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

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

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

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)

In order to compare the prognoses of patients with diffuse malignant lymphomas on the basis of histology and immunophenotypes, we retrospectively studied 62 cases of diffuse lymphoma arising in lymph nodes. We also evaluated the reactivity patterns of monoclonal antibodies (MoAb) LN1, LN2 and LN3 to determine the criteria for making a differential diagnosis in B cell lymphomas. The immunologic phenotypes were determined by the avidin biotin peroxidase complex method, using frozen or paraffin fixed tissues. The majority (66.3%) were B cell with the remaining 20.9% being T cell and 12.9% were non-B, non-T cell lineage. Immunological heterogeneity was found especially in the mixed small and large cell and the immunoblastic lymphomas. There was no significant difference between B- and T-cell lymphomas with respect to survival and death (P > 0.05). Histologically 79% (49/62) of the lymphoma was large cell and 21% (13/62), small cell lymphoma. There was a difference in prognosis between low, intermediate and high-grade of lymphomas. However there were no significant differences among the subtypes of the diffuse aggressive lymphomas. Factors associated with poor prognosis were advanced stages (P < 0.025) and histology of the malignant lymphomas. MoAb LN1, LN2 and LN3 gave positive staining in 83.3%, 91.7% and 60% of B cell lymphomas, respectively. The most common phenotypic pattern in B cell lymphomas was LN1+, LN2+, LN3+/-, suggestive of follicular center cell origin. As a panel, phenotypic patterns of MoAb LN1, LN2 and LN3 may be useful in differentiation of follicular center cell lymphoma from others.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD/biosynthesis ; Antigens, Differentiation, B-Lymphocyte/biosynthesis ; Child ; Female ; Follow-Up Studies ; Histocompatibility Antigens Class II/biosynthesis ; Humans ; Immunoenzyme Techniques ; Immunophenotyping ; Lymphoma, B-Cell/immunology ; Lymphoma, Non-Hodgkin/immunology/*pathology ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Sialyltransferases/biosynthesis

OBJECTIVETo study the mechanism of macrophage injury after trauma-hemorrhagic shock.

METHODSWistar male rats underwent trauma (closed bone fracture) and hemorrhage (mean arterial blood pressure of 35 mm Hg+/-5 mm Hg for 60 minutes, following fluid resuscitation). Rats without trauma, hemorrhage or fluid resuscitation served as controls. Peritoneal macrophages were harvested at 6 hours and 1, 2, 3, 7 days after traumatic hemorrhagic shock to determine the effects of pertussis toxin (PTX, as a specific inhibitor to Gi(alpha) and cholera toxin (CTX, as a stimulant to Gs(alpha) on macrophage-Ia expression and TNF-alpha production and levels of Gi(alpha) and Gs(alpha).

RESULTSThe macrophages from the injured rats revealed a significant decrease of Ia positive number and TNF-alpha release in response to LPS. Wi th pretreatment with PTX 10-100 ng/ml Ia positive cells and LPS-induced TNFalpha production in both control and impaired macrophages populations were dos e dependently increased. Both macrophages populations were not responding to CTX treatment (10-100 ng/ml). Western blot analyses showed that the levels of Gi(alpha) protein expression increased as much as 116.5%-148.8% of the control level fro m 6 hours through 7 days after traumatic hemorrhage. The levels of Gs protein expression were reduced at 6 hours and decreased to the lowest degree; 36% o f the control at day 1, began to return at day 2 and returned to the normal level at day 7, following traumatic hemorrhagic shock.

CONCLUSIONSPTX-sensitive G-protein may participate in th e modulation of macrophage-Ia expression and TNF-alpha release following traumatic hemorrhagic shock. Analyses of the alteration of Gi(alpha) and Gs protein express ions further supports the concept that G-protein is involved in trauma-induced macrophage signal transduction pathways.

Analysis of Variance ; Animals ; GTP-Binding Proteins ; immunology ; metabolism ; Histocompatibility Antigens Class II ; immunology ; Immunoblotting ; Lipopolysaccharides ; pharmacology ; Macrophages, Peritoneal ; immunology ; metabolism ; Male ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; blood ; immunology ; Signal Transduction ; Tumor Necrosis Factor-alpha ; biosynthesis

The heart transplantation-associated accelerated graft arteriosclerosis (AGAS) is one of the major causes of cardiac allograft failure. We investigated the early time-course of expresssion patterns of cytokines, transcription factor, and its inhibitor in the intraabdominally transplanted mice hearts that differed only in the D locus of class I histocompatibility antigen. The allograft hearts were harvested at 1-3, 5, 7, 14, 28, and 42 days after the transplantation, and the expressions of NF-kappaB/I-kappaB and cytokines (TNF-alpha , INF-gamma) were examined in these specimens. The expressions of TNF-alpha and INF-gamma were observed on day 1, peaking on day 5 and 7, respectively. Activated NF-kappaB (p65) expression was present on the cytoplasm and perinuclear area in the endothelial cells of coronary arteries on day 1. The peak of translocation of NF-B from cytoplasm to nucleus appeared on day 5 in the endothelial cells, myocytes, and leukocytes within the vessels, and remained elevated until day 42. The I-kappaB expression gradually increased from day 1 until day 5, but a remarkable decrease was detected on day 7. Our data suggest that the increased expressions of NF-kappaB/I-kappaB and cytokines (TNF-alpha, INF-gamma) play an important role in inducing immune responses in the donor allograft heart and hence the blockage of the expressions might be mandatory to avoid a potential graft failure.
Animal ; Chronic Disease ; Coronary Arteriosclerosis/etiology/*metabolism ; Cytokines/*biosynthesis ; *Graft Rejection ; *Heart Transplantation ; Histocompatibility Antigens Class I/analysis ; Intercellular Adhesion Molecule-1/biosynthesis ; Interferon Type II/biosynthesis ; Mice ; NF-kappa B/*biosynthesis ; Transplantation, Homologous ; Tumor Necrosis Factor/biosynthesis ; Vascular Cell Adhesion Molecule-1/biosynthesis

OBJECTIVETo investigate whether there is an association between the expression of B7-H1 in HBV transgenic mice and the immune tolerance to HBV.

METHODST cells stimulatory capacity of DC was analyzed using mixed lymphocyte reaction. Expression of MHC-II, CD80, CD86, B7-H1 on DC was detected by Flow Cytometry. IL-2, IFNgamma, IL-10 production of T cells were determined by using ELISA. B7-H1 mRNA and protein expression in liver tissue were detected by RT-PCR and Western blotting respectively.

RESULTSThe ability of DC cells from HBV transgenic mice to stimulate T cell proliferation was significantly impaired compared with DC cells from control mice (t = 16.674, 19.674, 21.712, P less than 0.01). Expression of MHC-II, CD80 on DC was markedly decreased in transgenic mice (t = 7.910, 6.413, P less than 0.05). Meanwhile, the expression of CD86 and B7-H1 on DC cells in HBV transgenic mice were not significantly different from that in control mice. The levels of IL-2, IFNgamma, IL-10 in supernatant of T cells was significantly lower compared with controls (t = 18.712, 18.712 and 11.683, P less than 0.05). There was no significant difference in B7-H1 expression at mRNA and protein levels in liver tissue compared with controls.

CONCLUSIONSFunctional defect of DC, partly due to decreased expression of MHC-II, CD80, but not related to B7-H1 expression, is the cause for immune tolerance to HBV in HBV transgenic mice.

Animals ; Antigens, CD ; biosynthesis ; genetics ; Cell Proliferation ; Cytokines ; biosynthesis ; Dendritic Cells ; immunology ; metabolism ; Flow Cytometry ; Hepatitis B virus ; genetics ; immunology ; Histocompatibility Antigens Class II ; metabolism ; Immune Tolerance ; Liver ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA, Messenger ; genetics ; metabolism ; Spleen ; immunology ; metabolism ; T-Lymphocytes ; immunology ; metabolism

OBJECTIVETo investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.

METHODSBy using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.

RESULTSThe results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.

CONCLUSIONMIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).

Antigens, Differentiation, B-Lymphocyte ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Matrix ; metabolism ; Histocompatibility Antigens Class II ; genetics ; metabolism ; Macrophage Migration-Inhibitory Factors ; genetics ; metabolism ; Peptide Fragments ; genetics ; Protein Interaction Domains and Motifs ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Two-Hybrid System Techniques

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.
Animals ; Antigens, CD/immunology/metabolism ; Bacterial Outer Membrane Proteins/*pharmacology ; Cells, Cultured ; Female ; Histocompatibility Antigens Class II/immunology/metabolism ; Host-Pathogen Interactions ; Interleukin-12/biosynthesis ; Interleukin-6/biosynthesis ; Legionella pneumophila/*immunology/metabolism ; Legionnaires' Disease/immunology/metabolism ; Lipoproteins/*pharmacology ; Macrophage Activation/drug effects/immunology ; Macrophages, Peritoneal/drug effects/immunology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Toll-Like Receptor 2/*metabolism ; Tumor Necrosis Factor-alpha/biosynthesis