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Capture Microdissection (capture + microdissection)
Kinds of Capture Microdissection Selected AbstractsIdentification of erythroid-enriched gene expression in the mouse embryonic yolk sac using microdissected cellsDEVELOPMENTAL DYNAMICS, Issue 2 2008Latasha C. Redmond Abstract Little is known about the genes that control the embryonic erythroid program. Laser capture microdissection was used to isolate primitive erythroid precursors and epithelial cells from frozen sections of the embryonic day 9.5 yolk sac. The RNA samples were amplified and labeled for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. Ninety-one genes are expressed significantly higher in erythroid than in epithelial cells. Ingenuity pathway analysis indicates that many of these erythroid-enriched genes cluster in highly significant biological networks. One of these networks contains RBTN2/LMO2, SCL/TAL1, and EKLF/KLF1, three of the very few genes required for primitive erythropoiesis. Quantitative real-time polymerase chain reaction was used to verify that platelet factor 4, reelin, thrombospondin - 1, and muscleblind - like 1 mRNA is erythroid-enriched. These genes have established roles in development or differentiation in other systems, and are, therefore, good candidates for regulating primitive erythropoiesis. These results provide a catalog of genes expressed during primitive erythropoiesis. Developmental Dynamics 237:436,446, 2008. © 2008 Wiley-Liss, Inc. [source] Laser capture microdissection and microarray analysis of dividing neural progenitor cells from the adult rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2007Ulf Gurok Abstract Neural progenitor cells reside in the hippocampus of adult rodents and humans and generate granule neurons throughout life. Knowledge about the molecular processes regulating these neurogenic cells is fragmentary. In order to identify genes with a role in the proliferation of adult neural progenitor cells, a protocol was elaborated to enable the staining and isolation of such cells under RNA-preserving conditions with a combination of immunohistochemistry and laser capture microdissection. We increased proliferation of neural progenitor cells by electroconvulsive treatment, one of the most effective antidepressant treatments, and isolated Ki-67-positive cells using this new protocol. RNA amplification via in vitro transcription and subsequent microarray analysis revealed over 100 genes that were differentially expressed in neural progenitor cells due to electroconvulsive treatment compared to untreated control animals. Some of these genes have already been implicated in the functioning of neural progenitor cells or have been induced by electroconvulsive treatment; these include brain-derived neurotrophic factor (Bdnf), PDZ-binding kinase (Pbk) and abnormal spindle-like microcephaly-associated (Aspm). In addition, genes were identified for which no role in the proliferation of neurogenic progenitors has been described so far, such as enhancer of zeste homolog 2 (Ezh2). [source] Trans-species hair growth induction by human hair follicle dermal papillaeEXPERIMENTAL DERMATOLOGY, Issue 4 2001C. A. B. Jahoda Abstract: A series of experimental bioassays has shown that the dermal papilla of the adult rodent vibrissa hair follicle retains unique inductive properties. In view of the many phenotypic and functional differences between specific hair follicle types, and the growing interest in hair follicle biology and disease, it remains important to establish that the human hair follicle dermal papilla has equivalent capabilities. In this study we tested the ability of human hair follicle papillae to induce hair growth when implanted into transected, athymic mouse vibrissa follicles. The implanted papillae that interacted with mouse follicle epithelium created new fibre-producing follicle end bulbs. The origin of the papillae in the recombinant structures was confirmed using laser capture microdissection and human specific gender determination by PCR. The demonstration that intact adult human dermal papillae can induce hair growth has implications for molecular analysis of basic hair growth mechanisms, particularly since the study involved common epithelial,mesenchymal signalling and recognition properties across species. It also improves the prospects for a cell-based clinical approach to hair follicle disorders. [source] Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brainGLIA, Issue 16 2008Li Hong Shen Abstract The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is up-regulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by an injection of 3 × 106 rat BMSCs (n = 16) or phosphate-buffered saline (n = 15) into the tail vein 24 h later. Animals were sacrificed at 8 days after stroke. Immunostaining analysis showed that reactive astrocytes were the primary source of neurocan, and BMSC-treated animals had significantly lower neurocan and higher growth associated protein 43 expression in the penumbral region compared with control rats, which was confirmed by Western blot analysis of the brain tissue. To further investigate the effects of BMSCs on astrocyte neurocan expression, single reactive astrocytes were collected from the ischemic boundary zone using laser capture microdissection. Neurocan gene expression was significantly down-regulated in rats receiving BMSC transplantation (n = 4/group). Primary cultured astrocytes showed similar alterations; BMSC coculture during reoxygenation abolished the up-regulation of neurocan gene in astrocytes undergoing oxygen-glucose deprivation (n = 3/group). Our data suggest that BMSCs promote axonal regeneration by reducing neurocan expression in peri-infarct astrocytes. © 2008 Wiley-Liss, Inc. [source] Different signaling pathways in the livers of patients with chronic hepatitis B or chronic hepatitis C,HEPATOLOGY, Issue 5 2006Masao Honda The clinical manifestations of chronic hepatitis B (CH-B) and chronic hepatitis C (CH-C) are different. We previously reported differences in the gene expression profiles of liver tissue infected with CH-B or CH-C; however, the signaling pathways underlying each condition have yet to be clarified. Using a newly constructed cDNA microarray consisting of 9614 clones selected from 256,550 tags of hepatic serial analysis of gene expression (SAGE) libraries, we compared the gene expression profiles of liver tissue from 24 CH-B patients with those of 23 CH-C patients. Laser capture microdissection was used to isolate hepatocytes from liver lobules and infiltrating lymphoid cells from the portal area, from 16 patients, for gene expression analysis. Furthermore, the comprehensive gene network was analyzed using SAGE libraries of CH-B and CH-C. Supervised and nonsupervised learning methods revealed that gene expression was correlated more with the infecting virus than any other clinical parameters such as histological stage and disease activity. Pro-apoptotic and DNA repair responses were predominant in CH-B with p53 and 14-3-3 interacting genes having an important role. In contrast, inflammatory and anti-apoptotic phenotypes were predominant in CH-C. These differences would evoke different oncogenic factors in CH-B and CH-C. In conclusion, we describe the different signaling pathways induced in the livers of patients with CH-B or CH-C. The results might be useful in guiding therapeutic strategies to prevent the development of hepatocellular carcinoma in cases of CH-B and CH-C. (HEPATOLOGY 2006;44:1122,1138.) [source] Gene expression profiling during rat mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthraceneINTERNATIONAL JOURNAL OF CANCER, Issue 6 2009Masakazu Souda Abstract 7,12-Dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma is a well-recognized model; however, the genetic alterations during its carcinogenesis have yet to be determined. We used laser capture microdissection to specifically isolate cells from terminal end buds (TEBs), the origin of carcinoma, at 2 weeks after sesame oil treatment (control) or DMBA treatment (DMBA-TEBs), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (MC). Using an oligonucleotide microarray representing 20,600 rat probe sequences, we analyzed gene expression profiles and validated mRNA and protein levels of genes of interest byreal-time quantitative PCR and immunohistochemistry. The number of differentially expressed genes dramatically increased from DMBA-TEBs (63) to DCIS (798) and MC (981). Only the expression of PEP-19, an anti-apoptotic gene, showed significant increases in DMBA-TEBs (4-fold), DCIS (10-fold) and MC (16-fold). MMP-13 expression was increased markedly in DCIS (19-fold) and MC (61-fold) while OPN expression was increased 6-fold in DCIS and 8-fold in MC. MMP-7 expression was increased 4-fold in MC. Nidogen-1; a participant in the assembly of basement membranes, TSP-2; an inhibitor of angiogenesis and COUP-TFI; a transcription repressor showed significant decreases in DCIS (4-, 9- and 17-fold, respectively) and MC (10-, 37- and 100-fold). Network analyses with IPA software revealed that the most significant network included Akt groups in DCIS and ERK groups in MC. The present findings provide us with a better understanding of the molecular alteration that occur during mammary carcinogenesis and suggest the importance of PEP-19 overexpression in the very early stage of mammary carcinogenesis. © 2009 UICC [source] Molecular determinants of irinotecan efficacyINTERNATIONAL JOURNAL OF CANCER, Issue 10 2006Daniel Vallböhmer Abstract Molecular markers predicting the efficacy of CPT-11 based chemotherapies in patients with colorectal cancer (CRC) are unknown. Therefore, we investigated whether mRNA levels of drug targets (Topoisomerase I, TS), enzymes involved in 5-FU metabolism (DPD), in angiogenesis (EGFR, IL-8, VEGF) and in DNA-repair/drug detoxification (ERCC1, GST-P1) are associated with the clinical outcome of patients with CRC treated with first-line CPT-11 based chemotherapy. Thirty three patients with metastatic CRC were included in the study. Intratumoral gene expression levels were assessed from paraffin-embedded tissue samples, using laser capture microdissection and quantitative Real-Time PCR. Complete response was observed in 1 patient, partial response in 12 patients, stable disease in 13 patients and progressive disease in 6 patients. Response was inevaluable for 1 patient. Patients with complete response or partial response were classified as responders, while patients with stable disease or progressive disease were classified as nonresponders. High intratumoral mRNA levels of EGFR, ERCC1 and GSPT-P1 were each significantly associated with response to CPT-11 based chemotherapy. Recursive partitioning analysis showed that mRNA levels of EGFR and ERCC1 are primarily responsible for delineating responders from nonresponders. Also, the combination of high intratumoral gene expression levels of both EGFR and ERCC1 was significantly associated with progression-free survival. The mRNA levels of EGFR had a significant correlation with expression levels of ERCC1, GST-P1 and VEGF. This small retrospective study suggests that gene expression levels of EGFR, ERCC1 and GST-P1 may be useful in predicting the clinical outcome of patients with metastatic CRC treated with first-line CPT-11 based chemotherapy. © 2006 Wiley-Liss, Inc. [source] Increasing cyclooxygenase-2 (cox-2) gene expression in the progression of Barrett's esophagus to adenocarcinoma correlates with that of Bcl-2INTERNATIONAL JOURNAL OF CANCER, Issue 4 2006Daisuke Shimizu Abstract Previous studies from our laboratory and others have suggested that increased expression of cox-2 is important in the genesis of esophageal adenocarcinoma. In vitro studies suggest that cox-2 regulates expression of the anti-apoptotic protein bcl-2, thus possibly accounting for reduced apoptosis in carcinogenesis. The aim of this study was to investigate the relationship of these 2 genes in the development of Barrett's-associated adenocarcinoma. Histologic sections from endoscopic biopsies or esophagectomy specimens were classified as non-dysplastic Barrett's (n = 30), intraepithelial neoplasia (n = 12) and adenocarcinoma (n = 48). The desired tissue was isolated by laser capture microdissection and expression levels of cox-2 and bcl-2 were measured by quantitative real-time PCR (Taqman®). Gene expression levels were compared to samples of the distal esophageal squamous epithelium (n = 55) and reflux-esophagitis (n = 25), without Barrett's or cancer. Expression of both bcl-2 and cox-2 were increased in non-dysplastic Barrett's (p = 0.0077, p = 0.0037), intraepithelial neoplasia (p = 0.0053, p = 0.0220) and adenocarcinoma (p < 0.0001, p < 0.0001) compared to squamous epithelium or reflux-esophagitis. Furthermore, there is a significant correlation between these two genes, especially in carcinoma (p < 0.0001). © 2006 Wiley-Liss, Inc. [source] Overexpression of malignancy-associated laminins and laminin receptors by angiotropic human melanoma cells in a chick chorioallantoic membrane modelJOURNAL OF CUTANEOUS PATHOLOGY, Issue 12 2009Claire Lugassy Background: As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin-derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor. Design: C8161 human melanoma cells in a shell-less chick CAM assay were used to study EVMM associated with the presence of vascularized angiotropic melanoma areas. For both high-quality histomorphology and RNA preservation in paraffin-embedded tissue, we used a methanol-based fixative coupled with microwave-assisted rapid tissue processing as previously described. Using laser capture microdissection, angiotropic melanoma areas as well as avascular areas were microdissected. Using quantitative real time polymerase chain reaction (QRT-PCR), six genes have been studied: LAMC2 (laminin ,2 chain), LAMA4 (laminin ,4 chain), ITGB1 (integrin ,1), ITGB3 (integrin ,3), RSPA (ribosomal protein), and MMP2 (matrix metallopeptidase 2). QRT-PCR data were normalized to human GAPDH housekeeping gene and values were compared against Human Total RNA. Final results were expressed as percentage of expression. Results: All tumors demonstrated a similar pattern, i.e. EVMM of angiotropic melanoma cells. The microdissected histopathological sections presented both angiotropic areas and avascular areas. All genes were overexpressed in angiotropic melanoma areas vs. avascular melanoma areas, especially LAMC2, LAMA4 and ITGB3 (respectively, 165.18, 208.86, and 483.69%). Conclusion: This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM. [source] Improved detection of clonality in cutaneous T-cell lymphomas using laser capture microdissectionJOURNAL OF CUTANEOUS PATHOLOGY, Issue 8 2003Amir S. Yazdi Background:, The diagnosis of cutaneous T-cell lymphoma is a challenge for both the pathologist and the clinician. This is particularly true for distinguishing early-stage mycosis fungoides from dermatitis. In this clinical setting, the presence of a clonal T-cell population supports lymphoma. Methods:, Usually, routinely processed paraffin-embedded material is available for gene rearrangement analysis, and polymerase chain reaction (PCR)-based methods to assess clonality can be performed. One drawback of this approach is that sensitivity is suboptimal in biopsy specimens in which the lymphocytic infiltrate represents only a small percentage of all cells present. Another drawback is that DNA extraction from routinely processed, paraffin-embedded tissue is a time-consuming and labor-intensive procedure which can take up to 5 days in our laboratory. To bypass these problems, we used laser capture microdissection (LCM) to obtain lymphocytic infiltrates from tissue sections of formalin-fixed, paraffin-embedded skin biopsy specimens. This approach allows for more specific PCR assessment of the lymphocytic infiltrate and for rapid DNA extraction and PCR analysis. Results:, Using the LCM approach, we could demonstrate clonal T-cell receptor , gene rearrangements in biopsy specimens that did not show clonality using DNA extracted by conventional methods from full tissue sections. In addition, DNA extraction and PCR analysis can be performed in 11 h. Conclusion:, In conclusion, applying LCM to clonality analysis of cutaneous lymphocytic infiltrates is rapid and more sensitive than conventional methods, and we recommend introducing this approach into the routine diagnostic setting. [source] Gene expression in skeletal tissues: application of laser capture microdissectionJOURNAL OF MICROSCOPY, Issue 1 2005D. Benoyahu Summary Tissue differentiation is based on the expression of transcription factors, receptors for cytokines, and nuclear receptors that regulate a specific phenotype. The purpose of this study was to select cells from various skeletal tissues in order to analyse differential gene expression of cells in the native environment in vivo. It is a difficult task to obtain cells from skeletal tissues, such as cartilage, periost, bone and muscle, that are structured together and do not exist as individual organs. We used laser capture microdissection which permits the selection and isolation of individual cells from tissue sections. The RNA isolated from these tissues was used for reverse transcriptase-polymerase chain reactions for molecular analysis. We analysed the expression of transcription factors (cFOS, cbfa1, MyoD), receptors for cytokines, nuclear receptors, alkaline phosphatase and the structural proteins osteocalcin and collagen II. The results obtained demonstrate differential patterns of gene expression according to the tissue arrangement in their native in vivo environment, with reliable interpretation of the functions of the analysed genes in the context of intact skeletal tissue physiology. [source] Neurosteroid transport by the organic solute transporter OST,,OST,JOURNAL OF NEUROCHEMISTRY, Issue 1 2010Fang Fang J. Neurochem. (2010) 115, 220,233. Abstract A variety of steroids, including pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulfate (DHEAS) are synthesized by specific brain cells, and are then delivered to their target sites, where they exert potent effects on neuronal excitability. The present results demonstrate that [3H]DHEAS and [3H]PREGS are relatively high affinity substrates for the organic solute transporter, OST,,OST,, and that the two proteins that constitute this transporter are selectively localized to steroidogenic cells in the cerebellum and hippocampus, namely the Purkinje cells and cells in the cornu ammonis region in both mouse and human brain. Analysis of Ost, and Ost, mRNA levels in mouse Purkinje and hippocampal cells isolated via laser capture microdissection supported these findings. In addition, Ost, -deficient mice exhibited changes in serum DHEA and DHEAS levels, and in tissue distribution of administered [3H]DHEAS. OST, and OST, proteins were also localized to the zona reticularis of human adrenal gland, the major region for DHEAS production in the periphery. These results demonstrate that OST,,OST, is localized to steroidogenic cells of the brain and adrenal gland, and that it modulates DHEA/DHEAS homeostasis, suggesting that it may contribute to neurosteroid action. [source] Expression of insulin-like growth factor system genes during the early postnatal neurogenesis in the mouse hippocampusJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2007Jihui Zhang Abstract Insulin-like growth factor-1 (IGF-1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF-1 and -2) signaling is mediated primarily by the type 1 IGF receptor (IGF-1R) and modulated by six high-affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF-2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP-1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF-1, IGFBP-2, -3, and -5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF-2, IGF-2R, and IGFBP-6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF-1R and IGFBP-4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF-1, IGF-1R, IGF-2R, IGFBP-2, and -4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF-1 and IGF-1R (by 130% and 86%, respectively), lower levels of IGF-2R (by 72%), and similar levels of IGFBP-4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu. © 2007 Wiley-Liss, Inc. [source] Analysis of neuronal gene expression with laser capture microdissectionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2002Valerie A.M. Vincent Abstract The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimer's disease. © 2002 Wiley-Liss, Inc. [source] Expression of contactin associated protein-like 2 in a subset of hepatic progenitor cell compartment identified by gene expression profiling in hepatitis B virus-positive cirrhosisLIVER INTERNATIONAL, Issue 1 2010Huafeng Wang Abstract Background: Hepatic progenitor cells (HPC), a cell compartment capable of differentiating into hepatocytic and biliary lineages, may give rise to the formation of intermediate hepatobiliary cells (IHBC) or ductular reactions (DR). Aims: The aim of this study was to analyse the gene expression profiles of DR in cirrhosis and further investigate novel proteins expressed by HPC and their intermediate progeny. Methods: DR in hepatitis B virus (HBV)-positive cirrhotic liver tissues adjacent to hepatocellular carcinoma and interlobular bile ducts (ILBDs) in normal liver tissues were isolated by laser capture microdissection and then subjected to microarray analysis. Differential gene expression patterns were verified by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry on serial sections. HPC and their intermediate progeny were recognized by immunostaining with hepatocytic and biliary markers [HepPar1, cytokeratin (CK)7, CK19, neural cell adhesion molecule (NCAM), epithelial cell adhesion molecule (EpCAM)]. Results: A total of 88 genes showed upregulation in DR compared with ILBDs. Gene ontology analyses revealed that these upregulated genes were mostly associated with cell adhesion, immune response and the metabolic process. Contactin associated protein-like 2 (CNTNAP2) was first confirmed to be a novel protein expressed in a subpopulation of DR that was positive for CK7, NCAM or EpCAM. In addition, immunoreactivity for CNTNAP2 was also noted in a subset of isolated CK7-positive HPC as well as some ductular IHBC positive for CK19 and HepPar1 in DR. Conclusion: CNTNAP2 is specifically associated with the emergence of ductular populations and may be identified as a novel protein for defining a subset of HPC and their intermediate progeny in cirrhosis. [source] Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection,A technical reportMICROSCOPY RESEARCH AND TECHNIQUE, Issue 12 2009Tomoatsu Kaneko Abstract Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [source] Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell typesNEW PHYTOLOGIST, Issue 3 2008Jin Nakashima Summary ,,Independent antisense down-regulation of 10 individual enzymes in the monolignol pathway has generated a series of otherwise isogenic alfalfa (Medicago sativa) lines with varying lignin content and composition. These plants show various visible growth phenotypes, and possess significant differences in vascular cell size and number. ,,To better understand the phenotypic consequences of lignin modification, the distributions of lignin content and composition in stems of the various alfalfa lines at the cellular level were studied by confocal microscopy after staining for specific lignin components, and by chemical analysis of laser capture dissected tissue types. ,,Although all antisense transgenes were driven by the same promoter with specificity for vascular, fiber and parenchyma tissues, the impact of down-regulating a specific transgene varied in the different tissue types. For example, reducing expression of ferulate 5-hydroxylase reduced accumulation of syringyl lignin in fiber and parenchyma cells, but not in vascular elements. ,,The results support a model for cell type-specific regulation of lignin content and composition at the level of the monolignol pathway, and illustrate the use of laser capture microdissection as a new approach to spatially resolved lignin compositional analysis. [source] Design and assessment of a tissue-engineered model of human phalanges and a small jointORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 4 2005WJ Landis Structured Abstract Authors ,, Landis WJ, Jacquet R, Hillyer J, Lowder E, Yanke A, Siperko L, Asamura S, Kusuhara H, Enjo M, Chubinskaya S, Potter K, Isogai N. Objectives ,, To develop models of human phalanges and small joints by suturing different cell-polymer constructs that are then implanted in athymic (nude) mice. Design ,, Models consisted of bovine periosteum, cartilage, and/or tendon cells seeded onto biodegradable polymer scaffolds of either polyglycolic acid (PGA) or copolymers of PGA and poly-L-lactic acid (PLLA) or poly- , -caprolactone (PCL) and PLLA. Constructs were fabricated to produce a distal phalanx, middle phalanx, or distal interphalangeal joint. Setting and Sample Population ,, Studies of more than 250 harvested implants were conducted at the Northeastern Ohio Universities College of Medicine. Experimental Variable ,, Polymer scaffold, cell type, and implantation time were examined. Outcome Measure ,, Tissue-engineered specimens were characterized by histology, transmission electron microscopy, in situ hybridization, laser capture microdissection and qualitative and quantitative polymerase chain reaction analysis, magnetic resonance microscopy, and X-ray microtomography. Results ,, Over periods to 60 weeks of implantation, constructs developed through vascularity from host mice; formed new cartilage, bone, and/or tendon; expressed characteristic genes of bovine origin, including type I, II and X collagen, osteopontin, aggrecan, biglycan, and bone sialoprotein; secreted corresponding proteins; responded to applied mechanical stimuli; and maintained shapes of human phalanges with small joints. Conclusion ,, Results give insight into construct processes of tissue regeneration and development and suggest more complete tissue-engineered cartilage, bone, and tendon models. These should have significant future scientific and clinical applications in medicine, including their use in plastic surgery, orthopaedics, craniofacial reconstruction, and teratology. [source] Signal pathway profiling of prostate cancer using reverse phase protein arraysPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2003Robert L. Grubb Abstract Reverse phase protein arrays represent a new proteomics microarray technology with which to study the fluctuating state of the proteome in minute quantities of cells. The activation status of cell signaling pathways controls cellular fate and deregulation of these pathways underpins carcinogenesis. Changes in pathway activation that occur between early stage prostatic epithelial lesions, prostatic stroma and the extracellular matrix can be analyzed by obtaining pure populations of cell types by laser capture microdissection (LCM) and analyzing the relative states of several key phosphorylation points within the cellular circuitry. We have applied reverse phase protein array technology to analyze the status of key points in cell signaling involved in pro-survival, mitogenic, apoptotic and growth regulation pathways in the progression from normal prostate epithelium to invasive prostate cancer. Using multiplexed reverse phase protein arrays coupled with LCM, the states of signaling changes during disease progression from prostate cancer study sets were analyzed. Focused analysis of phospho-specific endpoints revealed changes in cellular signaling events through disease progression and between patients. We have used a new protein array technology to study specific molecular pathways believed to be important in cell survival and progression from normal epithelium to invasive carcinoma directly from human tissue specimens. With the advent of molecular targeted therapeutics, the identification, characterization and monitoring of the signaling events within actual human biopsies will be critical for patient-tailored therapy. [source] FGFR3 protein expression and its relationship to mutation status and prognostic variables in bladder cancer,THE JOURNAL OF PATHOLOGY, Issue 1 2007DC Tomlinson Abstract FGFR3 is frequently activated by mutation in urothelial carcinoma (UC) and represents a potential target for therapy. In multiple myeloma, both over-expression and mutation of FGFR3 contribute to tumour development. To define the population of UC patients who may benefit from FGFR-targeted therapy, we assessed both mutation and receptor over-expression in primary UCs from a population of new patients. Manual or laser capture microdissection was used to isolate pure tumour cell populations. Where present, non-invasive and invasive components in the same section were microdissected. A screen of the region of the highest tumour stage in each sample yielded a mutation frequency of 42%. Mutations comprised 61 single and five double mutations, all in hotspot codons previously identified in UC. There was a significant association of mutation with low tumour grade and stage. Subsequently, non-invasive areas from the 43 tumours with both non-invasive and invasive components were analysed separately; 18 of these had mutation in at least one region, including nine with mutation in all regions examined, eight with mutation in only the non-invasive component and one with different mutations in different regions. Of the eight with mutation in only the non-invasive component, six were predicted to represent a single tumour and two showed morphological dissimilarity of fragments within the block, indicating the possible presence of distinct tumour clones. Immunohistochemistry showed over-expression of FGFR3 protein in many tumours compared to normal bladder and ureteric controls. Increased expression was associated with mutation (85% of mutant tumours showed high-level expression). Overall, 42% of tumours with no detectable mutation showed over-expression, including many muscle-invasive tumours. This may represent a non-mutant subset of tumours in which FGFR3 signalling contributes to the transformed phenotype and which may benefit from FGFR-targeted therapies. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Genome analysis and gene expression profiling of neuroblastoma and ganglioneuroblastoma reveal differences between neuroblastic and Schwannian stromal cellsTHE JOURNAL OF PATHOLOGY, Issue 3 2005Simona Coco Abstract Neuroblastic tumours are a group of paediatric cancers with marked morphological heterogeneity. Neuroblastoma (Schwannian stroma-poor) (NB-SP) is composed of undifferentiated neuroblasts. Ganglioneuroblastoma intermixed (Schwannian stroma-rich) (GNBi-SR) is predominantly composed of Schwannian stromal (SS) and neuroblastic (Nb) cells. There are contrasting reports suggesting that SS cells are non-neoplastic. In the present study, laser capture microdissection (LCM) was employed to isolate SS and Nb cells. Chromosome 1p36 deletion and MYCN gene amplification were found to be associated in two out of seven NB-SPs, whereas no abnormalities were observed in five GNBi-SRs. In some cases, loss of heterozygosity (LOH) at 1p36 loci was detected in Nb cells but not in the bulk tumour by LCM; furthermore, LOH was also identified in both SS and tumour tissue of a GNBi-SR. DNA gain and loss studied by comparative genomic hybridization were observed at several chromosome regions in NB-SP but in few regions of GNBi-SR. Finally, gene expression profiles studied using an oligo-microarray technique displayed two distinct signatures: in the first, 32 genes were expressed in NB-SP and in the second, 14 genes were expressed in GNBi-SR. The results show that NB-SP is composed of different morphologically indistinguishable malignant cell clones harbouring cryptic mutations that are detectable only after LCM. The degree of DNA imbalance is higher in NB-SP than in GNBi-SR. However, when the analysis of chromosome 1p36 is performed at the level of microdissection, LOH is also observed in SS cells. These data provide supportive evidence that SS cells have a less aggressive phenotype and play a role in tumour maturation. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] A Review of Molecular Contrasts Between Arresting and Viable Porcine Attachment SitesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2007Jocelyn M. Wessels Significant spontaneous fetal loss of unknown cause occurs in North American commercial swine. About 30% of conceptuses, thought to be genetically normal, are lost during the peri-attachment period. An additional 20% are lost at mid-pregnancy. Littermate endometrial and trophoblast biopsies were studied by quantitative real-time PCR for gene expression, and immunohistochemistry for protein expression at gestation day (gd)15,23 and 50. RNA analyses were also conducted on endometrial lymphocytes and arterial endothelial cells removed from biopsies by laser capture microdissection. Genes were selected for study from human literature and cloned as required. As in humans, angiogenic, cytokine, chemokine and chemokine decoy receptor gene expression occurs at the porcine maternal,fetal interface. In each tissue studied, distinct patterns of expression are found between early and mid-pregnancy, as well as between viable and arresting conceptus attachment sites. These changes involve both endometrial lymphocytes and dendritic cells. Restriction in endometrial angiogenesis, reduction in expression of the chemokine decoy receptor D6, and reduction in dendritic cell numbers contribute to fetal arrest. In peri-attachment loss, interferon-, is more abundantly transcribed than tumor necrosis factor-,, but this ratio is reversed during midgestation failure. Further characterization of spontaneous fetal loss in pigs will identify targets for modification by hog producers and may provide a model for identification of antecedents to fetal loss in humans. [source] Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.)THE PLANT JOURNAL, Issue 3 2007Kazuhiro Ohtsu Summary All above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up- and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed. [source] Laser capture microdissection for the analysis of gene expression during embryogenesis of ArabidopsisTHE PLANT JOURNAL, Issue 1 2005Stuart Casson Summary It is during embryogenesis that the body plan of the developing plant is established. Analysis of gene expression during embryogenesis has been limited due to the technical difficulty of accessing the developing embryo. Here we demonstrate that laser capture microdissection can be applied to the analysis of embryogenesis. We show how this technique can be used in concert with DNA microarray for the large-scale analysis of gene expression in apical and basal domains of the globular-stage and heart-stage embryo, respectively, when critical events of polarity, symmetry and biochemical differentiation are established. This high resolution spatial analysis shows that up to approximately 65% of the genome is expressed in the developing embryo, and that differential expression of a number of gene classes can be detected. We discuss the validity of this approach for the functional analysis of both published and previously uncharacterized essential genes. [source] Heterogeneous gene methylation patterns among pre-invasive and cancerous lesions of the prostate: A histopathologic study of whole mount prostate specimensTHE PROSTATE, Issue 1 2004Karen Woodson Abstract BACKGROUND Gene methylation may contribute to prostate carcinogenesis through the silencing of gene transcription. We report on the methylation status of several genes shown to be silenced at different stages of progression using whole mount prostate specimens and laser capture microdissection. This is the first study to evaluate gene methylation patterns across multiple pre-cancerous and invasive cancer foci from the same prostate gland. METHODS Real-time PCR was used to evaluate methylation of five genes (GSTP1, RASSF1A, RAR,2, CD44, and EDNRB) across normal epithelium, high-grade prostatic intraepithelial neoplasia (HGPIN), and multiple tumor foci from each of 11 prostate cancer patients. RESULTS Gene methylation was not found in normal epithelium. To our knowledge, this is the first report of RASSF1A and RAR,2 methylation in HGPIN lesions (30% prevalence for each gene). In addition, RASSF1A, RAR,2, and GSTP1 methylation was highly prevalent in tumor foci (>75% for all three genes). Methylation of CD44 and EDNRB was observed in 41 and 38% of tumors but was not present in HGPIN. CONCLUSIONS These data suggest that genes may be methylated at different points in the histopathologic progression of prostate cancer and these differences can be found in various histologic foci from the same gland. © 2004 Wiley-Liss, Inc. [source] Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/BAMERICAN JOURNAL OF TRANSPLANTATION, Issue 9 2006G. Einecke One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis. [source] Extensive astrocyte infection is prominent in human immunodeficiency virus,associated dementia,ANNALS OF NEUROLOGY, Issue 2 2009Melissa J. Churchill PhD Astrocyte infection with human immunodeficiency virus (HIV) is considered rare, so astrocytes are thought to play a secondary role in HIV neuropathogenesis. By combining double immunohistochemistry, laser capture microdissection, and highly sensitive multiplexed polymerase chain reaction to detect HIV DNA in single astrocytes in vivo, we showed that astrocyte infection is extensive in subjects with HIV-associated dementia, occurring in up to 19% of GFAP+ cells. In addition, astrocyte infection frequency correlated with the severity of neuropathological changes and proximity to perivascular macrophages. Our data indicate that astrocytes can be extensively infected with HIV, and suggest an important role for HIV-infected astrocytes in HIV neuropathogenesis. Ann Neurol 2009;66:253,258 [source] Clonal origin of multifocal hepatocellular carcinomaCANCER, Issue 17 2010Kurt B. Hodges MD Abstract BACKGROUND: Hepatocellular carcinoma is the most common primary tumor of the liver. Patients frequently have multiple histologically similar, but anatomically separate tumors. The clonal origin of multiple hepatocellular carcinomas is uncertain. METHODS: The authors analyzed 31 tumors from 12 different patients (11 women, 1 man), who had multiple hepatocellular carcinomas involving 1 or both lobes. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue using laser capture microdissection. DNA was analyzed for loss of heterozygosity (LOH), X chromosome inactivation status, and TP53 gene mutations. RESULTS: Ten (83%) of the 12 patients showed LOH in at least 1 of the analyzed microsatellite markers. Concordant LOH patterns between separate hepatocellular carcinomas in individual patients were seen in 8 (80%) of 10 cases, whereas discordant patterns were seen in 2 (20%) of 10 cases. Five (50%) of 10 informative female patients showed identical nonrandom X chromosome inactivation patterns in multiple tumors; 1 case showed discordant nonrandom X chromosome inactivation pattern. TP53 mutations were identified in 8 (67%) of 12 patients. Tumors in 7 (88%) of these 8 patients showed different point mutations. Three patients (Cases 4, 5, and 10) had tumors with additional TP53 point mutations, indicating additional genetic abnormalities in these tumors. CONCLUSIONS: The data suggested that the significant proportion of patients with multifocal hepatocellular carcinomas have tumors of common clonal origin. Cancer 2010. © 2010 American Cancer Society. [source] Prostate carcinoma tissue proteomics for biomarker discoveryCANCER, Issue 12 2003Yaxin Zheng M.D. Abstract BACKGROUND The advent of the prostate-specific antigen (PSA) test has had a profound impact on the diagnosis and treatment of prostate carcinoma. However, the use of PSA levels alone for screening for prostate carcinoma was compromised by the variations in the amount of PSA produced by the benign prostatic tissue specimens. Proteins were involved in various pathways that determine the behavior of a cell. Therefore, information regarding proteins may reveal drug targets and/or markers for early detection. METHODS The authors used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to determine the protein profiles from fresh tissues of the prostate. Laser capture microdissection was performed to isolate pure populations of cells. RESULTS The authors identified a protein with an average m/Z of 24,782.56 ± 107.27 that was correlated with the presence of prostate carcinoma. Furthermore, using laser capture microdissection, they demonstrated that the origin of this protein, which the authors designated PCa-24, was derived from the epithelial cells of the prostate. PCa-24 expression was detected in 16 of 17 (94%) prostate carcinoma specimens but not in paired normal cells. In addition, this protein was not expressed in any of the 12 benign prostatic hyperplasia specimens that were assayed. CONCLUSIONS PCa-24 may be useful a marker for prostate carcinoma. Cancer 2003;98:2576,82. © 2003 American Cancer Society. [source] Quantitative analysis of hTERT mRNA levels in cells microdissected from cytological specimensCANCER SCIENCE, Issue 11 2008Hayato Fujita Clinicians frequently require cytopathological assessment of tumor samples for preoperative diagnosis, but in some specimens, diagnosis remains inconclusive after cytological examination. To date, several molecular markers, including human telomerase reverse transcriptase (hTERT), have been assessed for the ability to detect malignancy. However, analyses using whole cytological specimens are generally affected by contamination of untargeted cells. The present study investigated the feasibility of more sensitive examination by quantitative mRNA analysis of target cells microdissected from cytological specimens. Laser capture microdissection (LCM) was used to obtain target cells from cytological specimens. hTERT mRNA levels were then measured in target cells by quantitative real-time RT-PCR (qRT-PCR). The effect of RNA fragmentation on qRT-PCR was also assessed. Total RNA from cytological specimens was sometimes fragmented to a large degree. To avoid the effect of RNA fragmentation, gene specific priming and PCR primers generating short PCR products were used and no difference in delta Ct values between fragmented and non-fragmented RNA were found. hTERT mRNA levels were measured in cells microdissected from 33 cytological specimens. The levels of hTERT mRNA were significantly higher in malignant cases compared to those in non-malignant cases (P = 0.0003). The sensitivity was 96.2%, even when the specificities were 100%. High levels of hTERT mRNA were also found in three cases that were not diagnosed as malignant by cytological examination. Quantitative assessment of hTERT mRNA levels in cells microdissected from cytological specimens is a potential diagnostic tool to potentiate cytological examination in diagnosing malignancy. (Cancer Sci 2008; 99: 2244,2251) [source] | |||||||||||||||||||||||||||||||