Platelet and immune characteristics of immune thrombocytopaenia patients non-responsive to therapy reveal severe immune dysregulation.

Multifactorial mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) might condition the ability of patients with ITP to respond to treatments. Examining their platelet and immune features, we aimed to detect singular characteristics of patients with ITP who do not respond to any treatment. We studied patients with chronic primary ITP who had been without treatment, or untreated (UT-ITP), for at least six months; included were responders to agonists of thrombopoietin receptors (TPO-RA), patients who showed no response to first- and second-line treatments (NR-ITP), and healthy controls. Platelets from NR-ITP patients exposed a reduced amount of sialic acid residues. Increased loss of platelet surface sialic acid residues was associated with increased platelet apoptosis. NR-ITP patients had an increased fraction of naive lymphocyte (L) B cells and a reduced LTreg (Lymphocyte T-regulator) subset. They also presented an anomalous monocyte and NK (Natural Killer) cells distribution. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls. In conclusion, our results indicate a severe deregulation of the immune system of NR-ITP. The inverse correlation between loss of sialic acid and LTreg count suggests a potential relationship between glycan composition on the platelet surface and immune response, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP.

Apoptosis and its relation with clinical course in patients with Crimean-Congo hemorrhagic fever.

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.

Association between serum levels of caspase-cleaved cytokeratin-18 and early mortality in patients with severe spontaneous intracerebral hemorrhage.

BACKGROUND: Apoptotic changes after cerebral hemorrhage in brain samples of humans have been found. Caspase-cleaved cytokeratin (CCCK)-18 could be detected in the bloodstream during apoptosis. Higher circulating CCCK-18 levels have been associated with 6-month mortality in patients with basal ganglia hemorrhage. The aim of our study was to determine whether there is an association between serum CCCK-18 levels and early mortality of spontaneous intracerebral hemorrhage (SIH) patients. We performed an observational, prospective and multicentre study. There were included patients with severe SIH defined as Glasgow Coma Scale (GCS) lower than 9. We determined serum CCCK-18 levels at the severe SIH diagnosis moment. RESULTS: We found that non-surviving SIH patients (n = 46) showed lower GCS, and higher serum CCCK-18 levels and APACHE-II score than survivor ones (n = 54). In ROC analysis was found that the area under the curve of serum CCCK-18 levels for 30-day mortality prediction was 90% (95% CI 82-95%; p < 0.001). In the multiple logistic regression analysis, we found an association between serum CCCK-18 levels and 30-day mortality (OR 1.034; 95% CI 1.013-1.055; p = 0.002). CONCLUSIONS: The novel finding of our study was that there is an association between high serum CCCK-18 levels and 30-day mortality in severe SIH patients.

Caspase-cleaved keratin-18 fragments increase during alcohol withdrawal and predict liver-related death in patients with alcoholic liver disease.

Noninvasive assessment of disease activity in patients with nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) is still unsettled, but essential for the evaluation of disease progression. We here studied the association of total (M65) and caspase-cleaved (M30) serum keratin-18 fragments (n = 204) with histological parameters (n = 106) in heavy drinkers primarily admitted for alcohol withdrawal before and after alcohol detoxification. An age-, sex-, and fibrosis-stage matched NAFLD cohort (n = 30) was used for comparison. The prognostic value of M30 and M65 levels were assessed in an additional prospectively followed-up cohort of 230 patients with alcoholic cirrhosis (AC) using competing risk analyses. Among the histological parameters, both M30/65 correlated significantly and better than any other serum marker with apoptosis and liver damage, such as ballooning (r = 0.65; P < 0.001), followed by lobular inflammation (0.48; P < 0.001), steatosis (0.46; P < 0.001), but less with fibrosis (0.24; P < 0.001). Area under the receiver operating characteristics curves to detect ballooning, steatosis, or steatohepatitis (SH) were slightly better for M30 (P < 0.005). Optimal M30 cut-off values for mild and severe ballooning were 330 and 420 U/L, and 290 and 330 U/L for SH grades 1 and 2. No significant differences of M30/65 were found between the matched NAFLD and ALD cohort. In contrast to aspartate-amino-transferase and M65, M30 levels increased significantly from 391 to 518 U/L during alcohol detoxification. Moreover, levels of M30 and M65 predicted non-hepatocellular carcinoma liver-related mortality in patients with AC during a mean observation interval of 67.2 months. CONCLUSION: Our data suggest M30 as highly specific marker of liver apoptosis both in ALD and NAFLD. In addition, hepatocellular apoptosis, as determined by M30 levels, occurs during alcohol withdrawal, and survival data point toward a novel underestimated role of apoptosis in patients with ALD. (Hepatology 2017;66:96-107).

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

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.

Circulating levels of apoptotic markers and oxidative stress parameters in women with polycystic ovary syndrome: a case-controlled descriptive study.

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.

[Features of a necrotic and inflammatory process in different forms of nonalcoholic fatty liver disease]. / Osobennosti nekroticheski-vospalitel'nogo protsessa pri raznykh formakh nealkogol'noi zhirovoi bolezni pecheni.

AIM: To identify the features of development of a necrotic and inflammatory process in different forms of nonalcoholic fatty liver disease (NAFLD), by comparatively analyzing a full set of clinical and laboratory parameters, including the cytokine status and the expression level of enzyme genes controlling the apoptosis of peripheral leukocytes. SUBJECTS AND METHODS: 86 patients with NAFLD, including 8 (9.3%) with hepatic steatosis (HS), 70 (81.4%) with nonalcoholic steatohepatitis (NASH), 40, 19, and 11 with mild, moderate, and high disease activity, respectively, and 8 (9.3%) with liver cirrhosis (LC), were examined. A control group consisted of 34 healthy donors. Clinical and biochemical blood indices, cytokine profile, and the level of caspase gene transcripts in the peripheral blood leukocytes (PBL) were estimated. RESULTS: As compared to the controls, the patients with HS had higher tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels and lower caspase 3, 6, and 8 mRNA in PBL. The concentration of IL-10 in NASH was higher than that in steatosis and positively correlated with the level of proinflammatory cytokines. The levels of TNF-α and IL6 were higher in the patients with NASH than in the controls. Those of C-reactive protein, γ-globulin, IL-6, and cytokeratin-18 fragment increased with the progression of NASH. In the latter, the transcriptional activity of caspase-3 gene decreased relative to the reference value and negatively correlated with the level of proinflammatory cytokines. In the patients with LC, the gene expression profile of caspases in PBL was similar to that in the control group; the level of IL-6 was higher than that in steatosis and NASH, that of IL-1ß was higher than in HS and positively correlated the concentration of IL-6 and the activity of alanine aminotransferase and aspartate aminotransferase. CONCLUSION: The features of a necrotic and inflammatory process were identified in different forms of NAFLD. When the latter progressed, the cytokine profile and gene expression levels of caspases in PBL altered along with a change in the general clinical picture.

Periodontal disease level-butyric acid amounts locally administered in the rat gingival mucosa induce ER stress in the systemic blood.

Periodontal diseases have long been postulated to contribute to systemic diseases and, likewise, it has been proposed that periodontal disease treatment may ameliorate certain systemic diseases. Short-chain fatty acids (SCFA) are major secondary metabolites produced by oral anaerobic bacteria and, among the SCFAs, butyric acid (BA) in high amounts contribute to periodontal disease development. Periodontal disease level-butyric acid (PDL-BA) is found among patients suffering from periodontal disease and has previously shown to induce oxidative stress, whereas, oxidative stress is correlated to endoplasmic reticulum (ER) stress. This would imply that PDL-BA may likewise stimulate ER stress, however, this was never elucidated. A better understanding of the correlation between PDL-BA and systemic ER stress stimulation could shed light on the possible systemic effects of PDL-BA-related periodontal diseases. Here, PDL-BA was injected into the gingival mucosa and the systemic blood obtained from the rat jugular was collected at 0, 15, 60, and 180 min post-injection. Collected blood samples were purified and only the blood cytosol was used throughout this study. Subsequently, we measured blood cytosolic GADD153, Ca(2+), representative apoptotic and inflammatory caspases, and NF-κB amounts. We found that PDL-BA presence increased blood cytosolic GADD153 and Ca(2+) amounts. Moreover, we observed that blood cytosolic caspases and NF-κB were activated only at 60 and 180 min post-injection in the rat gingival mucosa. This suggests that PDL-BA administered through the gingival mucosa may influence the systemic blood via ER stress stimulation and, moreover, prolonged PDL-BA retention in the gingival mucosa may play a significant role in ER stress-related caspase and NF-κB activation. In a periodontal disease scenario, we propose that PDL-BA-related ER stress stimulation leading to the simultaneous activation of apoptosis and inflammation may contribute to periodontal disease pathogenesis.

Simultaneous targeting of IL-1 and IL-18 is required for protection against inflammatory and septic shock.

RATIONALE: Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. OBJECTIVES: To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1ß, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. METHODS: LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. MEASUREMENTS AND MAIN RESULTS: Interestingly, deficiency of both IL-1ß and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1ß and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. CONCLUSIONS: Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

[EFFICIENCY OF URSODEOXYCHOLIC ACID APPLICATION IN NONALCOCHOLIC STEATOHEPATITIS].

AIM: to estimate the efficiency of ursodeoxycholic acid (UDHC) in nonalcocholic steatohepatitis (NASH) by analysis of conventional clinical datas, apoptosis and liver perfusion parameters. MATERIALS AND METHODS: UDHC was used as monotherapy in treatment of 92 NASH patients in daily dose 10-15 mg/kg. We have observed 44 (47.8%) males, 48 (52.2%) females, age was 56.8 ± 7.2 years, BMI was 28.4 ± 2.3 kg/m2, waist circumference was 93.8 ± 8.3 cm. Functional liver tests (ALAT, ASAT, alcaline phosphatase--APh, gamma-glutamyltranspeptidase--GGTP), abdominal ultrasonography and dopplerography of liver blood flow, kaspase-3, 6, 8, 9 genes expression in blood leucocytes were estimated. Periods of controls research and UDCA treatment were: 4-8 weeks in 92 patients, 20-24 weeks in 18 (19.6%) patients and 40-48 weeks in 13 (14.1%) patients. RESULTS: Significant positive dynamics of liver functional tests and decrease of kaspase-3, 6, 9 genes expression in blood leucocytes were observed over 4-8 weeks, normalization of liver tests--over 20-24 weeks and significant amelioration of venous and arterial liver perfusion parameters--over 40-48 weeks. CONCLUSION: Ursodeoxycholic acid in daily dose of 10-15 mg/kg in nonalcocholic steatohepatitis caused positive dynamics of cytolytic and cholestasis parameters, leucocytic apoptosis and venous and arterial liver blood flow parameters.

Sirtuin 1-mediated inhibition of p66shc expression alleviates liver ischemia/reperfusion injury.

OBJECTIVES: Ischemia/reperfusion is a leading cause of liver damage after surgical intervention, trauma, and transplantation. It has been reported that the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 attenuates myocardial, cerebral, and renal ischemia/reperfusion damage. This study aimed to investigate the involvement of sirtuin 1-mediated p66shc inhibition in liver ischemia/reperfusion and explore the effect of carnosic acid and ischemic preconditioning on liver ischemia/reperfusion-induced damage. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats and HepG2 cells. INTERVENTIONS: The rats were subjected to 45 minutes of ischemia to 70% of the liver, followed by 3-hour reperfusion. The HepG2 cells were subjected to hypoxia/reoxygenation-induced injury. MEASUREMENTS AND MAIN RESULTS: In the rats with liver ischemia/reperfusion injury, carnosic acid pretreatment and ischemic preconditioning dramatically reduced the serum aminotransferase activity and proinflammatory chemokine levels and improved the liver histological evaluations. Carnosic acid and ischemic preconditioning also increased manganese superoxide dismutase and Bcl-xL, but down-regulated cleaved caspase-3. Interestingly, the protective effect of carnosic acid and ischemic preconditioning was positively associated with sirtuin 1 activation. By contrast, p66shc, a kinase that promotes oxidative injury and apoptosis, was inhibited by carnosic acid and ischemic preconditioning. Sirtuin 1 small interfering RNA knockdown experiments confirmed that carnosic acid increased sirtuin 1-mediated repression of p66shc in HepG2 cells and that the protective effect of carnosic acid against hypoxia/reoxygenation injury was inhibited by the sirtuin 1 inhibitor nicotinamide. These results suggest that carnosic acid protects hepatocytes from hypoxia/reoxygenation damage through sirtuin 1-mediated p66shc suppression. To support this notion, we further demonstrated that the sirtuin 1 activator resveratrol achieved a protective effect similar to that of carnosic acid against hypoxia/reoxygenation injury, whereas sirtuin 1 small interfering RNA and nicotinamide had the opposite effect. CONCLUSIONS: Carnosic acid and ischemic preconditioning protect against ischemia/reperfusion-induced liver injury. Mechanistically, the protective effect involves the sirtuin 1-mediated inhibition of p66shc, suggesting that this pathway is a novel potential therapeutic target for protecting the liver from ischemia/reperfusion injury.

How does colistin-induced nephropathy develop and can it be treated?

Colistin is an old antibiotic used in the treatment of Gram-negative infections. It was once suspended because of its nephrotoxic effect but has since been reintroduced due to multidrug-resistant bacterial infections. The pathogenesis of colistin-associated nephropathy has not been clarified, and there is currently no effective therapeutic or prophylactic agent available. The aim of this study was to investigate the roles of caspase-associated apoptosis and caspase 1, calpain 1, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) expression in the pathogenesis of colistin-associated nephrotoxicity and the effect of grape seed proanthocyanidin extract (GSPE) in preventing it. Twenty-four rats were divided into three groups: control, colistin, and colistin plus GSPE (colistin+GSPE). Colistin-associated nephropathy was induced by the administration of 300,000 IU/kg of body weight/day colistin intraperitoneally for 7 days. The experiment was discontinued on the seventh day. Blood was collected for measurements of blood urea nitrogen (BUN) and creatinine levels. Histopathological examination of kidney tissue and caspase 1 and 3, iNOS, eNOS, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), and calpain 1 staining was also performed. Significant increases in BUN levels; creatinine levels; renal histopathological scores; and TUNEL, caspase 1 and 3, calpain 1, iNOS, and eNOS staining were observed for the colistin group compared to the control group. Significant decreases in BUN levels; creatinine levels; renal histopathological scores; and TUNEL, caspase 1 and 3, calpain 1, iNOS, and eNOS staining were observed in the colistin+GSPE group compared to the colistin group. Our study shows, for the first time in the literature, that caspase-mediated apoptosis, iNOS, caspase 1, and calpain 1 are involved in the pathogenesis of colistin-associated nephropathy. GSPE had a renoprotective effect, as shown by the lowered levels of these mediators.

Activation of caspases-9, -3 and -8 in human platelets triggered by BH3-only mimetic ABT-737 and calcium ionophore A23187: caspase-8 is activated via bypass of the death receptors.

Platelet apoptosis and activation have been studied in human platelets treated with BH3-only mimetic ABT-737 and calcium ionophore A23187, agents triggering apoptosis through the intrinsic mitochondrial pathway. Platelet apoptosis was determined as activation of crucial apoptosis-associated caspases, initiator caspase-9 of intrinsic apoptosis pathway, executioner caspase-3 and initiator caspase-8 of extrinsic death receptor pathway, and platelet activation was detected by P-selectin (CD62) exposure on the platelet surface. We found that ABT-737 predominantly induced activation of caspases-9, -3 and -8 rather than CD62 exposure, whereas A23187 induces both caspases activation and CD62 exposure. Caspase-8 activation was stimulated independently of the extrinsic apoptosis pathway via mitochondrial membrane permeabilization and depolarization. These data suggest that (i) caspase-8 activation is triggered in ABT-737- and A23187-treated anucleate platelets through the mitochondria-initiated caspase activation cascade bypassing the death receptors, and (ii) ABT-737-treated platelets are a useful experimental tool for discerning the role of platelet apoptosis in platelet function and survival.

Changes in expression of membrane TNF, NF-{kappa}B activation and neutrophil apoptosis during active and resolved inflammation.

BACKGROUND: Tumour necrosis factor (TNF) is central to the pathophysiological process of rheumatoid arthritis (RA), whether as soluble cytokine or membrane-expressed pro-TNF (mTNF). OBJECTIVES: To determine whether neutrophils, which can express TNF, are activated in the blood of patients with RA compared with healthy controls. To investigate, by focusing on mTNF expression, if the functions of RA neutrophils change in response to therapeutic TNF inhibition. METHODS: TNF was measured by flow cytometry and qPCR in neutrophils from 20 patients with RA before and after the start of TNF inhibitor therapy. Apoptosis was measured by morphology, and western blotting of pro- and antiapoptotic proteins in cell lysates. Nuclear factor κB (NF-κB) activation was determined by western blotting of phosphorylated NF-κB (p65). RESULTS: Before treatment RA neutrophils exhibited increased TNF mRNA expression, elevated mTNF levels and NF-κB activity compared with controls. They also underwent delayed apoptosis as shown by altered expression of anti- and proapoptotic proteins, such as Mcl-1 and caspases. Neutrophil TNF expression returned to baseline levels during successful treatment with anti-TNF biological agents, and there was a close correlation between clinical disease improvement and changes in neutrophil function. CONCLUSIONS: Neutrophils express elevated levels of TNF in RA and the transcription factor, NF-κB, a target of TNF, is activated. This mechanism could lead to a self-sustained inflammatory process. These data point to an important role of neutrophils in the abnormal TNF signalling pathways activated in RA and provide new evidence that neutrophils actively contribute to altered cytokine signalling in inflammatory diseases.

Genotype-, aging-dependent abnormal caspase activity in Huntington disease blood cells.

Huntington's Disease (HD) is caused by trinucleotide CAG repeat expansion >36 in huntingtin (htt), a protein with several documented functions. The elongated polyglutamine (polyQ) stretch in the N-terminal region of htt leads to dysfunctional and degenerative events in neurons and peripheral tissues. In this study, by extending the analysis to several caspase activities (i.e. caspase 2, 3, 6, 8 and 9), we describe genotype- and time- dependent caspase activity abnormalities, decreased cell viability and a large set of alterations in mitochondria morphology, in cultured blood cells from HD patients. Patients homozygous for CAG repeat mutations and heterozygous with high size mutations causing juvenile onset (JHD) presented significantly increased caspase 2, 3, 6, 8 and 9 activities, decreased cell viability and pronounced morphological abnormalities, compared with cells carrying low mutation size and controls. After cyanide treatment, all caspases increased their activities in homozygous and highly expanded heterozygous cells, caspase 8 and 9 increased also in those cells carrying low-size mutations, remarking their key role as 'caspase initiators' in HD. The remarkable ageing-dependent abnormalities in peripheral cells carrying particularly toxic mutations (i.e. homozygotes' and JHD's blood cells) points out the potential dependence of clinical HD development and progression on either mutated htt dosage or missing wild type htt. Peripheral tissues (i.e. blood cells) may theoretically represent an important tool for studying HD mechanisms and searching for new biomarkers, according to the patients' genotype.

Survivin and caspases serum protein levels and survivin variants mRNA expression in sepsis.

Sepsis is a dysregulated host response to infection related to devastating outcomes. Recently, interest has been shifted towards apoptotic and antiapoptotic pathobiology. Apoptosis is executed through the activation of caspases regulated by a number of antiapoptotic proteins, such as survivin. The survivin and caspases' responses to sepsis have not yet been elucidated. This is a multicenter prospective observational study concerning patients with sepsis (n = 107) compared to patients with traumatic systemic inflammatory response syndrome (SIRS) (n = 75) and to healthy controls (n = 89). The expression of survivin was quantified through real-time quantitative polymerase chain reaction for the different survivin splice variants (wild type-WT, ΔEx3, 2B, 3B) in peripheral blood leukocytes. The apoptotic or antiapoptotic tendency was specified by measuring survivin-WT, caspase-3, and -9 serum protein concentrations through enzyme-linked immunosorbent assay. The survivin-WT, -2B, -ΔΕx3 mRNA, survivin protein, and caspases showed an escalated increase in SIRS and sepsis, whereas survivin-3B was repressed in sepsis (p < 0.05). Survivin correlated with IL-8 and caspase-9 (p < 0.01). For discriminating sepsis, caspase-9 achieved the best receiver operating characteristic curve (AUROC) of 0.95. In predicting mortality, caspase-9 and survivin protein achieved an AUROC of 0.70. In conclusion, specific apoptotic and antiapoptotic pathways might represent attractive targets for future research in sepsis.

Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells.

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

Immune Thrombocytopenia Plasma-Derived Exosomes Impaired Megakaryocyte and Platelet Production through an Apoptosis Pathway.

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.

p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils.

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

Proteasome enzymatic activities in plasma as risk stratification of patients with acute myeloid leukemia and advanced-stage myelodysplastic syndrome.

PURPOSE: Cytogenetic abnormalities are currently the most important predictors of response and clinical outcome for patients with acute myeloid leukemia (AML) or advanced-stage myelodysplastic syndrome (MDS). Because clinical outcomes vary markedly within cytogenetic subgroups, additional biological markers are needed for risk stratification. EXPERIMENTAL DESIGN: We assessed the utility of measuring pretreatment proteasome chymotrypsin-like, caspase-like, and trypsin-like activities in plasma to predict response and survival of patients with AML (n = 174) or advanced-stage MDS (n = 52). RESULTS: All three enzymatic activities were significantly (P < 0.001) increased in the plasma of patients with AML and MDS compared with normal controls. Both chymotrypsin-like and caspase-like activities, but not trypsin-like activity, correlated with outcome. Chymotrypsin-like and caspase-like activities, but not trypsin-like activity, predicted response in univariate analysis (P = 0.002). However, only chymotrypsin-like activity was independent predictor of response from age grouping (<70 versus > or =70 years), cytogenetics, and blood urea nitrogen in multivariate analysis. Similarly, both chymotrypsin-like and caspase-like activities, but not trypsin-like activity, were predictors of overall survival in univariate analysis (P < 0.0001), but only chymotrypsin-like activity was independent of cytogenetics, age, performance status, blood urea nitrogen, and beta(2)-microglobulin in multivariate Cox regression models. Chymotrypsin-like activity was also a strong independent predictor of survival in patients with intermediate karyotype (n = 124). CONCLUSIONS: Measuring plasma chymotrypsin-like activity may provide a powerful biomarker for risk stratification in patients with AML and advanced-stage MDS, including those with normal karyotype.