Fresh rat corneas as well as corneas preserved in several different corneal preservation media were stained with Avidin-Biotin-peroxidase Complex method in order to evaluate major histocompatibility complex (MHC) antigen expression. In fresh corneas, class I antigen was identified in corneal epithelium, stroma and endothelium. Class II antigen was identified only in stroma. In corneas preserved in the media which contained chondroitin and dextran for 7 days, class I antigen was somewhat decreased but class II antigen was increased. In corneas preserved in the medium which contained insulin or epidermal growth factor for 7 days, class II antigens seemed to be increased compaired to the fresh cornea. Expression of MHC antigens of corneas in the medium with fetal bovine serum were similar to those of fresh corneas.
Animals ; Cornea/*metabolism ; Culture Media ; Histocompatibility Antigens Class I/*biosynthesis ; Histocompatibility Antigens Class II/*biosynthesis ; Immunoenzyme Techniques ; Major Histocompatibility Complex ; Organ Preservation/methods ; Rats ; Rats, Inbred Lew

Chlamydia ; immunology ; pathogenicity ; physiology ; Histocompatibility Antigens Class I ; biosynthesis ; immunology ; Humans ; Immune Tolerance ; immunology ; Muramidase ; immunology

No abstract available.
Cytokines/pharmacology* ; Gene Expression Regulation ; Genes, MHC Class I* ; Histocompatibility Antigens Class I/biosynthesis* ; Interferons/pharmacology ; Lymphotoxin/pharmacology ; Models, Genetic ; Signal Transduction ; Tumor Necrosis Factor/pharmacology

To obtain large quantity of human leukocyte antigen F (HLA-F) and cluster of differentiation 8alpha homodimers (CD8alphaalpha) proteins and to study their relationship, HLA-F and CD8alpha genes with rare codon in Escherichia coli were cloned using an N-terminal synonymous mutation method. High-efficiency expression protein inclusion bodies were acquired. The proteins were refolded using the dilution method and purified with gel-filtration and anion exchange chromatography. The results of gel-filtration and native-PAGE indicate that HLA-F interacts with CD8alphaalpha. This interaction may affect the binding between CD8alphaalpha and other MHC molecules to regulate immune responses. These results provide a basis for further research of HLA-F.
CD8 Antigens ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Histocompatibility Antigens Class I ; biosynthesis ; genetics ; Humans ; Mutation ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

CD8-Positive T-Lymphocytes ; immunology ; Cell Transplantation ; adverse effects ; Female ; Graft Rejection ; immunology ; prevention & control ; Hepatocytes ; transplantation ; Histocompatibility Antigens Class I ; biosynthesis ; Histocompatibility Antigens Class II ; biosynthesis ; Humans ; Liver Failure ; surgery ; Male

Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.
Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Histocompatibility Antigens Class I ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; beta 2-Microglobulin ; biosynthesis ; genetics

Rhesus macaque (Macaca mulatta) is the best model to study of human immunodeficiency virus (HIV) infection and to develop acquired immunodeficiency syndrome (AIDS) vaccine. The crystal structure of its major histocompatibility antigen complex (MHC) is helpful to understand the mechanism of HIV immune evasion. In this study, we cloned the light chain (beta2m) of MHC class I allele of rhesus macaques, Mamu-A*02, and inserted it into pET21a(+) vector. We transfected the recombinant plasmid pET21a(+)-Mamu-beta2m and pET21a(+)-Mamu-alpha into BL21(DE3). Mamu-A*02 and beta2m were expressed in the form of inclusion bodies in BL21 (DE3). We co-refolded the inclusion bodies of Mamu-alpha and Mamu-beta2m with SIV nonapeptide YY9 and obtained the correct refolded protein complex. Then we purified the protein complex by the gel filtration and anion-exchange column. With hanging-drop method, we screened and optimized for the protein crystal. We managed to collect a X-ray diffraction with the resolution to 2.8 angstroms in the condition of 0.1 mol/L BIS-TRIS (pH5.5), 2.0 mol/L(NH4)2SO4. This crystal belong to perpendicular space group P2(1)2(1)2(1), with unit-cell parameters a = 128.99 angstroms, b = 129.01 angstroms, c = 129.03 angstroms. This data is available for the structure determination.
Animals ; Crystallography, X-Ray ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Histocompatibility Antigens Class I ; biosynthesis ; genetics ; Macaca mulatta ; Oligopeptides ; biosynthesis ; genetics ; Simian Immunodeficiency Virus ; immunology

OBJECTIVEThe aim of this study was to investigate the mRNA expression levels of human leukocyte antigen Class I at different progressive stages of human oral squamous cell carcinomas.

METHODSThe expression of mRNA of human leukocyte antigen--B/C was detected in 23 primary tumors, 10 metastatic focuses and 11 histological normal oral epithelia using in situ hybridization method with a digoxigenin--labeled DNA probe. The probe was human leukocyte antigen--B/C locus specific.

RESULTSThe hybridization signals were present in the cytoplasm of either normal epithelia or tumor cells. The integrated optical density values of the hybridization signals were detected with the aid of an image analysis system. The results showed that the average integrated optical density values of the primary tumors were statistically lower than the normal oral epithelia (P < 0.05), but there was no significant difference between metastatic tumors and the primary tumors or the normal epithelia. The integrated optical density values measured in the metastatic tumors also did not show statistically differences compared with the primary tumors of the same patients.

CONCLUSIONImpaired regulation of human leukocyte antigen--B/C transcription could occur but might not be directly associated with metastasis of oral squamous cell carcinomas.

Carcinoma, Squamous Cell ; genetics ; immunology ; HLA-B Antigens ; biosynthesis ; genetics ; HLA-C Antigens ; biosynthesis ; genetics ; Histocompatibility Antigens Class I ; biosynthesis ; genetics ; Humans ; In Situ Hybridization ; Mouth Neoplasms ; genetics ; immunology ; RNA, Messenger ; biosynthesis ; genetics ; Transcription, Genetic

OBJECTIVESTo study the relationship between the mutation of Leu60Val in HBV core region and the cellular immunity in patients with chronic hepatitis B (CHB).

METHODSHBV DNA C gene mutation was confirmed by polymerase chain reaction (PCR) and sequencing the products directly. The cytokines (IFN-gamma, TNF-alpha and IL-2) levels in serum were measured by enzyme linked immunosorbent assay (ELISA). The distribution of T-lymphocyte subpopulations in peripheral blood was detected by flow cytometry (FCM).

RESULTSThe mutation of Leu60Val was found in 19 out of the 91 CHB patients. With the CHB severity, the mutation rate was getting higher, especially in the severe hepatitis group. The IFN-gamma and TNF-alpha levels were much higher in mutant strain group than those in wild strain group (t=2.584, 4.766, P<0.01), so was the ratio of CD4+/CD8+ (t=2.275, P<0.05).

CONCLUSIONThe mutant strain of 60Val may increase affinity to HLA-I molecule, or up-regulate the expression of HLA-I molecule, resulting in the activation of CTL to release the cytokines and cause immune response in liver.

Adult ; Aged ; CD4-CD8 Ratio ; Flow Cytometry ; Hepatitis B Core Antigens ; genetics ; Hepatitis B, Chronic ; immunology ; virology ; Histocompatibility Antigens Class I ; metabolism ; Humans ; Interferon-gamma ; biosynthesis ; Middle Aged ; Mutation ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis

Tumor immunity is primarily mediated by cells as CD8+ cytotoxic T lymphocytes (CTL) recognize tumor antigen by MHC class I molecules. But most tumors are associated with a decreased expression of MHC class I to escape the antitumor immunity of the host. Our previous data have demonstrated that MPL has an antitumor effect on metastatic lung cancer of B16 melanoma with enhancing cytotoxicity due to increase of IFN-gamma and IL-2, and decrease of IL-4, which indicates the stimulation of type 1 helper T cells (Th1). To determine the effects of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha on MHC class I expression of B16 melanoma cells, we evaluated the expression of MHC class I molecules with treatments of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha by flow cytometry. The supernatant of MPL-treated spleen cells in vitro upregulated the expression of MHC class I molecules of B16 melanoma cells compared to the control supernatant of spleen cells. The MHC class I expression of B16 melanoma cells treated with IFN-gamma, but not TNF-alpha or IL-1 alpha, increased in a time-dependent manner. In conclusion, MPL upregulated MHC class I expression of B16 melanoma cells by activating spleen cells via IFN-gamma. These data suggest that increased IFN-gamma by MPL is responsible for the upregulation of MHC class I expression to augment cytotoxicity. Therefore, we suggest that MPL could play an important role in immunotherapy.
Adjuvants, Immunologic/pharmacology* ; Animal ; Histocompatibility Antigens Class I/biosynthesis* ; Interferon Type II/pharmacology ; Interferon Type II/biosynthesis* ; Lipid A/pharmacology ; Lipid A/analogs & derivatives* ; Mice ; Mice, Inbred C57BL ; Tumor Cells, Cultured ; Up-Regulation (Physiology)