Abstract:Objective CD8 + T cells play an important role in some viral diseases，but the role of CD8 + T cells is not yet fully clear in the asymptomatic phase of HIV infection. The aim of this study was to elucidate the impact of CD8 + T cell in SHIV-infected monkeys with in vivo CD8 + T cells depletion，and to further understand the pathogenesis of AIDS. Methods Eight SHIV-infected rhesus monkeys in the asymptomatic phase were selected and randomly divided into two groups. Anti-CD8 + T antibody cM-T807 were injected intravenously into four monkeys on days 0，3，and 7. Then the CD8 + T cell number in peripheral blood and lymph nodes in the monkeys were detected by flow cytometry，the plasma viral load by real-time RT-PCR，and the cellular immunity by IFN-γ Elispot assay. Results The viral load of the four monkeys was elevated to above of detected after the CD8 + T cell depletion，but the reactions were not uniform. CD4 + T cells，the HIV-1 target cells，rebound and dropped slightly. The infection of monkeys with CD8 + T cell depletion was lighter than that after the SHIV virus first infection ( plasma viral load and CD4 cells) ，which was consistent with the Elispot results. Conclusions CD8 + T cells play an important role in the asymptomatic phase of HIV-1 infection，but with some interindividual variation.

Abstract:Objective To evaluate the virological and immunological changes in rhesus macaques infected with SHIVCHN19P4 virus，isolate and prepare SHIVCHN19P4 virus stock suitable for passaging in rhesus macaques. Methods Two adult rhesus macaques were infected with SHIVCHN19P4 virus intravenously. For serial passages，10 mL of whole blood from each of the P1 rhesus macaques at 60 d was transfused intravenously into two native rhesus macaques. To analyze the changes of features in those monkeys by flow cytometry，PCR，binding antibody and sequence alignment. Simultaneously，SHIVCHN19P4 virus stock was propagated by PBMCs co-culture. Results All of the four rhesus macaques established systemic infection，the infectivity of the virus was considerably enhanced after passaging. The sequence analysis revealed that SHIVCHN19P4 acquired adaptive changes. Conclusions Our findings provide a firm basis for establishing a SHIV infection model of Chinese rhesus macaques. This model may be very useful for HIV-1 subtype C vaccine and pathogenesis studies.

Abstract:Objective To determine the viral concentration of SHIV1157ipd3N4 to infect Chinese-origin rhesus macaques intravenously， and to clarify the viral replication and immunological impairment in the monkeys after SHIV1157ipd3N4 inoculation. Methods 10 Chinese-origin rhesus macaques were used in this study. Firstly，all the monkeys were screened by serological test to ensure that they were free of SIV，SRV，and STLV. Then they were infected with 1 mL of 10-fold serial dilution of SHIV1157ipd3N4 separately. Finally to make blood routine examination and measure the viral loads of the monkeys by real-time RT-PCR. Results More than 5 TCID50 /mL of SHIV1157ipd3N4 can infect the Chinese-origin rhesus macaques i. v. successfully. Conclusions Our findings of this study provide a firm basis for establishing a SHIV1157ipd3N4 infection model of Chinese rhesus macaques intravenously. This model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.

Abstract:Objective To study the characteristics of RT-SHIV passaging in Chinese rhesus macaques and to establish a RT-SHIV /rhesus animal model providing an animal platform in evaluation of the effectiveness of HIV-1 drugs.Methods Four healthy rhesus macaques selected were negative to SIV，SRV and STLV-1. Two monkeys of them were infected with RT-SHIV intravenously. Blood samples were collected from the infected monkeys at the acute phase and CD8 － PBMC were isolated，and then propagated RT-SHIV. The propagated RT-SHIV with PBMC were injected intravenously into the other two monkeys. Viral load in plasma，CD4 + /CD8 ratio and absolute number of CD4 + T lymphocytes and B lymphocytes were detected，and the RT gene variation was analyzed. Results Systemic infection was established in all of the four rhesus macaques，and the two passaged animals demonstrated more severe changes at the acute phase. There were three amino acid changed during infection and passages. Conclusion The results of our study provide basic information for the establishment of RT-SHIV /Chinese-origin rhesus monkeys models.

Abstract:Objective To prepare an inactivated HIV-1 virus with configurational integrity and more than 90% purity for HIV vaccine research. Methods HIV-1 virus was inactivated by 2，2’-dithiodipyridine ( aldrithiol-2，AT-2 )with complete immunogenicity. Ultracentrifugation and washing was conducted to remove the chemical reagent used for virus inactivation. The residual impurity proteins in the inactivated virus was removed by molecular sieve technology. Results For chemical inactivation，HIV-1 virus was treated with 250 μM AT-2 for 1 h at 37℃ . Chemically inactivated noninfectious SIV with conformationally and functionally intact envelope glycoproteins was produced by treatment with AT-2 as describedelsewhere［1 － 2］. No AT-2 residue was detected in the purified inactivated virus. Residual bovine serum albumin was detected less than 50 ng /mL. Conclusions In this study HIV-1 virus was successfully inactivated with AT-2 and reached a purity of 95. 6% . It can meet the requirements as an immune stimulating agent in HIV vaccine research.

Abstract:Objective To explore the role of ulinastatin (UTI) in the treatment course of ventilator-induced lung injury (VILI) by investigating the effect of UTI on expression of Clara cell secretory protein (CC16) in the lung of rats in high tidal volume mechanical ventilation. Methods Twenty-four male Wistar rats were randomly divided into three groups:control group，high tidal volume ventilation group and UTI pretreatment group. The levels of MDA in the lung and total protein in bronchoalveolar lavage fluid ( BALF) were measured. The expression of CC16 mRNA in the lung was detected by reverse transcription polymerase chain reaction (RT-PCR) . The number of Clara cells and synthesis of CC16 were detected by immunohistochemistry. Results Comparing with the control group，the levels of MDA in lung and total proteinin bronchoalveolar lavage fluid were very significantly increased ( P < 0. 01 ) in the high tidal volume ventilation group，and the expression of CC16 was significantly decreased ( P < 0. 01 ) . Comparing with the high tidal volume ventilation group，the levels of MDA in the lung and total protein in bronchoalveolar lavage fluid were significantly decreased (P < 0. 01 ) in the UTI-pretreatment group，and the expression of CC16 mRNA and protein increased significantly. Conclusions CC16 plays an important role in ventilator-induced lung injury (VILI) . The expression of CC16 mRNA and protein are reduced in a VILI rat model. Ulinastatin may effectively prevent the inflammatory process and ventilator-induced lung injury by increasing the level of CC16 and inhibit the lipid peroxidation.

Abstract:Objective To establish a human gastric cancer orthotopic xenograft model labeled with luciferase.Methods Luciferase labeled MGC803 cells were cloned with G418 selection ( MGC803-Luc) . MGC803-Luc cells were implanted into the stomach wall of BALB / cA-nu mice to establish the tumor-bearing mouse models. Whole-body optical imaging technique was used to monitor the growth of the implanted tumor. Results The successful rate of orthotopic transplantation was 100% . The whole-body optical imaging found that the fluorescence could be detected at 7 days after transplantation. The tumor growth became logarithmic after 21 days. However，the mean fluorescent intensity tended to decline when the tumor had apparent necrosis after 28 days. Ultrasonic imaging found a tumor on the stomach，approximately 8. 39 mm in diameter and 28. 92 mm2 in area. Conclusions It is possible to real-time monitoring the growth of human gastric cancer labeled with luciferase in animals.

Abstract:Objective To analyze the therapeutic effects of 12 herbal monomers on APPswe /PS1 ΔE9 transgenic mice，and to explore promising candidates for treatment of Alzheimer disease. Methods APPswe /PS1 ΔE9 transgenic mice at age of 5 months were randomized into 14 groups. One group was vehicle group ( placibo) and one group was Aricept group as positive control. The other 12 groups were used for treatment with the 12 herbal monomers，respectively. The littermates of no-negative mice were used as normal control. Morris water-maze test was performed at 4 weeks after treatment to assess the nervous function. Results The evodiamine-treated mice showed significantly decurtated latency and increased number of crossings of the removed-platform area in Morris water maze test，compared with that of the vehicle group. Conclusions Evodiamine treatment can improve the learning and memory ability in APPswe /PS1 ΔE9 double-transgenic mice.

Abstract:Objective The aim of this study was to identify the variations of canine distemper viruses (CDV) obtained from clinical samples，and provide a theoretical basis for the prevention and control of CDV infection. Methods The segments of hemagglutinin protein (H) gene，fusion protein (F) gene and nucleocapsid protein (N) gene were amplified by RT-PCR. The amplicons were cloned into pGEM-T-easy vector and the recombination clones were sequenced. Subsequently，the nucleotide and amino acid sequences were analyzed. Results The nucleotide lengths of H，F and N genes for the virus were 1863 bp，1653 bp and 2234 bp，respectively. Sequence analysis did not present any nucleotide insertion or deletion. The three sequences determined in this study showed the high amino acid identities corresponding with three Chinese highly pathogenic CDV isolates BJ080127 (H gene)，GN (F gene) and HL (N gene)，which were 97. 3% ，97. 7% and 99. 3% ，respectively. Meanwhile，each of them displayed the nucleotide identity of 94. 4% ，93. 8% and 97. 2% ，when compared with the CDV prototypic strain A75-17. In addition，comparing with the CDV vaccine strain CDV3，they displayed the nucleotide similarities of 88. 5% ，88. 8% and 93. 9% ，respectively. The glycosylation sites analyses revealed a glycosylation site aimed at the 3 ～ 5 amino acids of F gene. The phylogenetic tree displayed that the virus had the closest relationship with the highly pathogenic CDV isolates identified in China.Conclusion In this study，we cloned the H，F and N genes of a CDV determined in canine samples and identified that the virus is a highly pathogenic CDV，which contains a novel glycosylation site in F gene. This study provides evidence in studies on the molecular mutation epidemiology of CDV infection.

Abstract:Objective To observe the changes of nitric oxide ( NO) and hydrogen sulfide ( H2 S) levels in the liver tissue of rabbit with nonalcoholic fatty liver disease ( NAFLD ) ，and explore the effect of NOand H2 S in the pathogenesis of NAFLD. Methods Forty Japanese white rabbits were randomly divided into severe NAFLD group，mild NAFLD group，and blank control group. The severe NAFLD group was given high fat diet 160 g /rabbit. day，the mild NAFLD group was given high fat diet 80 g /rabbit. day and normal diet 80 g /rabbit. day，and the blank control group was given normal diet160 g /rabbit. day. The raising cycle was 13 weeks. Plasma samples were collected before and after the experiment，and the concentration of triglyceride ( TG) ，cholesterol ( TC) ，and the levels of NO and H2 S in the liver tissue were determined. Pathological changes of the liver tissue were examined by light microscopy with HEstaining. Results (1) TC and TG levels: There was no significant difference between the mild and blank control groups ( P > 0. 05) . After the high diet feeding，the TC and TG were ( 32. 12 ± 1. 25 ) and ( 6. 02 ± 2. 12 ) nmol /L in the severe NAFLD group，(18. 34 ± 2. 10) and (4. 39 ± 1. 93) nmol /L in the mild NAFLD group，respectively，before experiment ( P < 0. 01) ，and those in the severe were also significantly higher than thosein the mild NAFLD group (P < 0. 01) . (2) The NO level in the liver tissue: It was (132. 4 ± 20. 7)μmol / g. pro in the severe，(95. 4 ± 19. 8)μmol / g. pro in the mild NAFLD group，and (74. 9 ± 34. 7) μmol / g. pro in the blank control group，with a very significant difference between the severe and mild NAFLD groups，and between the two NAFLD and the blank control groups. (3) The H2S level in the liver tissue of the severe and mild NAFLD groups was significantly decreased，compared with that in the blank control group ( P < 0. 01) ，and between the severe and mild NAFLD groups ( P < 0. 05) . (4) Liver pathology: Moderate changes were seen in the severe NAFLD group，slight to moderate changes in the mild NAFLD group，and normal histological appearance in the liver tissue of the blank control group. Conclusions NO and H2S participate the pathogenesis of nonalcoholic fatty liver disease. It is possible that intervention of the NO and H2S may become another future direction in prevention of this disease.

Abstract:Objective To get expression of Bordetella bronchiseptic recombinant protein DNT1 and establish an indirect ELISA for detection of recombinant protein DNT1. Method According to a pair of specific primers designed to amplify DNT gene of Bordetella bronchiseptica in swines promulgated by Genbank，the fragment of the target gene was amplified by PCR and then constructed with prokaryotic expression vector PET-28a ( + ) . The recombinant expression plasmids were transfected into BL21(DE3) strain. The optimal concentration of coated antigen recombinant protein DNT1 and serum dilution was determined to develop the ELISA technique. Results DNT1 gene of Bordetella bronchiseptica was successfully cloned and expressed. The SDS-PAGE and Western blot analysis unfolded the excellent immunogenicity of the recombinant proteins which were used as coating antigens to develop the ELISA method for Bb-specific antibody diagnosis.The peridium consistency of the recombinant protein DNT1 determined in this experiment was 6. 25 μg /mL，and the optimal testing serum dilution was 1:100. Conclusion The development of DNT1 indirect ELISA offers a simple and practical way for monitoring antibody of Bb，and provides much information for laying a basis for development of a Bb diagnosis kit.

Abstract:Objective To construct fused gene Ag85B-Esat6-HspX of Mycobacterium tuberculosis and investigate its expression in eukaryotic cells in vitro. Methods The gene encoding Ag85B，ESAT6 and HspX protein was amplified by PCR from genome of Mycobacterium tuberculosis H37Rv strain，and was inserted into cloning vector pUC19-T. After the sequence was confirmed，the fused gene was subcloned to eukaryotic expression vector pcDNA3. 1( － ) . After identified by restriction enzymec digestion and verified by DNA sequencing again，the recombinant plasmid pcDNA-Ag85B-Esat6-HspX was transfected into 293T cells with MegaTran 1. 0. Western blot was used to detect the expression of goal protein. Result A 65 kDa protein was detected with specific antibodies. Conclusion The eukaryotic expression vector pcDNAAg85B-Esat6-HspX has been constructed successfully and the fusion protein could be expressed specifically in 293T cells.

Abstract:Objective To study the possible influence of transportation stress on some physiological parameters in Beagle dogs based on noninvasive telemetry measurement. Methods Sixteen Beagle dogs were randomly divided into two groups ( n = 8 in each) : control group and transportation stress group. 24 h consecutive monitoring electrocardiogram (ECG) ，activity，skin temperature and respiratory parameters were recorded in the control group and after transportation stress for 4 h in transportation group，from conscious and unrestrained beagle dogs using a noninvasive telemetry system.Results Heart rate，RR interval，QT interval，activity，skin temperature and respiration had significant circadian rhythm changes (P < 0. 01 ) . Compared with the control group，heart rate，activity，respiratory frequency，minute volume and tidal volume were significantly increased in the transportation stress group (P < 0. 01) ，while RR interval，PR interval and skin temperature were significantly decreased ( P < 0. 01 ) . Correlation analysis showed that there was remarkable correlation between transportation stress and heart rate，activity，skin temperature and respiratory frequency (P < 0. 05，P< 0. 01) . Conclusions Transportation stress for 4 h may cause significant changes of physiological parameters in Beagle dogs，but except for skin temperature，it does not impair the circadian rhythm change in Beagle dogs. Application of noninvasive telemetry technology can be useed to establish an ideal living model of Beagle dogs，and it is helpful in researches of animal welfare and pharmacology and toxicology.

Abstract:Objective To measure the body weight and weight of major organs in wild adult tree shrews，and calculate the organ coefficient. Methods Sixty wild adult tree shrews (35 male and 25 female) were chosen，and the body weight and the weight of 11 organs were determined，then the organ coefficients were calculated. Finally the data were statistically analyzed. Results Between female and male tree shrews，the heart and lung weights showed a very significant difference (P < 0. 01) and the difference of weight of brain，adrenal gland and pancreas was significant ( P < 0. 05) ，the coefficient of the adrenal and pancreas had very significant difference ( P < 0. 01 ) and the difference of heart，lung and kidney was significant (P < 0. 05) . The growth of major organs of animal community was in a good harmony determined through the analysis of Kendall coefficients. Conclusions Significant sex differences exist in the weights of heart，lung，brain，adrenal gland，pancreas，and coefficients of adrenal gland，pancreas，heart，lung，kidney between female and male wild adult tree shrews.

Abstract:Objective To determine the values of anatomical parameters of HBK-SPF ducks at age of 7 and 56 days old and compare the differences between the male and female ducks. Methods The values of anatomical parameters of 7- and 56-day-old HBK-SPF ducks were measured and the results were analyzed by t-test. Results There was a very significant difference between the left and right kidney values (P < 0. 01) ，and a significant difference of shank girth and cecum 2 values (P < 0. 05) between male and female ducks at the age of 7 days. At the age of 56 days，a very significant difference existed only of the thymus values (P < 0. 01) ，and a significant difference of the cecum 1 and rectum values (P< 0. 05) between the male and female ducks. Conclusion The values of some anatomical parameters are significantly different between male and female HBK-SPF ducks at age of 7 and 56 days.

Abstract:Objective To analyze the performance of Beagle dogs bred in two kinds of breeding houses. Methods Performance of Beagle dogs bred in different breeding houses ( house Ⅰ and house Ⅱ) such as litter situation，birth weight，survival rate and body weight of weaning dogs 60-days old， and weight gain per month was compared.Results The birth weights of Beagle dogs bred in house Ⅰ and house Ⅱ were statistically not significant different( P >0. 05) ，while the survival rate of weaning dogs 60 days old bred in house Ⅱ was 18. 4% higher than that in house Ⅰ (P< 0. 01) and the weight of weaning dogs 60-day old bred in house Ⅱ was 0. 62 ± 0. 13 kg higher than that in house Ⅰ (P< 0. 01) ，as well as weight gain per month of the dogs bred in house Ⅱ was 0. 29 ± 0. 14 kg higher than that in house Ⅰ(P = 0. 08 > 0. 05) . Conclusion The House Ⅱ is better than house Ⅰ.

Abstract:Under the impulse of society need and scientific progress，3R principles and alternative methods is on the way shifted from humane conception to experimental technology. Seeing from the point of view of the global science development，confront the current statues and future prospect of alternative methods in China，the consensus platform of Chinese Center for Alternatives Research and Evaluation ( CCARE) was established using MSSQLServer2000，to control the information database and connect the CCARE with internet WWW ( WorldWide Web) by WEB technology，which constructed the dynamic information platform and internet search system. The platform of CCARE provided a convenience resource for techniques engaging in alternative research，and building a bridge between overseas and Chinese researchers.