[Effects of acupuncture at "Zusanli" (ST 36) on eosinophil apoptosis and related gene expression in rats with asthma].

OBJECTIVE: To observed efficacy differences of acupuncture at "Zusanli" (ST 36) in rats with asthma and asthma with spleen-deficency, so as to investigate the therapeutic mechanism. METHODS: Sixty SD rats were randomly divided into 5 groups according to their weight, named as an asthma with spleen-deficency group (group A), an acupuncture on asthma with spleen-deficency group (group B), an asthma group (group C), an acupuncture on asthma group (group D) and a control group. The rat models with spleen-deficiency in the first two groups were set up by TCM, then the rats of asthma model in the first four groups were induced by egg albumin, but the control group was treated by the same dose of saline. The group B and the group D were both treated with acupuncture at "Zusanli" (ST 36), once each day for 8 days, and the other groups remained unhandled. The mRNA expressions of Fas and Bcl-2 in the lung tissues were detected by hybridization in situ and apoptosis was detected by TUNEL (terminal dexynucleotidyl transferase-mediated dutp nick end labeling). RESULTS: Compared with the control group, in both the group A and the group C, the expression of Fas mRNA significantly decreased, but the expression of Bcl-2 mRNA significantly increased (all P < 0.01), and eosinophils (EOS) counts significantly increased, but EOS apoptosis rate significantly decreased (all P < 0.01). Compared with the group C, in the group A, the expressions of Fas mRNA significantly decreased, but the expressions of Bcl-2 mRNA and EOS counts significantly increased (all P < 0.01). At the same time, compared with the corresponding asthma groups, in both acupuncture groups, Fas mRNA expression obviously increased, Bcl-2 mRNA expression was significantly reduced (all P < 0.01), EOS counts remarkably decreased and EOS apoptosis rate significantly increased (all P < 0.01). There were no significant differences in the expressions of Fas mRNA and Bcl-2 mRNA between the two acupuncture groups (both P > 0.05), but compared with group B,in the group D, EOS counts significantly decreased and EOS apoptosis rate significantly increased (both P < 0.01). CONCLUSION: Acupuncture at "Zusanli" (ST 36) can regulate the disorders of Fas mRNA and Bcl-2 mRNA expression in the state of both asthma and asthma with spleen-deficency, promote EOS apoptosis so as to inhibit the development of inflammatory reaction of asthma, showing that acupuncture at "Zusanli" (ST 36) has certain advantages on regulation of related gene of EOS in asthma with spleen-deficency.

Enhancing mda-7/IL-24 therapy in renal carcinoma cells by inhibiting multiple protective signaling pathways using sorafenib and by Ad.5/3 gene delivery.

We have determined whether an adenovirus that comprises the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, more effectively infects and kills renal carcinoma cells (RCCs) compared to a serotype 5 virus, Ad.5-mda-7. RCCs are a tumor cell type that generally does not express the receptor for the type 5 adenovirus; the coxsackie and adenovirus receptor (CAR). Ad.5/3-mda-7 infected RCCs to a much greater degree than Ad.5-mda-7. MDA-7/IL-24 protein secreted from Ad.5/3-mda-7-infected RCCs induced MDA-7/IL-24 expression and promoted apoptosis in uninfected "bystander" RCCs. MDA-7/IL-24 killed both infected and bystander RCCs via CD95 activation. Knockdown of intracellular MDA-7/IL-24 in uninfected RCCs blocked the lethal effects of conditioned media. Infection of RCC tumors in one flank, with Ad.5/3-mda-7, suppressed growth of infected tumors and reduced the growth rate of uninfected tumors implanted on the opposite flank. The toxicity of the serotype 5/3 recombinant adenovirus to express MDA-7/IL-24 was enhanced by combined molecular or small molecule inhibition of MEK1/2 and PI3K; inhibition of mTOR, PI3K and MEK1/2; or use of the multi-kinase inhibitor sorafenib. In RCCs, combined inhibition of cytoprotective cell signaling pathways enhanced the MDA-7/IL-24-induction of CD95 activation, with greater mitochondrial dysfunction due to loss of MCL-1 and BCL-XL expression, and tumor cell death. Treatment of RCC tumors in vivo with sorafenib also enhanced Ad.5/3-mda-7 toxicity and prolonged animal survival. Future combinations of these approaches hold promise for developing a more effective therapy for kidney cancer.

Sodium valproate, a histone deacetylase inhibitor, decreases the secretion of soluble Fas by human osteosarcoma cells and increases their sensitivity to Fas-mediated cell death.

PURPOSE: Effects of valproic acid (VPA), a histone deacetylase inhibitor, on the susceptibility to cell death induced by agonistic anti-Fas antibody were examined using four human osteosarcoma cell lines. METHOD: Cell growth, secretion of soluble Fas, expression of cell-surface Fas, and sensitivity to Fas-mediated cell death were examined using cell proliferation assay, flow cytometry, enzyme-linked immunosorbent assay, and agonistic anti-Fas antibody, respectively. RESULTS: VPA suppressed the growth of all the four osteosarcoma cell lines and the secretion of soluble Fas from these cells. VPA showed no or slight suppressive effect on the expression of cell-surface Fas in the four osteosarcoma cell lines, but increased the sensitivity of three of four osteosarcoma cell lines to Fas-mediated cell death. CONCLUSION: VPA enhances the susceptibility of human osteosarcoma cells to Fas-ligand-induced cell death by decreasing the secretion of soluble Fas and increasing the sensitivity to Fas-mediated cell death.

Apoptosis supercedes necrosis in mitochondrial DNA-depleted Jurkat cells by cleavage of receptor-interacting protein and inhibition of lysosomal cathepsin.

In the present study, we used mitochondrial DNA-depleted Jurkat subclones (rho0 cells) to demonstrate that Fas agonistic Ab (CH-11), at the concentrations that evoke apoptotic death of the parental Jurkat cells, induced necrosis mainly through generation of excess reactive oxygen species, lysosomal rupture, and sequential activation of cathepsins B and D, and in minor part through activation of receptor-interacting protein (RIP). In the rho0 cells treated with CH-11, ATP supplementation converted necrosis into apoptosis by the formation of the apoptosome and subsequent activation of procaspase-3. In these ATP-supplemented rho0 cells (ATP-rho0), generation of excess ROS and lysosomal rupture were still seen, yet cathepsins B and D were inactivated and RIP was degraded. The conversion of necrosis to apoptosis, RIP degradation, and cathepsin inactivation in ATP- rho0 cells were blocked by caspase-3 inhibitors. Activities of cathepsins B and D in the lysate of necrotic rho0 cells were inhibited by the addition of apoptotic parental Jurkat cell lysate. Thus, apoptosis may supercede necrosis.

Immunomodulatory effects of blood transfusions: the synergic role of soluble HLA Class I free heavy-chain molecules detectable in blood components.

BACKGROUND: Over the past decades, the weight of the published literature demonstrates that blood transfusions can induce clinically significant immunosuppression in recipients. Several studies showed significant improved clinical outcomes in the patients receiving leukoreduced transfusions, compared with control patients who received nonleukoreduced transfusions. Moreover, the immunosuppressive potential of blood products grows with the time of their storage and becomes highest in nonleukoreduced blood products stored for a long time. STUDY DESIGN AND METHODS: The interest was previously focused on the determination of immunomodulatory soluble molecules such as soluble HLA Class I (sHLA-I) and soluble Fas ligand (sFasL) in different blood components and on the evaluation of their immunomodulatory activities. On this basis, whether soluble beta2-microglobulin free HLA Class I heavy chains (sHLA-beta2fHC) could be detected and immunochemically characterized in different blood components was evaluated. Immunomodulatory activity of detectable sHLA-beta2fHC molecules was evaluated by apoptosis inducing capacity in interleukin-2-activated antigen-specific cytotoxic T lymphocytes (CTL). RESULTS: Double-determinant immunoenzymatic assay indicates that sHLA-beta2fHC levels in red blood cells stored for up to 30 days and in random-donor platelets are significantly (p < 0.001) higher than in other blood components, and the immunochemical characterization suggests that the major source of sHLA-beta2fHC molecules might be the residual white cells that undergo membrane damage during storage. Finally, allogeneic CD8+ CTL apoptosis induction confirmed biofunctionality of sHLA-beta2fHC molecules. CONCLUSION: These data are comparable with those previously reported dealing with contaminant soluble molecules in allogeneic and autologous blood components, suggesting that sHLA-beta2fHC molecules could contribute to the immunosuppressive effects of blood transfusions.

Antiproliferative activity of aucubin is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells.

Aucubin, an iridoid glycoside isolated from the leaves of Aucuba japonica, inhibits human non-small cell lung cancer A549 cells by blocking cell cycle progression in the G(0)/G(1) phase and inducing apoptosis. An ELISA showed that the G(0)/G(1) phase arrest is due to p53-mediated induction of p21. Enhancement of Fas and its two ligands, membrane-bound and soluble Fas ligand, may be responsible for the apoptotic effect induced by aucubin. The present study shows, for the first time, that the induction of p53 and activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of aucubin in A549 cells.

The hepatic vagus nerve attenuates Fas-induced apoptosis in the mouse liver via alpha7 nicotinic acetylcholine receptor.

BACKGROUND & AIMS: Although accumulating evidence has recently shown that the efferent vagus nerve attenuates systemic inflammation, it remains unclear whether or not the vagus nerve can affect Fas-induced liver apoptosis. We investigated the effect of the vagus nerve by using a selective hepatic vagotomy. METHODS: We assessed the mortality and apoptosis in Fas-induced fulminant hepatitis in sham-operated and vagotomized male C57BL/6 mice. To determine how the nerve influences hepatocyte apoptosis, hepatitis was preceded by pretreatment with nicotine; PNU-282987, an alpha7 nicotinic acetylcholine receptor (AChR) agonist; liposome-encapsulated dichloromethylene diphosphonate (lipo-Cl(2)MDP), a macrophage eliminator; and Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), an oxidative inhibitor. RESULTS: Mortality in the vagotomized mice was significantly higher than that in the sham-operated mice following intravenous administration with the anti-Fas antibody Jo-2. This result was also supported by the data from both terminal deoxynucleotidyl-transferase mediated dUTP nick-end labeling and caspase-3 assay, in which vagotomized livers showed a significant elevation in the number of apoptotic hepatocytes and increased caspase-3 activity following Jo-2 treatment compared with the sham-operated livers. Supplementation with nicotine and PNU-282987 dose dependently inhibited this detrimental effect of the vagotomy. Moreover, the vagotomy-triggered exacerbation of Fas-induced hepatitis was completely blocked by lipo-Cl(2)MDP. Similarly, pretreatment with MnTBAP also completely suppressed the vagotomy-triggered exacerbation. CONCLUSIONS: The hepatic vagus nerve appears to play an important role in attenuating Fas-induced hepatocyte apoptosis through alpha7 nicotinic AChR, perhaps by causing the Kupffer cells to reduce their generation of an excessive amount of reactive oxygen species.

[Effects of radix Astragali injection on apoptosis of lymphocytes and immune function in patients with systemic lupus erythematosus].

OBJECTIVE: To investigate the effect of Radix Astragali Injection on apoptosis of lymphocytes and immune function in treating patients with systemic lupus erythematosus (SLE). METHODS: Eighty SLE patients were randomly assigned into the routine treatment group (RT) treated with conventional therapy and the Radix Astragali treated group (RA) treated with Radix Astragali Injection besides routine treatment. The expressions of Fas and Bcl-2 antigen on lymphocytes and the changes of T lymphocyte subsets in peripheral blood before and after treatment were observed. RESULTS: After treatment, the expression of Fas antigen on lymphocytes significantly lowered (P < 0.01), and that of Bcl-2 antigen, CD4+ lymphocyte subset and CD4+ / CD8+ ratio significantly increased in both groups (all P < 0.01). However, the changes of Fas antigen expression, CD4+ and CD4+ / CD8+ ratio were more significant in the RA group than those in the RT group (P < 0.05). CONCLUSION: Radix Astragli Injection can enhance the inhibitory function of corticosteroid/immunosuppressant on apoptosis, and regulate the ratio and function of T lymphocyte subsets to normal range, which may be a useful approach for enhancing the efficacy of treatment to SLE.

Arsenic trioxide: A promising novel therapeutic agent for lymphoproliferative and autoimmune syndromes in MRL/lpr mice.

MRL/lpr mice develop a human lupuslike syndrome and, as in autoimmune lymphoproliferative syndrome (ALPS), massive lymphoproliferation due to inactivation of Fas-mediated apoptosis. Presently, no effective therapy exists for ALPS, and long term, therapies for lupus are hazardous. We show herein that arsenic trioxide (As2O3) is able to achieve quasi-total regression of antibody- and cell-mediated manifestations in MRL/lpr mice. As2O3 activated caspases and eliminated the activated T lymphocytes responsible for lymphoproliferation and skin, lung, and kidney lesions, leading to significantly prolonged survival rates. This treatment also markedly reduced anti-DNA autoantibody, rheumatoid factor, IL-18, IFN-gamma, nitric oxide metabolite, TNF-alpha, Fas ligand, and IL-10 levels and immune-complex deposits in glomeruli. As2O3 restored cellular reduced glutathione levels, thereby limiting the toxic effect of nitric oxide, which is overproduced in MRL/lpr mice. Furthermore, As2O3 protected young animals against developing the syndrome and induced almost total disease disappearance in older affected mice, thereby demonstrating that it is a novel promising therapeutic agent for autoimmune diseases.

Involvement of the TNF-alpha autocrine-paracrine loop, via NF-kappaB and YY1, in the regulation of tumor cell resistance to Fas-induced apoptosis.

Many tumors are resistant to Fas ligand (FasL)-induced apoptosis. This study examined the role of tumor-derived TNF-alpha, via an autocrine/paracrine loop, in the regulation of tumor-cell resistance to FasL-induced apoptosis. We have reported that Fas expression and sensitivity to FasL is negatively regulated by the transcription repressor factor Yin Yang 1 (YY1). Thus, we hypothesized that tumor-derived TNF-alpha induces the activation of NF-kappaB and the transcription repressor YY1, both of which negatively regulate Fas expression and sensitivity to FasL-induced apoptosis. This hypothesis was tested in PC-3 prostate cancer cells which synthesize and secrete TNF-alpha and express constitutively active NF-kappaB and YY1. Treatment of PC-3 cells with TNF-alpha (10 units) resulted in increased NF-kappaB and YY1 DNA-binding activity, upregulation of YY1 expression, downregulation of surface and total Fas expression and enhanced resistance of PC-3 to apoptosis induced by the FasL agonist antibody CH-11. In contrast, blocking the binding of secreted TNF-alpha on PC-3 cells with soluble recombinant sTNF-RI resulted in significant inhibition of constitutive NF-kappaB and YY1 DNA-binding activity, downregulation of YY1 expression, upregulation of Fas expression and sensitization of tumor cells to CH-11-induced apoptosis. The regulation of YY1 expression and activity by NF-kappaB was demonstrated by the use of the NF-kappaB inhibitor Bay 11-7085 and by the use of a GFP reporter system whereby deletion of the YY1-tandem binding site in the promoter significantly enhanced GFP expression. The direct role of YY1 expression in the regulation of PC-3 resistance to CH-11-induced apoptosis was shown in cells transfected with siRNA YY1 whereby such cells exhibited upregulation of Fas expression and were sensitized to CH-11-induced apoptosis. These findings demonstrate that the TNF-alpha autocrine-paracrine loop is involved in the constitutive activation of the transcription factors NF-kappaB and YY1 in the tumor cells and this loop leads to inhibition of Fas expression and resistance to FasL-induced apoptosis. Further, these findings identify new targets such as TNF-alpha, NF-kappaB and YY1, whose inhibition can reverse tumor cell resistance to FasL-mediated apoptosis.

Frequency and resistance of CD95 (Fas/Apo-1) gene-transfected tumor cells to CD95-mediated apoptosis by the elimination and methylation of integrated DNA.

It is important for more effective gene therapies to clarify the mechanisms by which cDNA integrated into cells can maintain or lose its function in vivo. We evaluated genetic and epigenetic events leading to alternation of the introduced CD95 (Fas/Apo-1) gene as a model of gene therapy. Solid tumors formed by CD95 cDNA-transfected hepatoma cells (F6b) were almost completely cured by a single treatment of anti-CD95 monoclonal antibody (mAb) but recurred in gld/gld lpr/lpr mice after initial complete response. Recurred tumors were resistant to repeated mAb treatment. The ratio of resistant cells in tumors was estimated as 4.2 cells per 10(6) cells. The CD95-resistant tumor contained CD95-vanished and CD95-decreased cells. CD95-vanished cells were due to the deletion of CD95cDNA. However, CD95-decreased cells retained CD95cDNA, which was highly methylated when determined with methylation-dependent enzymes and a demethylation reagent, indicating that DNA methylation was responsible for the reduced CD95 expression and resistance to mAb. CD95-decreased cells reduced the CD95 expression further but did not delete cDNA after a second in vivo treatment with anti-CD95 mAb, suggesting that the elimination of cDNA is not induced after its methylation and that cells containing methylated genes became more resistant by further methylation. Thus, the elimination and methylation of integrated cDNA appear to occur through different mechanisms. Our study of resistant tumor cells, which arose by both mutational and epigenetic modifications of the introduced CD95 plasmid, provides important and fundamental information about the fate of introduced cDNA, augmenting the efficiency of gene therapy.

Rituximab (chimeric anti-CD20) sensitizes B-NHL cell lines to Fas-induced apoptosis.

Rituximab (chimeric anti-CD20 monoclonal antibodies) is currently being used in the treatment of B non-Hodgkin's lymphoma (NHL). We have recently reported that rituximab triggers and modifies various intracellular signaling pathways in NHL B-cell lines, resulting in reverting the chemoresistant phenotype to a sensitive phenotype. This study investigated whether rituximab also modifies intracellular signaling pathways resulting in the sensitization of NHL cells to Fas-induced apoptosis. Treatment of the Fas-resistant NHL cell lines (2F7, Ramos and Raji) with rituximab sensitized the cells to CH-11 (FasL agonist mAb)-induced apoptosis and synergy was achieved. Fas expression was upregulated by rituximab as early as 6 h post-treatment as determined by flow cytometry, reverse transcriptase-polymerase chain reaction and Western blot. Rituximab inhibited both the expression and activity of the transcription repressor Yin-Yang 1 (YY1) that negatively regulates Fas transcription. Inhibition of YY1 resulted in the upregulation of Fas expression and sensitization of the tumor cells to CH-11-induced apoptosis. The downregulation of YY1 expression was the result of rituximab-induced inhibition of both the p38 mitogen-activated protein kinase (MAPK) signaling pathway and constitutive nuclear factor kappa of B cells (NF-kappaB) activity. The involvement of NF-kappaB and YY1 in the regulation of Fas expression was corroborated by the use of Ramos cells with a dominant-active inhibitor of NF-kappaB (Ramos IkappaB-estrogen receptor (ER) mutant) and by silencing YY1 with YY1 siRNA, respectively. Further, the role of rituximab-mediated inhibition of the p38 MAPK/NF-kappaB/YY1 pathway in the regulation of Fas and sensitization to CH-11-induced apoptosis was validated by the use of specific chemical inhibitors of this pathway and which mimicked rituximab-mediated effects. These findings provide a novel mechanism of rituximab-mediated activity by sensitizing NHL cells to Fas-induced apoptosis.

Rituximab-induced inhibition of YY1 and Bcl-xL expression in Ramos non-Hodgkin's lymphoma cell line via inhibition of NF-kappa B activity: role of YY1 and Bcl-xL in Fas resistance and chemoresistance, respectively.

Rituximab treatment of B non-Hodgkin's lymphoma (NHL) cell lines inhibits the constitutive NF-kappaB activity and results in the sensitization of tumor cells to both chemotherapy and Fas-induced apoptosis. Cells expressing dominant active IkappaB or treated with NF-kappaB-specific inhibitors were sensitive to both drugs and Fas agonist mAb (CH-11)-induced apoptosis. Down-regulation of Bcl-xL expression via inhibition of NF-kappaB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL-overexpressing Ramos cells, Ramos hemagglutinin (HA)-Bcl-x, which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent; Ramos HA-Bcl-x cells were as sensitive as the wild type to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor yin-yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab or the NO donor DETANONOate or after transfection with YY1 small interfering RNA resulted in up-regulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two mechanisms underlying the chemosensitization and immunosensitization of B-NHL cells by rituximab via inhibition of NF-kappaB. The regulation of chemoresistance by NF-kappaB is mediated via Bcl-xL expression, whereas the regulation of Fas resistance by NF-kappaB is mediated via YY1 expression and activity. The potential clinical significance of these findings is discussed.

Neurokinin-1 receptor antagonists protect mice from CD95- and tumor necrosis factor-alpha-mediated apoptotic liver damage.

Previously, we have shown that primary afferent neurons are necessary for disease activity in immune-mediated liver injury in mice. These nerve fibers are detectable by substance P (SP) immunocytochemistry in the portal tract of rodent liver. Antagonists of the neurokinin-1 receptor (NK-1R), which is the prime receptor of SP, prevented liver damage by suppressing the synthesis of proinflammatory cytokines. Here, we investigated the influence of primary afferent nerve fibers, SP, and NK-1 receptor antagonists on hepatocyte apoptosis in vivo induced by administration of activating anti-CD95 monoclonal antibody (mAb) to mice. Depletion of primary afferent nerve fibers by neonatal capsaicin treatment prevented CD95-mediated activation of caspase-3, measured as enzymatic activity in liver homogenates or by demonstration of hepatocellular immunoreactivity for active caspase-3 in liver slices, and liver damage. This effect was reversed by administration of SP to anti-CD95 mAb-treated mice depleted from primary afferent neurons. The presence of the NK-1R on mouse hepatocytes was demonstrated by immunocytochemistry and flow cytometry. Intraperitoneal pretreatment with the NK-1 receptor antagonists (2S,3S)-cis-2-(diphenylmethyl)-N-([2-methoxyphenyl]-methyl)-1-azabicyclo(2.2.2.)-octan-3-amine (CP-96,345) or (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperadine (L-733,060) dose dependently protected mice from CD95-mediated liver injury. Similar results were obtained when apoptotic liver damage was induced by administration of tumor necrosis factor-alpha to d-galactosamine-sensitized mice. In conclusion, SP, probably by binding to its receptor on hepatocytes, might aggravate apoptotic signals in these cells. Because NK-1 receptor antagonists not only suppress the proinflammatory cytokine response in the liver but also prevent liver cell apoptosis in vivo, they might be suitable drugs for treatment of immune-mediated liver disease.

Osthole prevents anti-Fas antibody-induced hepatitis in mice by affecting the caspase-3-mediated apoptotic pathway.

Fas (Apo-1/CD95) ligand, which is a type II membrane protein, is a major inducer of apoptosis. Osthole is a coumarin derivative present in medicinal plants. The effect of osthole on hepatitis induced by anti-Fas antibody in mice was studied. Pretreatment of mice with osthole (10, 50, and 100 mg/kg, i.p.) prevented the elevation of plasma alanine aminotransferase (ALT) caused by anti-Fas antibody (175 microg/kg, i.v.). Administration of osthole to mice even at a dose of 10 mg/kg significantly inhibited of anti-Fas antibody-induced elevation of plasma ALT. Capase-3 is a cysteine protease, and treatment of mice with anti-Fas antibody caused an elevation of caspase-3 activity at 3.5 and 6 hr. Pretreatment of mice with osthole (100 mg/kg, i.p.) inhibited the elevation of caspase-3 activity caused by anti-Fas antibody. However, the addition of osthole (up to 10(-4)M) to a liver cytosol fraction isolated from mice treated with anti-Fas antibody did not inhibit caspase-3 activity in vitro. Thus, treatment of mice with osthole inhibited caspase-3 activity by an effect upstream of caspase-3 activation. The livers of mice treated with anti-Fas antibody contained apoptotic and dead cells; osthole attenuated the development of this apoptosis and cell death. The present results show that osthole prevented anti-Fas antibody-induced hepatitis by inhibiting the Fas-mediated apoptotic pathway.

A role for oxidative stress in apoptosis: oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fas-mediated apoptosis.

Exposure of phosphatidylserine (PS) on the surface of apoptotic cells has been suggested to serve as an important recognition signal for macrophages. In this work we show that triggering of the death receptor Fas on Jurkat cells results in the generation of reactive oxygen species with oxidation and externalization of PS but not of the other major aminophospholipid, phosphatidylethanolamine. These cells were readily ingested by several classes of macrophages, whereas Raji cells, which are defective for Fas-induced PS exposure, remained unengulfed. However, when Raji cells were incubated with the thiol-reactive agent N-ethylmaleimide to induce PS exposure in the absence of other features of apoptosis, these cells were also engulfed by macrophages. Phagocytosis of Fas-triggered Jurkat cells was inhibited by superoxide dismutase and catalase, which prevent oxidation of PS while allowing PS to remain externalized on these cells. Moreover, liposomes containing oxidized PS (PS-OX) were more potent inhibitors of phagocytosis than those containing its nonoxidized counterpart. Finally, enrichment of the plasma membrane of Jurkat or Raji cells, or myeloid leukemic HL-60 cells, with exogenous PS resulted in phagocytic cell clearance, and this process was further enhanced when PS was substituted for by PS-OX. Taken together, our data suggest that the presence of PS-OX in conjunction with nonoxidized PS on the cell surface is an important signal for macrophage clearance of apoptotic cells.

6-Methylprednisolone does not impair anti-thymocyte globulin (ATG) immunosuppressive activity in non-human primates.

BACKGROUND: Induction treatments with anti-thymocyte globulin (ATG) in solid organ transplantation may enhance the efficacy of maintenance immunosuppressive therapy. Since ATG can trigger Fas (CD95) mediated T cell apoptosis, a process antagonized in vitro by corticosteroids, an important issue is whether corticosteroids could interfere with T cell depleting and immunosuppressive activities of ATG. METHODS: MHC mismatched skin allografts were performed on cynomolgus and rhesus monkeys treated with ATG (20 mg/kg) associated or not with 6-methylprednisolone (10 mg/kg). RESULTS: There was no difference between the two immunosuppressive regimens as regards the intensity and duration of peripheral T lymphocyte depletion and the appearance of anti-ATG antibodies. Skin graft survival was increased in monkeys treated with 6-methylprednisolone as compared with ATG alone. CONCLUSIONS: In vivo, corticosteroids do not interfere with ATG ability to induce massive T cell depletion and to delay skin allograft rejection in non-human primates.

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells.

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-gamma (IFN-gamma) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-gamma, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-gamma-differentiated U937 cells. Western blot analysis revealed that, in IFN-gamma-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-gamma-differentiation, compared to that of the control. These results suggest that the IFN-gamma-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.

Development and application of a GFP-FRET intracellular caspase assay for drug screening.

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.

Yang-gan-wan protects mice against apoptosis induced by anti-Fas antibody (Jo2).

We investigated the protective effect of yang-gan-wan (YGW, Pro-Liver pill), a Chinese herbal remedy, against liver apoptosis (programmed cell death) induced by anti-Fas antibody (Jo2). Mice were pretreated daily with 200 mg/kg YGW for 14 days before treatment with 10 microg/20 kg body weight Jo2. YGW significantly reduced the elevated activity of alanine aminotransferase (ALT) and also reduced the elevated activity of sorbitol dehydrogenase (SDH) produced 4 hrs after treatment with Jo2. YGW also increased the survival of mice treated with Jo2 by about 1 hr in 11 out of 16 mice (69%). Most of the mice died 9 hrs after Jo2 injection. This study demonstrates that YGW has protective effects against liver apoptosis induced by Jo2.