fraction, which could provide valuable references for safe and effective clinical use of EGF.

Animals ; Asteraceae ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; toxicity ; Flowers ; chemistry ; Humans ; Liver ; drug effects ; Male ; Rats ; Rats, Wistar

OBJECTIVETo investigate the dynamic changes of myocardial matrix metalloproteinases (MMPs) activities in mice with viral myocarditis (VM) and their relationships with cardiac function and myocardial collagen amount and to explore the role of MMPs in the pathologic lesion of VM.

METHODSSixty-five six-week-old male DBA/2 mice were obtained from the Chinese Academy of Medical Sciences. They were divided into two groups randomly. Mice in infected group (n=50) were inoculated intraperitoneally with 0.14 ml of coxsackievirus B3 (CVB3, Nancy strain). Control mice (n=15) were inoculated intraperitoneally with 0.14 ml of Eagle's solution. Eight infected mice were sacrificed on day 3, 7, 10, 21 and 30, respectively and fifteen control mice were killed on day 30 after inoculation. Total protein concentration was determined according to the method of Bradford, while MMPs activities were measured with SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Echocardiographic studies were performed under anesthesia with 3% chloralhydrate intraperitoneally (0.01-0.015 ml/g). Cardiac systolic function indexes, such as peak velocity of aorta (Vp) and flow velocity integral of aorta (Vi) were determined by echocardiography. Histological cross sections of hearts were stained with hematoxylin-eosin and myocardial histopathologic scores were counted under optical microscope. Myocardial collagen amount was measured by determination of hydroxyproline quantification.

RESULTSIn virus-infected mice, both MMP-2 and MMP-9 activities were increased significantly compared with those in controls and reached the peak on day 10 (P < 0.01). On day 10, cardiac systolic function indexes (Vp and Vi) were all significantly lower than those at other stages after virus inoculation and in control group (P < 0.05). There was no obvious elevation in myocardial collagen amount in mice with VM at acute stage (P > 0.05). While the myocardial collagen amount in infected group at recovery stage (on day 21 and 30) increased significantly compared with controls. MMP-2 and MMP-9 activities positively correlated with myocardial histopathological scores, respectively (r =0.801, 0.821 P < 0.01), while they negatively correlated with Vp (r = -0.649, -0.683, P < 0.01) and Vi, respectively (r = -0.711, -0.755, P < 0.01). However, Vp and Vi negatively correlated with myocardial histopathological scores (r = -0.756, -0.584, P < 0.01).

CONCLUSIONSIn mice with VM, the activities of myocardial MMP-2 and MMP-9 at acute stage increased significantly, then myocardial collagen amount elevated in recovery stage. These changes were associated with myocardial remodeling and cardiac dysfunction. Myocardial MMP activities are important markers of myocardial pathologic lesion. They are of value in the evaluation of the severity of myocardial damage and cardiac dysfunction in mice with VM.

Animals ; Collagen ; metabolism ; Coxsackievirus Infections ; complications ; Disease Models, Animal ; Echocardiography ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Matrix Metalloproteinases ; metabolism ; Mice ; Mice, Inbred DBA ; Myocarditis ; diagnostic imaging ; pathology ; physiopathology ; virology ; Myocardium ; metabolism ; pathology ; Systole ; Ventricular Dysfunction ; diagnostic imaging ; physiopathology ; Ventricular Remodeling

Objective To analyze the magnetic resonance imaging (MRI) features of spinal epidural angiolipoma to improve the diagnostic accuracy of this tumor. Methods The MRI findings in 2 patients with histopathologically confirmed spinal extradural angiolipoma were analyzed retrospectively. Results In both of the cases, the tumors were located in the extradural space of the thoracic intraspinal canal. The spindle-shaped tumor mass extended parallel to the longitudinal axis of the spine. The lesions presented with fat and vascular signal features. One of the tumors grew through the intervertebral foramen with a dumbbell appearance, invading the adjacent diaphragma, vertebral body, pedicle of the vertebral arch and the spinous process. The other tumor consisted of some tortuous and dilated blood vessels with flow void signals. Conclusion Spinal epidural angiolipoma presents characteristic features on MRI, which is considered the best diagnostic modality for this tumor.

OBJECTIVETo assess the efficacy and safety of balloon dilatation through flexible bronchoscopy in the management of inflammatory stenosis of grade 4-5 bronchus.

METHODThirty patients with inflammatory bronchial stenosis caused by mycoplasmal pneumonia complicated with pulmonary atelectasis were treated with balloon dilatation through fiberoptic bronchoscopy. Before the procedure and after the last operation, therapeutic effect on pulmonary atelectasis were evaluated with CT and all of the patients were followed-up for 1 - 6 months.

RESULTOne to three operations were required to achieve satisfactory dilatation. After balloon dilatation, the average airway diameter increased obviously and the farther airways were opened after the therapy with irrigation. In 25 of 30 cases satisfactory immediate effects were obtained, a narrow airway diameter above expansion significantly increased as compared with preoperative diameter. In 5 children treated with balloon dilatation, the stenosis could not be improved significantly. In 3 patients with hyperplasia of granulation tissue, cryotherapy had to be applied. The operations were ineffective in the other two patients whose course of disease exceeded 3 months. After follow-up periods of 1 - 6 months, chest CT manifestation of expanded sites was improved in 28 patients and atelectasis disappeared. No severe complication was found in any patients.

CONCLUSIONBronchoplasty by balloon dilatation through flexible fiberoptic bronchoscopy is a simple, effective and safe method to treat childhood tracheobronchial stenosis after pulmonary infections.

Bronchial Diseases ; therapy ; Bronchoscopy ; Catheterization ; methods ; Child ; Child, Preschool ; Female ; Humans ; Male ; Pneumonia, Mycoplasma ; therapy ; Pulmonary Atelectasis ; microbiology ; therapy ; Tracheal Stenosis

The study was aimed to investigate the curative effects and adverse effects of amifostine in the treatment of patients with myelodysplastic syndrome (MDS). Amifostine (AMF) was used alone (4/12) or combined with recombinant human erythropoietin (rh-EPO) (8/12) in 12 MDS patients. The therapeutic regimen was adopted with AMF 0.4 g/day for 5 days, then took a break of 2 days and then went on for 3 weeks consecutively, that was reputed as one treatment cycle. rh-EPO 6 000 U was used for 3 days per week. The results showed that 12 patients all attained hematological improvement in peripheral blood. 11 cases showed major effective response rate (91.7%), while 1 case showed minor response rate (8.3%). The effective response rate of hemoglobin, leukocytes and platelets was 100%, 75% and 58.3% respectively. The intervals of red cell transfusions (RCT) in 2 cases living on red cell transfusion before AMF treatment were prolonged after AMF treatments, and the amount of each RCT was decreased obviously. The side effect was usually discomfort of digestive system, but all patients can endure. In conclusion, Amifostine is a potential drug in the treatment of MDS patients with safety especially to those elder patients who often suffered from other multiple organ disfunctions, and the curative effect will be improved by more treatment cycles.
Adult ; Aged ; Aged, 80 and over ; Amifostine ; adverse effects ; therapeutic use ; Drug Therapy, Combination ; Erythropoietin ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; drug therapy ; Recombinant Proteins