OBJECTIVETo study the significance of Fas and Fas-L expression in adenocarcinoma of uterine cervix.

METHODSBoth carcinoma tissue and their surrounding tissues from 36 patients with adenocarcinoma of uterine cervix, previously untreated either by radiation or chemotherapy, were studied for the expression of Fas and Fas-L by immunohistochemical stain with DNA apoptosis fragment detected by TUNEL.

RESULTSThe TUNEL labeling index was negatively correlated with differentiation of adenocarcinoma of cervix. Compared to highly differentiated and moderately differentiated tumor, the TUNEL labeling index was reduced obviously in poorly differentiated adenocarcinoma (P < 0.01). Fas expression was detected in 31 cases (86%) while there were only 3 weakly stained in the normal endocervical glands around the carcinoma. The 5 unstained carcinomas were 3 highly differentiated and 2 moderately differentiated. The positively stained Fas was associated with differentiation; the stronger the stain, the less differentiation there was. The Fas-L expression was detected in all adenocarcinomas while there was only 1 weakly stained in the normal ones. No significant difference was found in the expression of Fas-L in carcinomas with different degrees of differentiation. No correlation was observed between Fas and Fas-L expression.

CONCLUSIONSThe Fas expression is positively correlated with the different degrees of differentiation and Fas-L expression may be associated with the escape from of immunal surveillance.

Adenocarcinoma ; diagnosis ; metabolism ; Apoptosis ; physiology ; Biomarkers, Tumor ; biosynthesis ; Cell Differentiation ; physiology ; Fas Ligand Protein ; Female ; Humans ; Immunohistochemistry ; Membrane Glycoproteins ; biosynthesis ; Uterine Cervical Neoplasms ; diagnosis ; metabolism ; fas Receptor ; biosynthesis

The possibility of immunotherapy for lymphoma by single FasL-Fas way was investigated. After pBillneo-mFasL was transformed into competent E. coli DH5alpha and amplified, the plasmid DNA was prepared and purified from the DH5alpha. To determine the primary structure and inserting direction of mFasL cDNA gene in pBillneo-mFasL, the plasmid DNA was cleaved by restriction enzyme, and the mFasL cDNA of pBillneo-mFasL was amplified by polymerase chain reaction (PCR), the DNA sequence of the PCR product was analysed by automatic DNA sequencing. After pBillneo-mFasL was transfected into COS-7 cells by liposome, the COS-7 cells were selected with G418 selective medium, and the expressing levels of mFasL cDNA on the COS-7 cell membrane was assayed by Western Blot. After the COS-7 cells higher expressing mFasL protein and mouse lymphoma cell line Yac-1 expressing Fas were cocultured for 5 hours, the suspending Yac-1 cells were collected and labeled by annexin V/PI kit. The apoptosis rate of the Yac-1 cells was tested by flow cytometry. The EcoRI cleaving products of pBillneo-mF asL included 920 bp and 7227 bp fragments. Its Hind III cleaving products included 1293 bp and 6807 bp fragments. These results showed: (1) the length of DNA sequence containing mFasL cDNA within pBillneo-mFasL is the same as theoretical length; (2) the inserting of mFasL cDNA in pBillneo-mFasL was in positive orientation. The expected 890 bp DNA fragments of mFasL cDNA (from ATG to +36 bp following TAA) emerged in PCR product with pBillneo-mFasL as a template. The sequencing result of the PCR product equaled the known mFasL cDNA sequence in the gene bank. The COS-7 cells transfected by pBillneo-mFasL and selected with G418 culture medium expressed more mFasL membrane protein assayed by Western Blot. After the COS-7 cells were cocultured with Fas(+) Yac-1 cells in different E:T ratios (1:1, 5:1 and 10:1) for 5 hours, the apoptosis rates of Yac-1 cells were (22 +/- 4.8)%, (32.18 +/- 7.8)%, and (51.8 +/- 5.4)%, respectively. These were obviously different from the control group (P < 0.01), in which the COS-7 cell was transfected by pBillneo (not carrying mFasL gene). It was concluded that lymphoma cells highly expressing Fas can be effectively killed through single Fas-FasL way in vitro.
Animals ; Apoptosis ; COS Cells ; Fas Ligand Protein ; Immunotherapy ; Lymphoma ; pathology ; therapy ; Membrane Glycoproteins ; genetics ; physiology ; Mice ; Transfection ; Tumor Cells, Cultured ; fas Receptor ; analysis

The purpose of this study was to investigate the expression of Fas, Fas ligand (FasL) and CD80 and function of FasL on the surface of acute myelomonocytic leukemia cells from WEHI-3 line. The expression of Fas, FasL and CD80 on the surface of WEHI-3 were detected by flow cytometry, the apoptosis of YAC-1 cell induced by FasL on the surface of WEHI-3 were detected by (3)H-TdR incorporation. The results showed that the expression rate of Fas, FasL and CD80 on the surface of WEHI-3 cells were (6.75 +/- 2.31)% (n = 5), (63.73 +/- 5.23)% (n = 5) and (5.06 +/- 0.41)% (n = 5) respectively. The apoptosis rate of YAC-1 cells (target cells) co-cultured with WEHI-3 cells (Effector cells) at the rate of 1:3, 1:10 and 1:30 were (26 +/- 4.5)%, (35 +/- 3.2)% and (43 +/- 2.7)% (n = 5) respectively. It is concluded that WEHI-3 cells have high expression of FasL and low expression of Fas and CD80 on their cell membrane, and can induce the apoptosis of Fas(+) YAC-1 cells.
Apoptosis ; physiology ; B7-1 Antigen ; biosynthesis ; Cell Membrane ; metabolism ; Fas Ligand Protein ; biosynthesis ; Humans ; Leukemia, Myelomonocytic, Acute ; metabolism ; pathology ; Tumor Cells, Cultured ; fas Receptor ; biosynthesis

OBJECTIVETo determine the involvement of FasL/Fas pathway in apoptosis of J774A.1 cells induced by Leptospira interrogans.

METHODSThe cell infection model was established with mouse monocyte-macrophage J774A.1 cells infected by L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601. The morphological characteristics of apoptotic J774A.1 cells were observed by DAPI staining method, and the apoptosis rate was quantitatively determined by flow cytometry. FasL neutralizing antibody was applied to block the apoptosis. Expression of FasL or Fas in the L.interrogans strain 56601-infected J774A.1 cells was detected by flow cytometry using PE-conjugated monoclonal antibody.

RESULTChromatin condensation and marginalization were found in J774A.1 cells infected by L.interrogans strain 56601 for 4 h, which became more predominant for 24 h and karyorrhexis was present in some cells. When J774A.1 cells were infected for 4 h and 24 h, the apoptosis rates were 53.6% and 64.31%, respectively. However, the apoptosis rates were decreased to 10.27% and 15.9% after the cells were pre-treated with FasL neutralizing antibody. When J774A.1 cells were infected for 4 h and 24 h, FasL expression rates were increased to 21.69% and 65.70% from that of 4.19% before infection, and Fas expression rates were risen to 91.96% and 88.01% from that of 12.88% before infection.

CONCLUSIONInducement of cell apoptosis is an important mechanism of L.interrogans strain 56601 injuring J774A.1 cells. The strain of L.interrogans is able to up-regulate FasL/Fas expression levels of host cells and induce apoptosis of the cells via FasL/Fas pathway.

Animals ; Apoptosis ; physiology ; Cell Line ; Fas Ligand Protein ; metabolism ; Leptospira interrogans ; pathogenicity ; Macrophages ; microbiology ; pathology ; Mice ; Up-Regulation ; fas Receptor ; metabolism

At angle of cell apoptosis, the excessive less of hematopeitic cell apoptosis is a reason of hematopoietic cell accumulation. The Fas/FasL system as an important pathway inducing cell apoptosis participates in occurrence and development of leukemia. Leukemia cells generally are not sensitive or are resistant to Fas/FasL-mediated apoptosis, while it is one of important reasons resulting in immunoescape and unsensitivity of leukemia cells to chemotherapy. In recent years studies related to mechanisms of leukemia cell resistance to Fas/FasL-mediated apoptosis such as Fas and FasL mutation and expression abnormality, Fas signaling transduction pathway abnormality, and regulatory affect of apoptotic regulatory genes on Fas/FasL system, as well as strategies replying to antiapoptosis of leukemia cells including NF-kappab, XIAP, membrane receptor CD28 and matrix metalloproteinase 7 obtained some progresses. The above-mentioned issues were reviewed in this article.
Apoptosis ; physiology ; CD28 Antigens ; metabolism ; Fas Ligand Protein ; physiology ; Humans ; Leukemia ; pathology ; Matrix Metalloproteinase 7 ; metabolism ; NF-kappa B ; metabolism ; Tumor Cells, Cultured ; X-Linked Inhibitor of Apoptosis Protein ; metabolism ; fas Receptor ; physiology

To investigate the value of apoptosis of the allo-antigen specific T cells induced by Fas/FasL pathway in order to prevent GVHD in allo-transplant, the CD34(+) cells were transfected with FasL or not, used as effector cells, mixed with allo-antigen specific T lymphocytes with presence or absence of IFN-gamma or IL-2. After 5 days, apoptosis of T cells was detected by TdT nick end mediated dUTP labeling (TUNEL) and flow cytometry. The effects of IFN-gamma or IL-2 on apoptosis of CD34(+) cells of graft induced by Fas/FasL pathway observed as controls. The apoptosis incidence of T cells was (12.1 +/- 1.5)% when CD34(+) cells transfected with FasL were used as effector cells, that was much higher than that T cells with CD34(+) cells non-transfected (p < 0.01). In the presence of IFN-gamma or IL-2, apoptosis incidence reached to (20.1 +/- 2.3)% or (17.6 +/- 1.3)% respectively (p < 0.01). When sFasL was added to CD34(+) cells freshly isolated or induced with IFN-gamma or IL-2, the incidence or apoptosis of CD34(+) cells was (7.8 +/- 0.8)%, (18.7 +/- 1.6)% (p < 0.01) or (7.9 +/- 1.0)% (P > 0.05) respectively. The results suggest that it is possible to induce apoptosis of the allo-antigen specific T cells in grafts activated by allo-antigen by exogenous Fas ligand expressed on receptor cells and that may hopefully provide a new method to prevent GVHD
Animals ; Antigens, CD34 ; analysis ; Apoptosis ; Cell Communication ; physiology ; DNA, Complementary ; genetics ; Fas Ligand Protein ; Humans ; Interferon-gamma ; pharmacology ; Membrane Glycoproteins ; genetics ; physiology ; Mice ; T-Lymphocytes ; cytology ; physiology ; Transfection ; fas Receptor ; biosynthesis

OBJECTIVETo analyze the significance of Fas-FasL in NOD insulitis and to explore the mechanism of the autoimmune diabetes.

METHODSThirty-two female NOD mice, 3-32 weeks of age, were selected. The blood glucose concentrations were recorded. The pathological data were obtained from the HE staining of the pancreatic sections and the immunohistochemical staining, in which insulin, Fas, FasL, CD8 were detected.

RESULTSDiabetes was found from the age of 14 weeks. In normal islets, insulin + cells accounted for (59.37 +/- 1.21)%, and some islet cells were observed expressing Fas. At the age of 6 weeks, insulitis lesions could be found. The average score of insulitis tended to rise with the increasing age (P < 0.0005). Meanwhile, insulin + cells decreased (P < 0.0005), and correlated negatively with scoring (P < 0.05). Fas+ islet cells increased (P < 0.0005), correlated positively with scoring (P < 0.01). In insulitis lesions, islet cells expressed FasL that increased gradually (P < 0.0005) and correlated positively with scoring (P < 0.01). The infiltrating cells were all Fas negative. But these mononucleated cells showed the expression of FasL and CD8, both increasing gradually (P < 0.0005). Furthermore, there was certain correlation between the expression of some antigens: in islet cells, between Fas and insulin (negative, P < 0.01), insulin and FasL (negative, P < 0.01), and Fas and FasL (positive, P < 0.01). In the infiltrating cells, the expression of CD8 was correlated with FasL (positively, P < 0.01); it was also found that there was a negative correlation between Fas+ islet cells and CD8+ mononucleated cells (P < 0.05).

CONCLUSIONTo sum up, there may be some important and complicated effects by Fas-FasL on the damage of beta cells and the regulation of autoreactive T cells in NOD insulitis, which will facilitate further studies in human type 1 diabetes.

Animals ; Autoimmune Diseases ; etiology ; immunology ; metabolism ; Diabetes Mellitus, Experimental ; etiology ; immunology ; Fas Ligand Protein ; Female ; Inflammation ; immunology ; metabolism ; Insulin ; blood ; Islets of Langerhans ; immunology ; metabolism ; Membrane Glycoproteins ; physiology ; Mice ; Mice, Inbred NOD ; fas Receptor ; physiology

Animals ; Apoptosis ; Blood Glucose ; analysis ; CD8 Antigens ; analysis ; Diabetes Mellitus, Type 1 ; pathology ; Fas Ligand Protein ; Female ; Insulin ; analysis ; Islets of Langerhans ; pathology ; Membrane Glycoproteins ; physiology ; Mice ; Mice, Inbred NOD ; fas Receptor ; physiology

The aim of study was to explore the effects of NF-kappaB inhibitor Bay 11 - 7082 on Fas/FasL system and Fas-mediated apoptosis in HL-60 cells. The mRNA and protein expression levels of Fas, FasL and XIAP after treatment with Bay 11 - 7082 were detected by RT-PCR and FCM respectively. The level of sFasL was detected by ELISA before and after treatment with Bay 11 - 7082; apoptosis was detected by FCM before and after treatment with Bay 11 - 7082. The results showed that after treating HL-60 cells with Bay 11 - 7082, the mRNA and protein levels of FasL and XIAP were lower than that of controls, the difference was significant by statistic analysis (p < 0.05). Neither the mRNA and protein levels of Fas, nor the level of sFasL changed significantly (p > 0.05). Apoptotic rate of HL-60 cells treated with Bay 11 - 7082 was significantly higher as compared with controls (p < 0.05). It is concluded that Bay 11 - 7082 can enhance Fas-mediated apoptosis in HL-60 cells by down-regulation of FasL and XIAP levels.
Apoptosis ; drug effects ; Down-Regulation ; Fas Ligand Protein ; physiology ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; NF-kappa B ; antagonists & inhibitors ; Nitriles ; pharmacology ; RNA, Messenger ; metabolism ; Sulfones ; pharmacology ; X-Linked Inhibitor of Apoptosis Protein ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the immune privilege induced by the Fas ligand (FasL) expressed by cotransplanted testicular Sertoli cells in islet allografts, and the effect of FasL gene transfection on islet cells in pancreatic islet allografts.

METHODSAllogeneic islets and testicular cells were cotransplanted into diabetic recipients. Pancreatic islets were infected with the recombinant adenovirus, AdV-FasL, and transplanted into diabetic recipients. Allograft survival, islet function, apoptosis of infiltrative lymphocytes in allografts and gene transfected islet allografts were analyzed.

RESULTSAll animals receiving islet allograft alone returned to a diabetic state in a few days (mean survival time 6.3 +/- 0.6 days). When the quantity of testicular cells cotransplanted with islets increased to 1 x 10(7), all animals remained normoglycemic throughout the follow-up period (60 days). FasL expression by cotransplanted Sertoli cells induced apoptosis of activated lymphocytes. Rejection of allografts in the FasL gene transfer group was accelerated and allograft survival was shortened to 3.4 +/- 0.2 days (P < 0.05). Pancreatic islets infected with AdV-FasL demonstrated positive staining for FasL at 24h after transplantation, with increased intensity at 48h. Apoptosis assays of pancreatic islet allografts at 24h and 48h revealed apoptosis of transfected islets.

CONCLUSIONSFasL-expressing testicular Sertoli cells can induce apoptosis of activated lymphocytes. Cotransplantation of testicular cells allows long-term survival of allogeneic islets because of immune privilege, but the direct expression of FasL on islet allografts infected with AdV-FasL accelerates islet rejection via islet apoptosis and granulocyte infiltration.

Animals ; Apoptosis ; Fas Ligand Protein ; Immunohistochemistry ; Islets of Langerhans Transplantation ; mortality ; Male ; Membrane Glycoproteins ; genetics ; physiology ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Transplantation, Homologous