SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.

Objective To detect the expression level of aquaporin 8 (AQP8) in patients with functional constipation(FC) or constipated irritable bowel syndrome (IBS-C),and the correlation between the expression of AQP8 and clinical features.Methods From March to December 2014,a total of 16 patients with IBS-C and 19 patients with FC met Rome Ⅲ criteria were collected,and nine healthy individuals were assigned to control group at the same period.The ascending and decending colonic tissues mucosa of FC,IBS-C and control group were taken under endoscope.The expression of AQP8 at mRNA and protein level was detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC).The differences in AQP8 mRNA expression and AQP8 relative area were analyzed by Kruskal-Wallis test among groups,and Pearson correlation coefficient was performed for correlation analysis between the expression and clinical features.Results The relative expressions of AQP8 mRNA of ascending colon and descending colon of FC patients (1.38,0.61 to 4.09;2.65,0.82 to 7.52) and IBS-C patients (2.23,0.82 to 4.67;1.35,0.51 to 2.03) were higher than those of control group (0.56,0.19 to 0.97;0.38,0.21 to 1.19),and the differences were statistically significant (ZFc =-2.435,-3.149,ZIBS-C =-2.690,-2.152;all P＜0.05).AQP8 mRNA expression of descending colon in patients with FC was higher than that of patients with IBS-C,and the difference was statistically significant (Z =-2.003,P =0.045).The expression of AQP8 in patients with FC and IBS-C was positively correlated with disease course (ascending colon r=0.57 and 0.53;descending colon r=0.49 and 0.54,all P＜0.05),and was negatively correlated with frequency of defecation (ascending colon r=-0.82 and-0.61;descending colon r=-0.49 and-0.53,all P＜0.05).There was no correlation between the expression of AQP8 and age,gender,onset age,presence of abdominal symptoms of the patients (all P＞ 0.05).Most of AQP8 of FC group was expressed in cytoplasm of colonic mucosa epithelial cells,while that of IBS-C group and control group was mostly expressed at apical membrane and basal membrane of epithelial cells.The results of semi-quantification demonstrated that AQP8 relative area of descending colon of FC and IBS-C group increased compared with that of control group (3.42％ (1.24％ to 5.61％),2.45％(1.72％ to 4.27％) vs 1.18％ (0.35％ to 2.81％);Z=-2.534,-2.151,both P＜0.05).Meanwhile,AQP8 relative area of ascending colon of FC group increased compared with that of control group (2.46％(1.48％ to 4.18％) vs 1.14％(1.29％ to 2.15％) Z=-2.041,P＜0.05).Conclusion There are differences in AQP8 expression quantity and location in cells of descending colon between patients with FC and patients with IBS-C,which is a way for differentiation these two diseases.

Objective To study the expression and prognostic significance of neuroendocrine differentiation markers chromogranin A(CgA)and synaptophysin(SYN) in patients with resectable non-small cell lung cancer(NSCLC).Methods From January 2000 to January 2003,123 patients with NSCLC who received operations were investigated.The resected specimens and clinical data were collected.Immunohistochemical Elivison method was used to detecte the expression of CgA and SYN.Kaplan-Meier survival curve and Cox proportional hazard model multivariate analysis were applied for the prognostic factors. Results The positive expression rates of CgA and SYN were 22%,17.9%,respectively.The expression of SYN was associated with histological differentiation(P=0.001).No significant association was found between NSCLC with neuroendocrine differentiation(NSCLC-ND) and sex,age,smoke index,TNM Stage and pathology classification.No evidence showed the patients with positive expression of CgA or SYN could be tolerant with more cycles of chemotherapy(P=0.406).Kaplan-Meier survival curve indicated the survival had a relation with the expression of CgA and SYN.It was revealed by Cox analysis that SYN(P=0.001),TNM stage(P=0.02)and the maximal diameter of tumor(P=0.049) were independent prognostic factors. Conclusion The patients with NSCLC-ND had a poorer prognosis.SYN may be one of the prognostic factors in patients with NSCLC.

Objective To study the effects of different level of iodine nutrition on the thyroid function in women of reproductive age. Methods A total of 100 (50 from each) women of reproductive age but not pregnant were collected from iodine deficient and adequate areas. The questionnaire was obtained individually with items concerning personal history of thyroid diseases, goiters and category of edible salt and drinking water based on the project design. The household salt and drinking water were collected for measuring iodine content, and blood samples were obtained for TSH, FT4 and FT3 testing. Results The coverage of iodized salt and the median level of urinary iodine in iodine deficient women(72.0% and 95.5 μg/L) were obviously lower than that in iodine sufficient women(100.0% and 167.4 μg/L, χ2=16.28, U = 632.00, P < 0.01). Median level of serum TSH in iodine deficient women (2.56 m U/L) appeared in an increasing tendency compared to the iodine sufficient women (1.88 mU/L), but there was no significance (U=990.50, P > 0.05). Serum FT4 mean level in iodine deficient women [(14.7±2.0) pmol/L]was lower than that in iodine sufficient women[(17.0±3.8)pmoI/L, t=3.76, P<0.01]. There was no difference in serum FT3 between two group women[(5.1±1.4), (4.8±0.5)pmoI/L, t = 1.59, P > 0.05]; but FT3/FT4 ratio in iodine deficient women(0.33±0.04) was markedly higher than that in the iodine sufficient women(0.30±0.04, t=3.13, P<0.01). The percentage of thyroid dysfunction in iodine deficient women[20.0% (10/50)]was higher compared with the iodine sufficient women[8.0%(4/50)], but without significance(χ2=2.99, P>0.05). Conclusions Iodine deficiency is a primary cause leading to hypothyroid in women of reproductive age. Long term of iodized salt usage is an efficient way to correct iodine deficiency.

Objective :To study effects of early rehabilitation after percutaneous coronary intervention (PCI) on car‐diac rehabilitation in patients with coronary heart disease (CHD).Methods :A total of 100 CHD patients undergoing PCI in our hospital were randomly and equally divided into routine nursing group and early rehabilitation group . Clinical indexes and incidence rate of major adverse cardiovascular events (MACE) after PCI ,scores of Morningside rehabilitative state scale (MRSS) ,self‐rating anxiety scale (SAS) ,self‐rating depression scale (SDS) ,social disabili‐ty screening schedule (SDSS) ,short‐form 36 health survey questionnaire (SF‐36) before and after treatment and pa‐tient satisfaction were compared between two groups before discharge .Results : Compared with routine nursing group ,there were significant reductions in first‐time get out of bed time 、 exercise time ,complete self‐care time , hospitalization time ,postoperative incidence rate of MACE (52% vs .18%) in early rehabilitation group , P＝0. 001 all ;After treatment ,compared with routine nursing group , there were significant reductions in scores of MRSS [ (5.87 ± 1.21) scores vs .(2.69 ± 1.01) scores] ,SAS [ (49.96 ± 3. 98) scores vs .(44.56 ± 3.12 ) scores] ,SDS [ (49.89 ± 3.85) scores vs .(45.38 ± 3.15) score] and each dimension score of SDSS ,and significant rise in SF‐36 score [ (89.76 ± 7.23) scores vs .(93.98 ± 8. 09) scores] and total satisfaction rate of patients (70% vs.90%) in early rehabilitation group , P＜0. 05 or ＜0.01. Conclusion :Early rehabilitation after PCI can significantly improve cardiac rehabilitation level ,psychological state ,social function and quality of life ,shorten postoperative recovery time and comprehensively improve patient satisfaction in CHD patients .

BACKGROUNDRevision anterior cruciate ligament (ACL) surgery can be expected to become more common as the number of primary reconstruction keeps increasing. This study aims to investigate the factors causing instability after primary ACL reconstruction, which may provide an essential scientific base to prevent surgical failure.

METHODSOne hundred and ten revision ACL surgeries were performed at our institute between November 2001 and July 2012. There were 74 men and 36 women, and the mean age at the time of revision was 27.6 years (range 16 - 56 years). The factors leading to instability after primary ACL reconstruction were retrospectively reviewed.

RESULTSFifty-one knees failed because of bone tunnel malposition, with too anterior femoral tunnels (20 knees), posterior wall blowout (1 knee), vertical femoral tunnels (7 knees), too posterior tibial tunnels (12 knees), and too anterior tibial tunnels (10 knees). There was another knee performed with open surgery, where the femoral tunnel was drilled through the medial condyle and the tibial tunnel was too anterior. Five knees were found with malposition of the fixation. One knee with allograft was suspected of rejection and a second surgery had been made to take out the graft. Three knees met recurrent instability after postoperative infection. The other factors included traumatic (48 knees) and unidentified (12 knees).

CONCLUSIONTechnical errors were the main factors leading to instability after primary ACL reconstructions, while attention should also be paid to the risk factors of re-injury and failure of graft incorporation.

Adolescent ; Adult ; Anterior Cruciate Ligament Reconstruction ; adverse effects ; Female ; Humans ; Joint Instability ; etiology ; Male ; Middle Aged ; Retrospective Studies

Objective To evaluate the efficacy and safety of acute ureteroscopy for the treatment of ureterie stones during middle and late pregnancy.Methods From June 1998 to March 2005,17 pregnant women(mean age,27 years;age range,21-35 years)with ureteric stones were treated by ureteroscopy when the fetus was at 20-36 weeks of gestation(mean,29 weeks).All the cases presented with urgent symptoms such as recurrent renal colic(11 cases),fever(4)or acute obstructive anuria(2).Among 17 cases,the stones(between 6 mm?7 mm and 13 mm?21 mm)were located in the upper(8 cases),middle(5)or lower ureter(4);and on the left side(5 eases),on the right(10)and on both(2)of the lower ureter. Mild hydronephrosis were observed in 6 cases and moderate hydronephrosis in 11,Of the 17 cases,14 under- went ureteroscopic pneumatic lithotripsy;in 1 case the calculi were pushed to the renal pelvis;and 2 cases were treated by Double-J catheter drainage.Results All the urgent symptoms in 17 cases were relieved after treatment.The stone-free rate of initial treatment was 82.4%(14 of 17).Three cases with residual stones were treated by Douhle-J catheters,which were replaced every 3 months until the calculi were re- moved.No abortion,premature delivery or complications such as ureter perforation occurred.Mild renal colic occurred in 1 case after insertion of Douhle-J catheter,and it was relieved 3d later;gross hematuria occurred in l case and disappeared 6 d later without treatment.All 17 patients had normal delivery and gave birth to healthy children.Conclusions Ureteroscopy is a safe and reliable method for the treatment of ureteric calculi during middle and late pregnancy.

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.
ATP-Binding Cassette, Sub-Family B, Member 1 ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Antibody Specificity ; genetics ; physiology ; Blotting, Western ; CD3 Complex ; immunology ; Chromatography, Gel ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Flow Cytometry ; Humans ; Jurkat Cells ; drug effects ; metabolism ; K562 Cells ; drug effects ; metabolism ; Mice ; Polymerase Chain Reaction ; Radioimmunoassay ; Trifluoperazine ; pharmacology ; Verapamil ; pharmacology

OBJECTIVETo study the specific targeting cytotoxicity to drug-resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody.

METHODSThe diabody was purified by affinity chromatography and identified by SDS-PAGE and FACS. The effect of the anti-Pgp/anti-CD3 diabody mediated lysis of Pgp-expressing tumor cells was assayed by human leukemia nude mice xenograft model in vivo.

RESULTSThe diabody was produced in E.coli in a soluble functional form and could bind both Jurkat cells (CD3(+)) and K562/A02 cells (Pgp(+)). The binding rates were 86.25% and 86.26%, respectively. It could inhibit tumor growth by 98.57% and prolonged the survival of mice bearing xenografted K562/A02 cells.

CONCLUSIONThe diabody was proved to be a potent agent for mediating T lymphocyte cytotoxicity to lyse Pgp expressing tumor cells in vitro and in vivo.

ATP-Binding Cassette, Sub-Family B, Member 1 ; immunology ; Animals ; Antibodies, Bispecific ; immunology ; pharmacology ; CD3 Complex ; immunology ; Cytotoxicity, Immunologic ; drug effects ; Drug Resistance, Neoplasm ; immunology ; Humans ; Jurkat Cells ; Mice ; Mice, Nude ; T-Lymphocytes ; drug effects ; immunology ; Xenograft Model Antitumor Assays

OBJECTIVETo establish a real-time RT-PCR based plasma virus quantification method and monitor the plasma viral load of SHIV-CN97001 during its in vivo passages in rhesus macaques.

METHODSViral RNA standards were prepared by in vitro transcription and one-tube real-time RT-PCR were established and optimized using TaqMan EZ RT-PCR CORE REAGENTS and TaqMan probes and primers directed to the 91 bases within the conserved gag region of SHIV. Plasma viral RNA of 126 plasma samples from rhesus macaques of different viral passages was quantified.

RESULTSThe PCR system was optimized by using serial dilution of standards, and the viral RNA load was detected. The lowest limit of the standard curve reached 2x10(-2) copies/ml. The correlation (r>0.99) and the repetition (CV=4.14 percent) also met the requirement. It was revealed that the viral RNA load of third passage was the highest. Generally, the viral load peaks (10(5)-10(6) copies/ml) appeared at the fourteenth day after the infection or inoculation.

CONCLUSIONThe method of one-tube real-time RT-PCR was established successfully, which may provide a sensitive way to qualify SHIV viral load. This will contribute to the establishment and application of SHIV/rhesus macaque models. It was also found that the replicative ability of SHIV-CN97001 was enhanced during the first 2 in vivo passages.

Animals ; HIV ; genetics ; isolation & purification ; HIV Infections ; virology ; Humans ; Macaca mulatta ; RNA, Viral ; blood ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Serial Passage ; Simian Acquired Immunodeficiency Syndrome ; virology ; Simian Immunodeficiency Virus ; genetics ; isolation & purification ; Viral Load