Kinetic assay for the determination of the oxidative stress biomarker, advanced oxidation protein products (AOPP) in the human blood plasma

A number of human diseases and pathological conditions were found to be associated with increased oxidative stress. In the literature several techniques are available for the assessment of oxidative stress, but most of them are not applicable for a routine medical laboratory due to the complex methodology and/or financial reasons. We report here on a simple, inexpensive, kinetic assay for the determination of the oxidative stress biomarker, advanced oxidation protein products (AOPP) in the human blood plasma. METHODS: This study involved 70 patients (47M/23F; mean age: 64.6 y; range: 16-85) admitted to our Department with a wide range of cardiovascular and peripheral vascular diseases. Three critically ill patients were assigned for monitoring purposes. Plasma AOPP were simultaneously determined using an end-point assay as reference method and by a kinetic method developed in our laboratory. Plasma fibrinogen concentration was measured according to the Clauss method. RESULTS: There was a highly significant correlation (r2 = 0.588; p < 0.0001) between AOPP concentration (reference method) and AOPP reactivity (kinetic method). Both AOPP concentration and AOPP reactivity also significantly correlated with plasma fibrinogen concentration (r2 = 0.780; p < 0.0001; r2 = 0.564; p < 0.0001). The three representative cases presented appear to support the relevance of our novel method in the monitoring of critically ill patients. CONCLUSIONS: This simple and inexpensive kinetic assay can be widely used in any routine laboratory interested in oxidative stress research. It is especially recommended for monitoring critically ill or other patients.     

The Chitinase-like Protein YKL-40 Is Secreted by Airway Epithelial Cells at Base Line and in Response to Compressive Mechanical Stress

The chitinase-like protein YKL-40, encoded by the CHI3L1 gene, is a biomarker and functional effector of chronic inflammatory and allergic diseases. In the lung it is associated with asthma severity and reduced lung function. The cellular sources of YKL-40 in human airways and the mechanisms regulating YKL-40 expression are poorly understood. We previously showed that mechanical stress similar to that experienced during bronchoconstriction triggers epithelial cell signaling through epidermal growth factor receptor (EGFR), fibrotic mediator release, and goblet cell hyperplasia consistent with airway remodeling in asthma. We now show that well differentiated normal human bronchial epithelial cells express CHI3L1 and secrete YKL-40 under base-line culture conditions. Mechanical stress (30-cm H₂O transcellular compressive stress) applied for 3 h induces CHI3L1 expression by ∼4-fold compared with time matched controls, resulting in increased secretion of YKL-40 by 3.6-fold 24 h after onset of the 3-h stimulus. Inhibition of EGFR or MEK1/2 (ERK kinase) significantly but incompletely attenuates mechanical stress-induced up-regulation of CHI3L1 expression in normal human bronchial epithelial cells. Direct activation of EGFR utilizing EGF-family ligands induces CHI3L1 expression. Our results reveal that human airway epithelial cells are a source of YKL-40 and demonstrate that mechanical stress potently induces CHI3L1 expression leading to increased secretion of YKL-40 protein in an EGFR and MEK1/2-dependent pathway. In the asthmatic airway mechanical stress may contribute to enhanced YKL-40 levels.     

Prognostic Value of Blood Zinc, Iron, and Copper Levels in Critically Ill Children with Pediatric Risk of Mortality Score III

We aimed to explore the association of blood Zn, Fe, and Cu concentrations and changes in the pediatric risk of mortality (PRISM) score in critically ill children, to predict prognosis. We included 31 children (22 boys and 9 girls, 1 month to 5 years old), who had been admitted to the intensive care unit of our hospital and who were critically ill according to PRISM score of III. Another 20 children (12 boys, 8 girls, 3 months to 5 years old) who were brought to the hospital for a health checkup were included as controls. We recorded clinical data, time in the intensive care unit, prognosis, and PRISM III score for critically ill children. Blood Cu, Zn, and Fe values were measured by inductively coupled plasma atomic emission spectrophotometry. Zn and Fe levels were significantly lower in patients than in controls (all p?<?0.05). Cu levels differed between patients and controls, but not significantly (p?>?0.05). In ill children, blood Zn and Fe concentrations were inversely correlated with PRISM III score (Zn: r?=??0.36; Fe: r?=??0.50, both p?<?0.05), with no significant correlation of blood Cu level and PRISM III score (r?=??0.13, p?>?0.05). Serious illness in children may lead to decreased Zn and Fe blood concentrations. Zn and Fe supplements may be beneficial for critically ill children.     

Impaired plasma antioxidative defense and increased nontransferrin-bound iron during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation

To analyze the effects of radiochemotherapy on the pro-oxidative/antioxidative balance in plasma, we measured the total radical antioxidant parameter of plasma (TRAP) and single plasma antioxidants (uric acid, sulfhydryl groups, alpha-tocopherol, ubiquinone-10/total coenzyme-Q10 ratio, ascorbate, and bilirubin) every 12 h during high-dose chemotherapy and radiochemotherapy preceding bone marrow transplantation (BMT). Nontransferrin-bound iron (NTBI) was monitored as a potential pro-oxidant. Plasma levels of polyunsaturated fatty acids (PUFA) were measured as substrates, and thiobarbituric acid-reactive substances (TBARS) were measured as products of lipid peroxidation. Allantoin was analyzed as the product of uric acid oxidation. Patients receiving busulfan, VP-16, and cyclophosphamide (BU/VP/CY) (n = 8) were compared with those receiving total body irradiation in addition to VP-16 and cyclophosphamide (TBI/VP/CY) (n = 8). TRAP values were within the normal range before therapy and decreased after BU/VP/CY by 37% (p <. 02) and after TBI/VP/CY by 39% (p <.02). During TBI and after VP-16, a temporary increase in TRAP values occurred, which was not related to changes in individual antioxidants. In vitro experiments confirmed that VP-16 had an antioxidative effect. The concentration of uric acid declined in both groups and correlated with TRAP (BU/VP/CY: r =.80, p <.001; TBI/VP/CY: r =.84, p <.001). Levels of NTBI, which is normally not found in plasma, increased rapidly during conditioning therapy (p <.02 in both groups) and correlated inversely with TRAP (weighted intraindividual Spearman rank correlation coefficient for both groups: NTBI and TRAP: r = -.59, p <.001) and PUFA (in the radiochemotherapy group: r = -.67, p <.001). Whereas PUFA declined (p <.02 in both groups), TBARS increased (p <. 05 in both groups). Furthermore, an increase of allantoin and ubiquinone-10/total coenzyme-Q10 ratio in the BU/VP/CY group was found (allantoin: p <.02; ubiquinone-10/total coenzyme-Q10 ratio: p <.05). Antioxidants only partially recovered to baseline values until day 14 after BMT. Our findings indicate oxidative stress after high-dose radiochemotherapy and suggest a contribution of NTBI therein.     

Urinary excretion of 8-hydroxydeoxyguanosine and malondialdehyde after high dose radiochemotherapy preceding stem cell transplantation

The urinary excretion of the hydroxylated DNA base 8-hydroxydeoxyguanosine (8-OHdG) and the lipid peroxidation product malondialdehyde (MDA) was monitored in 11 patients with hematological malignancies undergoing total body irradiation and high-dose chemotherapy preceding bone marrow transplantation. Nine patients showed a prompt increase in urinary 8-OHdG (8-25 times the initial baseline level) on days 0-7 after irradiation onset; the excretion then decreased during the aplastic period and increased again when engraftment took place (in 7 patients). A significant positive correlation was found between urinary 8-OHdG and whole blood leukocyte count, both on day 5 (p =.04, r =.72) and on day 22 (p =.009, r =.80) after irradiation onset. One patient who lacked the first peak of 8-OHdG excretion showed low blood leukocyte counts (less than 2 x 10(9)/l) before therapy onset; this patient, however, later had a successful engraftment and then also showed considerable increases in both 8-OHdG excretion and leukocyte count. These observations suggest leukocytes play a part in the excretion of 8-OHdG after conditioning therapy preceding bone marrow transplantation. As opposed to the biphasic 8-OHdG excretion, the excretion of MDA showed a single peak appearing on days 11-19 after radiochemotherapy onset, i.e., during the period in which the patients suffered from cytopenia, mucositis, and other side effects of the treatment. It is suggested, therefore, that these clinical manifestations are associated with increased lipid peroxidation. Altogether, these findings illustrate the utility of serial urinary samples for monitoring oxidative stress due to conditioning therapy in clinical practice. They also demonstrate that different oxidative stress markers may behave quite differently regarding their appearance in the urine after whole-body oxidative stress.     

Impaired High-Density Lipoprotein Anti-Oxidant Function Predicts Poor Outcome in Critically Ill Patients

Introduction

Oxidative stress affects clinical outcome in critically ill patients. Although high-density lipoprotein (HDL) particles generally possess anti-oxidant capacities, deleterious properties of HDL have been described in acutely ill patients. The impact of anti-oxidant HDL capacities on clinical outcome in critically ill patients is unknown. We therefore analyzed the predictive value of anti-oxidant HDL function on mortality in an unselected cohort of critically ill patients.

Method

We prospectively enrolled 270 consecutive patients admitted to a university-affiliated intensive care unit (ICU) and determined anti-oxidant HDL function using the HDL oxidant index (HOI). Based on their HOI, the study population was stratified into patients with impaired anti-oxidant HDL function and the residual study population.

Results

During a median follow-up time of 9.8 years (IQR: 9.2 to 10.0), 69% of patients died. Cox regression analysis revealed a significant and independent association between impaired anti-oxidant HDL function and short-term mortality with an adjusted HR of 1.65 (95% CI 1.22–2.24; p = 0.001) as well as 10-year mortality with an adj. HR of 1.19 (95% CI 1.02–1.40; p = 0.032) when compared to the residual study population. Anti-oxidant HDL function correlated with the amount of oxidative stress as determined by Cu/Zn superoxide dismutase (r = 0.38; p<0.001).

Conclusion

Impaired anti-oxidant HDL function represents a strong and independent predictor of 30-day mortality as well as long-term mortality in critically ill patients.     

Hsp72 Is a Novel Biomarker to Predict Acute Kidney Injury in Critically Ill Patients

Background and Objectives

Acute kidney injury (AKI) complicates the course of disease in critically ill patients. Efforts to change its clinical course have failed because of the fail in the early detection. This study was designed to assess whether heat shock protein (Hsp72) is an early and sensitive biomarker of acute kidney injury (AKI) compared with kidney injury molecule (Kim-1), neutrophil gelatinase-associated lipocalin (NGAL), and interleukin-18 (IL-18) biomarkers.

Methods

A total of 56 critically ill patients fulfilled the inclusion criteria. From these patients, 17 developed AKI and 20 were selected as controls. In AKI patients, Kim-1, IL-18, NGAL, and Hsp72 were measured from 3 days before and until 2 days after the AKI diagnosis and in no-AKI patients at 1, 5 and 10 days after admission. Biomarker sensitivity and specificity were determined. To validate the results obtained with ROC curves for Hsp72, a new set of critically ill patients was included, 10 with AKI and 12 with no-AKI patients.

Results

Urinary Hsp72 levels rose since 3 days before the AKI diagnosis in critically ill patients; this early increase was not seen with any other tested biomarkers. Kim-1, IL-18, NGAL, and Hsp72 significantly increased from 2 days before AKI and remained elevated during the AKI diagnosis. The best sensitivity/specificity was observed in Kim-1 and Hsp72: 83/95% and 100/90%, respectively, whereas 1 day before the AKI diagnosis, the values were 100/100% and 100/90%, respectively. The sensibility, specificity and accuracy in the validation test for Hsp72 were 100%, 83.3% and 90.9%, respectively.

Conclusions

The biomarker Hsp72 is enough sensitive and specific to predict AKI in critically ill patients up to 3 days before the diagnosis.     

Two distinctly altered cellular responses to DNA double-strand breaks in human neuroblastoma

Neuroblastoma (NB), the most common extracranial solid tumors in children, presents with numerous genetic abnormalities that accumulate in a very short lifetime. To better understand this process, we have induced DNA double-strand breaks in NB cell lines and analyzed the activation of the ATM-H2AX/Chk2-p53 signaling pathway. We have found that NB cells could be classified into two distinct groups. The first group strongly expressed activated Chk2, displayed an important sub-G1 population, expressed very low levels of p21, and exhibited an attenuated G1 arrest. Conversely, the second group weakly expressed Chk2 pT68, displayed no sub-G1 cell population, strongly expressed p21, and exhibited a functional G1 arrest. These findings were independent of the MYCN amplification or p53 status of the NB cell lines tested. Interestingly, most p21 weakly expressing NB cells expressed neuron-specific enolase and Bcl2, two markers of N-type NB cells, but did not express vimentin, a marker of S-type NB cells. The expression profile was reversed in the p21 strongly expressing NB cells which highly expressed vimentin. Along with additional data, our findings lead us to propose that N-type-like NB cells would survive under stress conditions by antagonizing the Chk2-dependent apoptosis pathway, whereas S-type-like NB cells would survive by down-regulating Chk2 expression to facilitate the crossing of the senescence barrier.     

人EEN蛋白的结构预测和功能分析

采用基于神经网络的算法预测了我们自行克隆的新的白血病相关蛋白ＥＥＮ（ｅｘｔｒａ ｅｌｅｖｅｎｎｉｎｅｔｅｅｎ, ＥＥＮ）全长分子的二级结构,结果表明：ＥＥＮ 蛋白可能有三个结构域,Ｎ 端由三段α螺旋和短β折叠组成,中间为四段α螺旋组成的四螺旋结构,Ｃ端为ＳＨ３结构域,类似于在受体酪氨酸激酶信号传导途径中起重要作用的ＳＥＭ－５／ＧＲＢ２ Ｃ端ＳＨ３结构域;利用同源蛋白结构模拟的方法,模拟了ＥＥＮ ＳＨ３结构域的三维结构,结果表明：ＥＥＮ ＳＨ３结构域与ＳＥＭ－５／ＧＲＢ２ ＳＨ３结构域具有相近的结构,构成脯氨酸结合区的氨基酸非常保守．上述结果提示：ＥＥＮ 蛋白可能为新的信号蛋白,可能涉及新的信号传导途径或新的信号传导旁路,ＳＨ３结构域是其功能区域．     

Effects of glutamine supplementation, GH, and IGF-I on glutamine metabolism in critically ill patients

During critical illness glutamine deficiency may develop. Glutamine supplementation can restore plasma concentration to normal, but the effect on glutamine metabolism is unknown. The use of growth hormone (GH) and insulin-like growth factor I (IGF-I) to prevent protein catabolism in these patients may exacerbate the glutamine deficiency. We have investigated, in critically ill patients, the effects of 72 h of treatment with standard parenteral nutrition (TPN; n = 6), TPN supplemented with glutamine (TPNGLN; 0.4 g x kg(-1) x day(-1), n = 6), or TPNGLN with combined GH (0.2 IU. kg(-1). day(-1)) and IGF-I (160 microg x kg (-1) x day(-1)) (TPNGLN+GH/IGF-I; n = 5) on glutamine metabolism using [2-(15)N]glutamine. In patients receiving TPNGLN and TPNGLN+GH/IGF-I, plasma glutamine concentration was increased (338 +/- 22 vs. 461 +/- 24 micromol/l, P < 0.001, and 307 +/- 65 vs. 524 +/- 71 micromol/l, P < 0.05, respectively) and glutamine uptake was increased (5.2 +/- 0.5 vs. 7.4 +/- 0.7 micromol x kg(-1) x min(-1), P < 0.05 and 5.2 +/- 1.1 vs. 7.6 +/- 0.8 micromol x kg(-1) x min(-1), P < 0.05). Glutamine production and metabolic clearance rates were not altered by the three treatments. These results suggest that there is an increased requirement for glutamine in critically ill patients. Combined GH/IGF-I treatment with TPNGLN did not have adverse effects on glutamine metabolism.     

Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure

Lymphopenia and lymphoid depletion occur in adults dying of sepsis. Prolactin increases Bcl-2 expression, suppresses stress-induced lymphocyte apoptosis, and improves survival from experimental sepsis. We hypothesized that prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia occur in children dying with sepsis and multiple organ failure (MOF). Fifty-eight critically ill children with and 55 without MOF admitted to a university hospital pediatric intensive care unit were enrolled in a prospective, longitudinal, observational clinical study. Prolactin levels and absolute lymphocyte count were measured on days 1, 3, 7, 14, and 21. Lymph node, thymus, and spleen autopsy specimens were examined for lymphoid depletion, with immunohistochemical staining for CD4, CD20, and CD21 and for lymphoid apoptosis. Prolonged lymphopenia (absolute lymphocyte count < 1000 for >7 days) occurred only in children with MOF (29 vs 0%, p < 0.05) and was associated independently with nosocomial infection (odds ratio (OR), 5.5, 95% confidence interval (CI), 1.7-17, p < 0.05), death (OR, 6.8, 95% CI, 1.3-34, p < 0.05), and splenic and lymph node hypocellularity (OR, 42, 95% CI, 3.7-473, p < 0.05). Lymphocyte apoptosis and ante/postmortem infection were observed only in children with lymphoid depletion. Prolonged hypoprolactinemia (>7 days) was more common in children with MOF (17 vs 2%, p < 0.05) and was associated independently with prolonged lymphopenia (OR, 8.3, 95% CI, 2.1-33, p < 0.05) and lymphoid depletion (OR, 12.2, 95% CI, 2.2-65, p < 0.05). Prolonged lymphopenia and apoptosis-associated depletion of lymphoid organs play a role in nosocomial sepsis-related death in critically ill children. Prolonged hypoprolactinemia is a previously unrecognized risk factor for this syndrome.     

NSC-87877 inhibits DUSP26 function in neuroblastoma resulting in p53-mediated apoptosis

Dual specificity protein phosphatase 26 (DUSP26) is overexpressed in high-risk neuroblastoma (NB) and contributes to chemoresistance by inhibiting p53 function. In vitro, DUSP26 has also been shown to effectively inhibit p38 MAP kinase. We hypothesize that inhibiting DUSP26 will result in decreased NB cell growth in a p53 and/or p38-mediated manner. NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid), a novel DUSP26 small molecule inhibitor, shows effective growth inhibition and induction of apoptosis in NB cell lines. NB cell lines treated with small hairpin RNA (shRNA) targeting DUSP26 also exhibit a proliferation defect both in vitro and in vivo. Treatment of NB cell lines with NSC-87877 results in increased p53 phosphorylation (Ser37 and Ser46) and activation, increased activation of downstream p38 effector proteins (heat shock protein 27 (HSP27) and MAP kinase-activated protein kinase 2 (MAPKAPK2)) and poly ADP ribose polymerase/caspase-3 cleavage. The cytotoxicity resulting from DUSP26 inhibition is partially reversed by knocking down p53 expression with shRNA and also by inhibiting p38 activity with SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine). In an intrarenal mouse model of NB, NSC-87877 treatment results in decreased tumor growth and increased p53 and p38 activity. Together, these results suggest that DUSP26 inhibition with NSC-87877 is an effective strategy to induce NB cell cytotoxicity in vitro and in vivo through activation of the p53 and p38 mitogen-activated protein kinase (MAPK) tumor-suppressor pathways.Neuroblastoma (NB) remains the most common extracranial solid tumor in children and is associated with a very poor prognosis in high-risk patients.¹ Current treatment strategy for this subgroup of patients includes intense myeloablative chemotherapy, radical surgical resection of primary tumor, radiation therapy and stem cell rescue. In spite of these therapies, survival in high-risk NB patients is <50% at 5 years from diagnosis.² These therapies confer major long-term toxicities in over 90% of long-term survivors; therefore, efforts are underway to identify more specific biologic therapies with less toxicity and better efficacy at targeting NB.A therapeutic strategy that is gaining much interest is utilizing small molecule inhibitors to activate innate, non-mutated cell senescence and death pathways, such as the p53 tumor suppressor. Mutations in the p53 gene are seen in over 60% of adult cancers; however, pediatric solid tumors, particularly NB, do not exhibit frequent p53 mutations and actually have an intact pathway that is suppressed by other mechanisms.³ Mouse double minute 2 (MDM2) inhibition is a strategy to activate p53 using compounds such as Nutlin-3a, RITA and RG7112, which has already been tested in a phase I clinical trial in adults.^{4, 5, 6} The p38 stress kinase, MAP kinase, pathway is another tumor-suppressive pathway that is upstream from p53 and can function through p53-dependent and -independent mechanisms to induce apoptosis. Although described as oncogenic in some cancers, there is evidence that p38 activation leads to tumor cell apoptosis in NB.^{7, 8, 9, 10} Both of these tumor-suppressive pathways are regulated through phosphorylation and dephosphorylation events by an array of kinases and phosphatases.Phosphatase targeting in NB has had very limited application because of the limited number of phosphatases found to have an oncogenic role. Protein phosphatase 2A (PP2A), protein tyrosine phosphatase receptor delta (PTPRD) and dual specificity protein phosphatase 12 (DUSP12) have been found to be involved in NB cell differentiation and tumor suppression.^{11, 12, 13, 14} First discovered in breast cancer, PPM1D, or Wip-1 phosphatase, is active in NB, and small molecule inhibition results in p53 activation and chemosensitivity.^{15, 16, 17} In this report, we show DUSP26 functions by inhibiting p53 and p38 function to promote growth of NB tumor cells.DUSP26 (MKP-8, LDP-4) was originally described as a dual specifity phosphatase with enzymatic activity against p38 MAP kinase resulting in dephosphorlyation of the primary p38 activation sites, Thr180/Tyr182.^{18, 19} Song et al.²⁰ showed that NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid), a small molecule phosphatase inhibitor of SHP-1 (src homology region 2 domain-containing phosphatase), specifically inhibits DUSP26 phosphatase activity at a much lower IC₅₀ than other phosphatases resulting in increased p38 phosphorylation in vitro. Yu et al.²¹ have shown that DUSP26 is overexpressed in anaplastic thyroid cancer tissue samples and functions by inhibiting the p38 MAP kinase pathway.A novel DUSP26 function shown in NB is enzymatic regulation of the p53 tumor suppressor.²² We demonstrated that DUSP26 physically binds p53, dephosphorylates p53 at Ser20 and Ser37, and causes inhibition of downstream p53 signaling. DUSP26 activity leads to increased chemoresistance to doxorubicin and VP-16 (etoposide) treatment, and overexpression was seen in high-risk NB tumor tissue samples correlating with a worse prognosis in these patients. Here, we show how DUSP26 has pro-proliferative effects in NB by inhibiting the p53 tumor-suppressor pathway, as well as the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition with small hairpin RNA (shRNA) targeting DUSP26 or NSC-87877 results in decreased proliferation and cell viability in NB cell lines in vitro and in vivo. Inhibiting the p53 or p38 pathways reverses this phenotype seen with inhibition of DUSP26. These data establish DUSP26 inhibition as a promising novel therapeutic approach for NB.     

Use of intravenous peramivir for treatment of severe influenza A(H1N1)pdm09

Oral antiviral agents to treat influenza are challenging to administer in the intensive care unit (ICU). We describe 57 critically ill patients treated with the investigational intravenous neuraminidase inhibitor drug peramivir for influenza A (H1N1)pdm09 [pH1N1]. Most received late peramivir treatment following clinical deterioration in the ICU on enterically-administered oseltamivir therapy. The median age was 40 years (range 5 months-81 years). Common clinical complications included pneumonia or acute respiratory distress syndrome requiring mechanical ventilation (54; 95%), sepsis requiring vasopressor support (34/53; 64%), acute renal failure requiring hemodialysis (19/53; 36%) and secondary bacterial infection (14; 25%). Over half (29; 51%) died. When comparing the 57 peramivir-treated cases with 1627 critically ill cases who did not receive peramivir, peramivir recipients were more likely to be diagnosed with pneumonia/acute respiratory distress syndrome (p?=?0.0002) or sepsis (p?=?<0.0001), require mechanical ventilation (p?=?<0.0001) or die (p?=?<0.0001). The high mortality could be due to the pre-existing clinical severity of cases prior to request for peramivir, but also raises questions about peramivir safety and effectiveness in hospitalized and critically ill patients. The use of peramivir merits further study in randomized controlled trials, or by use of methods such as propensity scoring and matching, to assess clinical effectiveness and safety.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

Lysosomal-Associated Protein Multispanning Transmembrane 5 Gene (LAPTM5) Is Associated with Spontaneous Regression of Neuroblastomas

Background

Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors.

Principal Findings

We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells.

Conclusions

We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.     

Essential amino acids and muscle protein recovery from resistance exercise

This study tests the hypothesis that a dose of 6 g of orally administered essential amino acids (EAAs) stimulates net muscle protein balance in healthy volunteers when consumed 1 and 2 h after resistance exercise. Subjects received a primed constant infusion of L-[(2)H(5)]phenylalanine and L-[1-(13)C]leucine. Samples from femoral artery and vein and biopsies from vastus lateralis were obtained. Arterial EAA concentrations increased severalfold after drinks. Net muscle protein balance (NB) increased proportionally more than arterial AA concentrations in response to drinks, and it returned rapidly to basal values when AA concentrations decreased. Area under the curve for net phenylalanine uptake above basal value was similar for the first hour after each drink (67 +/- 17 vs. 77 +/- 20 mg/leg, respectively). Because the NB response was double the response to two doses of a mixture of 3 g of EAA + 3 g of nonessential AA (NEAA) (14), we conclude that NEAA are not necessary for stimulation of NB and that there is a dose-dependent effect of EAA ingestion on muscle protein synthesis.     

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.ConclusionsDFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration

Clinicaltrials.gov NCT#01059071     

Urinary excretion of three nucleic acid oxidation adducts and isoprostane F(2)alpha measured by liquid chromatography-mass spectrometry in smokers, ex-smokers, and nonsmokers

To assess novel liquid chromatography/mass spectrometric methods for measuring oxidative damage to nucleic acids and lipids, we compared urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 5-hydroxymethyl-2'-deoxyuridine (5-OHmU), and 8-hydroxyguanosine (8-OxoG), and an isoprostane, 8-iso-prostaglandin F(2)alpha (IsopF(2)alpha) in 234 healthy men (n = 113) and women (n = 121), 80 current smokers, 96 never-smokers), and 58 ex-smokers (no tobacco use for 3 years). The 8-OHdG and 8-OxoG did not differ significantly by group; 5-OHmU was higher in smokers, compared with ex- (p <.003) and never- (p <.0001) smokers and in ex- vs. never-smokers (p =.014) at, respectively, 13.5 +/- 0.7, 11.3 +/- 1.0, and 8.7 +/- 0.3 microg/g creatinine. IsopF(2)alpha was higher in smokers, compared with ex- (p =.007) and never-smokers (p <.0001) and in ex- vs. never- smokers (p =.002) at, respectively, 1.1 +/- 0.10; 0.74 +/- 0.07, and 0.51 +/- 0.04 microg/g creatinine. There were significant correlations among all three nucleic acid adducts and between IsopF(2)alpha and both 5-OHmU and 8-OHdG. Many smokers and ex-smokers had high levels of either 5-OHmU excretion or IsopF(2)alpha excretion, but not both. We conclude that 5-OHmU and IsopF(2)alpha are more discriminating of oxidative stress from tobacco smoke than the other two compounds measured. Whether characteristic patterns of excretion of these indicators forecast differential disease risk should be explored in future research.     

Retinoic acid confers resistance to p53-dependent apoptosis in SH-SY5Y neuroblastoma cells by modulating nuclear import of p53.

Many cell lines derived from neuroblastoma (NB) carry the wild-type p53 gene with a p53-dependent apoptotic pathway that is responsive to DNA damaging agents. A recent study has demonstrated that retinoic acid (RA) pretreatment of NB cells promotes chemoresistance to apoptosis induced by chemotherapeutic agents. We examine here the possible contribution of the p53 pathway to the chemoresistance response associated with the RA treatment in NB cells. Upon treatment with RA (1-10 microM) for 4 days, the human NB cells, SH-SY5Y, developed resistance selectively to p53-dependent apoptotic stimuli including gamma-irradiation, etoposide, and 1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine (H-7). Interestingly, RA affected the ability of H-7 to induce nuclear accumulation of the p53 protein without altering its effect on elevating the steady-state level of p53, suggesting that drug-induced up-regulation and nuclear accumulation of the wild-type p53 protein are separable processes. The modulation of nuclear import of p53 protein by RA may thus represent a potential mechanism by which certain tumor cells with the wild-type p53 gene develop resistance to chemotherapeutic agents.     

Activated protein synthesis and suppressed protein breakdown signaling in skeletal muscle of critically ill patients

Background

Skeletal muscle mass is controlled by myostatin and Akt-dependent signaling on mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β) and forkhead box O (FoxO) pathways, but it is unknown how these pathways are regulated in critically ill human muscle. To describe factors involved in muscle mass regulation, we investigated the phosphorylation and expression of key factors in these protein synthesis and breakdown signaling pathways in thigh skeletal muscle of critically ill intensive care unit (ICU) patients compared with healthy controls.

Methodology/Principal Findings

ICU patients were systemically inflamed, moderately hyperglycemic, received insulin therapy, and showed a tendency to lower plasma branched chain amino acids compared with controls. Using Western blotting we measured Akt, GSK3β, mTOR, ribosomal protein S6 kinase (S6k), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), and muscle ring finger protein 1 (MuRF1); and by RT-PCR we determined mRNA expression of, among others, insulin-like growth factor 1 (IGF-1), FoxO 1, 3 and 4, atrogin1, MuRF1, interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and myostatin. Unexpectedly, in critically ill ICU patients Akt-mTOR-S6k signaling was substantially higher compared with controls. FoxO1 mRNA was higher in patients, whereas FoxO3, atrogin1 and myostatin mRNAs and MuRF1 protein were lower compared with controls. A moderate correlation (r² = 0.36, p<0.05) between insulin infusion dose and phosphorylated Akt was demonstrated.

Conclusions/Significance

We present for the first time muscle protein turnover signaling in critically ill ICU patients, and we show signaling pathway activity towards a stimulation of muscle protein synthesis and a somewhat inhibited proteolysis.