OBJECTIVETo confirm the relations between the expression of cyclin E, p16ink4, ki67 and HPV16/18 infection using cervical exfoliated cells, and evaluate the usefulness of cyclin E, p16ink4 and ki67 as biomarkers for screening of cervical carcinomas.

METHODSThe expression of cyclin E, p16ink4 oncoproteins and ki67 proliferative activity was evaluated immunohistochemically in 78 cervical exfoliated epithelial specimens. Human papillomavirus type16 and 18 (HPV16/18) infection was assessed by polymerase chain reaction (PCR) using type specific primers.

RESULTSCyclin E, p16ink4 and ki67 were all overexpressed in cervical preneoplasia and neoplasia cells, compared with little expressed in ASCUS (P less than 0.005). Overexpression of cyclin E was observed in CIN, (P less than 0.01), p16ink4 and ki67 overexpressed in invasive carcinoma(100 percent and 90.9 percent respectively). The degree of p16ink4 and ki67 expression correlated well with the degree of cervical neoplasia (P less than 0.005). HPV16 infection was assessed at all stages of cervical neoplasia samples, and a significant relationship with the degree of cervical epithelial lession was observed at the same time. The expression level of p16ink4 and ki67 seemed to be more closely associated with HPV16 infection than cyclin E did (rs=1.0 vs rs=0.4). HPV18 was found positive in only 1 case in CIN1 and in 4 cases in CIN2-3. Therefore no significance was found on statistical analysis (P less than 0.005).

CONCLUSIONCyclin E, p16ink4 and ki67 should be regarded as useful biomarkers of HPV-related cervical neoplasias, and be used for screening patients at high risk for developing cervical carcinomas. Moreover, cyclin E might be a significant cytologic marker for the primary screening of cervical carcinomas.

Adult ; Aged ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Cervix Uteri ; cytology ; metabolism ; virology ; Cyclin E ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; DNA, Viral ; genetics ; Female ; Host-Pathogen Interactions ; Human papillomavirus 16 ; genetics ; physiology ; Human papillomavirus 18 ; genetics ; physiology ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; biosynthesis ; Middle Aged ; Papillomavirus Infections ; metabolism ; pathology ; virology ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the specific anti-tumor immunity induced by gene immunization with ectopic hCG encoding gene.

METHODSBALB/c mice were immunized with plasmid TR421-hCGbeta coding for hCGbeta and mock DNA for 3 times at 3 weekly intervals. The level of specific anti-hCGbeta IgG antibody in the serum was determined by ELISA at the indicated time in the two groups. The growth inhibitory activity of the sera against tumor cells was examined in vitro by [(3)H]-Thymidine incorporation assay. Specific lympho-proliferation versus hCGbeta was detected by [(3)H]-Thymidine incorporation assay with hCGbeta protein or inactivated SP2/0-hCGbeta cells as specific stimulating antigen. Cytotoxic T lymphocyte (CTL) activity of the splenocytes derived from the immunized mice was measured by [(3)H]-Thymidine release assay. Protective assay was performed by subcutaneous inoculation of SP2/0-hCGbeta cells into the immunized mice. The weight and formation rate of the tumor were evaluated after challenge.

RESULTSAll mice immunized with plasmid TR421-hCGbeta developed high level of anti-hCGbeta antibodies, which could inhibit the growth of Hela cells and SP2/0-hCGbeta cells compared with the serum from animals immunized with mock DNA (P < 0.05). The high-level specific lympho-proliferation against hCGbeta protein or/and inactivated SP2/0-hCGbeta cells were shown in TR421-hCGbeta immunized mice, whereas no significant proliferative activity was found in mock DNA immunized animals (P < 0.01). A strong cytotoxic activity against SP2/0-hCGbeta in TR421-hCGbeta immunized mice was found. Inoculation of SP2/0-hCGbeta cells into the mice immunized with mock DNA developed large tumors within 25 days. But a marked reduction of tumor weight and formation rate was found after the tumor cells challenge in the mice immunized with TR421-hCGbeta plasmid DNA (P < 0.01).

CONCLUSIONThe gene immunization of ectopic hCGbeta encoding gene, eliciting high-level of specific humoral and cellular immune responses, could inhibit the growth of tumor cells harboring ectopic hCGbeta in vitro and in vivo.

Animals ; Antibody Formation ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chorionic Gonadotropin, beta Subunit, Human ; immunology ; Cytotoxicity, Immunologic ; Female ; Genetic Therapy ; HeLa Cells ; drug effects ; Humans ; Immune Sera ; pharmacology ; Immunization ; Mice ; Mice, Inbred BALB C ; Multiple Myeloma ; pathology ; prevention & control ; Neoplasm Transplantation ; Plasmids ; T-Lymphocytes, Cytotoxic ; immunology

SIP24/24p3 is a secreted murine acute phase protein which has been speculated to play an anti-inflammatory role in vivo. Recently SIP24/24p3 has been found to be able to specifically induce apoptosis in leukocytes. By using (35)S metabolic labeling method, we studied the regulation of SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines IL-6 and TNF-alpha in cultured Balb/c 3T3 and BNL cells. The following results were observed: (1) dexamethasone induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells, the induction was more significant in BNL cells; (2) dexamethasone and IL-6 synergistically induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells; (3) in Balb/c 3T3 cells dexamethasone and TNF-alpha acted synergistically to induce the expression of SIP24/24p3, whereas in BNL cells dexamethasone and TNF-alpha induced the expression of SIP24/24p3 in an additive manner; (4) dexamethasone and IL-6/TNF-alpha acted synergistically in Balb/c 3T3 cells and additively in BNL cells to induce the expression of SIP24/24p3. The inducibility of SIP24/24p3 by multiple factors will help to explain its highly specific expression in vivo. The difference in the expression patterns of SIP24/24p3 in different cell types is also suggestive to its expression and regulation in hepatic and extrahepatic tissues. Finally, the fact that SIP24/24p3 protein can be induced by both pro-inflammatory as well as anti-inflammatory factors is indicative of the important role of SIP24/24p3 in the entire acute phase response process.
Acute-Phase Proteins ; biosynthesis ; genetics ; Animals ; BALB 3T3 Cells ; Carrier Proteins ; biosynthesis ; genetics ; Cytokines ; pharmacology ; Dexamethasone ; pharmacology ; Drug Synergism ; Gene Expression Regulation ; Interleukin-6 ; pharmacology ; Lipocalin-2 ; Lipocalins ; Mice ; Mice, Inbred BALB C ; Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

Objective To study the clinical significance of PI3Kp110αexpression in breast cancer. Methods The expressions of PI3Kp110α, HER2, PTEN, p?Akt and Ki?67 in invasive ductal carcinoma ( IDC ) , adjacent ductal carcinoma in situ ( DCIS ) and normal breast tissue were detected by immunohistochemistry in 102 cases of breast cancer. The expression of PI3Kp110α in IDC was compared with those in DCIS and normal breast tissues. Correlations between expression of PI3Kp110αand expression of HER2, PTEN, p?Akt and Ki?67 index in IDC were analyzed. Correlation between expression of PI3Kp110αand stage of IDC was studied as well. Results In the normal breast tissues, there were 97 cases (95.1%) with low level and 5 cases (4.9%) with high level expression of PI3Kp110α. In the DCIS tissues, there were 67 cases ( 65. 7%) with low level and 35 cases ( 34. 3%) with high level expression of PI3Kp110α. In the IDC tissues, there were 14 cases (13.7%) with low level and 88 cases (86.3%) with high level expression of PI3Kp110α. The difference between expression of PI3Kp110α in normal breast tissue, DCIS and IDC was significant ( P<0. 001 ) . In the IDC tissues, expression of PI3Kp110α was negatively correlated with expression of HER2 ( rs=-0.213,P=0.032) and PTEN ( rs=-0.197,P=0.047) , while was not significantly correlated with expression of p?Akt ( P=0.119) and Ki?67 index ( P=0.636) . In contrast, expressions of HER?2 and p?Akt were positively correlated with Ki?67 index ( P=0. 001, P=0.035), while expression of HER2 was not correlated with p?Akt (P=0.177). Expression of PI3Kp110αwas negatively correlated with T stage and TNM stage (P=0.003, P=0.016), while not correlated with N stage and M stage(both P>0.05). Expression of HER?2 was positively correlated with T stage (P=0.037), while not correlated with N stage, M stage and TNM stage ( P>0. 05 for all ) . Neither Ki?67 index nor expression of PTEN and p?Akt (P=0.194) were correlated with T stage, N stage, M stage and TNM stage. Conclusions Expression of PI3Kp110α plays an important role in oncogenesis and development of breast cancer. Based on the fact that expression of PI3Kp110αis negatively correlated with expression of HER2 and PTEN and with T stage and TNM stage, we may conclude that increased expression of PI3Kp110αis another independent pathway in breast cancer development, and breast cancer patients with high expression of PI3Kp110α may have a better prognosis.

Objective To study the clinical significance of PI3Kp110αexpression in breast cancer. Methods The expressions of PI3Kp110α, HER2, PTEN, p?Akt and Ki?67 in invasive ductal carcinoma ( IDC ) , adjacent ductal carcinoma in situ ( DCIS ) and normal breast tissue were detected by immunohistochemistry in 102 cases of breast cancer. The expression of PI3Kp110α in IDC was compared with those in DCIS and normal breast tissues. Correlations between expression of PI3Kp110αand expression of HER2, PTEN, p?Akt and Ki?67 index in IDC were analyzed. Correlation between expression of PI3Kp110αand stage of IDC was studied as well. Results In the normal breast tissues, there were 97 cases (95.1%) with low level and 5 cases (4.9%) with high level expression of PI3Kp110α. In the DCIS tissues, there were 67 cases ( 65. 7%) with low level and 35 cases ( 34. 3%) with high level expression of PI3Kp110α. In the IDC tissues, there were 14 cases (13.7%) with low level and 88 cases (86.3%) with high level expression of PI3Kp110α. The difference between expression of PI3Kp110α in normal breast tissue, DCIS and IDC was significant ( P<0. 001 ) . In the IDC tissues, expression of PI3Kp110α was negatively correlated with expression of HER2 ( rs=-0.213,P=0.032) and PTEN ( rs=-0.197,P=0.047) , while was not significantly correlated with expression of p?Akt ( P=0.119) and Ki?67 index ( P=0.636) . In contrast, expressions of HER?2 and p?Akt were positively correlated with Ki?67 index ( P=0. 001, P=0.035), while expression of HER2 was not correlated with p?Akt (P=0.177). Expression of PI3Kp110αwas negatively correlated with T stage and TNM stage (P=0.003, P=0.016), while not correlated with N stage and M stage(both P>0.05). Expression of HER?2 was positively correlated with T stage (P=0.037), while not correlated with N stage, M stage and TNM stage ( P>0. 05 for all ) . Neither Ki?67 index nor expression of PTEN and p?Akt (P=0.194) were correlated with T stage, N stage, M stage and TNM stage. Conclusions Expression of PI3Kp110α plays an important role in oncogenesis and development of breast cancer. Based on the fact that expression of PI3Kp110αis negatively correlated with expression of HER2 and PTEN and with T stage and TNM stage, we may conclude that increased expression of PI3Kp110αis another independent pathway in breast cancer development, and breast cancer patients with high expression of PI3Kp110α may have a better prognosis.

To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.
Animals ; Bacteriophage T7 ; genetics ; Cell Line ; DNA-Directed RNA Polymerases ; genetics ; Gene Targeting ; Promoter Regions, Genetic ; genetics ; Viral Proteins ; genetics

T cell anergy has been successfully induced under different conditions in cloned CD4(+) T cells, but induction of T cell anergy in vivo has been difficult and controversial. Due to the low frequency of naturally occurring T cell population with specificity to a defined antigen, it is very difficult to study anergy of naïve T cells without prior in vivo priming which complicates the interpretation of experimental data. To solve this problem, we adopted the HNT-TCR transgenic mice which have homogeneous antigen specific CD4(+) T cell population. In this study, we generated an influenza virus hemagglutinin (HA) peptide-specific CD4(+) T cell clone from the HNT-TCR transgenic mice and induced anergy using APCs which were treated with the crosslinker, ECDI (1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide). The proliferative response of the cloned or freshly purified naïve CD4(+) transgenic T cells after treatment with ECDI-treated APCs and the HA peptide antigen was monitored as the index of anergy induction. The results showed that anergy was successfully induced in the cloned HNT-TCR transgenic CD4(+) T cells. It was determined that the induced anergy was antigen- and MHC-specific. By contrast, anergy was not observed in freshly purified naïve CD4(+) transgenic T cells under the same conditions. The results suggest that naïve CD4(+) T cells may have different anergy inducing requirements, or that cloned CD4(+) T cells may have certain priming or in vitro cloning artifact which makes them more susceptible to anergy induction. We propose that induction of T cell anergy may depend on the T cell growth, activation and differentiation state or cloning conditions. The results from the present study may have important implications for the study of the mechanism(s) underlying T cell anergy induction in vivo and for applications of immune tolerance based therapy.
Animals ; Antigen-Presenting Cells ; immunology ; metabolism ; Antigens, CD ; genetics ; immunology ; metabolism ; CD4 Antigens ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; Clonal Anergy ; genetics ; immunology ; Clone Cells ; immunology ; Epitopes, T-Lymphocyte ; biosynthesis ; Immune Tolerance ; genetics ; Major Histocompatibility Complex ; immunology ; Mice ; Mice, Transgenic ; Receptors, Antigen, T-Cell ; physiology

Objective To understand the current situation of potential exposure to rabies among the rural habitants in Hunan province,and to study the impact related to familial factors on post rabies exposure vaccination.Methods In total,40 villages from 20 townships of 4 counties were selected by multistage sampling method.Study samples were selected from these villagers and familial basic information and vaccination post rabies exposures were recorded through questionnaires.Data were statistically analyzed by SPSS 17.0.Results Among 3042 villagers from 864 households being surveyed,124 person-time exposures were found from January,2009 to October,2010,with a total exposure rate as 4.08％,and the annual average exposure rate as 2.33％.Data from univariante analysis showed that the rates on post rabies exposure vaccination were statistically correlated with the following four factors:knowledge on the score of rabies prevention (x2 =8.260,P =0.042),whether being involved in the new type of rural cooperative medical care (P =0.035),family disposable cash income in the year of 2009 (x2 =10.831,P =0.031),distance between the households and the health facilities in towns and townships (x2 =9.071,P=0.033).Results from logistic regression analysis indicated that the score of knowledge on rabies prevention (OR=1.420,95％CI:1.055-1.905) and the annual disposable cash income of the family in 2009 (OR=1.480,95％ CI:1.044-2.098) were independent factors that influencing the rabies vaccination.Conclusion Strengthening the education programs on rabies prevention in rural habitants and increasing family income were feasible way to increase the rate of rabies vaccination in rural areas of Hunan province.

OBJECTIVETo investigate the pathophysiological role of CXCL16 in immunological liver injury induced by Bacille de Calmette et Guerin (BCG) and lipopolysaccharides (LPS).

METHODSImmunological liver injury was induced by BCG and LPS in mice, and the expression of CXCL16 was detected in the liver tissues by real-time quantitative PCR and immunohistochemical examination. The relationship of the expression of CXCL16 and the extent of hepatic necrosis was investigated histopathologically and immunohistochemically. Mononuclear cells were isolated from the liver tissues and their numbers were counted; T lymphocytes populations in the liver tissue were also analyzed with FACS.

RESULTSThe immunological liver injury model was successfully created. Up-regulation of CXCL16 in injured livers correlated with the extent of liver injury and the amountmononuclear cell infiltrations.

CONCLUSIONThese findings suggest that up-regulation of CXCL16 was closely correlated with liver injury extent during the immunological liver injury induced by BCG-LPS in mice, and intrahepatic recruitment of specific lymphocytes might be an important mechanism of liver injury.

Animals ; Chemical and Drug Induced Liver Injury ; Chemokine CXCL16 ; Chemokine CXCL6 ; Chemokines, CXC ; biosynthesis ; genetics ; Lipopolysaccharides ; Liver Diseases ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mycobacterium bovis ; Receptors, Scavenger ; biosynthesis ; genetics

OBJECTIVETo investigate effects of anti-dsDNA autoantibodies on growth of tumor in vitro and in vivo.

METHODSBALB/c mice were inoculated with inactivated tumor cells and challenged s.c. with SP 2/0 and Wehi 164 tumor cells four weeks after the last inoculation. The naïve mice were inoculated with SP 2/0 tumor cells immediately after incubating with sera derived from the immunized mice at week 6. Then the tumor size was examined. In vitro, the cytotoxicity of anti-dsDNA autoantibodies to tumor cells was analysed. Furthermore, apoptosis of SP 2/0 and Wehi 164 tumor cells induced by anti-dsDNA autoantibodies was examined by FACS.

RESULTSIn vivo study showed that the growth of SP 2/0 and Wehi 164 tumors were inhibited in mice with anti-dsDNA autoantibodies, but not in mice lack of anti-dsDNA autoantibodies. In vitro, apoptosis of SP 2/0 and Wehi 164 tumor cells was induced when the tumor cells were incubated with the sera containing anti-dsDNA autoantibodies. Statistical analysis showed that the ability of anti-dsDNA autoantibodies to induce apoptosis of SP 2/0 and Wehi 164 tumor cells was significantly correlated with affinity (r = 0.990, P < 0.01 and r = 0.901, P < 0.05).

CONCLUSIONAnti-dsDNA autoantibodies have inhibitory effect on tumor cells via inducing apoptosis.

Animals ; Antibodies, Neoplasm ; biosynthesis ; immunology ; Apoptosis ; Autoantibodies ; biosynthesis ; immunology ; Cell Line, Tumor ; DNA ; immunology ; Fibrosarcoma ; pathology ; prevention & control ; Immune Sera ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred DBA ; Multiple Myeloma ; pathology ; prevention & control ; Neoplasm Transplantation