OBJECTIVETo investigate the expressions of CD1a and CD83 of Langerhans cells (LC) in the lesions of epidermodysplasia verruciformis (EV) patients.

METHODSWe used immunohistochemical method to detect the expressions of CD1a and CD83 in the lesions of 10 patients with EV lesions and in the skins of 10 normal subjects.

RESULTSNo CD83 + LCs was detected in all EV patients and normal controls, but CD1a + LC was found in all cases. The quantity of CD1a + LCs in the lesions of EV patients was significantly lower than that in the normal skin (P < 0.01); furthermore, the distribution of LCs in EV lesions was uneven.

CONCLUSIONThe functions of LCs may be inhibited in EV patients.

Antigens, CD ; biosynthesis ; genetics ; Antigens, CD1 ; biosynthesis ; genetics ; Epidermodysplasia Verruciformis ; immunology ; pathology ; Humans ; Langerhans Cells ; immunology ; Leukocyte Immunoglobulin-like Receptor B1 ; Receptors, Immunologic ; biosynthesis ; genetics ; Skin ; immunology ; pathology

Animals ; Antigens, CD ; biosynthesis ; genetics ; immunology ; B7-H1 Antigen ; CD28 Antigens ; biosynthesis ; genetics ; Humans ; Immune Tolerance ; Liver ; immunology ; RNA, Messenger ; biosynthesis ; genetics ; T-Lymphocytes ; immunology

To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.
Antigens, CD ; biosynthesis ; genetics ; immunology ; Antigens, Differentiation ; biosynthesis ; genetics ; immunology ; CTLA-4 Antigen ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Adult ; Aged ; Antigens, CD ; biosynthesis ; genetics ; B7-H1 Antigen ; CD28 Antigens ; biosynthesis ; genetics ; Female ; Hepatitis B, Chronic ; immunology ; Humans ; Leukocytes, Mononuclear ; immunology ; Male ; Middle Aged

OBJECTIVETo study the major histocompatibility complex class II (MHC II) antigen and costimulatory molecules expression on the surface of breast cancer cells.

METHODSMHC II antigen and costimulatory molecule expression on five breast cancer cell lines including MCF-7, SK-BR-3, T47D, MDA-MB-435 and ZR-75-30 were detected through flow cytometery analysis, with their expression level compared with that of normal mammary cell line HBL-100.

RESULTSThe MHC II expression level of the five breast cancer cell lines were significantly different from that of HBL-100 (P < 0.05). MHC II antigen expression of MCF-7 cells which was about 20 percent of HBL-100 was the lowest. MDA-MB-435 and ZR-75-30 cell expression levels were twice as much as that of HBL-100, with the fluorescence intensity of MDA-MB-435 the highest of all cells. CD40 molecule expression on the surface of MDA-MB-435 cells was the lowest, which was nearly ten percent of that of MCF-7 and HBL-100 cells. CD80 and CD86 molecule expression showed no difference in MDA-MB-435 or HBL-100 cell (P > 0.05), and those of the other four breast cancer cells were lower than that of HBL-100 (P < 0.05).

CONCLUSIONMHC II antigen and costimulatory molecule expression on the surface of breast cancer cells is abnormal, with different molecule expression in different cells. Breast cancer cells can escape immune surveillance through abnormal molecule expression.

Antigens, CD ; biosynthesis ; B7-1 Antigen ; biosynthesis ; B7-2 Antigen ; Breast Neoplasms ; immunology ; CD40 Antigens ; biosynthesis ; Cell Membrane ; immunology ; Female ; Histocompatibility Antigens Class II ; biosynthesis ; Humans ; Membrane Glycoproteins ; biosynthesis ; Tumor Cells, Cultured

Neutrophils are also known to acquire the characteristics of dendritic cells (DCs) under the appropriate conditions. In this study, neutrophils were cultivated in vitro in the presence or absence of compounds modulating their survival in an attempt to characterize the expression profile of the DC markers. Higher MHC-II, CD80, CD86, CD83, and CD40 expression levels were detected on the surface of the cultured neutrophils for 24 h than on the freshly isolated cells. The annexin V-positive cells showed a higher expression level of the DC markers than the annexin V-negative cells. The population of neutrophils double stained with annexin V and the DC markers increased after being incubated with agonistic anti-Fas Ab. LPS, the anti-apoptotic compound, decreased the CD86 and MHC-II expression levels but 50-60% of the DC marker-positive cells were detected in the annexin V-positive cells. In contrast, CD80, CD86, CD83, and HLA-DR mRNA levels increased in the GM-CSF-treated neutrophils but not in the anti-Fas Ab-treated neutrophils. T cell proliferation was inhibited by co-culturing them with anti-Fas Ab- or LPS-treated neutrophils at a high neutrophil:T cell ratio. However, the superantigen-mediated T cell proliferation was increased by the LPS-treated neutrophils but decreased by the anti-Fas Ab-treated neutrophils. There was a lower level of interferon-gamma production in the T cells co-cultured with anti-Fas Ab-treated neutrophils than with the LPS-treated neutrophils. This suggests that apoptotic neutrophils express DC markers on their surface and the differential expression of DC markers might have a detrimental effect on the immune reaction.
Antigen Presentation ; Antigens, CD/biosynthesis ; Antigens, CD95/pharmacology ; Antigens, Differentiation/*biosynthesis ; *Apoptosis ; Cells, Cultured ; Dendritic Cells/*metabolism ; Humans ; Lipopolysaccharides/pharmacology ; Lymphocyte Activation ; Neutrophils/*metabolism/physiology ; T-Lymphocytes/immunology

Animals ; Antigens, CD ; immunology ; Cytokines ; metabolism ; Hepacivirus ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Hepatitis C Antibodies ; biosynthesis ; immunology ; Hepatitis C Antigens ; immunology ; Humans ; Killer Cells, Natural ; immunology ; T-Lymphocytes ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; metabolism ; Viral Proteins ; immunology

It is known that CD28, a positive costimulatory receptor, plays a very important role in inducing the optimal stimulation of T lymphocytes. CTLA-4 (CD152), however, acts as a negative regulator in T lymphocyte activation. The effect of an feline immunodeficiency virus (FIV) infection on the expression of feline CD28 and CTLA-4 was studied with FIV-infected and uninfected peripheral blood mononuclear cells (PBMC) using a competitive PCR assay. The nature of CD28 and CTLA-4 expression was also examined with fresh and antigen-stimulated PBMC. FIV infection induced a lower expression of CD28, but a higher expression of CTLA-4 in the infected PBMC than in the uninfected PBMC. Relatively high levels of CD28 expression were demonstrated in both the fresh and the antigen-stimulated PBMC. The expression level of CTLA-4 in the freshly isolated PBMC was rather low, however, FIV antigen stimulation induced a relatively high expression of CTLA-4 in feline PBMC.
Animals ; Antigens, CD ; Antigens, CD28/*biosynthesis ; Antigens, Differentiation/*biosynthesis ; Antigens, Viral/*immunology ; Cats ; Cell Survival ; Cells, Cultured ; Gene Expression ; Immunodeficiency Virus, Feline/immunology/*physiology ; Leukocytes, Mononuclear/immunology/*virology ; Polymerase Chain Reaction/veterinary

To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.
AC133 Antigen ; Animals ; Antibodies ; genetics ; Antigens, CD ; immunology ; Antigens, CD34 ; immunology ; Base Sequence ; Cadherins ; immunology ; Female ; Glycoproteins ; immunology ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Library ; Peptides ; immunology

OBJECTIVETo construct and express human CD96 gene outer membrane domain (hCD96om) in prokaryotic cells and prepare rabbit polyclonal antibody of hCD96om.

METHODShCD96om was amplified by RT-PCR from the peripheral blood of patients with acute myeloid leukemia and inserted into prokaryotic expression vector pET32a(+) to construct the recombinant plasmid pET32-CD96. The expression of hCD96om was induced by IPTG in BL21(DE3) cells, and the expression product was identified by Western blotting. The anti-hCD96 polyclonal antibody was prepared by immunization of rabbits with the fusion protein. The specificity of anti-hCD96 antibody was determined by Western blotting.

RESULTShCD96om protein was expressed in E.coli BL21(DE3) cells in the form of inclusion body, with a relative molecular mass around 37 kD. Western blotting showed a specific reaction of the prepared antiserum with the 70 kD protein extracted from human leukemia cell line HL-60 cells and with the 37 kD hCD96om fusion protein.

CONCLUSIONThe CD96 gene of human has been successfully cloned and expressed in BL21(DE3) cells, and its rabbit polyclonal antibody has been obtained.

Animals ; Antibodies ; immunology ; metabolism ; Antigens, CD ; biosynthesis ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immune Sera ; biosynthesis ; Immunization ; Leukemia, Myeloid, Acute ; immunology ; Molecular Sequence Data ; Neoplastic Stem Cells ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology