OBJECTIVETo establish a method for increasing T regulatory cells (Treg) in the graft by using in vivo treatment with dexamethasone (Dex) plus IL-2 and observe its suppressing effect on graft-versus-host-disease (GVHD) in mice.

METHODSAfter treatment of male C57BL/6N mice (donor) with Dex (5 mgxkg(-1)xd(-1)) combined with IL-2 (300 000 IUxmouse(-1)xday(-1)) for three days, spleen mononuclear cells were isolated for flow cytometry analysis of CD4(+)CD25(+) POXP3(+) Treg cells. The allogeneic lymphocytes were transplanted from male C57BL/6N mice to female BALB/c mice aged 8-12 weeks. GVHD and survival time were investigated after transplantation. Donor-derived hematopoiesis reconstituted in recipient mice was detected by Y-chromosome-specific PCR and H-2K(b) by flow cytometry.

RESULTSAdministration of Dex and IL-2 markedly expanded functional CD4(+)CD25(+)FOXP3(+) Treg cells in murine spleen, the number of which in treated group was (24.2 +/- 7.6)% while in control group was (4.0 +/- 0.8)% (P = 0.01). The ratio of Treg to effector T cells (Teff) increased obviously in the treated group (0.43 +/- 0.15 vs 0.14 +/- 0.01, P = 0.01). In a murine allogeneic lymphocyte transplantation model, the grafts from donor with combined treatment of Dex and IL-2 led to a longer survival time than that from the control group (median survival time > 60 d vs 12 d, P = 0.0045), while the mortality rate was decreased (29.4% vs 71.4%, P < 0.05).

CONCLUSIONCostimulation with Dex and IL-2 can selectively expand the functional CD4(+)CD25(+)FOXP3(+) Treg in vivo, which can suppress acute GVHD.

Animals ; Dexamethasone ; Forkhead Transcription Factors ; Graft vs Host Disease ; Humans ; Interleukin-2 ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory

Objective To establish a method of detecting hepatitis B virus x antigen (HBxAg) and antibody to HBxAg (anti-HBx) and to demonstrate its clinical significance of HBxAg and anti-HBx in sera from patients of chronic hepatitis B (CHB),liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Methods Full length HBx gene was cloned into pET30a(+),a prokaryotic expression vector,named pET30a-X.It was transformed into Escherichia coli BL21 (DE3),followed the fusion protein of HBx-His was induced by IPTG.The purified fusion protein was used to immunize rabbit as an antigen to generate polyclonal antibody to HBx protein.The method of enzyme-linked immunosorbent assay (ELISA) was established by using purified fusion protein and generated antibody,which was used to detect HBxAg and anti-HBx in sera from patients of CHB,LC,HCC and normal healthy people.Results The positive rates of HBxAg/anti-HBx were 8.7%/10.4% for CHB,17.9%/40.6% for LC,and 9.8%/34.4% for HCC, respectively.In statistics,the positive rates of anti-HBx in LC and HCC were higher than that in CHB (P

Objective:To analyze the reason which cause the different change of airway appearance after laparoscopic radical resection and transabdominal radical resection for cervical cancer.Methods:Patients with ASAⅠ-Ⅱ,twenty undergoing laparoscopic radical resection of cervical cancer and twenty with transabdominal radical resection of cervical cancer were enrolled.The parameters of Mallampati class,class of upper-lip-bite test,and neck circumference were measured pre-surgery, 1 h after and 24 h after surgery.The correlation between these three parameters and airway pressure or central venous pressure were analyzed.Results:The parameters of Mallampati class,class of upper-lip-bite test,and neck circumference showed no statistical significance between laparoscopic group and transabdominal group at each time.The statistical significances were found between the parameter of Mallampati class (P= 0.001)and class of upper-lip-bite test (P= 0.003)1 h after surgery and before surgery,while no parameters significantly changed between 24 h after surgery and before surgery.The parameter of neck circumference in laparoscopic treatment group statistically changed 1 h ([34.18 ± 2.50 ]cm)and 24 h ([32.98 ± 2.30]cm)after surgery than before surgery ([32.48 ± 2.58 ]cm,P<0.05 ).In transabdominal group,no statistical difference was found in the parameters of Mallampati class ,class of upper-lip-bite test and neck circumference at 1 h or 24 h after surgery.In the group of laparoscopic treatment,Mallampati class 1 h after surgery was positively related to the airway pressure at 1 h and 2 h after beginning of surgery and central venous pressure at 1 h after beginning of surgery(P<0.05). There were also positive correlation of class of upper-lip-bite test 1 h after surgery and airway pressure at 1 h,2 h and 3 h after beginning of surgery and central venous pressure at 2 h after beginning of surgery (P<0.05).Conclusions:Compared with the transabdominal radical resection,in laparoscopic radical resection,there are significant changes of airway at 1 h after surgery, which related to the airway pressure and central venous pressure at the beginning of surgery.

OBJECTIVETo investigate the effect of N-desulfated heparin on tumor metastasis, tumor angiogenesis and basic fibroblast growth factor(bFGF) gene expression of orthotopically implanted human gastric carcinoma in NOD-SCID mice.

METHODSHuman gastric cancer SGC-7901 tissues were orthotopically implanted into the stomach of the NOD-SCID mice. Twenty mice were randomly divided into two groups which received either intravenous injection of 0.9% NaCl solution(0.9%NaCl solution group) or 10 mg/kg N-desulfated heparin (N-desulfated heparin group) twice a week for three weeks. Mice were sacrificed six weeks after tumor implantation. Tissues from stomach and other organs were obtained for histopathological evaluation. The intratumoral microvessel density (MVD) in tumor was evaluated immunohistochemically. Real time PCR was used to detect bFGF mRNA expression.

RESULTSThe tumor metastasis rates were 9/10 in 0.9% NaCl solution group and 2/10 in N-desulfated heparin group(P<0.05).MVD was 9.1+/-3.4 in 0.9% NaCl solution group and 4.7+/-1.8 in N-desulfated heparin group (t=3.617,P<0.05). bFGF mRNA expression was lower in N-desulfated heparin group(2.60+/-0.56%)than that in 0.9% NaCl solution group(30.65+/-6.84%).

CONCLUSIONN-desulfated heparin can inhibit the metastasis of gastric cancer through inhibiting tumor bFGF gene expression and tumor angiogenesis with no obvious anticoagulant activity.

Animals ; Fibroblast Growth Factor 2 ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; genetics ; Heparin ; analogs & derivatives ; pharmacology ; therapeutic use ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Metastasis ; Neoplasm Transplantation ; Neovascularization, Pathologic ; drug therapy ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; metabolism ; Stomach Neoplasms ; blood supply ; drug therapy ; genetics

OBJECTIVERho, a ras homologous gene, encodes a group of GTP-binding proteins. Our previous study suggested that one member of the Rho gene family, RhoC, was related to the progression of human hepatocellular carcinoma (HCC). This study is to elucidate correlation of Rho overexpression with invasion and metastasis of HCC.

METHODSThe expression level of RhoC mRNA and protein in 25 cases of HCC and adjacent non-cancerous liver tissue was examined by RT-PCR and Western blot. Mutation of RhoC gene was examined by PCR-SSCP.

RESULTSThe expression of RhoC mRNA and protein was found in all HCC and adjacent non-cancerous liver tissue. The expression level of RhoC mRNA and protein was significantly higher in tumor tissue than in adjacent non-cancerous liver tissue (1.8 +/- 1.1 vs 1.0 +/- 0.7; 33 992 +/- 10 384 vs 17 342 +/- 9998, P < 0.01). The degree of RhoC overexpression was even more marked in metastatic lesions than in primary tumors (P < 0.01). Overexpression of the rhoC gene was significantly correlated with such clinic-pathological findings as cell differentiation, portal vein invasion, number of primary tumor nodules and metastatic lesions (P < 0.05). Mutation of RhoC gene was found in none of the HCC specimens examined.

CONCLUSIONOverexpression of RhoC gene may play an important role in carcinogenesis and progression of HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Differentiation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Liver ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Invasiveness ; Portal Vein ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; rho GTP-Binding Proteins ; biosynthesis ; genetics ; rhoC GTP-Binding Protein

BACKGROUNDWhite blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review. However, this last step is time consuming and requires experienced personnel. We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials.

METHODSA total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials. WBC differentials of 622 samples were also determined using both a cell counter and FCM. These results were then confirmed using manual microscopic methods.

RESULTSThe probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%. The resulting WBC differentials were well correlated between FCM and the cell counter (r > 0.88, P < 0.001), except in the case of basophils. Neutrophils, lymphocytes, and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r > 0.80, P < 0.001). The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%, respectively) were higher than those of the cell counter method (44.92% and 11.11%, respectively). The specificities of FCM were all above 85%, substantially better than those of the cell counter method.

CONCLUSIONThese five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Leukocyte Count ; methods ; Leukocytes ; cytology ; Male ; Middle Aged ; Young Adult

OBJECTIVETo explore the correlation between the expression of GATA-3 and the level of local cytokines (IL-5, IL-6 and IL-8).

METHODSThe levels of IL-5, IL-6 and IL-8 in ethmoid sinus mucosa were titrated in 45 patients with chronic rhinosinusitis and 11 normal subjects by ELISA. Patients were divided into AR group (with allergic rhinitis) and NAR group (without allergic rhinitis) . Semi-quantitative RT-PCR and immunohistochemical staining were used to examine the GATA-3 expression in nasal mucosa. The correlation between the expression of GATA-3 and the levels of cytokines was evaluated.

RESULTSIL-5, IL-6 and IL-8 levels in both AR and NAR groups were significantly elevated compared with normal group (all P < 0.01 for AR group; P < 0.05, 0.05, 0.01 for NAR group, respectively), and they were much higher in AR group in comparison with NAR group (P < 0.01, 0.05, 0.01, respectively). Semi-quantitative RT-PCR showed that AR and NAR groups had markedly greater level of GATA-3 mRNA than that in control group (P < 0.01, respectively), and the level of GATA-3 mRNA in AR group was further higher than that in NAR group (P < 0.01). Immunohistochemical staining illustrated that GATA-3 was primarily presented in cytoplasma and the GATA-3 positive cells were mainly infiltrating inflammatory cells in submucosa. The mean GATA-3 positive-staining rate was (27. 90 +/- 16.75)% and (10.22 +/- 0.05)% in AR and NAR group, which were markedly higher than (1.30 +/- 1.78)% in control group (P < 0.01, respectively). Pearson correlation analysis demonstrated that GATA-3 positive-staining rate was closely correlated with IL-5 level, but not IL-6 and IL-8. The correlation coefficient was 0. 712 for GATA-3 and IL-5 (P < 0.01), 0.200 for GATA-3 and IL-6 (P > 0.05), 0.089 for GATA-3 and IL-8 (P > 0.05).

CONCLUSIONSActivation of GATA-3 might be one of the mechanisms for induction of IL-5 expression in chronic rhinosinusitis . Concomitance of allergic rhinitis with chronic rhinosinusitis further increased expression of GATA-3, and subsequently enhanced IL-5 expression. Chronic sinusitis may be related to allergy, and GATA-3 may play a key role in the pathogenesis of chronic sinusitis.

Adolescent ; Adult ; Aged ; Chronic Disease ; Female ; GATA3 Transcription Factor ; metabolism ; Humans ; Interleukin-5 ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Male ; Middle Aged ; Nasal Mucosa ; metabolism ; Sinusitis ; metabolism ; Young Adult

OBJECTIVETo evaluate the incidence of precore mutation in HBeAg negative HBV infected patients and the therapeutic effect of the immune therapy (levamisole + HBV vaccine + dipyridamole) on patients chronically infected by HBV with precore mutation.

METHODSThe precore region of HBV from the HBeAg (-) chronic hepatitis patients was sequenced and the patients suffered from HBV with precore mutation were treated with immune therapy.

RESULTSThe precore mutation rate was 10/12. The therapeutic effect of the immune therapy on the precore mutation patients (5/7) was better than that on the HBsAg(+), HBeAg(+) patients (2/11), P less than 0.05.

CONCLUSIONThe precore mutation rate was quite high in the HBsAg(+), HBeAg(-) patients we studied. The immune-therapy has some therapeutic effects on the patients with precore mutation. But the number of cases was too small, further study is needed.

Adolescent ; Adult ; Child ; Combined Modality Therapy ; DNA, Viral ; blood ; Dipyridamole ; therapeutic use ; Hepatitis B Vaccines ; therapeutic use ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; therapy ; virology ; Humans ; Immunotherapy ; Lamivudine ; therapeutic use ; Levamisole ; therapeutic use ; Middle Aged ; Mutation

Objective To develop a rapid test for the detection of F1 antigen of Yersinia Pestis based on gold-immunochromatography.Methods F1 antibodies were coupled with colloidal gold to prepare collidal gold reagent,which was used to detect F1 antibodies based on double antigen sandwich.The collidal gold reagent was estimated for its sensitivity specificity and stablity in labs and 1798 samples were detected in 17 surveillance spots.Results The reagent was sensitive to 0.0010 g/L F1 antigens.The reagens kept stable when it had been placed at 4℃ or room-temperature for 12 months and did not react to Yersinia pseudotuberculosis and Yersinia enterolitica.In 17 surveillance labs the reagent was used to test 1798 viscera samples from animal.resulting an accordance rate of 97.11%(1746/1798)to bacterial culture and 96.83%(1741/1798)accordance to reverse indirect hemagglutination assay(RIHA),showing a higher detection rate[9.23%(166/1798)]compared with RIHA[6.79%(122/1798)]and bacterial culture[6.28%(113/1798)].Conclusions The collidal gold reagent,sensitive and specific in diagnosing Yersinia pestis infection of both human and animals,is a rapid method in surveillance spot.

OBJECTIVETo clone UreB gene of Helicobacter pylori (H. pylori) isolated from children to pGEX-4T-1 expression plasmid, and do sequence analysis.

METHODSA pair of specific primer was designed according to H. pylori UreB gene in the GenBank. Using H. pylori strains isolated from children as a template, a UreB gene was obtained by PCR. After EcoR I and Not I digestion, the PCR production was linked with pGEX-4T-1 which was digested with the same enzymes. The recombinant plasmid was transformed into E.coli BL21 and identified by double enzyme digestion and sequence analysis. The sequence results were compared with the gene sequence in the GenBank.

RESULTSA UreB gene was successfully amplified from children's H. pylori strain GZCH1. It was 1710 bp in size. The objective band was identified by double enzyme digestion. DNA sequence showed that UreB was in the correct open reading frame. The sequence comparison analysis showed that DNA and amino acid sequence identities of UreB gene with other strains were 98%. The sequence of UreB of H. pylori strain GZCH1 was submitted to GenBank (accession number:FJ455126).

CONCLUSIONSUreB of H. pylori strain GZCH1 is successfully cloned to pGEX-4T-1, which provides a basis for research of oral H. pylori vaccine.

Amino Acid Sequence ; Bacterial Vaccines ; immunology ; Child ; Cloning, Molecular ; Helicobacter pylori ; enzymology ; immunology ; Humans ; Male ; Molecular Sequence Data ; Urease ; chemistry ; genetics ; immunology