Successful mild brain hypothermia therapy followed by targeted temperature management for pediatric hemorrhagic shock and encephalopathy syndrome.

OBJECTIVE: Hemorrhagic shock and encephalopathy syndrome (HSES) is the most severe form of acute encephalopathy that progresses rapidly, often resulting in death or severe neurological sequelae. We report the case of a 4-year-old girl with HSES with shock and impaired consciousness. PATIENT AND METHODS: Blood test results showed hypercytokinemia, and the 4-year-old patient was immediately admitted to the intensive care unit. Within 4 h of symptom onset, she received mild brain hypothermia therapy with a target body temperature of 35°C. Methylprednisolone pulse, high dose immunoglobulin, and large doses of circulatory drugs were administered. RESULTS: After 72 h of brain hypothermia therapy, targeted temperature management with a target body temperature between 36°C and 37°C was continued for 96 h. The patient was diagnosed with HSES based on acute encephalopathy with shock, hypercytokinemia, low platelet count, coagulation disorder, renal damage, and intestinal bleeding. Magnetic resonance imaging results revealed no signs of any specific acute encephalopathy. She was discharged without neurological sequelae 28 days after symptom onset. CONCLUSIONS: Mild brain hypothermia therapy initiated in the early stages followed by targeted temperature management may be an effective way to improve neurological outcomes in children suffering from HSES.

Post-occlusional hyperemia for fractional flow reserve assessment and pull-back curve analysis.

Balloon occlusion is a potential method for inducing hyperemia to measure post-percutaneous coronary intervention (PCI) fractional flow reserve (FFR). The objective of this study was to determine the clinical usefulness of post-occlusional hyperemia. FFRs measured using post-occlusional hyperemia caused by 30 (FFRoccl30) and 60 s (FFRoccl60) of balloon occlusion after PCI were compared in 60 lesions from 60 patients. The duration of hyperemia was also measured. There was a strong correlation between FFRoccl30 and FFRoccl60 (r = 0.969, p < 0.01). The duration of hyperemia was significantly longer with FFRoccl60 than with FFRoccl30 (68 ± 23 vs. 37 ± 15 s, p < 0.01). The time required for pullback curve analysis was around 45 s. However, in 7 (12%) cases, the duration of hyperemia with FFRoccl60 was < 45 s, which was not enough for pull-back curve analysis. To predict the duration of hyperemia with FFRoccl60 ≥ 45 s, the receiver operating characteristic curve analysis revealed a cut-off value of 25 s of hyperemia with FFRoccl30. FFRoccl30 is sufficient for diagnostic purposes. FFRoccl60 is suitable for pull-back curve analysis in select cases based on predictions made using the duration of hyperemia with FFRoccl30.

Arf tumor suppressor disrupts the oncogenic positive feedback loop including c-Myc and DDX5.

Tumor suppressor protein p19(ARF) (Arf; p14(ARF) in humans) functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals caused by proto-oncogene activation, but its p53-independent activities remain poorly understood. Using the tandem affinity purification-tag technique, we purified Arf-containing protein complexes and identified p68 DEAD-box protein (DDX5) as a novel interacting protein of Arf. In this study, we found that DDX5 interacts with c-Myc, and harbors essential roles for c-Myc-mediated transcription and its transforming activity. Furthermore, when c-Myc was forcibly expressed, the expression level of DDX5 protein was drastically increased through the acceleration of protein synthesis of DDX5, suggesting the presence of an oncogenic positive feedback loop including c-Myc and DDX5. Strikingly, Arf blocked the physical interaction between DDX5 and c-Myc, and drove away DDX5 from the promoter of c-Myc target genes. These observations most likely indicate the mechanism by which Arf causes p53-independent tumor-suppressive activity.

Pharmacokinetic modelling of serum and bronchial concentrations for clarithromycin and telithromycin, and site-specific pharmacodynamic simulation for their dosages.

WHAT IS KNOWN AND OBJECTIVE: Clinical pharmacokinetic profiles of clarithromycin and telithromycin in bronchopulmonary sites have not been fully characterized. This study aimed to describe in more detail the pharmacokinetics of the two macrolides in epithelial lining fluid (ELF) of human bronchi and to evaluate their pharmacodynamic target attainment at this site. METHODS: Previously reported drug concentration data for serum and ELF were simultaneously fitted to a three-compartment pharmacokinetic model using nonmem program. The model parameter estimates were used for site-specific pharmacodynamic simulation. RESULTS AND DISCUSSION: Population mean parameters for clarithromycin were as follows: distribution volumes of central, peripheral and ELF compartments (V1 /F, V2 /F and V3 /F) = 204·7, 168·9 and 67·1 L; clearance (CL/F) = 34·4 L/h; absorption rate constant (Ka ) = 0·680 1/h; transfer rate constants connecting compartments (K12 , K21 , K13 and K31  = 0·0193, 0·434, 0·667 and 0·260 1/h, respectively). Mean parameters for telithromycin were as follows: V1 /F, V2 /F and V3 /F = 370·3, 290·3 and 213·8 L; CL/F = 89·5 L/h; Ka  = 0·740 1/h; K12 , K21 , K13 and K31  = 0·0026, 1·044, 0·758 and 0·158 1/h, respectively. Using these parameters, the mean ELF/serum ratio in the area under drug concentration-time curve (AUC) was 7·80 for clarithromycin and 8·05 for telithromycin. Clarithromycin achieved a ≥ 90% probability of attaining a pharmacodynamic target [AUC/minimum inhibitory concentration (MIC) = 100] in ELF against bacterial isolates for which MICs were ≤0·5 and ≤1 mg/L for twice-daily doses of 250 and 500 mg, respectively. For telithromycin, once-daily doses of 600 and 800 mg achieved a ≥90% probability in ELF against Streptococcus pneumoniae, Staphylococcus aureus and Moraxella catarrhalis isolates but not Haemophilus influenzae isolates. WHAT IS NEW AND CONCLUSION: These results should provide a better understanding of the bronchial pharmacokinetics of clarithromycin and telithromycin, while also providing useful information about their dosages for respiratory tract infections based on site-specific pharmacodynamic evaluation. Further studies in a large number of patients are needed to confirm our findings and clarify their therapeutic implications.

Proteasome inhibitors exert cytotoxicity and increase chemosensitivity via transcriptional repression of Notch1 in T-cell acute lymphoblastic leukemia.

The Notch signaling pathway has been recognized as a key factor for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high incidence of activating mutations of Notch1. Notch inhibition could serve as a new treatment strategy for T-ALL; however, the attempts to perturb Notch signaling pathways have been unsuccessful so far. In this study, we found that proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive activation of Notch1 to a similar extent as myeloma cells. The proteasome inhibitor bortezomib repressed the transcription of Notch1 and downstream effectors including Hes1, GATA3, RUNX3 and nuclear factor-κB (NF-κB) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by NICD overexpression. The synergy of bortezomib and DEX was confirmed in vivo using a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL.

HOX-mediated LMO2 expression in embryonic mesoderm is recapitulated in acute leukaemias.

The Lim Domain Only 2 (LMO2) leukaemia oncogene encodes an LIM domain transcriptional cofactor required for early haematopoiesis. During embryogenesis, LMO2 is also expressed in developing tail and limb buds, an expression pattern we now show to be recapitulated in transgenic mice by an enhancer in LMO2 intron 4. Limb bud expression depended on a cluster of HOX binding sites, while posterior tail expression required the HOX sites and two E-boxes. Given the importance of both LMO2 and HOX genes in acute leukaemias, we further demonstrated that the regulatory hierarchy of HOX control of LMO2 is activated in leukaemia mouse models as well as in patient samples. Moreover, Lmo2 knock-down impaired the growth of leukaemic cells, and high LMO2 expression at diagnosis correlated with poor survival in cytogenetically normal AML patients. Taken together, these results establish a regulatory hierarchy of HOX control of LMO2 in normal development, which can be resurrected during leukaemia development. Redeployment of embryonic regulatory hierarchies in an aberrant context is likely to be relevant in human pathologies beyond the specific example of ectopic activation of LMO2.

BCR-ABL regulates death receptor expression for TNF-related apoptosis-inducing ligand (TRAIL) in Philadelphia chromosome-positive leukemia.

Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(-) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL.

γ-Secretase inhibitor enhances antitumour effect of radiation in Notch-expressing lung cancer.

BACKGROUND: Notch receptor has an important role in both development and cancer. We previously reported that inhibition of the Notch3 by γ-secretase inhibitor (GSI) induces apoptosis and suppresses tumour proliferation in non-small-cell lung cancer. Although radiation is reported to induce Notch activation, little is known about the relationship between radiation and Notch pathway. METHODS: We examined the effect of combining GSI and radiation at different dosing in three Notch expressing lung cancer cell lines. The cytotoxic effect of GSI and radiation was evaluated using MTT assay and clonogenic assay in vitro and xenograft models. Expressions of Notch pathway, mitogen-activated protein kinase (MAPK) pathway and Bcl-2 family proteins were investigated using western blot analysis. RESULTS: We discovered that the antitumour effect of combining GSI and radiation was dependent on treatment schedule. γ-Secretase inhibitor administration after radiation had the greatest growth inhibition of lung cancer in vitro and in vivo. We showed that the combination induced apoptosis of lung cancer cell lines through the regulation of MAPK and Bcl-2 family proteins. Furthermore, activation of Notch after radiation was ameliorated by GSI administration, suggesting that treatment with GSI prevents Notch-induced radiation resistance. CONCLUSION: Notch has an important role in lung cancer. Treatment with GSI after radiation can significantly enhance radiation-mediated tumour cytotoxicity.

HDAC inhibitors augment cytotoxic activity of rituximab by upregulating CD20 expression on lymphoma cells.

Anti-CD20 antibody rituximab is now essential for the treatment of CD20-positive B-cell lymphomas. Decreased expression of CD20 is one of the major mechanisms underlying both innate and acquired resistance to rituximab. In this study, we show that histone deacetylase (HDAC) inhibitors augment the cytotoxic activity of rituximab by enhancing the surface expression of CD20 antigen on lymphoma cells. HDAC inhibitors, valproic acid (VPA) and romidepsin, increased CD20 expression at protein and mRNA levels in B-cell lymphoma cell lines with relatively low CD20 expression levels. The VPA-mediated increase in CD20 expression occurred at 1 m, which is clinically achievable and safe, but insufficient for inducing cell death. Chromatin immunoprecipitation assays revealed that HDAC inhibitors transactivated the CD20 gene through promoter hyperacetylation and Sp1 recruitment. HDAC inhibitors potentiated the activity of rituximab in complement-dependent cytotoxic assays. In mouse lymphoma models, HDAC inhibitors enhanced CD20 expression along with histone hyperacetylation in transplanted cells, and acted synergistically with rituximab to retard their growth. The combination with HDAC inhibitors may serve as an effective strategy to overcome rituximab resistance in B-cell lymphomas.

Bortezomib overcomes cell-adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma.

Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

Simultaneous blockade of AP-1 and phosphatidylinositol 3-kinase pathway in non-small cell lung cancer cells.

c-Jun is a major constituent of AP-1 transcription factor that transduces multiple mitogen growth signals, and it is frequently overexpressed in non-small cell lung cancers (NSCLCs). Earlier, we showed that blocking AP-1 by the overexpression of a c-Jun dominant-negative mutant, TAM67, inhibited NSCLC cell growth. The phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathway is important in transformation, proliferation, survival and metastasis of NSCLC cells. In this study, we used NCI-H1299 Tet-on clone cells that express TAM67 under the control of inducible promoter to determine the effects of inhibition of AP-1 and PI3K on cell growth. The PI3K inhibitor, LY294002, produced a dose-dependent inhibition of growth in H1299 cells and that inhibition was enhanced by TAM67. TAM67 increased dephosphorylation of Akt induced by LY294002 and reduced the TPA response element DNA-binding of phosphorylated c-Jun. TAM67 increased G1 cell cycle blockade induced by LY294002, which was partially associated with cyclin A decrease and p27(Kip1) accumulation. Furthermore, TAM67 and LY294002 act, at least additively, to inhibit anchorage-independent growth of the H1299 cells. These results suggest that AP-1 and PI3K/Akt pathways play an essential role in the growth of some NSCLC cells.

Growth inhibition of non-small cell lung cancer cells by AP-1 blockade using a cJun dominant-negative mutant.

cJun, a major constituent of AP-1 transcription factor transducing multiple mitogen growth signals, is frequently overexpressed in non-small cell lung cancers (NSCLCs). The purpose of this study is to determine the effects of AP-1 blockade on the growth of NSCLC cells using a cJun dominant-negative mutant, TAM67. Transiently transfected TAM67 inhibited AP-1 transcriptional activity in NSCLC cell lines, NCI-H1299 (H1299), A549 and NCI-H520 (H520). The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not. To elucidate the effects of TAM67 on the growth of H1299, we established H1299 clone cells that expressed TAM67 under the control of a doxycycline-inducible promoter. In the H1299 clone cells, the induced TAM67 inhibited anchorage-dependent growth by promoting G1 cell-cycle block, but not by apoptosis. The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A. TAM67 also inhibited anchorage-independent growth of these cells. Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice. These results suggest that AP-1 plays an essential role in the growth of at least some of NSCLC cells.

The FLT3 inhibitor PKC412 exerts differential cell cycle effects on leukemic cells depending on the presence of FLT3 mutations.

PKC412 is a staurosporine derivative that inhibits several protein kinases including FLT3, and is highly anticipated as a novel therapeutic agent for acute myeloblastic leukemia (AML) carrying FLT3 mutations. In this study, we show that PKC412 exerts differential cell cycle effects on AML cells depending on the presence of FLT3 mutations. PKC412 elicits massive apoptosis without markedly affecting cell cycle patterns in AML cell lines with FLT3 mutations (MV4-11 and MOLM13), whereas it induces G2 arrest but not apoptosis in AML cell lines without FLT3 mutations (THP-1 and U937). In MV4-11 and MOLM13 cells, PKC412 inactivates Myt-1 and activates CDC25c, leading to the activation of CDC2. Activated CDC2 phosphorylates Bad at serine-128 and facilitates its translocation to the mitochondria, where Bad triggers apoptosis. In contrast, PKC412 inactivates CDC2 by inducing serine-216 phosphorylation and subsequent cytoplasmic sequestration of CDC25c in THP-1 and U937 cells. As a result, cells are arrested in the G2 phase of the cell cycle, but do not undergo apoptosis because Bad is not activated. The FLT3 mutation-dependent differential cell cycle effect of PKC412 is considered an important factor when PKC412 is combined with cell cycle-specific anticancer drugs in the treatment of cancer and leukemia.

Compensation of effective field in the field-induced superconductor kappa-(BETS)2FeBr4 observed by 77Se NMR.

We report results of 77Se NMR frequency shift in the normal state of the organic charge-transfer salt kappa-(BETS)2FeBr4 which shows magnetic field-induced superconductivity (FISC). From a simple mean-field analysis, we determined the field and the temperature dependences of the magnetization m(pi) of the pi conduction electrons on BETS molecules. We found that the Fe spins are antiferromagnetically coupled to the pi electrons and determined the exchange field to be J = -2.3T/microB. The exchange field from the fully saturated Fe moments (5 microB) is compensated by an external field of 12 T. This is close to the central field of the FISC phase, consistent with the Jaccarino-Peter local field-compensation mechanism for FISC [Phys. Rev. Lett. 9, 290 (1962)].

Transfection of antisense core 2 beta1,6-N-acetylglucosaminyltransferase-1 cDNA suppresses selectin ligand expression and tissue infiltration of B-cell precursor leukemia cells.

B-cell precursor (BCP) leukemia cells infiltrate into peripheral organs and the disease often relapses. Inhibition of tissue infiltration may improve the treatment outcome of BCP-leukemia patients. Selectin ligand has been suggested to play an important role in the infiltration of leukemia cells. However, the regulation mechanisms and involvement in tissue infiltration of selectin ligand expression in BCP-leukemia cells are not fully understood. In this study, we report that BCP-leukemia cells express selectin ligand on O-sialoglycoproteins. Core 2 beta1,6-N-acetylglucosaminyltransferase-1 (C2GnT-1) is mainly expressed in BCP-leukemia cells. Transfection of the antisense C2GnT-1 cDNA resulted in a significant reduction of either selectin ligand expression or selectin-dependent cell adhesion in BCP-leukemia cell line KM3 cells. Migration ability into mouse peripheral organs was reduced significantly in the antisense transfectant. These findings suggest that C2GnT-1 regulates selectin ligand expression. Downregulation of the selectin ligand expression level inhibits tissue infiltration of BCP-leukemia cells. C2GnT-1 may be a candidate of therapeutic target for the inhibition of infiltration of leukemia cells.

Biodegradation of an endocrine-disrupting chemical, di-2-ethylhexyl phthalate, by Bacillus subtilis No. 66.

A bacterial strain capable of rapidly degrading di-2-ethylhexyl phthalate (DEHP) was isolated from soil and identified as Bacillus subtilis. The organism also utilized di-butyl phthalate, di-ethyl phthalate, di-pentyl phthalate, di-propyl phthalate, and phthalic acid as sole carbon sources; and their biodegradation ratio was over 99%, when the incubation was performed for 5 days at 30 degrees C. The microorganism degraded di-2-ethylhexyl phthalate and di-butyl phthalate through the intermediate formation of mono-2-ethylhexyl phthalate and mono-butyl phthalate, which were then metabolized to phthalic acid and further by a protocatechuate pathway, as evidenced by oxygen uptake studies and GC-MS analysis. The decontamination of soil polluted with di-2-ethylhexyl phthalate by B. subtilis was investigated. Experimental results showed that the strain could degrade about 80% of 5 mM DEHP simply by adding 8% culture medium to soil, indicating that the degradation can occur even when other organisms are present.

Endobronchial ultrasonography with guide-sheath for peripheral pulmonary lesions.

The usefulness of endobronchial ultrasonography (EBUS) with guide-sheath (GS) as a guide for transbronchial biopsy (TBB) for diagnosing peripheral pulmonary lesions (PPL)s and for improving diagnostic accuracy was evaluated in this study. EBUS-GS-guided TBB was performed in 24 patients with 24 PPLs of < or =30 mm in diameter (average diameter=18.4 mm). A 20-MHz radial-type ultrasound probe, covered with GS was inserted via a working bronchoscope channel and advanced to the PPL in order to produce an EBUS image. The probe with the GS was confirmed to reach the lesion by EBUS imaging and X-ray fluoroscopy. When the lesion was not identified on the EBUS image, the probe was removed and a curette was used to lead the GS to the lesion. After localising the lesion, the probe was removed, and TBB and bronchial brushing were performed via the GS. Nineteen peripheral lesions (79.2%) were visualised by EBUS. All patients whose PPLs were visible on EBUS images subsequently underwent an EBUS-GS-guided diagnostic procedure. A total of 14 lesions (58.3%) were diagnosed. Even when restricted to PPLs <20 mm in diameter, the diagnostic sensitivity was 53%. In conclusion, endobronchial ultrasonography with guide sheath-guided transbronchial biopsy was feasible and effective for diagnosing peripheral pulmonary lesions.

Expression of human coagulation factor VIII in adipocytes transduced with the simian immunodeficiency virus agmTYO1-based vector for hemophilia A gene therapy.

We demonstrate that transduction of adipocytes with a simian immunodeficiency virus agm TYO1 (SIVagm)-based lentiviral vector carrying the human coagulation factor VIII gene (SIVhFVIII) resulted in expression of the human FVIII transgene in vitro and in db/db mice in vivo. Cultured human adipocytes were transduced with the SIVagm vector carrying the GFP gene in a dose-dependent manner and transduction of adipocytes with SIVhFVIII resulted in efficient expression of human coagulation factor VIII (hFVIII; 320 +/- 39.8 ng/10(6) adipocytes/24 h) in vitro. Based upon successful transduction of adipocytes by SIV vectors carrying the lacZ gene in vivo in mice, the adipose tissue of db/db mice was transduced with SIVhFVIII. There was a transient appearance of human FVIII in mouse plasma (maximum 1.8 ng/ml) on day 11 after the injection. Transcripts of human FVIII transgene and human FVIII antigen also were detected in the adipose tissue by RT-PCR and immunofluorescence, respectively, on day 14. Emergence of anti-human FVIII antibodies 14 days after the injection of SIVhFVIII may explain the disappearance of human FVIII from the circulation. These results suggest that transduction of the adipocytes with vectors carrying the human FVIII gene may be potentially applicable for gene therapy of hemophilia A.