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 effect of natural antioxidant ?-carotene(?-C) combined with vitamin E and C supplement on cellular oxidative damage in aged people. Method:Three hundred subjects aged from 60 to 75y,128 males and 172 females were randomized into 5 groups:group 1(VE 200mg/d+VC 300 mg/d+?-C 16.7 mg/d) ,group 2(VE 200 mg/d+VC 300 mg/d+?-C 8.4 mg/d) ,group 3(VE 200 mg/d+VC 300 mg/d+?-C 5.6 mg/d) ,group 4(VE 200mg/d+VC 300mg/d) ,group 5(VE 5 mg/d) . The trial lasted 16 weeks. All subjects were asked to record the nutrients intake daily. The investigators followed-up and supervised the subjects irregularly. The samples of blood and urine were collected at the beginning and end of the trial. The proliferation of lymphocytes was detected by MTT and H2O2-induced erythrocyte hemolytic degrees were measured before and after the trial. Thefluidity of erythrocyte membrane was evaluated by fluorescence polarization(?) and microviscosity(?) . O6-MeG was analyzed by high performance capillary zone electrophoresis. Results:The proliferation of lymphocytes was increased and H2O2-induced erythrocyte hemolytic degrees were decreased from group 1 to group 4 at the end of trial with significant difference compared with control group 5. The ? and ? values were lower in 1 to 4 groups at the end of trial. O6-MeG was significantly lower in group 1 to 4 group at the end of trial,and group 1 lower than group 4. Conclusion:Different doses of natural antioxidant ?-carotene supplement combined with vitamin E and C could improve the function of erythrocyte and the proliferation of lymphocytes,and decrease DNA alkylating damage.

The pol gene of HIV-1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no anti-IN drug was approved. HIV-1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multi-drug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′-processing step and the 5′-strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV-1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wild-type enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV-1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV-1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET-28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was puriied sy the Co+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV-1 integrase.

Adverse environmental stimulation in the neonatal intensive care unit (NICU) can affect neurodevelopment through epigenetic modification and thus has adverse effects on the long-term developmental outcome of preterm infants. Developmental care can reverse epigenetic changes in genes and promote neurodevelopment in preterm infants. This article reviews the influence of environmental stress in the NICU and developmental care on neurodevelopment in preterm infants, as well as related epigenetic effects, in order to provide a reference for epigenetic studies of preterm infants.
Epigenesis, Genetic ; Humans ; Infant ; Infant, Newborn ; Infant, Premature ; Intensive Care Units, Neonatal

Single cell gel electrophoresis assay (SCGE), also named as alkaline comet assay, was a simple, rapid and sensitive method to evaluate DNA damage. In this study SCGE technique was used to monitor DNA damage difference in tumor patients caused by chemotherapy, DNA damage distribution frequency and DNA damage characters were analyzed by komet image analysis system (KIAS). The results showed that cyclophosphamide greatly caused DNA damage in lymphocytes of tumor patients. There was significant difference of peripheral blood lymphocyte DNA damage between tumor patients and healthy controls. Tail length of lymphocytes were 33.69 +/- 7.56 micro m, and tail DNA% we re 31.51 +/- 5.4 6% in 10 cancer patients treated with cyclophosphamide, while Tail length were 1 6.2 +/- 1.5 micro m and tail DNA% were 7.46 +/- 1.15% in healthy controls. there was great significant difference on tail length and tail DNA% values between cancer patients and healthy controls (P < 0.01). In conclusion, the successful measurement of DNA damage caused by Cyclophosphamide treatment means that the alkaline comet assay as a valuable tool can be very useful in cancer epideminology study, and also be valuable to evaluate DNA damage status of patients in clinic.
Comet Assay ; Cyclophosphamide ; adverse effects ; DNA Damage ; Electrophoresis, Agar Gel ; Humans ; Lymphocytes ; drug effects ; ultrastructure ; Neoplasms ; drug therapy ; genetics

OBJECTIVETo investigate the clinical therapeutic effects of endovascular repair for patients with DeBakey III aortic dissection.

METHODSFrom December 2002 to June 2007, endovascular TALENT stent-graft exclusion was performed in 75 (65 males, mean age 54.4 +/- 12.6 years) patients with DeBakey III aortic dissection (1 young woman due to Ehlers-Danlos syndrome, 2 young men due to primary aldosteronism and trauma respectively). All patients were diagnosed by contrast enhanced computed tomography (CT) or MRI. Stent-grafts were deployed via femoral artery to exclude the tear of dissection. Aortic angiography was performed immediately after procedure.

RESULTSEighty-one stent-grafts were installed in 75 patients successfully without operation related dissection. Endoleakage immediately after stent-graft deploying was evidenced in 25 patients and disappeared after stent placements (n = 6) or balloon dilation (n = 19). Two patients died from aortic rupture within 2 days after procedure. Iliac artery was torn in a female patient with Ehlers-Danlos syndrome, this patient developed hemorrhagic shock after stent-graft placement and recovered after anti-shock treatments and iliac artery replacement with synthetic artery. During the follow-up of 1 - 24 months, 2 patients (including the woman with Ehlers-Danlos syndrome) suddenly died half a year after procedure. The remaining patients were alive and well. Repeat CT during follow up showed that reduced lumen size and thrombosis in the false lumen. There was no aortic rupture, endoleak and stent migration during the follow-up period except descending aortic dissection distal of the stent-graft in 1 patient 1 year after procedure and the patient were successfully treated surgically without complication.

CONCLUSIONSEndovascular repair is a safe and effective treatment for patients with DeBakey III aortic dissection, suitable for old patients with high risk of surgery. Ehlers-Danlos syndrome should be considered in young DeBakey III aortic dissection patients without hypertension. Further studies are warranted on endovascular repair therapy for artery complication of Ehlers-Danlos syndrome.

Adult ; Aged ; Aged, 80 and over ; Aneurysm, Dissecting ; surgery ; Blood Vessel Prosthesis Implantation ; methods ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Stents

OBJECTIVETo investigate the role of AdeABC efflux pump in carbapenems resistance of Acinetobacter baumannii in light of the phenotype and genetype of the efflux pump.

METHODSThe phenotype of the efflux pump was detected in 138 clinical isolates of A.baumannii using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The mRNA expression of pump-encoding gene adeB in the strains was detected using quantitative real-time RT-PCR.

RESULTSOf the 138 strains, 28 showed positivities for AdeABC efflux pump identified by Mueller-Hinton Broth with CCCP. Of the 39 strains resistant to meropenem, 15 (38.4%) showed positive results in CCCP assay, a rate significantly higher than that among the 99 sensitive strains (13.1%, 13/99) (X(2)=12.477(b), P=0.01). The mRNA expression of efflux pump-encoding gene adeB was detected by real-time RT-PCR at a level of 0.899∓∓1.172 in meropenem-sensitive strains, significantly lower than the level of 21.101∓∓21.443 in meropenem-resistant strains (t=4.403, P=0.000).

CONCLUSIONSEfflux plays a role in carbapenems resistance in the clinical isolates of A. baumannii. The AdeABC efflux pump may be an important factor in reducing carbapenems sensitivity in A. baumannii.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Bacterial Proteins ; genetics ; metabolism ; Carbapenems ; pharmacology ; Humans ; Membrane Transport Proteins ; genetics ; metabolism ; beta-Lactam Resistance ; genetics

This study was aimed to investigate expression of vascular endothelial growth factor mRNA (VEGF mRNA) and its relationship with leukemic cell apoptosis after VEGF antisense oligonucleotide (VEGF ASODN) transferred into HL-60 cells. The phosphorothiate VEGF ODN was transferred into HL-60 cells in vitro by using cation poly mediated method, the inhibitory rate of cell proliferation was assayed by MTT, expression of VEGF mRNA was measured by RT-PCR, cell apoptosis was detected by cell morphology observation, DNA agarose gel electrophoresis and flow cytometer (FCM). The results showed that difference of the inhibitory rate of cell proliferation and the relative expression of VEGF mRNA between ASODN group and MSODN or control groups under the same condition (p < 0.05) was statistic significant, but no significant difference (p > 0.05) was found between MSODN and control. The number of clusters of cells in ASODN group decreased; the morphology features of apoptotic cells involved cell shrinking, more granulation in cytoplasm, nuclear contracting and many fragments of cells. In MSODN and control groups, however, cells were plump and clear, and grow healthly. The result of electrophoresis revealed DNA ladder in ASODN group, while only one band of DNA in control groups. The rate of cell apoptosis was 19.46% in ASODN group with a significant difference as compared with MSODN groups and control (p < 0.05). The rate of HL-60 cell apoptosis in combination of VEGF ASODN with VP16 was significantly higher than that in VP16 alone (p < 0.05) and showed time- and dose- dependence. It is concluded that VEGF ASODN can down-regulate expression of VEGF mRNA of HL-60 cells, induces the apoptosis, inhibits the proliferation of HL-60 cells and enhances VP16-induced apoptosis in HL-60 cells, the VEGF ASODN in combination with VP16 shows additive effect.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Etoposide ; pharmacology ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

BACKGROUNDAdoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice.

METHODSC57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body irradiation (TBI), or 7 Gy TBI plus bone marrow transplantation. Tumor areas were measured every 3 days to assess the influence of irradiation treatment on tumor regression. B16-melanoma bearing mice were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11, and 13 after irradiation. White blood cell levels were measured and transforming growth factor β1 (TGF-b1) and interleukin 10 (IL-10) levels in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed to test TGF-b1, IL-10 and Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) in spleens. B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2) or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment, tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs adoptive transfer; histologic analyses of eyes, skin, liver, lungs, and intestine were then performed.

RESULTSIrradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor regression compared to the control group; however, it down-regulated the proportion of Tregs in spleens and the TGF-b1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant difference among the groups.

CONCLUSIONAdoptive transfer of haploidentical tumor-specific T cells in irradiation-pretreated B16-melanoma bearing mice preserved antitumor capacity without causing a GVHD response.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Graft vs Host Disease ; Immunotherapy, Adoptive ; methods ; Male ; Melanoma, Experimental ; metabolism ; therapy ; Mice ; Mice, Inbred C57BL ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes ; immunology ; T-Lymphocytes, Regulatory ; immunology