Granzyme M directly cleaves inhibitor of caspase-activated DNase (CAD) to unleash CAD leading to DNA fragmentation.

Granzyme (Gzm)M is constitutively highly expressed in NK cells that may play a critical role in NK cell-mediated cytolysis. However, the function of GzmM has been less defined. Just one report showed GzmM induces a caspase-independent death pathway. In this study, we demonstrate a protein transfection reagent Pro-Ject can efficiently transport GzmM into target cells. GzmM initiates caspase-dependent apoptosis with typical apoptotic nuclear morphology. GzmM induces DNA fragmentation, not DNA nicking. GzmM can directly degrade inhibitor of caspase-activated DNase to release the nuclease activity of caspase-activated DNase for damaging DNA. Furthermore, GzmM cleaves the DNA damage sensor enzyme poly(ADP-ribose) polymerase to prevent cellular DNA repair and force apoptosis.

A combination of E. coli DNA fragments and modified lipopolysaccharides as a cancer immunotherapy.

The use of Escherichia coli DNA or lipopolysaccharide (LPS) as an immunotherapy is often associated with unacceptable toxicity and insufficient therapeutic effects. In this study, we investigated the efficacy of using a combination of bacterial DNA fragments and LPS as an anticancer agent. LPS was isolated from an E. coli strain expressing short-carbohydrate-chain-containing LPS and subjected to alkaline hydrolysis to remove lipid A. The ability to induce tumor necrosis factor-alpha (TNF-alpha) release in human whole blood cells was significantly lower for the LPS devoid of lipid A than for its parent form. The immunostimulating activity of E. coli DNA fragments of various sizes were tested. Those of 0.2-0.5 kb in size exhibited the highest activity in whole blood assays, whereas those of size 0.5-2.0 kb exhibited the highest adjuvant activity in mice. A combination of 0.5-2.0-kb DNA fragments and modified LPS at a ratio of 100:1, designated CIA07, exhibited higher immunostimulating activity than each substance alone, and its antitumor activity was significantly higher than that of Bacillus Calmette-Guerin in a mouse bladder cancer model. An intraperitoneal injection of CIA07 at a dose of 25mg/kg body weight caused no apparent adverse effects in mice and guinea pigs. Taken together, these data demonstrate that CIA07 exhibits potent immunostimulating activity with no apparent toxicity, and therefore warrant the further development of CIA07 as an immunotherapy for cancer treatment.

Neutrophil depletion reduces myocardial apoptosis and attenuates NFkappaB activation/TNFalpha release after ischemia and reperfusion.

BACKGROUND: This study tested the hypothesis that depletion of neutrophils (PMNs) reduces myocardial apoptosis via reducing oxidant generation and inhibiting NFkappaB-mediated signaling pathways after ischemia/reperfusion. METHODS: Anesthetized rats were randomly divided into one of four groups: CONTROL: 30 min ischemia and 3 h of reperfusion; PMN depletion: anti-PMN serum was injected 6 h before ischemia; N-acetylcysteine (NAC): NAC was given twice before ischemia and at reperfusion. Sham: the ligature was placed without coronary occlusion. Apoptosis was detected by TUNEL staining and DNA fragmentation. PMN accumulation was studied by immunohistochemical staining. Levels of TNF-alpha, IL-6, and caspase-3 were detected by Elisa kits. Expression in NFkappaB, Bcl-2, and Bax was assessed by Western blotting analysis. RESULTS: Relative to CONTROL, depletion of PMNs or NAC treatment reduced levels of plasma TNFalpha (567 +/- 130* and 231 +/- 72* versus 1994 +/- 447 pg/ml) and IL-6 (791 +/- 473* and 666 +/- 300* versus 3724 +/- 1233, pg/ml), accompanying a reduction in PMN accumulation (12 +/- 1* and 13 +/- 0.6* versus 20 +/- 1 mm2 myocardium) in ischemic myocardium. Both groups showed a reduction in expression of nuclear NFkappaB relative to CONTROL (62 +/- 9* and 67 +/- 8* versus 124 +/- 16 arb.u), consistent with reduced NFkappaB binding activity. The number of apoptotic cells (%) in area at risk myocardium was comparably reduced in anti-PMN and NAC groups relative to CONTROL (12 +/- 1* and 14 +/- 0.9* versus 20 +/- 1), consistent with reduced appearance of DNA ladders. Furthermore, activated caspase-3 was significantly reduced and Bcl-2 was increased relative to CONTROL. No difference in all parameters measured was detected during the course of experiment in the Sham group. CONCLUSION: These data suggest that the oxidants generated from activated PMNs after ischemia/reperfusion trigger myocardial apoptosis, which is further supported by an anti-oxidant therapy with NAC, potentially mediated by enhanced NFkappaB-TNFalpha signaling pathway, activated caspase-3 and down-regulated Bcl-2. *P < 0.05 versus CONTROL.

Exposure of lymphocytes to high doses of Actinobacillus actinomycetemcomitans cytolethal distending toxin induces rapid onset of apoptosis-mediated DNA fragmentation.

We have shown previously that treatment of human lymphocytes with the Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) results in dose-dependent G2 arrest, followed 24 h later by apoptotic cell death. Here we demonstrated that for Jurkat cells exposed to high concentrations of Cdt (>0.2 ng/ml) there was a dose-dependent increase in the level of S-phase cells and a concomitant decrease in the level of G2 cells. Fluorescence-activated cell sorter analysis demonstrated that the S-phase cells did not incorporate BrdU and likely represented cells that arrested in G2 and underwent significant DNA fragmentation. Analysis of the kinetics of the appearance of both S-phase cells and apoptotic cells supported this interpretation. Cells exposed to low doses of toxin exhibited G2 arrest at 24 h, but at 48 and 72 h there were also decreases in the level of G2 cells and concomitant increases in the levels of S, G0/G1, and sub-G0 cells; these changes were paralleled by increased numbers of apoptotic cells. Cells exposed to high doses of toxin exhibited these changes 24 to 48 h earlier. We also examined the relationship between G2 arrest, DNA fragmentation, and activation of the apoptotic cascade. We employed two inhibitors of apoptosis, overexpression of Bcl-2 and the caspase-3 inhibitor zvad. Both inhibitors blocked Cdt-induced apoptosis, Cdt-induced DNA fragmentation, and phosphorylation of the histone H2AX. However, the cells retained the ability to undergo G2 arrest in the presence of the toxin. Thus, it appears that high doses of Cdt induce rapid onset of DNA degradation resulting from activation of the apoptotic cascade.

Virulent clinical isolates of Mycobacterium tuberculosis grow rapidly and induce cellular necrosis but minimal apoptosis in murine macrophages.

In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosis representing a range of virulence for their capacity to grow in bone marrow-derived macrophages. The rate of growth of each of the isolates in macrophages reflected their known virulence, but the most virulent isolates strongly induced production of the cytokine tumor necrosis factor alpha. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains after several days in culture. Staining of cell monolayers for DNA fragmentation indicative of apoptosis showed that this was minimal and only evident to any degree in macrophages infected with the most virulent strains. In contrast, electron microscopy revealed damage of macrophages consistent with cell necrosis. These results suggest that rapid intracellular growth rate and induction of necrotic cell death within host macrophages are virulence factors of M. tuberculosis in the early stages of bacterial infection. They further imply that infected cell apoptosis, regarded as a defense mechanism or cross-priming mechanism, plays a minimal role.

Circulation DNA: biological implications for cancer metastasis and immunology.

Circulation cell free DNA (cf-DNA) is of considerable interest to oncology researchers seeking to isolate specific cancer markers. Here, we focus on the origin and biological implications of cf-DNA, exploring its potential roles in cancer biology and medicine. We hypothesize that cf-DNA is primarily released by living cancer cells in addition to apoptotic or necrotic cancer cells for three reasons: (1) following radiotherapy, cf-DNA quantities are significantly reduced in a high percentage of patients although radiation-induced massive apoptosis is expected; (2) cancer cell DNA concentration in cultured supernatants increases with cell proliferation when few apoptotic or necrotic cells are present; and (3) DNA concentration increases in normal lymphocyte cultures following stimulation with phytohemagglutinin, lipopolysaccharide or antigen. Our hypotheses have major biological implications in cancer biology. First, cancer cf-DNA may transform normal cells and form adjacent or remote metastases or second primary cancer. In this context, we also have raised an alarming advice that the cancer may be potentially infectious. Secondly, if a normal cf-DNA contains cytokine sequence, it may behave like an intrinsic DNA vaccine, producing therapeutic cytokine. If normal cf-DNA contains a sequence of a non-mutated oncogene or tumor suppressor gene, homologous recombination with the cancer genome may occur leading to knock out mutated oncogene or tumor suppressor gene that could thus elicit a spontaneous remission of cancer.

Nuclear autoantigen translocation and autoantibody opsonization lead to increased dendritic cell phagocytosis and presentation of nuclear antigens: a novel pathogenic pathway for autoimmunity?

Autoreactivity in lupus requires the delivery of autoantigens to APCs in a proinflammatory context. It has been proposed that apoptotic cells are a source of lupus autoantigens and targets for autoantibodies. Using a histone H2B-GFP fusion protein as traceable Ag, we show here that lupus autoantibodies, directed against nuclear autoantigens, can opsonize apoptotic cells, enhance their uptake through induction of proinflammatory Fc gammaR-mediated phagocytosis, and augment Ag-specific T cell proliferation by increasing Ag loading. Apoptotic blebs and bodies seemed to be a preferred target of DC phagocytosis, via both "eat-me signals" and Fc gammaR-mediated mechanisms; furthermore, inhibition of nuclear Ag redistribution, by blockade of chromatin fragmentation, could stop binding and opsonization of apoptotic cells by autoantibodies, and inhibited Fc gamma-R-mediated enhancement of phagocytosis. Our results suggest that DC uptake of opsonized histones and other nuclear Ags from apoptotic cells is a novel pathway for the presentation of nuclear Ags in a highly inflammatory context. Blockade of chromatin fragmentation in lupus is a potential therapeutic approach, which could theoretically limit DC access to autoantigens delivered in proinflammatory context, while leaving available for tolerization those delivered in a noninflammatory context.

Infliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model.

OBJECTIVE: To examine the toxic effects of tumor necrosis factor alpha (TNF-alpha) on ejaculated spermatozoa and evaluate the ability of infliximab to reverse these effects. DESIGN: Prospective controlled study. SETTING: Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Thirty-one healthy sperm donors. INTERVENTION(S): Exposure of human spermatozoa to varying concentrations of TNF-alpha (100, 300, 400, 500 pg/mL, and 2.5 microg/mL) and infliximab (400 microg/mL). MAIN OUTCOME MEASURE(S): Sperm motility, functional integrity of plasma membrane, and DNA fragmentation. RESULT(S): Spermatozoa quality declined following incubation with TNF-alpha in a dose-dependent and time-dependent manner. Sperm motility and membrane integrity were higher in the samples incubated with TNF-alpha plus infliximab than in the samples treated with TNF-alpha only. These parameters improved significantly and were comparable with both controls and sperm incubated with infliximab alone. Similarly, the percentage of spermatozoa with DNA fragmentation improved significantly following incubation with TNF-alpha plus infliximab and again was comparable with both controls and sperm incubated with infliximab alone. CONCLUSION(S): Spermatozoa may be exposed to abnormal levels of TNF-alpha in the male reproductive tract or during their passage into the female reproductive tract (in cases of endometriosis). Exposing spermatozoa to pathological concentrations of TNF-alpha can result in significant loss of their functional and genomic integrity. Infliximab could potentially be used to help treat female infertility caused by endometriosis in those with elevated levels of TNF-alpha in their peritoneal fluid.

Role of host's antitumor immunity in exercise-dependent regression of murine T-cell lymphoma.

We have reported that the ascitic growth of a transplantable T cell lymphoma of spontaneous origin, designated as Dalton's lymphoma (DL), is associated with a concomitant immunosuppression. We have also reported that progressive in vivo growth of DL resulted in an inhibition of macrophage functions. In present investigation we report that physical exercise by DL-bearing mice, on a treadmill on a daily basis for various time durations for 10 days, increased the life span along with an inhibition of tumor growth. A significant decrease in the volume of ascitic fluid and number of cells in the tumor was obtained in mice, which underwent exercise. DL cells obtained from exercised groups showed a decreased proliferation in vitro. An augmentation in the percent of cells showing apoptotic morphology and percent specific DNA fragmentation was observed, suggesting that physical exercise increased the incidence of apoptosis in tumor cells. Moreover, macrophages obtained from tumor-bearing mice, which underwent exercise training, showed an augmented tumoricidal activity and production of tumoricidal molecules like interleukin-1 (IL-1), tumor necrosis factor (TNF) and nitric oxide (NO). On the basis of this study it is suggested that the regression of tumor growth consequent to physical exercise training of tumor bearing host, may be due to an exercise-dependent augmentation of macrophage tumoricidal functions.

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells.

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.

Role of cysteine proteinase of Entamoeba histolytica in target cell death.

The bacterial flora of the intestine plays an important role in the virulence caused by Entamoeba histolytica. Cysteine proteinase (CP), an amoebic virulence factor, plays a major role in host cell destruction. The mechanism of increased virulence following bacterial co-association is not understood. We studied CP of E. histolytica HM1:IMSS which was co-associated with Escherichia coli K12 strain pre-incubated with GalNAc or CP specific inhibitor E 64. Co-association of E. histolytica with bacteria enhanced CP activity 3-6-fold as assessed by azocasein assay and substrate gel electrophoresis showed bands at molecular weights of 28, 35 and 56 kDa. Northern and Western blot analysis showed increase in ehcp2 and ehcp5 gene expression. Trophozoites co-associated with E. coli showed greater cytotoxicity of BHK cells by a 51Cr release assay than trophozoites that had not been co-associated; this enhancement was abolished by E-64 treatment. The killing of BHK 21 targets by E. histolytica was characterized by DNA laddering which was not inhibited with E-64. GalNAc pre-incubation of trophozoites reduced cytotoxicity and DNA laddering, while E. coli co-associated E. histolytica showed smearing with faint laddering of BHK implicating both necrosis and apoptosis. Hence, bacterial co-association increases CP activity and CP gene expression and contributes to the necrosis of the target cell.

Occurrence of apoptosis, secondary necrosis, and cytolysis in eosinophilic nasal polyps.

The paradigm states that inflammatory cells disappear from airway tissues through apoptosis and phagocytosis. However, cells may also be cleared through primary cytolysis, necrosis secondary to apoptosis, or transepithelial migration. This study examines the occurrence of apoptosis, secondary necrosis, and cytolysis of eosinophils in human nasal polyps in vivo and blood eosinophils in vitro. Eosinophils abounded in subepithelium and in paracellular epithelial pathways. Macrophages commonly occurred but without engulfed eosinophils. Scattered cells, including epithelial cells, were stained by antibody to the caspase cleavage product of poly(ADP-ribose) polymerase. Few cells were apoptotic (stained by terminal deoxy RNase nick end labeling). Of more than 3,000 examined tissue eosinophils, 110 were caspase cleavage positive, but only one was apoptotic. Transmission electron microscopy analysis of more than 500 eosinophils revealed viable and cytolytic eosinophils but not apoptosis, secondary necrosis, or engulfment of eosinophils. Plasma cells but neither epithelial cells nor eosinophils exhibited apoptotic ultrastructural morphology. Eosinophils in vitro exhibited different stages of apoptosis, ending with secondary necrosis distinct from in vivo eosinophil cytolysis. Our results show that the clearance of eosinophils from nasal polyps largely occurs through nonapoptosis pathways, including cytolysis and paraepithelial migration, and they challenge the belief that apoptosis is important for clearance of eosinophils from respiratory tissues.

Single base oligodeoxyguanosine upregulates Fas ligand release by nonspecific cytotoxic cells.

Nonspecific cytotoxic cells (NCC) are a type of teleost NK-like cell. In the present study a novel stimulus secretion model is described for catfish NCC utilizing single base oligodeoxyguanosine. Binding of guanosine 20-mers (dG20) to NCC up-regulated expression of cytosolic FasL detected by an anti-human FasL monoclonal antibody (mab). In vitro treatment of purified NCC with dG20 produced a 7-fold increase in expression of soluble Fas ligand (sFasL) after 3 h. Antibody binding to NCC was saturable and approximately 30-35% of total NCC were positive for sFasL expression. The teleost FasL equivalent produced programmed cell death of appropriate FasR positive targets. Supernatants from dG20 activated NCC produced hypoploidy and annexin-V binding by FasR bearing HL-60 cells. Treatment of activated supernatants with immobilized anti-FasL mab neutralized these activities. These studies demonstrated that an NK like cell (NCC) produces and secretes sFasL following binding by single base oligodeoxyguanosine.

Cathepsin-dependent apoptosis triggered by supraoptimal activation of T lymphocytes: a possible mechanism of high dose tolerance.

High doses of Ag can paradoxically suppress immune responses in vivo. This Ag-specific unresponsiveness (termed high dose tolerance) involves extrathymic mechanisms in mature T lymphocytes. To investigate these mechanisms, we used the in vitro model of PBL activated with anti-CD3 or PHA. In these conditions, increasing mitogen concentrations resulted in a reduction of the proliferative response, associated with an increased percentage of apoptotic cells. Apoptosis did not require prior exposure to IL-2, it was not the consequence of CD178/CD95 or TNF/TNFR interactions, and was therefore clearly distinct from activation-induced cell death. Although the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) decreased DNA fragmentation, cytochrome c release and caspase-9 and caspase-3 activation were not implicated, suggesting that this apoptosis did not primarily involve the intrinsic mitochondrial pathway. E64d, a cysteine protease inhibitor, as well as specific inhibitors of cathepsin B and cathepsin L conferred protection. We further demonstrated that cathepsin B and cathepsin L were released from the lysosomes and catalytically active in the cytosol. Release of cathepsin B and cathepsin L was the consequence of lysosomal membrane permeabilization without complete disruption of the cytosol-lysosome pH gradient. These results demonstrate a role for cathepsins in supraoptimal activation-induced apoptosis in vitro and suggest their possible participation in high dose tolerance in vivo.

Listeriolysin O from Listeria monocytogenes is a lymphocyte apoptogenic molecule.

Infection of mice with Listeria monocytogenes caused marked lymphocyte apoptosis in the white pulp of the spleen on day 2 postinfection. We prove in this study that listeriolysin O (LLO), a pore-forming molecule and a major virulence factor of Listeria, could directly induce murine lymphocyte apoptosis both in vivo and in vitro at nanomolar and subnanomolar doses. Induction of apoptosis by LLO was rapid, with caspase activation seen as early as 30 min post-treatment. T cells lost their mitochondrial membrane potential and exposed phosphatidylserine within 8 h of treatment. Incubation of lymphocytes with a pan-caspase inhibitor blocked DNA laddering and caspase-3 activation, but did not block phosphatidylserine exposure or loss of mitochondrial membrane potential. We describe a novel function for LLO: induction of lymphocyte apoptosis with rapid kinetics, effected by both caspase-dependent and -independent pathways.

ECH, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity.

CTL eliminate cells infected with intracellular pathogens and tumor cells by two distinct mechanisms mediated by Fas ligand (FasL) and lytic granules that contain perforin and granzymes. In this study we show that an epoxycyclohexenone derivative,(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one (ECH) specifically inhibits the FasL-dependent killing pathway in CTL-mediated cytotoxicity. Recently, we have reported that ECH blocks activation of procaspase-8 in the death-inducing signaling complex and thereby prevents apoptosis induced by anti-Fas Ab or soluble FasL. Consistent with this finding, ECH profoundly inhibited Fas-mediated DNA fragmentation and cytolysis of target cells induced by perforin-negative mouse CD4+ CTL and alloantigen-specific mouse CD8+ CTL pretreated with an inhibitor of vacuolar type H+-ATPase concanamycin A that selectively induces inactivation and proteolytic degradation of perforin in lytic granules. However, ECH barely influenced perforin/granzyme-dependent DNA fragmentation and cytolysis of target cells mediated by alloantigen-specific mouse CD8+ CTL. The components of lytic granules and the granule exocytosis pathway upon CD3 stimulation were also insensitive to ECH. In conclusion, our present results demonstrate that ECH is a specific nonpeptide inhibitor of FasL-dependent apoptosis in CTL-mediated cytotoxicity. Therefore, ECH can be used as a bioprobe to evaluate the contributions of two distinct killing pathways in various CTL-target settings.

Natural human polyreactive IgM induce apoptosis of lymphoid cell lines and human peripheral blood mononuclear cells.

Natural polyreactive IgM autoantibodies, encoded by unmutated germline Ig V genes, represent a major fraction of the normal circulating IgM repertoire. We have previously shown that therapeutic preparation of pooled IgM exerts immunomodulatory effects as assessed by in vitro and in vivo studies. Here, we show that the IgM preparation induces cell death in lymphoblastoid cell lines and in human peripheral blood mononuclear cells. The IgM-induced cell death involved classical features of apoptosis such as nuclear fragmentation and activation of caspases. Treatment of leukemic cells with IgM resulted in the cleavage of poly-(A)DP ribose polymerase, a substrate of caspase, and in a reduction in mitochondrial transmembrane potential during the early period of apoptosis induction. Natural IgM-induced apoptosis was inhibited by soluble Fas molecules and affinity-purified Fas antibodies from pooled IgM preparation induced apoptosis in lymphoblastoid cells, suggesting the involvement of the Fas receptor. Our results suggest a role for normal IgM in controlling cell death and proliferation, and imply a possible therapeutic role for IgM in autoimmune and lymphoproliferative disorders.

Extremely rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro.

Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin's lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab')(2) of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-kappaB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.

Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai.

Larval endoparasitoids can avoid the immune response of the host by the function of polydnavirus (PDV) and venom. PDV infects hemocytes and affects the hemocyte function of the host. In this paper, we investigated how PDV and venom affect the hemocyte population of the host. Cotesia kariyai, the larval endoparasitoid, lowers the hemocyte population of the noctuid host larvae soon after parasitization. The reduction in the number of circulating hemocytes is caused by the breakdown of the circulating hemocytes and of the hematopoietic organ which generates the circulating hemocytes. The decrease in the number of hemocytes shortly after parasitization is a response to the venom. However, the decrease in hemocyte population on and after 6 h post-parasitization appears to be caused by the PDV. Apoptosis in circulating hemocytes was observed on and after 6 h post-injection of PDV plus venom. It was revealed through cytometry that mitosis of circulating hemocytes was halted within 24 h after the injection of PDV plus venom. Apoptosis in the hematopoietic organ was induced 12 h after the injection of PDV plus venom. Furthermore, the plasma from the hosts injected with PDV plus venom depressed the number of hemocytes released from the hemotopoiteic organs.

DNA fragmentation and detachment of enterocytes induced by anti-CD3 mAb-activated intraepithelial lymphocytes.

To elucidate the role of intraepithelial lymphocytes (IEL) and enterocytes in the defense mechanism of the small intestine, we designed experiments to stimulate the IEL by anti-CD3epsilon, anti-TCRalphabeta, or anti-TCRgammadelta monoclonal antibodies (mAbs), and to examine the subsequent changes to the enterocytes. The enterocytes of the duodenum and jejunum, but not of the ileum, showed massive DNA fragmentation 30 min after intraperitoneal injection of anti-CD3 mAb. These responses were also induced by anti-TCRgammadelta mAb, but not by anti-TCRalphabeta mAb, and were completely inhibited by cyclosporin A. Nearly half of the enterocytes of the villi in the duodenum and jejunum were exfoliated into the lumen 4 h after the injection of the mAb. Administration of anti-CD3 mAb also induced DNA fragmentation in Fas-deficient MRL/lpr mice, indicating that the Fas-Fas ligand system was not involved in these events. The anti-CD3 mAb treatment also induced massive DNA fragmentation in the intestinal epithelium of the duodenum and jejunum in TNF-receptor-1-deficient mice, whereas TNF-alpha induced only the detachment of intestinal epithelium of wild-type mice, implying the dissociation of two independent factors and/or mechanisms for DNA fragmentation and the subsequent epithelial cell detachment in the murine duodenum and jejunum. The mAb failed to exfoliate the epithelium in TNF-R1-deficient mice. Thus, TCRgammadelta(+) IEL, when treated with anti-CD3 or anti-TCRgammadelta mAbs, induced rapid DNA fragmentation and subsequent detachment of the duodenal and jejunal epithelia, but not in the ileum ("the silent ileum"), partly because of the paucity of TCRgammadelta(+) IELs in the ileum.