Increased Cerebrospinal Fluid Adenosine 5'-Triphosphate Levels in Patients with Guillain-Barré Syndrome and Chronic Inflammatory Demyelinating Polyneuropathy.

Background: Extracellular adenosine 5'-triphosphate (ATP) acts as a signaling molecule in the peripheral nerves, regulating myelination after nerve injury. The present study examined whether the cerebrospinal fluid (CSF) ATP levels in patients with Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are related to disease severity. Methods: CSF ATP levels in 13 patients with GBS and 18 patients with CIDP were compared with those in a control group of 16 patients with other neurological diseases (ONDs). In patients with CIDP, CSF ATP levels were compared before and after treatment. The correlations between CSF ATP levels and other factors, including clinical data and CSF protein levels, were also evaluated. Results: Median CSF ATP levels were significantly higher in patients with GBS and CIDP than in those with ONDs. When patients with CIDP were classified into two groups depending on their responsiveness to immunotherapy, median CSF ATP levels were significantly higher in good responders than in ONDs. CSF ATP levels tended to decrease after treatment in patients with CIDP. In patients with CIDP, there is a negative correlation between CSF ATP and CSF protein levels. Conclusions: CSF ATP levels were increased in patients with GBS and CIDP. In particular, CSF ATP levels tended to decrease following treatment in patients with CIDP. CSF ATP levels may be useful biomarkers for the diagnosis or monitoring of therapeutic effects in patients with GBS and CIDP.

Establishing Monoclonal Gammopathy of Undetermined Significance as an Independent Pre-Disease State of Multiple Myeloma Using Raman Spectroscopy, Dynamical Network Biomarker Theory, and Energy Landscape Analysis.

Multiple myeloma (MM) is a cancer of plasma cells. Normal (NL) cells are considered to pass through a precancerous state, such as monoclonal gammopathy of undetermined significance (MGUS), before transitioning to MM. In the present study, we acquired Raman spectra at three stages-834 NL, 711 MGUS, and 970 MM spectra-and applied the dynamical network biomarker (DNB) theory to these spectra. The DNB analysis identified MGUS as the unstable pre-disease state of MM and extracted Raman shifts at 1149 and 1527-1530 cm-1 as DNB variables. The distribution of DNB scores for each patient showed a significant difference between the mean values for MGUS and MM patients. Furthermore, an energy landscape (EL) analysis showed that the NL and MM stages were likely to become stable states. Raman spectroscopy, the DNB theory, and, complementarily, the EL analysis will be applicable to the identification of the pre-disease state in clinical samples.

Increased cerebrospinal fluid adenosine 5'-triphosphate in patients with amyotrophic lateral sclerosis.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) has been suggested to cause neuroinflammation and motor neuron degeneration by activating microglia and astrocytes in amyotrophic lateral sclerosis (ALS). Since we have developed a highly sensitive ATP assay system, we examined cerebrospinal fluid (CSF) ATP levels in patients with ALS whether it can be a useful biomarker in ALS. METHODS: Forty-eight CSF samples from 44 patients with ALS were assayed for ATP with a newly established, highly sensitive assay system using luciferase luminous reaction. CSF samples from patients with idiopathic normal pressure hydrocephalus (iNPH) were assayed as a control. Patients were divided into two groups depending on their disease severity, as evaluated using the Medical Research Council (MRC) sum score. Correlations between the CSF ATP levels and other factors, including clinical data and serum creatinine levels, were evaluated. RESULTS: CSF ATP levels were significantly higher in patients with ALS than in the iNPH (716 ± 411 vs. 3635 ± 5465 pmol/L, p < 0.01). CSF ATP levels were significantly higher in the more severe group than in the iNPH group (6860 ± 8312 vs. 716 ± 411 pmol/L, p < 0.05) and mild group (6860 ± 8312 vs. 2676 ± 3959 pmol/L, p < 0.05) respectively. ALS functional rating scale-revised (ALSFRS-R) (37.9 ± 5.7 vs. 42.4 ± 2.8, p < 0.01) and serum creatinine levels (0.51 ± 0.13 vs. 0.68 ± 0.23 mg/dL, p < 0.05) were significantly lower in the severe group than in the mild group respectively. A negative correlation of CSF ATP levels with MRC sum score was demonstrated in the correlation analysis adjusted for age and sex (r = -0.3, p = 0.08). CONCLUSIONS: Extracellular ATP is particularly increased in the CSF of patients with advanced ALS. CSF ATP levels may be a useful biomarker for evaluating disease severity in patients with ALS.

Infected simple renal cyst due to Streptococcus pneumoniae rapidly diagnosed by the melting temperature mapping method: a case report.

BACKGROUND: Spontaneous infection of preexisting solitary renal cysts has been documented in adults but is extremely rare in children. To date, no cases of simple renal cysts infected with Streptococcus pneumoniae have been described. Recently, reports have described the diagnosis of bacterial infection using the 16 S rRNA gene as well as the accompanying antimicrobial stewardship for microorganisms that are difficult to culture and for culture-negative cases after preceding antibacterial administration. CASE PRESENTATION: A four-year-old Japanese girl who had a pleuroperitoneal shunt inserted to drain a right pleural effusion due to occlusion of the hepatic portion of the inferior vena cava at three years old visited our hospital due to fever and respiratory discomfort. She was incidentally found to have a right simple renal cyst 10 months before admission. The patient was suspected to have pneumonitis or catheter-related blood stream infection on chest X-ray, which showed right-side pleural effusion. She was diagnosed with invasive pneumococcal infection, as Streptococcus pneumoniae was detected from blood culture on admission. Transient improvements in her symptoms and decreases in the white blood cell count and C-reactive protein level were observed after effective antibiotic administration, but her respiratory condition deteriorated. Enhanced CT showed right renal cyst enlargement and enhancement and thickening of the surrounding wall. Using the melting temperature (Tm) mapping method, S. pneumoniae was rapidly detected directly from pus 4.5 hours after drainage. The specimen culture was negative, but the extracted 16 S rDNA sequence revealed 100 % identity for S. pneumoniae from the same specimen the subsequent day. We successfully performed optimal treatment and reduced medical cost based on the positive Tm mapping method result. CONCLUSIONS: We report the first case of a S. pneumoniae-infected simple renal cyst. The drainage culture was negative, but the Tm mapping method rapidly detected S. pneumoniae directly from the drainage. The Tm mapping method may have great impacts on rapid diagnosis and effective antimicrobial stewardship.

Cerebrospinal fluid ATP as a potential biomarker in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes (MELAS).

Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is caused by defective oxidative phosphorylation in the cerebral parenchyma, cerebral blood vessels, and leptomeningeal tissue. Although increased blood and cerebrospinal fluid (CSF) lactate level has been used as a diagnostic biomarker in patients with MELAS, no biomarkers reflecting disease activity exist. Since we have developed a highly sensitive ATP assay system using luciferase luminous reaction, we examined CSF ATP in patients with MELAS and found that it negatively correlates with disease activity and that it reflects the efficacy of the treatment. CSF ATP might be a novel disease monitoring marker for MELAS.

A Rapid ATP Bioluminescence-based Test for Detecting Levofloxacin Resistance Starting from Positive Blood Culture Bottles.

Administering appropriate antimicrobial therapy as early as possible is important for rescuing bacteremic patients. Therefore, rapid antimicrobial susceptibility tests in positive blood culture specimens have been diligently sought. Adenosine triphosphate (ATP) bioluminescence-based methods have been used for rapid antimicrobial susceptibility tests. However, blood culture specimens have not been examined in many studies, possibly due to abundant intracellular ATP in blood corpuscles resulting in false-susceptible results. In this study, we developed a rapid ATP bioluminescence-based method for detecting antibiotic resistance starting from positive blood culture. To minimize background ATP originating from blood corpuscles, specimens were centrifuged and the supernatant diluted with broth, and an ATP-eliminating reagent was then added to the bacterial suspension at the beginning of incubation. This newly devised procedure reduced the background ATP by more than five orders of magnitude. In a pilot study using levofloxacin, no false-susceptible results were observed in 15 clinical specimens. Furthermore, the results indicated that the rapid method provided additional information about bacterial activities with high resolution, in contrast to the less-thorough findings with the conventional turbidity method. Therefore, our approach will contribute to the treatment of infectious diseases as a rapid antimicrobial susceptibility test.

Factor X heterozygous mutation in a patient with potential risk of bleeding: A case report.

RATIONALE: Factor X (FX) deficiency is a rare autosomal recessive bleeding disorder. The majority of patients carry a missense mutation in F10, and patients with bleeding disorders are either homozygous or compound heterozygous for F10. Nonsense mutations are exceptionally rare, and a heterozygous nonsense mutation is not considered to cause bleeding disorders. PATIENT CONCERNS: A 35-year-old Japanese female with an incidental hemorrhage after gynecologic polypectomy was referred to our hospital. DIAGNOSES: Following differential diagnostic workup, including cross-mixing test, congenital FX deficiency was strongly suspected. INTERVENTION: Coagulation tests and mutation analyses were conducted for the patient and her parents. OUTCOMES: Mutation analysis revealed that she carried a heterozygous nonsense mutation in F10. Pedigree analysis revealed that the mutation was inherited from her mother although there was no familial history of bleeding or hemostatic disturbance. LESSONS: Hemostatic disturbance may occur even in a patient with heterozygous F10. Because heterozygous nonsense mutation in F10 is expected to be hidden in an apparently healthy population, as observed in our patient, unexpected hemostatic disturbance may occur, particularly during the use of direct oral anticoagulant (DOAC)-targeting factor Xa for thrombotic diseases. FX activity should be evaluated before prescribing DOACs to patients.

Spondylodiscitis due to Parvimonas micra diagnosed by the melting temperature mapping method: a case report.

BACKGROUND: It has been suggested that more than 100 bacterial species can be identified using only seven universal bacterial primer sets in the melting temperature (Tm) mapping method and that these findings can be obtained within 3 h of sterile site collection. CASE PRESENTATION: A 67-year-old Japanese man with type 2 diabetes visited our hospital complaining of progressive lower back pain for 2 months. The patient was suspected to have spondylodiscitis on magnetic resonance imaging of the spine. Blood culture and transcutaneous vertebral biopsy were subsequently performed. Using the Tm mapping method, Parvimonas micra was detected from a transcutaneous vertebral biopsy specimen in 3 h. Gram-positive cocci were also detected by Gram staining and P. micra was identified directly from the anaerobic blood culture by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Four days after admission, the biopsy specimen culture isolate was identified as P. micra. CONCLUSIONS: The Tm mapping method may be useful for the diagnosis of bacterial infections where diagnosis is challenging because of the difficulty of culturing.

Thrombomodulin alfa treatment in patients with acute promyelocytic leukemia and disseminated intravascular coagulation: a retrospective analysis of an open-label, multicenter, post-marketing surveillance study cohort.

INTRODUCTION: Patients with acute promyelocytic leukemia (APL) can develop disseminated intravascular coagulation (DIC) that results in life-threatening hemorrhagic complications. Studies regarding the safety and efficacy of thrombomodulin alfa (TM-α; recombinant human soluble thrombomodulin) in patients with APL and DIC are limited. MATERIALS AND METHODS: A retrospective evaluation was performed on a cohort of 172 patients with APL from an open-label, multicenter, post-marketing surveillance study of TM-α. RESULTS: Of the 172 patients, 31 were relapse/refractory APL patients, and 141 were newly diagnosed APL patients. Within the first 30 days, 24 patients (14.0%) died, and six of those deaths (3.5%) were due to hemorrhage. In total, 12 patients (7.0%) had severe hemorrhagic complications. Both the early death rate due to hemorrhage as well as the severe hemorrhage rate did not exceed those in some recent population-based studies of patients with APL. Forty-nine patients received TM-α prior to the initiation of antileukemic treatment, and one patient experienced hemorrhagic early death (ED), suggesting that early TM-α treatment appeared to result in a reduction in the hemorrhagic ED rate. Moreover, TM-α improved coagulopathy regardless of concomitant all-trans retinoic acid treatment. CONCLUSIONS: This study confirmed the safety and efficacy of TM-α in daily clinical practice for patients with APL and DIC. TM-α appeared to reduce hemorrhagic early deaths due to DIC in patients with APL who were receiving antileukemic treatment.

Post-marketing surveillance of thrombomodulin alfa, a novel treatment of disseminated intravascular coagulation - safety and efficacy in 1,032 patients with hematologic malignancy.

INTRODUCTION: Post-marketing surveillance of thrombomodulin alfa (TM-α) was performed to evaluate safety and efficacy in patients with disseminated intravascular coagulation (DIC) with hematologic malignancy. MATERIALS AND METHODS: All patients treated with TM-α from May 2008 to April 2010 in Japan were included. Information about baseline characteristics, safety, and efficacy were collected. The DIC resolution rate, survival rate on Day 28 after the last TM-α administration, and changes in DIC score and coagulation tests were evaluated. RESULTS: The underlying diseases associated with DIC were acute myeloid leukemia (except for acute promyelocytic leukemia, n=350), lymphoma (n=199), acute promyelocytic leukemia (n=172), acute lymphoblastic leukemia (n=156), myelodysplastic syndromes (n=61), and other (n=94). The incidence rates of bleeding-related adverse events and adverse drug reactions were 17.8% and 4.6%, respectively. In subjects with bleeding symptoms at baseline, 55.0% were assessed as disappeared or improved based on symptoms after TM-α treatment. The DIC resolution and survival rates were 55.9% and 70.7%, respectively. The DIC score and coagulation tests including thrombin-antithrombin complex (TAT) were significantly improved. Coagulation tests were significantly improved after TM-α treatment even in subjects whose clinical course of underlying disease was assessed as unchanged or exacerbated. CONCLUSIONS: This surveillance confirmed the safety and efficacy of TM-α in clinical practice, thus TM-α may be an ideal treatment for patients with DIC based upon hematologic malignancy.

N-terminal pro-brain natriuretic peptide as a predictor of reoperation in children with surgically corrected tetralogy of fallot.

BACKGROUND: Most patients with surgically corrected tetralogy of Fallot (TOF) are faced with multiple residua and sequelae such as pulmonary regurgitation (PR), resulting in reoperation for pulmonary valve replacement (PVR). Plasma brain natriuretic peptide (BNP) level and serum N-terminal pro-BNP (NT-pro-BNP) level are useful as diagnostic objective markers of chronic heart failure (CHF). The aim of the study was to examine whether these markers have predictive ability for reoperation in children with surgically corrected TOF. METHODS AND RESULTS: Fifty-eight patients (38 male, 20 female) aged 1-18 years (median, 7 years) were enrolled. Serum NT-pro-BNP in TOF patients was significantly higher than in age-matched hospital controls without CHF (359.5±449.7pg/ml vs. 86.1±45.1pg/ml, respectively; P<0.0001). BNP and NT-pro-BNP had a better correlation with CHF index, RVEDP, and LVEDV in TOF groups. Children with surgically corrected TOF who had indication for PVR had higher BNP and NT-pro-BNP and more severe PR than those without indication for PVR. On multivariate logistic regression analysis, NT-pro-BNP was the strongest predictor for reoperation in patients with surgically corrected TOF. Area under the curve of NT-pro-BNP for reoperation was 0.950 (P<0.001) with a sensitivity of 88.9% and specificity of 91.8%. CONCLUSIONS: NT-pro-BNP is a good biomarker for monitoring CHF, and is a good predictor of PVR in children with surgically repaired TOF.

[Points of revision of the guideline JSLM 2012].

The revised version of the guideline JSLM 2012 was published in December 2012 by the Japanese Society of Laboratory Medicine. This version included new sections, such as blood gas analysis, sleep apnea syndrome, interstitial lung disease, liver and pancreas cancer, chronic kidney disease, acute kidney disease, gout and hyperuricemia, bone metabolism abnormality, malignant lymphoma, and rheumatoid arthritis. The guideline committee was composed of specialists in each field of internal medicine, who were responsible for selecting and requesting the authors and also in promoting and proofreading the manuscripts. In special program I at the 60th annual meeting, each specialist gave lectures concerning the points of the revision in their fields. The questionnaire surveys were performed using FAX or the Internet. Analysis of eighty-seven (2.5%) responses from 3,500 individuals/facilities, which were sent the guideline, revealed that this guideline was graded as excellent by 38 readers, fair by 42, and average by in 6. Significant opinions on the guideline were obtained from the readers, and they will be the bases for the next revision. The main subjects of this guideline were confirmed to be the residents and general physicians, by whom it is hoped the routine laboratory tests will be properly utilized. Therefore, based on the section of 'approach of the laboratory test results', which is a representative characteristic of this guideline, the sections of 'symptoms' will be fulfilled for the next version. This guideline needs to be periodically revised with advances in medicine.

Impact of recombinant soluble thrombomodulin (thrombomodulin alfa) on disseminated intravascular coagulation.

INTRODUCTION: We assessed the safety and effectiveness of recombinant soluble thrombomodulin (thrombomodulin alfa, TM-α) in the treatment of disseminated intravascular coagulation (DIC) in a post-marketing surveillance. METHODS: The cases of 3548 patients with DIC caused by infection (n=2516, Infection-DIC) or hematological malignancy (n=1032, Hemat-DIC) were analyzed and compared to the results of a phase III (P-III) study. RESULTS: The DIC scores were significantly decreased in the Infection-DIC and Hemat-DIC groups with TM-α treatment (both P<0.001). The incidences of critical bleeding adverse drug reactions (ADRs) in the Infection-DIC and Hemat-DIC groups were 2.6% and 2.4%, and the survival rates were 64.1% and 70.7%, respectively. Patients with DIC were subcategorized into three groups (Infection-DIC-1 or Hemat-DIC-1, P-III criteria-matched patients; Infection-DIC-2 or Hemat-DIC-2, P-III criteria-non-matched patients treated solely with TM-α; and Infection-DIC-3 or Hemat-DIC-3, P-III criteria-non-matched patients treated with TM-α and other concomitant anticoagulants). Subcategory analysis revealed that the incidences of critical bleeding ADRs of Hemat-DIC-2 and Hemat-DIC-3 were significantly higher and their survival rates were significantly lower than those of Hemat-DIC-1. By multivariate analysis in Hemat-DIC, younger age (odds ratio: 2.629, P=0.0033) and pre-existing bleeding (odds ratio: 2.044, P=0.019) were found to affect bleeding ADRs and the severity of underlying disease was the most important factor for survival rate (odds ratio: 0.288, P<0.001). CONCLUSIONS: This surveillance provided real-world data for the safety and effectiveness of TM-α in the treatment of Infection-DIC and Hemat-DIC in general practice settings.

A novel Y231del mutation of HFE in hereditary haemochromatosis provides in vivo evidence that the Huh-7 is a human haemochromatotic cell line.

Hereditary haemochromatosis (HH), which is mainly associated with a C282Y polymorphism in HFE, is common among Caucasians of north European descent, but is very rare among Asians. Herein, we report a 43-year-old Japanese man who was diagnosed as having HH. A laboratory examination revealed an elevated serum iron level (280 µg/dl), hyperferritinemia (1698 ng/ml) and a low serum level of hepcidin-25 (4.0 ng/ml). Abdominal magnetic resonance imaging revealed findings suggestive of iron accumulation in the liver and pancreas. HFE gene sequencing in the patient revealed a novel homozygous TAC nucleotide deletion (c. 691_693del) responsible for the loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This homozygous Y231del mutation was recently found in the Huh-7 hepatoma cell line and was shown to prevent the translocation of HFE to the cell surface. This clinical case provides in vivo evidence suggesting that Huh-7 is undoubtedly a human haemochromatotic cell line and, as such, is a valuable tool for investigating the pathogenesis of HFE-related HH in humans.

Aberrant calcium/calmodulin-dependent protein kinase II (CaMKII) activity is associated with abnormal dendritic spine morphology in the ATRX mutant mouse brain.

In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the sucrose-nonfermenting 2 family, cause several mental retardation disorders, including α-thalassemia X-linked mental retardation syndrome. We generated ATRX mutant mice lacking exon 2 (ATRX(ΔE2) mice), a mutation that mimics exon 2 mutations seen in human patients and associated with milder forms of retardation. ATRX(ΔE2) mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). Consistent with other mouse models of mental retardation, ATRX(ΔE2) mice exhibited longer and thinner dendritic spines compared with wild-type mice without changes in spine number. Interestingly, aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRX(ΔE2) mice. Increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-guanine nucleotide exchange factors (GEFs) T-cell lymphoma invasion and metastasis 1 (Tiam1) and kalirin-7, known substrates of CaMKII. We confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRX(ΔE2) mice. In cultured cortical neurons, PP1 inhibition by okadaic acid increased CaMKII-dependent Tiam1 and kalirin-7 phosphorylation. Together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes plus elevated Rac1-GEF/PAK signaling seen in ATRX(ΔE2) mice may contribute to mental retardation syndromes seen in human patients.

Venous thromboembolism after spine surgery: changes of the fibrin monomer complex and D-dimer level during the perioperative period.

OBJECT: The goal of this study was to determine the incidence of deep venous thrombosis (DVT) and pulmonary embolism (PE) after spine surgery. Another purpose was to clarify the rapid changes of the fibrin monomer complex (FMC) and D-dimer levels during the perioperative period of spine surgery for early diagnosis of venous thromboembolism (VTE). METHODS: The participants were 72 patients who underwent spine surgery between September 2007 and March 2008. The FMC and D-dimer levels were measured 6 times: 1) at induction of general anesthesia; 2) just after implantation or during surgery; 3) immediately following surgery; 4) 1 day after surgery; 5) 3 days postsurgery; and 6) 7 days after surgery. All patients received mechanical prophylaxis, including compression stockings and intermittent pneumatic compression devices, and all were examined with duplex ultrasonography assessments of both lower extremities and with lung perfusion scintigraphy 7-10 days after surgery. If DVT or PE was suspected, the patient underwent multidetector CT venography. RESULTS: There were no patients with clinical signs of DVT and PE, but 6 (8.3%) showed VTE, among whom 5 had DVT and 3 had PE. Patients with VTE had significantly higher FMC levels 1 day after surgery, compared with those without VTE (55.9 ± 17.2 µg/ml vs 11.1 ± 2.89 µg/ml; p < 0.01). Patients with VTE had significantly higher D-dimer levels 7 days postsurgery, compared with those without VTE (12.5 ± 2.95 µg/ml vs 4.3 ± 0.39 µg/ml; p < 0.01). Receiver operating characteristic analysis showed that the FMC result was more useful than the D-dimer assay for diagnosis of VTE. When the cutoff value was set to 20.8 µg/ml for FMC, sensitivity was 100% and specificity was 86.3%. CONCLUSIONS: In this study the prevalence of VTE after spine surgery was 8.3%. The FMC measured 1 day after spine surgery is considered to be useful as an indicator of VTE.

Induction of autophagic cell death and radiosensitization by the pharmacological inhibition of nuclear factor-kappa B activation in human glioma cell lines.

OBJECT: The intrinsic radioresistance of certain cancer cells may be closely associated with the constitutive activation of nuclear factor-kappa B (NF-kappaB) activity, which may lead to protection from apoptosis. Recently, nonapoptotic cell death, or autophagy, has been revealed as a novel response of cancer cells to ionizing radiation. In the present study, the authors analyzed the effect of pitavastatin as a potential inhibitor of NF-kappaB activation on the radiosensitivity of A172, U87, and U251 human glioma cell lines. METHODS: The pharmacological inhibition of NF-kappaB activation was achieved using pitavastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Growth and radiosensitivity assays were performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Hoechst 33258 staining, supravital acridine orange staining, and electron microscopy were performed utilizing 3 glioma cell lines with or without pitavastatin pretreatment to identify apoptosis or autophagy after irradiation. RESULTS: The growth of these 3 glioma cell lines was not significantly inhibited by pitavastatin at a concentration of up to 1 microM. Treatment with 0.1 microM of pitavastatin enhanced radiation-induced cell death in all glioma cell lines, with different sensitivity. Apoptosis did not occur in any pretreated or untreated (no pitavastatin) cell line following irradiation. Instead, autophagic cell changes were observed regardless of the radiosensitivity of the cell line. An inhibitor of autophagy, 3-methyladenine suppressed the cytotoxic effect of irradiation with pitavastatin, indicating that autophagy is a result of an antitumor mechanism. Using the most radiosensitive A172 cell line, the intracellular localization of p50, a representative subunit of NF-kappaB, was evaluated through immunoblotting and immunofluorescence studies. The NF-kappaB of A172 cells was immediately activated and translocated from the cytosol to the nucleus in response to irradiation. Pitavastatin inhibited this activation and translocation of NF-kappaB. CONCLUSIONS: Autophagic cell death rather than apoptosis is a possible mechanism of radiation-induced and pitavastatin-enhanced cell damage, and radiosensitization by the pharmacological inhibition of NF-kappaB activation may be a novel therapeutic strategy for malignant gliomas.

Changes of the soluble fibrin monomer complex level during the perioperative period of hip replacement surgery.

BACKGROUND: Venous thromboembolism (VTE) is a critical complication after hip replacement surgery, so both early diagnosis and prophylaxis are important. The purpose of this study was to clarify the rapid changes of the fibrin monomer complex (FMC) and soluble fibrin (SF) during the perioperative period of hip replacement surgery. METHODS: The subjects were 32 patients (7 men, 25 women) who underwent elective hip replacement surgery between November 2004 and January 2006. Their ages ranged between 34 to 82 years (mean 56.8 years). According to their thromboembolic risk, the patients received different prophylaxis: unfractionated heparin (4 patients), danaparoid sodium (14 patients), or mechanical therapy only (14 patients). RESULTS: FMC and SF became rapidly elevated during the operation and just after surgery but declined to preoperational levels 3 days after surgery; they were higher in lupus anticoagulant (LA)-positive patients. In contrast, FDP and D-dimer had gradually become elevated 3 and 7 days after surgery. According to venous ultrasonography and lung perfusion scintigraphy, VTE occurred in 7 patients overall (21.9%). The incidence of VTE was 7.1% in the danaparoid group, whereas it was 35.7% in the mechanical therapy group. We also found that danaparoid sodium rapidly decreased FMC and SF within 3 days. CONCLUSIONS: FMC and SF were rapidly elevated during hip replacement surgery and differentiated in LA-positive and LA-negative patients.

Effects of different statins on endothelial nitric oxide synthase and AKT phosphorylation in endothelial cells.

BACKGROUND: In the present study, we examined effects of pitavastatin and cerivastatin on NO production and their mechanisms in EC. METHODS: HUVEC cells (1x10(4) cells/well) were seeded into 96-well plates in 100 microl of culture medium for overnight, and then treated with various concentrations of pitavastatin or cerivastatin for 48 h. The cytotoxicity was evaluated using a WST-8 assay; The cells were cultured for 6 h in 200 microl of fresh medium containing increasing doses of pitavastatin or cerivastatin at 37 degrees C for 6 h, the NO production was detected by diaminofluoresceins (DAFs) assay; Simultaneously, The cells (1 x 10(5) cells/well) were seeded into 96-well plates in medium for overnight, and then treated with reagents at 37 degrees C for 30 min, cGMP level was measured by enzyme-immunoassay. The cells were cultured in 2 ml of fresh medium containing increasing doses of pitavastatin or cerivastatin at 37 degrees C for 30 min, the phosphorylations of eNOS and Akt were detected by Western blotting. RESULTS: We found that pitavastatin not only induced NO production, but also increased cGMP level in HUVECs. Furthermore, EC were incubated with pitavastatin or cerivastatin for 30 min, Western blot analysis showed that pitavastatin (0.1 microM) significantly upregulated the phosphorylation of eNOS and Akt about 1.4-fold or 1.3-fold compared with control, however, cerivastatin (0.1 microM) did not have any effects on them. CONCLUSION: Low dose of pitavastatin (0.1 microM) involves Akt pathway, activates eNOS activity, increases cGMP level and produces NO in EC, which is higher than that of cerivastatin.