SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Laser-assisted microdissection is a powerful tool for the analysis of morphologically defined cell populations. We report here that the combination of microdissection and real-time RT-PCR technologies together with an optimized RNA microscale extraction procedure allows the reproducible determination of gene expression levels in archival formalin-fixed, paraffin-embedded tissue samples.
The aims of this study were to investigate the tissues from uncemented Mittelmeier alumina ceramic-on-ceramic total hip replacements using histological methods and to isolate and characterise the ceramic wear debris using laser capture microdissection and electron microscopy. Tissues from around 10 non-cemented Mittelmeier alumina ceramic on ceramic THRs were obtained from patients undergoing revision surgery. Tissues were also obtained from six patients who were undergoing revisions for aseptic loosening of Charnley, metal-on-polyethylene prostheses. Tissue sections were analysed using light microscopy to determine histological reactions and also the location and content of alumina ceramic wear debris. Tissue samples were extracted from sections using laser capture microdissection and the characteristics of the particles subsequently analysed by TEM and SEM. The tissues from around the ceramic-on-ceramic prostheses all demonstrated the presence of particles, which could be seen as agglomerates inside cells or in distinct channels in the tissues. The tissues from the ceramic-on-ceramic retrievals had a mixed pathology with areas that had no obvious pathology, areas that were relatively rich in macrophages and over half of the tissues had in the region of 60% necrosis/necrobiosis. In comparison, the Charnley tissues showed a granulomatous cellular reaction involving a dense macrophage infiltrate and the presence of giant cells and < 30% necrosis/necrobiosis. The tissues from the ceramic prostheses also showed the presence of neutrophils and lymphocytes, which were not evident in the tissues from the Charnley retrievals. There were significantly more macrophages (p < 0.05), and giant cells (p < 0.01) in the Charnley tissues and significantly more neutrophils (p < 0.01) in the ceramic-on-ceramic tissues. TEM of the laser captured tissue revealed the presence of very small alumina wear debris in the size range 5-90 nm, mean size + SD of 24 +/- 19nm whereas SEM (lower resolution) revealed particles in the 0.05-3.2 microm size range. This is the first description of nanometre sized ceramic wear particles in retrieval tissues. The bi-modal size range of alumina ceramic wear debris overlapped with the size ranges commonly observed with metal particles (10-30 nm) and particles of ultra-high molecular weight polyethylene (0.1-1,000 microm). It is possible that the two size ranges of contributed to the mixed tissue pathology observed. It is speculated that the two types of ceramic wear debris are generated by two different wear mechanisms in vivo, under normal articulating conditions, relief polishing wear and very small wear debris is produced. while under conditions of microseparation of the head and cup and rim contact, intergranular and intragranular fracture and larger wear particles are generated.
At present, the molecular mechanisms of hepatocellular carcinogenesis are not well understood. It is known, however, that cancer development and progression are accompanied by profound changes at the cellular and subcellular level, involving RNA/DNA and protein structure and function. Therefore, high-throughput, proteomic techniques targeting these biological molecules may provide novel insights into HCC genesis and prognosis. We characterized tissue protein profiles from 10 HCC patients using ProteinChip technology (SELDI) which is able to detect minute amounts of proteins and moreover to analyze complex protein pattern. Therefore, after histopathological examination, proteins from kryostat sections of non-tumorous hepatic tissue as well as from central and peripheral tumor areas were isolated from complete histological sections or from selected and microdissected tissue areas. Analysis on the SAX and WCX ProteinChip Arrays revealed 14-26, and 25-29 differentially expressed peaks respectively, which characterized non-tumorous and tumor tissue (p< or =0.05). One feature which allows differentiation between central tumor and peripheral tumor regions could only be detected in microdissected tissue. Using ProteinChip technology in combination with tissue microdissection it is possible to investigate complex changes at the protein level in hepatocellular cancer associated with tumor development and progression.
Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.
BACKGROUND AND PURPOSE: Ureteropelvic junction obstruction (UPJO) is defined as an impediment to urinary flow from the renal pelvis into the ureter. The exact cause remains an enigma despite investigations along embryologic, anatomic, and histologic lines. Our goal was to investigate in vivo the expression profile of cytokines in hyperplastic urothelial cells as a means of determining the source of UPJO. MATERIALS AND METHODS: Cellular proteomes of matched normal and hyperplastic urothelial cells were analyzed by laser capture microdissection (LCM) and tissue microdissection and human cytokine proteomic chips. All specimens (N = 9) were surgically obtained from patients undergoing laparoscopic dismembered pyeloplasty and were immediately embedded in O.C.T. solution and flash-frozen in liquid nitrogen. Tissue sections (6 microm) were mounted on uncoated glass slides using a cryostat, fixed in 70% ethanol, stained with hematoxylin and eosin, and sequentially dehydrated in ethanol and xylene. Typically, paired samples of normal and hyperplastic urothelial cells were procured on separate caps from serial sections of each specimen by the Arcturus PixCell II LCM system using 3000 laser pulses and a spot diameter of 30 microm. Total proteins were harvested and quantitated. Differential expression profile analysis of 43 cytokines in normal and hyperplastic cells were performed using human protein chips. Briefly, the membranes were initially probed with protein (150 ng) from normal or hyperplastic cells and sequentially reacted with a cocktail of biotinylated cytokine antibodies and horseradish peroxidase-conjugated streptavidin. The membranes were developed using enhanced chemiluminescence and analyzed by densitometry. RESULTS: Comparative densitometric analysis revealed twofold to fourfold upregulation of growth-related oncogene alpha (GRO-alpha), interleukin (IL)-1alpha, interferon (INF)-gamma, IL-8, tumor necrosis factor (TNF)-alpha, RANTES, and macrophage inflammatory protein-1beta (MIP-1beta) and twofold to fourfold downregulation of transforming growth factor (TGF)-beta and IL-10 in hyperplastic urothelial cells compared with paired control cells. CONCLUSIONS: We here report the efficient application of LCM and proteomic array chips for expression profile analysis of cytokines in vivo. Because the etiology and pathogenesis of UPJO are still fragmentary, the marked heterogeneity of the observed cytokine alterations reported here may be of significance. Further studies are required to elucidate the functional significance of the differentially expressed cytokines in the pathogenesis of the disease.
Tissue microdissection is potentially one of the most useful techniques in molecular pathology. Laser-assisted microdissection has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with multiplex molecular approaches, it is now feasible to study genetic alterations and isolate genes and proteins in defined cell populations from complex normal and diseased tissues. The fundamental advantage of this technique is the possibility of capturing single cells from which high-quality DNA and mRNA can be isolated for analysis of sequence and quantitation of expression. Moreover, the integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers for disease. The advent of laser-assisted microdissection is likely to have a profound impact on molecular pathology.
Gene expression analysis using total RNA of bulk tissue usually cannot assign specific messages to particular cell types. Cell-specific RNA expression profiling, though, may be crucial for a better understanding ofthe role of each distinct cell type within a physiological or pathophysiological setting. RNA profiling based on laser-controlled microdissection (LCM) of defined cells of a tissue now provides a useful tool for studying molecular crosstalk between different cell types within a tissue. The LCM technique allows for efficient isolation of single cells with no or very low contamination of surrounding tissue components, simultaneously leaving the intracellular structure and molecules intact. In this review, different issues of the LCM technique and the RNA amplification procedure for microarray analysis are discussed. An exemplary summary of results obtained from gene profiling of epithelial and stromal cells from human prostate tumors is presented, demonstrating the power of LCM-based molecular analysis. Finally, we discuss the potential use of the LCM technique i) to study the transcriptome of distinct cells from formalin-fixed and paraffin-embedded tissues in subcellular RNA profiling and ii) high resolution proteomic and metabolistic studies.
Human breast carcinoma is thought to develop through progressive stages from atypical hyperplasias to in situ carcinoma and finally to invasive and metastatic cancer. In situ breast carcinoma consists of small, isolated neoplastic foci which cannot be selectively studied by conventional methods. In this study, we used tissue microdissection to examine the loss of heterozygosity (LOH) of chromosome 11q13 in both in situ and invasive lesions of the breast, as compared to normal breast epithelium from the same patients. Forty-one cases of sporadic breast cancer were analyzed. Tissue microdissection allows for procurement and PCR-based analysis of small lesions using either frozen or formalin-fixed, paraffin-embedded tissue sections. LOH on chromosome 11q13 was found in 24 of 36 (67%) of the informative invasive breast cancer cases using two polymorphic DNA markers specific for this region (INT2 and PYGM). Twenty-one of the cases which demonstrated LOH in the invasive tumor also contained in situ carcinoma in the same tissue section. Seventy-one % (15 of 21) of the microdissected in situ lesions showed LOH of chromosome 11q13. Every case (15 of 15) of in situ tumor with LOH showed loss of the same allele in the corresponding invasive tumor cells. The results of this study suggest that a tumor suppressor gene located on chromosome 11q13 may play an important role in the early stages of development of sporadic human breast cancer. This finding provides molecular genetic support for the hypothesis that invasive breast cancer arises from in situ lesions.
Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.
A differential susceptibility phenotype to the organotropic colon carcinogen azoxymethane (AOM) has been described in mice. The following studies were undertaken to test the hypothesis that intraspecific susceptibility can be accounted for by the specific complement of genetic alterations acquired by precancerous colon lesions referred to as aberrant crypt foci (ACF). As an initial approach to this question, mutations in codons 12 and 13 of the Ki-ras proto-oncogene were assessed in ACF, normal-appearing AOM-treated colonic epithelium, and tumors from A/J and SWR/J (susceptible) as well as AKR/J (resistant) mice. Four-week-old male mice were injected intraperitonealy, with AOM once a week for a total of 6 wk and killed 4 and 24 wk after the last injection. DNA was isolated from microdissected tissue, and polymerase chain reaction (PCR)-amplified products of Ki-ras exon 1 (codons 12 and 13) were directly sequenced from microdissected tissues. At 4 wk after AOM exposure, there was no significant difference in the frequency of Ki-ras activation (20-33%) between the three strains. Ki-ras mRNA expression was also evaluated by reverse transcription (RT)-PCR analysis and was comparably reduced (40-50%) in all three strains at the 4 wk time point. However, Ki-ras expression returned to normal by 24 wk after treatment. Finally, to gain further insight into the molecular pathogenesis underlying this experimental tumor model, analysis of the adenomatous polyposis coli (APC) protein within the colonic epithelium was undertaken by using an immunohistochemical approach. Although the APC protein was lost to a varying extent in tumors from A/J and SWR/J mice, the full-length form of the protein was still present in precancerous ACF isolated from each of the three strains, regardless of the degree of dysplasia of the lesion. A further molecular genetic analyses of ACF will be required to gain a more complete understanding of the molecular basis of tumor susceptibility phenotype in this murine model.
Laser microdissection enables the contamination-free isolation of morphologically defined pure cell populations from archival formalin-fixed paraffin-embedded tissue specimens. Cells isolated by this method have been characterized by a wide variety of qualitative molecular assays, e.g. loss of heterozygosity, point mutations, clonality and lineage origin. The recently introduced real-time PCR technology renders the reliable quantification of very small amounts of nucleic acids possible. Several groups including our own showed that this technique can be successfully applied for the quantification of DNA and RNA isolated from microdissected archival tissue sections, even after immunohistochemical staining. The exact analysis of quantitative changes of nucleic acids during the course of pathological alterations has thus become possible. In many situations these quantitative changes can be expected to be more important than qualitative changes. The new technology for the quantification of structural genomic alterations and changes in the gene expression pattern in conjunction with microdissection have equipped morphologists with a powerful tool to study reactive and neoplastic changes of tissues.
Global genome amplification from formalin-fixed tissues is still problematic when performed with low cell numbers. Here, we tested a recently developed method for whole genome amplification termed "SCOMP" (single cell comparative genomic hybridization) on archival tissues of different ages. We show that the method is very well suited for formalin-fixed paraffin-embedded samples obtained by nuclei extraction or laser microdissection. The polymerase chain reaction (PCR) products can be used for subsequent comparative genomic hybridization, loss of heterozygosity studies, and DNA sequencing. To control for PCR-induced artifacts we amplified genomic DNA isolated from 20 nuclei of archival formalin-fixed, paraffin-embedded nonpathological lymph nodes. Subsequent comparative genomic hybridization revealed the expected balanced profiles. For loss of heterozygosity analysis by microsatellite PCR 60 to 160 cells were sufficient. In comparative experiments the approach turned out to be superior to published degenerated oligonucleotide-primed-PCR protocols. The method provides a robust and valuable tool to study very small cell samples, such as the genomes of dysplastic cells or the clonal evolution within heterogeneous tumors.
Identification of proteomic alterations associated with early stages in the development of prostate cancer may facilitate understanding of progression of this highly variable disease. Matched normal, high-grade prostatic intraepithelial neoplasia (hPIN) and prostate cancer cells of predominantly Gleason grade 3 were procured by laser capture microdissection from serial sections obtained from snap-frozen samples dissected from 22 radical prostatectomy specimens. From these cells, protein profiles were generated by surface-enhanced laser desorption/ionization-time of flight mass spectrometry. A 24-kDa peak was observed at low or high intensity in profiles of prostate cancer cells in 19 of 27 lesions and at low intensity in 3 of 8 hPIN lesions but was not detectable in matched normal cells. SDS-PAGE analysis of prostate cancer and matched normal epithelium confirmed expression of a prostate cancer-specific 24-kDa protein. Mass spectrometry and protein data-based analysis identified the protein as the dimeric form of mature growth differentiation factor 15 (GDF15). The increased expression of mature GDF15 protein in prostate cancer cells cannot be explained on the basis of up-regulation of GDF15 mRNA because reverse transcription-PCR analysis showed similar amounts of transcript in normal, hPIN, and prostate cancer cells that were obtained by laser capture microdissection in the same set of serial sections from which the protein profiles were obtained. Our findings suggest that early prostate carcinogenesis is associated with expression of mature GDF15 protein.
PURPOSE: The glucocorticoid-induced TNF-related receptor (GITR) plays a pivotal role in regulating the suppressive function of CD4+CD25+ regulatory T cells. GITR is also involved in the inhibition of T-cell receptor-induced apoptosis and the upregulation of inducible nitric oxide synthase (iNOS). GITR expression on CD4+ T cells has been shown to correlate with the disease course of noninfectious uveitis. The current study was conducted to examine the expression of glucocorticoid-induced TNF-related receptor ligand (GITRL) and its regulation by ocular tissue. METHODS: Immunohistochemistry and confocal immunofluorescence microscopy were performed on human ocular tissue to examine the in vivo protein expression of GITRL. The regulation of GITRL was investigated by culturing retinal pigment epithelium (RPE) with proinflammatory cytokines and performing immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The in vivo mRNA expression of GITRL was studied by RT-PCR on RNA from microdissected tissue sections. RESULTS: Both immunohistochemistry and confocal immunofluorescence microscopy demonstrated that GITRL was expressed constitutively on RPE and photoreceptor inner segments. Immunocytochemistry demonstrated that in vitro stimulated RPE cells expressed GITRL at the protein level, and RT-PCR showed that GITRL was upregulated at 24 hours by proinflammatory cytokines. Constitutive GITRL mRNA expression in vivo was confirmed by RT-PCR analysis of microdissected tissue. CONCLUSIONS: GITRL is expressed constitutively on RPE and in high levels on photoreceptor inner segments. The upregulation of GITRL by proinflammatory cytokines suggests that GITRL may play an important role in ocular immunity. The high level of constitutive GITRL expression on photoreceptor inner segments suggests that photoreceptors participate in the regulation of ocular inflammation.
The impact of high frequency oscillatory ventilation (HFOV) compared with intermittent mandatory ventilation (IMV) on oxygenation and pulmonary inflammatory response was studied in a surfactant depleted piglet model. After establishment of lung injury by bronchoalveolar lavage, piglets either received HFOV (n =5) or IMV (control; n = 5) for eight hours. PaO(2) was higher and mean pulmonary arterial pressure (MPAP) was lower with HFOV (HFOV versus control, mean +/- SEM; endpoint PaO(2): 252 +/- 73 versus 68 +/- 8.4 mm Hg; p < 0.001; MPAP: 22 +/- 2.3 versus 34 +/- 2.5 mm Hg; p < 0.01). mRNA expression of interleukin (IL)-1 beta, IL-6, IL-8, IL-10, TGF-beta 1, Endothelin-1, and adhesion molecules (E-selectin, P-selectin, ICAM-1) in lung tissue was quantified by real time PCR normalized to beta-actin and hypoxanthine-guanine-phosphoribosyl-transferase (HPRT). mRNA expression of all cytokines and adhesion molecules/HPRT was higher in controls (e.g.: HFOV versus control, mean +/- SEM; IL-1 beta/HPRT: 1.6 +/- 0.3 versus 23.1 +/- 8.6 relative units (RU), p < 0.001; IL-8/HPRT: 8.5 +/- 2.0 versus 63.5 +/- 15.2 RU, p < 0.001). IL-8/HPRT gene expression was quantified in microdissected single cells. With HFOV, IL-8 gene expression was highly reduced in alveolar macrophages: 10 +/- 3.4 copies IL-8 mRNA/copy HPRT mRNA versus 356 +/- 142; p < 0.05 (bronchiolar epithelial cells: 33 +/- 16 versus 208 +/- 108; alveolar septum: 2.1 +/- 1.3 versus 26 +/- 11; bronchiolar smooth muscle cells: 1.3 +/- 0.3 versus 2.8 +/- 1.0; vascular smooth muscle cells: 0.7 +/- 0.3 versus 1.1 +/- 0.4). In conclusion, HFOV improved oxygenation, reduced pulmonary arterial pressure and attenuated pulmonary inflammatory response.
The architecture and compartmentalization of the kidney has stimulated the development of an array of microtechniques to study the functional differences between the distinct nephron segments. With the vast amounts of genomic sequence data now available, the groundwork has been laid for a comprehensive characterization of the molecular pathways defining the differences in nephron function. With the development of sensitive gene expression techniques the tools for a comprehensive molecular analysis of specific renal microenvironments have been provided: Quantitative RT-PCR technologies now allow the analysis of specific mRNAs from as little as single microdissected renal cells. A more global view of gene expression regulation is a logical development from the application of large scale profiling techniques. In this review, we will discuss the power and pitfalls of these approaches, including their potential for the functional characterization of nephron heterogeneity and diagnostic application in renal disease.
Prostate carcinomas are one of the most common malignancies in western societies. The pathogenesis of this tumor is still poorly understood. These tumors present with two characteristic features: epithelial-mesenchymal interactions, which play a pivotal role for tumor development and most of clinically manifest cancers arise in prostate proper compared to a minority of tumors which develop in the transitional zone. Deciphering the epithelial-mesenchymal cross talk and identification of molecular pecularities of the sub-populations of cells in different zones can therefore help understanding carcinogenesis and development of new, non-invasive tools for the diagnosis and prognosis of prostate carcinomas which has remained a challenge until today. A ProteinChip array technology (SELDI = surface enhanced laser desorption ionization) has been developed recently by Ciphergen Biosystems enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the analysis of approximately 500-1000 freshly obtained prostate cells by SELDI-TOF-MS (surface enhanced laser desorption ionization time-of-flight mass spectrometry). Pure cell populations of stroma, epithelium and tumor cells were selected by laser assisted microdissection. Multiple specific protein patterns were reproducibly detected in the range from 1.5 to 30 kDa in 28 sub-populations of 4 tumorous prostates and 1 control. A specific 4.3 kDa peak was increased in the prostate tumor stroma compared to normal prostate proper and transitional zone stroma and increased in prostate tumor glands compared to normal prostate proper and transitional zone glands. Coupling laser assisted microdissection with SELDI provides tremendous opportunities to identify cell and tumor specific proteins to understand molecular events underlying prostate carcinoma development. It underlines the vast potential of this technology to better understand pathogenesis and identify potential candidates for new specific biomarkers in general which could help to screen for and distinguish disease entities, i.e. between clinically significant and insignificant carcinomas of the prostate.
Explore the source record for details and available documents.