Spontaneously hyperactive MEK-Erk pathway mediates paradoxical facilitation of cell proliferation in mild hypoxia.

BACKGROUND: Oxygen is important for common eukaryotic cells to generate ATP. Pathophysiological conditions such as ischemic diseases cause tissue hypoxia. In addition, oxygen availability in deep tissues is supposed to be far lower than surrounding atmosphere even in healthy animals, and the oxygen partial pressures in most normal tissues are estimated to be around 40-50mmHg, so-called mild hypoxia. Recent studies have demonstrated that mild hypoxia has distinct effects on living cells from severe hypoxia. For instance, mild hypoxia was reported to promote cell reprogramming. Although severe hypoxia is known to inhibit cell proliferation, mild hypoxia has been paradoxically demonstrated to increase cell proliferation. However, it has not been clarified by which molecular mechanisms mild hypoxia evokes the discontinuous increment of cell proliferation. METHODS: We established experimental conditions showing the opposite influences of mild and severe hypoxia on cell proliferation using undifferentiated Caco2 human colon carcinoma cells in order to clarify the underlying molecular mechanism. RESULTS: The basal activity of Erk, which is a typical mediator of mitogenic signals, is spontaneously increased specifically in cells exposed to mild hypoxia, and inhibition of MEK, an upstream kinase of the Erk, completely inhibited the mild hypoxia-induced enhancement of cell proliferation. CONCLUSIONS: Spontaneous hyperactivation of the MEK-Erk pathway by mild hypoxia should be the plausible molecular mechanism of the paradoxical promotion of cell proliferation. GENERAL SIGNIFICANCE: Our findings will provide clues to the molecular basis of mild hypoxia-evoked phenomena such as cell reprogramming.

Characteristics of and risk factors for interstitial lung disease induced by chemotherapy for lung cancer.

BACKGROUND: Drug-induced interstitial lung disease (DILD) is generally a serious adverse effect and almost always necessitates the discontinuation of the offending drug. Cancer pharmacotherapy is strongly associated with DILD, and the risk of DILD has been suggested to be higher in patients with lung cancer because of preexisting pneumonic disease. OBJECTIVE: The aim of this retrospective study was to identify the risk factors and prognostic factors for early death from interstitial lung disease (ILD) induced by chemotherapy for lung cancer. METHODS: The medical records of 459 patients who underwent chemotherapy for lung cancer between April 2007 and March 2013 were analyzed with regard to patient background and DILD development, initial symptoms, and prognosis. RESULTS: A total of 33 patients (7.2%) developed chemotherapy-induced ILD. The most frequently observed initial symptom was dyspnea (94.3%). Preexisting ILD was identified as a risk factor for DILD (odds ratio [OR] = 5.38; 95% CI = 2.47-11.73; P < 0.01). Among the 33 patients who developed DILD, 10 patients suffered an early death despite steroid therapy. Poor prognostic factors included epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) use (OR = 9.26; 95% CI = 1.05-82.0; P < 0.05) and 2 or more prior chemotherapy regimens (OR = 6.95; 95% CI = 1.14-42.3; P < 0.05). CONCLUSIONS: Many lung cancer patients have coexisting ILD, and these patients have a high risk of developing chemotherapy-induced ILD. In addition, patients with DILD who underwent EGFR-TKI therapy and 2 or more prior chemotherapy regimens had a higher risk of fatal outcome.

A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.

Identification of the risk factors associated with hypocalcemia induced by denosumab.

Denosumab, a fully human monoclonal antibody that inhibits the receptor activator of nuclear factor-κB ligand, inhibits the activation of osteoclasts. Some clinical trials have shown that denosumab suppresses bone resorption in patients with advanced cancer, but hypocalcemia has been reported as a serious adverse effect after the administration of denosumab. It is difficult to predict hypocalcemia in such cases because the risk factors for denosumab-induced hypocalcemia have not been reported. Accordingly, the aim of the present study was to identify the risk factors for hypocalcemia induced by denosumab. We retrospectively reviewed the records of patients who had received denosumab at Tokushima University Hospital between April 2012 and May 2013. Fifty-three patients were analyzed and eleven patients had hypocalcemia after administration of denosumab. Univariate logistic regression analysis revealed that the patients who had not been administered zoledronic acid before receiving denosumab or had lower creatinine clearance (CCr) appeared to have a higher risk of hypocalcemia (p<0.05). The cut off value of CCr was 50.4 mL/min calculated by receiver-operator characteristics curves. Moreover, multivariate logistic regression analysis revealed that non-administration of zoledronic acid (odds ratio 10.43, p<0.05) and CCr less than 50.0 mL/min (odds ratio 5.90, p<0.05) were independent risk factors for denosumab-induced hypocalcemia. These findings provide useful information regarding the monitoring of hypocalcemia in patients receiving denosumab.

Iron reduction by deferoxamine leads to amelioration of adiposity via the regulation of oxidative stress and inflammation in obese and type 2 diabetes KKAy mice.

Iron is an essential trace metal for most organisms. However, excess iron causes oxidative stress through production of highly toxic hydroxyl radicals via the Fenton/Haber-Weiss reaction. Iron storage in the body is reported to be associated with fat accumulation and type 2 diabetes mellitus. We investigated the role of iron in adiposity by using KKAy mice and obese and diabetic model mice. Eight-week-old KKAy mice were divided into two groups and treated with deferoxamine (DFO), an iron chelator agent, or a vehicle for 2 wk. DFO treatment diminished fat iron concentration and serum ferritin levels in KKAy mice. Fat weight and adipocyte size were reduced significantly in DFO-treated mice compared with vehicle-treated mice. Macrophage infiltration into fat was also decreased in DFO-treated mice compared with vehicle-treated mice. Superoxide production and NADPH oxidase activity in fat, as well as urinary 8-hydroxy-2'-deoxyguanosine excretion, were decreased in KKAy mice after DFO treatment while p22(phox) expression in adipose tissue was diminished in such mice. Ferritin expression in the fat of DFO-treated KKAy mice was decreased. In addition, F4/80-positive cells also presented through both p22(phox) and ferritin expression. The mRNA expression levels of inflammatory cytokines were also reduced in fat tissue of DFO-treated mice. These findings suggest that reduction of iron levels ameliorates adipocyte hypertrophy via suppression of oxidative stress, inflammatory cytokines, and macrophage infiltration, thereby breaking a vicious cycle in obesity.

Plasma dipeptidyl peptidase 4 activity correlates with body mass index and the plasma adiponectin concentration in healthy young people.

We previously found that plasma dipeptidyl peptidase 4 (DPP4) activity was associated with the development of obesity, type 2 diabetes, and type 1 diabetes using animal models. In this study, we investigated whether DPP4 activity is correlated with the clinical parameters of obesity and/or diabetes in healthy young subjects. Body mass index (BMI), plasma DPP4 activity, total cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides, fasting blood glucose, adiponectin concentration, and body fat were measured in 165 subjects (110 males and 55 females, age 23.2 ± 2.4 years). In correlation analyses, DPP4 activity displayed strong positive correlations with BMI (p = 5.5 × 10(-5)) and total cholesterol (p = 0.0014), and a negative correlation with the plasma adiponectin concentration (p = 0.013), but not fasting blood glucose. Our findings suggest that plasma DPP4 activity is correlated with the clinical parameters of obesity rather than diabetes in young people.

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca(2+) in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells.

BACKGROUND: It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca(2+) mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca(2+) release from Ca(2+) stores in adrenal chromaffin cells. METHODS: We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca(2+) in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2h. RESULTS: Exposure to the magnetic field significantly reduced the increase in [Ca(2+)]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca(2+) release by the exposure was unaffected. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca(2+) release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca(2+)]i.

Altered dipeptidyl peptidase-4 activity during the progression of hyperinsulinemic obesity and islet atrophy in spontaneously late-stage type 2 diabetic rats.

Altered dipeptidyl peptidase-4 (DPP4) activity during the progression of late-stage type 2 diabetes was measured in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Compared with OLETF rats subjected to 30% food restriction, food-satiated OLETF rats exhibited spontaneous hyperphagic obesity, insulin resistance, hyperglycemia, hyperinsulinemia, and increased plasma DPP4 activity during the early phase of the experiment (up to ∼30 wk). Subsequently, their plasma DPP4 activity as well as their body weight, body fat, and plasma insulin concentration declined to control levels during the late phase, resulting in excessive polyuria, proteinuria, dyslipidemia, pancreatic islet atrophy, hypoinsulinemia, and diabetes, which changed from insulin-resistant diabetes to hypoinsulinemic diabetes secondary to progressive islet insufficiency, and their fasting blood glucose level remained high. Since plasma DPP4 activity demonstrated significant positive correlations with body weight and the fasting plasma insulin level but not with the fasting blood glucose level during the late stage of diabetes, body fat and fasting plasma insulin levels may be useful factors for predicting the control of plasma DPP4 activity. In contrast, pancreatic DPP4 activity was significantly increased, and the expression of pancreatic insulin was significantly reduced in late-stage diabetic OLETF rats, suggesting that a relationship exists between the activation of pancreatic DPP4 and insulin depletion in pancreatic islet atrophy. In conclusion, it is suggested that plasma DPP4 activity changes in accordance with the progression of hyperinsulinemic obesity and pancreatic islet atrophy. DPP4 activity may play an important role in insulin homeostasis.

Identification of TMEM45B as a protein clearly showing thermal aggregation in SDS-PAGE gels and dissection of its amino acid sequence responsible for this aggregation.

SDS-PAGE is one of the most powerful experimental techniques used for the separation of proteins, and most proteins are separated according to their molecular size by this technique. However, exceptional proteins showing abnormal behavior in SDS-PAGE gels are known to exist. Thermal aggregation, rarely observed with membrane proteins, is one of the exceptional behaviors of proteins during SDS-PAGE, but detailed characterization of this aggregation has not yet been achieved. In the present study, we found that a putative membrane protein, TMEM45B, very clearly showed properties of thermal aggregation when it was expressed in COS7 cells and subjected to SDS-PAGE. We dissected the region of TMEM45B responsible for this aggregation, and found that of the seven putative transmembrane domains, a region comprising the 4th to 7th ones was essential for the thermal aggregation properties. We also demonstrated that these transmembrane domains, 4th to 7th, of TMEM45B conferred thermal aggregation properties on other proteins, by fusing this amino acid sequence to target proteins. The molecular mechanism causing thermal aggregation by TMEM45B is still uncertain, but TMEM45B could be utilized as a nice model to show clear thermal aggregation in SDS-PAGE gels.

Pharmacology in health food: metabolism of quercetin in vivo and its protective effect against arteriosclerosis.

Quercetin, a member of the bioflavonoids family, has been proposed to have anti-atherogenic, anti-inflammatory, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. It was recently demonstrated that quercetin 3-O-ß-D-glucuronide (Q3GA) is one of the major quercetin conjugates in human plasma, in which the aglycone could not be detected. Although most of the in vitro pharmacological studies have been carried out using only the quercetin aglycone form, experiments using Q3GA would be important to discover the preventive mechanisms of cardiovascular diseases by quercetin in vivo. Therefore we examined the effects of the chemically synthesized Q3GA, as an in vivo form, on vascular smooth muscle cell (VSMC) disorders related to the progression of arteriosclerosis. Platelet-derived growth factor-induced cell migration and proliferation were inhibited by Q3GA in VSMCs. Q3GA attenuated angiotensin II-induced VSMC hypertrophy via its inhibitory effect on JNK and the AP-1 signaling pathway. Q3GA scavenged 1,1-diphenyl-2-picrylhydrazyl radical measured by the electron paramagnetic resonance method. In addition, immunohistochemical studies with monoclonal antibody 14A2 targeting the Q3GA demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may have preventative effects on arteriosclerosis relevant to VSMC disorders.

Expression of soluble vascular endothelial growth factor receptor-1 in human monocyte-derived mature dendritic cells contributes to their antiangiogenic property.

The soluble form of vascular endothelial growth factor receptor-1 (sVEGFR-1) is produced from endothelial cells by alternative splicing of VEGFR-1 mRNA, and can inhibit angiogenesis by blocking the biological effects of VEGF. In this study, we show the expression of a large amount of sVEGFR-1 in human monocyte-derived mature dendritic cells (mDCs). As compared with monocytes and immature DCs, mDCs generated by TNF-alpha or soluble CD40L with IFN-gamma, but not LPS or other stimuli, preferentially produce sVEGFR-1. We also detected the mRNA of sVEGFR-1 generated by alternative splicing of VEGFR-1 mRNA in mDCs induced by TNF-alpha. The production of sVEGFR-1 showed a distinct contrast to those of VEGF in each DC matured with various stimuli. The supernatant of DCs matured with TNF-alpha or soluble CD40L with IFN-gamma showed inhibition of the tube formation of HUVECs, which was neutralized by anti-VEGFR-1 Ab, indicating that sVEGFR-1 secreted from mDCs was biologically active. Interestingly, the supernatant of mDCs generated with LPS increased HUVEC capillary-like formation in vitro. The ratio of sVEGFR-1 to VEGF clearly reflected the net angiogenic property of mDCs. Administration of mDCs induced by TNF-alpha into the s.c. tumor of PC-14 cells implanted in SCID mice demonstrated the inhibition of tumor growth via reduction of the number of CD31-positive vessels, indicating their in vivo antiangiogenic potential. These results suggest that sVEGFR-1 produced by mDCs contribute to their antiangiogenic property, and the ratio of sVEGFR-1 to VEGF might be a useful tool for evaluating their ability to regulate angiogenesis mediated by VEGF.

Angiotensin II receptor blocker attenuates PDGF-induced mesangial cell migration in a receptor-independent manner.

BACKGROUND: Clinical studies have shown that angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are able to provide renoprotection independent of their blood pressure lowering effects. ARBs also are reported to suppress oxidative stress, inflammation and certain other cellular responses in a receptor-independent manner. We investigated the effects of an ARB, olmesartan, on the cell migration induced by platelet-derived growth factor (PDGF), a major mitogen involved in the pathogenesis of glomerulonephritis in rat mesangial cells (RMCs). METHODS: Cell migration was determined by a modified Boyden chamber assay. The intracellular signalling pathway was examined by western blotting. AT1 receptor expression was knocked down by small interfering RNAs. The intracellular reactive oxygen species (ROS) was measured by using a fluorescent probe. The O(2)(.-) scavenging activities were studied by the electron paramagnetic resonance-spin trapping method. RESULTS: PDGF-induced cell migration was inhibited by olmesartan in AT1 receptor knockdown RMCs. Olmesartan attenuated big mitogen-activated protein (MAP) kinase 1 (BMK1) and Src activation by PDGF in AT1 receptor knockdown RMCs. PDGF-induced BMK1 activation was suppressed by the Src family tyrosine kinase inhibitors, indicating that Src exists upstream of BMK1. The NADPH oxidase inhibitors inhibited not only PDGF-induced BMK1 and Src activation but also RMC migration. The elevation in ROS generation induced by PDGF was decreased by olmesartan. Olmesartan displayed neither directly ROS scavenging activity nor the inhibition of ROS-mediated intracellular signalling in RMCs. CONCLUSIONS: Olmesartan attenuates ROS generation by PDGF, leading to the subsequent inhibition of Src/ BMK1/migration in an AT1 receptor-independent manner in RMCs.

HM1.24 (CD317) is a novel target against lung cancer for immunotherapy using anti-HM1.24 antibody.

HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. We examined the expression of HM1.24 antigen in lung cancer cells and the possibility of immunotherapy with anti-HM1.24 antibody which can induce antibody-dependent cellular cytotoxicity (ADCC). The expression of HM1.24 antigen in lung cancer cells was examined by flow cytometry as well as immunohistochemistry using anti-HM1.24 antibody. ADCC was evaluated using a 6-h (51)Cr release assay. Effects of various cytokines on the expression of HM1.24 and the ADCC were examined. The antitumor activity of anti-HM1.24 antibody in vivo was examined in SCID mice. HM1.24 antigen was detected in 11 of 26 non-small cell lung cancer cell lines (42%) and four of seven (57%) of small cell lung cancer cells, and also expressed in the tissues of lung cancer. Anti-HM1.24 antibody effectively induced ADCC in HM1.24-positive lung cancer cells. Interferon-beta and -gamma increased the levels of HM1.24 antigen and the susceptibility of lung cancer cells to ADCC. Treatment with anti-HM1.24 antibody inhibited the growth of lung cancer cells expressing HM1.24 antigen in SCID mice. The combined therapy with IFN-beta and anti-HM1.24 antibody showed the enhanced antitumor effects even in the delayed treatment schedule. HM1.24 antigen is a novel immunological target for the treatment of lung cancer with anti-HM1.24 antibody.

Chimeric and humanized anti-HM1.24 antibodies mediate antibody-dependent cellular cytotoxicity against lung cancer cells.

HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. The aim of this study was to evaluate the anti-tumor activity of mouse-human chimeric and humanized anti-HM1.24 monoclonal antibodies (mAbs) against lung cancer cells in vitro. Human peripheral blood lymphocytes and monocytes separated from mononuclear cells (PBMCs) were used as effector cells. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of chimeric and humanized anti-HM1.24 mAbs against lung cancer cells were determined by chromium-release assay. In some experiments, target or effector cells were pretreated with various cytokines. Chimeric and humanized anti-HM1.24 mAbs effectively induced ADCC against lung cancer cells mediated more efficiently by lymphocytes than monocytes. The cytotoxic activity correlated with the level of HM1.24 expression on lung cancer cells. Natural killer cells were identified as the major effector cells in ADCC mediated by the anti-HM1.24 mAb. The treatment of lymphocytes or monocytes with IL-2, IL-12, IL-15, M-CSF, or IFN-gamma significantly increased the ADCC activity. Moreover, the culture of lung cancer cells with IFN-beta or IFN-gamma augmented their susceptibility to ADCC and CDC. PBMCs from patients with lung cancer induced a level of ADCC comparable to that induced by PBMCs from healthy donors. Chimeric or humanized anti-HM1.24 mAbs have potential as a new therapeutic tool in lung cancer, and in combination with interleukins and interferons, could be useful for enhancing ADCC.

Effects of a time-varying strong magnetic field on transient increase in Ca2+ release induced by cytosolic Ca2+ in cultured pheochromocytoma cells.

Exposure of pheochromocytoma (PC 12) cells to a time-varying 1.51 T magnetic field inhibited an increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by addition of caffeine to Ca(2+)-free medium. This inhibition occurred after a 15-min exposure and was maintained for at least 2 h. [Ca2+]i sharply increased in cells loaded with cyclic ADP-ribose, and 2-h exposure significantly suppressed the increase. Addition of ATP induced a transient increase in intracellular Ca2+ release mediated by IP3 receptor, and this increase was strongly inhibited by the exposure. Results indicated that the magnetic field exposure strongly inhibited Ca2+ release mediated by both IP3 and ryanodine receptors in PC 12 cells. However, thapsigargin-induced Ca2+ influx (capacitative Ca2+ entry) across the cell membrane was unaffected. The ATP content was maintained at the normal level during the 2-h exposure, suggesting that ATP hydrolysis was unchanged. Therefore, Mg2+ which is known to be released by ATP hydrolysis and inhibit intracellular Ca2+ release may not relate the exposure-caused inhibition. Eddy currents induced in culture medium appear to change cell membrane properties and indirectly inhibit Ca2+ release from endoplasmic reticulum and other Ca2+ stores in PC 12 cells.

Dual effects of endothelin-1 (1-31): induction of mesangial cell migration and facilitation of monocyte recruitment through monocyte chemoattractant protein-1 production by mesangial cells.

We previously found that human chymase selectively cleaves big endothelin-1 (ET-1) at the Tyr31-Gly32 bond and produces 31-amino acid endothelins, ET-1 (1-31), without any further degradation products. In this study, we investigated the effect of ET-1 (1-31) on the migration of cultured rat mesangial cells (RMCs) and on cells of the human monocytic cell line, THP-1. In addition, we examined the interaction between RMCs and THP-1 cells using conditioned media from ET-1 (1-31)-stimulated RMCs. ET-1 (1-31) caused an increase in RMC migration in a concentration-dependent manner, and the degree of increase was similar to those by ET-1 and angiotensin II (All). The ET-1 (1-31)-induced increase in RMC migration was inhibited by BQ123, an endothelin ETA receptor antagonist, but not by BQ788, an endothelin ETB receptor antagonist. ET-1 (1-31) alone did not cause significant migration of THP-1 cells. However, significant recruitment of THP-1 cells was observed with conditioned media taken from ET-1 (1-31)-stimulated RMCs. The conditioned media-induced migration of THP-1 cells was inhibited by BQ123, but not by BQ788. Western blotting analysis of the lysate of RMCs revealed that the expression of monocyte chemoattractant protein-1 (MCP-1) protein in RMCs was increased by treatment with ET-1 (1-31). The addition of neutralizing antibody for MCP-1 to the medium inhibited the migration of THP-1 cells induced by conditioned media from ET-1 (1-31)-stimulated RMCs. These findings suggest that ET-1 (1-31) play a role in glomerulonephritis (GN) via dual effects that directly cause the migration of mesangial cells (MCs) and may be responsible for the recruitment of mononuclear cells mediated through the activation of MCs. Since human chymase has been reported to be involved in glomerular disease, ET-1 (1-31) may be among the mediators.

Improvement of solubility and oral bioavailability of a poorly water-soluble drug, TAS-301, by its melt-adsorption on a porous calcium silicate.

The aim of the present study was to improve the solubility and oral bioavailability of a poorly water-soluble drug, 3-bis(4-methoxyphenyl) methylene-2-indolinone (TAS-301), by its melt-adsorption on a porous calcium silicate, Florite RE (FLR), without any solvents. The melt-adsorbed products were prepared by two methods: the small-scale batch method and the twin screw extruder method. The drug was melted and adsorbed on FLR (i.e., "melt-adsorption"), above its melting point. Crystallinity of the drug in the melt-adsorbed product was estimated by differential scanning calorimetry (DSC) and powder X-ray diffraction analysis. The dissolution test was conducted by the JP XIII paddle method. Oral absorption of the melt-adsorbed product was studied in fasted and fed dogs. The melt-adsorbed products prepared by the two methods were in powder forms. The drug existed in an amorphous state in the product and hardly recrystallized even after storing at a stressed condition (60 degrees C/80% RH for 3 days). The TAS-301 dissolution rate from the melt-adsorbed product was markedly enhanced compared with drug crystals. The area under the plasma concentration-time curve (AUC) and peak concentration (C(max)) values of the drug after dosing the melt-adsorbed product were significantly greater than those after dosing the drug crystals. The solubility and bioavailability of TAS-301 were improved by its melt-adsorption on FLR. The present findings suggest melt-adsorption is a useful technique for improving solubility and bioavailability of poorly water-soluble drugs.

A single institutional subset analysis of the WJLCG study comparing concurrent and sequential chemoradiotherapy for stage III non-small-cell lung cancer.

PURPOSE: To supplement findings of the West Japan Lung Cancer Group (WJLCG) study, treatment outcomes in our institution were reviewed from the perspective of radiation oncology. MATERIALS AND METHODS: Chemotherapy consisted of cisplatin (80 mg/m2 on days 1 and 29), vindesine (3 mg/m2 on days 1, 8, 29, and 36), and mitomycin (8 mg/m2 on days 1 and 29). In the concurrent arm, radiation therapy began on day 2 with a dose of 56 Gy in 28 fractions over 6.8 weeks, with an interval of 10 days at 28 Gy. In the sequential arm, radiation therapy began on day 50 with a dose of 56 Gy in 28 fractions over 5.6 weeks, without an interval. RESULTS: Twenty-four patients in the concurrent arm and 25 patients in the sequential arm in our institution were eligible for the WJLCG study. In the concurrent arm, three patients could not receive the full dose of radiation therapy and 12 patients required interruption of radiation therapy for more than 4 days. The median survival time among per-protocol patients and in those with interruption or with incomplete radiation therapy was 28.9 months and 14.1 months, respectively (p = 0.02). In the sequential arm, one patient could not receive the full dose of radiation therapy and none of the patients required such interruption. Local relapse and distant metastases as the first site of relapse occurred in 12 (11 in-field, 1 marginal) and five patients, respectively, in the concurrent arm, and in eight (7 in-field, 1 marginal) and 11 patients, respectively, in the sequential arm. CONCLUSION: In the concurrent regimen, noncompletion or interruption of radiation therapy was frequent, and the prognosis of such patients was poor.

Inhomogeneity correction in radiotherapy for lung cancer in multicenter clinical trials.

PURPOSE: Dose distribution in patients in past multicenter clinical trials was reviewed from the perspective of clinicians to evaluate the quality of treatment and to improve the quality of future clinical trials. MATERIALS AND METHODS: Thirty patients with stage III lung cancer, who had undergone radical radiation therapy in multicenter clinical trials were retrospectively reviewed. A two-dimensional treatment planning system using Clarkson integration was used to calculate correction factors at the primary lesions and at the mediastinal lymph nodes. RESULTS: Correction factors at the primary lesions ranged from 1.00 to 1.12 (mean, 1.06) and from 1.02 to 1.14 (mean, 1.06) in the AP/PA fields and in the oblique fields, respectively. The lowest values of correction factors at the mediastinal lymph nodes on the axial plane including the primary lesions ranged from 0.93 to 1.04 (mean, 0.98) and from 0.97 to 1.10 (mean, 1.02) in the AP/PA fields and in the oblique fields, respectively. In 14 patients whose primary tumor was located in the upper lung field, the correction factors at the subcarinal lymph nodes ranged from 0.90 to 1.01 (mean, 0.96) in the AP/PA fields. CONCLUSION: Delivered doses lower than those prescribed at the mediastinal lymph nodes should be taken into consideration to improve the quality of multicenter clinical trials.

Serious adverse events and compensation in registration trials: a review of data from a Japanese university hospital.

BACKGROUND: Clinical trials leading to regulatory approval, or registration trials, play a central role in the development of drugs and medical devices. The contribution of support staff, such as the clinical research coordinator (CRC) and administrative officers, in registration trials is now widely recognized. Attending to serious adverse events is an important duty of the CRC and investigators alike, and managing these complications and compensation constitutes a key responsibility. We retrospectively examined the frequency of serious adverse events and compensation events reported from 2007 through 2011 at Tokushima University Hospital, an academic hospital in rural Japan. We present herein the results of our analysis. RESULTS: Over the five-year period, 284 subjects participating in 106 registration trials experienced a total of 43 serious adverse events, and eight compensation events were documented. Among the serious adverse events, 35 (81.4%) were considered not related to the investigational drug, and 17 (39.5%) resulted in withdrawal of the study drug. Patients with malignant diseases experienced serious adverse events significantly more frequently compared to those with non-malignant diseases (28.3% versus 8.2%, respectively; P < 0.01). CONCLUSIONS: The CRC should be vigilant for serious adverse events in oncology clinical trials due to the generally higher frequency of these complications in subjects with malignancy. However, on an individual basis, the CRC may be seldom involved in the process for compensating serious adverse events. Therefore, the CRC's ability to share such experiences may serve as an opportunity for educating clinical trial support staff at the study site as well as those at other sites. However, further study is warranted to determine the role of the clinical trial support staff in optimizing methods for managing adverse events requiring compensation in registration trials.