Phase I study of GSK461364, a specific and competitive Polo-like Kinase 1 (PLK1) inhibitor in patients with advanced solid malignancies.

Purpose: GSK461364 is an ATP-competitive inhibitor of polo-like kinase 1 (Plk1). A phase I study of two schedules of intravenous GSK461364 was conducted. Experimental Design: GSK461364 was administered in escalating doses to patients with solid malignancies by two schedules, either on days 1, 8, and 15 of 28-day cycles (schedule A) or on days 1, 2, 8, 9, 15, and 16 of 28-day cycles (schedule B). Assessments included pharmacokinetic and pharmacodynamic profiles, as well as marker expression studies in pretreatment tumor biopsies. Results: Forty patients received GSK461364: 23 patients in schedule A and 17 in schedule B. Dose-limiting toxicities (DLT) in schedule A at 300 mg (2 of 7 patients) and 225 mg (1 of 8 patients) cohorts included grade 4 neutropenia and/or grade 3–4 thrombocytopenia. In schedule B, DLTs of grade 4 pulmonary emboli and grade 4 neutropenia occurred at 7 or more days at 100 mg dose level. Venous thrombotic emboli (VTE) and myelosuppression were the most common grade 3–4, drug-related events. Pharmacokinetic data indicated that AUC (area under the curve) and C max (maximum concentration) were proportional across doses, with a half-life of 9 to 13 hours. Pharmacodynamic studies in circulating tumor cells revealed an increase in phosphorylated histone H3 (pHH3) following drug administration. A best response of prolonged stable disease of more than 16 weeks occurred in 6 (15%) patients, including 4 esophageal cancer patients. Those with prolonged stable disease had greater expression of Ki-67, pHH3, and Plk1 in archived tumor biopsies. Conclusions: The final recommended phase II dose for GSK461364 was 225 mg administered intravenously in schedule A. Because of the high incidence (20%) of VTE, for further clinical evaluation, GSK461364 should involve coadministration of prophylactic anticoagulation.

Dietary intake of fruits and vegetables improves microvascular function in hypertensive subjects in a dose-dependent manner

Background— Observational evidence has consistently linked increased fruit and vegetable consumption with reduced cardiovascular morbidity; however, there is little direct trial evidence to support the concept that fruit and vegetable consumption improves vascular function. This study assessed the dose-dependent effects of a fruit and vegetable intervention on arterial health in subjects with hypertension.

Methods and Results— After a 4-week run-in period during which fruit and vegetable intake was limited to 1 portion per day, participants were randomized to consume either 1, 3, or 6 portions daily for the next 8 weeks. Endothelium-dependent and -independent arterial vasodilator responses were assessed by venous occlusion plethysmography in the brachial circulation before and after intervention. Compliance was monitored with serial contemporaneous 4-day food records and by measuring concentrations of circulating dietary biomarkers. A total of 117 volunteers completed the 12-week study. Participants in the 1-, 3-, and 6-portions/d groups reported consuming on average 1.1, 3.2, and 5.6 portions of fruit and vegetables, respectively, and serum concentrations of lutein and ß-cryptoxanthin increased across the groups in a dose-dependent manner. For each 1-portion increase in reported fruit and vegetable consumption, there was a 6.2% improvement in forearm blood flow responses to intra-arterial administration of the endothelium-dependent vasodilator acetylcholine (P=0.03). There was no association between increased fruit and vegetable consumption and vasodilator responses to sodium nitroprusside, an endothelium-independent vasodilator.

Conclusions— The present study illustrates that among hypertensive volunteers, increased fruit and vegetable consumption produces significant improvements in an established marker of endothelial function and cardiovascular prognosis.

Genome-wide association study identifies five new schizophrenia loci

We examined the role of common genetic variation in schizophrenia in a genome-wide association study of substantial size: a stage 1 discovery sample of 21,856 individuals of European ancestry and a stage 2 replication sample of 29,839 independent subjects. The combined stage 1 and 2 analysis yielded genome-wide significant associations with schizophrenia for seven loci, five of which are new (1p21.3, 2q32.3, 8p23.2, 8q21.3 and 10q24.32-q24.33) and two of which have been previously implicated (6p21.32-p22.1 and 18q21.2). The strongest new finding (P = 1.6 x 10(-11)) was with rs1625579 within an intron of a putative primary transcript for MIR137 (microRNA 137), a known regulator of neuronal development. Four other schizophrenia loci achieving genome-wide significance contain predicted targets of MIR137, suggesting MIR137-mediated dysregulation as a previously unknown etiologic mechanism in schizophrenia. In a joint analysis with a bipolar disorder sample (16,374 affected individuals and 14,044 controls), three loci reached genome-wide significance: CACNA1C (rs4765905, P = 7.0 x 10(-9)), ANK3 (rs10994359, P = 2.5 x 10(-8)) and the ITIH3-ITIH4 region (rs2239547, P = 7.8 x 10(-9)).

Restoration of adipose function in obese, glucose-tolerant men following pioglitazone treatment is associated with CCAAT enhancer-binding protein β upregulation

Obese AT (adipose tissue) exhibits increased macrophage number. Pro-inflammatory CD16+ peripheral monocyte numbers are also reported to increase with obesity. The present study was undertaken to simultaneously investigate obesity-associated changes in CD16+ monocytes and ATMs (AT macrophages). In addition, a pilot randomized placebo controlled trial using the PPAR (peroxisome-proliferator-activated receptor) agonists, pioglitazone and fenofibrate was performed to determine their effects on CD14+/CD16+ monocytes, ATM and cardiometabolic and adipose dysfunction indices. Obese glucose-tolerant men (n=28) were randomized to placebo, pioglitazone (30 mg/day) and fenofibrate (160 mg/day) for 12 weeks. A blood sample was taken to assess levels of serum inflammatory markers and circulating CD14+/CD16+ monocyte levels via flow cytometry. A subcutaneous AT biopsy was performed to determine adipocyte cell surface and ATM number, the latter was determined via assessment of CD68 expression by IHC (immunohistochemistry) and real-time PCR. Subcutaneous AT mRNA expression of CEBPß (CCAAT enhancer-binding protein ß), SREBP1c (sterol-regulatory-element-binding protein 1c), PPAR?2, IRS-1 (insulin receptor substrate-1), GLUT4 (glucose transporter type 4) and TNFa (tumour necrosis factor a) were also assessed. Comparisons were made between obese and lean controls (n=16) at baseline, and pre- and post-PPAR agonist treatment. Obese individuals had significantly increased adipocyte cell surface, percentage CD14+/CD16+ monocyte numbers and ATM number (all P=0.0001). Additionally, serum TNF-a levels were significantly elevated (P=0.017) and adiponectin levels reduced (total: P=0.0001; high: P=0.022) with obesity. ATM number and percentage of CD14+/CD16+ monocytes correlated significantly (P=0.05). Pioglitazone improved adiponectin levels significantly (P=0.0001), and resulted in the further significant enlargement of adipocytes (P=0.05), without effect on the percentage CD14+/CD16+ or ATM number. Pioglitazone treatment also significantly increased subcutaneous AT expression of CEBPß mRNA. The finding that improvements in obesity-associated insulin resistance following pioglitazone were associated with increased adipocyte cell surface and systemic adiponectin levels, supports the centrality of AT to the cardiometabolic derangement underlying the development of T2D (Type 2 diabetes) and CVD (cardiovascular disease).

Deep sequencing reveals predominant expression of miR-21 amongst the small non-coding RNAs in retinal microvascular endothelial cells

The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non-coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well-characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR-2284/2285 family were detected. Several similar to 30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one-third of all mapped reads. Inhibition of miR-21 with an LNA inhibitor significantly reduced proliferation, migration, and tube-forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR-21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature. J. Cell. Biochem. 113: 20982111, 2012. (C) 2012 Wiley Periodicals, Inc.

The incidences of trisomy 8, trisomy 9 and D20S108 deletion in polycythaemia vera: an analysis of blood granulocytes using interphase fluorescence in situ hybridization:an analysis of blood granulocytes using interphase fluorescence in situ hybridization

We have used interphase fluorescence in situ hybridization (IFISH) to detect trisomy 8, trisomy 9 and 20q deletion in circulating granulocytes from patients with polycythaemia vera (PV). Out of 64 PV patients, 15 (23%) exhibited an abnormality. Two patients had trisomy 9, three had trisomy 8 and 10 patients had hemizygous deletion of D20S108 (a locus in the 20q common deleted region). Aberrant nuclei ranged from 10% to 80% in these 15 cases. There was no correlation between the presence of a marker and sex, age, interval between presentation and IFISH analysis, neutrophil or platelet count or therapy. Conventional marrow cytogenetic karyotype results were available in 23 cases and there was concurrence between these and blood IFISH in 16 cases (13 normal and three with 20q/D20S108 deletion by both methods). Three patients with D20S108 deletion by IFISH were normal by previous marrow cytogenetic testing and four cases with 20q deletion by previous marrow cytogenetics had normal blood granulocytes according to IFISH. Thus, we confirm that trisomies 8 and 9 and deletion of 20q are diagnostically useful markers of PV. IFISH analysis of blood granulocytes is a practical method for detecting these markers, but as an adjunct to, not as a substitute for, conventional marrow cytogenetics.

Temporal distribution of porcine circovirus 2 genogroups recovered from postweaning multisystemic wasting syndrome-affected and -nonaffected farms in Ireland and Northern Ireland

Porcine circovirus type 2 (PCV2) is now recognized as the essential infectious component of porcine postweaning multisystemic wasting syndrome (PMWS). PMWS was first recognized in high-status, specific pathogen-free pigs in Canada in 1991 and is now an economically important disease that affects the swine industry around the world. Recently, reports of genomic studies on PCV2 viruses indicated that 2 distinctive genogroups of PCV2 exist.(4,10) This report involves the results of a study on the distribution of predominant PCV2 genogroups recovered from samples taken from PMWS-affected and PMWS-nonaffected farms on the island of Ireland over a 9-year period and the results of a study on PCV2 genogroup recovery from fecal samples taken from a farm in Northern Ireland from 2003 to 2005 that was first diagnosed as PMWS positive in August 2005. The results indicate that, although at least 2 distinct genogroups of PCV2 have been circulating on pig farms on the island of Ireland, there does not appear to be a direct relationship between infection with these different genogroups of PCV2 and the development of PMWS.

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types

Background: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported.

Results: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p).

Conclusion: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

Detection of undiagnosed coeliac disease with atypical features using antireticulin and antigliadin antibodies

Eighteen patients with a variety of non-gastrointestinal symptoms were incidentally found to have circulating antireticulin antibody and on subsequent testing were also positive for antigliadin antibody. They prospectively underwent jejunal biopsy to determine whether or not they had coeliac disease. Their age range was 21-79 years (mean 42 years). Enteropathy was present in 13 (72 per cent) and was always associated with circulating IgA antigliadin antibody. Enteropathy was not present in the five cases who had only IgG antibody. Clinical improvement occurred in eight of 11 patients who complied with a gluten-free diet and was paralleled by an improvement in the mucosal histology in seven of eight who were re-biopsied. The most remarkable cases were two patients who presented with severe debility and no apparent haematological or biochemical abnormalities, and who subsequently made a dramatic recovery on a gluten-free diet. It is concluded that antireticulin antibody detected by routine autoantibody screening and confirmed to have IgA antigliadin antibody specificity is a useful indicator of an otherwise undiagnosed enteropathy. This serves to emphasize that the condition can sometimes be associated with atypical features and significant morbidity.

Gliadin antibody detection in gluten enteropathy

Circulating antigliadin antibody has been described in patients with gluten enteropathy although the prevalence varies in different studies. It has been suggested that the investigation for antigliadin antibody might be useful as a screening test. The object of the present study was to evaluate two different techniques for assaying these antibodies - an indirect immunofluorescent method and an enzyme-linked immunosorbent assay (ELISA). Antibodies were assayed in the sera of 102 patients in whom jejunal biopsies were also obtained. The specificity of both tests was greater than 95%, and the correlation between the presence of antibody and histology was significant (p <0.005), though the sensitivity of each test was less than 70%.

CD11b+ bone marrow-derived monocytes are the major leukocyte subset responsible for retinal capillary leukostasis in experimental diabetes in mouse and express high levels of CCR5 in the circulation

We investigated the phenotype of cells involved in leukostasis in the early stages of streptozotocin-induced diabetes in mice by direct observation and by adoptive transfer of calcein-AM-labeled bone marrow-derived leukocytes from syngeneic mice. Retinal whole mounts, confocal microscopy, and flow cytometry ex vivo and scanning laser ophthalmoscopy in vivo were used. Leukostasis in vivo and ex vivo in retinal capillaries was increased after 2 weeks of diabetes (Hb A(1c), 14.2 ± 1.2) when either donor or recipient mice were diabetic. Maximum leukostasis occurred when both donor and recipient were diabetic. CD11b(+), but not Gr1(+), cells were preferentially entrapped in retinal vessels (fivefold increase compared with nondiabetic mice). In diabetic mice, circulating CD11b(+) cells expressed high levels of CCR5 (P = 0.04), whereas spleen (P = 0.0001) and retinal (P = 0.05) cells expressed increased levels of the fractalkine chemokine receptor. Rosuvastatin treatment prevented leukostasis when both recipient and donor were treated but not when donor mice only were treated. This effect was blocked by treatment with mevalonate. We conclude that leukostasis in early diabetic retinopathy involves activated CCR5(+)CD11b(+) myeloid cells (presumed monocytes). However, leukostasis also requires diabetes-induced changes in the endothelium, because statin therapy prevented leukostasis only when recipient mice were treated. The up-regulation of the HMG-CoA reductase pathway in the endothelium is the major metabolic dysregulation promoting leukostasis.

Characterization of Transmission Losses

In this paper, characterizing transmission losses according to their origin is carried out. Transmission loss is decomposed into three components. The first is due to the current flow from generators to loads. The second is due to the circulating current between generators. The third represents the contribution of network structure and controls to increasing or decreasing transmission losses. Analytical proofs of the proposed loss decomposition are presented along with methods of allocating each component to the parties contributing to it. Illustration on simple dc and ac systems is presented. Results of application of the proposed method compared with other methods are also presented.

Stem cells, vascular smooth muscle cells and atherosclerosis

Stem cells have the ability to differentiate into a variety of cells to replace dead cells or to repair tissue. Recently, accumulating evidence indicates that mechanical forces, cytokines and other factors can influence stem cell differentiation into vascular smooth muscle cells (SMCs). In developmental process, SMCs originate from several sources, which show a great heterogenicity in different vessel walls. In adult vessels, SMCs display a less proliferative nature, but are altered in response to risk factors for atherosclerosis. Traditional view on SMC origins in atherosclerotic lesions is challenged by the recent findings that stem cells and smooth muscle progenitors contribute to the development of atherosclerotic lesions. Vascular progenitor cells circulating in human blood and the presence of adventitia in animals are recent discoveries, but the source of these cells is still unknown. The present review gives an update on the progress of stem cell and SMC research in atherosclerosis, and discusses possible mechanisms of stem/progenitor cell differentiation that contribute to the disease process.

Expression of Toll-Like Receptors 2 and 4 is Upregulated During Hospital Admission in Traumatic Patients:Lack of Correlation with Blunted Innate Immune Responses

Background: There are reports with conflicting results on the expression of toll-like receptors (TLRs) in trauma patients. In addition, these studies analyzed TLR expression only at patients hospital admission but not later when complications usually arise. Objectives: To analyze the surface expression of TLR2 and TLR4 on circulating monocytes from trauma patients during the hospitalization period and to correlate this with cytokine production after stimulation with TLR2 and TLR4 agonists. The phagocytic capacity of monocytes was analyzed at the same time points of TLR expression analysis; to correlate these molecular findings with the presence or absence of infections. Methods: Prospective and observational study from June 2005 to June 2007. In all analysis, a control group composed of healthy subjects was included. Results: We studied 70 trauma patients admitted to the intensive care unit (ICU) of a tertiary hospital, and 30 healthy volunteers. Blood samples were collected at hospital admission, on day 7 and 14. Forty-four patients (63%) developed at least one episode of infection. Monocytes from trauma patients expressed higher levels of TLR2 and TLR4 than monocytes from control subjects at all time points. Expression of TLR2 and TLR4 in monocytes from those patients who developed any infection was significantly lower than in those patients without infection but still significantly higher than in control subjects. Cellular responses to TLR4 agonist were impaired. Monocytes from traumatic patients phagocytosized less efficiently than monocytes from control subjects. Conclusions: These results indicate that trauma patients present a dysregulation of the innate immune system that persists during the first 14 days after hospital admission. Copyright © 2010 by Lippincott Williams & Wilkins.

Lack of effect of glutamine administration to boost the innate immune system response in trauma patients in the intensive care unit

The use of glutamine as a dietary supplement is associated with a reduced risk of infection. We hypothesized that the underlying mechanism could be an increase in the expression and/or functionality of Toll-like receptors (TLR), key receptors sensing infections. The objective of this study was to evaluate whether glutamine supplementation alters the expression and functionality of TLR2 and TLR4 in circulating monocytes of trauma patients admitted to the intensive care unit (ICU).