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

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

OBJECTIVETo investigate the effect of mitogen Phorbol 12-myristate 13-Acetate (PMA) on CD3, CD4 and CD8 expression of human T-lymphocytes.

METHODSPeripheral blood mononuclear cells from 37 blood samples stimulated in vitro with PMA at different concentrations (2,5,10,20 and 50 ng/ml for 4 hours) and time (10 ng/ml for 2,4 and 6 hours) were analyzed by 4-color flow cytometry (FCM).

RESULTSUnder different PMA stimulation protocols,significant CD4 down-regulation was observed,which was negatively correlated with intracellular cytokine secretion (r= 0.601,P<.001), except for PMA stimulation at 10 ng/ml for 2-hours which showed no significant intracellular cytokine secretion. The expressions of CD3 and CD8 molecules after PMA activation were not significantly affected as compared with pre-activation. Among CD3 positive T lymphocytes, CD4/CD8 double-negative cells only account for 5.52%.

CONCLUSIONPMA has a significant down-regulation effect on CD4 molecules of Th cells, without altering the CD3 and CD8 expression. For quantitative analysis of Th1/Th2 variation, indirect method such as CD3(+)CD8( ) T cells can be used to define CD4(+) Th cells stimulated by PMA in the future.

CD3 Complex ; blood ; CD4 Antigens ; blood ; CD8 Antigens ; blood ; Child ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; chemistry ; drug effects ; Tetradecanoylphorbol Acetate ; pharmacology ; Th1 Cells ; immunology ; Th2 Cells ; immunology

CD8-Positive T-Lymphocytes ; metabolism ; Case-Control Studies ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Interferon-gamma ; biosynthesis ; Lymphocytes ; immunology ; Oligodeoxyribonucleotides ; pharmacology

OBJECTIVETo study the influence of percutaneous microwave ablation (PMA) and surgical resection for patients with small primary hepatocellular carcinoma (PHC) on dissemination of tumor cells in peripheral blood determined by AFP mRNA.

METHODSForty patients with small PHC (The maximal diameter < or = 5 cm) confirmed histologically were included in this study. All the patients had single tumor nodule only without metastasis. Of the 40 patients, 19 were treated by PMA and 21 by surgical resection. Blood samples were collected and tested immediately before treatment, 30 min after the mass ablated/resected, 1 d and 7 d later by RTD-Nested-RT-PCR for AFP mRNA. The CD3, CD4, CD8 and CD4/CD8 in blood, and hepatic function were tested at the same time points as well.

RESULTSAfter treatment, ALT and AST in peripheral blood increased in both groups, but more intensely in the surgical group. The CD3, CD4 and CD4/CD8 in peripheral blood decreased at 30 min, 1 day and 7 days after surgical resection, and the lowest value was at 30 min after surgery. The immune function was kept at the same level as pre-treatment in the PMA group. AFP mRNA copies in blood could be detected in 27 of 40 patients (67.5%) in two groups before treatment, and the copy number was increased after treatment. There was no significant difference between the two groups. The patients were followed up for 1 - 16 months. AFP mRNA copies in blood could be detected persistently in the 4 patients with extrahepatic metastasis or liver recurrence.

CONCLUSIONSurgical resection and microwave ablation may cause PHC cells dissemination into the blood circulation in patients with small PHC, and there was no difference between the two treatment groups. The cellular immune function in peripheral blood is decreased after surgical resection, but is maintained at the same level as pre-treatment in the PMA group. The impairment of liver function is less severe after PMA treatment than surgical resection. PMA may provide certain value for clinical management of small hepatocellular carcinoma.

Adult ; Aged ; CD3 Complex ; blood ; CD4 Antigens ; blood ; CD4-CD8 Ratio ; CD8 Antigens ; blood ; Carcinoma, Hepatocellular ; blood ; surgery ; therapy ; Catheter Ablation ; methods ; Female ; Follow-Up Studies ; Hepatectomy ; Humans ; Liver Neoplasms ; blood ; surgery ; therapy ; Male ; Microwaves ; therapeutic use ; Middle Aged ; Neoplasm Recurrence, Local ; RNA, Messenger ; biosynthesis ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

Severe chronic active Epstein-Barr virus (EBV) infection is a rare and life-threatening illness. Although the criteria for diagnosis include chronic or recurrent infectious mononucleosis-like symptoms lasting more than 6 months and high titers of anti-EBV antibodies, clinical and laboratory findings may be heterogeneous and flexible application of those criteria is necessary in cases showing typical clinical and pathologic findings. We report a case of severe chronic active EBV infection in a 62-yr-old female patient who showed classical clinical findings with infiltration of EBV-infected T lymphocytes in the bone marrow, spleen, and lymph nodes, and died four months after presentation.
Antigens, CD3/biosynthesis ; Antigens, CD4/biosynthesis ; Antigens, CD8/biosynthesis ; Bone Marrow Cells/virology ; Epstein-Barr Virus Infections/*diagnosis/*mortality ; Female ; Herpesvirus 4, Human/genetics ; Human ; Immunohistochemistry ; Lymph Nodes/virology ; Lymphocytes/metabolism ; Middle Aged ; Organ Weight ; Spleen/pathology/virology ; Support, Non-U.S. Gov't ; T-Lymphocytes/virology

The study was to investigate the biological characteristics of hyperleucocyte acute leukemia (HAL) and its clinical significance. Immunophenotyping was performed in 48 HAL patients and 73 NHAL patients by three-color flow cytometry analysis using CD45/SSC gating, meanwhile the cytogenetic analysis was performed in 74 patients. The results showed that as compared with NHAL group, HAL group had lower proportion of eryth-lineage in bone marrow (P < 0.05); in AML, the CD14 expression of HAL group was apparently higher than that of NHAL group (P < 0.05); in ALL, HAL group had higher expression of CD8 and lower expression of CD22, cCD79a compared with NHAL group (P < 0.05); the two groups had no significant difference in expression of special lineage antigens and overlapping lineage antigens (P > 0.05). The CR rate of HAL group was lower than that of NHAL group. It is concluded that bone marrow inhibition of HAL group is more severe than that of NHAL group. In AML, monocytic leukemia is easier to become into HAL than other leukemias. In ALL, T-lineage antigens of HAL group are more easily expressed than those of NHAL group; the leukemia cells of HAL group are naiver than those of NHAL group, meanwhile the prognosis of HAL is poor.
Adolescent ; Adult ; Aged ; CD79 Antigens ; biosynthesis ; genetics ; CD8 Antigens ; biosynthesis ; genetics ; Child ; Child, Preschool ; Female ; Humans ; Immunophenotyping ; Leukemia, Myeloid, Acute ; drug therapy ; mortality ; pathology ; Leukocyte Count ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; mortality ; pathology ; Prognosis ; Sialic Acid Binding Ig-like Lectin 2 ; biosynthesis ; genetics

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

OBJECTIVETo study the efficacy and explore the mechanism of the anti-tumor immunity elicited by heat shock protein 70-peptide complexes (HSP70-PC) derived from tumor cells.

METHODSCells culture, flow cytometric analysis, affinity chromatography for protein purification, SDS-PAGE, Western-blotting and animal experiment were used.

RESULTSHSP70-PC immunization rendered protective effect to both naive tumorl-bearing mice. All of the naive mice obtained complete resistance to Hcaf cell attack; 40% of the tumor-bearing mice survived for over 90 days, whereas the mice of control group died within 2 weeks (P < 0.01). CD8+ subset of T lymphocytes in the peripheral blood of immunized mice increased by 12%.

CONCLUSIONHSP70-PC induces anti-tumor immunity via activation of cytotoxic T lymphocytes (CTLs), and it possesses strong tumor vaccine effect. Our research adds more evidence to support the clinical use of HSP70-PC to fight human cancers.

Animals ; CD8 Antigens ; blood ; Cell Membrane ; chemistry ; HSP70 Heat-Shock Proteins ; biosynthesis ; isolation & purification ; therapeutic use ; Liver Neoplasms, Experimental ; chemistry ; drug therapy ; immunology ; pathology ; Mice ; Neoplasm Transplantation ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured