The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34+ cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF + FL could up-regulate the expression of CD95 in vitro culture and tumor necrosis factor-alpha (TNF-alpha) and interon-gamma (IFN-gamma) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis were analyzed by incubation of CD34+ CB cells in the presence of anti-CD95 monoclonal antibodies (McAbs). The effects of anti-CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU-C assays. A decrease of viable cells, CFU-C and LTIC numbers were observed in the presence of anti-CD95 McAbs and TNF-alpha or IFN-gamma. However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking to CD95 caused low apoptosis of CD34+ cells. The correlation of increased intracytoplasmic levels of bcl-2 and the presence of CD95 on fresh CB CD34+ cells suggested that bcl-2 might be involved in protecting against CD95-mediated apoptosis of CB CD34+ cells.
Antigens, CD34 ; Antigens, CD95/*metabolism ; Apoptosis ; Fetal Blood/*cytology ; Hematopoiesis ; Hematopoietic Stem Cells/*metabolism ; Leukocytes, Mononuclear/metabolism ; Proto-Oncogene Proteins c-bcl-2/*metabolism

BACKGROUND/AIMS: Apoptosis via Fas/FasL system is thought to be involved in the development of hepatocyte death in viral hepatitis B. In chronic hepatitis C, sFas/sFasL system was reported to control liver injury induced by Fas/FasL mediated apoptosis. To determine the role of sFas/sFasL system in chronic hepatitis B, we analyzed serum sFas/sFasL in 58 HBV patients and 29 healthy controls. METHODS: HBV patients were categorized into two groups; normal ALT (< or =40 IU/L) and elevated ALT (>40 IU/L). Serum sFas/sFasL levels in HBV patients were measured by ELISA and was compared with those in 29 healthy controls. Serum ALT levels, histological activity, and Fas/FasL expression of liver were compared. RESULTS: Chronic hepatitis B patients with elevated ALT had significantly higher serum sFas levels than those in healthy controls (P<0.01). Serum sFasL levels, however, were significantly lower than those in healthy controls (P<0.01). Patients with moderate to marked degree of inflammation and fibrosis had significantly higher serum sFas levels than those in healthy controls (P<0.05). Serum sFasL levels had no correlation with the hepatic histological activity. Serum sFas/sFasL levels also had no significant correlation with the Fas/FasL expression of liver. CONCLUSIONS: Serum sFas/sFasL levels play a possible role in the pathogenesis of chronic hepatitis B. These results suggest that serum sFas levels might serve as a marker for estimating the degree of hepatic histological activity.
Adult ; Antigens, CD95/analysis/*blood ; Fas Ligand Protein/analysis/*blood ; Female ; Hepatitis B virus/isolation & purification ; Hepatitis B, Chronic/*diagnosis ; Humans ; Male ; Middle Aged

Deregulation of soluble apoptosis stimulating fragment (sFas) plays an important role in glomerulonephritis (GN). The study assed the influence of immunosuppressive treatment on serum and urine sFas in patients with proliferative (PGN) and non-proliferative (NPGN) GN, and evaluated the potential of sFas measurements in predicting outcomes. Eighty-four patients with GN (45 males and 39 females) were included. Serum concentration (ng/mL) and urinary excretion (ng/mg of urinary creatinine) of sFas were measured before and after the treatment. After 12 months of therapy with steroids and cyclophosphamide, patients were divided into two subgroups according to the treatment results: Responders (R) and Non-Responders (NR). The sFas urinary excretion was reduced after treatment in both PGN and NPGN (from 17.12 +/- 15 to 5.3 +/- 4.2, P = 0.008 and from 10.11 +/- 6.1 to 3.4 +/- 3.0, P = 0.039; respectively) whereas the sFas serum concentration remained unchanged. In PGN, pre-treatment urinary sFas concentration was significantly lower in the Responders than in Non-Responders (2.3 +/- 3.1 vs 19.4 +/- 14.1, P = 0.003), and was lower still than in both R (P = 0.044) and NR (P = 0.042) subgroups with NPGN. The immunosuppressive treatment reduced sFas urinary excretion in proliferative and non-proliferative GN and results suggest that the lower urinary sFas may be linked with favorable therapy outcomes in patients with PGN.
Adult ; Antigens, CD95/blood/*urine ; Cyclophosphamide/therapeutic use ; Female ; Glomerulonephritis/*drug therapy/metabolism/pathology ; Humans ; Immunosuppressive Agents/*therapeutic use ; Male ; Middle Aged ; Steroids/therapeutic use ; Treatment Outcome

Interleukin 15 (IL-15) is an important regulatory cytokine in cellular immunity. In vitro replacement of IL-15 has been shown to enhance immunity in Human immunodeficiency virus type 1 (HIV-1) infected lymphocytes. We evaluated the effect of IL-15 on the survival of peripheral blood mononuclear cells of HIV patients by examining in vitro lymphocyte apoptosis, and correlated the process with Bcl-2 and Fas gene regulation. Peripheral blood mononuclear cells (PBMC) from 21 HIV-infected adults and 24 HIV-seronegative healthy individuals were isolated and cultured to determine the effect of escalating doses of IL-15 (0, 1, 10, 100, 1000 ng/mL) on apoptosis. Lymphocyte proliferation assay with (3H) TdR was measured and Bcl-2 and Fas gene regulation was observed. The results were as follows: 1) IL-15 reduced culture induced lymphocyte apoptosis in HIV patients in a dose dependent manner, and reached a plateau level at a concentration of 100 ng/ml; 2) IL-15 significantly reduced the level of apoptosis after 3 days (14%) and 5 days (15%) of culture in HIV patients, while no difference was observed in HIV (-) donors; 3) The percentage of viable cells among the total number of lymphocytes was significantly enhanced by 25% in HIV patients with IL-15; 4) Bcl-2 expression was decreased in HIV patients (53.9 +/- 12.3%) compared to HIV (-) donors (93.0 +/- 3.7%), and IL-15 increased Bcl-2 expression by 21.2 +/- 5.2% in HIV patients; 5) Fas expression was increased in HIV patients (70.2 +/- 4.6%) compared to HIV (-) donors (32.4 +/- 4.3%), and IL-15 increased Fas expression by 8.4 +/- 1.2% in HIV (-) donors. Our findings indicate that IL-15 may influence immunologic abnormalities in HIV infection, particularly its ability to prevent apoptosis of lymphocytes by suppressing the down-modulation of Bcl-2. This may provide an experimental basis for IL-15 immunotherapy.
Antigens, CD95/genetics ; Apoptosis/drug effects* ; Cells, Cultured ; Gene Expression Regulation/physiology ; Genes, bcl-2/genetics ; HIV Infections/blood* ; Human ; Interleukin-15/pharmacology* ; Monocytes/drug effects*

OBJECTIVES: To quantitate apoptosis and Fas antigen expression of T lymphocytes by activation in aplastic anemia (AA) and compare with that of normal controls and completely-recovered AA, and to investigate the apoptotic sensitivity to anti-fas antibody of activated T lymphocytes in AA. METHODS: We studied the expression of Fas antigen on fresh T lymphocytes of twenty patients with AA [13 newly diagnosed, 7 recorvered AA after immunosuppressive therapy (IST)], and investigated the activation-induced cell death (AICD) and Fas expression by activation [interleukin-2 (200 U/ml) and phytohemagglutinin (50 micrograms/ml)] in 5 newly-diagnosed AA, 5 normal controls and 5 AA in complete response (CR). Apoptotic sensitivity to anti-Fas antibody was assessed by the time-course kinetics of induction of cell death by anti-Fas antibody (500 ng/ml). RESULTS: There was no significant difference of Fas antigen expression on freshly-isolated T lymphocytes among newly-diagnosed severe AA, normal control s and patients with AA in CR after IST. In normal controls, T lymphocytes death was greatly increased at 3 days of activation, and Fas antigen expression on T lymphocytes was increased above baseline at day 1 of activation. In contrast, in newly-diagnosed AA, T lymphocytes showed delayed cell death, which correlated with a slowed increase of Fas antigen expression by activation. Also, anti-Fa s antibody sensitivity of activated T lymphocytes was decreased in newly-diagnosed AA. In completely recovered AA, these abnormal AICD and Fas antigen expressions by activation were recovered to normal range. CONCLUSIONS: Abnormal AICD plays a role in the immune pathophysiology of AA, and defective Fas system is involved in this process.
Anemia, Aplastic/pathology ; Anemia, Aplastic/immunology* ; Antigens, CD95/blood ; Apoptosis ; Case-Control Studies ; Human ; In Vitro ; Lymphocyte Transformation ; T-Lymphocytes/pathology* ; T-Lymphocytes/immunology* ; Time Factors

BACKGROUND: During ex vivo expansion of cord blood (CB) CD34+ cells, differentiation of the expanded cells happened and hematopoietic potential of the progenitor cells decreased. In this study, we evaluate the effect of the expression of Fas antigen, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during ex vivo expansion. METHODS: CD34+ and AC133+ cells isolated from human CB were cultured in serum free medium supplemented with several cytokines for 7 days. After expansion culture, we re isolated CD34+ and AC133+ cells and compared the numbers of granulocyte-macrophage colony-forming units (CFU-GM) and granulocyte, erythrocyte, monocyte, and macrophage colony-forming units (CFU-GEMM), and expression of Fas antigen, Bcl-2, and Bax with unexpanded cells. RESULTS: CFU-GM was expanded 23.94 fold in CD34+ cells and 15.22 fold in AC133+ cells at day 7 of culture but CFU-GEMM was not expanded. The expression of Fas antigen and Bax was 7.44% and 2.75%, respectively, in fresh isolated CD34+ cells and increased to 19.71 % and 33.67%, respectively, in expanded CD34+ cells at day 7 culture, but Bcl-2 was not changed. In case of AC133+ cells, the expression of Fas antigen and Bax were also increased from 5.87% and 6.19% to 24.85% and 22.83%, respectively, and Bcl-2 was slightly decreased. Apoptosis was not changed in CD34+ cells and AC133+ cells during ex vivo expansion. CONCLUSION: These results indicate that the nature of expansion was similar between CD34+ and AC133+ cells, and expression of Fas antigen and Bax increased on CD34+ and AC133+ cells during ex vivo expansion. Selection of the expanded progenitor cells without apoptosis may be useful for the hematopoietic stem cell transplantation.
Antigens, CD95* ; Apoptosis* ; Cytokines ; Erythrocytes ; Fetal Blood ; Granulocyte-Macrophage Progenitor Cells ; Granulocytes ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells* ; Humans ; Macrophages ; Monocytes ; Myeloid Progenitor Cells ; Stem Cells