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Background: Vildagliptin is a drug for the treatment of diabetes. DPP-IV inhibitor represents a new class of oral antihyperglycemic agents to treat patients with type 2 diabetes. Several RP-HPLC methods have been reported to determine Vildagliptin alone. However, it has been noted that there are no available stability-indicating methods in pharmacopeias (USP/BP/EP/JP) nor in the available literature to quantify known and unknown impurity patterns for vildagliptin in vildagliptin tablets. Objective: The aim of this study is to develop a new single, sensitive, robust and specific gradient RP-HPLC method to quantify known and unknown impurities and degradants of Vildagliptin in Vildagliptin tablets. Methods: Chromatographic separation has been accomplished on the Hypersil ODS column (250 x 4.6) mm, 5 μm with a mobile phase consisting of a mixture of Perchloric acid Buffer, methanol, acetonitrile and Triethylamine delivered at a flow rate of 1.0 mL minute-1 and the detection wavelength 210 nm. The developed method was validated as per ICH guidelines. Results: Vildagliptin was found degraded significantly under oxidative and alkaline stress conditions. The degradation products were well resolved from Vildagliptin and its impurities. An analytical method was found linear, accurate and precise from LOQ (Limit of Quantification) level to 150% of impurity specification limit (0.5%). Conclusion: The method found sensitive, rapid and accurate quantification of known, unknown impurities and degradants. The peak purity results confirmed that the Vildagliptin peak was homogeneous and pure in all stress samples, thus proving the stability-indicating nature of the method.

Objective: The aim of the research work to develop a simple, sensitive, rugged, robust and specific novel gradient stability indicating reverse phase HPLC method for quantitative determination of known and unknown impurities profiling of Carvedilol pharmaceutical dosage forms (Tablets). Methods: Chromatographic separation has been achieved on an Inertsil ODS 3V column (150 mm x 4.6 mm, 5μm) with mobile phase consisting Mobile phase-A (Water, Acetonitrile and Trifluroacetic acid in the ratio of 80:20:0.1 v/v/v respectively and pH adjusted to 2.0 with dilute potassium hydroxide solution) and Mobile phase-B (Water and acetonitrile in the ratio of 100:900 v/v respectively) delivered at flow rate of 1.0 ml min-1 and the detection wavelength 240 nm. The column compartment temperature maintained at 40 °C. Results: Resolution between Carvedilol and its impurities has been achieved greater than 1.5. The developed method was validated as per ICH guidelines. Analytical method found Precise, Linear, accurate, specific, rugged and robust. Conclusion: Developed and validated novel analytical method can be used to for impurity profile analysis of Carvedilol Pharmaceutical dosage form (Tablets).

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lacks specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first “humanized” zebrafish that expresses human hematopoietic-specific cytokines (granulocyte-monocyte colony-stimulating factor, stem cell factor, and stromal cell-derived factor 1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation which more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.

Background Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. Methods Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4–11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. Results Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. Conclusions Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

Background T-cell Acute Lymphoblastic Leukemia (T-ALL) / Lymphoblastic Lymphoma (LBL) represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Development of CAR-T cell therapies for T-cell cancers has been complicated by induction of fratricide and the high risk of malignant cell contamination of the drug product in the autologous setting. Previously, Cooper et. al., demonstrated that CRISPR/Cas9 gene-editing to delete CD7 prevented self-killing, and deletion of the T-cell receptor alpha constant (TRAC) enabled the use of healthy donor-derived T-cells to manufacture CD7-targeted CAR-T cells without risk of malignancy and mitigating the risk of GvHD. Here we present preclinical data supporting the safety and efficacy of WU-CART-007, an IND ready, off-the-shelf, and fratricide resistant CD7-targeted CAR-T cell for the treatment of CD7+ T-cell malignancies. Methods WU-CART-007 was manufactured using T cells isolated from healthy donors by deletion of CD7 and TRAC, followed by CAR transduction, cell expansion, depletion of residual TCRa/b+ cells and cryopreservation. Donors were confirmed negative for a panel of adventitious viruses. CD7/TRAC deletion and CAR transduction were confirmed by flow cytometry. Off-target editing profile was assessed by GUIDE-Seq. The binding kinetics to human CD7 were conducted by bio-layer interferometry and CD7 selectivity was confirmed by cell microarray with a library of HEK-293 cells expressing approximately 6000 human proteins. The in vitro activity of WU-CART-007 was interrogated by co-culture with human CD7+ CCRF-CEM T-ALL cells and the potential on-target, off-tumor activity was assessed by co-culture with a panel of immune and non-immune primary human cells. In vivo anti-tumor functionality was confirmed in immunocompromised NSG mice after the establishment of low or high tumor burden CCRF-CEM xenografts engineered to express green fluorescent protein (GFP) and click beetle red (CBR) luciferase. The impact of WU-CART-007 on normal hematopoiesis was assessed using CD34+ humanized NCG mice. Results Several successful full-scale manufacturing runs were completed with consistently high dual CD7/TRAC deletion, transduction efficiency, and cell viability. Drug product was primarily composed of a T cell memory phenotype. Off- target nuclease analysis by GUIDE-seq and targeted NGS confirmed no evidence of off-target editing events. The WU-CART-007 scFv exhibited high affinity and exquisite specificity for human CD7. In vitro co-incubation experiments confirmed strong cytotoxicity against CD7-expressing cells including CCRF-CEM T-ALL cells, primary T and NK cells, but not CD7- cells such as myeloid cells, B cells, hepatocytes, astrocytes, cardiomyocytes, epithelial cells, and endothelial cells. Importantly, no cytotoxicity was observed against hematopoietic progenitor cells in human bone marrow or cord blood following co-incubation with WU-CART-007. Similarly, WU-CART-007 treatment of a non-tumor bearing humanized mouse model resulted in transient reductions in CD7+ cells (T-cells and NK cells) but not CD7- cells (myeloid and B cells), and the impacted cells recovered after circulating WU-CART-007 cells were no longer detectable. Assessment of in vivo anti-tumor efficacy revealed that WU-CART-007 effectively inhibited tumor progression (>99% TGI) in both low and high burden CCRF-CEM tumor models and improved survival in a dose-dependent manner, while CAR- cells were inactive, confirming CD7-dependent activity. Conclusions These preclinical studies support the use of WU-CART-007 in clinical trials and highlight the potential of WU-CART-007 to be a well-tolerated and active therapy for patients with CD7+ T-cell malignancies. A first in human Phase 1/2 trial in patients with R/R T-ALL/LBL is currently open for enrollment (NCT# 04984356). Disclosures No relevant conflicts of interest to declare.

Data from high-income countries suggest that women receive less intensive diagnostic and therapeutic management than men for acute coronary syndrome (ACS). There is a paucity of such data in the Indian population, which is 69% rural and prior studies focused mostly on urban populations. The objective of the present study was to identify the gender based differences in ACS management, if any, in a predominantly rural population.Data from 35 hospitals across Himachal Pradesh covering90% of state population were collected for one year (July 2015-June 2016). A total of 2118 ACS subjects met inclusion criteria and baseline characteristics, in-hospital treatments and mortality rates were analyzed.Women constituted less than one-third of ACS population. Women were older compared to men and were more likely to present with NSTEMI/UA. Misinterpretation of initial symptoms and late presentation were also common in women. Fewer women received optimal guideline based treatment and PCI (0.9% vs 4.2%, p0.01). Compare to men, women more often had Killip class1 (27.3% vs 20.4%, p0.01) and higher in-hospital mortality (8.5% vs 5.6%, p=0.009). On multivariate analysis the association between female gender and mortality was attenuated (adjusted odds ratio [OR]=1.36 [0.77-2.38]).The present study from India, is the first of its kind to evaluate the gender based differences among ACS patients, in a predominantly rural population. Our analysis demonstrates a significant gender based difference between symptom awareness and delay in presentation, management and in-hospital outcome. Further studies are warranted across other parts of country to investigate this gender disparity.

Existing metrics for grading mitral regurgitation (MR) are limited and fraught with high interobserver variability. We developed and evaluated a Doppler-based, semiquantitative novel index (Mitral Regurgitation Severity Index [MRSI]) of MR severity. In a total of 125 patients (70 in the derivation cohort and 55 in the validation cohort), MRSI was calculated as a ratio of time velocity integral of mitral inflow (continuous-wave Doppler-TVI MV) to the time velocity integral of the left ventricle outflow (pulse-wave Doppler-TVI LVOT). Inter-rater agreement for MRSI and predictive ability of the MRSI were then assessed. In the derivation cohort, MRSI differed significantly between patients with severe MR (2.6 ± 0.51) and mild-moderate (nonsevere) MR (1.4 ± 0.18) and a cutoff of ≥1.8 was associated with optimal diagnostic accuracy. In the validation cohort, MRSI exhibited excellent agreement between a level II and a level III reader with a mean difference of -0.14 (95% confidence limit of agreement: -0.80 to 0.53), correlation coefficient of 0.88 (p

Overexpression of the multiple myeloma set domain (MMSET) Wolf-Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t(4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138+ tumor cells from t(4;14)-positive and -negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t(4;14)-negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11-driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid-derived 2)-like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11-driven increases in ROS; however, transcriptional up-regulation of some of NRF2’s antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.—Mahajan, N., Wu, H.-J., Bennett, R. L., Troche, C., Licht, J. D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Hypertrophic cardiomyopathy (HCM) affects the right ventricle (RV) because of the anatomically hypertrophied septum and plausibly by extension of the myopathic process to the RV. We sought to investigate RV strain in patients with left ventricular hypertrophy secondary to either HCM or hypertension (H-LVH). Our cross-sectional study included 32 patients with HCM, 21 patients with H-LVH, and 11 healthy subjects, who were evaluated with transthoracic echocardiography. Using a dedicated software package, bi-dimensional acquisitions were analyzed to measure segmental longitudinal strain in apical views. Right ventricular global longitudinal strain (GLS) was calculated by averaging septal and right free wall strains. The HCM and H-LVH groups were comparable for age and demographic characteristics. Right ventricular tricuspid annular plane systolic excursion was not significantly different between HCM and H-LVH subjects. Moreover, RV GLS, septal and lateral RV myocardial strain were significantly impaired in patients with HCM (all p0.001). Regional and global RV strain parameters were not significantly impaired in H-LVH compared to healthy controls An RV GLS cut-off value of14.9% differentiated HCM and H-LVH with a 90% sensitivity and a 95% specificity (p0.001). RV strain parameters are impaired in patients with HCM. Assessment of two-dimensional RV strain parameters could help differentiate between HCM and H-LVH.

The current definition of peripartum cardiomyopathy (PC) is restricted to patients with left ventricular systolic dysfunction (ejection fraction [EF]45%). Data on peripartum heart failure (HF) with normal EF are sparse. We describe clinical characteristics of patients with normal (≥55%) and patients with low (45%) left ventricular ejection fractions (LVEFs). Electronic medical records (2006 to 2013) of our tertiary care center were retrospectively screened to identify peripartum HF with normal EF, defined as an entity meeting Framingham criteria for HF with symptom onset during the last month of pregnancy or up to 5 months after delivery and with an EF of ≥55%. Clinical characteristics, echocardiographic parameters, and outcomes of these patients were compared with age-matched control patients with traditionally defined PC (EF45%). A total of 25 patients with PC and EF≥55% were identified. Exclusion of hypertension (n=9), preeclampsia (n=1), and diabetes mellitus (n=2) yielded 13 patients with PC and EF≥55%. Age-matched patients with traditional PC (EF45%) constituted controls (n=16). Compared with patients with PC and low LVEF, patients with PC and normal LVEF had lower B-type natriuretic peptide levels, systolic and diastolic left ventricular dimensions, left atrial size, and incidence of decompensated HF during delivery (p0.05). Compared with historical age-matched controls, patients with normal LVEF exhibited attenuated E' mitral annular velocities. On follow-up, these patients were associated with a lower New York Heart Association functional class. In conclusion, peripartum HF with normal LVEF appears to be a distinct entity.

Precursor T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogenous hematologic malignancy resulting from accumulation of molecular lesions in a multistep process. Although survival rates have improved considerably, event free survival for patients with T-ALL are generally inferior compared to B-cell ALL. Genetic alterations are important determinants of responsiveness to therapy and serve as targets for molecularly tailored therapies. More than 75 chimeric fusion genes have been reported in T-ALL, the majority of which encode factors involved in transcriptional regulation, while only a smaller percentage codes for tyrosine kinases. We report a case of relapsed T-ALL, despite low risk stratification at the time of diagnosis, harboring a novel fusion protein SPTAN1-ABL1. Primary bone marrow specimen collected at diagnosis was transplanted in NSG-B2m mice and propagated as a patient-derived xenograft (PDX) line. For transcriptomic characterization, RNA isolated from primary and PDX samples was subjected to error-corrected targeted next-generation sequencing using ArcherDX FusionPlex HemeV2 kit. Bioinformatics analysis identified the novel SPTAN1-ABL1 gene fusion in which exon 2 of SPTAN1 was fused with exon 4 of ABL1. This fusion was confirmed by Sanger sequencing. Translation of the fusion product sequence showed in-frame fusion leading to the generation of a chimeric protein containing N-terminal SPTAN1 and C-terminal ABL1 with intact kinase domain. SPTAN1 encodes non-erythryocytic-1-spectrin-alpha protein, an actin-binding protein, with N-terminal domain possessing oligomerization activity. Because oligomerization of ABL1 promotes its kinase activity, it is possible that SPTAN1-ABL1 possesses constitutive kinase activity. The full-length SPTAN1-ABL1 fusion protein was cloned in a mammalian expression vector and expressed in BaF3 cells. SPTAN1-ABL1 fusion was detected at similar allelic frequencies in primary and PDX samples indicating the concordance between the two. Furthermore, treatment of engrafted mice with dasatinib (Qd10, 5 mg/Kg, p.o.) significantly prolonged survival compared to untreated mice (n=5 each, P Citation Format: Anilkumar Gopalakrisnapillai, Erin Crowgey, Demetria Ruhl, Darcy Hamill, Nitin Mahajan, Todd Druley, E. Anders Kolb, Sonali P. Barwe. Identification of a novel fusion protein SPTAN1-ABL1 in a child with T-cell acute lymphoblastic leukemia: Functional characterization and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-322.

Background Peripartum cardiomyopathy (PPCM) is a rare life-threatening condition of unclear etiology. Data on the use of cardiac magnetic resonance (CMR) imaging to characterize PPCM are limited. This study was done to assess the role of CMR imaging for the diagnosis and prognostication of patients with PPCM. Methods Medical records of a tertiary medical center were screened for PPCM patients with CMR imaging done within the past 5 years (2006-2011). Images were reviewed by 2 expert radiologists (blinded to clinical data) using cine sequences for chamber function and size, T2-weighted images for the determination of edema (T2-ratio), and late gadolinium enhancement (LGE) sequences for myocardial tissue characterization. Results Ten PPCM patients (aged 28 ± 6 years, 90% African American) had a total of 15 CMR examinations: 4 in the acute phase (within 7 days of diagnosis) and 11 during follow-up (median, 12 months; range, 1-72 months). Left ventricular ejection fraction was decreased in all 4 initial scans. Elevated T2 ratio (> 2) seen in 1 patient decreased on follow-up imaging. LGE was seen in 1 of the 4 acute-phase scans and in 4 of the 11 follow-up phase scans. These 4 patients had multiple readmissions because of heart failure exacerbations and persistently low left ventricular ejection fraction on subsequent echo- cardiograms. Conclusions LGE seems to be associated with a poor prognosis in the setting of PPCM. CMR imaging seems to have promising practical implications in the diagnosis and prognostication of PPCM patients.

Henoch-Schönlein purpura (HSP) is an acute systemic vasculitis with unknown etiology, although several studies have found HSP to be related to cytokines such as tumor necrosis factor α, interleukin (IL)-1, and adhesion molecules. In the present study we determined the levels of cytokines such as IL-18 and endothelin-1 (ET-1) in children with HSP. Subjects were divided into three groups (group 1, 20 subjects with HSP; group 2, 10 subjects belonging to group 1 during their follow-up 4 to 6 months later; and group 3, 16 controls who were healthy siblings of the subjects). IL-18 and ET-1 levels were determined using enzyme immunoassay and expressed as mean ± standard deviation. We observed higher IL-18 levels in children with HSP (767.6 ± 145.1 pg/mL) than in controls (614.6 ± 66.54 pg/mL, p 0.05), but IL-18 levels were found to be significantly lower in subjects with HSP in remission (502.7 ± 60.81 pg/mL) than in those who were in an active phase (1,050 ± 244.5 pg/mL, p 0.05, n = 10). ET-1 levels were found to be significantly higher in subjects with HSP (1.93 ± 0.19 pg/mL) than in controls (1.10 ± 0.13 pg/mL, p 0.05), although no significant difference was observed in ET-1 levels between subjects in group 1 (1.88 ± 0.30 pg/mL) and group 2 (1.91 ± 0.120, p 0.05, n = 10). A positive correlation was observed between IL-18 and ET-1 levels in subjects with HSP (correlation coefficient [r] = 0.5254, p 0.01). These results suggest that levels of IL-18 and ET-1 are worth monitoring during the clinical course of the disease, but caution must be exercised in extrapolating data based on small study samples.

Maspin is a member of the serine protease inhibitor (serpin) superfamily and displays tumor-suppressing activity by controlling cell migration, proliferation, apoptosis, and adhesion. Here, we provide evidence that maspin acts as a reactive oxygen species (ROS) scavenger through oxidation of three structurally exposed cysteine thiols to sulfenic acid. Ablation of these cysteine residues in maspin resulted in a significant increase in total ROS production in mouse mammary TM40D cells. Also, cells containing a triple-cysteine mutant of maspin showed elevated ERK1/2 activity, a downstream target of ROS, and enhanced proliferation and colony formation. These findings establish a novel mechanism by which maspin utilizes its cysteine thiols to inhibit oxidative stress and cell growth.

Aim: The aim is to review the English literature for post-traumatic superolateral dislocation of mandibular condyle (SDMC),discuss their dynamics and clinical management and to propose to modify the existing classification of SDMC. Patients and Methods: A literature search was carried at Pubmed, Sciencedirect, Google and references from reported articles were crosschecked to look for the cases of SDMC from 1969 to 2015 in English language. Also, we have reviewed 11 of our patients with total of 18 superolateral dislocated intact or sagittal split condyles ,who visited our unit in the previous two years. Results: In our retrospective analysis 58 cases of SDMC were found in the literature, of which 38 had intact mandibular condyles and 20 had sagittal split. Early and intact SDMC were successfully managed conservatively with closed reduction, whereas old cases and largely fractured condyles necessitated open reduction. Additionally, we observed an unusual dislocation associated with fracture of contralateral posterior mandible(angle) in our series which did not gratify the existing classification. Conclusion: Alteration of the existing classification was required to accommodate the unusual type of dislocation.

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA-binding domain and have diverse biological functions including tumor suppressor as well as tumor promoter functions. They are regulated via a complex and diverse number of mechanisms and control key cellular processes. Prostate-derived Ets transcription factor (PDEF), a unique member of the ETS family, is present in tissues with high epithelial content are hormone-regulated, such as prostate, breast, salivary glands, ovaries, colon, airways, and stomach tissues. PDEF (prostate-derived Ets factor) is also referred to as SPDEF (SAM pointed domain containing Ets transcription factor), PSE (mouse homolog), or hPSE (human PSE) in the literature and is the sole member of the PDEF ETS sub-family. The role of PDEF in cancer development is still not fully elucidated though. The present article focuses on the key findings about the PDEF's biological functions, interacting proteins, and its target genes. There is a strong urge to focus on the clinical studies in larger cohort, which elucidate the regulation of PDEF and its target genes, to determine the potential of PDEF as biomarker. Based on the studies discussed in the present article, one can anticipate that PDEF offers a great potential for developing therapeutics against cancer.

Objectives The purpose of this study was to estimate the effect of long-term exposure to lower plasma low-density lipoprotein cholesterol (LDL-C) on the risk of coronary heart disease (CHD). Background LDL-C is causally related to the risk of CHD. However, the association between long-term exposure to lower LDL-C beginning early in life and the risk of CHD has not been reliably quantified. Methods We conducted a series of meta-analyses to estimate the effect of long-term exposure to lower LDL-C on the risk of CHD mediated by 9 polymorphisms in 6 different genes. We then combined these Mendelian randomization studies in a meta-analysis to obtain a more precise estimate of the effect of long-term exposure to lower LDL-C and compared it with the clinical benefit associated with the same magnitude of LDL-C reduction during treatment with a statin. Results All 9 polymorphisms were associated with a highly consistent reduction in the risk of CHD per unit lower LDL-C, with no evidence of heterogeneity of effect (I 2 = 0.0%). In a meta-analysis combining nonoverlapping data from 312,321 participants, naturally random allocation to long-term exposure to lower LDL-C was associated with a 54.5% (95% confidence interval: 48.8% to 59.5%) reduction in the risk of CHD for each mmol/l (38.7 mg/dl) lower LDL-C. This represents a 3-fold greater reduction in the risk of CHD per unit lower LDL-C than that observed during treatment with a statin started later in life (p = 8.43 × 10 −19 ). Conclusions Prolonged exposure to lower LDL-C beginning early in life is associated with a substantially greater reduction in the risk of CHD than the current practice of lowering LDL-C beginning later in life.

Takayasu's arteritis (TA) is an inflammatory fibrosing arteritis affecting predominately the aorta and its main branches. Pathogenesis of this disease remains enigmatic. Despite the numerous studies, the role of adventitia in vascular lesion formation in the setting of TA has been ignored. Virtually nothing is known about the mechanism regulating inflammation in the adventitia in the setting of TA. The present study included subjects with Takayasu's arteritis and normal healthy control subjects. Isolated T cells from peripheral blood mononuclear cells (PBMCs) using nylon wool and HUT-78 (human cutaneous T lymphoma cell line) were stimulated with PHA for 24 h. Stimulated cell were fixed with paraformaldehyde and fractionated into membrane, cytosolic and nuclear fractions. These cellular fractions were co-cultured with human fibrosarcoma cell line (HT-1080) and transcriptional expression of matrix metalloproteinases (MMP-1, 3, 9 and TIMP-1) was determined using semiquantitative RT-PCR. Stimulation of MMPs-TIMP synthesis by HT-1080 cells was mimicked by a membranous fraction derived from activated T-cell isolated from TA subjects and activated HUT-78 cells, whereas cytosolic and nuclear fractions were ineffective. In conclusion, for the first time we provide evidence for the presence of a cell surface-specific antigenic moiety on T-cells of TA subjects, which is responsible for activation of fibroblasts (cells predominantly present in adventitia) to enhance MMP production and, therefore, may lead to extracellular matrix degradation.

BACKGROUND Despite the high prevalence of rheumatic heart disease (RHD) in developing countries such as India, data on characteristics, complications, and treatment practices are lacking. The HP-RHD (Himachal Pradesh Rheumatic Heart Disease) registry aimed at reporting these parameters in patients with RHD from a northern state of India. METHODS A total of 2,005 consecutive patients of RHD were enrolled over a period of 6 years (2011 to 2016) in the present study. The clinical characteristics, complications, and treatment practices were systematically recorded. RESULTS The mean age for patients with RHD was 40.3 ± 14.3 (range 5 to 83 years). RHD predominantly affected females (72.3%) and population from rural background (92%). Multivalvular involvement was frequent (43.2%), mitral valve was the commonest affected valve (83.3%). The majority of the patients had moderate-to-severe valvular dysfunction (69.3%). Mitral and tricuspid valve involvement was more frequent in female subjects compared with more frequent aortic valve involvement in male subjects (p

Background Receptor for advanced glycation end products (RAGE) may play an important role in inflammatory processes and endothelial activation. Extracellular newly identified RAGE binding protein (EN-RAGE), natural pro-inflammatory ligand for RAGE. The role of C-reactive protein (CRP) as a mediator in inflammation and atherosclerosis is the subject of recent investigations worldwide. In the present study, we investigated the effect of CRP on RAGE and EN-RAGE gene expression in THP-1 monocytic cell line. MAP kinases (ERK, p38 and JNK) were exploited as possible signaling pathways involved in the signal transduction by CRP. Further, atorvastatin was used as a therapeutic modality for modulation of these genes in the presence of CRP. Materials and methods Time and dose-dependent experiments were carried out in the presence of CRP. Specific MAPK pathways inhibitors were used to elucidate the signaling pathways involved. Effect of atorvastatin was also determined in the presence of CRP on the expression of these genes. Results Time and dose-dependent experiments revealed that, treatment of THP-1 cells with 100 μg of CRP/ml/10 6 cells for 24 h, augmented the expression of RAGE and EN-RAGE genes by 2.5–3.5 folds and 3.5–4.5 folds respectively. CRP acted via FcγRII and utilized ERK, p38 and JNK pathways to transduce signals. Atorvastatin in a dose of 20 μM, was able to attenuate up-regulation of CRP-induced genes ( p Conclusion Our data strongly suggests that blockade of RAGE–EN-RAGE by statins at an early stage may prevent inflammation in atherosclerosis and counteract the harmful effects mediated by CRP.

Aim: Accelerated extracellular matrix breakdown caused by the increased activity of matrix metalloproteinases (MMPs) has been implicated in several rheumatological disorders and systemic vasculitides, especially Takayasu’s arteritis and Kawasaki disease. Therefore, the aim of the present study was to investigate the potential role of MMPs in Henoch–Schonlein purpura (HSP), an acute type of systemic vasculitis in children. Methods: We studied the activity of MMP-2 and MMP-9 in the sera using gelatin zymography and the transcriptional expression in peripheral blood mononuclear cells using semi-quantitative RT-PCR in 20 patients with HSP in acute and convalescent phase and in 20 healthy children, who were siblings of the subjects with same age group. Results: All 20 children with HSP showed increased levels of serum activity of MMP-2 and MMP9 in acute phase as compared with their convalescent phase [MMP-2 (p > 0.05); MMP-9 (p > 0.05)] and their control counterparts [MMP-2 (p < 0.001); MMP-9 (p < 0.001)]. Similarly, transcriptional expression of MMPs was found to be higher in the acute phase of HSP than in convalescent phase [MMP-2 (p < 0.05); MMP-9 (p < 0.001)] and in their healthy controls [MMP-2 (p < 0.001); MMP-9 (p < 0.01)]. Conclusion: The presence of excessive transcriptional expression and gelatinolytic activity of MMPs may be downstream to the actual aetiopathogenetic factors.

Chronic underlying inflammation is involved in the pathophysiology of coronary artery disease (CAD). Polyalthia longifolia var. pendula bark extract (PLE) is known to exhibit anti-inflammatory activity and has high content of phytosteroids. Since phytosteroids mimic estrogen structurally, we postulated that PLE may provide protection in postmenopausal women against CAD. Thus the effect of PLE has been explored on expression of estrogen receptors (ERalpha and ERbeta) and inflammatory inducible nitric oxide synthase (iNOS) genes in vitro in peripheral blood mononuclear cells (PBMCs) obtained from postmenopausal women. A total of 20 postmenopausal women were included in the present study. Group I (N = 10) included women with angiographically proven CAD, and group II (N = 10) is composed of equal number of age-matched healthy postmenopausal females as controls. Significantly low levels of serum 17-beta estradiol were observed in subjects of group I as compared to group II (p0.01). A marked increase in L: -citrulline levels (p0.05) and significantly augmented levels of reactive nitrogen intermediates (p0.05) were observed in group I subjects. PLE significantly attenuated PMA-induced expression of both ERalpha and ERbeta receptors and inflammatory iNOS gene in vitro in a dose- and time-dependent manner and had an additive effect on these genes when compared with tamoxifen. Ours is the first report to demonstrate that PLE contains certain bioactive principles, which possess anti-inflammatory and estrogenic properties, and thereby hold the promise to be screened for their anti-atherogenic potential in experimental animals to favorably alter several other markers of cardiovascular risk.

Inflammation participates in atherosclerosis from its inception onwards. RAGE (receptor for advanced glycation end products) and its natural pro-inflammatory ligand, EN-RAGE (extracellular newly identified RAGE-binding protein) have been implicated in various inflammatory diseases. In present study, we determined the expression of RAGE and EN-RAGE in peripheral blood mononuclear cells (PBMCs) of subjects with pre-mature coronary artery disease (CAD) for the first time.The study patients were angiographically proven non-diabetic patients with pre-mature CAD (Group I; N=100) and control group comprised of subjects with coronary risk factors and without coronary artery lesions (Group II; N=40). Semi-quantitative RT-PCR was performed to determine transcriptional expression of RAGE and EN-RAGE in PBMCs. Soluble RAGE (sRAGE) and C-reactive protein (hsCRP) levels were determined in serum of all study subjects using immunoassays. A significantly increased transcriptional expression of RAGE and EN-RAGE in PBMCs (p0.01) of Group I patients was observed. Increased circulating hsCRP (p0.01) levels and decreased sRAGE (p0.01) levels were observed in Group I as compared with the Group II subjects. Severity of disease determined by Gensini score was found to be positively correlated with transcriptional expression of RAGE (r=0.530) and EN-RAGE (r=0.323). EN-RAGE expression revealed a strong association with RAGE (r=0.326), hsCRP (r=0.251) and a negative association with sRAGE (r=-0.222).Increased expression of RAGE and EN-RAGE in non-diabetic pre-mature CAD and various associations discussed may amplify several cellular perturbations and thus significantly contribute to the pathophysiology of CAD.

Background Left main disease (LMD), defined as ≥ 50% lesion stenosis, occurs in 3% to 5% of patients undergoing catheterization. Limited data on the value of single-photon emission computed tomography (SPECT) imaging for diagnosis of LMD exists. Hypothesis This study sought to evaluate the diagnostic accuracy of SPECT imaging in the diagnosis of LMD. Methods A total of 74 consecutive patients with LMD, identified from our catheterization lab database (January 2003–December 2007) with gated exercise (15 patients) or adenosine (59 patients), thallium 201, or Tc-99m SPECT imaging within 6 months of index angiography were included. Group 1 (Gp 1) included 33 patients with isolated LMD. Group 2 (Gp 2) consisted of 41 patients with LMD and 1-vessel disease (6); LMD and 2-vessel disease (24); and LMD and 3-vessel disease (11). Results Reversible perfusion defects (PD) were absent in 6 (18%) of Gp 1 and 8 (20%) of Gp 2 patients. Among Gp 1 patients, PD in 1-vessel, 2-vessel, 3-vessel distribution were seen in 20 (61%), 5 (15%), and 2 (6%) patients respectively. In comparison, PD in 1-vessel, 2-vessel, 3-vessel distribution were observed in 20 (49%), 12 (29%), and 1 (2%) of Gp 2 patients respectively. Left main (LM) pattern was noted in 6 patients (Gp 1: 2, Gp 2: 4). Transient ischemic dilatation (TID) was encountered in 34 patients (Gp 1: 17, Gp 2: 17) and in 4 patients with normal perfusion scans. Conclusions These data represent the largest analysis of patients with “isolated” LMD. Approximately 19% of patients with LMD have no reversible PD on SPECT. While LM pattern is rare, 1-vessel distribution PD and TID represent the most frequent SPECT abnormalities. Copyright © 2009 Wiley Periodicals, Inc.

Aortic valve calcification is common in the elderly and in patients with congenital bicuspid aortic valve but unlike calcific mitral valve disease it is not a well recognized risk factor for stroke. Although autopsy studies have revealed evidence of systemic embolism in one-third of cases with calcific aortic valves, there is conflicting data from larger clinical studies examining the association between calcific aortic valve and stroke. There are only 8 reported cases of symptomatic stroke from spontaneous cerebral thromboembolism associated with calcific aortic valve in the literature. Computerized tomography (CT) angiography and CT without contrast are modalities of choice to diagnose calcific embolism, while MRI may be useful in delineating the extent of ischemia. Ideal management strategy, the role of antiplatelet therapy, anticoagulation or recommendations for valve replacements are poorly defined. We present a focused literature review on this topic.

Background: Acute coronary syndrome (ACS) resulting from culprit lesion in left main coronary artery (LMCA) can cause rapid hemodynamic deterioration. It is important to identify these patients early to facilitate timely revascularization. ST segment elevation in aVR greater than or equal to V 1 (aVR-V 1 ≥ 0) has been suggested as a sensitive predictor of LMCA disease. As a result of balanced forces, we hypothesized that ST deviation in V 6 greater than or equal to ST deviation in V 1 (V 6 -V 1 ≥ 0) might be a good determinant of LMCA disease. Methods: We compared admission 12-lead ECGs of ACS resulting from culprit LMCA lesion (n = 75, group I) with ACS resulting from culprit left anterior descending lesion (n = 81, group II). Group I was selected over a period of 10 years. We compared V 6 -V 1 ≥ 0 to aVR-V 1 ≥ 0 in both groups. We also looked at ratios of ST deviations in V 6 ,V 1 (V 6 /V 1 ≥ 1) and aVR,V 1 (aVR/V 1 ≥ 1) in patients where ST segment in V 1 was not isoelectric (group I = 54 and group II = 55). Results: ST deviation in V 6 was significantly greater in group I as compared to group II (P < 0.001). The reliabilities of V 6 -V 1 ≥ 0, V 6 /V 1 ≥ 1, aVR-V 1 ≥ 0, and aVR/V 1 ≥ 1 in predicting LMCA disease were determined. Conclusion: This is the largest series of ECG analysis on ACS resulting from culprit LMCA lesion. V 6 -V 1 ≥ 0 and V 6 /V 1 ≥ 1 were more sensitive in predicting LMCA as culprit vessel in comparison to previously reported greater ST segment elevation in aVR than V 1 .

Purpose Normal tissue toxicity remains a dose limitation for cancer radiotherapy and chemoradiotherapy. Growth factors offer a novel means of mitigating normal tissue radiotoxicity. In particular, keratinocyte growth factor (rHuKGF), whose proliferative activity is restricted to epithelial cells, holds promise on the basis of the findings of preclinical models of epithelial cytoprotection and the clinical developments to date. We report the radioprotection of murine lung by an increase in tissue cellularity after rHuKGF-induced proliferation. Methods and materials Flow cytometric and image analysis techniques after bromodeoxyuridine labeling were used to estimate proliferative parameters. Our specialized analytical methods measure not only labeling indexes, but also the durations of S and G 2 +M phases, potential doubling times, and the net cell production rate. Image analysis techniques were used to identify the specific cell types that were proliferating (type II pneumocytes). Results Lung labeling index control values (0.5%) rose to a maximum (5.5%) at 3 days after intratracheal rHuKGF, returning to normal by Day 7. The potential doubling time fell from 66 days to 4.4 days. The net cell production rate rose from a control value of 1%/d to >15%/d by Day 3. This resulted in a nearly twofold increase in alveolar epithelial cellularity, which remained significantly elevated on Day 7. Saline-treated control animals exhibited no significant changes in the proliferative parameter values or cellularity. On the basis of these data, mice were irradiated, solely to the thorax, with ranges of single doses of 250 kVp X-rays 7 days after either intratracheal administration of 5 mg/kg rHuKGF or phospate-buffered saline. This interval was chosen because the proliferative response of the type II cells was finished but the cellularity of the lung remained increased. Pretreatment with rHuKGF extended the latent period before onset of pneumonitis after all radiation doses. rHuKGF treatment 7 days before thoracic irradiation significantly protected against pneumonitis (median effective dose 13.7 Gy, 95% confidence limit 13.4–14.0) compared with the control pretreatment with phosphate-buffered saline (median effective dose 12.8 Gy, 95% confidence limit 12.6–13.1). Conclusion The data showed that an increase in tissue cellularity, caused by rHuKGF treatment before irradiation, protected the lung from damage due to pneumonitis.

The goal of this study was to assess the clinical applicability of molecular genetic techniques in the study of minimal residual disease (MRD) in pediatric acute myeloid leukemia (AML) via DNA and RNA-based methods. Currently, multi-parametric flow cytometry (MPFC) for surface immunophenotypes is the gold standard for MRD with a limit of detection between 0.001-0.002. However, approximately one-third of children with no detectable MRD by MPFC after first induction still relapse and suffer inferior outcomes. One potential reason is that a majority of refractory or recurrent AML clones will present with altered immunophenotypes compared to diagnostic specimens. In contrast, DNA mutations present at diagnosis will remain in refractory disease and therefore, a gene-based MRD platform surveying many genes could enable targeted, gene-specific therapy. Recently, the heterogeneous and unique genomic landscape of pediatric AML was characterized in the TARGET project and highlights the potential for leveraging companion molecular screens in the analysis of AML MRD. Contrary to adult AML, pediatric AML is not primarily characterized by single nucleotide variants (SNVs), but rather by complex structural variants (SV) that are difficult to detect using standard next generation sequencing techniques and analysis pipelines. The need to detect these complex SV and SNVs, all of which are at low allelic ratios (AR) at the remission state, demands assays that are capable of analyzing both DNA and RNA molecules at levels of detection comparable to MPFC. As proof of principal, we leveraged a unique capture technique (ArcherDx; CoreAML) to detect an important SV in pediatric AML, the internal tandem duplication in Fms related tyrosine Kinase (FLT3-ITD). Via a serial dilution of cells with known FLT3-ITD allelic ratio (MV4-11 cells), to determine a limit of detection, we were able to detect the mutation as low as 0.001. Next, we bench-marked the technique using diagnostic and relapse samples, and successfully detected FLT3-ITD at AR To complement the DNA-based approach, we compared two error-corrected (EC) RNA assays (a Druley lab developed protocol and the ArcherDx; HemeV2 panel) to further assay SVs that cause novel gene fusions and aberrant exon usage, which are not detectable via DNA assays. Compared to existing cytogenetic and RNA-seq data, this new platform was capable of detecting known and novel cryptic translocations. Finally, we integrated the results from the FLT3-ITD assay and the RNA assay with our custom error-corrected sequencing data to create a novel bioinformatics workflow for assessing the biological implications of MRD clones detected. Collectively, our data support that EC sequencing, at both the DNA and RNA level, enable accurate detection of low allelic variants that could be used for improved clinical MRD diagnostics, prognostication and therapeutic selection. Citation Format: Erin L. Crowgey, Nitin Mahajan, Wing H. Wong, Edward A. Kolb, Todd Druley. Sensitive and specific DNA and RNA sequencing techniques for detecting minimal residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4878. doi:10.1158/1538-7445.AM2017-4878

The retinoblastoma gene (RB1) has been implicated as a tumor suppressor in multiple myeloma (MM), yet its role remains unclear because in the majority of cases with 13q14 deletions, un-mutated RB1 remains expressed from the retained allele. To explore the role of Rb1 in MM, we examined the functional consequences of single- and double-copy Rb1 loss in germinal center B cells, the cells of origin of MM. We generated mice without Rb1 function in germinal center B cells by crossing Rb1(Flox/Flox) with C-γ-1-Cre (Cγ1) mice expressing the Cre recombinase in class-switched B cells in a p107(-/-) background to prevent p107 from compensating for Rb1 loss (Cγ1-Rb1(F/F)-p107(-/-)). All mice developed normally, but B cells with two copies of Rb1 deleted (Cγ1-Rb1(F/F)-p107(-/-)) exhibited increased proliferation and cell death compared with Cγ1-Rb1(+/+)-p107(-/-) controls ex vivo. In vivo, Cγ1-Rb1(F/F)-p107(-/-) mice had a lower percentage of splenic B220+ cells and reduced numbers of bone marrow antigen-specific secreting cells compared with control mice. Our data indicate that Rb1 loss induces both cell proliferation and death in germinal center B cells. Because no B-cell malignancies developed after 1 year of observation, our data also suggest that Rb1 loss is not sufficient to transform post-germinal center B cells and that additional, specific mutations are likely required to cooperate with Rb1 loss to induce malignant transformation.

Takayasu’s arteritis (TA) was reported as an eye disease in the year 1905 and later was confirmed as a vasculitis. Since then, the etiology of the disease remains unknown; however, characteristic clinical features suggest multiple causative factors. Recent progress in vascular biology and other disciplines enlightens the pathophysiology of TA and demonstrated induction of various nonspecific inflammatory symptoms and destruction of the arterial wall, which leads to aneurysms and rupture of the affected arteries. Matrix metalloproteinases (MMPs) as an enzyme family have well-established roles in several vascular pathologies including intima formation, atherosclerosiss and aneurysms. MMPs have been proposed to be one of the molecules with a potential of having dual role in the course of TA, first as an active participant in pathophysiology and secondly as a diagnostic biomarker for TA disease. The desire to improve our understanding of the importance of MMPs and their endogenous inhibitors (TIMPs) in TA disease and for the development of therapeutic agents has inspired basic and clinical scientists for over a decade. In the present paper, we summarized the scientific rationale which highlights the signatures of matrix metalloproteinases and their endogenous inhibitors in pathophysiology as well as their being a potential candidate as biomarker for Takayasu’s arteritis.

Systolic blood pressure (SBP) rises approximately linearly with age in most societies. The cause of this rise is unclear. We tested the hypothesis that SBP is causally associated with the rate of rise in SBP with age by evaluating the effect of 12 polymorphisms associated with lower SBP on the age-related rate of rise in SBP in a series of meta-regression analyses involving ≤199 477 participants in 63 studies. We then evaluated the effect of these polymorphisms on the odds of coronary heart disease in 22 223 case and 64 762 control subjects and compared it with the effect of lower SBP observed in both prospective cohort studies and blood pressure–lowering randomized trials. All 12 polymorphisms were associated with both lower SBP and a slower age-related rise in SBP. The weighted mean effect of these 12 polymorphisms was associated with a 0.32-mm Hg lower SBP ( P =1.79×10 −7 ) and a 0.093-mm Hg/decade slower age-related rise in SBP ( P =3.05×10 −5 ). The effect of long-term exposure to lower SBP on coronary heart disease mediated by these polymorphisms was 2-fold greater than that observed in prospective cohort studies ( P =0.006) and 3-fold greater than that observed in short-term blood pressure treatment trials ( P =0.001). We conclude therefore that SBP seems to be causally associated with the rate of rise in SBP with age and has a cumulative effect on the risk of coronary heart disease.

Background Oxidative stress and activation of matrix metalloproteinases (MMPs) together have been widely implicated in various inflammatory vascular disorders. As Takayasu's arteritis (TA) is an inflammatory disorder characterized by destruction of elastic fibers, we hypothesized that oxidative stress and MMPs might play an important role in pathophysiology of the disease. Methods 40 patients with clinically defined TA and equal number of normal, healthy, controls were enrolled in the study. Subjects with TA were further divided into clinically active disease or in remission phase of the disease. Results Augmented levels of 8-isoPGF 2α (238.9±125.7 vs 79.5±29.1ng/ml), nitrite (NO 2 − ) (14.9±15.6 vs 3.3±1.7μM) were observed in TA patients as compared to the controls ( p 2α (272.2±117.3 vs 105.9±43.5ng/ml) and NO 2 − (17.9±16.1 vs 3.1±2.5μM) levels were higher in subjects with active disease vs those in remission ( p p p >0.05). Further, gelatinolytic activity showed a positive correlation with 8-iso-PGF 2α and NO levels in patients with TA. Conclusions Increased oxidative stress and MMP activity may play an active role in the progression of Takayasu's arteritis disease.

Background Congestive heart failure in sinus rhythm ranks second after atrial fibrillation (AF) among cardiogenic risk-factors for stroke. Clinical and echocardiographic predictors of stroke in this high-risk population remain poorly defined. Methods Retrospective screening of 1886 consecutive patients with severe systolic dysfunction (LVEF ≤35%) at a tertiary medical center echocardiography database (Nov 2005–Sep 2008) identified 83 patients in sinus rhythm with cardioembolic stroke. Patients with AF on follow-up, prosthetic valve, ventricular arrhythmia and lack of consensus between reviewing neurologists were excluded ( n =10). Consecutive age and gender-matched controls in sinus rhythm formed GpII ( n =73). Results The incidence of stroke was 3.9% (73/1886) over 35 months in this study. There were no significant differences in prevalence of established clinical risk-factors for stroke. There was a significantly higher prevalence of LV non-compaction ( p =0.02), aneurysm ( p p p Conclusions LV non-compaction, aneurysm, spontaneous echo-contrast and pulmonary hypertension are associated with an increased risk of stroke. While anticoagulation of these high-risk subgroups appears reasonable, further study in a prospective randomized clinical trial merits consideration.

Hypertension is a disorder controlled by multiple genes and inflammation and vascular remodelling of arteries have been implicated in pathogenesis of this disease. Green tea polyphenols (GrTPs) are rich in antioxidants and are known to inhibit inflammatory responses. A significant time-dependent increase in mRNA expression of both IL-6 and MMP-9 were observed in THP-1 macrophages when cultured in normocholesterolaemic hypertensive sera (P

Objectives: Essential hypertension (EH) is a multifactorial disease and oxidative stress has been demonstrated to play an important role in its pathophysiology. Among several genes implicated in the pathophysiology of hypertension, endothelin-2 (ET-2), a potent vasoconstrictor, has been incriminated as a candidate gene for essential hypertension. To find out correlation of in vivo biomarkers of oxidative stress [oxidized low density lipoprotein (Ox-LDL), 8-iso- Prostaglandin F2α (8-iso-PGF2α)] and Nitrite (NO2-) are related to endothelin-2 levels and to determine their value as diagnostic markers in essential hypertension. Methods: 100 subjects with essential hypertension and 150 age and sex-matched normotensive controls were enrolled to determine levels of ET-2, Ox-LDL, 8-iso-PGF2α and NO2-. Results: We observed significantly augmented levels of ET-2 (P

The importance of epigenetic alterations such as DNA methylation, histone modification and nucleosome remodeling in breast cancer is well established. Epigenetic alterations are reversible, and much research has been focused on understanding these alterations with the aim of developing effective therapies. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors and has long been under investigation for its key role in tumor development and progression. To date, no studies have been conducted to elucidate the epigenetic modifications of PDEF in cancer progression. Using breast and prostate cancer cells, we investigated the effect of the methylation inhibitor 5' azacytidine (AZA) on the expression of PDEF in these cells. The inhibition of methylation observed was specific to breast cancer cells as experiments with prostate cells did not exhibit any significant change. Notably, the expression of p21, a cyclin-dependent kinase (CDK) inhibitor 1 and also a target gene of PDEF, was found to be positively correlated with PDEF expression following 5'AZA treatment. Inhibition of methylation led to a decrease in the proliferation rate of MDA-MB-468 cells as revealed by MTT proliferation assay. Other epigenetic alterations such as histone modifications were not observed in these breast cancer cells following treatment with specific HDAC inhibitors. Our data suggest the possibility of epigenetic modification of PDEF due to DNA methylation and involvement of the cell cycle inhibitor p21. Future studies on the epigenetic alterations of PDEF in correlation with p21 or other targets may facilitate the development of effective therapies for the treatment of breast cancer.

Historically, the receptor for advanced glycation end products (RAGE) was thought to exclusively play an important role under hyperglycemic conditions. However, more and more evidence suggests that RAGE in fact is an inflammation perpetuating multi-ligand receptor and participates actively in various vascular and inflammatory diseases even in normoglycaemic conditions. Various ligands include advanced glycation end products (AGEs), S100 proteins and amphoterins etc. Besides full-length RAGE, numerous truncated forms of the receptor have also been described including the well-characterized soluble RAGE (sRAGE). sRAGE has an ability to act as a decoy to avoid interaction of RAGE with its pro-inflammatory ligands. Ligand engagement of RAGE activates multiple signaling pathways and also forms a positive feedback loop for its own enhanced expression. This review will discuss the role of multi-ligand receptor i.e. RAGE in context to various vascular diseases, which have a pathophysiologically important inflammatory component in normoglycaemic conditions.

Coronary atherosclerosis is a chronic progressive disease that begins early in life and progresses slowly over several decades before becoming clinically manifest. The causal relationship between low-density lipoprotein cholesterol (LDL-C) and the risk of coronary atherosclerosis is well established. Multiple randomized trials have demonstrated that lowering LDL-C levels during treatment with a statin reduces the risk of major atherosclerotic coronary events. However, individuals being treated with a statin continue to experience a high residual risk of events. Here we review the evidence that lowering LDL-C levels beginning earlier in life, and therefore earlier in the atherosclerotic disease process, can prevent or substantially delay the development of atherosclerosis and thereby substantially improve the clinical benefit of therapies that lower LDL-C levels. We focus on providing a critical appraisal of the naturally randomized evidence that is emerging from recently conducted genetic association studies.

Takayasu's arteritis (TA) is a rare primary and granulomatous large vessel vasculitis of unknown origin that predominantly affects the aorta and itsmajor branches. It has been shown toaffect individuals of anyage, gender and a wide variety of ethnic and racial population worldwide. The pathophysiology of TA is complex and multi-factorial and the exact pathogenesis remains to be elucidated; however, participation of autoimmunity, inflammation and oxidative stress has potentially been implicated [1–3]. Receptor for advanced glycation endproducts (RAGE) is amemberof the immunoglobulin superfamily and is expressed on the surface of various cells e.g. endothelium, mononuclear phagocytes, lymphocytes and smooth muscle cells. Extracellular newly identified RAGE-binding protein (EN-RAGE or S100A12), an inflammatory ligand of RAGE, is a member of the S100 protein family. Since its discovery twodecades back [4], the inflammatory role of RAGE and EN-RAGE interaction is nowwell established in various chronic inflammatory disorders. RAGE has a circulating truncated variant isoform and soluble RAGE (sRAGE), corresponds to its extracellular domain only. By competing with cell-surface RAGE for ligand binding, sRAGE contributes to the removal/neutralization of circulating ligands, thus, functioning as a decoy [5–7]. Previously, we demonstrated low sRAGE levels showing inverse correlation with MMPs in a group of forty TA subjects as compared to controls [8]. Keeping in mind the importance of RAGE biology in various diseases, we thought it worth to determine the expression of RAGE and its inflammatory ligand EN-RAGE at transcriptional level in peripheral blood mononuclear cells (PBMCs) fromTA subjects. For this, 24 subjectswith TA and 24 normal healthy controls were recruited as described previously [3,8,9]. The Institutional Ethics Committee approved the present study. Venous blood was collected from overnight fasted individuals in the morning from antecubital vein and processed as described previously [5,8]. Semi-quantitative RT-PCR was performed for determining the transcriptional expression of RAGE andEN-RAGE in PBMCs [5]. Circulating levels of sRAGE were determined in all the study subjects using commercially available enzyme-linked immunoassay as per manufacturer's instructions (DRG00, RD p=0.003, Fig. 1E). However, no significant correlation of sRAGE levels was observed with either RAGE or EN-RAGE mRNA in TA subjects. This is the first report in literature which demonstrates that mRNA levels of RAGE and its ligand EN-RAGE are up-regulated in subjects with TA. This study also demonstrates a positive correlation of RAGE and ENRAGE, in subjectswith TA for thefirst time. Our data is supported by other reports, which demonstrate that RAGE expression increases stably in certain pathological processes, and is associated with ligand-rich microenvironments [6,7]. In a previous study, we demonstrated that RAGE and EN-RAGE genes are augmented in non-diabetic CAD subjects and showed their positive correlation with the severity of disease [5]. Ligation of this receptor with its ligands activates the downstream signaling pathways such as NF-κB [10]. In addition, RAGE and EN-RAGE interactions could further induce the expression of adhesion molecules, migration and proliferation of leukocytes and augment cytokine release from the lymphocytes. Excessive expression of these genes at sites of inflammation may feasibly form a positive feedback loop that may further aggravate underlying inflammation. In corroboration to this, other reports in literature and our work have clearly demonstrated a potential participation of various cytokines, chemokines and MMPs in the pathophysiology of TA [3,8,9,11]. Significantly increased RAGE–EN-RAGE expression in TA further confirms a prevalent oxidative stress and inflammatory milieu in such a situation. Presence of various functional splice variants of RAGE is another important characteristic of this particular receptor. The production of endogenous secretory RAGE by alternative splicing may influence the actions of ligands on the full form of RAGE, located on the plasma membrane [12]. Finding increased RAGE mRNA levels in TA subjects, one

Background Left main disease (LMD) and three-vessel disease (3VD) have important prognostic value in patients with coronary artery disease. However, uncertainties still exist about their prevalence and predictors in patients with acute coronary syndrome (ACS) and also in patients with stable coronary disease. Thus the aim of this study was to perform an international collaborative systematic review and meta-analysis to appraise the prevalence and predictors of LMD and 3VD. Methods Medline/PubMed were systematically searched for eligible studies published up to 2010, reporting multivariate predictors of LMD or 3VD. Study features, patient characteristics, and prevalence and predictors of LMD and 3VD were abstracted and pooled with random-effect methods (95% CIs). Results 17 studies (22 740 patients) were included, 11 focusing on ACS (17 896 patients) and six on stable coronary disease (4844 patients). In the ACS subgroup, LMD or 3VD occurred in 20% (95% CI 7.2% to 33.4%), LMD in 12% (95% CI 10.5% to 13.5%), and 3VD in 25% (95% CI 23.1% to 27.0%). Heart failure at admission and extent of ST-segment elevation in lead aVR on 12-lead ECG were the most powerful predictors of LMD or 3VD. In the stable disease subgroup, LMD or 3VD was found in 36% (95% CI 18.5% to 48.8%), with the most powerful predictors being transient ischaemic dilation during the imaging stress test, extent of ST-segment elevation in aVR and V1 during the stress test, and hyperlipidaemia. Conclusions This meta-analysis demonstrated that severe coronary disease—that is, LMD or 3VD—is more common in patients with ACS or stable coronary disease than generally perceived, and that simple and low-cost tools may help in the selection of the most appropriate therapeutic approach.

Although there appears to be a role for statins in reducing cerebrovascular events, the exact role of different lipid fractions in the etiopathogenesis of cerebrovascular disease (CVD) is not well understood. A secondary analysis of data collected for the placebo arm (n = 2,078) of the Cholesterol and Recurrent Events (CARE) trial was performed. The CARE trial was a placebo-controlled trial aimed at testing the effect of pravastatin on patients after myocardial infarction. Patients with histories of CVD were excluded from the study. A Cox proportional-hazards model was used to evaluate the association between plausible risk factors (including lipid fractions) and risk for first incident CVD in patients after myocardial infarction. At the end of 5 years, 123 patients (6%) had incident CVD after myocardial infarction (76 with stroke and 47 with transient ischemic attack). Baseline non–high-density lipoprotein (HDL) cholesterol level emerged as the only significant lipid risk factor that predicted CVD; low-density lipoprotein cholesterol and HDL cholesterol were not significant. The adjusted hazard ratios (adjusted for age, gender, hypertension, diabetes mellitus, and smoking) for CVD were 1.28 (95% confidence interval [CI] 1.06 to 1.53) for non-HDL cholesterol, 1.14 (95% CI 0.96 to 1.37) for low-density lipoprotein cholesterol, and 0.90 (95% CI 0.75 to 1.09) for HDL cholesterol (per unit SD change of lipid fractions). This relation held true regardless of the level of triglycerides. After adjustment for age and gender, the hazard ratio for the highest natural quartile of non-HDL was 1.76 (95% CI 1.05 to 2.54), compared to 1.36 (95% CI 0.89 to 1.90) for low-density lipoprotein cholesterol. In conclusion, non-HDL cholesterol is the strongest predictor among the lipid risk factors of incident CVD in patients with established coronary heart disease.

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA binding domain that controls key cellular processes. Prostate-derived Ets transcription factor (PDEF), a member of the Ets family, is reported to be present in tissues with high epithelial content, notably breast and prostate. However, the role of PDEF in cancer development is not fully understood. To gain insight into the molecular mechanisms associated with prostate cancer progression, we employed iTRAQ labeling followed by mass spectrometric (MS) analysis to identify candidate proteins that are differentially expressed in prostate cancer cells with or without PDEF. To this end, we overexpressed PDEF in PC3 human prostate cells using a tetracycline inducible system (Tet-On). Many differentially expressed proteins which play important roles in various cellular and biological processes were identified. Among them, stathmin (STMN), which is a microtubule (MT)-destabilizing protein, was found to be downregulated in multiple analyses. We demonstrated that re-expression of STMN reversed the antitumor properties of PDEF in PDEF-overexpressing PC3 cells. Using in vitro functional assays, we showed that STMN overexpression counteracted PDEF's effects against cell proliferation, colony formation and tumor migration. Similar results were further confirmed with the prostate cancer cell line CWR22rv1. In conclusion, many differentially expressed proteins were identified and STMN was found to be downregulated by PDEF. These results suggest that PDEF may inhibit prostate cancer progression by transcriptional downregulation of oncogenic STMN expression. Analyzing the association among differentially expressed proteins may provide a basis to better understand the molecular mechanisms underlying the process of cancer progression and development and further aid in designing therapeutics in the future.

stent thrombosis: strut coverage as a marker of endothelialization. Circulation 2007;115:2435–41. [4] Takebayashi H, Mintz GS, Carlier SG, et al. Nonuniform strut distribution correlates with more neointimal hyperplasia after sirolimus-eluting stent implantation. Circulation 2004;110:3430–4. [5] Turco MA, Ormiston JA, Popma JJ, et al. Polymer-based, paclitaxel-eluting TAXUS Liberte stent in de novo lesions: the pivotal TAXUS ATLAS trials. J Am Coll Cardiol 2007;49:1676–83. [6] TakanoM, Ohba T, Inami S, et al. Angioscopic differences in neointimal coverage and in persistence of thrombus between sirolimus-eluting stents and bare metal stents after a 6-months implantation. Eur Heart J 2006;27:2189–95. [7] YamamotoM, Okamatsu K, Inami S, et al. Relationship between neointimal coverage of sirolimus-eluting stents and lesion characteristics: a study with serial coronary angioscopy. Am Heart J 2009;158:99–104. [8] Takano M, Yamamoto M, Murakami D, et al. Lack of association large angiographic late loss and low risk of in-stent thrombus: angioscopic comparison between paclitaxeland sirolimus-eluting stent. Circ Cardiovasc Intervent 2008;1:20–7. [9] Coats AJ. Ethical authorship and publishing. Int J Cardiol 2009;131:149–50.

Background and Aims C-reactive protein (CRP) serves not only as a biomarker for the risk of cardiovascular disease and underlying inflammation but also functions as an active mediator of atherosclerosis by promoting activation of endothelial cells and monocytes. Peroxisome proliferator activated receptor-gamma (PPAR-γ) transcription factor has been recognized to regulate the expression of many genes involved in inflammation, lipid metabolism and vascular remodeling. Therefore, in the present study we tried to explore the role of CRP as a possible mediator of atherosclerosis by determining its effect on PPAR-γ and its effector genes, i.e., liver X receptor-α (LXR-α) and matrix metalloproteinase-9 (MMP-9) in THP-1 cells. Methods Semi-quantitative RT-PCR was used to determine mRNA expression. Results CRP upregulates the expression of PPAR-γ and LXR-α at lower doses (5–25 μg/mL), which were further declined at higher doses (50–100 μg/mL). However, a dose-dependent increase was observed for MMP-9 expression. Atorvastatin (10–20 μM) was able to significantly accelerate the CRP-induced expression of PPAR-γ and LXR-α and attenuate MMP-9 expression. Conclusions For the first time we demonstrate that CRP modulates PPAR-γ and its effector genes and reinforces the mechanistic link of CRP as a possible mediator in atherosclerosis and also advocate atorvastatin as a therapeutic modality.

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