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Reduced megakaryocyte (MK) apoptosis and insufficient platelet production play important roles in the pathogenesis of immune thrombocytopenia (ITP). The contribution of plasma-derived exosomes to the decreased platelet count in ITP has not been entirely understood. Here, we found the percentage of apoptotic MKs in patients with ITP was significantly lower than those in healthy volunteers. In the presence of ITP plasma-derived exosomes (ITP-Exo), the apoptosis of MKs was reduced during the process of MK differentiation in vitro, which contributed to the reduced platelet production by Bcl-xL/caspase signaling. Furthermore, in vivo study demonstrated that ITP-Exo administration led to significantly delayed platelet recovery in mice after 3.5 Gy of irradiation. All these findings indicated that ITP-Exo, as a regulator of platelet production, impaired MK apoptosis and platelet production through Bcl-xL/caspase signaling, unveiling new mechanisms for reduced platelet count in ITP., Competing Interests: None declared., (Thieme. All rights reserved.)

Crimean-Congo hemorrhagic fever (CCHF) is a tick-mediated viral infection. Patients with CCHF may show various clinical presentations. The cause of this difference in the clinical course is not completely understood. Apoptosis is programmed cell death and plays an important role in regulating the immune system. Our knowledge of the role of apoptosis in CCHF disease is limited. We investigated the role of apoptosis and their relationship with the severity of the disease in CCHF. Thus, in 30 patients with CCHF and 30 healthy individuals, we analyzed the serum levels of cytochrome C, apoptotic protease activating factor-1 (Apaf 1), caspase 3, caspase 8, caspase 9, sFas, sFasL, perforin, granzyme B, and CK18 by enzyme-linked immunosorbent assay. This is the first study that research the serum levels of the mentioned apoptosis markers in adult patients with CCHF. We found that the serum levels of sFasL, cytochrome C, Apaf 1, caspase 3, caspase 8, caspase 9, perforin, granzyme B, and M30 were statistically significantly different in the acute phase of the disease compared with healthy individuals and patients in convalescent period. There was no association between the clinical severity of the disease and apoptosis markers. In conclusion, the results of our study suggested that the extrinsic and intrinsic apoptosis pathway play an important role in CCHF., (© 2019 Wiley Periodicals, Inc.)

Background: Cardiorenal syndrome type 1 (CRS type 1) is characterized by a rapid worsening of cardiac function leading to acute kidney injury (AKI). Its pathophysiology is complex and not completely understood. In this study, we examined the role of apoptosis and the caspase pathways involved., Material and Methods: We enrolled 40 acute heart failure (AHF) patients, 11 of whom developed AKI characterizing CRS type 1. We exposed the human cell line U937 to plasma from the CRS type 1 and AHF groups and then we evaluated apoptotic activity by annexin-V evaluation, determination of caspase-3, -8 and -9 levels, and BAX, BAD, and FAS gene expression., Results: We observed significant upregulation of apoptosis in monocytes exposed to CRS type 1 plasma compared to AHF, with increased levels of caspase-3 (p < 0.01), caspase-9 (p < 0.01), and caspase-8 (p < 0.03) showing activation of both intrinsic and extrinsic pathways. Furthermore, monocytes exposed to CRS type 1 plasma had increased gene expression of BAX and BAD (intrinsic pathways) (p = 0.010 for both). Furthermore, strong significant correlations between the caspase-9 levels and BAD and BAX gene expression were observed (Spearman ρ = - 0.76, p = 0.011, and ρ = - 0.72, p = 0.011)., Conclusion: CRS type 1 induces dual apoptotic pathway activation in monocytes; the two pathways converged on caspase-3. Many factors may induce activation of both intrinsic and extrinsic apoptotic pathways in CRS type 1 patients, such as upregulation of proinflammatory cytokines and hypoxia/ischemia. Further investigations are necessary to corroborate the present findings, and to better understand the pathophysiological mechanism and consequent therapeutic and prognostic implications for CRS type 1., (© 2018 S. Karger AG, Basel.)

Context: Apoptotic dysregulation plays a role in the pathogenesis of polycystic ovary syndrome (PCOS)., Objective: To evaluate circulatory apoptotic markers and oxidative stress in patients with PCOS., Materials and Methods: Forty-four women with PCOS, and 44 healthy women as controls were enrolled in the study. Oxidative stress parameters and caspases levels were measured in serum., Results: The caspase 9 level was significantly lower and related with oxidant status in patients with PCOS, while the circulating levels of caspases 3 and 7 were statistically similar in both groups., Discussion: This study is the first report demonstrating the circulating levels of apoptotic markers and their relationship with oxidant status in PCOS., Conclusion: The circulating caspase 9 and oxidant status might contribute to apoptotic dysregulation in PCOS.

Objective: Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo., Approach and Results: Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus., Conclusions: Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus., (© 2017 American Heart Association, Inc.)

Increased apoptotic cell death in uremic patients has been confirmed by a variety of studies. The present study aimed to investigate the effect of uremic toxins and duration of hemodialysis (HD) therapy on apoptosis by means of measuring serum caspase cleaved CK18 (CCCK-18) levels. Seventy chronic HD patients were recruited and divided into three groups with differing periods of HD, from 6 months to 10 years. Twelve healthy subjects served as controls. Serum CCCK-18 level was found significantly higher in HD patient groups (Group 2; 189 ± 71 IU/L, Group 3; 182 ± 65 IU/L, Group 4; 204 ± 111 IU/L) as compared to the control group (122 ± 20 U/L) (P < 0.05). When all hemodialysis patients considered together serum CCCK-18 showed positive correlation with serum uric acid and phosphorus (P < 0.05). In conclusion, our results suggest that apoptosis is enhanced in HD patients, phosphorus and uric acid might play a role in this increment, but duration of HD therapy has no effect on apoptosis., (© 2017 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

Patients with myelodysplastic syndromes (MDS) have a defect in the differentiation of bone marrow multipotent progenitor cells. Thrombocytopenia in MDS patients may be due to premature megakaryocyte death, but platelet apoptotic mechanisms may also occur. This study aimed to study function and apoptotic state of platelets from MDS patients with different platelet count. Reticulated platelets, platelet activation, activated caspases and annexin-V binding were evaluated by flow cytometry. Pro-apoptotic Bax and Bak proteins were determined by western blots and plasma thrombopoietin by ELISA. Microparticle-associated procoagulant activity and thrombin generation capacity of plasma were determined by an activity kit and calibrated automated thrombography, respectively. High plasma thrombopoietin levels and low immature circulating platelet count showed a pattern of hypoplastic thrombocytopenia in MDS patients. Platelets from MDS patients showed reduced activation capacity and more apoptosis signs than controls. Patients with the lowest platelet count showed less platelet activation and the highest extent of platelet apoptosis. On this basis, patients with thrombocytopenia should suffer more haemorrhagic episodes than is actually observed. Consequently, we tested whether there were some compensatory mechanisms to counteract their expected bleeding tendency. Microparticle-associated procoagulant activity was enhanced in MDS patients with thrombocytopenia, whereas their plasma thrombin generation capacity was similar to control group. This research shows a hypoplastic thrombocytopenia that platelets from MDS patients possess an impaired ability to be stimulated and more apoptosis markers than those from healthy controls, indicating that MDS is a stem cell disorder, and then, both number and function of progeny cells, might be affected.

Introduction: Chronic obstructive pulmonary disease (COPD) is a worldwide burden and a major cause of death. The disease is accompanied by chronic inflammation and increased cellular turnover that is partly due to an overwhelming induction of apoptosis. In this study, we hypothesized that systemic markers of apoptosis are altered in patients with mild-to-severe COPD., Materials and Methods: A total number of 64 patients and controls were enrolled in the study. Lung function parameters of all groups (nonsmoker, healthy smoker, COPD GOLD I&II, COPD GOLD III&IV) were evaluated at the time of inclusion. Enzyme-linked immunosorbent assays were used to quantify protein levels in serum samples., Results: Serum contents of apoptotic end-products caspase-cleaved cytokeratin-18 and histone-associated-DNA-fragments were increased in patients with COPD, whereas anti-inflammatory soluble ST2 showed a peak in patients with COPD I&II (P=0.031) compared to healthy smokers. Levels of pro-inflammatory caspase-1/ ICE correlated significantly with the number of pack years (R=0.337; P=0.007)., Discussion: Our results indicate a systemic release of apoptosis-specific proteins as markers for increased cellular turnover accompanied by progression of COPD. Furthermore, soluble ST2 seems to have a critical role in the anti-inflammatory regulatory mechanism at early stages of the disease.

The effects of gamma-hexachlorocyclohexane, one of the endocrine disrupters, widely used for agricultural and medicinal purpose, on apoptosis in PC12 cells were investigated using western blotting analysis and reverse transcriptase-DNA polymerase chain reaction (RT-PCR) method. Apoptosis is a fundamental process necessary for development of individuals and organs. Although gamma -HCH at high concentration did not have an effect on cell viability and apoptosis, DNA fragmentation was slightly enhanced, and apoptotic factors; Bax, Bad, cytochrome c and caspase-3 showed tendency to increase by the addition of a low dose of gamma-HCH to the cell medium. However these changes were not statistically significant. It was concluded that gamma -HCH did not affect on apoptosis in the PC12 cell line system, although gamma -HCH has been reported to induce apoptosis in other cell lines.

Background: Disturbed polymorphonuclear leukocyte (PMNL) apoptosis contributes to the dysregulation of the non-specific immune system in uraemia. Intracellular Ca(2+) modulates PMNL apoptotic cell death. We investigated the effect of para-hydroxy-hippuric acid (PHA), an erythrocyte plasma membrane Ca(2+)-ATPase inhibitor accumulating in uraemic sera, and of cyclopiazonic acid (CPA), an inhibitor of the sarko/endoplasmatic Ca(2+)-ATPase, on PMNL apoptosis., Methods: Apoptosis of PMNLs from healthy subjects and from haemodialysis (HD) patients was assessed after incubation for 20 h by evaluating morphological features under the fluorescence microscope and by measuring the DNA content and caspase activities by flow cytometry. The intracellular calcium concentration ([Ca(2+)](i)) was determined by measurement of fura-2 fluorescence using the 340/ 380 nm dual wavelength excitation., Results: Spontaneous apoptosis of PMNLs from healthy subjects and from HD patients did not differ. PHA significantly attenuated, while CPA increased, the apoptotic cell death of PMNLs from healthy subjects. The PHA effect was not observed with PMNLs from HD patients, irrespective of whether the blood was drawn before or after HD treatment. Baseline [Ca(2+)](i) was increased in PMNLs obtained from HD patients before dialysis but reversed after dialysis. The PHA effects were not mediated via [Ca(2+)](i). The chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induced a [Ca(2+)](i) increase and reduced PMNL survival. Extracellular Ca(2+) did not affect CPA- and fMLP-induced apoptosis., Conclusions: PHA, without affecting [Ca(2+)](i), attenuates apoptosis of healthy but not of uraemic PMNLs. CPA and fMLP enhance PMNL apoptosis independently of Ca(2+) influx.

Caspase-cleaved proteins are released from disintegrated apoptotic cells and can be detected in the circulation. We here addressed whether caspase-cleaved cytokeratin 18 (CK18-Asp396) can be used as a serum biomarker for assessment of the clinical efficiency of chemotherapy in hormone-refractory prostate cancer (HRPC). A total of 82 patients with HRPC were evaluated during 751 treatment cycles, either with estramustine (EMP)/vinorelbine or with EMP/docetaxel. The levels of CK18-Asp396 and of total CK18 were measured in patient serum before and during therapy by ELISA. Docetaxel induced significant increases in serum CK18-Asp396 (P<0.0001) and total CK18 (P<0.0002), suggesting induction of apoptosis. Similarly, vinorelbine induced increases in both CK18-Asp396 and CK18 (P<0.001 and 0.011). In contrast, EMP induced increases in total serum CK18 (P<0.0001), but not in CK18-Asp396 (P=0.13). The amplitudes of docetaxel-induced increases were associated with baseline prostate-specific antigen (PSA) and CK18 serum levels in these patients, consistent with tumoral origin of caspase-cleaved fragments. Docetaxel induced significant increases in CK18-Asp396 during second-, third- and fourth-line therapy and induced increased levels of CK18-Asp396 during treatment cycles 1-8. In contrast, vinorelbine induced significant increases only during cycles 1-3. In a subgroup of 32 patients that received EMP/vinorelbine in second line followed by EMP/docetaxel in third line, docetaxel induced stronger increases than vinorelbine (P=0.008). These results show that the CK18-Asp396 serum marker can be used to assess tumour apoptosis in vivo and suggest that the clinical efficiency of docetaxel in HRPC is due to induction of apoptosis during multiple treatment cycles.

Changes in the oxidative status of erythrocytes can reduce cell lifetime, oxygen transport, and delivery capacity to peripheral tissues and have been associated with a plethora of human diseases. Among reactive oxygen and nitrogen species of importance in red blood cell (RBC) homeostasis, superoxide and nitric oxide radicals play a key role. In the present work, we evaluated subcellular effects induced by peroxynitrite, the product of the fast reaction between superoxide and nitric oxide. Peroxynitrite induced 1) oxidation of oxyhemoglobin to methemoglobin, 2) cytoskeleton rearrangement, 3) ultrastructural alterations, and 4) altered expression of band-3 and decreased expression of glycophorin A. With respect to control cells, this occurred in a significantly higher percentage of human RBC (approximately 40%). The presence of antioxidants inhibited these modifications. Furthermore, besides these senescence-associated changes, other important modifications, absent in control RBC and usually associated with apoptotic cell death, were detected in a small but significant subset of peroxynitrite-exposed RBC (approximately 7%). Active protease cathepsin E and mu-calpain increased; activation of caspase 2 and caspase 3 was detected; and phosphatidylserine externalization, an early marker of apoptosis, was observed. Conversely, inhibition of cathepsin E, mu-calpain, as well as caspase 2 and 3 by specific inhibitors resulted in a significant impairment of erythrocyte "apoptosis" Altogether, these results indicate that peroxynitrite, a milestone of redox-mediated damage in human pathology, can hijack human RBC toward senescence and apoptosis by a mechanism involving both cysteinyl and aspartyl proteases.

Study Objective: Idiopathic pulmonary fibrosis (IPF) is a chronic, usually fatal lung disease of unknown etiology. A common feature is the presence of microscopic areas of epithelial cell dropout. Increased apoptosis of these cells could elucidate the speculative pathogenesis of the disease. Therefore, the aim of our study was to examine the expression of p53, p21, bcl-2, bax, and caspase-3 in association with DNA strand breaks in bronchial and alveolar epithelial cells in lung specimens from IPF patients and control subjects., Patients and Methods: We examined by immunohistochemistry the expression of p53, p21, bax, bcl-2, and caspase-3 in association with DNA strand breaks detected by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) in bronchial and alveolar epithelial cells in lung specimens taken by biopsy in 12 IPF patients and 10 control subjects. An independent tissue evaluation by two pathologists graded semiquantatively the degree of staining present., Results: TUNEL was positive in epithelial cells in all IPF patients and only in one control subject. The expression of p53, p21, bax, and caspase-3 was up-regulated in IPF patients compared to control subjects. Bcl-2 was expressed less in IPF patients than in control subjects., Conclusions: These results confirm that apoptotic hyperplastic epithelial cells are present in patients with IPF and that the expression of p53, p21, bax, and caspase-3 appears to be up-regulated and that of bcl-2 down-regulated in these cells. The increased expression of proapoptotic molecules in epithelial cells in IPF may be involved in the inadequate and delayed reepithelialization, which in turn contributes to fibroblast proliferation.

Objective: Activation-induced cell death (AICD) is a major mechanism in the regulation of peripheral tolerance and its impairment can determine the development of autoimmunity. In the present study, in order to evaluate the role of caspase-3 in type 1 diabetes mellitus (T1DM) AICD, caspase-3 expression was analyzed in peripheral blood lymphocytes from 37 new onset T1DM patients and from 36 normal control subjects (NC) in resting conditions and after anti-Fas-triggered apoptosis., Methods: Caspase-3 expression was determined by semiquantitative RT-PCR and Western blot. Apoptosis was induced in activated lymphocytes by anti-Fas monoclonal antibody and quantified by flow cytometry and morphological analysis., Results: Caspase-3 mRNA expression was reduced in resting lymphocytes in 18/37 T1DM patients and in 1/36 NC (P < 0.01). Patients studied for both Fas-mediated AICD and caspase-3 mRNA expression revealed that a reduced caspase-3 mRNA expression in resting lymphocytes occurred in all patients showing resistance to Fas-mediated apoptosis (T1DM vs NC, P < 0.02) with the exception of 3 patients who exhibited normal caspase-3 expression levels. Caspase-3 protein analysis confirmed mRNA data and showed an impaired expression of caspase-3 active form in T1DM subjects compared with NC., Conclusions: Our data show that defective expression and function of caspase-3 in peripheral lymphocytes of T1DM patients may contribute to the development of AICD resistance in type 1 diabetes.

The aim of this study is to explore the mechanism by which diabetes exaggerates cerebral stroke and its outcome. Since ischemia can be related to not only necrosis but apoptosis as well, we compared the development of apoptosis in STZ-diabetic rats and STZ-diabetic rats subjected to occlusion of the middle cerebral artery (MCA). 24-48 hr following MCA occlusion the animals were killed, the brain removed and prepared for evaluation by several indexes of apoptosis: nucleosomal DNA fragmentation, TUNEL staining, activation of caspase-3 and alteration in the expression of Bax and Bcl2. DNA fragmentation was not detected in the cortex of normal and diabetic animals, but was evident following MCA occlusion in diabetic rats. Bax expression was increased in the cortex of normal rats following MCA occlusion and this expression was further increased in the cortex of MCA occluded diabetic rats. Bcl2 expression was not changed in any of the groups. In the hippocampus, DNA fragmentation was not evident in control rats but was observed in diabetic rats. Ischemic injury did not enhance DNA laddering in diabetic animals. The expression of Bax was increased in diabetic rats but was not increased following MCA occlusion. Bcl2 expression was not changed by ischemia in any of the animal models. These data suggest that diabetes may enhance the development of stroke via increased cortical apoptotic activity but this was not additive in the hippocampus following ischemic injury.

Eosinophils are one of the major effector cells in asthma, and controlling the number and survival of eosinophils might attenuate the severity of asthma. This result could be achieved by inducing eosinophil apoptosis. Apoptosis allows the removal of cells without inducing an inflammatory response. Our knowledge of the factors involved in regulating eosinophil apoptosis remains limited. CD30 molecule has been associated with T cell-negative selection and in TCR-mediated apoptosis. In this study we examined the expression and role of CD30 in apoptosis of human blood eosinophils. Percentage of apoptotic eosinophils was determined by annexin V-propidium iodide labeling, and CD30 expression was examined by flow cytometry. Spontaneous apoptosis was induced by serum deprivation, and survival was conferred by incubating cells with 10% FBS and IL-5. CD30 surface expression was up-regulated in eosinophils incubated for 24 h as compared with freshly isolated eosinophils, and both CD30 expression and eosinophil apoptosis increased in a time-dependent manner. We also measured CD30 mRNA expression by quantitative real-time RT-PCR and determined that CD30 transcripts increased in eosinophils undergoing apoptosis only under serum deprivation conditions. The agonistic CD30 Abs, Ber-H8 and HeFi-1, significantly enhanced eosinophil apoptosis. FBS and IL-5 failed to inhibit or suppress the CD30 agonistic-induced apoptosis. These data support the role of CD30 activation in eosinophil apoptosis. This research will help in furthering our understanding of eosinophil apoptosis and therefore might contribute to the development of better therapeutic modalities in the treatment and/or cure of allergic inflammation in bronchial asthma.

Phosphatidylserine exposure in platelets is required for normal haemostasis and is also a hallmark of apoptosis. It results from activation of a phospholipid scramblase, which has been shown to be differently stimulated by Ca(2+)-influx and during apoptosis, thus suggesting that mitochondria may be involved in phosphatidylserine exposure in platelets. It is also well known that local anaesthetics can expose phosphatidylserine in platelets and affect the mitochondrial metabolism in other cells. Thus, the present study was undertaken to evaluate the specific involvement of mitochondria in phosphatidylserine exposure in platelets. For this purpose, we stimulated phosphatidylserine exposure by local anaesthetics and avoided any external Ca(2+)-influx by performing all experiments in the absence of added Ca(2+). We report that phosphatidylserine exposure, induced by the lipophilic local anaesthetics dibucaine and tetracaine, was accompanied by depolarization of the mitochondrial membrane, cytochrome c release, calpain-processing of caspases 9 and 3 to active enzymes, as well as a prolonged increase in both cytosolic and mitochondrial Ca(2+) concentrations. In contrast, in the absence of extracellular Ca(2+), the Ca(2+)-ionophore A23187 induced a smaller transient increase in both cytosolic and mitochondrial Ca(2+) concentrations, but did not induce any other phenomena, nor phosphatidylserine exposure. However, phosphatidylserine exposure and depolarization induced by dibucaine still occurred in spite of inhibition of intracellular Ca(2+) elevation. Thus we conclude that phosphatidylserine exposure in platelets is associated with mitochondrial apoptotic-like events. Therefore, we propose that mitochondria engagement in an apoptotic pathway in platelets could lead to PS exposure without the participation of Ca(2+).

Homocysteine (Hcy) is a nonprotein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, it has been suggested that increased levels of plasma Hcy may play a role in the pathogenesis of various diseases, particularly at the cardiovascular level. The pathogenic mechanism of hyperhomocysteinemia, however, has not been clarified. Because oxygen radicals can be generated by the auto-oxidation of this amino acid, it has been suggested that Hcy may cause cellular damage through oxidative mechanisms, ultimately leading to apoptotic cell death. In this study, we sought to investigate the effects of Hcy on oxidative damage and antioxidant agent levels, as well as on apoptosis-related proteins and apoptosis occurrence in human cells. For this purpose, we measured levels of Bcl-2, caspase-3 and caspase-9 activity, Cu/Zn superoxide dismutase, reduced glutathione, lipid peroxidation [malondialdehyde and 4-hydroxy-2 (E)-nonenal concentrations], apoptotic single-stranded DNA and nuclear changes in human isolated lymphocytes exposed to increasing concentrations of Hcy. Incubation with Hcy did not induce significant changes in any of these biomarkers. Therefore, our results do not support the existence of a direct link between increased levels of Hcy and the occurrence of a pro-apoptotic state mediated by enhanced oxidative stress.

Background: Octreotide may prolong survival in patients with hepatocellular carcinoma, through an as yet unidentified mechanism. Kupffer cells play a key role in antitumour activity. Kupffer cell apoptosis is of major importance for the maintenance of this antitumour activity., Materials and Methods: We studied the in vitro effects of octreotide in the RNA expression of apoptotic and antiapoptotic proteins in isolated rat Kupffer cells, before and after exposure to lipopolysaccharide (LPS). Apoptotic and antiapoptotic gene expression was assessed using a semi-quantitative multiplex RT-PCR measuring bax, bcl-xS, bcl-2 and bcl-xL. LICE (caspase-3) mRNA was used as a measure of apoptosis., Results: Unstimulated Kupffer cells exhibited increased proapoptotic gene expression in a time-dependent manner, paralleled by a similar increase of LICE. LPS stimulation decreased the expression of proapoptotic bax and bcl-xS mRNA without effecting the antiapoptotic proteins. A decrease of LICE expression became significant at 48 hours. Octreotide showed a reduction of proapoptotic proteins, accompanied by an early increase and a late reduction of antiapoptotic proteins and a net decrease of LICE expression. A combination of LPS and octreotide produced a similar effect with the exception of a late increase of LICE expression, probably caused by a late increase of bax and bcl-xS., Conclusion: LPS and octreotide reverse the observed increased apoptosis of cultured Kupffer cells by influencing both proapoptotic and antiapoptotic proteins. Therefore, the antitumour effect of octreotide in hepatocellular carcinoma may, in part, be explained by its antiapoptotic effect on Kupffer cells.

Background: Viral hepatitis and autoimmune liver diseases cause hepatocyte apoptosis. Concanavalin A (Con A)-induced hepatitis resembles human viral hepatitis and autoimmune hepatitis. The role of nitric oxide (NO) in liver injury was controversial in different liver injury models. We hypothesize both endogenous and exogenous NO protect liver against Con A-induced liver injury. Molsidomine is metabolized into SIN-1 by the liver, and SIN-1 subsequently generates NO. So, molsidomine was used as a NO donor in this study., Study Design: To study a protective role of endogenous NO in Con A-induced liver injury, mice were pretreated with a specific inducible nitric oxide synthase (iNOS) inhibitor, L-N(6)-(1-iminoethyl)-lysine (L-NIL), before Con A challenge. To study a time-dependent protection against Con A-induced liver injury, animals were either given molsidomine, a NO donor, before or after Con A administration. Serum alanine aminotranferase (ALT) was analyzed. Liver samples were subjected to DNA fragmentation assay, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling stain, Western blot analysis, and caspase activity assays., Results: Animals pretreated with L-NIL had significantly increased serum ALT levels compared with those challenged with Con A alone; but pretreatment with molsidomine dramatically decreased ALT levels in L-NIL-pretreated animals or in animals that received Con A alone. Administration of molsidomine 30 minutes before or 1, 2, and 3 hours after Con A injection significantly reduced serum ALT levels and attenuated hepatocyte apoptosis from caspase inactivation. The ALT reduction was associated with inhibition of both caspase-3 and caspase-8 activation and reduction of hepatocyte apoptosis., Conclusions: Endogenous NO plays an important protective role against Con A-induced liver injury by reducing hepatocyte apoptosis. Administration of a NO donor early after Con A injection protects the liver from injury. This is the first study demonstrating a time-dependent inhibition of liver injury induced by Con A administration.

New agents are required for the treatment of chronic lymphocytic leukaemia (CLL). We show here that a protein kinase C inhibitor, bisindolylmaleimide IX, is a potent inducer of apoptosis in CLL cells, and investigate the mechanisms by which this is induced. Bisindolylmaleimide IX induced a conformational change and subcellular redistribution of Bax from the cytosol to the mitochondria, resulting in the release of the proapoptotic mediators cytochrome c, Smac and Omi/HtrA2 from the mitochondrial inner membrane space. This was followed by the activation of caspase-9 as the apical caspase and subsequent activation of effector caspases. CLL cells undergoing apoptosis showed a rapid caspase-mediated cleavage of Mcl-1, an antiapoptotic member of the Bcl-2 family implicated in CLL survival and poor prognosis. This cleavage was mediated primarily by caspase-3. Cleavage of Mcl-1 may provide a feed-forward amplification loop, resulting in the rapid induction of apoptosis. Bisindolylmaleimide IX or a related derivative may be of clinical use in the treatment of CLL.

Objectives: To observe different responsiveness of lymphocytes, eosinophils, and neutrophils from peripheral blood of asthmatic patients to dexamethasone and montelukast-induced apoptosis and to explore the roles of Fas antigen and caspase-3 in the heterogeneity of cell apoptosis., Methods: Lymphocytes, eosinophils, and neutrophils were isolated from peripheral blood of 18 asthmatic patients. Cells were incubated in vitro and treated with dexamethasone and leukotriene receptor antagonist montelukast respectively. Cell apoptosis rates and Fas expression rates were examined by flowcytometry whereas caspase-3 levels in these cells were detected by enzyme linked immunosorbent assay (ELISA)., Results: (1) Apoptosis rates: in vitro lymphocytes, eosinophils and neutrophils were compromised of spontaneous apoptosis at lower rates [(6.9 +/- 0.7)%, (31 +/- 11)% and (32 +/- 30)%, respectively]. With induction of dexamethasone, the apoptosis rates were (17.1 +/- 10.8)%, (44 +/- 22)% and (35 +/- 24)%. Montelukast markedly elevated the apoptosis rates of these three cells [(22.5 +/- 17.6)%, (50 +/- 27)% and (55 +/- 22)%, respectively] (compared to control, P < 0.01, < 0.05, > 0.05, respectively). (2) Fas expression: lymphocytes, eosinophils and neutrophils expressed low levels of Fas antigen at baseline [(1.50 +/- 0.07)%, (2.20 +/- 0.10)% and (1.21 +/- 0.09)%, respectively]. Dexamethasone induced Fas antigen expression levels of these cells of (6.58 +/- 2.10)%, (7.52 +/- 3.20)% and (3.24 +/- 2.34)%, and montelukast induced the expression levels of (5.06 +/- 1.66, 7.45 +/- 2.63, 3.03 +/- 2.47, P < 0.01, < 0.01, > 0.05, respectively). (3) caspase-3 levels: lymphocytes, eosinophils and neutrophils expressed constitutive caspase-3 levels of [(3.3 +/- 2.9) ng/L, (5 +/- 4) ng/L and (4.3 +/- 2.6) ng/L, respectively]. The dexamethasone induced caspase-3 levels were (6.7 +/- 3.1) ng/L, (6 +/- 3) ng/L and (3.1 +/- 1.8) ng/L. The montelukast induced levels were (5.2 +/- 3.7) ng/L, (8 +/- 4) ng/L, and (3.1 +/- 2.0) ng/L (compared to control, P < 0.01, < 0.01, > 0.05, respectively). It was demonstrated that dexamethasone and montelukast significantly induced apoptosis of lymphocytes and eosinophils which were assocreased with increased expression of Fas antigen and caspase-3. Dexamethasone was incapable of inducing neutrophils to apoptosis and had no significant effects on Fas expression and caspase-3 activity. Neutrophils underwent significant apoptosis after montelukast treatment, however, the induction was unlikely to be regulated by Fas and caspase-3 pathway., Conclusions: In asthmatic inflammatory modulating and effective cells, neutrophils is distinct from lymphocytes and eosinophils in profile of apoptosis induced by glucocorticoids and leukotriene receptor antagonist. The signal pathway contributing neutrophil apoptosis heterogeneity may involve deficient caspase cascade or Fas/FasL.

Background: PLTs for transfusion lose viability during storage in blood banking. This loss of viability is accelerated at 37 degrees C, as is the risk of bacterial contamination, and has led to the selection of 22 degrees C as the routine storage temperature. Because PLTs contain an intact apoptotic mechanism, we sought to determine whether PLTs undergo apoptosis during storage and whether storage at 37 degrees C accelerated this process., Study Design and Methods: PLT-rich plasma from PLT concentrates was stored at 37 or 22 degrees C in small aliquots or whole bags, with and without cell-permeable caspase inhibitors. Number of PLTs, pH, LDH level, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium activity were analyzed over time. PLT lysates were prepared and tested for the presence and activation of apoptotic proteins by enzyme assay and Western blotting., Results: PLT viability was greatly reduced after 1 to 2 days of storage at 37 degrees C; however, signs of apoptosis were evident by 3 hours after temperature shift. In temperature-stressed PLTs only, a gradual rise in caspase-3 activity was detected that correlated with the appearance of the 17- to 20-kDa cleavage products of caspase-3. Gelsolin, a caspase-3 substrate, underwent cleavage within the same time frame. Bcl-xL and caspase-2 also declined significantly; caspase-9 activity rose. Specific caspase inhibitors could prevent caspase activation but did not improve PLT cellular viability at 37 degrees C., Conclusions: PLTs contain apoptotic proteins that are activated during PLT storage at 37 degrees C and may account for the rapid decline in PLT cellular viability. Although ineffective here, inhibition of PLT apoptosis may improve PLT cellular viability.

Glycogen storage disease type 1b (GSD1b) is a rare autosomal recessive disorder characterized by hypoglycemia, hepatomegaly, and growth retardation, and associated-for unknown reasons- with neutropenia and neutrophil dysfunction. In 5 GSD1b patients in whom nicotin-amide adenine dinucleotide phosphate-oxidase activity and chemotaxis were defective, we found that the majority of circulating granulocytes bound Annexin-V. The neutrophils showed signs of apoptosis with increased caspase activity, condensed nuclei, and perinuclear clustering of mitochondria to which the proapoptotic Bcl-2 member Bax had translocated already. Granulocyte colony-stimulating factor (G-CSF) addition to in vitro cultures did not rescue the GSD1b neutrophils from apoptosis as occurs with G-CSF-treated control neutrophils. Moreover, the 2 GSD1b patients on G-CSF treatment did not show significantly lower levels of apoptotic neutrophils in the bloodstream. Current understanding of neutrophil apoptosis and the accompanying functional demise suggests that GSD1b granulocytes are dysfunctional because they are apoptotic.

Background: Platelets possess some of the machinery required for apoptotic cell death. However, disruption of mitochondria function, implicated in several models of cell death, has not been extensively studied in platelets. Mitochondrial viability and several other measures of apoptotic death in stored and experimentally stressed platelets were evaluated., Materials and Methods: Platelet mitochondrial transmembrane potentials (Deltapsim) were studied by staining platelets with JC-1, a dye that fluoresces at different wavelengths based on the state of mitochondrial polarization. Annexin V binding, a measure of phosphatidylserine (PS) exposure, and CD62P expression, an indicator of platelet activation, were determined by flow cytometry. Caspase-3 activity was measured with an enzyme assay and by Western blotting. Experimental platelet stressors included storage for 7 days, azide exposure, calcium ionophore stimulation, and plasma deprivation., Results: As measured by flow cytometry, Deltapsim values were similar in freshly drawn platelets and in platelet concentrates stored for up to 7 days. However, compared to fresh platelets, stored platelet concentrates had significantly increased PS exposure (3.1 vs. 5.1%, p = 0.015), CD62P expression (6.5 vs. 13.5%, p = 0.0067), and caspase-3 activity. Azide exposure, which decreased ATP release 20 to 30 percent, did not affect the Deltapsim. Stressed platelets exhibited higher degrees of mitochondrial depolarization in response to calcium ionophore stimulation than platelets that were not stressed. Plasma deprivation also resulted in significant alterations in Deltapsim, PS exposure, and CD62P expression., Conclusions: Platelet mitochondria maintain Deltapsim when stored for up to 7 days under standard blood bank storage conditions. Therefore, changes in platelet mitochondria Deltapsim do not correlate with downstream markers of apoptotic death such as caspase activation and PS exposure.

Chronic lymphocytic leukemia (CLL) cells develop chemo-resistance over time. Most anticancer agents function through induction of apoptosis, and therefore resistance against these agents is likely to be caused by selection for CLL cells with defects in the particular apoptosis pathway that is triggered by these drugs. Anticancer agents that function through alternative apoptotic pathways might therefore be useful in treating chemo-resistant CLL. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity. We examined the effects of CDDO (triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid) on CLL B cells in vitro. CDDO induced apoptosis in a dose-dependent manner in all (n = 30) CLL samples tested, including previously untreated and chemo-resistant CLL specimens. CDDO induced rapid proteolytic processing of caspase-8, but not caspase-9, in CLL B cells, suggesting activation of a mitochondria-independent pathway. CDDO-induced apoptosis of CLL B cells was blocked by cytokine response modifier A (CrmA), a suppressor of caspase-8, but not by X-linked inhibitor of apoptosis protein-baculovirus IAP repeat-3 (XIAP-BIR3), a fragment of XIAP, which selectively inhibits caspase-9. Examination of CDDO effects on expression of several apoptosis-relevant genes demonstrated significant reductions in the levels of caspase-8 homolog Fas-ligand interleukin-1-converting enzyme (FLICE)-inhibitory protein (c-FLIP), an endogenous antagonist of caspase-8. However, reductions of FLIP achieved by FLIP antisense oligonucleotides were insufficient for triggering apoptosis, indicating that CDDO has other targets in CLL B cells besides FLIP. These data suggest that the synthetic triterpenoid CDDO should be further explored as a possible therapeutic agent for treatment of chemo-resistant CLL.

Objective: The aim of this study was to determine whether granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are more or less intrinsically sensitive to spontaneous apoptosis than granulocytes from healthy individuals. Resistance to apoptosis has been suggested as an explanation for the proliferation or selection of PNH clones., Patients and Methods: Peripheral blood granulocytes from five patients with PNH, five patients with myelodysplastic syndrome (MDS), and five healthy volunteers were cultured in the absence of serum. Spontaneous apoptosis of the granulocytes was assessed every 6 hours by flow cytometry. The expression levels of CD16b, CD95, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor also were studied by flow cytometry, and caspase-3 activity was measured by fluorometry., Results: There were no significant differences in the proportion or absolute numbers of apoptotic and apoptotic/dead granulocytes between the cells from PNH patients and healthy individuals, whereas those from MDS patients showed significantly lower frequencies of apoptotic granulocytes compared with normal controls. The proportion of CD16b(-) granulocytes was not significantly different among the three groups during in vitro culture. CD95 and GM-CSF receptor was not significantly increased in cultured granulocytes or noncultured granulocytes from, respectively, patients with PNH and normal controls. Caspase-3 activity significantly decreased in cultured granulocytes from MDS patients, but not in granulocytes from PNH patients., Conclusions: Granulocytes from PNH patients did not display a reduced sensitivity to spontaneous apoptosis, suggesting that the apoptosis of blood cells in PNH may not be an important factor in proliferation or selection of PNH clones. These findings are in agreement with the normal lifespan of granulocytes in vivo.

Inhibition of neutrophil apoptosis has been identified as a prominent feature in chronic inflammation, parenchymal damage, and unresolved organ dysfunction. Lung injury animal models suggest that the neuropeptides vasoactive intestinal peptide and bombesin are protective. Therefore, in vitro effects of VIP and bombesin on apoptosis of normal human neutrophils were tested. For measuring effects on cell survival and apoptosis, trypan dye exclusion, colorimetric MTT assay to assess cell survival, and caspase-3 assay and annexin-V binding for analysing apoptosis rates were used. Foetal calf serum, Fas ligand, and tumour necrosis factor-alpha served as modulatory control agents; survival-promoting and apoptosis-inducing activities of the respective agents were confirmed. Vasoactive intestinal peptide and bombesin, however, failed to significantly affect cell death in neutrophils. Data suggest that direct regulation of neutrophil apoptosis is unlikely to be among the mechanisms of lung-protective actions of VIP and bombesin.

After 12, 18, and 24 h of oral administration of carbon tetrachloride (as a 1:1 mixture with mineral oil: 4 ml/kg body weight) to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group that was given mineral oil (4 ml/kg). In plasma, the activity of caspase-3 was barely detectable in the control rat, but increased significantly 24 h after drug administration along with a dramatic increase in glutamate oxaloacetate transaminase. These results indicate that carbon tetrachloride causes apoptosis in the liver by activating caspase-3, which is released to plasma by secondary necrosis. After 18 and 24 h of carbon tetrachloride administration, the liver concentration of hydrophilic vitamin C was decreased significantly, while that of hydrophobic vitamin E was not affected. The plasma concentration of vitamins C and E was not influenced significantly. These results suggest that carbon tetrachloride induces oxidative stress mainly in the aqueous phase of the liver cell.

Background: Although it is usually involved only in nucleated cells (NCs), artificially enucleated cells also lose viability by a programmed process of cell death called apoptosis. Because platelets undergo loss of viability during storage, an attempt was made to determine whether platelets contained the apoptotic mechanisms and whether it was activated during platelet storage., Study Design and Methods: Platelet viability was measured by reduction of a tetrazolium dye (MTS) and annexin V binding. Members of the death receptor, caspase, and Bcl-2 families were detected by RNase protection assay and Western blotting. Caspase 3 activation was measured by enzyme and Western blot assays and by cleavage of gelsolin., Results: After 5 days of storage under standard blood banking conditions, platelets display biochemical signs of apoptosis by losing MTS activity and increasing the amount of phosphatidylserine on their surface. The mRNA and the proenzyme for several members of the caspase, death receptor, and Bcl-2 families are expressed at high levels in platelets. An increase in caspase 3 activity and the amount of the biologically active p17 subunit of active caspase 3 were observed to coincide with the appearance of apoptotic markers during storage. These effects were not due to platelet activation. The caspase 3 substrate, gelsolin, began to undergo proteolysis after 3 to 4 days of storage, and the addition of the caspase inhibitor z-VAD-fmt substantially inhibited this process., Conclusion: Platelets contain many of the components of the apoptotic mechanism and show activation of caspase 3 and consequent cleavage of gelsolin during storage, independent of platelet activation. Evaluation of the mechanism of apoptosis in platelets may provide a basis for developing novel strategies to enhance platelet viability during storage.

Background: Ischemia followed by reperfusion (I/R) causes apoptosis, inflammation, and tissue damage leading to organ malfunction. Ischemic preconditioning can protect against such injury. This study investigates the contribution of the acute phase proteins alpha(1)-acid glycoprotein (AGP) and alpha(1)-antitrypsin (AAT) to the protective effect of ischemic preconditioning in the kidney., Methods and Results: Exogenous AGP and AAT inhibited apoptosis and inflammation after 45 minutes of renal I/R in a murine model. AGP and AAT administered at reperfusion prevented apoptosis at 2 hours and 24 hours, as evaluated by the presence of internucleosomal DNA cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and the determination of renal caspase-1- and caspase-3-like activity. AGP and AAT exerted anti-inflammatory effects, as reflected by reduced renal tumor necrosis factor-alpha expression and neutrophil influx after 24 hours. In general, these agents improved renal function. Similar effects were observed when AGP and AAT were administered 2 hours after reperfusion but to a lesser extent and without functional improvement. Moreover, I/R elicited an acute phase response, as reflected by elevated serum AGP and serum amyloid P (SAP) levels after 24 hours, and increased hepatic acute phase protein mRNA levels after 18 hours of renal reperfusion., Conclusions: We propose that the antiapoptotic and anti-inflammatory effects of AGP and AAT contribute to the delayed type of protection associated with ischemic preconditioning and other insults. This mechanism is potentially involved in the course of many clinical conditions associated with I/R injury. Moreover, exogenous administration of these proteins may provide new therapeutic means of treatment.

Acute pancreatitis (AP) may lead to the development of multiple organ dysfunction syndrome (MODS), especially in severe cases. Resolution of such inflammatory responses is dependent on neutrophil apoptosis. Delays in this apoptotic response are associated with persistent inflammation and subsequent tissue damage. The aim of this study is to determine the effects of AP on neutrophil apoptosis and to investigate the underlying mechanisms involved. Neutrophils and serum were isolated from control (n=10) and from patients with AP (mild, n=35, and severe, n=5). Neutrophil apoptosis was assessed by propidium iodide DNA staining using flow cytometry. Caspase, glutathione-S-transferase (GST), and Mcl-1 protein expression were assessed by SDS-PAGE western blotting. Serum interleukin (IL)-1beta and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were measured by ELISA. Neutrophils isolated from patients with AP show a significant delay in spontaneous neutrophil apoptosis. Serum factors contributed to this delay with increases in IL-1beta and GM-CSF. Isolated neutrophils were resistant to Fas antibody-induced apoptosis. Caspases represent a central mechanism for spontaneous and Fas antibody-induced neutrophil apoptosis. Procaspase 3 expression was decreased in mild and severe cases, but this effect was independent of serum factors. Increases in GST expression may also contribute to the antiapoptotic effect. Altered caspase expression may represent an additional factor contributing to delayed neutrophil apoptosis. This may contribute to the development of AP and its related complications.

Delayed neutrophil apoptosis is a feature of persistent acute inflammation. Neutrophil-mediated damage has been shown to be associated with the development of inflammatory bowel disease (IBD). Persistence of these cells both at the colonic site and circulation may further contribute to IBD. The aims of this study were to determine whether neutrophils isolated from IBD patients delay apoptosis and to investigate possible mechanisms involved in this delay. We studied 20 patients with IBD, 13 with Crohn's disease, and 7 with ulcerative colitis, all of whom were undergoing intestinal resection for symptomatic disease. Seventeen patients undergoing elective resection of colon cancer acted as operative controls. Systemic, mesenteric arterial, and mesenteric venous blood was harvested. Neutrophils isolated from patients with IBD showed decreased spontaneous apoptosis compared to cancer patients. Mesenteric venous serum of IBD patients contributed to this delay, which contained higher concentrations of interleukin-8 (IL-8). Pro-caspase 3 expression was also reduced in IBD neutrophils, which may contribute to decreased spontaneous and Fas antibody-induced apoptosis. Neutrophil apoptosis may be altered in Crohn's disease and ulcerative colitis through release of anti-apoptotic cytokines and altered caspase expression. The alterations in cell death mechanisms may lead to persistence of the inflammatory response associated with IBD.

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.

In this study, a mechanism is reported which determines the lifetime of polymorphonuclear neutrophils (PMN). In human PMN freshly isolated from the circulation, expression of bcl-Xl, bax-alpha, and bak, members of the bcl-2 family of apoptosis-associated genes, was found using the reverse transcription-polymerase chain reaction technique. In contrast, no expression of bcl-2 was seen in PMN, whereas the myeloid cell line HL-60 was positive for bcl-2 mRNA. Two gene products, Bcl-Xl and Bax-alpha, which are known to function as the regulatory machinery of programmed cell death (apoptosis), were detected at the protein level in PMN. Moreover, differential expression of these proteins was found upon induction or prevention of apoptosis by cytokines: Whereas induction of apoptosis by tumor necrosis factor-alpha was associated with a reduction of expression of the anti-apoptotic Bcl-Xl protein, prevention of apoptosis by granulocyte-macrophage colony-stimulating factor led to a downregulation of expression of the death-promoting Bax-alpha protein. This shift of balance of anti- and pro-apoptotic proteins was found to control caspase-3 activity which, in turn, downregulated Bcl-Xl expression in PMN undergoing apoptosis. Thus, cytokines can affect the ratio of Bax-alpha/Bcl-Xl expression in human PMN and modulate the subsequent activity of caspase-3, which functions as executer of the programmed cell death and may promote apoptosis by a positive feed-forward mechanism that downregulates Bcl-Xl.

We show that mouse sperm die spontaneously within 1-2 days in culture and that treatment with either staurosporine (STS) and cycloheximide (CHX) or a peptide caspase inhibitor does not accelerate or delay the cell death. Chicken erythrocytes, by contrast, are induced to die by either serum deprivation or treatment with STS and CHX, and embryonic erythrocytes are more sensitive than adult erythrocytes to both treatments. Although these erythrocyte deaths display a number of features that are characteristic of apoptosis, they are not blocked, or even delayed, by peptide caspase inhibitors, and most of the cells die without apparently activating caspases. A small proportion of the dying erythrocytes do activate caspase-3, but even these cells, which seem to be the least mature erythrocytes, die just as quickly in the presence of caspase inhibitors. Our findings raise the possibility that both mouse sperm and chicken erythrocytes have a death programme that may not depend on caspases and that chicken erythrocytes lose caspases as they mature. Chicken erythrocytes may provide a useful 'stripped down' cell system to try to identify the protein components of such a death programme, which may serve to back-up the conventional caspase-dependent suicide mechanism in many cell types.