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Results for “Tissue Array Analysis”

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PubMed2005

Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays.

Neural stem cells are the attractive cell source for functional regeneration of damaged central nervous tissues by means of cell transplantation or in situ induction of differentiated neural cells. Such stem cell therapies require the prospective identification and isolation of neural stem cells. However they are difficult due to limited information on surface markers. This study aimed at developing an antibody microarray that permits parallel analysis of multiple surface antigens expressed on neural stem cells present in a neurosphere-forming cell population. A microarray was prepared by micro-spotting antibodies directed to surface antigens and ligands for membrane-associated receptors onto the patterned monolayer of alkanethiols self-assembled on a gold-evaporated glass plate. Neurosphere-forming cells were subjected to a cell-binding assay on the microarray followed by immunofluorescent staining of nestin, an intracellular marker of neural stem cells. It was demonstrated that such a cell based assay facilitated to examine the specificity of surface antigens for nestin-positive neural stem cells. Furthermore, the microarray could also be used to assess proliferation capability of cells bound to individual spots. These results suggest that the microarray-based strategy will provide a useful tool for the parallel analysis of surface markers expressed on a specific cell type in a heterogeneous population.

Animals↗

PubMed2006

Accelerated intervertebral disc degeneration in scoliosis versus physiological ageing develops against a background of enhanced anabolic gene expression.

Molecular consequences of long-term deformation and altered mechanical loading of intervertebral disc (IVD) tissue in scoliosis have yet to be elucidated. We hypothesized that histological disc degeneration is faster in scoliosis than in normal ageing and that this is reflected by an altered gene expression profile. A semiquantitative histodegeneration score (HDS) revealed significantly enhanced degeneration in scoliosis (HDS 5.3) versus age-matched control IVDs (HDS 2.25; p = 0.001). Gene expression analysis by cDNA array and RT-PCR demonstrated higher mRNA levels for extracellular-matrix molecules like aggrecan, biglycan, decorin, lumican, chondromodulin, and COL2A1 in scoliotic discs versus normal discs of identical degeneration score. No differences were evident for catabolic molecules like MMP3, MMP13, MMP17, and TIMP1. In sum, morphologic disc degeneration was accelerated by about 2 decades in scoliosis versus physiological ageing and developed against a background of stronger anabolic matrix metabolism at younger age or in response to the altered mechanical environment of the tissue.

Adolescent↗

PubMed2005

Identification of the difference in extracellular matrix and adhesion molecules of cultured human gingival fibroblasts versus juvenile hyaline fibromatosis gingival fibroblasts using cDNA microarray analysis.

BACKGROUND: A difference from the normal range in collagen profile and perivascular hyaline deposition in the dermis and gingiva has been demonstrated histopathologically in juvenile hyaline fibromatosis (JHF), which is an autosomal recessive disease. The aim of this study was to understand the mechanism of gingival overgrowth in JHF, and to observe differences in the expression of genes regulating extracellular matrix organization. METHODS: Human gingival fibroblasts (GF) were obtained from individuals who have clinically healthy gingival tissue. JHF-GF were obtained from a patient who underwent a gingivectomy. Cultured fibroblast cells were examined visually using a phase contrast microscope. Total RNA from both cell types was isolated, and after biotin-deoxyuridine triphosphate (dUTP) labeling of cDNA, hybridization was performed with a pathway-specific gene expression profiling array membrane. Extracellular matrix (ECM) and adhesion molecule (AM) mRNA expressions in GF and JHF-GF were analyzed, and microarray data on genes modulating ECM remodeling were confirmed with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Cell morphology differences were observed between fibroblast types. Although type I collagen gene expression levels were almost the same, decreased type IV collagen expression was noted in JHF-GF versus GF. Decreased matrix metalloproteinase (MMP) and increased tissue inhibitor of matrix metalloproteinase (TIMP) transcripts were noted in JHF-GF versus GF. Increased fibronectin and decreased laminin mRNA expression were observed in JHF-GF when compared to GF. The present findings suggest that GF and JHF-GF differ not only morphologically but also in the expression level of ECM and AM genes involving connective tissue turnover and remodeling. CONCLUSIONS: Results from these analyses may be helpful to clarify the nature of overgrowth mechanisms, especially regarding enzymes and their inhibitors. This information is important in understanding the remodeling of ECM. The gingival overgrowth that is observed in JHF patients may be explained by a decreased level of MMPs and increased blockage of MMPs with TIMPs.

Adult↗

PubMed2000

Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis.

Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.

Animals↗

PubMed2002

Generation of diversity in the innate immune system: macrophage heterogeneity arises from gene-autonomous transcriptional probability of individual inducible genes.

Microbial products such as LPS stimulate macrophages to produce a wide diversity of inducible gene products needed for immediate host defense and priming of an appropriate acquired immune response. In this study, we have examined LPS-inducible gene expression in subclones of a mouse macrophage cell line, RAW264, using cDNA microarrays. Even archetypal target genes such as TNF-alpha were not induced in all subclones, and there was no absolute correlation between expression of pairs of genes. Nevertheless, the array analysis revealed clusters of genes that were more likely to be coexpressed. RAW264 cells stably transfected with luciferase reporter genes driven by LPS-responsive promoters revealed the same kind of clonal heterogeneity. The results indicate that each LPS-inducible gene has its own inherent probability of activation in response to LPS.

Animals↗

PubMed1991

Cepstral analysis of ultrasound in chronic liver disease--a preliminary study in the non-invasive evaluation of structural change.

To evaluate the morphological changes in chronic liver disease non-invasively we used the frequency-domain analysis of ultrasound. We assumed that liver tissue is a collection of semi-regularly arrayed small scatterers of ultrasound. We applied cepstral analysis to ultrasonic waveforms and evaluated the periodicity of scalloping of the power spectrum, caused by an interference effect among liver scatterers of a given spacing. In a phantom experiment, we could correctly measure the space among scatterers of a phantom consisting of a regularly spaced distribution of nylon filaments. In an experiment involving a freshly excised porcine liver, the distribution of the space among scatterers (SAS) determined on cepstral analysis reflected the size distribution of the hepatic lobules. In a human study, two statistical parameters, mode and kurtosis, were determined from the distribution of the SAS for each patient. In a previous study, we collected ultrasonic data during peritoneoscopy with a 10 MHz transducer directly contacting the liver and showed that we could evaluate morphological changes in chronic liver disease using cepstral analysis. In the present study, we obtained data in two ways: with a 3.5 MHz transducer through the abdominal wall and with a 10 MHz transducer directly contacting the liver during peritoneoscopy. The mode and kurtosis of the SAS obtained with the 3.5 MHz transducer were both well correlated with the mode and kurtosis of the SAS obtained with the 10 MHz transducer. These results suggest that we can evaluate morphological changes in chronic liver disease non-invasively.

Animals↗

PubMed2004

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Blotting, Northern↗

PubMed2005

Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.

PURPOSE: To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology. METHODS: RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis. RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26). CONCLUSIONS: RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

A DNA microarray survey of gene expression in normal human tissues.

BACKGROUND: Numerous studies have used DNA microarrays to survey gene expression in cancer and other disease states. Comparatively little is known about the genes expressed across the gamut of normal human tissues. Systematic studies of global gene-expression patterns, by linking variation in the expression of specific genes to phenotypic variation in the cells or tissues in which they are expressed, provide clues to the molecular organization of diverse cells and to the potential roles of the genes. RESULTS: Here we describe a systematic survey of gene expression in 115 human tissue samples representing 35 different tissue types, using cDNA microarrays representing approximately 26,000 different human genes. Unsupervised hierarchical cluster analysis of the gene-expression patterns in these tissues identified clusters of genes with related biological functions and grouped the tissue specimens in a pattern that reflected their anatomic locations, cellular compositions or physiologic functions. In unsupervised and supervised analyses, tissue-specific patterns of gene expression were readily discernable. By comparative hybridization to normal genomic DNA, we were also able to estimate transcript abundances for expressed genes. CONCLUSIONS: Our dataset provides a baseline for comparison to diseased tissues, and will aid in the identification of tissue-specific functions. In addition, our analysis identifies potential molecular markers for detection of injury to specific organs and tissues, and provides a foundation for selection of potential targets for selective anticancer therapy.

Cluster Analysis↗

PubMed1993

Simultaneous measurement of monoamines, metabolites and amino acids in brain tissue and microdialysis perfusates.

A high-performance liquid chromatographic method with coulometric array electrochemical detection is described for the simultaneous analysis of monoamines, their metabolites and o-phthalaldehyde (OPA)-derivatized amino acids. This method has been used to examine metabolite levels in both striatal tissue homogenates and striatal microdialysis perfusates. An aliquot of sample was initially analyzed for monoamines and metabolites by isocratic elution and electrochemical detection on a serial electrode array of eight coulometric flow-through graphite electrodes (0 to 490 mV; 70-mV increment). The remaining sample was derivatized pre-column with OPA-beta-mercaptoethanol and after column switching was analyzed for amino acids on a second isocratic system with electrochemical detection on four electrodes. Metabolites were then identified based on their retention time as well as electrochemical behavior across the arrays. The analysis, derivatization procedure, column switching, data reduction and peak identification were fully automated. The limit of detection for striatal tissue homogenates was approximately 1.38 ng/g wet weight for the monoamines and 8.25 ng/g wet weight for amino acids. The limit of detection for striatal perfusates was approximately 2.5 pg per 20-microliters sample for the monoamines and 15 pg per 20-microliters sample for the amino acids with analysis completed within 25 min making it ideal for microdialysis samples.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2002

Transcriptional profiling in human epilepsy: expression array and single cell real-time qRT-PCR analysis reveal distinct cellular gene regulation.

Highly parallel expression monitoring by microarrays is a powerful tool to study human brain disorders. In contrast to various nonneuronal tissues, the CNS is composed of a multitude of different cell types. Changed mRNA levels in neuropathological conditions may simply reflect altered tissue composition, rather than specific gene transcription regulation. Therefore, it is crucial, to supplement expression array data of histologically heterogeneous brain samples with a detailed analysis at the cellular level. Here, we have used a two-step approach to identify specific changes in hippocampal gene expression in patients with a hippocampal seizure focus (TLE) and marked neuronal damage. Using comparative expression array hybridization, 21 genes appeared to be differentially regulated. Expression alterations of a subset of these genes, i.e. (up-regulation of ataxin-3 and glial fibrillary acid protein (GFAP) as well as down-regulation of calmodulin) was confirmed in an extended series of individuals by real-time quantitative RT-PCR (qRT-PCR). In order to determine the cellular localization of these mRNAs, we performed real-time qRT-PCR of individual laser-microdissected neurons and glial cells. While ataxin-3 was expressed only in hippocampal neurons, GFAP was detected in reactive astrocytes. The differential calmodulin expression found on the tissue level was not observed in mRNA analyses from single neurons, suggesting that lower calmodulin mRNA levels are a consequence of segmental cell loss and do not indicate reduced cellular expression. Ataxin-3 has been related to neuronal maintenance. Its functional role for TLE has to be further evaluated.

Ataxin-3↗

PubMed2004

Using the gene ontology for microarray data mining: a comparison of methods and application to age effects in human prefrontal cortex.

One of the challenges in the analysis of gene expression data is placing the results in the context of other data available about genes and their relationships to each other. Here, we approach this problem in the study of gene expression changes associated with age in two areas of the human prefrontal cortex, comparing two computational methods. The first method, "overrepresentation analysis" (ORA), is based on statistically evaluating the fraction of genes in a particular gene ontology class found among the set of genes showing age-related changes in expression. The second method, "functional class scoring" (FCS), examines the statistical distribution of individual gene scores among all genes in the gene ontology class and does not involve an initial gene selection step. We find that FCS yields more consistent results than ORA, and the results of ORA depended strongly on the gene selection threshold. Our findings highlight the utility of functional class scoring for the analysis of complex expression data sets and emphasize the advantage of considering all available genomic information rather than sets of genes that pass a predetermined "threshold of significance."

Adolescent↗

PubMed2003

A microRNA array reveals extensive regulation of microRNAs during brain development.

Several hundred microRNAs (miRNAs) have recently been cloned from a wide range of organisms across phylogeny. Despite the high degree of conservation of miRNAs, their functions in general, and in mammals particularly, are just beginning to be defined. Here we show that an oligonucleotide DNA array can be successfully used for the simultaneous analysis of miRNA expression profiles from tissues or cells. From a subset of miRNAs expressed in the brain we designed an oligonucleotide array spotted with probes specific for 44 mature miRNAs. These arrays demonstrated precise regulation of miRNA expression at mammalian brain developmental epochs. About 20% of the probed miRNAs changed significantly in their expression during normal brain development, and two of them, miR-9 and miR-131, were dysregulated in presenilin-1 null mice exhibiting severe brain developmental defects. Transcripts with regulated expression patterns on the arrays were validated by Northern blots. Additionally, a bioinformatic analysis of developmentally regulated miRNAs suggested potential mRNA targets. The arrays also revealed miRNAs distributed to translating polyribosomes in primary neurons where they are likely to modulate translation. Therefore, oligonucleotide arrays provide a new tool for studying miRNA expression in a variety of biological and pathobiological settings. Creating clusters of coexpressed miRNAs will contribute to understanding their regulation, functions, and discovery of mRNA targets.

Animals↗

PubMed2002

Active, fulminant, lethal myocarditis associated with parvovirus B19 infection in an infant.

We report a case of fulminant myocarditis in an 11-month-old female infant who had no other clinical signs of parvovirus infection. The patient presented with severe respiratory distress and died in sudden cardiac arrest 3 h after admission. The clinical presentation was similar to that of an asthmatic attack. Autopsy revealed signs of acute lymphocytic myocarditis. Parvovirus DNA was demonstrated by polymerase chain reaction (PCR) analysis of tissue sections obtained from the heart, lungs, liver, kidneys, and spleen. Transmission electron microscopy of myocardial tissue showed crystalline arrays with the appearance of parvovirus. The results of immunohistochemical analysis for the detection of parvovirus antigens were negative, and no viral inclusions were demonstrable. We suggest that the current diagnostic procedure underestimates the prevalence of parvovirus-associated myocarditis. PCR analysis should be used as a complement in suspected cases, to enhance the rate of detection of the infection and to reach a correct diagnosis.

DNA, Viral↗

PubMed2001

Angiostatin effects on endothelial cells mediated by ceramide and RhoA.

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.

Angiostatins↗

PubMed2004

Modulation of the gene expression in the protective effects of electroacupuncture against cerebral ischemia: a cDNA microarray study.

In the present study, a cDNA microarray technology was used to analyze the gene expression profile in ischemia and in electroacupuncture (EA)-treated ischemia. A focal cerebral ischemia/reperfusion model on Macaca mulatta was performed with a modified middle cerebral artery occlusion method. Among the nearly 8000 genes, approximately 8% of the total number of genes examined were affected after ischemia/reperfusion injury. Major altered genes were downregulated. In EA-treated monkeys, approximately 10% of the total number of genes examined were affected. Major altered genes were upregulated, including signal transduction-, cell-cycle-, metabolism-, stress response-, DNA repair-related genes. One of the representative upregulated genes encodes insulin-like growth factor-1 (IGF-1) was confirmed using in situ hybridization. Results showed that after ischemia/reperfusion injury, IGF-1 mRNA expression decreased in ipsilateral striatum, whereas increased in ipsilateral hippocampus. No expression changes were observed in cortex. EA treatment could obviously upregulate the IGF-1 mRNA expression in striatum, and further enhance its expression in hippocampus. Therefore, the data presented suggest a possible mediator underlying the mechanisms of anti-ischemic effect of acupuncture. In conclusion, the protective mechanisms of EA against stroke include several related pathways and gene expressions. Microarray analysis may provide a framework for understanding these complicated mechanisms and yield valuable, clinically relevant insights and potentially therapeutic targets of stroke.

Animals↗

PubMed2004

A microarray model system identifies potential new target genes of the proto-oncogene HOX11.

HOX11 is a homeobox gene originally identified at a chromosomal breakpoint in T-cell acute lymphoblastic leukemia (T-ALL). It is one of the most frequently deregulated genes in T-ALL, although the precise role of HOX11 in leukemogenesis as well as in normal development remains obscure. To gain more insight into the functional role of HOX11, we utilized a microarray model system to characterize the gene expression network that it directs. Using one of our T-ALL cell lines that had been stably transfected to express HOX11 and high-density oligonucleotide HG-U95A arrays, we identified a large number of differentially expressed genes in response to the enforced expression of HOX11. We focused on examining genes found to be up-regulated according to the microarray analysis and selected three putative target genes, NFKB2, SMARCD3, and NR4A3, for further investigation. We could not only confirm the up-regulation of NR4A3 by an independent method in all clones expressing HOX11, but luciferase reporter assays demonstrated that the effect that HOX11 exerted on the proximal promoter of NR4A3 was dependent on the presence of an intact homeodomain, providing support for the idea that HOX11 manifests its regulatory function via its action as a transcription factor.

Cell Line, Tumor↗

PubMed2001

DNA array analysis of changes in preovulatory gene expression in the rat ovary.

During the periovulatory period, the mammalian ovary is the site of dramatic functional and structural changes, leading to oocyte maturation, follicle rupture, and corpus luteum formation. To a large extent, these processes result from changes in the transcriptome of various ovarian cell types. To develop a broader view of periovulatory changes in gene expression in the ovary and to identify further genes involved in periovulatory events, we used the recently developed DNA array technology. Immature female eCG-primed rats were killed either immediately before or 6 h after ovulation induction with hCG. Total ovarian RNA was isolated and used to prepare radiolabeled cDNA probes, which were hybridized to DNA arrays representing approximately 600 rat genes. Quantitative analysis identified a multitude of regulated gene messages, including several genes involved in extracellular matrix degradation and lipid/steroid metabolism previously reported to be induced by hCG. This screening also identified a group of candidate genes whose ovarian expression and gonadotropin regulation was hitherto unknown. The induction of three of these genes, encoding cutaneous fatty acid-binding protein, the interleukin-4 receptor alpha chain, and prepronociceptin, was confirmed and further characterized by Northern blot analysis. In addition, in situ hybridization analysis showed that hCG administration resulted in exclusive or predominant expression of all three genes in theca cells. These results demonstrate that DNA arrays can be used to identify genes regulated during the periovulatory period, thus contributing to a more detailed understanding of the molecular mechanisms of ovulation.

Animals↗