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.

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.

Determinants of Monocyte Apoptosis in Cardiorenal Syndrome Type 1.

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.

[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.

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).

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.

Alterations in Red Blood Cell Functionality Induced by an Indole Scaffold Containing a Y-Iminodiketo Moiety: Potential Antiproliferative Conditions.

We have recently proposed a new erythrocyte-based model of study to predict the antiproliferative effects of selected heterocyclic scaffolds. Starting from the metabolic similarity between erythrocytes and cancer cells, we have demonstrated how the metabolic derangement induced by an indolone-based compound (DPIT) could be related to its antiproliferative effects. In order to prove the validity of our biochemical approach, in the present study the effects on erythrocyte functionality of its chemical precursor (PID), whose synthesis we reported, were investigated. The influence of the tested compound on band 3 protein (B3), oxidative state, ATP efflux, caspase 3, metabolism, intracellular pH, and Ca(2+) homeostasis has been evaluated. PID crosses the membrane localizing into the cytosol, increases anion exchange, induces direct caspase activation, shifts the erythrocytes towards an oxidative state, and releases less ATP than in normal conditions. Analysis of phosphatidylserine externalization shows that PID slightly induces apoptosis. Our findings indicate that, due to its unique features, erythrocyte responses to exogenous molecular stimuli can be fruitfully correlated at structurally more complex cells, such as cancer cells. Overall, our work indicates that erythrocyte is a powerful study tool to elucidate the biochemical/biological effects of selected heterocycles opening considerable perspectives in the field of drug discovery.

Baseline caspase activity predicts progression free survival of temsirolimus-treated head neck cancer patients.

Squamous cell cancer of the head and neck (SCCHN) is a frequent aggressive malignancy with limited therapeutic options. Increasing evidence suggests that mammalian target of rapamycin (mTOR)-inhibitors might be effective in advanced SCCHN. However, non-invasive biomarkers for early prediction of treatment efficacy are not established in SCCHN. Highly proliferating tumours are characterised by enhanced cell turnover which is associated with enhanced apoptosis. During apoptosis of epithelial cells caspases cleave cytokeratin (CK)-18 can be detected in the blood. In this study we analysed sera from patients with relapsed or metastatic SCCHN patients who have been treated with temsirolimus for caspase-cleaved and total (caspase-cleaved and uncleaved) CK-18 by enzyme-linked immunosorbent assays (ELISAs). In addition, caspase-3 activity was detected by luminometric substrate assay. SCCHN patients revealed higher serum levels of those biomarkers compared to healthy controls. Importantly, patients with short progression-free survival (PFS) showed higher serum levels of caspase-3 activity compared to patients with longer PFS (⩾ 2months). Caspase-3 activity is inversely correlated with PFS. A cut-off value for caspase-3 activity was determined that correctly predicted PFS <2months with a sensitivity of 86% and a specificity of 67%. These data demonstrate that detection of serum caspase-3 activity might be a useful non-invasive biomarker for early identification of SCCHN patients not responding to treatment with novel targeted therapies such as mTOR-inhibitors.

An integrated epigenomic analysis for type 2 diabetes susceptibility loci in monozygotic twins.

DNA methylation has a great potential for understanding the aetiology of common complex traits such as Type 2 diabetes (T2D). Here we perform genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-seq) in whole-blood-derived DNA from 27 monozygotic twin pairs and follow up results with replication and integrated omics analyses. We identify predominately hypermethylated T2D-related differentially methylated regions (DMRs) and replicate the top signals in 42 unrelated T2D cases and 221 controls. The strongest signal is in the promoter of the MALT1 gene, involved in insulin and glycaemic pathways, and related to taurocholate levels in blood. Integrating the DNA methylome findings with T2D GWAS meta-analysis results reveals a strong enrichment for DMRs in T2D-susceptibility loci. We also detect signals specific to T2D-discordant twins in the GPR61 and PRKCB genes. These replicated T2D associations reflect both likely causal and consequential pathways of the disease. The analysis indicates how an integrated genomics and epigenomics approach, utilizing an MZ twin design, can provide pathogenic insights as well as potential drug targets and biomarkers for T2D and other complex traits.

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.

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.

Platelet apoptosis and agonist-mediated activation in myelodysplastic syndromes.

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.

Can determination of circulating endothelial cells and serum caspase-cleaved CK18 predict for response and survival in patients with advanced non-small-cell lung cancer receiving endostatin and paclitaxel-carboplatin chemotherapy? a retrospective study.

INTRODUCTION: Early prediction of the efficacy of a combination of an antiangiogenic drug with cytotoxic chemotherapy is a significant challenge. In that regard, circulating endothelial cells (CECs) and cytokeratins (CKs) seem to reflect their roles in both tumor angiogenesis and tumor cell death. METHODS: Patients with advanced, previously untreated non-small-cell lung cancer were randomly assigned to an endostatin treatment group (paclitaxel + carboplatin + endostatin) and a control group (paclitaxel + carboplatin + placebo). A total of 122 patients were evaluated, of whom 107 had measurements of blood CECs, CK8, caspase-cleaved CK18 (ccCK18), and uncleaved CK18 (CK18) before and at weeks 3 and 6 of treatment, respectively. RESULTS: Higher baseline CECs in patients with a tumor response (partial remission + stable disease, p = 0.002 for the entire group; p = 0.000 for the treatment group) were observed. The number of CECs decreased significantly after endostatin treatment (p = 0.000), whereas CK levels increased. Increased levels of ccCK18 and CK18, but not CK8, reached significance (p = 0.001 and p = 0.048, respectively) when compared with the baseline. Tumor response showed a strong correlation with reduction of CECs (p = 0.000) and increase of ccCK18 (p = 0.040) after endostatin therapy. Cutoff values of changes of CECs and ccCK18 for prediction of survival were 0.58/µl and 19.6 ng/ml, respectively. Reduction of CECs and increase of ccCK18 significantly correlated with longer median survival (p = 0.013 and p = 0.016 for progression-free survival; p = 0.009 and p = 0.012 for overall survival, respectively). CONCLUSIONS: CECs and CKs could be biomarkers for selecting patients with non-small-cell lung cancer who will benefit from treatment with endostatin in combination with paclitaxel plus carboplatin.

The expression of caspases is enhanced in peripheral blood mononuclear cells of autism spectrum disorder patients.

Autism and autism spectrum disorders (ASDs) are heterogeneous complex neuro-developmental disorders characterized by dysfunctions in social interaction and communication skills. Their pathogenesis has been linked to interactions between genes and environmental factors. Consistent with the evidence of certain similarities between immune cells and neurons, autistic children also show an altered immune response of peripheral blood mononuclear cells (PBMCs). In this study, we investigated the activation of caspases, cysteinyl aspartate-specific proteases involved in apoptosis and several other cell functions in PBMCs from 15 ASD children compared to age-matched normal healthy developing controls. The mRNA levels for caspase-1, -2, -4, -5 were significantly increased in ASD children as compared to healthy subjects. Protein levels of Caspase-3, -7, -12 were also increased in ASD patients. Our data are suggestive of a possible role of the caspase pathway in ASD clinical outcome and of the use of caspase as potential diagnostic and/or therapeutic tools in ASD management.

Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum.

The administration of biological therapeutics can potentially elicit the development of neutralizing antibodies (NAbs) to the therapeutic drug in patients, which could have a significant impact on drug efficacy and safety. A rigorous in vitro cell-based assay for the detection of NAbs is critical for the assessment of the immunogenicity profile of the therapeutic drug. Conatumumab is a fully human monoclonal agonist antibody directed against the extracellular domain of human TRAIL receptor 2 (TR-2). It is being investigated as a cancer treatment because it is able to induce apoptosis in sensitive tumor cells. This report demonstrates how statistically designed experiments could be employed effectively in different stages of a NAb bioassay life cycle in order to characterize, optimize and stabilize the assay with added benefit of resource efficiency. By combining the approach of design of experiments (DOE) with subject matter expertise and experience, we were able to understand thoroughly how assay parameters affect the performance of the assay individually and interactively, identify the key assay parameters, define assay operating ranges and finally achieve a robust and sensitive cell-based assay for the detection of NAbs to Conatumumab. With the goal of developing a cell-based bioassay that is highly optimized for sensitivity, specificity, precision, and robustness, we performed 2 DOE experiments for assay optimization and 1 DOE experiment to validate assay robustness. We evaluated key operating parameters of the assay such as cell number, percentage of serum matrix, concentration of the therapeutic drug, concentration of the cross-linker, length of various incubation steps, cell age, interval between cell subculture and bioassay time, and detection equipment.

Screening for circulating nucleic acids and caspase activity in the peripheral blood as potential diagnostic tools in lung cancer.

The focus of the current investigational study was to examine whether circulating nucleic acids (i.e., DNA and microRNAs) have the potential to become suitable blood-based markers for diagnosis and progression of lung cancer. The concentrations of cell-free DNA and four circulating microRNAs (miR10b, miR34a, miR141 and miR155) as well as the caspase activity were measured in serum of 35 lung cancer patients (19 non-small-cell lung cancer, 8 small cell lung cancer patients and 8 patients with indefinite cancer type), 7 patients with benign lung tumors and 28 healthy individuals by PicoGreen, TaqMan MicroRNA, and Caspase-Glo®3/7 assay, respectively. The data were correlated with the established risk factors for lung cancer progression. The concentrations of cell-free DNA (p = 0.0001), serum microRNAs (p = 0.0001) and caspase activities (p = 0.0001) significantly discriminated cancer patients from healthy individuals. Serum DNA, caspase activities and RNA levels could not distinguish between patients with benign lung disease and cancer patients. However, the levels of miR10b (p = 0.002), miR141 (p = 0.0001) and miR155 (p = 0.007) were significantly higher in lung cancer patients than those in patients with benign disease. As determined by the Spearman-Rho test, high levels of cell-free DNA significantly correlated with elevated circulating caspase activities (p = 0.0001). In lung cancer patients high serum miR10b values associated with lymph node metastasis (p < 0.03) and elevated levels of TPA (tissue polypeptide antigen, p = 0.01), whereas high serum miR141 values associated with elevated levels of uPA (urokinase plasminogen activator, p = 0.02). The findings of our pilot study suggest that the assays for circulating DNA, microRNAs and caspase activities in blood might become novel minimally invasive diagnostic tools for detection and risk assessment of lung cancer, provided that their clinical utility can be confirmed in larger prospective trials.

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.

Indication of participation of caspase-2 and caspase-5 in mechanisms of human cervical malignancy.

INTRODUCTION: When apoptosis is disrupted, the transformed cells can survive, proliferate, and evolve into a malignancy. The strictly conserved caspase genes and the reliable experimental data clearly show that some caspases play a crucial role in apoptosis even if some of them have no apoptotic activity and others exhibit both apoptotic and nonapoptotic properties. Although caspase-2 belongs to initiator caspases, its normal role remains unclear. Experimental studies have shown that it is primarily necessary for the execution of apoptosis in mutagenic cells. Human caspase-5 is classified as an inflammatory caspase, although its substrate has not been identified yet. In this research, the activities of caspase-2 and caspase-5 have been estimated during the progression of human cervical malignancy. METHODS: The experimental material includes human cervical tissue samples (normal and pathological) and blood serum samples of the corresponding tissue donors, where enzyme activities have been measured colorimetrically. RESULTS: Both caspases' activities showed the highest increase, statistically significant (P < 0.01, by t test) compared with the controls, in the low-grade squamous intraepithelial lesion tissues. Caspase-2 of all pathological tissues was proved more active than the controls. Serum caspases' activities were significantly lower than those of the tissues. Serum caspase-2's activity in patients with low-grade squamous intraepithelial lesion stage showed no statistically significant increase compared with the controls. Serum caspase-5's activity of all patients with malignancy stages was presented elevated, whereas that of the serum of patients with cervical cancer had the highest activity (P < 0.01, by t test). CONCLUSIONS: The changes of caspase-2 and caspase-5 activities could be indicative of their involvement in the cervical malignancy mechanisms.

Endogenous protection against oxidative stress caused by cisplatin: role of superoxide dismutase.

CONCLUSION: The administration of cisplatin induces the activation of superoxide dismutase (SOD) as a response to oxidative stress in the cochleae of Sprague-Dawley rats and this activation is proportional to the activation of the intrinsic pathway of apoptosis. OBJECTIVES: To determine the role of the antioxidant endogenous mechanism in the preservation of cochlear integrity and function in an experimental model of cisplatin ototoxicity. METHODS: Sixteen Sprague-Dawley rats were studied at 7 days after intraperitoneal injection of CDDP (n = 8) or 10 ml/kg NaCl 0.9% w/v in the control group (n = 8) by means of auditory steady-state responses. These findings were compared with the expression of SOD and caspase-3/7 and caspase-9 activities. RESULTS: Groups receiving cisplatin showed increased auditory thresholds after injection of cisplatin and control groups maintained normal hearing. Measurements of caspase-3/7 and caspase-9 showed a significant increase in cisplatin-treated rats. A significantly increased activity of total SOD in whole cochlear extracts was observed in animals from the CDDP groups vs control animals. Likewise, differences between CDDP groups were also statistically significant.

Systematic review: caspase-cleaved fragments of cytokeratin 18 - the promises and challenges of a biomarker for chronic liver disease.

BACKGROUND: Biomarkers hold great promise for detecting chronic liver disease without the use of liver biopsy. AIM: To review the usefulness of cytokeratin (CK) 18 fragments, a marker of hepatocyte apoptosis, to predict the presence of chronic liver injury. METHODS: Available literature identified from PubMed was reviewed. RESULTS: Levels of CK18 fragments have been shown to be elevated in hepatocellular carcinoma, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease and cholestatic liver disease. In the setting of nonalcoholic fatty liver disease, CK18 fragments may distinguish nonalcoholic steatohepatitis from simple fatty liver. CONCLUSIONS: Undoubtedly, the most promising application of CK18 fragments is currently in nonalcoholic fatty liver disease, and especially for distinguishing patients with nonalcoholic steatohepatitis vs. those with simple steatosis. Further investigations and technical improvements are required to cross the boundary from research to the clinical application of CK18 fragments as a marker of chronic liver disease.