Home About us Contact
|
Home About us Contact | ||
|
|
||
Real-time Quantitative Polymerase Chain Reaction (real-time + quantitative_polymerase_chain_reaction)
Selected AbstractsMolecular fingerprinting of TGFß-treated embryonic maxillary mesenchymal cellsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 4 2003M.M. Pisano Abstract The transforming growth factor-ß (TGFß) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGFß on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGFß in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGFß action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's AtlasTM Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGFß-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGFß-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImageTM analysis in order to determine differences in gene expression between control and TGFß-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGFß. Affected genes could be grouped into three general functional categories: transcription factors and general DNA-binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta-actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in response to TGFß treatment. Gene expression profiling results were verified by Real-Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGFß responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGFß in orchestrating craniofacial ontogeny. [source] The antiepileptic drug levetiracetam selectively modifies kindling-induced alterations in gene expression in the temporal lobe of ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2004Jessie Gu Abstract Gene expression profiling by microarrays is a powerful tool for identification of genes that may encode key proteins involved in molecular mechanisms underlying epileptogenesis. Using the Affymetrix oligonucleotide microarray, we have surveyed the expression levels of more than 26,000 genes and expressed sequence tags (ESTs) in the amygdala-kindling model of temporal lobe epilepsy. Furthermore, the effect of the antiepileptic drug levetiracetam (LEV) on kindling-induced alterations of gene expression was studied. Treatment of rats with LEV during kindling acquisition significantly suppressed kindling development. For gene expression profiling, six groups of rats were included in the present study: (i) and (ii) sham-operated rats treated with saline or LEV; (iii) and (iv) electrode-implanted but non-kindled rats treated with saline or LEV; (v) and (vi) kindled rats treated with saline or LEV. Treatment was terminated after 11 or 12 daily amygdala stimulations, when all vehicle-treated rats had reached kindling criterion, i.e. a stage 5 seizure. Twenty-four hours later, the ipsilateral temporal lobe was dissected for mRNA preparation. Six temporal lobe preparations from each group were analysed for differential gene expression. In control (non-kindled) rats, LEV treatment was devoid of any significant effect on gene expression. In saline-treated kindled rats, a large number of genes were observed to display mRNA expression alterations compared with non-kindled rats. LEV treatment induced marked effects on gene expression from kindled rats. Previously described epilepsy-related genes, such as neuropeptide Y (NPY), thyrotropin-releasing hormone (TRH) and glial fibrillary acidic protein (GFAP) were confirmed to be up-regulated by kindling and partially normalized by LEV treatment. Real-time quantitative polymerase chain reaction confirmed NPY, TRH and GFAP expression data from chip experiments. Furthermore, a number of novel genes were identified from the gene chip experiments. A subgroup of these genes demonstrated correlation between expression changes and kindled phenotype measurements. In summary, this study identified many genes with potentially important roles in epileptogenesis and highlighted several important issues in using the gene chip technology for the study of animal models of CNS disorders. [source] In Vivo and In Vitro Evidence of the Involvement of CXCL1, a Keratinocyte-Derived Chemokine, in Equine LaminitisJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 5 2009R.R. Faleiros Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans. Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis. Animals: Twenty adult horses. Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression. Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [P= .0001]) and DTP groups (21-fold [P= .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases (P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H2O2 (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA (P= .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes. Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated. [source] Gene expression analyses on embryonic external genitalia: identification of regulatory genes possibly involved in masculinization processesCONGENITAL ANOMALIES, Issue 2 2008Hisayo Nishida ABSTRACT Androgen plays a crucial role in initiating and maintaining the expression of male sexual characteristics in mammals. In humans and mice, any defects along the pathway of androgen functions result in congenital urogenital abnormalities. The genital tubercle (GT), an anlage of the external genitalia, differentiates into a penis in males and a clitoris in females. Although masculinization of the external genitalia is androgen-dependent, the molecular pathway of its potential downstream genes is largely unclear. To identify the genes involved in mouse GT masculinization, we performed gene expression analyses, such as real-time quantitative polymerase chain reaction and section in situ hybridization analysis. From our studies we have identified candidate genes, Cyp1b1, Fkbp51 and MafB as potential androgen targets during mouse GT masculinization. [source] Characterization and expression of AmphiBMP3,/3b gene in amphioxus Branchiostoma japonicumDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2010Yi Sun Bone morphogenetic proteins (BMPs) are responsible for regulating embryo development and tissue homeostasis beyond osteogenesis. However, the precise biological roles of BMP3 and BMP3b remain obscure to a certain extent. In the present study, we cloned an orthologous gene (AmphiBMP3/3b) from amphioxus (Branchiostoma japonicum) and found its exon/intron organization is highly conserved. Further, in situ hybridization revealed that the gene was strongly expressed in the dorsal neural plate of the embryos. The gene also appeared in Hatschek's left diverticulum, neural tube, preoral ciliated pit and gill slit of larvae, and adult tissues including ovary, neural tube and notochordal sheath. Additionally, real-time quantitative polymerase chain reaction (RTqPCR) analysis revealed that the expression displayed two peaks at gastrula and juvenile stages. These results indicated that AmphiBMP3/3b, a sole orthologue of vertebrate BMP3 and BMP3b, might antagonize ventralizing BMP2 orthologous signaling in embryonic development, play a role in the evolutionary precursors of adenohypophysis, as well as act in female ovary physiology in adult. [source] Enantioselective estrogenicity of o,p'-dichlorodiphenyltrichloroethane in the MCF-7 human breast carcinoma cell line,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2009Lumei Wang Abstract Research increasingly suggests that selectivity between enantiomers may exist in acute and chronic toxicological effects of chiral contaminants. In this study, we used the human breast carcinoma MCF-7 cell line to evaluate enantioselectivity of o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT). Baseline separation of o,p'-DDT enantiomers was achieved on the Chiralcel® OJ chiral column by high-performance liquid chromatography, and the absolute configuration and optical rotation of the resolved enantiomers were further identified. Significant differences in estrogenic potential were observed between the two enantiomers of o,p'-DDT in the MCF-7 cell proliferation assay (i.e., the E-Screen assay) and the real-time quantitative polymerase chain reaction (PCR). In the E-Screen assay, the relative proliferative effect ratios of R -(,)- o,p'-DDT and S -(+)- o,p'-DDT were 89.4 and 27.9%, respectively, and the relative proliferative potency ratios were 0.1 and 0.001%, respectively. Compared to the solvent control, R -(,)- o,p'-DDT induced the maximal increase of 2.31-fold at a concentration of 10,6 mol/L, while S -(+)- o,p'-DDT at 10,5 mol/L induced the maximal increase of 1.65-fold in estrogenic biomarker pS2 mRNA level. The maximal down-regulation of the transcription levels of estrogen receptor a (ER,) and ER, by R -(,)- o,p'-DDT were 49 and 40% at the concentration of 10,6 mol/L, while those by S -(+)- o,p'-DDT were 24 and 26% at the concentration of 10,5 mol/L. The cell proliferation, the up-regulation of pS2, and the down-regulation of ER, and ER, gene expressions induced by the racemate and enantiomers of o,p'-DDT were all reversed by cotreatment with 10,6 mol/L ICI 182,780. Therefore, the enantioselective estrogenicity of o,p'-DDT was likely through the ER, and ER, signaling pathways. Results from this study suggest the need for considering enantioselectivity of chiral contaminants in chronic ecological toxicities. [source] PRECLINICAL STUDY: Modulation of MDMA-induced behavioral and transcriptional effects by the delta opioid antagonist naltrindole in miceADDICTION BIOLOGY, Issue 3 2009Emilie Belkaï ABSTRACT The delta opioid system is involved in the behavioral effects of various drugs of abuse. However, only a few studies have focused on the possible interactions between the opioid system and the effects of 3,4-methylenedioxymethamphetamine (MDMA). In order to examine the possible role of the delta opioid system in MDMA-induced behaviors in mice, locomotor activity and conditioned place preference (CPP) were investigated in the presence of naltrindole (NTI), a selective delta opioid antagonist. Moreover, the consequences of acute and chronic MDMA administration on pro-enkephalin (Penk) and pro-opiomelanocortin (Pomc) gene expression were assessed by real-time quantitative polymerase chain reaction (QPCR). The results showed that, after acute MDMA administration (9 mg/kg; i.p.), NTI (5 mg/kg, s.c.) was able to totally block MDMA-induced hyperlocomotion. Penk gene expression was not modulated by acute MDMA, but a decrease of Pomc gene expression was observed, which was not antagonized by NTI. Administration of the antagonist prevented the acquisition of MDMA-induced CPP, suggesting an implication of the delta opioid receptors in this behavior. Following chronic MDMA treatment, only the level of Pomc was modulated. The observed increase was totally blocked by NTI pre-treatment. All these results confirm the interactions between the delta opioid system (receptors and peptides) and the effects of MDMA. [source] Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retinaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Heather D. VanGuilder Abstract Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real-time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin-diabetic and control Sprague,Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin-immunoreactive puncta were significantly smaller and fewer in sections from 1- and 3-month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1- and 3-month diabetic rats, and in synaptosomes from 1-month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1-month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for ,-actin and neuron-specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1-month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well-established defects in vision and the electrophysiological response of the retina in diabetes. [source] Upregulation of discoidin domain receptor 2 in nasopharyngeal carcinoma,HEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 4 2008Huey-Huey Chua PhD Abstract Background. Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and has high metastatic potential. Discoidin domain receptors (DDR1, DDR2) are receptor-type tyrosine kinases activated by collagen. Their ability to induce expression of matrix metalloproteinase is related with tumor invasion. Therefore, we aim to investigate DDRs gene expression and its regulation in NPC. Methods and Results. By use of real-time quantitative polymerase chain reaction (Q-PCR), DDR2 gene expression but not DDR1 was significantly higher in primary and metastatic NPC. DDR2 was predominantly distributed in NPC tumor cells rather than in infiltrating lymphocytes. EBV Z-transactivator (Zta) transfection may distinctly elevate DDR2 level. Furthermore, data from reporter assay indicate that Zta could transactivate DDR2 promoter activity, suggesting the possible upregulation mechanism. Conclusion. DDR2 was differentially upregulated in NPC and modulated by EBV Zta protein. DDR2 may play a role in NPC invasion and serve as a diagnostic and therapeutic target. © 2007 Wiley Periodicals, Inc. Head Neck, 2008 [source] Transforming growth factor- ,1 gene expression and cyclosporine A-induced gingival overgrowth: a pilot studyJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 5 2008gorzata Radwan-Oczko Abstract Aims: The relationship between gingival overgrowth (GO) induced by cyclosporine A (CsA) and transforming growth factor- ,1 (TGF- ,1) remains unclear. The aims of the present study were to evaluate TGF- ,1 gene expression under different immunosuppressive treatments and its association with TGF- ,1 gene functional polymorphism and GO in renal transplant recipients. Material and Methods: The study included 98 CsA-treated renal transplant recipients (with and without GO) and 44 tacrolimus-treated transplant patients (without GO). TGF- ,1 mRNA expression was measured using a real-time quantitative polymerase chain reaction assay. The levels were correlated with TGF- ,1 gene polymorphisms at codons 10 and 25, with different immunosuppressive treatment and GO. Results: The level of TGF- ,1 gene expression was insignificantly lower in the CsA-treated group compared with the tacrolimus group, and significantly lower in the group with GO compared with patients without GO. In tacrolimus- and CsA-treated patients, but not in patients with GO, the level of TGF- ,1 gene expression was associated with functional phenotypes of TGF- ,1. The incidence, degree and extent of GO were higher in recipients with lower TGF- ,1 gene expression. Conclusions: Lower level TGF- ,1 gene expression, not functional polymorphism, in patients treated with CsA may be considered to be a risk factor for GO. [source] Reassessment of microarray expression data of porokeratosis by quantitative real-time polymerase chain reactionJOURNAL OF CUTANEOUS PATHOLOGY, Issue 3 2010Zheng-Hua Zhang Background: Porokeratosis (PK) is a heterogeneous group of keratinization disorders that exhibit similarities with psoriasis at both the clinical and molecular levels. Methods: The transcript levels of keratin (KRT) 6A, 16, 17, S100A7, A8, A9, p53 and three candidate genes (i.e. SART3, SSH1 and ARPC3) were reassessed in pairwise lesional and uninvolved skin from nine patients with PK by real-time quantitative polymerase chain reaction (RTQ,PCR). Results: The results of RTQ,PCR confirmed that KRT6A, 16, S100A7, A8 and A9 (p = 0.008) were mostly up-regulated in the lesional skin when compared with uninvolved skin. Different from the microarray data, there was no significant difference observed in KRT17 expression patterns between lesional and normal-appearing skin (p = 0.066). No statistical difference was observed in p53 and three candidate genes' expression patterns between lesional and uninvolved skin. Conclusions: In the present study, 9 of the 10 gene expression measured by RTQ,PCR in PK were statistically comparable to microarray data. KRT6A was identified as specific biomarker for porokeratotic keratinocytes, as it was the most significantly up-regulated gene in the nine patient samples. Zhang Z-H, Wang Z-M, Crosby ME, Kang KF, Luan J, Huang W, Xiang L-H, Zheng Z-Z. Reassessment of microarray expression data of porokeratosis by quantitative real-time polymerase chain reaction. [source] Acute Activation of Hippocampal Glucocorticoid Receptors Results in Different Waves of Gene Expression Throughout TimeJOURNAL OF NEUROENDOCRINOLOGY, Issue 4 2006M. C. Morsink Abstract Several aspects of hippocampal cell function are influenced by adrenal-secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)-induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR-mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR-activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down-regulated genes 1 h after GR-activation to both up and down regulated genes 3 h after GR-activation. After 5 h, the response was almost back to baseline. Additionally, real-time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR-responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid-mediated effects on hippocampal cell function. [source] Vascular endothelial growth factor, fms-like tyrosine kinase-1 (Flt-1) and soluble Flt-1 gene expressions in Korean pre-eclamptic placentasJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 4 2010Jong-Sik Park Abstract Aim:, To assess the expressions of vascular endothelial growth factor (VEGF), fms-like tyrosine kinase-1 (Flt-1), and soluble Flt-1 (sFlt-1) genes in healthy normotensive and pre-eclamptic placentas of Korean women. Methods:, We investigated 12 healthy normotensive pregnant women and 10 pre-eclamptic pregnant women at Eulji University Hospital. The obtained placental tissues were analyzed using reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction. Results:, The sFlt-1 messenger ribonucleic acid (mRNA) level was elevated 2.6 times more in pre-eclamptic placentas than in normal control placentas. However, the VEGF mRNA level of pre-eclamptic placentas was decreased. There was no difference in the Flt-1 mRNA level between control and pre-eclamptic placentas. Conclusions:, Our study showed that expressions of genes relating to angiogenesis were altered in Korean pre-eclamptic placentas. These results suggest that the alteration in expressions of sFlt-1 and VEGF genes might be associated with the pathogenesis of pre-eclampsia. [source] cDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic achilles tendinosisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2003Håkan Alfredson The aetiology and pathogenesis of chronic painful Achilles tendinosis are unknown. This investigation aimed to use cDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique to study tendinosis and control tissue samples. Five patients (females mean age 57.1 ± 4.3 (years ±SD)) with chronic painful Achilles tendinosis were included. From all patients, one biopsy was taken from the area with tendinosis and one from a clinically normal area (control) of the tendon. The tissue samples were immediately immersed in RNAlater and frozen at ,80°C until RNA extraction. Portions of pooled RNA from control and tendinosis sites, respectively, were transcribed to cDNA, radioactively labelled (32P), hybridized to cDNA expression arrays, and exposed to phosphoimager screens over night. Expressions of specific genes, shown to be regulated in the cDNA array analysis, were analyzed in the individual samples using real-time PCR. cDNA arrays showed that gene expressions for matrix-metalloproteinase-2 (MMP-2), fibronectin subunit B (FNRB), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase p38 (MAPKp38) were up-regulated, while matrix-metalloproteinase-3 (MMP-3) and decorin were down-regulated, in tendinosis tissue compared with control tissue. Using real-time PCR, , and , patients showed up-regulation of MMP-2 and FNRB mRNA, respectively. For decorin, VEGF, and MAPKp38, real-time PCR revealed a great variability among patients. Interestingly, the mRNAs for several cytokines and cytokine receptors were not regulated, indicating the absence of an inflammatory process in chronic painful Achilles tendinosis. In conclusion, cDNA-arrays and real-time PCR can be used to study differences in gene expression levels between tendinosis and control tendon tissue. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Effect of Chronic Ethanol on Enkephalin in the Hypothalamus and Extra-Hypothalamic AreasALCOHOLISM, Issue 5 2010Guo-Qing Chang Background:, Ethanol may be consumed for reasons such as reward, anxiety reduction, or caloric content, and the opioid enkephalin (ENK) appears to be involved in many of these functions. Previous studies in Sprague,Dawley rats have demonstrated that ENK in the hypothalamic paraventricular nucleus (PVN) is stimulated by voluntary consumption of ethanol. This suggests that this opioid peptide may be involved in promoting the drinking of ethanol, consistent with our recent findings that PVN injections of ENK analogs stimulate ethanol intake. To broaden our understanding of how this peptide functions throughout the brain to promote ethanol intake, we measured, in rats trained to drink 9% ethanol, the expression of the ENK gene in additional brain areas outside the hypothalamus, namely, the ventral tegmental area (VTA), nucleus accumbens shell (NAcSh) and core (NAcC), medial prefrontal cortex (mPFC), and central nucleus of the amygdala (CeA). Methods:, In the first experiment, the brains of rats chronically drinking 1 g/kg/d ethanol, 3 g/kg/d ethanol, or water were examined using real-time quantitative polymerase chain reaction (qRT-PCR). In the second experiment, a more detailed, anatomic analysis of changes in gene expression, in rats chronically drinking 3 g/kg/d ethanol compared to water, was performed using radiolabeled in situ hybridization (ISH). The third experiment employed digoxigenin-labeled ISH (DIG) to examine changes in the density of cells expressing ENK and, for comparison, dynorphin (DYN) in rats chronically drinking 3 g/kg/d ethanol versus water. Results:, With qRT-PCR, the rats chronically drinking ethanol plus water compared to water alone showed significantly higher levels of ENK mRNA, not only in the PVN but also in the VTA, NAcSh, NAcC, and mPFC, although not in the CeA. Using radiolabeled ISH, levels of ENK mRNA in rats drinking ethanol were found to be elevated in all areas examined, including the CeA. The experiment using DIG confirmed this effect of ethanol, showing an increase in density of ENK-expressing cells in all areas studied. It additionally revealed a similar change in DYN mRNA in the PVN, mPFC, and CeA, although not in the NAcSh or NAcC. Conclusions:, While distinguishing the NAc as a site where ENK and DYN respond differentially, these findings lead us to propose that these opioids, in response to voluntary ethanol consumption, are generally elevated in extra-hypothalamic as well as hypothalamic areas, possibly to carry out specific area-related functions that, in turn, drive animals to further consume ethanol. These functions include calorie ingestion in the PVN, reward and motivation in the VTA and NAcSh, response-reinforcement learning in the NAcC, stress reduction in the CeA, and behavioral control in the mPFC. [source] Effect of Ethanol on Hypothalamic Opioid Peptides, Enkephalin, and Dynorphin: Relationship With Circulating TriglyceridesALCOHOLISM, Issue 2 2007Guo-Qing Chang Background: Recent evidence has demonstrated that ethanol intake can stimulate the expression and production of the feeding-stimulatory peptide, galanin (GAL), in the hypothalamic paraventricular nucleus (PVN), and that PVN injection of this peptide, in turn, can increase the consumption of ethanol. To test the hypothesis that other feeding-related systems are involved in ethanol intake, this study examined the effect of ethanol on the hypothalamic opioid peptides, enkephalin (ENK), and dynorphin (DYN). Method: Adult, male Sprague,Dawley rats were trained to voluntarily drink increasing concentrations of ethanol, up to 9% v/v, on a 12-hour access schedule or were given a single injection of ethanol (10% v/v) versus saline vehicle. The effect of ethanol on GAL, ENK, and DYN mRNA was measured using real-time quantitative polymerase chain reaction and radiolabeled in situ hybridization, while radioimmunoassay was used to measure peptide levels. In addition to blood alcohol, circulating levels of triglycerides (TG), leptin, and insulin were also measured. Results: The data demonstrated that: (1) rats voluntarily drinking 9% v/v ethanol (approximately 2.0 g/kg/d) show a significant increase in GAL, ENK, and DYN mRNA in the PVN compared with water-drinking rats; (2) voluntary consumption of ethanol also increases peptide levels of ENK and DYN in the PVN; (3) acute injection of 10% ethanol (1.0 g/kg of 10% v/v) similarly increases the expression of GAL, ENK, and DYN in the PVN; and (4) ethanol consumption and injection, while having little effect on leptin and insulin, consistently increase circulating levels of TG as well as alcohol, both of which are strongly, positively correlated with peptide expression in the PVN. Conclusions: These findings, together with published studies, suggest a possible role for hypothalamic opioid peptides in the drinking of ethanol. Based on evidence that dietary fat and lipid injections stimulate the PVN peptides and injection of the opiates and GAL increase ethanol intake, it is proposed that both TG and alcohol in the circulation, which are elevated by the ingestion or injection of ethanol, are involved in stimulating these peptides in the PVN, which in turn promote further consumption of ethanol. [source] Hepatitis B viral X protein alters the biological features and expressions of DNA repair enzymes in LO2 cellsLIVER INTERNATIONAL, Issue 2 2010Bin Cheng Abstract Objectives: This study aimed at examining the effects of hepatitis B viral X protein (HBx) on the biological features and the expression of DNA repair enzymes in non-tumour human hepatic LO2 cells in vitro. Methods: The HBx gene was transfected into LO2 cells to establish stably HBx-expressing LO2/HBx cells. The morphological features, cell growth, cell cycle, apoptosis and colony formation of LO2/HBx cells, vector-transfected LO2/pcDNA3.1 cells and unmanipulated LO2 cells were studied. The expressions of DNA repair enzymes and DNA oxidative stress-related 8-hydroxydeoxyguanosine (8-OHdG) were determined by a real-time quantitative polymerase chain reaction assay and high-performance liquid chromatography coupled with electrochemical detection respectively. Results: In comparison with controls, significant morphological changes, faster growth, higher frequency of cells at the S phase, but lower at G0/G1 and M/G2 phases, a lower frequency of natural cell apoptosis and a higher percentage of colony formation were observed in the LO2/HBx cells. Furthermore, significantly higher levels of intracellular 8-OHdG and lower levels of human DNA glycosylase , (hMYH,) mRNA transcripts, but no significant change in human 8-oxoguanine DNA glycosylase 1 (hOGG1), were detected in the LO2/HBx cells. Conclusions: Our data indicated that HBx promoted growth and malignant transformation of non-tumour hepatic LO2 cells in vitro, which was associated with the downregulation of hMYH, expression and accumulation of mutagenic DNA adduct 8-OHdG. [source] Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative densityMOLECULAR ECOLOGY, Issue 9 2006N. CHAUVATCHARIN Abstract Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia -associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia -infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia -infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 × 105± 1.61 × 105 orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible. [source] Gene expression normalization in a dual-compartment system: a real-time quantitative polymerase chain reaction protocol for symbiotic anthozoansMOLECULAR ECOLOGY RESOURCES, Issue 2 2009ANDERSON B. MAYFIELD Abstract Traditional real-time quantitative polymerase chain reaction protocols cannot be used accurately with symbiotic organisms unless the relative contribution of each symbiotic compartment to the total nucleic acid pool is known. A modified ,universal reference gene' protocol was created for reef-building corals and sea anemones, anthozoans that harbour endosymbiotic dinoflagellates belonging to the genus Symbiodinium. Gene expression values are first normalized to an RNA spike and then to a symbiont molecular proxy that represents the number of Symbiodinium cells extracted and present in the RNA. The latter is quantified using the number of genome copies of heat shock protein-70 (HSP70) amplified in the real-time quantitative polymerase chain reaction. Gene expression values are then normalized to the total concentration of RNA to account for differences in the amount of live tissue extracted among experimental treatments and replicates. The molecular quantification of symbiont cells and effect of increasing symbiont contributions to the nucleic acid pool on gene expression were tested in vivo using differentially infected sea anemones Aiptasia pulchella. This protocol has broad application to researchers who seek to measure gene expression in mixed organism assemblages. [source] The potato StLTPa7 gene displays a complex Ca2+ -associated pattern of expression during the early stage of potato,Ralstonia solanacearum interactionMOLECULAR PLANT PATHOLOGY, Issue 1 2009GANG GAO SUMMARY Although nonspecific lipid transfer proteins (nsLTPs) are widely expressed during plant defence responses to pathogens, their functions and regulation are not fully understood. In this article, we report the isolation of a cDNA for the new nsLTP, StLTPa7, from cultivated potato (Solanum tuberosum) infected with Ralstonia solanacearum. The cDNA was predicted to encode a type 1 nsLTP containing an N-terminal signal sequence and possessing the characteristic features of nsLTPs. A phylogenetic analysis showed that the encoded amino acid sequence of the nsLTP was similar to those of other previously reported plant nsLTPs, which contain a putative calmodulin-binding site consisting of approximately 12 highly conserved amino acid residues. The expression of the StLTPa7 gene was studied during the early stages of potato,R. solanacearum interaction using real-time quantitative polymerase chain reaction (qRT-PCR) and Northern analyses, and a complex calcium (Ca2+)-associated pattern of expression was observed with the following features: (i) transcripts of the StLTPa7 gene were systemically up-regulated by infection with R. solanacearum; (ii) the StLTPa7 gene was stimulated by salicylic acid, methyl jasmonate, abscisic acid and Ca2+; (iii) qRT-PCR showed that, during the early stage of R. solanacearum infection, nsLTP transcripts accumulated over a time course that paralleled that of Ca2+ accumulation, detected using environmental scanning electron microscopy and energy-dispersive X-ray (EDAX) spectrometry; and (iv) the Ca2+ channel blocker, ruthenium red, partially blocked R. solanacearum -induced StLTPa7 expression. This report represents the first use of EDAX analysis to establish a synchrony between Ca2+ accumulation and nsLTP expression in response to potato,R. solanacearum interactions. Collectively, these results suggest that StLTPa7 may be a pathogen- and Ca2+ -responsive plant defence gene. [source] Transcriptome analysis of root-knot nematode functions induced in the early stages of parasitism,NEW PHYTOLOGIST, Issue 2 2007G. Dubreuil Summary ,,Root-knot nematodes of the genus Meloidogyne are obligate biotrophic parasites able to infest > 2000 plant species. The nematode effectors responsible for disease development are involved in the adaptation of the parasite to its host environment and host response modulation. ,,Here, the differences between the transcriptomes of preparasitic exophytic second-stage juveniles (J2) and parasitic endophytic third-stage juveniles (J3) of Meloidogyne incognita were investigated. ,,Genes up-regulated at the endophytic stage were isolated by suppression subtractive hybridization and validated by dot blots and real-time quantitative polymerase chain reaction (PCR). ,,Up-regulation was demonstrated for genes involved in detoxification and protein degradation, for a gene encoding a putative secreted protein and for genes of unknown function. Transcripts of the glutathione S-transferase gene Mi-gsts-1 were 27 times more abundant in J3 than in J2. The observed Mi-gsts-1 expression in the oesophageal secretory glands and the results of functional analyses based on RNA interference suggest that glutathione S-transferases are secreted during parasitism and are required for completion of the nematode life cycle in its host. Secreted glutathione S-transferases may protect the parasite against reactive oxygen species or modulate the plant responses triggered by pathogen attack. [source] Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytesTHE JOURNAL OF GENE MEDICINE, Issue 2 2007Clare Selden Abstract Background Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. Methods We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /, growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. Results Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. Conclusions This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity. Copyright © 2007 John Wiley & Sons, Ltd. [source] ORIGINAL ARTICLE: Endometrial Osteopontin mRNA Expression and Plasma Osteopontin Levels are Increased in Patients with EndometriosisAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 4 2009SiHyun Cho Problem, The aim of this study was to evaluate osteopontin (OPN) mRNA expression in eutopic endometrium and plasma OPN levels in patients with endometriosis. Method of study, A total of 79 patients with histologically confirmed endometriosis and 43 patients without endometriosis participated in this study. OPN mRNA expression in endometrial tissues was measured by real-time quantitative polymerase chain reaction (PCR) and plasma concentrations of OPN were quantified using a specific commercial sandwich enzyme-linked immunosorbent assays (ELISA). Results, Osteopontin mRNA expression in endometrial tissue was significantly higher in women with endometriosis than in controls (P = 0.010). The mean plasma levels of OPN (mean ± S.E.M.) in patients with endometriosis and controls were 407.31 ± 37.80 ng/mL and 165.84 ± 19.29 ng/mL, respectively (P < 0.001). Receiver operating characteristic (ROC) analysis for plasma OPN revealed an area under the curve (AUC) of 0.894, with a sensitivity of 93.0%, specificity of 72.4%, positive likelihood ratio of 3.37, and negative likelihood ratio of 0.1 using a cut-off value of 167.68 ng/mL. Conclusion, Osteopontin may be involved in the pathogenesis of endometriosis and plasma OPN may be a useful non-invasive marker for the diagnosis of endometriosis. [source] Gene Expression Profiling of Nasal Polyps Associated With Chronic Sinusitis and Aspirin-Sensitive Asthma,THE LARYNGOSCOPE, Issue 5 2008Konstantina M. Stankovic MD Abstract Objective: To identify genes whose expression is most characteristic of chronic rhinosinusitis and aspirin-sensitive asthma through genome-wide transcriptional profiling of nasal polyp tissue. Study Design: Prospective, controlled study conducted at a tertiary care institution. Methods: Thirty genome-wide expression microarrays were used to compare nasal polyp tissue from patients with chronic rhinosinusitis alone (CRS, n = 10) or chronic rhinosinusitis and a history of aspirin-sensitive asthma (ASA, n = 10) to normal sinonasal mucosa from patients who underwent surgery for non-sinus related conditions (controls, n = 10). Genes found to be most characteristic of each polyp phenotype, as determined from bioinformatic analyses, were validated using real-time quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry in different patient sets. Results: The transcriptional signature of the control mucosa was distinctly different from that of either polyp phenotype. Genes most characteristic of the CRS phenotype included two upregulated genes,met proto-oncogene (MET) and protein phosphatase 1 regulatory subunit 9B (PPP1R9B),and two downregulated genes, prolactin-induced protein (PIP) and zinc alpha2-glycoprotein (AZGP1). The gene most characteristic of the ASA phenotype was periostin (POSTN), which was upregulated relative to controls. Differences between the CRS and ASA phenotypes were associated with alterations in the 6p22, 22q13, and 1q23 chromosomal regions. Conclusions: Nasal polyps appear to have characteristic transcriptional signatures compared to normal sinonasal mucosa. The five genes identified in this study likely play roles in the pathogenesis of polyps associated with CRS and ASA, and are therefore attractive targets for novel medical therapies for these common debilitating diseases. [source] Heme Oxygenase (HO)-1 Is Upregulated in the Nasal Mucosa With Allergic Rhinitis,THE LARYNGOSCOPE, Issue 3 2006Ahmed Elhini MD Abstract Background: Heme oxygenase (HO) is considered to be an antioxidant enzyme that catabolizes heme to produce carbon monoxide (CO) and biliverdin. Three isoforms of HO have been discovered. Recently, HO-1 has been found to be upregulated after allergic inflammations of the lower airway. Objective: The objective of this study was to address the expression of HO isoenzymes 1 and 2 in the nasal mucosa of patients with allergic rhinitis as well as normal control subjects. Methods: Nasal mucosa from 30 patients with persistent allergic rhinitis as well as from 10 normal volunteers was used in this study. We used immunofluorescent technique, Western blotting, and real-time quantitative polymerase chain reaction to localize and quantify the expression of these isoenzymes in normal and allergic human nasal tissues. Results: We found that HO-1 is expressed in the epithelial cells of seromucinous glands and macrophages with significant upregulation of its glandular expression in allergic rhinitis but with no difference in its macrophage expression between the study groups in contrast to HO-2 that is expressed in the vascular endothelial lining cells as well as macrophages with no marked difference between the study groups. Conclusion: We demonstrated that expression of HO-1, but not HO-2, was upregulated within the nasal tissues in allergic rhinitis inflammation, and understanding the induction of HO-1 expression may provide for better management of allergic rhinitis that involves oxidative stress. [source] Interferon-stimulated gene 15 (ISG15) conjugates proteins in dermatomyositis muscle with perifascicular atrophyANNALS OF NEUROLOGY, Issue 1 2010Mohammad Salajegheh MD Objective We investigated interferon-stimulated gene 15 (ISG15), a poorly understood ubiquitin-like modifier, and its enzymatic pathway in dermatomyositis (DM), an autoimmune disease primarily involving muscle and skin. Methods We generated microarray data measuring transcript abundance for approximately 18,000 genes in each of 113 human muscle biopsy specimens, and studied biopsy specimens and cultured skeletal muscle using immunohistochemistry, immunoblotting proteomics, real-time quantitative polymerase chain reaction, and laser-capture microdissection. Results Transcripts encoding ISG15-conjugation pathway proteins were markedly upregulated in DM with perifascicular atrophy (DM-PFA) muscle (ISG15 339-fold, HERC5 62-fold, and USP18 68-fold) compared with 99 non-DM samples. Combined analysis with publicly available microarray datasets showed that >50-fold ISG15 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM patients compared with 199 muscle samples from other muscle diseases. Free ISG15 and ISG15-conjugated proteins were only found on immunoblots from DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and ISG15 protein and conjugates. Laser-capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers. Interpretation A large-scale microarray study of muscle samples demonstrated that among a diverse group of muscle diseases DM was uniquely associated with upregulation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produced a molecular picture highly similar to that seen in human DM muscle. Perifascicular atrophic myofibers in DM were deficient in a number of skeletal muscle proteins including titin. ANN NEUROL 2010;67:53,63 [source] The infrapatellar fat pad in knee osteoarthritis: An important source of interleukin-6 and its soluble receptorARTHRITIS & RHEUMATISM, Issue 11 2009Emilie Distel Objective Obesity is a potent risk factor in knee osteoarthritis (OA). It has been suggested that adipokines, secreted by adipose tissue (AT) and largely found in the synovial fluid of OA patients, derive in part from the infrapatellar fat pad (IFP), also known as Hoffa's fat pad. The goal of this study was to characterize IFP tissue in obese OA patients and to compare its features with thigh subcutaneous AT to determine whether the IFP contributes to local inflammation in knee OA via production of specific cytokines. Methods IFP and subcutaneous AT samples were obtained from 11 obese women (body mass index ,30 kg/m2) with knee femorotibial OA. Gene expression was measured by real-time quantitative polymerase chain reaction. Cytokine concentrations in plasma and in conditioned media of cultured AT explants were determined by enzyme-linked immunosorbent assay or by Luminex xMAP technology. Results In IFP tissue versus subcutaneous AT, there was a decrease in the expression of genes for key enzymes implicated in adipocyte lipid metabolism, whereas the expression levels of genes for AT markers remained similar. A 2-fold increase in the expression of the gene for interleukin-6 (IL-6), a 2-fold increase in the release of IL-6, and a 3.6-fold increase in the release of soluble IL-6 receptor (sIL-6R) were observed in IFP samples, compared with subcutaneous AT, but the rates of secretion of other cytokines in IFP samples were similar to the rates in subcutaneous AT. In addition, leptin secretion was decreased by 40%, whereas adiponectin secretion was increased by 70%, in IFP samples versus subcutaneous AT. Conclusion Our results indicate that the IFP cytokine profile typically found in OA patients could play a role in paracrine inflammation via the local production of IL-6/sIL-6R and that such a profile might contribute to damage in adjacent cartilage. [source] Altered integrin mechanotransduction in human nucleus pulposus cells derived from degenerated discsARTHRITIS & RHEUMATISM, Issue 2 2009Christine Lyn Le Maitre Objective Several studies have demonstrated biologic responses of intervertebral disc (IVD) cells to loading, although the mechanotransduction pathways have not been elucidated. In articular chondrocytes, which have a phenotype similar to that of IVD cells, a number of mechanoreceptors have been identified, with ,5,1 integrin acting as a predominant mechanoreceptor. The purpose of this study was to investigate the role of integrin signaling in IVD cells during mechanical stimulation and to determine whether RGD integrins are involved. Methods Human nucleus pulposus (NP) cells derived from nondegenerated and degenerated discs were subjected to dynamic compressive loading in the presence of an RGD inhibitory peptide. Expression of the ,5,1 heterodimer in IVD tissue was examined by immunohistochemistry and possible alternative mechanoreceptors by real-time quantitative polymerase chain reaction. Results Aggrecan gene expression was decreased following loading of NP cells from nondegenerated and degenerated discs. This response was inhibited by treatment with an RGD peptide in cells from nondegenerated, but not degenerated, IVDs. Immunohistochemistry demonstrated that expression of the ,5,1 heterodimer was unaltered in degenerated IVD tissue as compared with normal IVD tissue. Conclusion Our results indicate that the mechanotransduction pathways are altered in cells from degenerated IVDs. Mechanosensing in NP cells from nondegenerated discs occurs via RGD integrins, possibly via the ,5,1 integrin, while cells from degenerated discs show a different signaling pathway that does not appear to involve RGD integrins. [source] Blocking vascular endothelial growth factor with soluble Flt-1 improves the chondrogenic potential of mouse skeletal muscle,derived stem cellsARTHRITIS & RHEUMATISM, Issue 1 2009Seiji Kubo Objective To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt-1 (sFlt-1), on chondrogenesis, using muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle. Methods The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real-time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP-4) were coimplanted with MDSCs transduced to express either VEGF or sFlt-1 (a VEGF antagonist) to provide a gain- and loss-of-function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups. Results Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt-1,treated) pellet cultures, but quantitative PCR revealed that sFlt-1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP-4 and transforming growth factor ,3 (TGF,3). In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt-1 improved the MDSC-mediated repair of articular cartilage, compared with BMP-4 alone. Conclusion Soluble Flt-1 gene therapy improved the BMP-4, and TGF,3-induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage. [source] Inflammatory cytokine gene expression in the urinary sediment of patients with lupus nephritisARTHRITIS & RHEUMATISM, Issue 5 2003Rebecca Wing-Yan Chan Objective Lupus nephritis is characterized by intrarenal inflammation and lymphocyte activation. In the present study, the expression of cytokine genes in the urinary sediment of patients with systemic lupus erythematosus (SLE) was examined. Methods We studied 3 SLE patient groups (25 with active lupus nephritis [active group], 25 with inactive SLE and previous renal involvement [remission group], 20 with inactive SLE and no history of renal involvement [nonrenal SLE group]) and 2 control groups (10 patients with noninflammatory renal diseases [non-SLE group] and 10 healthy volunteers [healthy group]). Cytokine gene expression in the urinary sediment was studied by real-time quantitative polymerase chain reaction. Results Expression of interferon-, (IFN,) in urinary sediment was significantly higher in the active group than in all other groups (P < 0.001 by Kruskal-Wallis test). Among the SLE patient groups, there was a close correlation between IFN, expression and the overall SLE Disease Activity Index (SLEDAI) score (Spearman's r = 0.590, P < 0.001) and the SLEDAI renal score (r = 0.642, P < 0.001). Urinary expression of interleukin-2 (IL-2) in the active group was significantly higher than that in the healthy group (P = 0.046) but not in the remission or nonrenal SLE groups. There was no difference in the levels of IL-4 expression among the SLE groups. Conclusion We found a predominance of Th1 cytokine in the urinary sediment of patients with active lupus nephritis. Measurement of cytokine gene expression in urinary sediment may be a useful noninvasive tool for assessing the severity of renal involvement in SLE. [source] | |||||||||||||||||||||||||