GAPDH Ratio Research Articles

Artemisinin Attenuates Isoproterenol-induced Cardiac Hypertrophy via the ERK1/2 and p38 MAPK Signaling Pathways.

Artemisinin (ART) is mainly derived from Artemisia annua, a traditional Chinese medicinal plant, and has been found to affect cellular biochemical processes, such as proliferation, angiogenesis, and apoptosis, in addition to its antimalarial properties. However, its effect on cardiac hypertrophy and the underlying mechanisms remain unclear. This study aimed to investigate the effect of ART on cardiac hypertrophy and explore its possible mechanisms. A rat model was established by intraperitoneal injection of isoproterenol (ISO) for 3 days, and the degree of myocardial hypertrophy was compared among 5 groups: a control (CON) group, an ISO group, and groups treated with different doses of ART (7 mg/kg/d, 35 mg/kg/d, and 75 mg/kg/d). Echocardiography was used to evaluate cardiac function and structure. The cross-sectional area of cardiomyocytes was measured by hematoxylin and eosin (H&E) staining. The heart weight (HW), body weight (BW), and tail length were measured, and the HW/tail length ratio and the HW/BW ratio were calculated. H9c2 rat cardiomyocytes were cultured, and different amounts of ART were added 2 hours before ISO stimulation. Phalloidin staining was used to evaluate the degree of cell hypertrophy. The levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were quantified in rat plasma and cell supernatant using enzyme-linked immunosorbent assay (ELISA), while the expression levels of p- ERK1/2, p-JNK, and p-p38 MAPK were assessed in the myocardium and H9c2 cells via western blot analysis. Intragastric administration of ART at a dosage of 35 mg/kg/d or over mitigated the early-stage cardiac hypertrophy induced by ISO in rats led to a reduction in left ventricular posterior wall diastolic thickness, interventricular septal thickness at diastole, lowered ANP and BNP levels, as well as a decrease in HW/tail length and HW/BW ratio. In vitro studies demonstrated that ART at a concentration of 100 μM inhibited ISO-mediated hypertrophy of H9c2 cells. The ISO group showed a higher p-ERK/GAPDH ratio and p-p38 MAPK/GAPDH ratio than the control group both in vivo and in vitro. Although the p-JNK/GAPDH ratio was increased in the ISO group, there was no statistical difference. The p-ERK/GAPDH and p-p38/GAPDH ratios were significantly lower in the ART group than in the ISO group. The mechanism of ART against cardiac hypertrophy was related to inhibition of the ERK1/2 and p38 MAPK signaling pathways.

The Clinical and Prognostic Significance of Ribonucleotide Reductase Subunits RRM1 and RRM2 mRNA Levels in Patients with Chronic Lymphocytic Leukemia

Ribonucleotide Reductase (RNR) converts ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of subunits M1 and M2. It has been studied as a prognostic factor in several solid tumors and in chronic hematological malignancies, but not in chronic lymphocytic leukemia (CLL). Peripheral blood samples were collected from 135 CLL patients. M1/M2 gene mRNA levels were measured and expressed as a RRM1-2/GAPDH ratio. M1 gene promoter methylation was studied in a patients’ subgroup. M1 mRNA expression was higher in patients without anemia (p = 0.026), without lymphadenopathy (p = 0.005) and 17p gene deletion (p = 0.031). Abnormal LDH (p = 0.022) and higher Rai stage (p = 0.019) were associated with lower M1 mRNA levels. Higher M2 mRNA levels were found in patients without lymphadenopathy (p = .048), Rai stage 0 (p = 0.025) and Trisomy 12 (p = 0.025). The correlation between RNR subunits and clinic-biological characteristics in CLL patients demonstrate RNR’s potential role as a prognostic factor.

Long noncoding RNA BACE1-antisense transcript plays a critical role in Parkinson’s disease via microRNA-214-3p/Cell death-inducing p53-target protein 1 axis

ABSTRACT This study aimed to analyze the function and latent mechanism of long noncoding RNA BACE1-antisense transcript (lncRNA BACE1-AS) in MPP+-induced SH-SY5Y cells. SH-SY5Y cells were cultivated in 1 mM MPP+ for 24 h to establish Parkinson’s disease (PD) model in vitro. TargetScan and luciferase reporter assay were conducted to predict and verify the interaction between microRNA (miR)-214-3p and CDIP1 (Cell death-inducing p53-target protein 1). Cell viability, lactate dehydrogenase (LDH) release, and cell apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT), LDH, and flow cytometer. The secretion of inflammatory factors and representative biomarkers of oxidative stress, including reactive oxygen species (ROS) and superoxide dismutase (SOD) were assessed using enzyme-linked immunosorbent assay (ELISA) and specific assay kits. Results suggested that lncRNA BACE1-AS was over-expressed and miR-214-3p was under-expressed in MPP+-stimulated SH-SY5Y cells. Further analyses revealed that MPP+ inhibited cell viability; enhanced cell apoptosis, Cleaved Caspase-3 expression and Cleaved Caspase-3/GAPDH ratio; induced oxidative stress and inflammation in SH-SY5Y cells were inhibited by lncRNA BACE1-AS-siRNA transfection; and all these inhibitions were reversed by miR-214-3p inhibitor. In addition, we found that CDIP1 was directly targeted by miR-214-3p and up-regulated in MPP+-stimulated SH-SY5Y cells. Further functional assays suggested that CDIP1-plasmid reversed the effects of miR-214-3p mimic on MPP+-stimulated SH-SY5Y cells. In conclusion, lncRNA BACE1-AS regulates SH-SY5Y cell proliferation, apoptosis, inflammatory response, and oxidative stress through direct regulation of miR-214-3p/CDIP1 signaling axis, and could be a potential candidate associated with the diagnosis and treatment of PD.

A Study of Ribonucleotide Reductase mRNA Expression and Its Prognostic Role in Patients with Chronic Lymphocytic Leukemia

BackgroundRibonucleotide Reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of two subunits, termed subunit 1 (RRM1) and 2 (RRM2). Imbalance in the regulation of RNR activity and control of dNTPs' pool leads to genomic instability and increases mutation rate. RNR expression has been associated with prognosis in pancreatic, non-small-cell lung, breast, and biliary tract cancer. However, RNR expression in chronic lymphocytic leukemia (CLL) and its possible prognostic role have not been investigated yet.AimIn this study we evaluate the possible prognostic role of RNR expression in CLL.MethodThe study comprised patients with immunophenotypically confirmed disease at the time of sample collection. Peripheral whole blood samples were collected from 84, 27, 15, and 9 patients before treatment, after one, two, and three lines of treatment respectively.RNA extraction and reverse transcription were carried out using standard protocols. A Taqman based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For both the housekeeping and target genes, a Taqman primer/probe mix was used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). RRM1 and RRM2 mRNA levels were expressed as an RRM1-2/GAPDH ratio. Western blot analysis was performed to quantify the RRM1 protein levels in a random sample of 41 patients. Antibodies used were: RRM1 #3388, β-actin #4967 and anti-rabbit IgG HRP-conjugated #7074 (Cell Signaling Technology, Danvers, MA, USA). Detection was done using the ECL western blotting reagents.Statistical analysis was conducted to study the possible correlations between the variables. All reported p values are two-tailed. Statistical significance was set at p<0.05 and analyses were conducted using SPSS statistical software (version 22.0).ResultsFrom 135 CLL patients included in the study 56.3% were female and the median age at diagnosis was 64 years. Peripheral blood was collected in 84 treatment-naïve patients (62.2%). Median follow up was 6.66 years (3.47 ─ 11.13) and median time from diagnosis until 1st line treatment was 23.1 months (IQR: 5.8 - 56.5 months). Out of 135 patients, 69 (51,1%) received 1 st line treatment and 35 patients (25,9%) 2 nd line treatment with median time between the two treatment lines being 26.5 months (IQR: 7.8 - 40.8 months). Furthermore, 48.5%, 33.8%, 12.3%, 3.1% and 2.3% of the patients had Rai score 0, I, II, III, IV respectively. The median mRNA expression of RRM1 was 0.04 (IQR: 0 - 0.09) and of RRM2 was 0.01 (IQR: 0 - 0.1).RRM1 mRNA expression was significantly higher in patients without anemia (p=.025) and without lymphadenopathy (p=.002). Higher values of ESR (r=-.30; p=.028), LDH (r=-.20; p=.026) and Rai score (r=-.18; p=.037) were associated with lower expression of RRM1 mRNA. In addition, TP53 gene deletion detected by FISH was associated with higher RRM1 mRNA expression (p=.036). Significantly higher RRM2 mRNA expression was reported in patients without lymphadenopathy (p=.021) and Rai score 0 (p=.003). Moreover, higher was the expression of RRM2 mRNA in cases with Trisomy 12 (p=.050).In samples collected before treatment, higher values of RRM1 mRNA expression were statistically significantly associated with lower RAI score (r=-.30; p=.005) and longer time periods between the first two lines of treatment (r=.95; p=.050).Western blot analysis confirmed detection of RRM1 protein but statistical correlation was not carried out due to lack of material from the whole group of patients.ConclusionFor the first time, mRNA expression of RRM1 and RRM2 is studied in patients with CLL. These results show RNR involvement in the pathophysiology of CLL. RRM1 and RRM2 mRNA higher expression found in 17p deletion and trisomy 12 cases respectively may be consistent with the existence of a methylation-depended mechanism proposed by other studies. Therefore, these results demonstrate RNR's potential role as a prognostic factor, and make it a probable therapeutic target. A study including a larger number of cases could further confirm our results. [Display omitted] DisclosuresKyrtsonis: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Panagiotidis: Abbvie: Research Funding; Pfizer: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Sandoz: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Research Funding; Astellas: Research Funding. Viniou: Sandoz: Research Funding; Takeda: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Roche: Research Funding; Astellas: Research Funding; Celgene: Research Funding.

Expression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 in Human Epicardial and Intramyocardial Coronary Arteries of Male Patients Undergoing Coronary Artery Bypass Grafting

Objectives: Atherosclerosis is almost absent in intramyocardial coronary arteries, while epicardial coronary arteries may show extensive occlusive disease. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression has been implicated in atherogenesis. We aimed to investigate differences in arterial wall LOX-1 expression between human epicardial and intramyocardial coronary arteries. Methods: Epicardial and intramyocardial total artery wall specimens were obtained from 13 male patients (aged 61.9 ± 10.3 years) undergoing coronary artery bypass graft surgery due to 3-vessel coronary artery disease. LOX-1 expression was examined by reverse transcription-polymerase chain reaction. Results: LOX-1 expression was significantly higher in the arterial wall of epicardial coronary arteries compared to intramyocardial coronary arteries. The LOX-1/GAPDH ratio was 0.48 ± 0.07 vs. 0.35 ± 0.03 (p < 0.001). Conclusions: Our findings may partially explain the atheroprotective effect of the intramyocardial course since arterial wall LOX-1 expression was lower in intramyocardial arteries and higher in epicardial coronary arteries.

P-003: Detection of bacterial and host proteins from a single colonic biopsy sample by a new mass spectrometry-based assay in pediatric ulcerative colitis

P-003: Detection of bacterial and host proteins from a single colonic biopsy sample by a new mass spectrometry-based assay in pediatric ulcerative colitis

P-209 YI Decreased Pregnane X Receptor (PXR) Expression in Inflamed Versus Non-Inflamed Small Bowel of Children with Crohn’s Disease

Human pregnane X receptor (PXR) is a xenobiotic nuclear receptor responsible for transcriptional regulation of specific drug metabolizing enzymes (e.g., CYP3A4) and transporters (e.g., ABCB1). PXR dysregulation has also been implicated in the pathogenesis of IBD in a well-established model of chemically-induced IBD in mice, as well as some human studies. Data on PXR expression in intestinal tissue of pediatric patients with Crohn’s Disease (CD) are limited, with no current conclusive information regarding PXR expression in healthy versus inflamed mucosa. The primary objective of this single center, retrospective, pilot study was to test the hypothesis that PXR expression is decreased in actively inflamed small intestinal tissue from pediatric patients with CD. RNA was extracted from archived formalin-fixed paraffin-embedded (FFPE) intestinal biopsy samples obtained during routine EGD/colonoscopy in pediatric patients, aged 7 to 18 years, with a diagnosis of CD (n = 18) and age- and sex-matched Controls without IBD (n = 12). Tissue samples with adequate mRNA quality, as determined by 3’:5’ GAPDH ratio <10, were included in the analysis of relative mRNA expression of PXR in inflamed terminal ileum (TI) versus non-inflamed duodenum. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) was used to determine relative mRNA expression. All data are reported as relative expression normalized against GAPDH, an established reference gene in the human small intestine, and are log transformed. Additionally, relative villin expression (an indicator of epithelial cell content) was determined across tissue types and disease states. Differences in relative mRNA expression were explored using a paired t-test; α = 0.05. In the cohort of children with CD, a statistically significant decrease in PXR expression was observed in inflamed TI versus non-inflamed duodenum (relative expression TI=1.88 ± 0.89 versus duodenum = 2.5 ± 0.67; P = 0.0003). In contrast, no difference in PXR expression was observed between non-inflamed TI versus non-inflamed duodenum in Controls (TI=2.11 ± 0.41 versus duodenum = 2.26 ± 0.61; P = 0.52). Similarly, villin expression was significantly decreased in the inflamed TI of children with CD compared to non-inflamed duodenum from the same children (TI = 3.79 ± 0.91 versus duodenum = 4.61 ± 0.52; P = 0.0002). This difference in villin expression was not observed in non-inflamed TI versus non-inflamed duodenum in Controls (TI = 4.30 ± 0.35 versus duodenum = 4.07 ± 0.4; P = 0.26). Finally, GAPDH expression did not differ with age, sex, or location along the small intestine (i.e., TI versus duodenum) in the CD or the Control group. Our findings demonstrate no apparent regional difference in PXR expression in the small intestine of children without IBD. In contrast, our data demonstrate a statistically significant decrease in PXR expression in actively inflamed versus non-inflamed small intestine of pediatric patients with CD. This differential pattern of PXR expression in CD may suggest a potential role for PXR in disease phenotype expression and, potentially, in the regulation of proteins important to drug disposition (i.e., CYP3A4, ABCB1). It is not yet clear how the observed differences in epithelial cell content contribute to the observed decrease in PXR, as the differences themselves may be the result of inflammation or disease state. Additional studies are currently underway.

Clinical Utility of PRV-1 mRNA Expression Levels in Polycythemia Vera and Essential Thrombocythemia.

AbstractAbstract 2846 Background:PRV-1 mRNA is overexpressed in most patients with polycythemia vera (PV) and a part of patients with essential thrombocythemia (ET). It is well known that PRV-1 expression levels correlate with JAK2 V617F mutation. While it was reported that JAK2 V617F clones can be significantly reduced or become undetectable by treatment with hydroxyurea (HU)[Girodon et al. Heamatologica 93:1723, 2008].The change of PRV-1 expression after treatment of myeloproliferative neoplasm (MPN) has been examined in only several small-scale studies with inconclusive results. Although PRV-1 overexpression is not included in the diagnostic criteria for MPN, it can be a potentially important biomarker in making diagnosis and predicting outcome. However, its clinical utility has not been fully investigated. Methods:From 1999 to 2011, 309 cases of PV and 451 cases of ET were registered in Chang Gung Memorial Hospital. The diagnosis was reclassified according to the 2008 WHO system. Among them, 203 cases of PV and 228 cases of ET were examined for PRV-1 overexpression and formed the basis of this study. Evolution from ET to PV during clinical follow-up (ET-PV) was observed in 41 patients and 16 of them have PRV-1 data available. Quantification of PRV-1 mRNA was performed by the real-time polymerase chain reaction with TaqMan assay on peripheral blood granulocytes. Data from fifty healthy donors served as normal controls. The results were compared between MPN subgroups (ET vs. PV vs. ET-PV) and correlated with JAK2 V617F allele burden, white blood cell counts, platelet counts and clinical thrombosis or bleeding events. PRV-1 mRNA expression levels of patients before and after HU treatment were compared. In addition, the pre-HU and post-HU data were analyzed in pairs to evaluate the impact of HU therapy on PRV-1 mRNA expression. Results:Levels of PRV-1 expression were significantly different between controls and ET ( P<0.01), and between ET and PV ( P<0.001). ET-PV patients’ pre-HU PRV-1 expression was comparable to other PV (P>0.05) but significantly higher than ET patients (P<0.01). PRV-1 overexpression, defined as pre-treatment PRV-1 /GAPDH ratio greater than 1.13, was found in 127 PV and 46 ET patients, which translated into 90.7% sensitivity and 75.3% specificity in differentiating PV from ET. When the post-HU PRV-1 /GAPDH ratio was used for differential diagnosis between PV and ET, the sensitivity was 90.5% and specificity 71.4%. PRV-1 expression levels were proportional to JAK2 V617F allele burden (r2=0.065, P=0.004). White blood cell counts were proportional to PRV-1 expression for both PV and ET (P<0.001 for PV, P<0.0001 for ET). The PRV-1 expression levels were divided into 4 quartiles, the top quartile had a risk ratio of 6.55 for developing thrombosis compared with the bottom quartile (95% CI 2.01–21.32, P=0.0008). In either PV or ET group, no difference was found between pre-HU and post-HU PRV-1 expression (P=0.579 for PV; P=0.253 for ET and P=0.143 for both subtypes combined). In analysis of the paired matched pre-HU and post-HU samples, there was no significant change of PRV-1 expression after HU treatment in any subtype (N=21, P=0.613 for PV; N=14, P=0.463 for ET and N=35, P=0.794 for both subtypes combined). Pre-treatment levels of PRV-1 expression were highly correlated with levels after HU (r2=0.20, P=0.007). Using 1.13 as the cut-off for definition of overexpression, the pre-HU and post-HU PRV-1 expression status was concordant in 72% of cases. The results of PRV-1 expression status largely unaffected by HU therapy have important clinical relevance in differential diagnosis when dealing with MPN patients who already received partial treatment. Conclusions:PRV-1 expression level is a useful predictor of risk of thrombosis, highly correlated with JAK2 V617F mutant levels and may serve as a surrogate marker for classification and prognostication of MPN. Disclosures:No relevant conflicts of interest to declare.

Abstract 276: Angiotensin AT1 Receptor Blockade Reverses Age-Related Changes in Myocardial Energy Metabolism and Increases Mitochondrial Biogenesis

Aging and mitochondrial function have been closely linked. We recently reported the identification of a mitochondrial angiotensin system. We hypothesized that angiotensin AT1 receptor blockade would increase energy production and mitochondrial biogenesis and reduce oxidative stress in aging hearts. We used Magnetic resonance spectroscopy to measure cardiac energy metabolism and function in young (20 wks old), aged (150 wks old) mice at baseline and after 4 weeks of losartan (50 mg/kg/day). For mitobiogenesis, qPCR was used to calculate CytB (mitochondrial gene)/GAPDH (nuclear gene) ratio and to measure mito-survival genes Sirt1, Sirt3, Nampt, and PGC-1α. Cardiomyocyte mitochondria from young, aged and treated mice were examined with electron microscopy. The expression of nitrotyrosine was quantified by immunohistochemistry. Older animals hearts (n=9) exhibited increase in LV mass (103±9 mg versus 120±8 mg, young (n=8) versus old (n=9), P&lt;0.002). The mean cardiac PCr/ATP was reduced in older animals (1.5±0.2) than that of young mice (2.0±0.3, P&lt;0.0004). Losartan abolished the LV mass increase in older animals (109±11 mg vs 101±7 mg, young versus old, P&lt;0.1) and improved the impaired energy metabolism of the older hearts increasing the PCr/ATP ratios towards those observed in younger animals (1.94±0.01 vs 1.87±0.4, control versus old, P&lt;0.7). Losartan increased EF in older animals (56±5% vs 63±5%, old versus old treated, P&lt;0.01). Losartan increased mitobiogenesis in the hearts of treated young and old mice (3.8+2.5 folds, P&lt;0.02 and 4.3+ 0.9 folds, P&lt;0.0001). Mito-survival genes in the heart were not increased but PGC-1α was up-regulated by 2.8+1.6-fold, P&lt;0.05 and 7+ 1.9-fold, P&lt;0.001 in young and old treated mice. Electron micrograph analysis revealed that aging was associated with swollen cardiac mitochondria and disrupted cristae, which were reversed by Losartan. Losartan in older animals significantly reduced oxidative damage as evidenced by less Nitrotyrosine staining score in cardiomyocytes (2.5±0.5 vs. 1.3±0.4, old versus old treated, P&lt;0.0009). Our results indicate that Losartan in aging increased mitobiogenesis, reduced oxidative stress, improved energy production and restored cardiac function to the healthy young adult level.

Inhibitory effects of 405 nm irradiation on Chlamydia trachomatis growth and characterization of the ensuing inflammatory response in HeLa cells

BackgroundChlamydia trachomatis is an intracellular bacterium that resides in the conjunctival and reproductive tract mucosae and is responsible for an array of acute and chronic diseases. A percentage of these infections persist even after use of antibiotics, suggesting the need for alternative treatments. Previous studies have demonstrated anti-bacterial effects using different wavelengths of visible light at varying energy densities, though only against extracellular bacteria. We investigated the effects of visible light (405 and 670 nm) irradiation via light emitting diode (LEDs) on chlamydial growth in endocervical epithelial cells, HeLa, during active and penicillin-induced persistent infections. Furthermore, we analyzed the effect of this photo treatment on the ensuing secretion of IL-6 and CCL2, two pro-inflammatory cytokines that have previously been identified as immunopathologic components associated with trichiasis in vivo.ResultsC. trachomatis-infected HeLa cells were treated with 405 or 670 nm irradiation at varying energy densities (0 – 20 J/cm2). Bacterial growth was assessed by quantitative real-time PCR analyzing the 16S: GAPDH ratio, while cell-free supernatants were examined for IL-6 and monocyte chemoattractant protein-1 (CCL2) production. Our results demonstrated a significant dose-dependent inhibitory effect on chlamydial growth during both active and persistent infections following 405 nm irradiation. Diminished bacterial load corresponded to lower IL-6 concentrations, but was not related to CCL2 levels. In vitro modeling of a persistent C. trachomatis infection induced by penicillin demonstrated significantly elevated IL-6 levels compared to C. trachomatis infection alone, though 405 nm irradiation had a minimal effect on this production.ConclusionTogether these results identify novel inhibitory effects of 405 nm violet light on the bacterial growth of intracellular bacterium C. trachomatis in vitro, which also coincides with diminished levels of the pro-inflammatory cytokine IL-6.

The cannabinoid receptor type 2 is time-dependently expressed during skeletal muscle wound healing in rats

The expression of the cannabinoid receptor type 2 (CB2R) was investigated by immunohistochemistry, Western blotting, and RT-PCR during wound healing of contused skeletal muscle in rats with attempt of its applicability to skeletal muscle wound age estimation. Furthermore, Macrophage Marker (MAC387) was utilized to identify macrophages recruited into injured skeletal muscle tissue. Co-localization of CB2R with Macrophage Marker was detected by confocal laser scanning microscopy. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, immunoreactivity of CB2R was detected in the sarcolemma and sarcoplasm of normal myofibers. In the contusion groups, a few polymorphonulcear cells, a large number of macrophages, and spindle-shaped fibroblastic cells showed a positive staining for CB2R in wounded zones. By Western blotting analysis, the average of CB2R to GAPDH ratios in 5-7 days post-injury groups was highest, and all the samples had ratios of >2.60. In the other groups, no samples showed ratios of >2.60 and the CB2R to GAPDH ratios ranged from 1.19 to 2.59. The expression tendency was also confirmed by RT-PCR. From the viewpoint of forensic pathology, these observations suggested that the ratio markedly exceeding 2.60 strongly indicated a wound age of 5-7 days. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R be involved in modulating macrophages in response to inflammatory event in rat skeletal muscle wound healing and CB2R be available as a marker for wound age determination.

Applicability of Real-Time PCR Methodology in the Neonatal Detection of Turner Syndrome

Turner syndrome (TS) is the complete or partial loss of the second sex chromosome, occurring in 1:5 000 girls. Early recognition allows appropriate therapy for short stature and puberty. Neonatal diagnosis of TS permits detection of associated malformations, minimizing sequels. Aiming to develop a molecular method for the diagnosis of TS we employed blood samples stored on filter paper. We evaluated 78 female controls, 25 TS girls with 45,X karyotype, and 32 TS patients with other karyotypes. After DNA extraction, samples were submitted to real-time PCR, using primers and probes directed to the study gene ARSE and to the control gene GAPDH. A ROC curve established the ARSE:GAPDH ratio with a cutoff value of 0.7. Low ARSE:GAPDH ratio of <0.7 was present in 100% of 45,X TS patients. This cutoff value presented a sensitivity of 100% and a specificity of 100% in detecting 45,X TS patients with a positive predictive value of 100% and a negative predictive value of 100%. The same cutoff value was able to identify only 56% of TS with other karyotypes, in which we observed a mean (SD) ARSE:GAPDH ratio=0.66 (0.2); and the interquartile range=0.4-0.8. Determination of ARSE:GAPDH ratio is a fast, sensitive, and specific method, with viable cost and feasible automation, which makes it potentially applicable in neonatal screening programs for the diagnosis of Turner syndrome 45,X.

Cumulative Episodes of Rejection Altered Myocardial Sarcoplasmic Reticulum Ca2+-ATPase and Ryanodine Receptor-2 mRNA Expression in Heart Transplant Recipients

The aim of the present study was to investigate the relationship between rejections and gene expression of Ca(2+)-handling proteins in heart transplant recipients. Thirty-seven heart transplant recipients underwent routine endomyocardial biopsy. Levels of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) and ryanodine receptor-2 mRNAs in endomyocardial tissue were quantified by a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Rejections were diagnosed according to the conventional International Society for Heart and Lung Transplantation criteria. Patients were classified as follows; group AR(+) (n = 9) with rejection grade of 2 or higher versus group AR(-) (n = 28) with rejection grade of 0, 1a or 1b at the time of biopsy, and group Rec-AR(+) (n = 6) with a history of more than 4 episodes of treatment required rejection versus group Rec-AR(-) (n = 31) without history of recurrent rejection. The mRNA levels of the SERCA2/GAPDH ratio and ryanodine receptor-2/GAPDH ratio were not different between group AR(+) and group AR(-); however, they were reduced in group Rec-AR(+) more than in group Rec-AR(-) (0.83 +/- 0.07 versus 0.90 +/- 0.07, P = 0.034, 0.74 +/- 0.06 versus 0.84 +/- 0.10, P = 0.027, respectively). A single episode of on-going rejection would not affect myocardial Ca(2+)-handling proteins; however, cumulative rejection episodes might alter the gene expression of myocardial Ca(2+)-handling proteins in heart transplant recipients.

Evaluation of HER-2 gene status in gastric carcinoma using immunohistochemistry, fluorescence in situ hybridization, and real-time quantitative polymerase chain reaction

HER-2 gene amplification and the overexpression of HER-2 protein have been observed in various solid tumors, including gastric carcinomas. HER-2 gene amplification has attracted research attention since the development of the new therapeutic agent trastuzumab. Here, we evaluated HER-2 status in the surgically resected tissues of 248 gastric carcinoma cases using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real-time quantitative polymerase chain reaction (q-PCR) and compared the results. In addition, we compared clinicopathologic characteristics with the presence of HER-2 gene amplification and with protein overexpression. Among the 248 cases, 56 (22.6%) cases showed HER-2 overexpression (2+ or 3+) by IHC and 19 cases (7.7%) showed HER-2 gene amplification by FISH. Four (2.1%) of the 192 cases negative (0 or 1+) by IHC showed amplification by FISH, whereas 15 (26.8%) of the 56 cases with HER-2 protein overexpression showed HER-2 amplification by FISH. The correlation between IHC and FISH results was statistically significant (P < .001). HER-2 protein overexpression and HER-2 gene amplification were common in cases with a well- or moderately differentiated histology according to the World Health Organization classification (P < .001) and in cases of intestinal type by the Lauren classification (P < .001). Real-time q-PCR results showed that calculated HER-2/GAPDH ratios were higher in amplified cases with 100.0% sensitivity and 96.9% specificity using FISH results as the standard. Measurements of HER-2 expression by FISH and real-time q-PCR and of HER-2 protein by IHC were found to be highly concordant at determining HER-2 status in gastric carcinoma.

Fibrotic response to angiotensin II is blunted in the kidney, but not in the heart, in insulin-sensitive long-lived transgenic dwarf rats

Insulin resistance is a characteristic feature of cardiovascular and renal diseases, and angiotensin II (Ang II) has been suggested to induce insulin resistance. The aims of this study were to elucidate the effect of chronic Ang II infusion on vascular reactivity and organ damage in insulin-sensitive rats. We confirmed the following three points. First, there was no significant difference in pressor response to chronic Ang II infusion (600 ng/kg/min) between insulin-sensitive transgenic rats (Tg) and control rats (C). Second, there was no significant difference in cardiac hypertrophy and fibrosis by chronic Ang II infusion between the two groups. However, third, fibrotic response to chronic Ang II infusion evaluated by histopathological scoring in the kidney was significantly decreased in insulin-sensitive transgenic rats (renal fibrosis and nephropathy score: C+Ang II vs Tg+Ang II; 2.5 vs 1.3; p<0.05). Furthermore, the expression of TGF-beta, a fibrosis indicator, was also significantly suppressed in the kidneys of the transgenic rats (TGF-beta1/GAPDH ratio: C+Ang II vs Tg+Ang II; 1.15 vs 0.81; p<0.05). This result indicates that the growth hormone/insulin-like growth factor-1 axis is critically involved in the development of renal injury and fibrosis, rather than hypertension, cardiac hypertrophy, and cardiac fibrosis induced by chronic Ang II administration.

Effects of Globin mRNA Reduction Methods on Gene Expression Profiles from Whole Blood

Excessive globin mRNA in whole blood RNA decreases transcript detection sensitivity and increases signal variation on microarrays. Hence, methods based on peptide nucleic acid inhibitory oligos and biotinylated DNA capture oligos have been developed to reduce globin mRNA. However, there is limited information about the effects of these two methods on gene expression profiles. Thus, we systematically compared the facility and effects of the two globin reduction methods on profile measurements from Jurkat cell line RNA with or without spiked globin mRNA and human blood RNA isolated using PAXgene collection tubes. We showed that the methods were efficient at increasing the sensitivity of transcript detection without loss of specificity, but neither method could recover a profile equivalent to that of an identical RNA sample without globin mRNA excesses. The capture oligo method had slightly better transcript detection sensitivity for cell line RNA, lowered signal variation for PAXgene RNA, and more similar profiles to controls than the inhibitory method. However, the capture method required larger amounts of initial high-quality RNA to yield sufficient cRNA amounts, and its procedures were more complex and time consuming than the inhibitory method. These results inform the selection of methods suitable for multicenter surveillance of gene expression profiles.

Inhibition of tumor necrosis factor-α-induced intercellular adhesion molecule-1 expression in diabetic rats: role of insulin

The effects of insulin on intercellular adhesion molecule (ICAM)-1-mediated leukocyte adhesion and migration were investigated. Diabetic rats (alloxan, 42 mg/kg, i. v., 42 days), matching controls, and insulin (NPH, 2 IU/day for 12 days) treated diabetic rats were used. The internal spermatic fascia of the animals was used for direct vital microscopy of the microcirculation, and for quantitation of ICAM-1 expression by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Experiments were performed 2 h after the local injection of recombinant rat tumor necrosis factor-alpha (5 ng). Relative to controls (C), diabetic (D) rats exhibited a reduced number of adhered (D: 2.2 +/- 0.4 and C: 14.1 +/- 0.6 cells/100 microm venule length, P < 0.001) and migrated leukocytes (D: 1.1 +/- 0.3 and C: 6.3 +/- 0.6 cells/1,000 microm (2), P < 0.001) accompanied by low expression of ICAM-1 in postcapillary venules (D: 18 +/- 4 and C: 51 +/- 7 arbitrary units, P < 0.001). There were no differences in ICAM-1 mRNA levels (D: 1.01 +/- 0.05 and C: 1.18 +/- 0.09 ICAM-1/GAPDH ratio, P > 0.05). Treatment of diabetic rats with insulin restored the number of adhered (10.9 +/- 1.2 cells/100 microm venule length), and migrated leukocytes (4.0 +/- 0.3 cells/1,000 microm (2)) as well as ICAM-1 expression (45 +/- 3 arbitrary units). Levels of mRNA for ICAM-1 remained unchanged after treatment (1.15 +/- 0.04 ICAM-1/GAPDH ratio). Insulin modulates TNF-alpha-induced ICAM-1 expression on microvascular endothelium controlling, therefore, leukocyte adhesion and migration.

Losartan reduces monocyte chemoattractant protein-1 expression in aortic tissues of 2K1C hypertensive rats

Background Previous study has demonstrated an arterial inflammatory response in aortic tissues in several hypertensive models which can not be fully explained by hemodynamic forces. This study sought to investigate the effect of angiotensin II (Ang II)and its subtype-1 receptor blocker Losartan on the chemokine expression of monocyte chemoattractant protein-1(MCP-1) in aortic tissues of acute stage of 2-kidney-1-clip (2K1C) hypertensive rats. Methods 2K1C renovascular hypertension was produced in male Wistar–Kyoto(WKY) rats by placing a silver clip with an internal diameter of 0.2 mm around the left renal artery. The MCP-1 mRNA on aortic wall was detected by in situ hybridization and reverse transcription-polymerase chain reaction(RT-PCR); the levels of Ang II in plasma and aorta were determined by radioimmunoassay. The concentration of MCP-1 in supernatant of cultured endothelial cells (ECV-304) was measured by ELISA. Results No MCP-1 was exhibited in aortic wall of normotensive rats both by RT-PCR and in situ hybridization; it would be expressed on the aortic wall of rats in 7 days after 2K1C hypertensive model was formed, especially in intima. The expression of MCP-1 in aortic wall was increased with the duration of hypertension and correlated with local Ang II activity ( r = 0.594, P = 0.02) other than those in plasma, it was decreased obviously after being treated by Losartan for 28 days (optical density of MCP-1 / GAPDH ratio: 0, 0.58 ± 0.10, 1.14 ± 0.09, 1.52 ± 0.20, 0.66 ± 0.07, respectively, P < 0.01). Ang II had increased the expression of MCP-1 in endothelial cells; the highest levels had been performed at 1 × 10 − 7 mol/l Ang II or after 4 h, respectively; and losartan markedly reduced the expression of MCP-1. Conclusions The expression of MCP-1 significantly increases in aortic tissues of the acute stage 2K1C hypertensive rats and is decreased markedly by treatment of losartan. These findings imply Ang II may be involved in facilitating MCP-1 production in hypertension, and may provide a molecular link between hypertension and the development of atherosclerosis.

Neutrophil and Eosinophil PRV-1 Expression in Atypical Myeloproliferative Disorders.

Background: Neutrophil PRV-1 over-expression has been strongly associated with polycythemia vera (PV) and its quantitative measurement might complement the diagnostic distinction between PV and secondary polycythemia (Klippel et al. Blood 2003; 102:3569). Furthermore, the PV-specificity of the particular abnormality had been suggested by studies that demonstrated increased PRV-1 expression in post-polycythemic but not agnogenic myeloid metaplasia (Tefferi et al. Blood 2003; 103:3547) as well as by other studies that suggested the inevitable progression into PV of essential thrombocythemia (ET) associated with PRV-1 over-expression (Griesshammer et al. Ann Hematol 2004; 83:364). Still others have suggested the possibile utility of the particular assay in differentiating clonal from reactive myeloproliferation (Cilloni et al. Blood 2004; 103:2428).Methods: The purpose of the current study was two-fold; i) to further evaluate the specificity of neutrophil PRV-1 over expression to either PV or clonal myeloproliferation by testing the assay in atypical myeloproliferative disorders (AMPD) and ii) to evaluate eosinophil PRV-1 expression in AMPD associated with eosinophilia. In a prospective study, 13 patients with AMPD and 2 with reactive eosinophilia were evaluated for neutrophil PRV-1 expression (table 1). PRV-1 transcript quantitation was performed according to published methods (Klippel et al. Blood 2003; 102:3569). PRV-1 gene expression was considered elevated in the presence of a PRV-1/GAPDH ratio of 1.17 or lower.Table 1PatientDiagnosisNeutrophil PRV-1/GAPDH ratioNeutrophil PRV-1 expression1Chronic myelomonocytic leukemia associated with myelofibrosis1.05Increased2FIP1L1/PDGFRA-positive eosinophilic disorder1.39Normal3FIP1L1/PDGFRA-positive eosinophilic disorder1.19Normal4FIP1L1/PDGFRA-positive eosinophilic disorder1.10Increased5Biphenotypic acute leukemia with prominent blood eosinophilia0.99Increased6Hypereosinophilic syndrome with cardiac involvement1.15Increased7Hypereosinophilic syndrome with cardiac involvement1.13Increased8Hypereosinophilic syndrome with sinus involvement1.29Normal9Reactive eosinophilia associated with atopic dermatitis1.09Increased10Reactive eosinophilia associated with T cell skin lymphoma1.22Normal11Aggressive systemic mastocytosis associated with chronic myelomonocytic leukemia1.08Increased12Aggressive systemic mastocytosis associated with myelofibrosis1.09Increased13Aggressive systemic mastocytosis with circulating mast cells1.29Normal14Indolent systemic mastocytosis1.20Normal15Indolent systemic mastocytosis1.18NormalIn addition, eosinophil PRV-1 expression was measured in 2 patients from the above cohort (patients 3 and 8) and 3 additional patients with hypereosinophilic syndrome (HES).Results: Table 1 outlines specific diagnoses as well as the corresponding neutrophil PRV-1 expression levels in all 15 patients tested (median age 54 years, range 20–74; 11 males). Eosinophil PRV-1 expression was normal in all 5 patients tested (range of PRV-1/GAPDH ratio between 1.20–1.39).Conclusion: The current study suggests that neutrophil PRV-1 over-expression is a non-specific marker of clonal myeloproliferation that is seen in a broad spectrum of both typical and atypical myeloid disorders. As has been the case in ET, only a subset of AMPD patients display the specific abnormality, thus undermining its diagnostic utility. The preliminary information from the current study does not suggest increased eosinophil PRV-1 expression in either HES or clonal eosinophilia.

Expression of LUN gene that encodes a novel RING finger protein is correlated with development and progression of non-small cell lung cancer

LUN is a novel RING finger protein that is highly expressed in the lung and might be a transcriptional regulator of E-cadherin [J. Biol. Chem. 276 (2001) 14004]. It might be possible that LUN plays important roles in the development and progression of lung cancer through regulating expression of E-cadherin, but no clinical study on LUN expression has been reported. In the present study, we quantitatively examined gene expression of the LUN in surgical specimens resected from non-small cell lung cancer (NSCLC) patients. In normal lung tissues, the LUN gene expression was down-regulated in smokers (the mean LUN/ GAPDH ratios, 0.222 for non-smokers and 0.144 for smokers; P=0.030). In addition, the mean LUN/ GAPDH ratio in lung cancer tissues was significantly lower than that in normal lung tissues (0.072 versus 0.162; P<0.001). In addition, the LUN gene expression was slightly down-regulated along with progression of primary tumors, and strongly down-regulated along with nodal metastases (the mean LUN/ GAPDH ratios, 0.091 for pN0, 0.073 for pN1, and 0.034 for pN2 diseases; P=0.001). These results suggested that LUN might play important roles in inhibition of nodal metastases as well as in suppression of smoking-related oncogenesis in NSCLC.