Association between blood caspase-8 levels and mortality of patients with malignant middle cerebral artery infarction.

OBJECTIVE: High concentrations of caspase-8 (main initiator caspase of apoptosis extrinsic pathway) have been found in brain tissue from traumatic brain injury patients and in blood of patients with different diseases. However, there are not data on blood caspase-8 concentrations in ischemic stroke patients. Therefore, the objective of this study was to determine whether there is an association between blood caspase-8 concentrations and the probability and speed of mortality at 30 days in patients with malignant middle cerebral artery infarction (MMCAI). DESIGN: Observational prospective study. SETTING: Five Intensive Care Units (ICU). PATIENTS: Patients with severe malignant middle cerebral artery infarction (MMCAI) defined as acute infarction in more than of 50% of that territory and Glasgow Coma Scale (GCS)<9. INTERVENTIONS: Determination of serum caspase-8 levels when MMCAI was diagnosed. MAIN VARIABLES OF INTEREST: Mortality at 30 days and time until this event. RESULTS: Severe MMCAI patients (n=28) compared to survivor patients (n=28) showed higher serum caspase-8 concentrations (p<0.001), lower platelet count (p=0.01) and lower GCS (p=0.002). We found an area under the curve for mortality prediction of 78% (95% CI=65%-91%; p<0.001) by serum caspase-8 levels. Kaplan-Meier analysis found higher mortality rate in patients with serum caspase-8 levels >62.8ng/mL (hazard ratio=11.2; 95% CI=4.4-28.4; p<0.001). CONCLUSIONS: The association of high blood caspase-8 concentrations with the rate and the velocity of 30-day mortality in MMCAI patients is the main new finding of our study.

Comparison of caspase-8, granzyme B and cytochrome C apoptosis biomarker levels in orthopedic trauma patients.

BACKGROUND: The primary objective of this study was to investigate whether or not apoptosis is induced following bone fracture, and if so, to investigate whether the extrinsic or intrinsic pathway of cell death is stimulated. METHODS: A total of 30 patients who presented at our clinic and were diagnosed with bone fracture following trauma were included in the study group. A control group was formed of 37 age and gender-matched volunteers. On the day after the fracture, blood samples taken from the patients were examined for cytochrome C, granzyme B and caspase-8 with the ELISA method. RESULTS: A total of 67 individuals were evaluated (fracture group: 30, control group: 37) in this study. Caspase-8 was found to be statistically significantly high in the patient group (0.37±0.06 ng/mL, p=0.002). No significant difference was determined between the groups in respect to cytochrome C values (p=0.173). The granzyme B values were determined to be significantly high in the patient group (52.56±8.51 pg/mL, p=0.007). CONCLUSION: These results obtained from patients with a long bone fracture demonstrated that serum caspase-8 and granzyme B levels were higher in patients than in the control group, thereby showing activation of the extrinsic pathway. However, no significant difference was determined between the groups concerning serum cytochrome C levels. This study may guide future studies designed for better understanding of the molecular pathways that govern the events during a fracture, which will be important for the future advancement of fracture treatment.

Prediagnostic circulating inflammation biomarkers and esophageal squamous cell carcinoma: A case-cohort study in Japan.

Esophageal squamous cell carcinoma (ESCC) is the predominant histologic subtype of esophageal cancer worldwide. Measurements of circulating inflammation-related biomarkers may inform etiology or provide noninvasive signatures for early diagnosis. We therefore examined levels of inflammation molecules for associations with ESCC risk. Using a case-cohort study designed within the Japan Public Health Center-based Prospective Study, we measured baseline plasma levels of 92 biomarkers using a multiplex assay in a subcohort of 410 randomly selected participants and 66 participants with incident ESCC (including four cases that occurred in the subcohort). ESCC hazard ratios (HRs) were calculated for 2-4 quantiles of each biomarker by Cox proportional hazards regression models with age as the time metric, adjusted for sex, smoking and alcohol use. Twenty analytes were undetectable in nearly all samples. Of the remaining 72, 12 biomarkers (FGF19, ST1A1, STAMBP, AXIN1, CASP8, NT3, CD6, CDCP1, CD5, SLAMF1, OPG and CSF1) were associated with increased ESCC risk (ptrend < 0.05) with HRs per quantile 1.28-1.65. Seven biomarkers (CXCL6, CCL23, CXCL5, TGFA, CXCL1, OSM and CCL4) were inversely associated with HRs 0.57-0.72. FGF19, CASP8, STAMBP, ST1A1 and CCL-4 met statistical significance with false discovery rate correction. Associations did not differ <5 vs. ≥5 years between blood collection and ESCC diagnosis. CASP8, STAMBP and ST1A1 were strongly correlated (p < 0.05). Our study expands the range of inflammation molecules associated with the development of this highly lethal neoplasia. Correlations among these novel biomarkers suggest a possible shared pathway. These findings need replication and could further delineate ESCCs molecular mechanisms of carcinogenesis.

Effect of maternal omega-3 fatty acids and vitamin E supplementation on placental apoptotic markers in rat model of early and late onset preeclampsia.

AIM: Disturbed placentation results in pregnancy complications like preeclampsia. Placental development is influenced by apoptosis during trophoblast differentiation and proliferation. Increased oxidative stress upregulates placental apoptosis. We have earlier reported increased oxidative stress, lower omega-3 fatty acids and vitamin E levels in women with preeclampsia. Current study examines effect of maternal omega-3 fatty acids and vitamin E supplementation on apoptotic markers across gestation in a rat model of preeclampsia. MAIN METHODS: Pregnant Wistar rats were randomly assigned to control; early onset preeclampsia (EOP); late onset preeclampsia (LOP); early onset preeclampsia + omega-3 fatty acid + vitamin E supplementation (EOP + O + E) and late onset preeclampsia + omega-3 fatty acid + vitamin E supplementation (LOP + O + E) groups. Animals (Control, EOP, EOP + O + E) were sacrificed at d14 and d20 of gestation while animals (LOP, LOP + O + E) were sacrificed at d20 to collect blood and placentae. Protein and mRNA levels of apoptotic markers were analyzed by ELISA and RT-PCR respectively. KEY FINDINGS: Protein levels of proapoptotic markers like Bcl-2 associated X-protein (BAX) (p < 0.05), caspase-8 and 3 (p < 0.01 for both) and malondialdehyde (p < 0.01) were higher only in the EOP group as compared to control. However, the antiapoptotic marker, B cell lymphoma 2 (Bcl-2) protein levels were lower in both the subtypes of preeclampsia (p < 0.01 for both). SIGNIFICANCE: Our findings suggest that supplementation was beneficial in reducing the caspase-8 and 3 in early onset preeclampsia but did not normalize BAX and Bcl-2 levels. This has implications for reducing placental apoptosis in preeclampsia.

Functional Analysis of Aberrantly Spliced Caspase8 Variants in Adult T-Cell Leukemia Cells.

The genetic and molecular alterations responsible for leukemogenesis and progression of HTLV-infected adult T-cell leukemia (ATL) have not been fully clarified. Previously, we reported that various genes are not only overexpressed but also abnormally spliced in ATL cells. Here, we identified various CASP8 transcript variants in PBMCs from a smoldering-type ATL patient, which encode aberrant truncated caspase 8 (Casp8) isoforms. Among those, we focus on the three transcript variants, CASP8L (including the first 136 bp of the intron 8 between exon 8 and exon 9), CASP8-ΔE4 (without the exon 4), and CASP8-ΔE7 (without the exon 7), because they encode isoforms, Casp8L, Casp8-ΔE4, and Casp8-ΔE7, respectively, without the C-terminal catalytic domains. In this study, we conducted in vitro characterization and functional analysis of those mutant Casp8 isoforms to clarify their changed functions compared with the wild-type (WT)-Casp8. We demonstrated that these abnormal Casp8 isoforms showed lower ability to induce apoptosis than WT-Casp8 due to their dominant-negative interactions with WT-Casp8, which impair WT-Casp8 homodimerization that is essential for induction of apoptosis. Moreover, Casp8L and Casp8-ΔE7, which have only two death-effector domains, significantly activated NFκB by forming filament-like structures, which probably function as scaffolds for the IKK complex formation. In view of increasing levels of these abnormal CASP8 transcripts in primary PBMCs from HTLV-1 carriers and patients with ATL, we propose a possibility that overexpression of those Casp8 mutants, with lower proapoptotic activities and higher NFκB-activating functions than WT-Casp8, may be one of the molecular abnormalities causing malignant transformation and growth of ATL cells. IMPLICATIONS: We describe naturally occurring CASP8 transcription variants in PBMCs from patients with ATL, which encode truncated Casp8-mutant isoforms with lower proapoptotic activities and higher NFκB-activating functions compared with WT-Casp8.

Receptor-Interacting Protein Kinase 3 (RIPK3) mRNA Levels Are Elevated in Blood Mononuclear Cells of Patients with Poor Prognosis of Acute-on-Chronic Hepatitis B Liver Failure.

Necroptosis refers to a programmed form of necrosis, which involves the receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). In this study, to investigate the role of necroptosis in the pathogenesis of acute-on-chronic hepatitis B liver failure (ACHBLF), we retrospectively analyzed 122 patients with ACHBLF, 131 patients with chronic hepatitis B (CHB), and 35 healthy controls (HCs). Using quantitative real-time polymerase chain reaction (RT-qPCR), we measured RIPK3 mRNA levels in peripheral blood mononuclear cells (PBMCs). ELISA was performed to measure the serum levels of MLKL, TNF-α and caspase-8. We found that RIPK3 mRNA levels were significantly higher in patients with ACHBLF than those with CHB or HCs. RIPK3 mRNA levels in patients with ACHBLF were positively correlated with serum levels of TNF-α or MLKL and negatively correlated with caspase-8 levels. Univariate and multivariate analysis revealed that RIPK3 mRNA level was predictive of 3-month mortality of ACHBLF. The area under receiver operating characteristic curve (AUC) of RIPK3 mRNA levels was 0.810 (95% CI: 0.729-0.876), which was higher than that of MELD scores (0.766, 95% CI: 0.681-0.838). The optimal cut-off point of 8.81 was determined for RIPK3 mRNA levels, which showed a sensitivity of 80.7% and a negative predictive value of 80.4%. These results indicate that elevated RIPK3 mRNA levels in PBMCs are associated with poor prognosis of ACHBLF. We thus propose that necroptosis may play an important role in pathogenesis of ACHBLF.

Brain-Derived Neurotrophic Factor Levels are Lower in Chronic Stroke Patients: A Relation with Manganese-dependent Superoxide Dismutase ALA16VAL Single Nucleotide Polymorphism through Tumor Necrosis Factor-α and Caspases Pathways.

The manganese-dependent superoxide dismutase (MnSOD) Ala16Val single nucleotide polymorphism (SNP) has shown to be associated to risk factors of vascular diseases. Brain-Derived Neurotrophic Factor (BDNF) plays an essential role in the plasticity and neuronal regeneration of the brain after vascular injuries. However, little is known about interaction between MnSOD Ala16Val SNP on stroke, a frequent neurologic disease that involves various interacting pathways, such as vascular dysfunctions, inflammation, and neurotrophic factors. In this sense, the objective of this study was to investigate the relationship between MnSOD Ala16Val SNP with BDNF levels on stroke and also its influence on nitrosative stress, inflammatory, apoptotic, and DNA damage parameters. For this, 88 subjects were investigated, 44 subjects poststroke and 44 healthy controls. Questionnaires were applied to clinical characteristics and after laboratorial exams were collected. We analyzed levels of oxidative/nitrosative stress, inflammatory, apoptotic, and DNA damage markers. We showed a higher proportion of VV genotype in poststroke as compared to healthy subjects. Nitrite/nitrate, Tumor Necrosis Factor-α, Caspase 3 (CASP 3) and 8 (CASP 8) activation, Acethylcholinesterase (AChE), and Picogreen levels were higher in VV poststroke group, as well as BDNF and ACh levels were lower in VV and AV poststroke. We may suggest that V allele carries a worse outcome profile after stroke, relating to nitrosative stress, inflammatory, and apoptotic response. These events associated to a BDNF reduction, probably, contribute to the appearance or reincidence of stroke.

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.

Plasma Concentration of Caspase-8 Is Associated With Short Sleep Duration and the Risk of Incident Diabetes Mellitus.

Context: The biological mechanism for the association between sleep duration and incident diabetes mellitus (DM) is unclear. Sleep duration and caspase-8, a marker of apoptotic activity, have both been implicated in ß-cell function. Objective: To investigate the associations between sleep duration and plasma caspase-8 and incident DM, respectively. Design: Prospective cohort study. Setting: The Malmö Diet and Cancer (MDC) Study is a population-based, prospective study run in the city of Malmö, Sweden. Participants: A total of 4023 individuals from the MDC Study aged 45 to 68 years at baseline without a history of prevalent DM and with information on habitual sleep duration. Main Outcomes: Incident DM. Results: Mean follow-up time was 17.8 years. Sleep duration was the only behavioral variable significantly associated with plasma caspase-8. Plasma caspase-8 was significantly associated with incident DM per standard deviation of its transformed continuous form [hazard ratio (HR) = 1.24; 95% confidence interval (CI), 1.13 to 1.36] and when dichotomized into high (quartile 4) (HR = 1.44; 95% CI, 1.19 to 1.74) compared with low (quartiles 1 to 3) concentrations. Caspase-8 interacted with sleep duration; compared with individuals who had 7 to 8 hours of sleep and low plasma caspase-8, those with high plasma caspase-8 and sleep duration <6 hours (HR = 3.54; 95% CI, 2.12 to 5.90), 6 to 7 hours (HR = 1.81; 95% CI, 1.24 to 2.65), and 8 to 9 hours (HR = 1.54; 95% CI, 1.09 to 2.18) were at significantly increased risks of incident DM. Conclusions: Sleep duration is associated with plasma caspase-8. Caspase-8 independently predicts DM years before disease onset and modifies the effect of sleep duration on incident DM. Future studies should investigate if change of sleep duration modifies plasma concentrations of caspase-8.

FADD, Caspase-3, and Caspase-8 and Incidence of Coronary Events.

OBJECTIVE: To investigate the relationship between 3 markers of apoptosis, that is, FADD (Fas-associated death domain-containing protein), caspase-3, and caspase-8, and incidence of coronary events (CEs) in a population-based cohort study. APPROACH AND RESULTS: In vitro experiments were performed to assess the response of the apoptotic biomarkers after Fas stimulation of peripheral blood mononuclear cells. The experiments showed significantly increased releases of FADD, caspase-3, and caspase-8 after Fas stimulation. The relationship between FADD, caspase-3, and caspase-8, respectively, and incidence of CEs was studied in 4284 subjects from the population-based Malmö Diet and Cancer Study. Cox' proportional hazards regression was used to examine the association between the apoptotic biomarkers and incidence of CE over a mean follow-up of 19 years. A total of 381 individuals had CE during the follow-up. High FADD at baseline was significantly associated with incident CE. In the highest compared with the lowest quartile of FADD, the risk factor adjusted hazards ratio for CE was 1.82 (95% confidence interval, 1.35-2.46; P for trend <0.001). A significant association was also found between caspase-8 and CE; the hazards ratio (Q4 versus Q1) was 1.90 (95% confidence interval, 1.39-2.60; P for trend <0.001) after adjustment for risk factors. No association was found between caspase-3 and CEs. CONCLUSIONS: High levels of FADD and caspase-8, but not caspase-3, were associated with increased incidence of CE in subjects from the general population. The in vitro experiments support the view that these biomarkers could reflect activation of the extrinsic apoptotic pathway.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL): a possible pathogenic role in chronic plaque psoriasis.

BACKGROUND: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine member of the tumour necrosis factor (TNF) family. Its role has been investigated in skin cancers and some inflammatory and/or immune-mediated skin diseases. An involvement of TRAIL in psoriasis pathogenesis has recently been hypothesized. We investigated the expression and localization of TRAIL and its receptors in psoriatic skin and measured serum TRAIL. The intracellular pathways activated by TRAIL were assessed to investigate its potential role in the pathogenesis of psoriasis. METHODS: Twenty-four consecutive patients with plaque psoriasis and age- and sex-matched healthy subjects were recruited. Serum TRAIL was measured by means of an enzyme-linked immunosorbent assay (ELISA). TRAIL and TRAIL receptors were evaluated by reverse transcription - polymerase chain reaction (RT-PCR) (RNA of lesional and non-lesional psoriatic skin) and by immunohistochemistry (lesional skin). Caspase 8 and NF-kB immunoexpression were also evaluated by immunohistochemistry. RESULTS: RT-PCR demonstrated increased synthesis of TRAIL and its receptors in lesional vs. non-lesional skin. Immunohistochemistry showed a strong staining of TRAIL and TRAIL receptors both in the epidermis and in the dermal infiltrate. Finally, a correlation emerged between caspase 8 and TRAIL immunoexpression in the dermis. CONCLUSIONS: Our findings suggest an involvement of TRAIL in psoriasis pathogenesis, probably through an action at the site of the inflammatory infiltrate, likely via caspase 8.

[Soluble apoptosis markers in obese patients with food intolerance].

For the soluble apoptosis markers study 151 patients with obesity (92 women and 59 men) aged between 18 and 63 years were examined. Diagnosis and degree of obesity was based on the body mass index (38.2 +/- 5.4 kg/m2). Generally food intolerance was identified in 36.4% of obese patients. Four groups of patients were formed: three groups of patients with obesity stage I (15 patients), II (18 patients) and III (22 patients), respectively, and with food intolerance, and a group of obese patients without food intolerance (control group, n = 31). Obese patients with food intolerance received standard version of hypocaloric diet with the exception of specific food allergens. Duration of observation was 39-43 days. Such soluble apoptosis markers as sFas-L, Caspase-9, Caspase-8 and sCD153 were significantly higher in stage III obesity patients compared obese patients without food allergy (0.120 +/- 0.030 vs 0.035 +/- 0.010; 13.2 +/- 3.2 vs 5.9 +/- 0.4; 1.4 +/- 0.18 vs 0.6 +/- 0.24; 0.123 +/- 0.010 vs 0.025 +/- 0.002 ng/ml respectively). Positive dynamic of sFas-L, Caspase-9 and Caspase-8 (decrease to 0.052 +/- 0.030; 7.7 +/- 2.2 and 0.4 +/- 0.18 ng/ml respectively) in patients with obesity stage III and intactness sCD153 during diet therapy course were revealed. Significant differences for only Caspase-9 in patients with obesity stage II were obtained. The data obtained are considered as normalization of apoptosis due to nutritional correction of immunological disorders. Study of sFas-L, Caspase-9 and Caspase-8 allows to predict the course of disease, as immunological research for early detection of food allergy makes possible to implement the principles of personalized diet therapy.