Diagnostic value of the CSF levels of D-Lactate and pro-inflammatory cytokines (TNF-alpha, IL-6, IL-8 and IL-17) in the patients with suspected nosocomial meningitis.

INTRODUCTION: This study aims to determine the diagnostic value of IL-6, IL-8, IL-17, TNF-α and D-lactate levels in the cerebrospinal fluid (CSF) in nosocomial meningitis. METHODS: CSF levels of cytokines and D-lactate were compared across 29 episodes who were diagnosed with nosocomial meningitis, 38 episodes with pleocytosis but without meningitis and 54 control subjects. RESULTS: CSF levels of IL-6, IL-8, and D-lactate were higher in the group with nosocomial meningitis compared to the control group and to the group with pleocytosis without meningitis (p<0.05). For the levels of IL-6, when the threshold was considered to be > 440 pg/mL, the sensitivity and specificity were 55.17% and 94.74%, respectively. For IL-8 levels, when the threshold was considered to be >1249 pg/mL, the sensitivity and specificity were 44.83% and 84.21%, respectively. In the patients with nosocomial meningitis, when the threshold of D-lactate levels was considered to be >1.05µmol/mL, the sensitivity and specificity were found to be 75.86% and 63.16%, respectively. In the pleocytosis without meningitis CSF samples and in the CSF samples diagnosed with nosocomial meningitis, the highest AUC was calculated for triple combination model of IL-6, IL-8, and D-lactate levels (AUC= 0.801, p<0.001), and double combination model IL-6 and IL-8 (AUC= 0.790) (p<0.001). CONCLUSION: In our study, we have concluded that IL-6, IL-8 and D-lactate levels could be diagnostic markers for nosocomial meningitis.

Can heat shock protein 32 be used for the early diagnosis of acute mesenteric ischemia?

OBJECTIVE: Acute mesenteric ischemia is a challenging and fatal disease. The aim of this study was to detect the heat shock protein 32 (HSP32) response in intestinal tissue and systemic blood to intestinal ischemia and ischemia/reperfusion to define a tool for the early diagnosis of acute mesenteric ischemia. MATERIAL AND METHODS: Thirty female Wistar albino rats were equally divided into 3 groups. Group 1 rats underwent simple laparotomy and closure (control). In Group 2 rats, 1-hour intestinal ischemia followed by 5-hour reperfusion was performed, and Group 3 rats were subjected to 6-hour intestinal ischemia. The experiment was repeated with a 24-hour waiting period. At the end of the waiting period, blood was withdrawn from the tail veins of the rats and the rats were sacrificed via cardiac puncture. Re-laparotomy was subsequently performed and intestinal tissue and luminal samples were obtained for biochemical and pathological investigations. The HSP32 levels of intestinal tissues, luminal contents and blood levels were compared among the groups. RESULTS: At the end of the 24-hour waiting period, the median tissue HSP32 levels were 0.43 (0-6.6) ng/mL for Group 1, 9.51 (2.5-49.9) ng/mL for Group 2 and 43.13 (6.3-121.3) ng/mL for Group 3 (p=0.001). The median blood HSP32 levels were 0.11 (0.1-1.4) ng/mL for Group 1, 0.42 (0.1-0.7) ng/mL for Group 2, and 0.25 (0.1-1.2) ng/mL for Group 3 (p=0.047). The HSP levels in the luminal contents were undetectable. CONCLUSION: Both ischemia and ischemia/reperfusion significantly raised intestinal tissue HSP32 levels in comparison with the control group. However, this change was not reflected in the circulating blood or luminal contents.

Anti-growth effect of a novel trans-dichloridobis[2-(2-hydroxyethyl)pyridine]platinum (II) complex via induction of apoptosis on breast cancer cell lines.

Breast cancer still continues to be the leading cause of cancer-related mortality in women worldwide. Although advances have been made in the treatment of this disease during the past decade, new approaches and novel compounds are urgently needed. The aim of this study was to evaluate the cytotoxic activity of trans-[PtCl2(2-hepy)2] [2-hepy=2-(2-hydroxyethyl) pyridine] on breast cancer cell lines, MCF-7 and MDA-MB-231. The platinum (II) complex was synthesized and characterized by our laboratory working group. Anti-growth effect was assayed by the MTT and ATP viability assays and also monitored real-time using xCELLigence system. The mode of cell death was evaluated by using the fluorescence microscopy (Hoechst 33342+Calcein-AM+Propidium iodide staining), Western blotting (cleaved PARP and caspase 3, total caspase 8), flow cytometry (quantitative analysis of live, early/late apoptotic, dead cells and caspase 3/7 activity) and the RT-PCR (the genes analyzed were BCL-2L10, BIK, BAX, BCL-2, FASLG, HRK, TNFRSF10B, and TNFRSF10A). The platinum (II) complex had anti-growth effect in a dose dependent manner in vitro. Cells were killed by apoptosis as evidenced by the pyknotic nuclei, cleavage of poly-(ADP-ribose) polymerase (PARP) and induction of active caspase-3. These results suggest that the complex might represent a potentially active novel drug for the breast cancer treatment and warrants further studies due to its promising cytotoxic activity.

The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18.

The primary aim of this study was to compare measurement of apoptosis by M30 immunoreactivity (a biomarker for apoptosis) to other apoptosis assays (morphological assessment of nuclei, Annexin-V-FITC staining, DNA fragmentation and PARP cleavage) in vitro. Caspase-cleaved cytokeratin 18 (M30, ccK18) is only produced in epithelial cells and is regarded as a pharmacodynamic biomarker of apoptotic cell death because it is released from cells during apoptosis induced by chemotherapeutic agents. However, we have observed false negative results using this assay in clinical samples. Therefore, we tested its ability to accurately detect apoptosis in a panel of lung cancer cell lines with a range of clinically approved chemotherapeutic drugs. Three different non-small cell lung cancer (NSCLC) cell lines (A549, H1299, PC3) were used to correlate M30 levels with alternate apoptosis assays. Following successful induction of apoptosis, the A549 cell line showed an increase in M30 levels along with other well-known features of apoptosis, whilst H1299 and PC3 cell lines did not show an increase in M30 levels, even when apoptosis was detected by other means. Further analysis showed that H1299 and PC3 cell lines expressed much lower levels of cytokeratin 18 protein compared to the A549 cell line. Our results suggest that reliable detection of apoptosis via the M30 assay only works when sufficient levels of cytokeratin 18 are present in the cells. This means that the M30 assay may result in false negative results for apoptosis, and as such, the ELISA should be used in conjunction with other assays.

Effects of off-pump versus on-pump coronary artery bypass grafting: apoptosis, inflammation, and oxidative stress.

BACKGROUND: It has been suggested that off-pump coronary artery bypass grafting (CABG) surgery reduces myocardial ischemia-reperfusion injury, postoperative systemic inflammatory response, and oxidative stress. The aim of this study was to measure serum malondialdehyde (MDA), high-sensitivity C-reactive protein (hs-CRP), M30, and M65 levels and to investigate the relationship between M30 levels and oxidative stress and inflammation in patients undergoing on-and off-pump CABG surgery. METHODS: Fifty patients were randomly assigned to on-pump or off-pump CABG surgery (25 patients off-pump and 25 on-pump CABG surgery), and blood samples were collected prior to surgery, and 30 minutes, 60 minutes, 6 hours, and 24 hours after CABG surgery. RESULTS: Compared to the on-pump group, serum MDA levels at 30 minutes, 60 minutes, 6 hours, and 24 hours after the CABG surgery were significantly lower in the off-pump group (P=.001, P=.001, P=.001, and P=.001, respectively). Serum M30 levels were found to be elevated in both groups, returning to baseline at 24 hours. When compared to baseline, the hs-CRP level reached its peak at 24 hours at 13.28±5.32 mg/dL in the on-pump group, and 15.44±4.02 mg/dL in the off-pump group. CONCLUSION: CABG surgery is associated with an increase in inflammatory markers and serum M30 levels, indicating epithelial/endothelial apoptosis in the early period.

Can safe and long-term exposure to extremely low frequency (50 Hz) magnetic fields affect apoptosis, reproduction, and oxidative stress?

PURPOSE: To determine whether 50 Hz extremely low frequency-magnetic fields (ELF-MF) affects apoptotic processes, oxidative damage, and reproductive characteristics such as sperm count and morphology in rat testes. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were used in the present study, which were divided into three groups (sham group, n = 10, and two experimental groups, n = 10 for each group). Rats in the experimental group were exposed to 100 and 500 µT ELF-MF (2 h/day, 7 days/week, for 10 months) corresponding to exposure levels that are considered safe for humans. The same experimental procedures were applied to the sham group, but the ELF generator was turned off. Tissues from the testes were immunohistochemically stained for active (cleaved) caspase-3 in order to measure the apoptotic index by a semi-quantitative scoring system. The levels of catalase (CAT), malondialdehyde (MDA), myeloperoxidase (MPO), total antioxidative capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) were also measured. Additionally, epididymal sperm count and sperm morphology was evaluated. RESULTS: There were no significant differences in the reproductive and oxidative stress parameters between the sham group and the exposed groups (p > 0.05). While no difference was observed between the final apoptosis score of the sham and the 100 µT ELF-MF group (p > 0.05), the final apoptosis score was higher in the 500 µT ELF-MF exposure group than in the sham group (p < 0.05). CONCLUSION: Long-term exposure to 100 µT and 500 µT ELF-MF did not affect oxidative or antioxidative processes, lipid peroxidation, or reproductive components such as sperm count and morphology in testes tissue of rats. However, long-term exposure to 500 µT ELF-MF did affect active-caspase-3 activity, which is a well-known apoptotic indicator.

Promising anti-growth effects of palladium(II) saccharinate complex of terpyridine by inducing apoptosis on transformed fibroblasts in vitro.

Fibrosarcoma is one of the fatal cancer types and there is still not satisfactory success in its treatment despite new drugs. Therefore, the search for a new compound has been going on. It is currently known that some palladium-based anti-cancer compounds seem to have powerful apoptosis-inducing effects in cancer cells. For this purpose, a palladium(II)-saccharinate complex containing terpyridine which was synthesized by our research group was investigated in terms of its anti-tumor effects against mouse embryonic fibroblast NIH/3T3 (normal cell line) and rat embryonic fibroblast 5RP7 (H-ras transformed cell line) in vitro. The MTT and ATP viability assays were used to determine anti-growth/cytotoxic effects. Cytotoxic activity was confirmed by real time cytotoxicity analysis system. Flow cytometry analysis was further used to determine the mode of cell death (apoptosis/necrosis). Apoptosis was confirmed by triple-staining the cells with Hoechst 33342/PI/Calcein-AM triple and evaluated with fluorescence microscopy. It was found that the compound showed significant anti-growth activity by inducing apoptosis in a dose dependent manner. In conclusion, taking into account the cytotoxic activity of the compound at even relatively lower doses, in vivo experiments to elucidate its potential use for the treatment of fibrosarcoma are warranted.

Serum fetuin A/α2HS-glycoprotein levels in patients with non-alcoholic fatty liver disease: relation with liver fibrosis.

BACKGROUND: Serum concentrations of fetuin A/α2HS-glycoprotein (AHSG) have been linked to human metabolic alterations and can serve as an indicator of liver cell function. We assayed serum levels of AHSG in patients with non-alcoholic fatty liver disease (NAFLD), a hepatic manifestation of the metabolic syndrome, and examined their association with clinical, biochemical and histological phenotypes. METHODS: Serum AHSG levels were determined by enzyme linked immunosorbent assay in 99 patients with biopsy-proven NAFLD and 75 age- and gender-matched controls. RESULTS: Serum AHSG levels were significantly higher in patients with NAFLD (940 ± 120 µg/mL) compared with healthy controls (800 ± 130 µg/mL, Student's t test, P < 0.001). Bivariate analyses (Spearman's rank correlation) in patients with NAFLD showed a statistically significant association between AHSG levels and insulin resistance as assessed by the HOMA (homeostasis model assessment) index (r = 0.31, P < 0.01) and the liver fibrosis score index (r = 0.36, P < 0.001). The association between AHSG and fibrosis remained statistically significant even after adjustment for potential confounders, including the HOMA index ([beta] = 1.65, t = 2.38, P < 0.05). CONCLUSION: Serum AHSG levels are significantly increased in adult patients with biopsy-proven NAFLD and are associated with insulin resistance. Importantly, our pilot data indicate that serum AHSG levels may identify NAFLD patients with higher fibrosis scores.

Comparative effects of pioglitazone and rosiglitazone on plasma levels of soluble receptor for advanced glycation end products in type 2 diabetes mellitus patients.

Low levels of soluble receptor for advanced glycation end products (sRAGE) have been associated with the occurrence of vascular complications in patients with type 2 diabetes mellitus. Preliminary evidence has suggested that thiazolidinediones have the ability to modulate circulating levels of this molecule in the hyperglycemic milieu. The aim of this pilot study was to assess the differential effect of 2 different thiazolidinediones-pioglitazone and rosiglitazone-on plasma levels of sRAGE in type 2 diabetes mellitus patients. Sixty type 2 diabetes mellitus subjects were randomly assigned to receive pioglitazone (30 mg/d, n = 19), rosiglitazone (4 mg/d, n = 20), or placebo (medical nutrition therapy, n = 21) for 12 weeks. Changes in plasma glucose, glycosylated hemoglobin, insulin resistance (homeostasis model assessment), total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and sRAGE were evaluated at baseline and after 12 weeks. At 12 weeks, the pioglitazone (P < .001) group had a significant increase from baseline in sRAGE values that was not seen in the medical nutrition therapy and rosiglitazone groups. We conclude that, in type 2 diabetes mellitus patients, pioglitazone-but not rosiglitazone-significantly raised sRAGE, which may contribute to its antiatherogenic effects.

Serial changes in circulating M30 antigen, a biomarker of apoptosis, in patients with acute coronary syndromes: relationship with the severity of coronary artery disease.

OBJECTIVES: Growing evidence has indicated the potential clinical usefulness of measuring different forms of cytokeratin 18 in patient sera (M30 antigen for apoptosis and M65 antigen for necrosis) for distinguishing different forms of cell death. Preliminary data have reported altered levels of cytokeratin 18 fragments in patients with acute coronary syndrome (ACS) and ischemic heart disease. In this study, serum levels of M30 and M65 were measured in 74 patients with ACS [including 17 cases with unstable angina and 57 patients with acute myocardial infarction (AMI)], 25 patients with stable angina, and 23 controls. METHODS: In patients with ACS, serial measurements of M30 and M65 were obtained, and for each patient, the following values were determined: (i) values at admission, (ii) values obtained 24 h after symptom onset, and (iii) values obtained at 48 h after symptom onset. The severity of coronary atherosclerosis was expressed using the Gensini score. RESULTS: On admission, M30 and M65 levels in ACS patients were similar to those observed in stable angina patients and control participants. In AMI patients, serum levels of M30 peaked at 24 h and declined thereafter at 48 h. Notably, serum levels of M30 measured at 24 h correlated significantly and positively with the extent of coronary artery disease as measured by the Gensini score in AMI patients (r = 0.253, P<0.05). CONCLUSION: Serum levels of the apoptotic marker M30 peak at 24 h after AMI and reflects the extent of coronary artery disease in this patient group.

Elevated serum levels of caspase-cleaved cytokeratin 18 (CK18-Asp396) in patients with nonalcoholic steatohepatitis and chronic hepatitis C.

BACKGROUND: Caspase-cleaved cytokeratin 18 (CK18-Asp396) is released from hepatocytes during apoptosis. Recent studies have indicated that serum levels of CK18-Asp396 could be a clinically useful biomarker of chronic liver disease. To shed more light on the rate of hepatocyte loss by apoptosis in chronic liver disease, serum levels of CK18-Asp396 were examined in patients with nonalcoholic steatohepatitis (NASH) and chronic hepatitis C. MATERIAL/METHODS: Apoptotic CK18-Asp396 levels were quantified in sera from 35 patients with nonalcoholic steatohepatitis (NASH), 21 patients with chronic hepatitis C (HCV), and 18 healthy controls. RESULTS: Analysis of serum CK18-Asp396 levels showed an increasing trend starting from healthy controls (median: 54.5 U/l), to HCV patients (80.1 U/l), to patients with NASH (144.1 U/l, Kruskall-Wallis test: P<0.001). Post hoc analyses revealed that CK18-Asp396 levels were significantly higher in the NASH patients than in both HCV patients (P=0.008) and healthy controls (P<0.001). Moreover, the levels were significantly higher in patients with HCV than in control individuals (P<0.05). In patients with chronic HCV infection there was a significant positive correlation between serum CK18-Asp396 levels and AST (r=0.442, P<0.05), the ultrasonographic grade of steatosis (r=0.446, P<0.05), and the histological steatosis score (r=0.759, P<0.001). CONCLUSIONS: Although subject to future confirmation, these pilot findings seem to indicate that serum levels of caspase-cleaved cytokeratin 18 (CK18-Asp396) are higher in patients with NASH than in those with chronic HCV infection. These data suggest that NASH patients have an increased hepatocyte loss by apoptosis compared with chronic hepatitis C patients.

Decreased plasma levels of soluble receptor for advanced glycation endproducts (sRAGE) in patients with nonalcoholic fatty liver disease.

OBJECTIVES: Levels of soluble receptor for advanced glycation endproducts (sRAGE) have been linked to several components of the metabolic syndrome. We tested the hypothesis that plasma levels of sRAGE may be associated with non-alcoholic fatty liver disease. DESIGN AND METHODS: We enrolled subjects with definite nonalcoholic steatohepatitis (NASH, n=40), borderline NASH (n=8), simple fatty liver (n=9) and healthy controls (n=14). Plasma levels of sRAGE were measured by ELISA. RESULTS: Concentrations of sRAGE were significantly lower in patients with definite NASH (1080+/-392 pg/mL, P<0.01) and borderline NASH (1050+/-278 pg/mL, P<0.05) compared to controls (1480+/-387 pg/mL). Levels of sRAGE were significantly and inversely correlated with ALT (r=-0.30, P<0.05) and AST (r=-0.23, P<0.05). CONCLUSION: Plasma levels of sRAGE are significantly reduced in definite and borderline NASH.

Association between serum neopterin, obesity and daytime sleepiness in patients with obstructive sleep apnea.

OBJECTIVE: Obesity and obstructive sleep apnea (OSA) and systemic inflammation may interact through biochemical pathways. Neopterin (NP) is a monocyte/macrophage activation marker produced by macrophages in response to interferon-gamma secreted by activated T-lymphocytes. This study examines the association between NP, obesity and OSA. PATIENTS AND METHODS: The study included 22 newly diagnosed OSA (+) patients and 18 OSA (-) patients. Subjects with history of coronary artery disease, transplant patients, history of alcohol and drug abuse, history of HIV and any other significant medical illnesses such as active infections, autoimmune disease, malignancy, liver disease, pulmonary disease (COPD, asthma,...), neuromuscular disease, patients on immunomodulating therapy or HMG-CoA reductase inhibitors were excluded. RESULTS: There were no significant differences in age, body mass index (BMI), and smoking habits of the OSA (+) patients and OSA (-) patients. Serum NP levels did not show any significant difference between the OSA (+) patients and OSA (-) patients, however, NP levels were positively correlated with BMI (r=0.320, p=0.044). There was no significant correlation between NP and any of the polysomnographic parameters. The result of stepwise regression analyses (r(2)=0.320, p<0.001) showed that high serum NP levels (p=0.004) and apnea-hypopnea index (AHI) were a risk factor for elevated Epworth sleepiness score, independent of BMI. CONCLUSION: We suggest that serum NP levels correlate with BMI. There was a significant relationship between serum NP levels and excessive daytime sleepiness in OSA patients.

The MTT assay yields a relatively lower result of growth inhibition than the ATP assay depending on the chemotherapeutic drugs tested.

Accurate assessment of the anti-growth effects of chemotherapeutics is immensely importance in cancer research with regard to drug discovery and toxicological safety. A number of in vitro cytotoxicity assays are used for these purposes. However, there is the possibility for different results in the assessments because the way they measure the viability of cancer cells is specific to each assay. In the present study, the performance of two common assays (MTT and ATP) in the assessment of anti-growth effects of chemotherapeutics on a lung cancer cell line (A549) was compared. The cells were treated with paclitaxel, docetaxel, gemcitabine, 5-fluorouracil (5-FU), etoposide, doxorubicin, epirubicin, cisplatin, 4-hydroperoxycyclophosphamide (4-HC) and carboplatin in six different concentrations. When taking all the drugs and inhibitions into account, a moderate correlation (r=0.670; p=0.01) between the assays was found. However, IC 50 values by the MTT assay were higher in 90% of the drugs than those found by the ATP assay. In addition to this, there was a statistically significant difference between the dose response curves of the assays, which was dependent on the drugs of choice. We recommend caution in comparing these assays to evaluate the anti-growth effects of chemotherapeutics because the MTT assay seem to give rise to relatively lower inhibition (higher viability) levels than the ATP assay, depending on the drugs of choice.