BACKGROUND: Dendritic cells, the most potent antigen presenting cells known at present, have been extensively used in the immunotherapy as adjuvant. OBJECTIVE: The present study was to construct Der p 2 eukaryotic expression vector and validate its expression in the mouse bone marrow-derived dendritic cells. DESIGN, TIME AND SETTING: A single sample observation was performed at the Institute of Respiratory Disease,Xinqiao Hospital, Third Military Medical University of Chinese PLA between May and December 2005. MATERIALS: C57BL/6 mice were included. Plambd-Der p 2 was the product of Heska Company, USA.pCI-neo plasmid was provided by the Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University of Chinese PLA.METHODS: Mouse bone marrow-derived dendritic cells were in vitro isolated and cultured.Complete Der p 2 cDNA was spliced from prokaryotic expression vector plambd-Der p 2, and then cloned into eukaryotic expression vector pCI-neo (pCI-neo-Der p 2).The positive recombinants pCl-neo-Der p 2 transfected into dendritic cells.Non-transfected and blank vector pCI-neo-transfected dendritic cells were used as controls. MAIN OUTCOME MEASURES: ①Identification of pCI-neo-Der p 2 recombinant plasmid.②Detection of Der p 2 mRNA and protein expression by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot techniques. RESULTS: Sequencing results showed Der p 2 cDNA in pCI-neo-Der p 2 was in coincidence with the sequence registrated in Gene Bank.RT-PCR and Western Blot results showed that expression of Der p 2 mRNA and protein could be detectable in the pCI-neo-Der p 2-transfected dendritic cells. CONCLUSION: The Der p 2 cDNA was successfully constructed into the eukaryotic expression vector, and Der p 2 gene and protein could be expressed efficiently in dendritic cells.

AIM: Site-specific functional neurons of brains were with different cellular morphology. It has not been fully understood whether the grafted neural stem cells could differentiate into the site-specific neurons. This experiment is to investigate the neuronal differentiation of the neural stem cells derived from a human fetal brain after transplanted into young rats' brains, to study the possibility of cell-replacement therapy for children's brain disorders with neural stem cells. METHODS: Experiments were performed at the Cell Laboratory of Naval General Hospital from April to July 2007. ①Human fetal brain tissues of 16 week gestation were provided by Department of Gynaecology and Obstetrics of Naval Hospital. Pregnant woman and family members signed an informed consent. Experimental intervention was approved by Hospital Ethical Committee. Fourteen clean brood young SD rats aged 10 days, irrespective of gender, were provided by Experimental Animal Center of Medical College of Peking University. Animal intervention met the animal ethical standards. ②The neural stem cell spheres were derived from the fetal brain tissues of 16 week gestation. The differentiation multipotency of the neurosphere was identified when cultured in a child's cerebrospinal fluid (CSF). The neurospheres cultured in vitro for 14 days were injected into the lateral ventricles of young rats of 10 days old. The rats were respectively killed at days 4, 7 and 14 after transplantation. The special immuno-fluorescent assays were performed using anti-human neurofilament (anti-hNF) to show the location and morphology of graft neurons. RESULTS: ①The typical floating neurospheres were obtained, with the potency to differentiate into neurons, astrocytes and oligodendrocytes. ②The neuronal differentiation of grafts was detected with the mixture of three monoclonal antibodies against human neurofilament. Four days after transplantation, the immune response positive cells lied within the granule cell layer of cerebral cortex were shown in the shape of granule cells, or within the pyramid cell layer in the shape of pyramid cells with long processes, and the interneuron-like cells also were seen. The Purkinje cells arranging in a monolayer were detected in the cerebellum. Compared the results at different time points, the location of grafts were the same. The graft cells were less and the processes were longer over time. CONCLUSION: The in vitro cultured neurosphere cells can migrate into brain tissues and differentiate into site-specific neurons in shape after transplanting into the lateral ventricles of young rats. It is suggested that the host brain tissue microenvironment played an important role in guiding the graft differentiation into neurons. The results have an important significance for understanding cell replacement of developing brain disorders.

Objective To improve the methods for in vitro generating dendritic cells (DCs) from mouse bone marrow and to identify it with morphological, phenotype determination. Methods Cells isolated from mice bone marrow were cultured in GM-CSF (20 ng/ml), differentiating into dendritic cells. Morphological changes were observed by optical phase contrast microscopy, and surface molecules including CD11 C, CD80, MHCⅡ were detected by FACS. Results A large number of typical DCs were observed after culturing for 10 d. FACS analysis showed that the amplified DCs could express CD11 C, CD80, MHCⅡ. Conclusion A large quantity of highly pure BM-DC can be obtained by this method.

ction of immune tolerance of T cells to allergens.

Objective To study the generation of metallo-β-lactamase(MBLs) and its related gene carrying situation in the clini-cal isolates of Pseudomonas aeruginosa .Methods Ceftazidime and imipenem were adopted to preliminarily screen MBLs of Pseudo-monas aeruginosa .The phenotypic confirmatory of imipenem-resistant and ceftazidime-resistant Pseudomonas aeruginosa was per-formed by using 2-mercaptopropionic acid (2-MPA) or EDTA synergy test and the MBLs genotypes of the positive strains in the preliminary screen were detected by PCR .Results The positive rate of the MBLs preliminary screen test in multi-resistant strains was 10 .9% ,and the positive rate of the MBLs in multi-resistant strains detected by CAZ/EDTA ,CAZ/2-MPA ,IMP/EDTA and IMP/2-MPA was 7 .5% ,7 .9% ,8 .8% and 9 .5% respectively .The positive rates of ipm1 and vim gene by PCR were 10 .4% and 8 .3% respectively .The strains with positive spm ,sim1 and gim were not found .Conclusion The MBLs test results detected by different methods are different ;MBLs genes carying ipm1 and vim are the main reason for carbapenem-resistant multi-drug resist-ant Pseudomonas aeruginosa in the hospital .

OBJECTIVETo compare the sensitivity, the specificity and the anti-jamming of several excrement occult blood experimental techniques. To evaluate the effect of transferrin (Tf) in the excrement in the digestive tract hemorrhage detection rate.

METHODSFor 600 patients of clinical suspicious digestive tract hemorrhage, take their excrement specimen, using the chemical process (pyramidon semi-quantitative examination law) to detect hemoglobin (Hb), and using monoclonal antibody colloidal gold method to detect Hb and Tf.

RESULTSFinally the hemoglobin chemical process (hereafter refers to as chemical process) to detect upper gastrointestinal hemorrhage with the positive rate 57.3%, and the detection of hemorrhage of lower digestive tract's positive rate is 44.8%; Hemoglobin monoclonal antibody colloidal gold method (hereafter refers to as colloid gold law) to examine upper gastrointestinal hemorrhage with a positive rate 60.4%, under examination hemorrhage with positive rate 77.6%; transferrin monoclonal antibody colloidal gold method (hereafter refer to as transferrin law) to examine upper gastrointestinal hemorrhage with a positive rate 82.3%, examination hemorrhage of lower digestive tract with a positive rate 66.4%; The union examination law (hemoglobin and transferrin to be detected twice, once positive that is positive) examines upper gastrointestinal hemorrhage the positive rate is 90.8%, hemorrhage of lower digestive tract's positive rate is 97.6%.

CONCLUSIONExcrement transferrin has the high detection rate in the upper gastrointestinal hemorrhage; Hb and the Tf combined examination may obviously raise the digestive tract hemorrhagic disease's positive detection rate.

Objective To investigate the effect of neridronate on osteoporosis-associated degeneration of lumbar disc in ovariectomized rats. Methods Totally 30 female SD rats, aged 3 months old, were divided into three groups randomly: Sham group, OVx (ovariectomy) +N (neridronate) group (receiving a subcutaneous injection of 15 μg/kg neridronate twice a week for 6 months), and OVx + PBO (placebo) group (receiving the same dosage of placebo). The rats were sacrificed and the bone mineral density (BMD), bone histomorphometry and biomechanical properties were measured 6 months later. The histological analysis and score were used to determine the process of lumbar disc degeneration, and the disc height index (DHI) and thickness of cartilage endplate (TCE) were also measured. The protein and mRNA expression levels of collagen type I (COL-I;), collagen type II (COL-II), matrix metalloproteinase-1(MMP-1), matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13) in the lumbar disc of ovariectomized rats were detected by Western blotting analysis and real-time RT-PCR, respectively. Results The BMD, bone histomorphometry and biomechanical properties were better in the OVx+N group compared with OVx+PBO group. Histological evaluation showed that the DHI of rats in the OVx+N group was shorter than that in the Sham group, and the TCE of rats in the OVx+N group was higher than that in the Sham group, but showing no significant difference, which indicated that neridronate could effectively maintain the DHI and delay the calcification of the cartilage endplate. The histological score of the OVx+N group was significantly lower than that of the OVx+PBO group (P<0.05), suggesting neridronate could delay the degeneration of lumbar disc. We also found that, compared with the OVx+PBO, the protein and mRNA expression levels of COL-I and COL-II in the OVx+N group were significantly higher and those of MMP-1, MMP-3 and MMP-13 were significantly reduced (P<0.05). Conclusion Neridronate can delay the process of lumbar disc degeneration in ovariectomized rats, which may be related to maintaining the integrity of lumbar, promoting COL-I and COL-II expression and suppressing MMP-1, MMP-3 and MMP-13 expression.

Objective To evaluate the effects of Jinlida granules on the renin-angiotensin system (RAS) in the kidney, heart and abdominal aorta of experimental diabetic rats, so as to explore the possible mechanism by which Jinlida granules protect the kidney and cardiovascular system. Methods The rats were randomized into 2 groups: normal contronl group (CON group, n=10) and diabetic model group Cn = 20). Rats in the control group were fed with regular chow; those in the diabetic model group were fed with high-fat diet for 4 weeks, and then were administered with streptozotocin (STZ; 30 mg/kg) by intraperitoneal injection to induce diabetic model. Successful diabetic models were further randomized into two groups with 10 in each; Jinlida granules treatment group (DM + JLG group) and non-treatment group (DM group). Rats in DM + JLG group were given Jinlida granules orally (3 g/kg per day) for 8 weeks. Angiotensin I (Ang I) and angiotensin P (Ang II) were measured by enzyme-linked immunosorbent assay (ELISA) in the homogenates of the kidney, heart and abdominal aortas the protein expression of angiotensin II type 1 receptor (ATR) and angiotensin II type 2 receptor (AT2R) was detected by Western blotting analysis. Results Compared with CON group, the kidney mass/body mass(KM/BM), heart mass/body mass (HM/BM), blood glucose (BG), and 24-hour urine proteins (24 h UP) were significantly increased in the DM group (P

Background: Carbohydrate antigen 72-4 (CA72-4) is generally recognized as a tumor marker of digestive system. However, elevated serum CA72-4 level is also evident in many benign diseases and healthy subjects, and its sensitivity in diagnosing malignant tumor is quite poor. Aims: To reassess the value of CA72-4 in tumor screening and diagnosis. Methods: Three cohorts were established in this study. Inpatients who underwent a serum CA72-4 measurement and had a definite final diagnosis were included into Cohort 1 (retrospective study). Inpatients with elevated serum CA72-4 level who had not been diagnosed as malignant tumor before admission were included into Cohort 2 (retrospective study). Individuals who underwent a serum CA72-4 measurement and willing to take a follow-up for at least 2 years were included into Cohort 3 (prospective study). Malignancies had been preliminarily excluded in all individuals in Cohort 3 before enrollment. Results: Among the 2 173 patients recruited in Cohort 1, the prevalence of positive serum CA72-4 was significantly higher in patients with malignancies than those without (16.4% vs. 7.4%, P<0.05). The sensitivity and specificity of CA72-4 for diagnosis of malignant tumor were 36.5% and 76.2%, respectively, at the cut-off value (2.955 U/mL) identified by ROC curve analysis. Among the 1 807 patients recruited in Cohort 2, most of the participants (76.5%) did not have malignancies. Serum CA72-4 level was associated with the histological classification, tumor differentiation and TNM staging of malignancies (P<0.05). Among the 376 individuals who underwent a follow-up for no less than 2 years in Cohort 3, elevated serum CA72-4 level did not increase the risk of malignant tumor (OR=1.268, 95% CI: 0.283-5.687). Conclusions: CA72-4 is not a sensitive marker for tumor screening, its value as an item in physical examination should be re-evaluated. In patients who had positive serum CA72-4 and malignant tumor was ruled out in initial examination, the necessity of long-term follow-up of serum CA72-4 needs to be discussed.

Narcolepsy is a rare disease that presents with sleep-wake disorder, which divided into narcolepsy type 1 (NT1) and narcolepsy type 2 (NT2). NT1 accounts for more than 75%, which is characterized by excessive daytime sleepiness (EDS), cataplexy attacks and nocturnal sleep symptoms (e.g. sleep paralysis, hallucinations, sleep disruptions, sleep movement disorders, etc.), accompanied by metabolic, psychiatric and emotional disturbances. The main clinical manifestation of NT2 is EDS, without cataplexy and nonspecific other symptoms of NT1. The treatments of narcolepsy mainly include the treatments of EDS and cataplexy, as well as the improvement of nocturnal sleep. This article will elaborate the advances in clinical manifestations and treatments of narcolepsy.