Electroacupuncture suppresses surgical trauma stress-induced lymphocyte apoptosis in rats

Cumulative evidences suggested that electroacupuncture (EA) could modulate immune function, but the mechanism needs further study. In the present study, the effect of EA on surgical trauma stress-induced lymphocyte apoptosis was investigated by using DNA gel electrophoresis, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and Western blot for Fas protein expression. The results showed that rats with surgical trauma stress exhibited a significant reduction in splenic cellularity. Increase in apoptotic cell death and Fas (CD95/Apo-1) expression in splenic lymphocytes was also observed. EA could suppress the increase of apoptosis and Fas protein expression in splenic lymphocytes induced by the surgical trauma stress. These results implied that EA could decrease splenic lymphocytes apoptosis via inhibiting Fas protein expression; consequently prevent deleterious immunological changes in the post-operative state.     

Apoptosis patterns in experimental Taenia solium and Taenia crassiceps strobilae from golden hamsters

Apoptosis or programmed cell death (PCD) patterns of two taeniid species, Taenia solium and Taenia crassiceps, were explored in adult tapeworms grown in golden hamsters. Animals were fed either ten viable T. solium cysticerci from naturally infected pigs or from T. crassiceps WFU strain maintained in Balb/c mice. Adult strobilae were recovered from the intestine at different times after infection and either frozen at –70°C or fixed in paraformaldehyde–glutaraldehyde. Frozen sections were processed using the DNA fragmentation fluorescent TUNEL reagents and examined in an epifluorescent microscope. Fixed tissues were processed for light and electron microscopy. Typical apoptotic cells were found in the central core of scolex and strobilar tissues, mainly in the germinal tissue and subtegumentary areas. By the TUNEL technique, cells exhibited the characteristic fluorescent images of condensed nuclear chromatin. By light microscopy of thick sections stained with toluidine blue, we found a number of small rounded cells which had lost their cytoplasmic bridges and had shrunken nuclei with aggregated chromatin, cells which were found interspersed with normal syncytial cells. Similar cell morphology was confirmed by electron microscopy. Stunted viable worms, recovered with longer mature specimens, had very short strobilae and exhibited a large number of apoptotic cells in the germinal neck tissues. The results are consistent with the syncytial nature of these parasites, and strongly suggest that cell proliferation and PCD in these adult cestodes are continuous processes of the germinal tissue and tegumentary cytons.     

Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.     

Anoikis potential of Entameba histolytica secretory cysteine proteases: evidence of contact independent host cell death

Mammalian epithelial, endothelial and various other cell types, upon their detachment from the extracellular matrix (ECM) undergo a specialized kind of apoptosis, known as anoikis. Entameba histolytica cysteine proteases have been implicated in degradation of the host ECM, which may induce anoikis in host cells. To explore this hypothesis, supernatant obtained from 2 h in-vitro cultivation of E. histolytica (SRP), was used as a source of cysteine proteases. MDA-MB-231 (human mammary epithelial adenocarcinoma) cells were treated with SRP and their detachment and apoptosis was evaluated. 25 μg/ml (with respect to protein concentration), SRP was found to be the optimal concentration to dislodge over 98% MDA-MB-231 cells from monolayer in 20 min. The detachment was followed by apoptosis of at least 41.2% cells, characterized by caspase-3 dependent inter-nucleosomal DNA fragmentation. The SRP-induced apoptosis was associated exclusively with the detached fraction. Moreover, detachment preceded apoptosis. E-64 (a cysteine protease inhibitor) abolished the SRP-induced detachment as well as inter-nucleosomal DNA fragmentation. Interestingly, SRP induced a 3.21 fold increase in the JNK activity, whilst SP600125 (a JNK inhibitor) blocked the SRP-induced inter-nucleosomal DNA fragmentation. Thus, it was concluded that spontaneously released cysteine proteases of E. histolytica can induce JNK dependent anoikis of MDA-MB-231 cells, which may be implicated in contact independent host cell death during amebiasis.     

Apoptosis induction by glucuronoxylomannan of Cryptococcus neoformans.

We studied the ability of glucuronoxylomannan (GXM), the major constituent of Cryptococcus neoformans capsular polysaccharide, to induce apoptosis in lymphocytes from normal rats. Spleen mononuclear cells (Smc) from normal rats treated with GXM for 24 h exhibited, in comparison with controls, an increased hypodiploidy in the DNA profile after staining with propidium iodide, as well as increased ladder-type DNA fragmentation in agarose gel electrophoresis and a high number of positive cells in the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) reaction. Furthermore, increased hypodiploidy in the DNA profile was also observed in Smc expressing T-cell receptor (TCR +). We also studied the induction of apoptosis in lungs and spleens from rats in the immunosuppressor period of disseminated cryptococcosis. TUNEL labeling of lungs and spleens from rats obtained 14 days after infection with C. neoformans showed a large number of apoptotic cells. Our results provide strong cytometric, molecular and morphological evidence that apoptosis could be a previously unrecognized immunosuppressive property of GXM in vitro. This programmed cell death may be involved in the immunosuppression observed during C. neoformans infection.     

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).     

Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.      

Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes after Thermal Injury

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72°C for 1 second. After exposure to temperatures of 58 to 59°C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66°C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72°C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59°C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.     

Programmed cell death in the interdigital tissue of the fetal mouse limb is apoptosis with DNA fragmentation

Background: Programmed cell death is an essential event during mammalian morphogenesis which eliminates unnecessary cells to accomplish histogenesis and organogenesis. Cell death in interdigital spaces of the developing limb is a classical example of morphogenetic cell death. We investigated whether classical programmed cell death in the interdigital tissue of the developing limb in mice is apoptosis with fragmentation of nuclear DNA and also examined sequentially the occurrence of programmed cell death and cell proliferation in the developing limb of mouse fetuses to analyze their interrelation. Methods: We examined the occurrence of apoptotic cell death in the developing limbs of mouse fetuses by using Nile blue sulphate staining, agarose gel electrophoresis for detecting DNA laddering, and a cytochemical labeling of DNA fragmentation. We also labeled proliferating cells using BrdU/anti-BrdU immunohistochemistry and examined the interrelation between apoptotic programmed cell death and cell proliferation. Results: DNA ladders, a biochemical evidence of apoptosis, were detected in DNA extracts from the interdigital tissue of day 13 mouse fetuses by agarose gel electrophoresis. Programmed cell death and DNA fragmentation were detected by Nile blue staining and cytochemical labeling of DNA fragmentation, respectively, in the interdigital mesoderm and in the regions of presumptive joints of the digit. BrdU/anti-BrdU immunohistochemistry for identifying proliferating S-phase cells revealed that interdigital mesenchymal cells cease DNA synthesis before programmed cell death and DNA fragmentation begin. Conclusions: We confirmed that both cytological apoptotic alterations and fragmentation of nuclear DNA occur in the interdigital tissue and presumptive joint areas of fetal mouse limbs, and they appear to play a significant role in the separation of digits as well as the formation of joint cavities. © 1995 Wiley-Liss, Inc.     

Triggering via the alternative CD2 pathway induces apoptosis in activated human T lymphocytes

Activation of immature thymocytes or transformed (i.e. leukemic) T lymphocytes via CD3/T cell receptor (TcR) signaling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TcR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral LT cells. We now report that interleukin-2 (IL-2) dependent human polyclonal T cell lines as well as T cell clones undergo programmed cell death when triggered via the alternative CD2-dependent activation pathway. In the presence of exogenous IL-2, a pair of mitogenic anti-CD2 mAb suppressed the IL-2-driven proliferative response. Growth inhibition was associated with cell death and DNA fragmentation as revealed by propidium iodide staining and gel electrophoresis, respectively. Induction of apoptosis by anti-CD2 mAb was prevented by cyclosporine A and FK 506. We conclude that programmed cell death can be initiated in activated human T cells by signaling via the CD2 pathway.     

Impairment of the inflammatory reaction on implanted Taenia solium metacestodes in mice by a T. solium RNA-peptide: a scanning electron microscopy study

Inhibition of inflammation by a Taenia solium RNA-peptide (metacestode factor, MF) was studied by scanning electron microscopy (SEM). Viable (96%) T. solium metacestodes obtained from a naturally infected pig were dissected and implanted in treated and control mice, removed at 6 and 12 days postimplantation (p.i.), and studied by SEM. At day 6, metacestodes in control mice showed vigorous inflammation, whereas in mice treated with MF they were apparently intact with exiguous inflammation. Mice immunized with T. solium metacestode antigens showed a moderate inflammation; those treated with both MF and T. solium antigens presented scanty inflammation. At day 12, metacestodes presented copious inflammation and severe damage to the sucker tissues in mice immunized with T. solium; in mice treated with either MF or MF and T. solium antigens there was only discrete inflammation. These observations illustrate the central role of MF in the inhibition of the early events leading to the parasite's destruction by means of an inflammatory response. Received: 17 February 1997 / Accepted: 19 August 1997     

Streptococcal Pyrogenic Exotoxin B Induces Apoptosis and Reduces Phagocytic Activity in U937 Cells

Treatment of U937 human monocyte-like cells with Streptococcus pyogenes led to an induction of apoptosis in these cells. A comparison between the wild-type strain and its isogenic protease-negative mutant indicated that the production of streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, caused a greater extent of apoptosis in U937 cells. Further study using purified SPE B showed that this protease alone could induce U937 cells to undergo apoptosis, which was characterized by morphologic changes, DNA fragmentation laddering on the gel, and an increase in the percentages of hypodiploid cells. The protease activity of SPE B was required for apoptosis to proceed, since treatment with cysteine protease inhibitor E64 or heat inactivation abrogated this death-inducing effect. The SPE B-induced apoptosis pathway was interleukin-1β converting enzyme (ICE) family protease dependent. Further experiments showed that the phagocytic activity of U937 cells was reduced by SPE B. Treatment with E64 and heat inactivation both abrogated this phagocytosis-inhibitory effect. Taken together, the present data show that SPE B not only possesses the ability to induce apoptosis in monocytic cells but also helps bacteria to resist phagocytosis by host cells.     

Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.     

Transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures

Summary.  Transmissible gastroenteritis virus (TGEV) is a coronavirus which causes severe gastroenteritis and atrophy of intestinal villous epithelial cells in piglets. However, the mechanism of cell death caused by TGEV is not known. In this study, we report that TGEV induces cell death by apoptosis. TGEV-induced apoptosis was demonstrated by agarose gel electrophoresis, electron microscopy, and terminal deoxytransferase digoxigenin-dUTP nick end labeling (TUNEL). Double labeling experiment confirmed the result from electron microscopy and showed that most of the apoptotic cells were bystander cells as they were negative for TGEV nucleic acids. Results of this study indicate that TGEV induces apoptosis in vitro and that most of the cells undergoing apoptosis are bystander cells, thus amplifying the cytopathic effect of TGEV. Received February 23, 1998 Accepted June 8, 1998     

Multiparametric assessment of bursal lymphocyte apoptosis.

When bursal lymphocytes are placed in cell culture, they undergo an apoptotic form of cell death that can be inhibited by phorbol esters and protein synthesis inhibitors. The goal of the current study was to evaluate the time course of this process and the inhibition of this process using several different assays to detect apoptosis: (1) terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) of lymphocyte DNA strand breaks with dUTP-FITC; (2) propidium iodide (PI) staining of lymphocyte chromatin; (3) chloromethyl-x-rosamine (CMX-Ros) binding to lymphocyte mitochondria; (4) merocyanine-540 (MC-540) binding to the lymphocyte plasma membrane; (5) flow cytometric analysis of light scatter from lymphocytes; (6) analysis of genomic DNA from lymphocytes by agarose gel electrophoresis; and (7) cellular caspase-3 activity of lymphocytes. When bursal lymphocyte apoptosis was analyzed as a function of time, or inhibited by phorbol esters or cycloheximide, all of these assays corroborated the apoptotic process. However, treatment of lymphocytes with a cytotoxic level of the proteinase inhibitor, n-ethylmaleimide (NEM) resulted in a putative, necrotic form of cell death that revealed discrepancies among the various assays in the detection of apoptotic cells. Specifically, the CMX-Ros and MC-540 assays erroneously detected the necrotic cells as being apoptotic cells following NEM treatment. These findings indicate the need for additional assays and appropriate treatment controls to verify the apoptotic process when using the CMX-Ros and MC-540 assays.     

Pseudorabies virus induces apoptosis in tissue culture cells

Summary.  The process of cell death as a result of exposure to pseudorabies virus (PRV) in cultured cells was examined and specific features characteristic of apoptosis were observed. At early times of infection, externalization of membrane phospholipid phophatidylserine was detected by flow cytometry analysis. During the infection process, caspase 3-like protease activity was induced and the activity increased in a time dependent manner. Cellular DNA degradation was demonstrated by agarose gel electrophoresis. Morphologic changes of the nucleus that included chromatin condensation and margination to the periphery of the nucleus were evident in electron microscopy analysis. These biochemical and morphologic changes demonstrated that, during PRV replication, the host cell was induced to undergo apoptosis. Received March 15, 2000 Accepted May 10, 2000     

Purification and characterisation of an extracellularly released protease of Trypanosoma brucei

Thrombocytopaenia, or platelet aggregation, is a serious complication of African trypanosomiasis. The biochemical basis is not clearly known. Proteases are known potent inducers of blood coagulation and platelet aggregation, and unknown factors released by Trypanosoma brucei have been shown to induce platelet aggregation. In attempts to define the biochemical mechanisms involved in thrombocytopaenia we purified and characterised a major proteolytic enzyme released extracellularly by T. brucei. Actively motile trypanosomes released proteins into the medium (phosphate/saline/glucose, pH 8.0) in which the organisms were incubated in vitro. The M_r of the released polypeptides ranged from 15 to >200 kDa, amongst which are proteases. One of the major protein bands, a 250 kDa protease, was purified to homogeneity by ammonium sulfate precipitation followed by diethylaminoethyl (DEAE)-cellulose chromatography and Sephacryl S-300 gel filtration. The protease migrated as a single band of 63 kDa upon electrophoresis in both denaturing and non-denaturing gel co-polymerised with gelatin. The enzyme was strongly active against Z-ARR-AFC peptide substrate, with a pH optimum of 7.0. The proteolytic activity was enhanced by dithiothreitol and inhibited by E-64, leupeptin, TPCK and antipain. The released proteolytic enzyme is putatively identified as a cathepsin B-like cysteine protease. Received: 18 May 1998 / Accepted: 30 September 1998     

Kainic acid-induced seizures produce necrotic, not apoptotic, neurons with internucleosomal DNA cleavage: implications for programmed cell death mechanisms

Prolonged seizures (status epilepticus) induced by kainic acid activate programmed cell death mechanisms, and it is believed that kainic acid-induced status epilepticus induces neuronal apoptosis. In order to test this hypothesis, adult rats were subjected to 3-h kainic acid-induced seizures, with 24- or 72-h recovery periods. Neuronal death was assessed by light microscopy with the Hematoxylin and Eosin stain and with in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL stain), by electron microscopy, and by agarose gel electrophoresis of DNA extracted from five vulnerable brain regions. Spontaneous and MK-801-induced apoptotic neurons from retrosplenial cortex of neonatal rats, evaluated by light and electron microscopy, were used as positive controls for apoptosis. Surprisingly, the large chromatin clumps of apoptotic neurons were TUNEL negative, whereas the cytoplasm showed light-to-moderate TUNEL staining, consistent with a lack of identifiable nuclear membranes ultrastructurally, and with intermingling of nuclear and cytoplasmic contents. Ultrastructurally, the acidophilic neurons produced by kainic acid-induced status epilepticus, identified with Hematoxylin and Eosin stain, were dark, shrunken and necrotic, with pyknotic nuclei containing small, dispersed chromatin clumps, and with cytoplasmic vacuoles, some of which were swollen, disrupted mitochondria. No apoptotic cells were seen. Acidophilic neurons were found in up to 20 of 23 brain regions examined and comprised 10-25% of the total number of neurons examined. A subset of these neurons (<10% of the total number of neurons in five of 23 regions) had TUNEL-positive nuclei 72h but not 24h after status epilepticus. Internucleosomal DNA cleavage (DNA "laddering") occurred in the four most damaged brain regions examined by electron microscopy 24h after SE and the three most damaged regions 72h after status epilepticus.Our results demonstrate that kainic acid-induced status epilepticus produces neuronal necrosis and not apoptosis in adult rats. The necrotic neurons show nuclear pyknosis, chromatin condensation and DNA laddering. Programmed cell death mechanisms activated by kainic acid-induced status epilepticus occur in neurons which become necrotic and could contribute to necrotic, as well as apoptotic, neuronal death.     

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-α and interferon-γ

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), we investigated the effects of these two cytokines on death of luteal cells in vitro. Methods: Mouse luteal cells were cultured in serum-free medium with TNF-α at 0,500,1,000,3,000, or 5,000 U/ml in the presence or absence of IFN-γ at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3′ end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. Results:On day 3 of culture, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500–5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-γ (1,000 U/ml) and TNF-α (5,000 U/ml) did. On day 6, IFN-γ alone at 1,000 U/ml or TNF-α alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-α alone at 5,000 U/ml did, and combinations of IFN-γ and TNF-α at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-α. On days 3–6 of culture, combinations of IFN-γ and TNF-α that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. Conclusions: The presence of IFN-γ modulates the ability of TNF-α to induce a reduction in the number of viable cells, although TNF-α alone at high concentrations can induce a reduction in the number of viable cells. © 1995 Wiley-Liss, Inc.