Thrombomodulin blocks calcineurin inhibitor-induced vascular permeability via inhibition of Src/VE-cadherin axis.

Recombinant human soluble thrombomodulin (rTM) counteracted capillary leakage and alleviated edema in individuals with sinusoidal obstruction syndrome and engraftment syndrome after hematopoietic stem cell transplantation. We previously showed that rTM increased levels of antiapoptotic protein Mcl-1 and protected endothelial cells from calcineurin inhibitor cyclosporine A (CsA)-induced apoptosis. However, the molecular mechanisms by which rTM enhances barrier function in vascular endothelial cells remain unknown. Here we show that exposure of vascular endothelial EA.hy926 cells to CsA induced phosphorylation of Src/vascular endothelial cadherin (VE-cadherin) and translocation of VE-cadherin from cell surface to cytoplasm, resulting in an increase in vascular permeability. In addition, CsA increased production of inflammatory cytokines, including interleukin (IL)-1ß and IL-6, associated with an increase in nuclear levels of nuclear factor-κB (NF-κB) which also enhanced vascular permeability. Importantly, the fourth and fifth regions of epidermal growth factor-like domain of TM (TME45) attenuated CsA-induced p-Src/VE-cadherin and vascular permeability in parallel with a decrease in nuclear levels of NF-κB and cytokine production in EA.hy926 cells. In conclusion, TM, especially TME45, maintains vascular integrity, at least in part, via Src signaling.

The fifth epidermal growth factor-like region of thrombomodulin exerts cytoprotective function and prevents SOS in a murine model.

The present study found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) possesses the cytoprotective function in association with an increase in levels of anti-apoptotic myeloid cell leukemia-1 protein in an activated protein C-independent manner in human umbilical vein endothelial cells (HUVECs). Importantly, TME5 counteracted calcineurin inhibitor-induced vascular permeability and successfully prevented monocrotaline-induced sinusoidal obstruction syndrome (SOS) in a murine model. Taken together, TME5 may be useful for preventing or treating lethal complications that develop after hematopoietic stem cell transplantation such as SOS and thrombotic microangiopathy in which endothelial cell damage has a role.

The novel function of CD82 and its impact on BCL2L12 via AKT/STAT5 signal pathway in acute myelogenous leukemia cells.

The aim of this study was to explore the biological functions of a tetraspanin family protein CD82 expressed aberrantly in chemotherapy-resistant CD34(+)/CD38(-) acute myelogenous leukemia (AML) cells. Microarray analysis of patient-isolated CD34(+)/CD38(-) AML cells revealed that the levels of anti-apoptotic protein BCL2L12 were downregulated after CD82 depletion by specific short hairpin RNA (shRNA). Western blot analysis indicated that BCL2L12 was aberrantly expressed in patient-isolated AML cells and AML cell lines. Furthermore, CD82 blockade by a specific antibody downregulated BCL2L12 in parallel with dephosphorylation of signal transducer and activator of transcription 5 (STAT5) and AKT, whereas pharmacological inhibition of STAT5 and AKT activation decreased BCL2L12 expression in leukemia cells. In addition, shRNA-mediated downregulation of BCL2L12 increased the levels of cleaved caspase-3 and suppressed proliferation of leukemia cells, impairing their engraftment in immunodeficient mice. Taken together, our results indicate that CD82 regulated BCL2L12 expression via STAT5A and AKT signaling and stimulated proliferation and engrafting of leukemia cells, suggesting that CD82 and BCL2L12 may be promising therapeutic targets in AML.

Thrombomodulin alleviates murine GVHD in association with an increase in the proportion of regulatory T cells in the spleen.

Recombinant human soluble thrombomodulin (rTM), a potent anticoagulant, has been used for the treatment of disseminated intravascular coagulation in Japan since 2008. Interestingly, rTM possesses anti-inflammatory and cytoprotective functions. This study examined whether rTM alleviates GVHD in a murine hematopoietic SCT (HSCT) model. Use of rTM significantly improved the survival of mice on day 28 of transplantation (survival rate 70% in rTM - treated mice vs 35% in control, P<0.05) in association with a significant decrease in plasma levels of IL-6, IFN-γ and high-mobility group B1 DNA-binding protein on day 7 of HSCT. Intriguingly, the proportion of regulatory T cells in the spleen was significantly increased in rTM-treated mice on day 7 of transplantation compared with control diluent-treated mice. In addition, elevated plasma levels of TM and fibrin/fibrinogen degradation product were noted in HSCT-recipient mice, suggesting coagulopathy caused by endothelial cell damage in this GVHD model. The use of rTM potently decreased these levels. Importantly, rTM did not hamper the anti-GVL and engraftment of hematopoietic cells. Taken together, the use of rTM may prevent GVHD and serve as a potential therapeutic strategy to improve clinical outcomes in individuals who receive HSCT.

Imatinib causes epigenetic alterations of PTEN gene via upregulation of DNA methyltransferases and polycomb group proteins.

We have recently reported the possible imatinib-resistant mechanism; long-term exposure of leukemia cells to imatinib downregulated levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) via hypermethylation of its promoter region (Leukemia 2010; 24: 1631). The present study explored the molecular mechanisms by which imatinib caused methylation on the promoter region of this tumor suppressor gene in leukemia cells. Real-time reverse transcription PCR found that long-term exposure of chronic eosinophilic leukemia EOL-1 cells expressing FIP1L1/platelet-derived growth factor receptor-α to imatinib induced expression of DNA methyltransferase 3A (DNMT3A) and histone-methyltransferase enhancer of zeste homolog 2 (EZH2), a family of polycomb group, thereby increasing methylation of the gene. Immunoprecipitation assay found the increased complex formation of DNMT3A and EZH2 proteins in these cells. Moreover, chromatin immunoprecipitation assay showed that amounts of both DNMT3A and EZH2 proteins bound around the promoter region of PTEN gene were increased in EOL-1 cells after exposure to imatinib. Furthermore, we found that levels of DNMT3A and EZH2 were strikingly increased in leukemia cells isolated from individuals with chronic myelogenous leukemia (n=1) and Philadelphia chromosome-positive acute lymphoblastic leukemia (n=2), who relapsed after treatment with imatinib compared with those isolated at their initial presentation. Taken together, imatinib could cause drug-resistance via recruitment of polycomb gene complex to the promoter region of the PTEN and downregulation of this gene's transcripts in leukemia patients.

Long-term exposure of leukemia cells to multi-targeted tyrosine kinase inhibitor induces activations of AKT, ERK and STAT5 signaling via epigenetic silencing of the PTEN gene.

Imatinib induces complete molecular response in patients with chronic myeloid leukemia (CML) and chronic eosinophilic leukemia (CEL). However, development of resistance to imatinib has emerged as an important clinical problem for molecular-targeted therapy in CML and CEL. In this study, we have established the imatinib-resistant CEL EOL-1 sub-lines (designated as EOL-1R) by culturing cells with increasing concentrations of imatinib for 6 months. Interestingly, EOL-1R cells showed epigenetic silencing of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene. Exposure of EOL-1R cells to imatinib failed to dephosphorylate AKT, ERK and STAT5, although PDGFRalpha was effectively inactivated. The forced expression of PTEN negatively regulated these signal pathways and sensitized EOL-1R cells to imatinib. Notably, hypermethylation of the promoter region of the PTEN gene in association with the downregulation of this gene's transcripts was identified in imatinib-resistant leukemia cells isolated from individuals with CEL, CML and Philadelphia-positive acute lymphoblastic leukemia. In addition, anti-epigenetic agents restored PTEN expression, resulting in the sensitization of EOL-1R cells to imatinib. Taken together, epigenetic silence of PTEN is one of the mechanisms that cause drug resistance in individuals with leukemia after exposure to imatinib. Anti-epigenetic agents may be useful for overcoming drug resistance in such a case.

A novel treatment strategy targeting polo-like kinase 1 in hematological malignancies.

Polo-like kinase1 (PLK1) belongs to the family of serine/threonine kinases and plays an important role in centrosome maturation, bipolar spindle formation, and cytokinesis during mitosis. We found in this study that PLK1 was aberrantly highly expressed in a variety of human leukemia cell lines (n=20), as well as, freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n=50) and acute lymphoblastic leukemia (n=15) compared with bone marrow mononuclear cells from healthy volunteers (n=13) (acute myelogenous leukemia, P=0.016; acute lymphoblastic leukemia, P=0.008), as measured by real-time RT-PCR. Downregulation of PLK1 by a small interfering RNA in NB4 acute myelogenous leukemia cells inhibited their proliferation. GW843682X is a novel selective PLK1 inhibitor. The compound-induced growth inhibition, caused accumulation of cells in the G2/M phase of the cell cycle and mediated apoptosis of human leukemia cells. Pre-treatment of cells with the caspase inhibitor Z-VAD-FMK attenuated the action of GW843682X in leukemia cells, indicating the involvement of the caspase pathway in the PLK1 inhibitor-mediated apoptosis. Furthermore, we found that the PLK1 inhibitor synergistically potentiated the growth inhibition and apoptosis of leukemia cells when combined with tubulin-depolymerizing agent vincristine. Taken together, targeting PLK1 may be a promising treatment strategy for individuals with leukemia.

Blockade of mTOR signaling potentiates the ability of histone deacetylase inhibitor to induce growth arrest and differentiation of acute myelogenous leukemia cells.

This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K) and p-S6K by western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analog RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27(kip1) and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by the expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce the expression of the myeloid differentiation-related transcription factor, CCAAT enhancer-binding protein-epsilon, in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of an HDACI and an mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.

Fludarabine induces growth arrest and apoptosis of cytokine- or alloantigen-stimulated peripheral blood mononuclear cells, and decreases production of Th1 cytokines via inhibition of nuclear factor kappaB.

Fludarabine is a purine analog that has demonstrated significant activity in B-cell malignancies, including CLL. Fludarabine also possesses an immunosuppressive effect and is being used to prevent GVHD in hematopoietic stem cell transplantation. However, the molecular mechanism by which fludarabine inhibits immunoreaction remains to be fully elucidated. This study found that fludarabine inhibited tumor necrosis factor alpha (TNF-alpha)-stimulated degradation of IkappaBalpha, resulting in blockade of nuclear translocation of nuclear factor kappaB (NF-kappaB) in Jurkat T cells, as measured by western blot analysis and immunocytochemistry. The ability of fludarabine to inhibit NF-kappaB was further confirmed by electrophoretic mobility shift assay. We also found that fludarabine induced growth arrest and apoptosis of alloreactive and TNF-alpha-stimulated PBMCs. In addition, fludarabine inhibited TNF-alpha-stimulated production of IL-2 and IFN-gamma, which play important roles in the onset of GVHD, in Jurkat cells. Taken together, fludarabine is useful for management of immune diseases, including GVHD, through inactivation of NF-kappaB.

Avaliação dos resultados clínicos após cirurgia descompressiva em cães com doença de disco intervertebral / Evaluation of clinical results of decompressive surgery in dogs with degenerative disk disease

Avaliaram-se os resultados clínicos após realização de cirurgia descompressiva em 45 cães com doença do disco intervertebral cervical ou toracolombar. Após a cirurgia, 35 cães (77,8 por cento) recuperaram-se totalmente, oito (17,8 por cento) parcialmente e dois (4,4 por cento) não apresentaram alteração do quadro inicial. Em oito cães com paraplegia e perda da sensibilidade dolorosa profunda houve completa melhora do quadro clínico, com recuperação total em 62,5 por cento dos casos. Em quatro cães com tetraparesia, a cirurgia foi eficaz. A cirurgia descompressiva (slot cervical e hemilaminectomia toracolombar), com a retirada do material do disco do interior do canal vertebral, foi uma forma efetiva de gerar melhora do quadro funcional

Clinical results after decompressive surgery were evaluated in 45 dogs with cervical or thoracolumbar intervertebral disk disease. After surgery, 35 dogs recovered totally, eight (17.8 percent) partially, and two (4.4 percent) did not present any change in clinical findings. Eight dogs with paraplegy and loss of deep pain perception showed improvement, with total recovering in 62.5 percent of cases. Surgery was effective in four dogs with tetraparesy. Decompressive surgery (cervical slot or hemilaminectomy), with removal of disk material from inside the vertebral canal, was an effective form to produce functional improvement in dogs with this disease

Avaliação dos resultados clínicos após cirurgia descompressiva em cães com doença de disco intervertebral

Clinical results after decompressive surgery were evaluated in 45 dogs with cervical or thoracolumbar intervertebral disk disease. After surgery, 35 dogs recovered totally, eight (17.8%) partially, and two (4.4%) did not present any change in clinical findings. Eight dogs with paraplegy and loss of deep pain perception showed improvement, with total recovering in 62.5% of cases. Surgery was effective in four dogs with tetraparesy. Decompressive surgery (cervical slot or hemilaminectomy), with removal of disk material from inside the vertebral canal, was an effective form to produce functional improvement in dogs with this disease.

Avaliaram-se os resultados clínicos após realização de cirurgia descompressiva em 45 cães com doença do disco intervertebral cervical ou toracolombar. Após a cirurgia, 35 cães (77,8%) recuperaram-se totalmente, oito (17,8%) parcialmente e dois (4,4%) não apresentaram alteração do quadro inicial. Em oito cães com paraplegia e perda da sensibilidade dolorosa profunda houve completa melhora do quadro clínico, com recuperação total em 62,5% dos casos. Em quatro cães com tetraparesia, a cirurgia foi eficaz. A cirurgia descompressiva (slot cervical e hemilaminectomia toracolombar), com a retirada do material do disco do interior do canal vertebral, foi uma forma efetiva de gerar melhora do quadro funcional.

Fludarabine induces apoptosis of human T-cell leukemia virus type 1-infected T cells via inhibition of the nuclear factor-kappaB signal pathway.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease in which the human T-cell lymphotropic virus type I (HTLV-I) has been recognized as the etiologic agent. Fludarabine is a purine analog that has demonstrated significant activity in B-cell malignancies, including chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma. This study explored the effects of fludarabine on HTLV-1-infected T cells (MT-1, -2, -4 and HUT102). Fludarabine induced growth arrest and apoptosis of these cells, as measured by 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell cycle analysis and annexin V staining. Moreover, exposure of HTLV-1-infected T cells to fludarabine decreased the levels of X-inhibitor of apoptosis protein in conjunction with inhibition of nuclear factor kappaB (NF-kappaB)/DNA-binding activity, as measured by Western blot analysis and electrophoretic mobility shift and reporter gene assays, respectively. Further studies found that fludarabine accumulated NF-kappaB and inhibitory subunit of NF-kappaB in cytosole in conjunction with downregulation of NF-kappaB in nucleus, suggesting that fludarabine blocked nuclear translocation of NF-kappaB. Taken together, fludarabine may be useful for treatment of individuals with ATL and other types of cancer in which NF-kappaB plays a role.

NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines.

HIV-1 protease inhibitor (PI), nelfinavir (NFV) induced growth arrest and apoptosis of NCI-H460 and -H520, A549, EBC-1 and ABC-1 non-small-cell lung cancer (NSCLC) cells in association with upregulation of p21waf1, p27kip1 and p53, and downregulation of Bcl-2 and matrix metalloproteinase (MMP)-2 proteins. We found that NFV blocked Akt signalling in these cells as measured by Akt kinase assay with glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) as a substrate. To explore the role of Akt signalling in NFV-mediated growth inhibition of NSCLC cells, we blocked this signal pathway by transfection of Akt small interfering RNA (siRNA) in these cells; transient transfection of Akt siRNA in NCI-H460 cells decreased the level of Bcl-2 protein and slowed their proliferation compared to the nonspecific siRNA-transfected cells. Conversely, forced-expression of Akt partially reversed NFV-mediated growth inhibition of these cells, suggesting that Akt may be a molecular target of NFV in NSCLC cells. Also, we found that inhibition of Akt signalling by NFV enhanced the ability of docetaxel to inhibit the growth of NCI-H460 and -H520 cells, as measured by MTT assay. Importantly, NFV slowed the proliferation and induced apoptosis of NCI-H460 cells present as tumour xenografts in nude mice without adverse systemic effects. Taken together, this family of compounds might be useful for the treatment of individuals with NSCLC.

[Estimation of daily dietary intake of aluminum].

The daily dietary intake of aluminum was estimated through a total diet study from 1996 to 1998. In ten institutes, total diet study samples were prepared and their aluminum concentration was determined. The average daily intake of aluminum was 3.5 mg and the range was 1.8-8.4 mg. The validity of the analytical result was supported by analyses of certified reference materials.

Relation between intellectual dysfunctioning and mortality in community-residing older people.

OBJECTIVE: To examine the relationship between intellectual dysfunctioning and mortality in a community-residing older population. DESIGN: Of the 1473 randomly selected people aged 65 years and older living in Settsu, Osaka Prefecture, in October 1992, 1405 were contacted. Data for assessment of intellectual dysfunctioning were obtained from 1383 people (98.4%), who constituted the study cohort. Follow-up for 42 months was completed for 1300 subjects (94.0%; 1117 living, 183 deceased). MEASURES: Data on general health status, history of health management, psychosocial conditions, and intellectual dysfunctioning were collected by means of interviews during home visits at the time of enrollment. Intellectual dysfunctioning was determined with the assessment instrument developed by the Social Survey Division of the Office of Population Censuses (OPCS) in Great Britain. RESULTS: The Kaplan-Meier analysis indicated that the estimated survival rates for men and women decreased with a decline in intellectual function in two age groups: 65 to 74 and 75 years and older. For both sexes, the log rank test showed that the curves among the four groups based on intellectual dysfunctioning (intact, mild, moderate, and severe) achieved statistical significance for the age group of 75 years and older. For both age groups and each of the levels of intellectual dysfunctioning, the estimated survival rate for men was lower than that for women. Application of the Cox proportional hazards model resulted in unadjusted hazard ratios of mild, moderate, and severe intellectual dysfunctioning for mortality of 1.68, 2.44, and 5.37, respectively. Multivariate analysis on the other hand, yielded adjusted hazard ratios of mild, moderate, and severe intellectual dysfunctioning of 1.19, 1.12, and 1.74, respectively, leaving severe dysfunctioning as the only statistically significant factor associated with mortality. Other factors such as sex, age, general health status, history of management, and psychosocial conditions were controlled. CONCLUSION: Intellectual dysfunctioning, as measured by an assessment instrument developed by OPCS, represents an increased risk factor for mortality among community-residing older people.

[Correlates and prognosis in relation to intellectual dysfunction in a community-residing elderly population].

To estimate the risk factors for intellectual dysfunction and examine its prognosis in a community-residing (non-institutionalized) elderly population, a randomly selected sample of 1,473 elderly people aged 65 years and over living in S city, Osaka Prefecture, was studied in October 1992, and data were obtained from 1,383, a response rate of 93.9%. A cohort of 1,383 was followed for 42 months and follow-up was completed for 1,300 (94.0%). The main results were as follows: 1) The prevalence of intellectual dysfunction did not differ significantly between sexes, and there was an increasing prevalence of intellectual dysfunction with age in both sexes. The prevalence of severe intellectual dysfunction was found to increase highly at age 85 and over. 2) By univariate analysis, odds ratios for age older than 75 years, low Activities of Daily Living (ADL), urinary and fecal incontinence, and no participation in social activities were significantly higher than 1 in any level of mild, moderate, and severe intellectual dysfunction. In the multivariate analysis using logistic regression, age older than 75 years and urinary and fecal incontinence showed significant higher odds ratios than 1 for severe intellectual dysfunction, and low ADL and treatment for hypertension also showed significant higher odds ratios than 1 for moderate intellectual dysfunction. 3) From analysis using the Kaplan-Meier method, the cumulative survival rates decreased with a decline in intellectual functioning in both age groups of 65-74 and 75 years and older. 4) Application of the Cox proportional hazards model resulted in adjusted hazard ratio for severe intellectual dysfunction of 1.79 (95% confidence interval, 1.02-3.12), controlling for other factors such as sex, age, general health status, incontinence and social activities.