B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in tumor immune escape by inducing T-cell apoptosis. In order to investigate the relationship between B7-H1 and immune escape of bladder cancer, B7-H1 expression in 50 cases of bladder cancer was detected by using immunohistochemical method. Survival curves were constructed using the Kaplan-Meier method and independent prognostic factors were evaluated using the Cox regression model. Our results showed that the positive rate of B7-H1 immunostaining in normal bladder tissue and bladder cancer was 0 and 72% respectively. The expression of B7-H1 was strongly associated with the pathological grade, clinical stage and recurrence (P<0.05). The survival rate was significantly lower in patients with B7-H1 positive group than in those with B7-H1 negative group and multi-variable analysis revealed that B7-H1 could be regarded as an independent factor in evaluating the prognosis of bladder cancer. It is concluded that the expression of B7-H1 is strongly associated with neoplastic progression and prognosis of bladder cancer. The manipulation of B7-H1 may become a beneficial target for immunotherapy in human bladder cancer.
Antigens, CD/genetics ; Antigens, CD/*metabolism ; Antigens, CD80/genetics ; Antigens, CD80/*metabolism ; Prognosis ; Tumor Escape/*genetics ; Urinary Bladder Neoplasms/*immunology ; Urinary Bladder Neoplasms/metabolism

Antigens, CD ; genetics ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Tetraspanin 24

OBJECTIVETo explore the role and mechanism of B7 molecules in T cell anergy.

METHODSAnti-B7-1 (CD(80)) and anti-B7-2 (CD(86)) monoclonal antibodies were used to induce T cell anergy. T cell proliferation were assayed by mixed lymphocyte reaction (MLR) with (3)H-TdR incorporation, and cytokine mRNA transcripts were analyzed with reverse transcriptase-polymerase chain reaction (RT-PCR). B7-transfected-CHO cells were used as artificial antigen presentation cells (APCs) in MLR to exclude the effects of other costimulatory molecules.

RESULTSMLR results showed that the proliferation of T cells was inhibited to various extents by anti-CD(80) or anti-CD(86) monoclonal antibody, the effect of anti-CD(86) antibody was greater than that of anti-CD(80) antibody, and the proliferation was totally blocked when the two were used together. The results of RT-PCR demonstrated that IL-2 and IFN-gamma mRNA transcripts decreased whereas IL-4 mRNA transcripts increased in T cell after treatment with anti-B7 antibo-dies for 24 hours. In MLR with artificial APC, signal one (DR7) alone could stimulate T cell proliferation at a certain threshold intensity. Costimulator B7-1 molecule could help signal one in T cell proliferation. This effect was blocked by anti-CD(80).

CONCLUSIONB7 molecules play an important role in T cell immune response. Blockade of B7 family resulted in T cell anergy. The role of CD(86) may be more important than that of CD(80). The conversion of cytokine profile from Th1's to Th2's reflected that anergetic T cells were differentiated into Th2 cells by anti-B7 suggesting that anergetic blockade of costimulator molecules may be one of the mechanisms of T cell.

Animals ; Antigens, CD ; genetics ; B7 Antigens ; B7-1 Antigen ; metabolism ; Cricetulus ; Lymphocyte Activation ; immunology ; Membrane Glycoproteins ; T-Lymphocytes ; immunology

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Swine ; Animals ; Induced Pluripotent Stem Cells/metabolism* ; Receptors, Cell Surface/genetics* ; Antigens, CD/metabolism* ; Porcine respiratory and reproductive syndrome virus/genetics*

Animals ; Antigens, CD ; metabolism ; CHO Cells ; Cricetinae ; Cricetulus ; Endocytosis ; Hepacivirus ; genetics ; pathogenicity ; physiology ; Tetraspanin 28 ; Viral Envelope Proteins ; genetics

OBJECTIVETo evaluate the expression of CD133 and its clinical significance in acute leukemia (AL) patients.

METHODSThe expression of CD133 and CD133 mRNA in leukemic blasts from 76 AL patients were detected by three-color flow-cytometry and hemi-quantitative RT-PCR respectively.

RESULTS(1) CD133 mRNA expression was highly correlated with CD133 expression in both of normal donors and AL patients groups. The expression of CD133 in AL patients was significantly higher than that in control group (P < 0.01). (2) The positive rates of CD133 and CD133 mRNA in AL group were 42.1% (32/76) and 46.1% (35/76) respectively. There was no significant difference in CD133 expression between AML-M(3) and normal control, AML and ALL, as well as T-ALL and B-ALL. The expression of CD133 in AML-M(4) were significantly higher than those in other AML subtypes (81.8% vs 43.7% and 81.8% vs 46.9% at CD133 and CD133 mRNA level, respectively, P < 0.01). (3) The expression of CD133 in AML was significantly correlated with the expression of CD34 and HLA-DR (P < 0.001). (4) The expression of CD133 had no relationship with the clinical prognostic factors such as cytogenetic or molecular aberrations, WBC counts, LDH, mdr1 expression and age. (5) There was a trend toward lower CR rate in CD133(+) cases, but only CD34/CD133(+) double positive cases had significant lower CR rate than that of negative ones (44.4% vs 71.4%, P < 0.05).

CONCLUSIONSAL had significantly higher CD133 expression compared to normal control. The detection of CD133 expression might help to identify AL type and predict therapeutic outcomes. Co-expression of CD133/CD34 might convey adverse prognosis of AL.

AC133 Antigen ; Acute Disease ; Adolescent ; Adult ; Aged ; Antigens, CD ; genetics ; Antigens, CD34 ; genetics ; Child ; Female ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; genetics ; HLA-DR Antigens ; genetics ; Humans ; Leukemia ; genetics ; pathology ; Male ; Middle Aged ; Peptides ; genetics ; Prognosis ; RNA, Messenger ; genetics ; metabolism

CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9 kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9 angstrom resolution and belonged to space group P21, with unit-cell parameters a = 35.1, b = 69.5, c = 49.6A, alpha=gamma=90 degrees, beta=105.4 degrees.
Antigens, CD ; biosynthesis ; genetics ; Crystallization ; Crystallography ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Protein Structure, Tertiary ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo study the expression of B7-H3 mRNA and B7-H3 protein in gastric carcinoma and their clinical significance.

METHODSThe expression of B7-H3 mRNA and B7-H3 protein in gastric carcinoma and the nearby normal tissue of 38 patients was detected by real-time RT-PCR and immunohistochemical assay respectively.

RESULTSB7-H3 mRNA was expressed both in gastric carcinoma and nearby normal tissue, but the expression level in gastric carcinoma was much lower than that in nearby normal tissue. There were no significant differences of B7-H3 mRNA expression among gender, age, histological type, tumor size, lymph node metastasis and invasive depth (all P >0.05). The positive rate of B7-H3 protein expressed in gastric carcinoma was 39.5%. There were no significant differences of B7-H3 protein expression among gender, age, histological type, tumor size, lymph node metastasis and invasive depth (all P >0.05), but there were significant differences among groups of clinical stage (P=0.022) and pathological grade (P=0.039). Kaplan-Meier analysis revealed that disease-free survival or overall survival of the patients with positive B7-H3 expression were significantly longer than those with negative B7-H3 expression (P=0.009 and P=0.010 respectively).

CONCLUSIONDetection of B7-H3 expression in gastric carcinoma will be beneficial to the judgment of the prognosis of gastric carcinoma and the choice of individualized treatment.

Antigens, CD ; genetics ; metabolism ; B7 Antigens ; Biomarkers, Tumor ; genetics ; metabolism ; Cytotoxicity, Immunologic ; Female ; Humans ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; genetics ; Receptors, Immunologic ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology

OBJECTIVETo investigate the mRNA expression of transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) in the full-term placenta from different maternal iron status, and explore the mechanism of placental iron transport and regulation.

METHODSThe mRNA level of TfR1 and IRP1 in full-term placentae was detected by reverse transcription polymerase chain reaction (RT-PCR) in normal group (N), iron deficiency group (ID) and iron deficiency anemia group (IDA).

RESULTS(1) The expression of TfR1 mRNA in N group was 0.4813 +/- 0.1891, in ID group was 0. 6647 +/- 0.2788, and in IDA group was 0.9767 +/- 0.2858. There was significant difference between IDA group and N group or ID group (t = 0.002, P < 0.01 or t = 0.028, P < 0.05), and was no difference between ID group and N group (t = 0.117, P > 0.05). (2) The expression of IRP1 mRNA in N group was 0.2616 +/- 0.0785, in ID group was 0.3696 +/- 0.1801, and in IDA group was 0.3971 +/- 0.0902 and was no difference among the three groups (F = 1.845, P = 0.179).

CONCLUSIONSThe expression of TfR1 mRNA is increased when maternal iron deficiency progressed while there is no change in the expression of IRP1 mRNA in the placentae of TfR1 mRNA indicated that IRP1 takes part in the regulation of placenta iron transport.

Anemia, Iron-Deficiency ; genetics ; Antigens, CD ; metabolism ; Female ; Humans ; Iron Regulatory Protein 1 ; metabolism ; Placenta ; metabolism ; Pregnancy ; RNA, Messenger ; metabolism ; Receptors, Transferrin ; metabolism

OBJECTIVETo investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells.

METHODSThe A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants.

RESULTSSpecific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX.

CONCLUSIONSCTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.

Animals ; Antigens, CD ; genetics ; immunology ; metabolism ; CTLA-4 Antigen ; Cricetinae ; Dental Caries ; prevention & control ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; genetics ; immunology