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Objective: To evaluate the cost-effectiveness of degarelix vs luteinizing hormone-releasing hormone analogue (triptorelin) plus short-term antiandrogen treatment for advanced prostate cancer., Methods: We developed a decision analytic model based on a clinical trial and literature review. The two interventions evaluated were: (i) monthly injection of degarelix and (ii) 3-monthly triptorelin therapy plus short-term flutamide, cyproterone or bicalutamide treatment. The model consisted of a decision tree monitoring a hypothetical cohort of patients aged 70 years from the start of hormonal treatment to the end of the first month, and a Markov model monitoring patients from the end of month 1 for a time horizon of 10 years (i.e. when 96% of patients are assumed to have died). The base-case analysis assumed patients present with asymptomatic metastatic prostate cancer. Costs and outcomes were collected over the model time horizon. Outcome measures were quality-adjusted life years (QALYs), lifetime costs and incremental cost-effectiveness ratios. Sensitivity analyses (one-way and multi-way) and probabilistic sensitivity analyses were conducted to explore the uncertainties around the assumptions., Results: In the base-case analysis, the incremental cost-effectiveness ratio (ICER) for degarelix vs triptorelin plus antiandrogen was £59,000 per QALY gained. The model was most sensitive to the rate of significant adverse events in the triptorelin plus antiandrogen group. The model was also sensitive to the assumed survival of patients with metastatic prostate cancer and the price of degarelix. The results of the probabilistic sensitivity analyses suggested that there was a low probability (9.6%) of degarelix being the most cost-effective treatment option when a willingness-to-pay threshold of £30,000 per QALY gained is assumed., Conclusion: Degarelix is unlikely to be cost-effective compared to triptorelin plus short-term antiandrogen in the management of advanced prostate cancer with respect to the usual thresholds of cost-effectiveness used in the UK: £20,000-30,000 per QALY gained (used by the National Institute for Health and Clinical Excellence)., (© 2011 THE AUTHORS. BJU INTERNATIONAL © 2011 BJU INTERNATIONAL.)

Objective: Vascular smooth muscle cells (VSMC) from type 2 diabetic db/db mice exhibit enhanced proinflammatory responses implicated in accelerated vascular complications. We examined the role of microRNA(miR)-200 family members and their target Zeb1, an E-box binding transcriptional repressor, in these events., Methods and Results: The expression levels of miR-200b, miR-200c, and miR-429 were increased, although protein levels of Zeb1 were decreased in VSMC and aortas from db/db mice relative to control db/+ mice. Transfection of miR-200 mimics into VSMC downregulated Zeb1 by targeting its 3'-UTR, upregulated the inflammatory genes cyclooxygenase-2 and monocyte chemoattractant protein-1, and promoted monocyte binding in db/+VSMC. In contrast, miR-200 inhibitors reversed the enhanced monocyte binding of db/dbVSMC. Zeb1 gene silencing with siRNAs also increased these proinflammatory responses in db/+VSMC confirming negative regulatory role of Zeb1. Both miR-200 mimics and Zeb1 siRNAs increased cyclooxygenase-2 promoter transcriptional activity. Chromatin immunoprecipitation showed that Zeb1 occupancy at inflammatory gene promoters was reduced in VSMC from type 2 diabetic db/db mice. Furthermore, Zeb1 knockdown increased miR-200 levels demonstrating a feedback regulatory loop., Conclusion: Disruption of the reciprocal negative regulatory loop between miR-200 and Zeb1 under diabetic conditions enhances proinflammatory responses of VSMC implicated in vascular complications.

TGF-β1 upregulates microRNA-192 (miR-192) in cultured glomerular mesangial cells and in glomeruli from diabetic mice. miR-192 not only increases collagen expression by targeting the E-box repressors Zeb1/2 but also modulates other renal miRNAs, suggesting that it may be a therapeutic target for diabetic nephropathy. We evaluated the efficacy of a locked nucleic acid (LNA)-modified inhibitor of miR-192, designated LNA-anti-miR-192, in mouse models of diabetic nephropathy. LNA-anti-miR-192 significantly reduced levels of miR-192, but not miR-194, in kidneys of both normal and streptozotocin-induced diabetic mice. In the kidneys of diabetic mice, inhibition of miR-192 significantly increased Zeb1/2 and decreased gene expression of collagen, TGF-β, and fibronectin; immunostaining confirmed the downregulation of these mediators of renal fibrosis. Furthermore, LNA-anti-miR-192 attenuated proteinuria in these diabetic mice. In summary, the specific reduction of renal miR-192 decreases renal fibrosis and improves proteinuria, lending support for the possibility of an anti-miRNA-based translational approach to the treatment of diabetic nephropathy.

CuI/4-hydroxy-L-proline catalyzed coupling of N-substituted o-bromobenzamides with formamide takes place at 80 °C, affording 3-substituted quinazolinones directly. Under these conditions other amides that were tested only provided simple coupling products, which can be converted into 2,3-disubstituted quinazolinones via HMDS/ZnCl(2) mediated condensative cyclization.

Double amination of ortho-substituted aryl bromides proceeded under mild conditions to afford 5-substituted 11-oxo-dibenzodiazepines, which revealed that there is a strong ortho-substituent effect caused by N-aryl aminocarbonyl groups during copper-catalyzed aryl amination.

Enhanced transforming growth factor-β1 (TGF-β1) expression in renal cells promotes fibrosis and hypertrophy during the progression of diabetic nephropathy. The TGF-β1 promoter is positively controlled by the E-box regulators, upstream stimulatory factors (USFs), in response to diabetic (high glucose) conditions; however, it is not clear whether TGF-β1 is autoregulated by itself. As changes in microRNAs (miRNAs) have been implicated in kidney disease, we tested their involvement in this process. TGF-β1 levels were found to be upregulated by microRNA-192 (miR-192) or miR-200b/c in mouse mesangial cells. Amounts of miR-200b/c were increased in glomeruli from type 1 (streptozotocin) and type 2 (db/db) diabetic mice, and in mouse mesangial cells treated with TGF-β1 in vitro. Levels of miR-200b/c were also upregulated by miR-192 in the mesangial cells, suggesting that miR-200b/c are downstream of miR-192. Activity of the TGF-β1 promoter was upregulated by TGF-β1 or miR-192, demonstrating that the miR-192-miR-200 cascade induces TGF-β1 expression. TGF-β1 increased the occupancy of activators USF1 and Tfe3, and decreased that of the repressor Zeb1 on the TGF-β1 promoter E-box binding sites. Inhibitors of miR-192 decreased the expression of miR-200b/c, Col1a2, Col4a1, and TGF-β1 in mouse mesangial cells, and in mouse kidney cortex. Thus, miRNA-regulated circuits may amplify TGF-β1 signaling, accelerating chronic fibrotic diseases such as diabetic nephropathy.

Registration or recognition systems for best-practice health promotion interventions may contribute to better quality assurance and control in health promotion practice. In the Netherlands, such a system has been developed and is being implemented aiming to provide policy makers and professionals with more information on the quality and effectiveness of available health promotion interventions and to promote use of good-practice and evidence-based interventions by health promotion organizations. The quality assessments are supervised by the Netherlands Organization for Public Health and the Environment and the Netherlands Youth Institute and conducted by two committees, one for interventions aimed at youth and one for adults. These committees consist of experts in the fields of research, policy and practice. Four levels of recognition are distinguished inspired by the UK Medical Research Council's evaluation framework for complex interventions to improve health: (i) theoretically sound, (ii) probable effectiveness, (iii) established effectiveness, and (iv) established cost effectiveness. Specific criteria have been set for each level of recognition, except for Level 4 which will be included from 2011. This point of view article describes and discusses the rationale, organization and criteria of this Dutch recognition system and the first experiences with the system.

TGF-β1-induced expression of extracellular matrix (ECM) genes plays a major role in the development of chronic renal diseases such as diabetic nephropathy. Although many key transcription factors are known, mechanisms involving the nuclear chromatin that modulate ECM gene expression remain unclear. Here, we examined the role of epigenetic chromatin marks such as histone H3 lysine methylation (H3Kme) in TGF-β1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-β1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-α1[Ι], and plasminogen activator inhibitor-1. Increased levels of chromatin marks associated with active genes (H3K4me1, H3K4me2, and H3K4me3), and decreased levels of repressive marks (H3K9me2 and H3K9me3) at these gene promoters accompanied these changes in expression. TGF-β1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-β1-induced ECM gene expression. Furthermore, a TGF-β1 antibody not only blocked HG-induced ECM gene expression but also reversed HG-induced changes in promoter H3Kme levels and SET7/9 occupancy. Taken together, these results show the functional role of epigenetic chromatin histone H3Kme in TGF-β1-mediated ECM gene expression in mesangial cells under normal and HG conditions. Pharmacologic and other therapies that reverse these modifications could have potential renoprotective effects for diabetic nephropathy.

Objective: Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice., Research Design and Methods: We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays., Results: We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2., Conclusions: These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

Increased accumulation of extracellular matrix proteins and hypertrophy induced by transforming growth factor-β1 (TGF-β) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy. Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-β increased microRNA-216a (miR-216a) levels in mouse MC, with parallel down-regulation of Ybx1, a miR-216a target and RNA-binding protein. TGF-β also enhanced protein levels of Tsc-22 (TGF-β-stimulated clone 22) and collagen type I α-2 (Col1a2) expression in MC through far upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-β, miR-216a mimics, or Ybx1 shRNA to increase Tsc-22 protein levels but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-β could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-immunoprecipitation assays revealed that TGF-β promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-β-induced Col1a2 in MC related to the pathogenesis of diabetic nephropathy.

Dementia with Lewy bodies (DLB) is characterized clinically by widespread cognitive loss, visual hallucinations, depression, anxiety and extrapyramidal signs (EPS). DLB is sensitive to typical neuroleptics. Repetitive transcranial magnetic stimulation (rTMS) has been studied as a potential treatment in many neurological disorders, which has been proved to have positive effect on a variety of cognitive functions, hallucinations of schizophrenia, major depression, anxiety, the Parkinson's disease. This report proposes that rTMS may represent an alternative strategy for the treatment of dementia with Lewy bodies.

Diabetes is associated with significantly accelerated rates of atherosclerosis, key features of which include the presence of excessive macrophage-derived foam cells in the subendothelial space. We examined the hypothesis that enhanced monocyte-vascular smooth muscle cell (VSMC) interactions leading to subendothelial monocyte retention and differentiation to macrophages under diabetic conditions may be underlying mechanisms. Human aortic VSMCs (HVSMCs) treated with diabetic stimuli high glucose (HG) or S100B, a ligand of the receptor for advanced glycation end products, exhibited significantly increased binding of THP-1 monocytic cells. Diabetic stimuli increased the expression of the adhesive chemokine fractalkine (FKN) in HVSMCs. Pretreatment of HVSMCs with FKN or monocyte chemoattractant protein-1 (MCP-1) neutralizing antibodies significantly inhibited monocyte-VSMC binding, whereas monocytes treated with FKN showed enhanced binding to VSMC. Mouse aortic VSMCs (MVSMCs) derived from type 2 diabetic db/db mice exhibited significantly increased FKN levels and binding to mouse WEHI78/24 monocytic cells relative to nondiabetic control db/+ cells. The enhanced monocyte binding in db/db cells was abolished by both FKN and MCP-1 antibodies. Endothelium-denuded aortas from db/db mice and streptozotocin-induced diabetic mice also exhibited enhanced FKN expression and monocyte binding, relative to respective controls. Coculture with HVSMCs increased CD36 expression in THP-1 cells, and this was significantly augmented by treatment of HVSMCs with S100B or HG. CD36 mRNA and protein levels were also significantly increased in WEHI78/24 cells after coincubation with db/db MVSMCs relative to control MVSMCs. These results demonstrate that diabetic conditions may accelerate atherosclerosis by inducing key chemokines in the vasculature that promote VSMC-monocyte interactions, subendothelial monocyte retention, and differentiation.

Unlabelled: Abnormalities of frontal white matter (WM) have been found in some children with ADHD. The purpose of this study was to explore the changes in WM in child patients with ADHD by DTI, which detects changes in WM microstructure based on properties of diffusion. We also expect to investigate the relationship between the changes in WM and executive function in child patients with ADHD. DTI was performed on 24 patients with ADHD and 20 healthy controls. A series of neuropsychological tests and a structural interview were conducted to assess the cognitive functions and clinical data of the ADHD patients and controls. Firstly, child patients with ADHD have higher fractional anisotropy (FA) values in WM in the right frontal region. Secondly, FA in right frontal WM is positively correlated with scores in the Stroop test., Conclusions: Increased FA of right frontal WM implies a higher degree of myelination and lower degree of neural branching in WM, contributing to the neurological deficits of ADHD.

Akt kinase is activated by transforming growth factor-beta1 (TGF-beta) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells. However, the mechanisms of Akt activation by TGF-beta are not fully understood. Here we show that TGF-beta activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-beta and miR-192 through E-box-regulated mechanisms, as shown previously. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-beta. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-beta. Due to the diversity of PTEN function, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Imaging studies of schizophrenia (SCZ) have repeatedly demonstrated volume differences in superior temporal gyrus (STG) and its subregions. Among them, some studies employed the Region of Interest (ROI) method. We carried out a systematic review of the published literature on STG volumetry MRI studies to examine the potential of ROI method for identifying specific structural differences and correlations with clinical variables including hallucinations and thought disorder symptoms in SCZ. Forty-six studies were identified as suitable for review and analysis including 1444 patients with SCZ and 1327 controls. Female and left-handed subjects are under-represented in the literature and insight from sex and handedness differences may be lost. Thirty-five studies reported significant differences in STG or subregional volumes including bilateral or unilateral ROI, and volume reduction was the most common change in SCZ. Thirty studies reported correlations between volume changes and clinical symptoms or syndromes and 18 found positive results. Among them, left STG or subregions appear to be more involved in the generation of hallucinations and thought disorder than right side. The majority of five follow-up studies found evidence of progressive changes in volumes. Clinical heterogeneity, MRI acquisition parameters, anatomical landmarks for ROI, and sample characteristics, are likely to be the main factors leading to heterogeneous results. Clearly this research links pathophysiological changes in the STG with the development of hallucinations and thought disorder in patients with SCZ, especially in the left side. There is a suggestion that these changes may be progressive but this requires more thorough and comprehensive assessment.

Objective: The 12/15-Lipoxygenase (12/15-LO) and its metabolite 12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] mediate proatherogenic responses in vascular smooth muscle cells (VSMCs). We examined the role of the nonreceptor tyrosine kinase Src in the signaling and epigenetic chromatin mechanisms involved in these processes., Methods and Results: Rat VSMCs (RVSMCs) were stimulated with 12(S)-HETE (0.1 micromol/L) in the presence or absence of the Src inhibitor PP2 (10 micromol/L). Src activation and downstream signaling events including inflammatory gene expression and chromatin histone H3-Lys-9/14 acetylation were examined by immunoblotting, RT-PCR, and chromatin immunoprecipitation assays, respectively. 12(S)-HETE significantly activated Src, focal adhesion kinase, Akt, p38MAPK, and CREB. Expression of monocyte chemoattractant protein-1 and interleukin-6 genes and histone H3-Lys-9/14 acetylation on their promoters were also increased by 12(S)-HETE. PP2 inhibited these responses as well as 12(S)-HETE-induced VSMC migration. Furthermore, dominant negative mutants of Src, CREB, and a histone acetyltransferase p300 significantly blocked 12(S)-HETE-induced inflammatory gene expression. In addition, growth factor induced Src signaling and downstream events including H3-Lys-9/14 acetylation and migration were significantly attenuated in VSMCs derived from 12/15-LO(-/-) mice relative to WT., Conclusions: Src kinase signaling plays a central role in the proatherogenic responses mediated by 12/15-LO and its oxidized lipid metabolite 12(S)-HETE in VSMCs.

Insulin resistance and type 2 diabetes are major risk factors for vascular complications. Vascular smooth muscle cells (VSMCs) derived from db/db mice, an established mouse model of type 2 diabetes, displayed enhanced inflammatory gene expression and proatherogenic responses. We examined the hypothesis that aberrant epigenetic chromatin events may the underlying mechanism for this persistent dysfunctional behavior and "memory" of the diabetic cells. Chromatin immunoprecipitation assays showed that levels of histone H3 lysine 4 dimethylation (H3K4me2), a key chromatin mark associated with active gene expression, were significantly elevated at the promoters of the inflammatory genes monocyte chemoattractant protein-1 and interleukin-6 in db/db VSMCs relative to db/+ cells. Tumor necrosis factor-alpha-induced inflammatory gene expression, H3K4me2 levels, and recruitment of RNA polymerase II at the gene promoters were also enhanced in db/db VSMCs, demonstrating the formation of open chromatin poised for transcriptional activation in diabetes. On the other hand, protein levels of lysine-specific demethylase1 (LSD1), which negatively regulates H3K4 methylation and its occupancy at these gene promoters, were significantly reduced in db/db VSMCs. High glucose (25 mmol/L) treatment of human VSMCs also increased inflammatory genes with parallel increases in promoter H3K4me2 levels and reduced LSD1 recruitment. LSD1 gene silencing with small interfering RNAs significantly increased inflammatory gene expression and enhanced VSMC-monocyte binding in nondiabetic VSMCs. In contrast, overexpression of LSD1 in diabetic db/db VSMCs inhibited their enhanced inflammatory gene expression. These results demonstrate novel functional roles for LSD1 and H3K4 methylation in VSMCs and inflammation. Dysregulation of their actions may be a major mechanism for vascular inflammation and metabolic memory associated with diabetic complications.

We previously showed that the 12/15-lipoxygenase (12/15-LO) pathway of arachidonate acid metabolism is involved in multiple events related to diabetic nephropathy (DN), including glomerular hypertrophy and extracellular matrix deposition (Kang SW, Adler SG, Nast CC, LaPage J, Gu JL, Nadler JL, Natarajan R. Kidney Int 59: 1354-1362, 2001; Kang SW, Natarajan R, Shahed A, Nast CC, LaPage J, Mundel P, Kashtan C, Adler SG. J Am Soc Nephrol 14: 3178-3187, 2003; Kim YS, Lanting L, Adler SG, Natarajan R. Kindney Int 64: 1702-1714, 2003; Reddy MA, Adler SG, Kim YS, Lanting L, Rossi JJ, Kang SW, Nadler JL, Shahed A, Natarajan R. Am J Physiol Renal Physiol 283: F985-F994, 2002). In this study, we investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting 12/15-LO can ameliorate renal injury and DN in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, we used double-stranded 12/15-LO siRNA oligonucleotides conjugated with cholesterol. Diabetic DBA/2J mice were injected subcutaneously with either cholesterol-tagged 12/15-LO siRNA, mismatched control siRNA, or vehicle alone, twice weekly for 7 wk. Relative to controls, mice that received 12/15-LO siRNA showed significant reduction in albuminuria, kidney-to-body weight ratios, glomerular mesangial matrix expansion, renal structural damage, and monocyte/macrophage infiltration. These effects were associated with lower renal cortical or glomerular levels of profibrotic markers transforming growth factor-beta, connective tissue growth factor, type I and type IV collagens, plasminogen activator inhibitor 1, and fibronectin. The diabetes-induced increase in glomerular cyclin-dependent kinase inhibitors that are associated with hypertrophy was also prevented by siRNA administration. Our results show for the first time that systemic delivery of cholesterol-tagged siRNAs targeting 12/15-LO has renoprotective effects under diabetic conditions and therefore could be a novel therapeutic approach for DN.

Objective: Ethnic differences in outcomes of outpatient diabetic care and the role of self-management behavior and its determinants in explaining observed differences., Methods: Face-to-face interviews were held with 102 Turkish or Moroccan, and 102 native Dutch diabetic patients to measure self-management behavior and determinants of self-management (as derived from the Attitudes-Social support self-Efficacy model, and Personal Models and Barriers). A medical record review was conducted to measure ethnic differences in outcomes of diabetes care. Data were analyzed using multiple linear regression., Results: Outcomes differed significantly with ethnic minorities having higher levels of lipids (risk difference=RD=0.7%; CI: 0.3-1.2) and HbA1c (RD=0.9%; CI: 0.4-1.4) than native Dutch patients. Differences in self-management could not explain the ethnic differences in outcomes. Self-efficacy explained 18% of the ethnic differences in HbA1c. Beliefs about seriousness of diabetes and social support regarding diabetes management together explained 47% of the ethnic differences in lipids., Conclusion: This study provides evidence for ethnic differences in outcomes of diabetes care. Self-efficacy is the most important determinant in explaining the differences in HbA1c., Practice Implications: For diabetes practice this suggests that strengthening patients' self-efficacy may improve the control of HbA1c and may result in a decrease of ethnic differences. The relationship between behavioral determinants like seriousness and social support and outcomes of diabetes care was differential by ethnic group, implying that caution is required when applying behavioral models to different ethnic groups.

Angiotensin II and its type 1 receptor (AT1R) play important roles in the pathogenesis of renal disease and diabetic nephropathy. The 12/15-lipoxygenase pathway of arachidonate metabolism and its lipid products have also been implicated in diabetic nephropathy. However, it is unclear whether 12/15-lipoxygenase regulates expression of AT1R. In cultured rat mesangial cells, we found that the 12/15-lipoxygenase product 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) increased AT1R mRNA and protein expression, primarily by stabilizing AT1R mRNA. Pretreatment with 12(S)-HETE also amplified the signaling effects of angiotensin II, likely due to the increased AT1R expression. Levels of AT1R protein expression decreased when 12/15-lipoxygenase was knocked down with specific short hairpin RNA (shRNA) compared with control cells. Similarly, levels of the AT1 receptor, but not the AT2 receptor, were significantly lower in mesangial cells and glomeruli derived from 12/15-lipoxygenase knockout mice compared with control mice. Reciprocally, stable overexpression of 12/15-lipoxygenase increased AT1R expression in cultured mesangial cells. In vivo, modified siRNA targeting 12/15-lipoxygenase reduced glomerular AT1R expression in a diabetic mouse model. Interestingly, angiotensin II induced greater levels of 12/15-lipoxygenase, TGF-beta1, and fibronectin (FN) in AT1R-overexpressing mesangial cells compared with control cells. Therefore, oxidized lipids generated by the 12/15-lipoxygenase-mediated metabolism of arachidonic acid can enhance AT1R expression in mesangial cells and augment the profibrotic effects of angiotensin II.

Increased expression and activity of 12/15-lipoxygenase (12/15-LO) in vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetes and vascular complications. However, the consequences of 12/15-LO overexpression for VSMC migration and inflammatory gene expression are not known. In this study, 12/15-LO was overexpressed using adeno- and baculoviral vectors in human VSMC (HVSMCs) and proatherogenic responses compared with control enhanced green fluorescent protein (EGFP)-expressing cells. HVSMCs transduced with 12/15-LO viruses expressed high levels of enzymatically active protein and produced increased levels of the LO product, 12(S)-hydroxyeicosatetraenoic acid. 12/15-LO-overexpressing HVSMCs exhibited increased oxidant stress, activation of p38 mitogen-activated protein kinase, migration and inflammatory gene expression relative to HVSMCs expressing EGFP. Furthermore, inflammatory gene expression induced by 12/15-LO overexpression was abolished by anti-oxidants, siRNAs targeting p65 (nuclear factor-kappaB), or new-generation baculoviruses expressing inhibitory IkappaBalpha or IkappaBalpha superrepressor mutant. Thus, we have used novel viral vector delivery systems, including baculoviruses, for the first time to deliver foreign genes into VSMCs and thereby demonstrated that 12/15-LO overexpression increases oxidant stress, mitogen-activated protein kinase activation, migration and inflammatory genes in VSMCs and that NF-kappaB is a key downstream effector. Enhanced proatherogenic responses in VSMCs triggered by increased 12/15-LO levels under pathological conditions may contribute to vascular dysfunction., (Copyright (c) 2007 S. Karger AG, Basel.)

Diabetic nephropathy (DN) remains a major complication in both type 1 and type 2 diabetes. Systemic administration of antitransforming growth factor-beta (TGF-beta) antibody has shown some promise in mouse models of DN. However, chronic blockade of the multifunctional TGB-beta could be problematic. Several downstream effects of TGF-beta are mediated by connective tissue growth factor (CTGF), which is up-regulated in several renal cells and secreted in the urine in the diabetic state. Using murine models of DN (type 1 and type 2) and a CTGF antisense oligonucleotide (ASO) of novel chimeric chemistry, we evaluated the specific role of this target in DN. In the type 1 model of DN, C57BL6 mice were made diabetic using streptozotocin injections and hyperglycemic animals were treated with CTGF ASOs (20 mg/kg/2 qw) for 4 months. ASO, but not mismatch control oligonucleotide, -treated animals showed significant reduction in target CTGF expression in the kidney with a concomitant decrease in proteinuria and albuminuria. Treatment with the CTGF ASO for 8 wk reduced serum creatinine and attenuated urinary albuminuria and proteinuria in diabetic db/db mice, a model of type 2 DN. The ASO also reduced expression of genes involved in matrix expansion such as fibronectin and collagen (I and IV) and an inhibitor of matrix degradation, PAI-1, in the renal cortex, contributing to significant reversal of mesangial expansion in both models of DN. Pathway analyses demonstrated that diabetes-induced phosphorylation of p38 MAPK and its downstream target CREB was also inhibited by the ASO. Our results strongly suggest that blocking CTGF using a chimeric ASO holds substantial promise for the treatment of DN.

Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-alpha 1 and -2 (Col1a1 and -2). Transforming growth factor beta1 (TGF-beta), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-beta increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, deltaEF1. TGF-beta treatment or short hairpin RNAs targeting deltaEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-beta also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to deltaEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-beta in MMC. TGF-beta treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3' UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with deltaEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-beta and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-beta-induced Col1a2 expression by down-regulating E-box repressors.

Diabetic nephropathy (DN) is characterized by mesangial cell (MC) expansion and accumulation of extracellular matrix proteins. TGF-beta is increased in MC under diabetic conditions and in DN and activates key signaling pathways, including the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. FoxO transcription factors play roles in cell survival and oxidative stress and are negatively regulated by Akt-mediated phosphorylation. We tested whether phosphorylation-mediated inactivation of FoxO3a by TGF-beta can mediate MC survival and oxidative stress. TGF-beta treatment significantly increased levels of p-Akt (activation) and p-FoxO3a (inactivation) in cultured MC. This FoxO3a inactivation was accompanied by significant decreases in the expression of two key FoxO3a target genes, the proapoptotic Bim and antioxidant manganese superoxide dismutase in MC. TGF-beta treatment triggered the nuclear exclusion of FoxO3a, significantly inhibited FoxO3a transcriptional activity, and markedly protected MC from apoptosis. A PI3K inhibitor blocked these TGF-beta effects. It is interesting that p-Akt and p-FoxO3A levels also were increased in renal cortical tissues from rats and mice at 2 wk after the induction of diabetes by streptozotocin, thus demonstrating in vivo significance. In summary, TGF-beta and diabetes can increase FoxO3a phosphorylation and transcriptional inactivation via PI3K/Akt. These new results suggest that Akt/FoxO pathway regulation may be a novel mechanism by which TGF-beta can induce unopposed MC survival and oxidant stress in early DN, thereby accelerating renal disease.

Diabetes is associated with enhanced inflammatory responses and cardiovascular complications such as atherosclerosis. However, it is unclear whether similar responses are present in cells derived from experimental animal models of diabetes. We examined our hypothesis that macrophages and short-term cultured vascular smooth muscle cells (VSMCs) derived from obese, insulin-resistant, and diabetic db/db mice would exhibit increased proatherogenic responses relative to those from control db/+ mice. We observed that macrophages from db/db mice exhibit significantly increased expression of key inflammatory cytokines and chemokines as well as arachidonic acid-metabolizing enzymes cyclooxygenase-2 and 12/15-lipoxygenase that generate inflammatory lipids. Furthermore, VSMCs derived from db/db mice also showed similar enhanced expression of inflammatory genes. Expression of inflammatory genes was also significantly increased in aortas derived from db/db mice. Both macrophages and VSMCs from db/db mice demonstrated significantly increased oxidant stress, activation of key signaling kinases, and transcription factors cAMP response element-binding protein and nuclear factor-kappaB, involved in the regulation of atherogenic and inflammatory genes. Interestingly, VSMCs from db/db mice displayed enhanced migration as well as adhesion to WEHI mouse monocytes relative to db/+. Thus, the diabetic milieu and a potential hyperglycemic memory can induce aberrant behavior of vascular cells. These new results demonstrate that monocyte/macrophages and VSMCs derived from db/db mice display a "preactivated" and proinflammatory phenotype associated with the pathogenesis of diabetic vascular dysfunction and atherosclerosis.

Coactivator-associated arginine methyltransferase-1 (CARM1) is known to enhance transcriptional activation by nuclear receptors through interactions with the coactivators p160 and cAMP response element binding protein-binding protein (CBP) and methylation of histone H3 at arginine 17 (H3-R17). Here, we show that CARM1 can act as a coactivator for the transcription factor nuclear factor-kappaB (NF-kappaB) and enhance NF-kappaB activity in a CBP (p300)-dependent manner. This enhancement in 293T cells was abolished by cotransfection with a specific short hairpin RNA targeted to knockdown CARM1. Chromatin immunoprecipitation demonstrated CARM1 recruitment in vivo to the promoters of NF-kappaB p65-regulated genes along with CBP and steroid receptor coactivator-1. This was accompanied by an increase in histone H3-R17 methylation as well as H3-K9 and H3-K14 acetylation, and a decrease in H3-citrulline. Immunoprecipitation with anti-p65 antibody revealed that CARM1 physically interacts with NF-kappaB p65. Furthermore, we demonstrated the physiological significance by observing that similar events occurred when THP-1 monocytic cells were stimulated with TNF-alpha or with S100b, a ligand for the receptor of advanced glycation end products, both of which are associated with diabetic complications and also known inducers of NF-kappaB and inflammatory genes in monocytes. These results demonstrate that CARM1 participates in NF-kappaB-mediated transcription through H3-R17 methylation and support a nonnuclear receptor-associated function for CARM1. They also demonstrate for the first time that CARM1 occupancy, histone H3-R17 methylation, and citrullination are regulated at the promoters of inflammatory genes in monocytes, thereby suggesting a novel role for histone arginine modifications in inflammatory diseases.

The receptor for advanced glycation end products (RAGE) and its ligands have been implicated in the activation of oxidant stress and inflammatory pathways in vascular smooth muscle cells (VSMCs) leading to the initiation and augmentation of atherosclerosis. Here we report that non-receptor Src tyrosine kinase and the membrane protein caveolin-1 (Cav-1) play a key role in the activation of RAGE by S100B in VSMCs. S100B increased the activation of Src kinase and tyrosine phosphorylation of caveolin-1 in VSMCs. A RAGE-specific antibody blocked both these effects. An inhibitor of Src kinase, PP2, significantly blocked S100B-induced activation of Src kinase, mitogen-activated protein kinases, transcription factors NF-kappaB and STAT3, superoxide production, tyrosine phosphorylation of Cav-1, VSMC migration, and expression of the pro-inflammatory genes monocyte chemotactic protein-1 and interleukin-6. Cholesterol depletion also inhibited S100B-induced effects indicating the requirement for intact caveolae in RAGE-specific signaling. Nucleofection of either a Src dominant negative mutant, or a Cav-1 mutant lacking the scaffolding domain, or Cav-1 short hairpin RNA significantly reduced S100B-induced inflammatory gene expression in VSMCs. Furthermore, VSMCs derived from insulin-resistant and diabetic db/db mice displayed increased RAGE expression, Src activation, and migration compared with those from control db/+ mice. The RAGE antibody blocked enhanced migration in db/db cells. These studies demonstrate for the first time that, in VSMCs, Src kinase and Cav-1 play important roles in RAGE-mediated inflammatory gene expression and migration, key events associated with diabetic vascular complications.

The 12/15-lipoxygenase (12/15-LO) and cyclooxygenase-2 (COX-2) pathways of arachidonate metabolism have been implicated in the pathogenesis of diabetic nephropathy (DN). In this study, we evaluated whether there is an interplay between 12/15-LO and COX-2 pathways in mesangial cells (MC). We utilized MC, microdissected glomeruli and renal cortical tissues. Transfections with cDNAs or short hairpin RNAs (shRNAs) were performed to overexpress or knockdown 12/15-LO and COX-2, respectively. Reverse transcription-polymerase chain reactions and Western blotting were used for evaluating mRNA and protein expression, respectively. We observed that the expression of both 12/15-LO and COX-2 were increased in high glucose stimulated rat MC relative to normal glucose, and also in cortical tissues from diabetic db/db and streptozotocin-injected mice relative to corresponding control mice. Treatment of rat MC with the 12/15-LO product, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), significantly increased COX-2 expression as well as levels of the COX-2 product, prostaglandin E(2) (PGE(2)). Interestingly, treatment of rat MC with PGE(2) led to a reciprocal increase in 12/15-LO expression as well as levels of 12(S)-HETE. The 12/15-LO shRNA could significantly attenuate COX-2 protein expression and vice versa. Furthermore, COX-2 expression levels were lower in MC and glomeruli from 12/15-LO knockout mice relative to control. Conversely, mouse MC stably overexpressing 12/15-LO had greater levels of COX-2 expression relative to mock-transfected cells. These new results indicate for the first time that 12/15-LO and COX-2 pathways can cross-talk and activate each other in MC. These novel interactions may amplify their effects on the progression of DN.

Vascular smooth muscle cells (VSMCs) express functional interleukin-18 receptors (IL-18Rs), composed of alpha and beta subunits. These subunits are elevated in VSMCs of atherosclerotic plaques and can be induced by inflammatory agents in cultured VSMC. Because both IL-18 and Angiotensin II (Ang II) are implicated in atherosclerosis, our objective was to analyze the role of IL-18 signaling and potential cross-talk with Ang II in VSMC. We observed that IL-18 activated Src kinase, protein kinase C, p38 and JNK MAPKs, Akt kinase, transcription factors NF-kB and AP-1, and induced expression of pro-inflammatory cytokines in VSMC. Pretreatment of VSMC with Ang II enhanced IL-18-induced NF-kB activation and cytokine gene expression. Interestingly, Ang II directly increased mRNA and cell surface protein levels of the IL-18Ralpha subunit. Functional relevance in an organ culture model was demonstrated by the observation that incubation of intact mouse aortas ex vivo with Ang II also significantly increased IL-18Ralpha expression. Furthermore, Ang II significantly stimulated transcription from a minimal IL-18Ralpha promoter containing putative binding sites for STAT and AP-1. Ang II also increased in vivo recruitment of STAT-3 on the IL-18Ralpha promoter. Finally, dominant negative STAT-3 mutant blocked Ang II-induced IL-18Ralpha promoter activation in CHO cells overexpressing AT1a receptor and IL-18Ralpha mRNA expression in HVSMC. Thus, Ang II enhances IL-18 induced inflammatory genes by increasing IL-18Ralpha expression. These results illustrate a novel mechanism wherein Ang II- mediated increases in inflammatory genes and proatherogenic effects in the vasculature are enhanced by a vicious loop and cross-talk with the IL-18 signaling pathway.

Background: Severe obesity can result in proteinuria and progressive glomerulosclerosis in humans and experimental animals. The associated renal disease is ameliorated by weight reduction and/or blockade of the renin-angiotensin system. Various growth factors, cytokines, and lipid mediators are implicated in the pathogenesis of renal disease. To explore the possible involvement of these mediators in obesity-induced renal disease, we examined the expression of key enzymes of arachidonate metabolism and inflammatory genes in untreated and losartan-treated obese Zucker rats, a model of obesity, insulin resistance, and renal injury., Methods and Results: Seven-week-old male obese Zucker rats were randomized to losartan-treated (100 mg/L drinking H2O) and untreated groups, with lean Zucker rats as controls. After 4 months, RNA and protein were obtained from renal cortical tissue for relative reverse transcription-polymerase chain reaction, Western blots, and immunohistochemistry. Compared with the lean controls, obese Zucker rats showed significant glomerular matrix expansion and increased mRNA expression of the extracellular matrix protein fibronectin, inflammatory mediators interleukin-6 and monocyte chemoattractant protein-1, and 2 major enzymes of arachidonate metabolism, namely, 12/15-lipoxygenase and cyclooxygenase-2. This was associated with significant increases in p38 and extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase activities and marked upregulation of angiotensin II type 1 receptor (AT1R) mRNA and protein expression. These abnormalities and the associated glomerulopathy and proteinuria were prevented by administration of the AT1R blocker losartan., Conclusions: These findings indicate that obesity-induced glomerulopathy is associated with upregulation of key inflammatory mediators. These events are associated with and perhaps in part due to upregulation of AT1R, as evidenced by their reversal with AT1R blocker treatment.

It is the intent of this paper to locate the activation point in Transcranial Magnetic Stimulation (TMS) efficiently. The schemes of coil array in torus shape is presented to get the electromagnetic field distribution with ideal focusing capability. Then an improved adaptive genetic algorithm (AGA) is applied to the optimization of both value and phase of the current infused in each coil. Based on the calculated results of the optimized current configurations, ideal focusing capability is drawn as contour lines and 3-D mesh charts of magnitude of both magnetic and electric field within the calculation area. It is shown that the coil array has good capability to establish focused shape of electromagnetic distribution. In addition, it is also demonstrated that the coil array has the capability to focus on two or more targets simultaneously.

The leukocyte-type 12/15-lipoxygenase (12/15-LO) has been implicated in the pathogenesis of atherosclerosis, hypertension, and diabetes. 12/15-LO and its products are associated with LDL oxidation, cellular growth, migration, adhesion, and inflammatory gene expression in monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs). Our objective, therefore, was to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance in macrophages and VSMCs. We used a PCR-based approach to rapidly identify effective siRNA target sites on mouse 12/15-LO and initially tested their efficacy on a fusion construct of 12/15-LO cDNA and enhanced green fluorescent protein. We then cloned these U6 promoter+siRNA PCR products into plasmid vectors [short hairpin siRNAs (shRNAs)] to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMCs. Furthermore, the functional effects of shRNA-mediated 12/15-LO knockdown were noted by the reduced oxidant stress and chemokine [monocyte chemoattractant protein-1 (MCP-1)] expression in a differentiated mouse monocytic cell line as well as by the reduced cellular adhesion and fibronectin expression in VMSCs. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, vascular cell adhesion molecule-1, and interleukin-6 in VSMCs. Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMCs and its relationship to oxidant stress and inflammation.

Diabetic nephropathy (DN) is characterized by mesangial cell (MC) hypertrophy and progressive accumulation of glomerular extracellular matrix (ECM). It was reported recently that 12/15-lipoxygenase (12/15-LO) expression is increased in high-glucose (HG)-stimulated MC and in experimental DN. 12-LO products could also directly induce MC hypertrophy and ECM expression and mediate growth factor effects, thus implicating the 12/15-LO pathway in DN. Because TGF-beta is a major player in the pathogenesis of DN, whether there is an interplay between the TGF-beta and 12/15-LO pathways in MC was evaluated. Treatment of rat MC (RMC) with TGF-beta significantly increased levels of the 12/15-LO product 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and also 12/15-LO mRNA and protein expression. HG-induced TGF-beta mRNA expression in RMC was inhibited by a specific ribozyme and siRNA targeted to knockdown rat 12/15-LO. It is interesting that direct treatment of RMC with 12(S)-HETE increased TGF-beta mRNA and protein levels, as well as p-Smad2/3, which are TGF-beta-specific target transcription factors. 12(S)-HETE also increased transcription from a minimal TGF-beta promoter. Furthermore, TGF-beta expression and p-Smad2/3 levels were lower in MC from 12/15-LO knockout mice relative to control mice. Reciprocally, mouse MC stably overexpressing 12/15-LO had greater TGF-beta mRNA and also nuclear p-Smad2/3 relative to mock-transfected cells. 12/15-LO and TGF-beta could functionally signal and increase ECM expression via the p38 mitogen-activated protein kinase signaling pathway. These results indicate for the first time that the 12/15-LO and TGF-beta pathways can cross-talk and activate each other. These novel interactions may amplify the signal transduction cascades and molecular events that lead to DN.

The 12/15-lipoxygenase (12/15-LO) pathway is activated in diabetes mellitus (DM), increasing 12(S)-hydroxyeicosatetraenoic acid (12-HETE). We showed that a 12-LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC) inhibited 12/15-LO activity in vivo and assessed the efficacy of another 12/15-LO inhibitor, N-benzyl-N-hydroxy-5-phenylpentamidine (BHPP), to diminish urinary 12-HETE and ameliorate diabetic nephropathy (DN) over 4 months. Rats studied were control (C, n=8), DM (n=6), and rats injected with BHPP (C+BHPP, n=4) and (DM+BHPP, n=5). BHPP 3 mg/kg/day decreased urinary (U) 12-HETE/creatinine (cr) by 30-50% after one injection and after 1 week of daily injections in DM rats. U 12-HETE/cr excretion increased paradoxically in controls given BHPP. There was a highly significant relationship between U 12-HETE/cr excretion and U alb/cr (r=0.79, P<10(-5)), demonstrating that renal 12/15-LO pathway activation is associated with albuminuria. BHPP did not inhibit glomerular collagen synthesis or improve histology. More sustained 12-LO inhibition may improve albuminuria in DN.

Objective: Vascular smooth muscle cells (VSMCs) may regulate monocyte functions within atherosclerotic lesions. We investigated the impact of VSMC/monocyte interactions on monocyte apoptosis and scavenger receptor CD36 expression, key events related to monocyte survival and differentiation., Methods and Results: Serum deprivation significantly increased THP-1 and human peripheral blood monocyte apoptosis. However, this was significantly reversed by physical binding to human VSMCs (HVSMCs). On binding to HVSMCs, antiapoptotic kinase Akt and its downstream targets were phosphorylated, and Bcl-2 expression was enhanced. Binding-mediated suppression of apoptosis and Akt phosphorylation were attenuated by a phosphoinositide 3-kinase inhibitor and also by an antibody to vascular cell adhesion molecule-1. CD36 expression was also significantly increased in THP-1 cells and in human peripheral blood monocytes after binding to HVSMCs, and this was mediated by both direct contact and soluble factors. Extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase phosphorylation was increased in THP-1 cells after HVSMC coculture. Furthermore, an ERK1/2 inhibitor blocked monocyte CD36 upregulation. Contact-dependent CD36 induction and ERK1/2 phosphorylation in monocytes were inhibited by blocking vascular cell adhesion molecule-1 on HVSMC, whereas soluble factor-induced CD36 expression was attenuated by a monocyte chemoattractant protein-1 neutralizing antibody., Conclusions: These data provide evidence of novel VSMC-dependent local regulation mechanisms for monocyte survival and differentiation in atherosclerosis.

Gemcitabine is a commonly used chemotherapy for biliary tree carcinomas, achieving response rates of 10% to 60%. Preclinical studies indicate that fixed dose rate infusion optimizes accumulation of gemcitabine triphosphate and may enhance the clinical activity of gemcitabine. We conducted a phase II study of fixed dose rate gemcitabine in 15 chemotherapy-naive patients with advanced cholangiocarcinoma and gallbladder carcinoma. Gemcitabine was administered at a dose of 1500 mg/m2 over 150 minutes weekly for 3 weeks every 28 days. Fourteen patients were evaluable for response. No complete or partial responses were observed. Two patients (13%) had stable disease lasting a median of 9 weeks. The median time to progression was 9 weeks; median survival was 20 weeks. There was considerable grade 3/4 hematologic toxicity, including neutropenia in 49% of patients, leukopenia in 40%, anemia in 27%, and thrombocytopenia in 27%. Grade 3/4 nonhematologic toxicities were minimal. We conclude that fixed dose rate gemcitabine results in significant myelosuppression and has minimal activity in patients with biliary tree carcinoma.

Adhesive interactions between monocytes and vascular smooth muscle cells (VSMC) may contribute to subendothelial monocyte-macrophage retention in atherosclerosis. We investigated the effects of angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB on VSMC-monocyte interactions. Treatment of human aortic VSMC (HVSMC) with ANG II or PDGF-BB significantly increased binding to human monocytic THP-1 cells and to peripheral blood monocytes. This was inhibited by antibodies to monocyte beta(1)- and beta(2)-integrins. The binding was also attenuated by blocking VSMC arachidonic acid (AA) metabolism by inhibitors of 12/15-lipoxygenase (12/15-LO) or cyclooxygenase-2 (COX-2). Conversely, binding was enhanced by overexpression of 12/15-LO or COX-2. Direct treatment of HVSMC with AA or its metabolites also increased binding. Furthermore, VSMC derived from 12/15-LO knockout mice displayed reduced binding to mouse monocytic cells relative to genetic control mice. Using specific signal transduction inhibitors, we demonstrated the involvement of Src, phosphoinositide 3-kinase, and MAPKs in ANG II- or PDGF-BB-induced binding. Interestingly, after coculture with HVSMC, THP-1 cell surface expression of the scavenger receptor CD36 was increased. These results show for the first time that growth factors may play additional roles in atherosclerosis by increasing monocyte binding to VSMC via AA metabolism and key signaling pathways. This can lead to monocyte subendothelial retention, CD36 expression, and foam cell formation.

Terminal deoxynucleotidyl transferase (TdT) is a unique intranuclear DNA polymerase that catalyzes the template-independent addition of deoxynucleotides to the 3'-hydroxyl terminus of oligonucleotide primers. The expression of TdT is restricted to lymphoid precursors. It is a useful marker in distinguishing acute lymphoblastic leukemia (ALL)from mature lymphoid neoplasms. Although TdT- T-cell ALL has been reported in the literature rarely, the frequency and significance of TdT-nonpositive (TdT(np) B-cell ALL have not been examined extensively. We reviewed the immunophenotypes of 186 new cases of pediatric B-cell ALL and found 5 TdT(np) cases (2.7%). They showed significantly higher frequencies of a WBC count of more than 50,000/microL (> 50.0 x 10(9)/L), CD10-, CD34-, and MLL gene rearrangement compared with those in TdT+ cases (3/5 [60%] vs 27/181 [14.9%], P = .03; 3/5 [60%] vs 11/181 [6.1%], P = .003; 4/5 [80%] vs 24/179 [13.4%], P = .002; 3/5 [60%] vs 9/181 [5.0%], P = .0019; respectively). These results indicate that nonpositive TdT does not rule out a diagnosis of ALL and suggest that TdT(np) B-cell ALL might be associated with CD10- and CD34- disease, a high WBC count, and MLL gene rearrangement.

The transcription factor NF-kappaB (NF-kappaB) plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators including histone acetyltransferases (HATs) such as CBP/p300 and p/CAF. Evidence shows that high glucose (HG) conditions mimicking diabetes can activate the transcription of NF-kappaB-regulated inflammatory genes. However, the underlying in vivo transcription and nuclear chromatin remodeling events are unknown. We therefore carried out chromatin immunoprecipitation (ChIP) assays in monocytes to identify 1) chromatin factors bound to the promoters of tumor necrosis factor-alpha (TNF-alpha) and related NF-kappaB-regulated genes under HG or diabetic conditions, 2) specific lysine (Lys (K)) residues on histone H3 (HH3) and HH4 acetylated in this process. HG treatment of THP-1 monocytes increased the transcriptional activity of NF-kappaB p65, which was augmented by CBP/p300 and p/CAF. ChIP assays showed that HG increased the recruitment of NF-kappaB p65, CPB, and p/CAF to the TNF-alpha and COX-2 promoters. Interestingly, ChIP assays also demonstrated concomitant acetylation of HH3 at Lys(9) and Lys(14), and HH4 at Lys(5), Lys(8), and Lys(12) at the TNF-alpha and COX-2 promoters. Overexpression of histone deacetylase (HDAC) isoforms inhibited p65-mediated TNF-alpha transcription. In contrast, a HDAC inhibitor stimulated gene transcription and histone acetylation. Finally, we demonstrated increased HH3 acetylation at TNF-alpha and COX-2 promoters in human blood monocytes from type 1 and type 2 diabetic subjects relative to nondiabetic. These results show for the first time that diabetic conditions can increase in vivo recruitment of NF-kappaB and HATs, as well as histone acetylation at the promoters of inflammatory genes, leading to chromatin remodeling and transcription.

Background: The 12/15-lipoxygenase (12/15-LO) enzyme has been implicated in the pathogenesis of diabetic nephropathy since lipoxygenase products induce cellular hypertrophy and extracellular matrix deposition in mesangial cells. In this study, in order to determine the potential in vivo functional role of 12/15-LO in kidney disease, we compared mouse mesangial cells (MMCs) derived from 12/15-LO knockout mice with those from genetic control wild-type mice., Methods: MMCs were isolated from wild-type and 12/15-LO knockout mice. Cellular growth, activation of mitogen-activated protein kinases (MAPKs), transcription factors, superoxide levels, and fibronectin expression were compared in the two cell types., Results: Levels of the 12/15-LO product and protein were lower in MMC from 12/15-LO knockout relative to wild-type. MMCs from 12/15-LO knockout mice grew slower than wild-type cells, and also showed lower rates of tritiated thymidine and leucine incorporation (21% and 15% of wild-type, respectively, P < 0.001). Levels of superoxide and the matrix protein fibronectin were also lower in 12/15-LO knockout mice cells. Serum and angiotensin II (Ang II)-stimulated activities of p38 or ERK1/2 MAPKs, and cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB) transcription factor were lower in 12/15-LO knockout relative to wild-type cells. In addition, DNA binding and transcriptional activities of activated protein-1 (AP-1) and CREB were lower in 12/15-LO knockout cells. Furthermore, stable 12/15-LO overexpression in MMC led to reciprocal increase in p38 MAPK activation and fibronectin expression., Conclusion: The differential activation of oxidant stress, specific signaling pathways, transcription factors, and growth and matrix genes may lead to reduced growth and growth factor responses in 12/15-LO knockout versus wild-type MMCs. These results provide ex vivo functional evidence for the first time that 12/15-LO activation plays a key role in mesangial cell responses associated with renal diseases such as diabetic nephropathy.

Cyclooxygenase-2 (COX-2) enzyme and its inflammatory products such as prostaglandin E2 (PGE2) have been implicated in the pathogenesis of several inflammatory diseases. However their role in diabetic vascular disease is unclear. Advanced glycation end products (AGEs) act via their receptor, RAGE, to play a major role in diabetic complications. In this study, we investigated the effect of AGEs and S100b, a specific RAGE ligand, on the expression of COX-2 and the molecular mechanisms involved in cultured THP-1 monocytes and human peripheral blood monocytes. S100b treatment of THP-1 cells led to a significant 3-5-fold induction of COX-2 mRNA (p < 0.001). COX-2 protein and its product PGE2 were also increased, whereas COX-1 expression was unaffected. In vitro prepared AGE also induced COX-2 mRNA. S100b-induced COX-2 mRNA was blocked by an anti-RAGE antibody and by inhibitors of NF-kappa B (Bay11-7082), oxidant stress, protein kinase C, ERK, and p38 MAPKs. S100b (4-h treatment) significantly increased transcription from a human COX-2 promoter-luciferase construct (4-fold, p < 0.001). Promoter deletion analyses and inhibition of transcription by an NF-kappa B superrepressor mutant confirmed NF-kappa B involvement. This was further supported by inhibition of S100b-induced PGE2 by Bay11-7082. Additionally, S100b-induced adherence of THP-1 monocytes to vascular smooth muscle cells was blocked by the COX-2 inhibitor NS-398, Bay11-7082, inhibitors of ERK and p38 MAPK, and protein kinase C thereby indicating functional relevance. S100b also increased COX-2 mRNA expression in human peripheral blood monocytes from healthy donors. Moreover, COX-2 mRNA levels were clearly evident in monocytes obtained from diabetic patients but not from normal subjects. These results show for the first time that AGEs can augment inflammatory responses by up-regulating COX-2 via RAGE and multiple signaling pathways, thereby leading to monocyte activation and vascular cell dysfunction.

Object: In this retrospective study the authors examined the aspect ratio (AR; the maximum dimension of the dome/width of the neck of an aneurysm) and compared the distribution of this ratio in a group of ruptured and unruptured aneurysms. A similar comparison was performed in relation to the maximum dimension of the aneurysm alone. The authors sought to evaluate the utility of these measures for differentiating ruptured and unruptured aneurysms., Methods: Measurements were made of 774 aneurysms in 532 patients at three medical centers. One hundred twenty-seven patients harbored only unruptured lesions, 290 only ruptured lesions, and 115 both ruptured and unruptured lesions. Cases were included if angiograms were available for measurement and the status of the individual patient's aneurysm(s) was known. The odds of a lesion falling in the ruptured aneurysm group increased with both the lesion's maximum size and the AR. The odds ratio for rupture rose progressively only for the AR. The distribution curves showed that ruptured aneurysms were larger and had greater ARs. The mean size of unruptured aneurysms was 7 mm and that of ruptured ones was 8 mm; the corresponding mean ARs were 1.8 and 3.4, respectively. The odds of rupture were 20-fold greater when the AR was larger than 3.47 compared with an AR less than or equal to 1.38. Only 7% of ruptured aneurysms had an AR less than 1.38 compared with 45% of unruptured lesions., Conclusions: The AR is probably a useful index to calculate. A high AR might reasonably influence the decision to treat actively an unruptured aneurysm independent of its maximum size. Prospective studies are warranted.

A cDNA library of male Chinese seahorse (Hippocampus kuda Bleeker) was constructed to investigate the molecular profile of seahorse as one of the most famous traditional Chinese medicine materials, and to reveal immunological and physiological mechanisms of seahorse as one of the most primitive vertebrates at molecular level. A total of 3372 expressed sequence tags (ESTs) consisting of 1911 unique genes (345 clusters and 1566 singletons) were examined in the present study. Identification of the genes related to immune system, paternal brooding and physiological regulation provides not only valuable insights into the molecular mechanism of immune system in teleost fish but also plausible explanations for pharmacological activities of Chinese seahorse. Furthermore, the occurrence of high prevalent C-type lectins suggested that a lectin-complement pathway might exert a more dominant function in the innate immune system of teleost than mammal. Carbohydrate recognition domain (CRD) without a collagen-like region in the lectins of seahorse was likely an ancient characteristic of lectins similar to invertebrates.

Background: Primary adenosarcoma arising in vaginal endometriosis poses a diagnostic challenge, especially in superficial vaginal biopsies., Case: A 56-year-old woman, with a prior diagnosis of ovarian endometriosis, presented with a rapidly enlarging mass of the vaginal vault. Two prior biopsies were benign and showed endometriosis. The third vaginal biopsy revealed benign endometriotic glands cuffed by a cellular stroma with moderate cytologic atypia, a histological appearance diagnostic of Müllerian adenosarcoma. A 16-cm vaginal mass that had infiltrated the pelvic structures was resected., Conclusions: Close clinical follow-up of extrauterine endometriosis and clinical-pathologic correlation is necessary. Histological features such as cellular stromal cuffing around benign endometriotic glands are critical in arriving at a timely diagnosis of adenosarcoma in patients with persistent extrauterine endometriosis, even in superficial vaginal biopsies.

Biochemical and genetic evidence support the involvement of leukocyte-type 12/15-lipoxygenase enzyme and its products in the atherogenic process. We recently showed that products of the 12/15-lipoxygenase pathway play an important role in mediating hypertrophy, matrix protein production, and inflammatory gene expression in vascular smooth muscle cells (VSMC) through activation of mitogen activated protein kinases and key transcription factors. The current study is aimed at establishing the in vivo role of 12/15-lipoxygenase in VSMC by comparing growth factor-induced responses in VSMC derived from 12/15-lipoxygenase knockout mice versus genetic control wild-type mice. In the lipoxygenase knockout cells, 12/15-lipoxygenase protein was not expressed, and levels of its product, 12(S)-hydroxyeicosatetraenoic acid, were reduced (51% of wild type). Knockout cells exhibited significantly lower rates of growth factor-induced migration, fibronectin production, and incorporation of 3H-thymidine and 3H-leucine (54%, 55%, 61%, and 57% of wild type, respectively). Growth factor-induced superoxide production and p38 mitogen-activated protein kinase activation were also reduced in knockout cells. Serum-stimulated AP-1 transcription factor activation was markedly reduced (50% of wild type), whereas cAMP response element binding protein activation was abrogated in knockout cells. Furthermore, growth factor-induced mRNA expression of immediate early genes and fibronectin were also greatly reduced. These results suggest that the modulation of specific signaling pathways and growth-responsive genes may be responsible for the altered growth factor responses in the lipoxygenase knockout cells. They also demonstrate the important in vivo role of vascular 12/15-lipoxygenase in VSMC growth, migration, and matrix responses associated with hypertension, atherosclerosis, and restenosis.

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage-dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.

Objectives: To determine whether an education program on prostate cancer could improve awareness and knowledge among African-American men. African-American men have the world's highest incidence of prostate cancer and more than twice the mortality compared with white men. Screening programs for prostate cancer have not been successful in attracting African-American participation. One explanation is a poor awareness and knowledge about the disease among this high-risk population., Methods: We surveyed 900 African-American adults attending prostate cancer education seminars in community settings throughout Illinois between March 1998 and January 2001. Participants were asked to complete a multiple-choice questionnaire on topics related to prostate cancer. The main outcome measures were a change in awareness and knowledge of prostate cancer after the 1-hour educational seminar., Results: The mean survey score improved from 26.0% before the seminar to 73.3% after it (P <0.0001). Every multiple-choice question was answered correctly more often after the seminar than before it. Increasing levels of education and income were associated with higher before and after scores (P <0.001). Men achieved a significantly greater score improvement (mean 48.1%) compared with women (mean 41.1%; P = 0.006). Previous screening for prostate cancer was reported by 23% of the participants. Using logistic regression analyses, higher levels of education and income correlated with higher rates of screening. After the seminar, 63.1% stated the intention to undergo screening., Conclusions: Our results demonstrate that prostate cancer awareness and knowledge can improve dramatically after a 1-hour seminar on the topic. Additional studies to evaluate the long-term retention of knowledge and impact on behavior are warranted.

To examine fluctuations in women's cigarette smoking during pregnancy and implications for the design of clinical interventions for pregnant smokers and research on the effects of fetal exposure to cigarettes. We examined changes in absolute smoking status in 1426 women who reportedly smoked during their last pregnancy in the National Health Interview Survey 1991 Pregnancy and Smoking Supplement and fluctuations in amount smoked in 60 pregnant smokers in the Family Health and Development Project. In the National Health Interview Survey 1991 Pregnancy and Smoking Supplement, a substantial proportion of women exhibited a pattern of repeated cessation and relapse. In multivariable logistic regression models, having more than a high school education was significantly associated with being an intermittent versus a continuous smoker (odds ratio = 1.55, P <.01) and with successful quitting versus continuously smoking or relapsing (odds ratio = 1.74, P <.01). Fluctuations in smoking intensity in the Family Health and Development Project were also substantial and, although 48% quit or reduced their smoking upon learning of their pregnancy, over half changed smoking intensity multiple times. We conclude that smoking during pregnancy is a complex and variable behavior for many women. Simple measures of smoking may lead to under-estimation of the impact of smoking on the fetus, and brief smoking cessation interventions early in pregnancy are likely to be inadequate for many smokers during pregnancy.

Purpose: Patients with cancer are increasingly incorporating complementary therapies into the overall treatment. We determine the prevalence and patterns of use of complementary therapies among patients with prostate cancer., Materials and Methods: Patients attending 6 urology institutions for prostate cancer management completed a self-administered questionnaire on complementary therapy. All men diagnosed with prostate cancer were eligible, regardless of age, stage of disease or treatment., Results: A total of 1,099 patients returned the questionnaire. The overall response rate was 78.5%. Complementary therapies had previously been or were currently being used by 23.5% (258) and 18.2% (200) of patients, respectively. Higher levels of education and income were associated with greater use of complementary therapy (p <0.002 by logistic regression). Patients with progressive disease or those primarily treated with hormones were most likely to use complementary therapy. Among the patients using complementary therapy 90% believed that it would help them live longer and improve quality of life, 60% believed it would relieve symptoms and 47% expected it to cure disease., Conclusions: Complementary therapies are used by a large number of patients with prostate cancer, particularly those with progressive disease or who have undergone multiple treatments. Health care providers need to recognize this growing pattern of use of complementary therapy. Among patients who use complementary therapy the perception of benefit is much greater than that supported by scientific data. Future research should aim to unravel the complex psychosocial dynamics that influence the decision to use complementary therapy by men with prostate cancer and to educate patients about the efficacy of such therapies.