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PubMed2006

Microdissection with PCR in situ.

In situ amplification techniques are designed to increase the mass of DNA in a fixed target, either whole cells or tissue sections. When combined with fluorescently labeled nucleotides, they can be used for locus detection. They also can be used to increase target mass for subsequent operations, such as cellular or chromosomal isolation by microdissection. When combined with chromosome microdissection, these techniques allow libraries to be made from single copies of chromosomes, chromosome fragments, or even bacteria.

Bacteria↗

PubMed1989

Distribution of a prolactinlike material in human eccrine sweat glands.

Because prolactin has been implicated in the transport of electrolytes in several mammalian tissues, we have looked for the presence of prolactin in human eccrine sweat glands where a primary isotonic secretion, rich in sodium chloride, is produced and subsequently modified by recovery of some sodium and chloride in excess of water. Sweat glands were microdissected from skin biopsies and then fixed overnight in phosphate-buffered 4% formaldehyde. The fixed tissue was dehydrated (to 95%) in ethanol and then embedded in glycol methacrylate. Sections were cut (5 microns) and immunostained with antihuman prolactin (NIDDK IC2) and the specifically-bound antibody was visualized using a biotinylated second antibody and Vector ABC reagents. Prolactinlike immunoreactivity was localized in the clear cells of the secretory coil and, to a much lesser extent, in the basal layer of duct cells. In many of the clear cells, the immunoreactive material appeared as a lateral strip and occasionally, in favorable sections, as a horseshoe of reaction product a few microns in from the apical and lateral membranes. In a subset of clear cells, with more euchromatic nuclei and a long, thin cellular profile, the immunostaining was more intense and was localized in a more juxtanuclear position. Controls for endogenous peroxidase, and those using normal serum or antihuman prolactin serum preabsorbed with purified human prolactin, gave no peroxidase localization in the tissue. These results are important because 1) they represent the first demonstration of prolactin or a prolactinlike substance in the sweat gland, 2) the prolactinlike material was localized to clear cells that are thought to be responsible for much of the fluid secretion, 3) the necessity for prolactin or pituitary extract in primary cultures of sweat gland epithelium is potentially explained, although not fully understood, and 4) it means that further studies concerned with the possible influence of prolactin on ion transport in the sweat gland are warranted.

Humans↗

PubMed1978

Tissue culture of isolated human glomeruli.

Glomerular cells have been grown in a reproducible manner from 5 normal human kidneys. A technique is described which combined mechanical disruption of renal cortex and microdissection, and provides large numbers of pure glomeruli within 30--45 mintues. Histological examination shows this technique produces intact glomeruli without cell disturbance. During tissue culture, glomeruli attach to the flask and the intrinsic cells migrate onto the flask and divide. Variations of culture conditions have shown that glomeruli are robust without fastidious culture requirements. Intact kidney tissue can be left at 4 degrees C for perios up to 24 hours prior to isolated of individual glomeruli without affecting subsequent cellular growth in culture. They grow in most commonly used media although the cells require 20% foetal calf serum for optimum growth. Their pH optimum is between 7.0 and 7.4 with temperature optimum of 37 degrees C. as glomeruli must attach prior to cell growth, minimum movement is critical to promote optimum growth. Under these optimum conditions a regular and predictable growth of cells of two distinct types, has been observed over 14 days; one of these types is probably epithelial.

Culture Techniques↗

PubMed1997

Glomerular Fc alphaR expression and disease activity in IgA nephropathy.

In this study, we examined the receptors for the Fc portion of immunoglobulin A (IgA) (Fc alphaR) in the glomeruli as well as circulating polymorphonuclear leukocytes and monocytes at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay and at the protein level by an immunohistochemistry/flow cytometry technique using a specific anti-Fc alphaR monoclonal antibody (My 43). Glomeruli were isolated from biopsy specimens of renal tissues from IgA nephropathy (IgAN; 20 cases) and non-IgA mesangial proliferative glomerulonephritis (PGN; 13 cases) patients, and from normal renal tissue specimens obtained from kidneys removed because of malignancies (five cases) applying the microdissection method. There was a relative increase in Fc alphaR in the circulating phagocytes from IgAN patients compared with those from PGN and healthy controls. Fc alphaR expression was present in approximately 40% of glomeruli samples from IgAN patients at the message levels. Fc alphaR-positive specimens were also strongly positive for expression of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 mRNA. Specimens from PGN patients and healthy controls did not show any detectable Fc alphaR message. Serum IgA levels and severity of hematuria were significantly higher in patients with positive Fc alphaR expression. A message for Fc alphaR was detected in the tissues that were more damaged histologically. Our data suggest that there is some in vivo induction of glomerular Fc alphaR expression, possibly mediated by a synergistic stimulus from IgA and inflammatory cytokines, and the expressed receptor is likely to be involved in the disease process of IgAN.

Antigens, CD↗

PubMed2005

Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas.

Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer cells. We have now used laser capture microdissection and real-time PCR to quantify the mRNA expression of components of matrix-degrading proteolytic systems in cancer and stromal areas of mouse mammary tumors genetically induced by the polyoma virus middle T (PyMT) antigen. We examined the mRNA levels of urokinase plasminogen activator, plasminogen activator inhibitor 1 and the matrix metalloproteases MMP-2, -3, -11, -13 and -14, and found that all these seven genes are predominantly expressed by stromal cells. Our results were qualitatively supported by in situ hybridization analysis of the expression of mRNAs for MMP-2, -3 and -13 in the PyMT tumors. Statistical analyses indicated that the quantitative expression patterns observed in cancer and stromal cells isolated from individual tumors from different PyMT mice are quite reproducible. The methodology described in this study provides excellent tools to study the possible interactions between cancer and stromal cells during the development of breast cancer, and the results suggest that stromal cells are involved in carcinogenesis and tumor progression, which may have important implications for the biology and therapy of cancer.

Animals↗

PubMed2003

[Frequent 4 977 bp deletion of mitochondrial DNA in tumor cell lines, solid tumors and precancerous lesions of human stomach].

OBJECTIVE: To clarify the frequency of mtDNA 4 977 bp deletion in tumor cell lines, solid tumors, patient's serum of gastric tumor and to find a easy and exact method to diagnose gastric tumor. METHODS: Primer-shift PCR method was used in 13 gastric tumor cell lines, 52 cases of gastric fresh tumor tissues matched the adjacent normal tissues, 40 cases of patient's serum of gastric tumor and 40 cases of normal serums for analysis of mtDNA deletion. RESULTS: Frequency of 4 977 bp mtDNA deletion was detected in 12 of 13 (92.3%) tumor cell lines, 38 of 52 (73.1%) cases of solid tumor tissues, 27 of 52 (52%) adjacent normal tissues, 17 of 40 (42.5%) patient's serum and 8 of 40 (20%) normal serums. Further more, in 2 of 10 pairs microdissected specimens, we found this deletion occurred not only in primary tumor but also in intestinal metaplasia comparing with no deletion was found in normal tissues. The frequency of this deletion was statistically significantly higher in the gastric tumor tissues and serums than in the adjacent normal tissues and serums. A good correlation between the deletion and young age of patients was representative in our data. CONCLUSION: mtDNA 4 977 bp deletion maybe play an important role in the carcinogenesis of human gastric mucous and maybe has happened in the tumor cell malignant transformation. To detect this deletion in patient's serum is a easy and exact method and maybe become a potential tumor marker.

Adenocarcinoma↗

PubMed2003

Improved detection of clonality in cutaneous T-cell lymphomas using laser capture microdissection.

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 gamma 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.

Clone Cells↗

PubMed2005

Aromatase expression in ovarian epithelial cancers.

Our study focused on aromatase cytochrome P450 (CYP19) expression in ovarian epithelial normal and cancer cells and tissues. Aromatase mRNA expression was analyzed by real-time PCR in ovarian epithelial cancer cell lines, in human ovarian surface epithelial (HOSE) cell primary cultures, and in ovarian tissue specimens (n=94), including normal ovaries, ovarian cysts and cancers. Aromatase mRNA was found to be expressed in HOSE cells, in BG1, PEO4 and PEO14, but not in SKOV3 and NIH:OVCAR-3 ovarian cancer cell lines. Correlation analysis of aromatase expression was performed according to clinical, histological and biological parameters. Aromatase expression in ovarian tissue specimens was higher in normal ovaries and cysts than in cancers (P<0.0001). Using laser capture microdissection in normal postmenopausal ovaries, aromatase was found to be predominantly expressed in epithelial cells as compared to stromal component. Using immunohistochemistry (IHC), aromatase was also detected in the epithelium component. There was an inverse correlation between aromatase and ERalpha expression in ovarian tissues (P<0.001, r=-0.34). In the cancer group, no significant differences in aromatase expression were observed according to tumor histotype, grade, stage and survival. Aromatase activity was evaluated in ovarian epithelial cancer (OEC) cell lines by the tritiated water assay and the effects of third-generation aromatase inhibitors (AIs) on aromatase activity and growth were studied. Letrozole and exemestane were able to completely inhibit aromatase activity in BG1 and PEO14 cell lines. Interestingly, both AI showed an antiproliferative effect on the estrogen responsive BG1 cell line co-expressing aromatase and ERalpha. Aromatase expression was found in ovarian epithelial normal tissues and in some ovarian epithelial cancer cells and tissues. This finding raises the possibility that some tumors may respond to estrogen and provides a basis for ascertaining an antimitogenic effect of AI in a subgroup of ovarian epithelial cancers.

Androstadienes↗

PubMed1982

Microdissection by ultrasonication after prolonged OsO4 fixation: a technique for scanning electron microscopy.

A new method of tissue preparation for electron microscopy is described for the selective dissection of mammalian tissues. This technique, based upon brittlization by prolonged osmication, dehydration to pure acetone and subsequent ultrasonication, has been especially useful for the SEM visualization of microcirculation and basal laminas. Epithelial cells of treated tissues were largely fragmented and removed, whereas collagen containing materials, notably epithelial and vascular basal laminas, withstood the vibratory insult. The extent of epithelium and basal lamina removal depended upon tissue type and duration of ultrasonic treatment. Alone or in conjunction with specific enzymatic pretreatment, this new technique offers a method to better visualize known and unrecognized histological relationships.

Animals↗

PubMed2004

Microdissection and SAGE as a combined tool to reveal gene expression in ductal carcinoma in situ of the breast.

The interplay between cancer cells and the normal surrounding tissue is believed to influence the biological behavior of the tumor. However, the presence of multiple cell types within the prelevated tumor specimen may attenuate changes that occur specifically in the malignant cells within their microenvironment. To study gene expression of the malignant cells in situ, we used a new microdissection method to separate ductal carcinoma in situ (DCIS) cells from the surrounding stroma, immunological infiltrates, and endothelial cells. We applied an adapted microSAGE protocol, without total mRNA amplification, to study their gene expression profile. Three thousand two hundred one different transcripts were identified in a total of 29 534 observed tags. Of these unique tags, 88.3% matched known GenBank sequences and 11.7% represented unknown transcripts. As compared to a total DCIS SAGE library, microdissection combined with SAGE revealed additional genes expressed only in normal surrounding, probably stromal, cells and not or significantly less in DCIS tumor cells. This study demonstrates that microdissection can be combined with SAGE as a tool to study transcriptomes. This approach provides important new information on differential gene expression both in tumor cells and normal surrounding tissue. Several of the observed differences indeed disappear when the total tumor mass is analyzed.

Adult↗

PubMed2000

Sporadic trichoepithelioma demonstrates deletions at 9q22.3.

BACKGROUND: Trichoepithelioma (TE) is a benign cutaneous tumor that originates from hair follicles and occurs either in multiple or solitary lesions. Multiple TE is transmitted as an autosomal dominant trait, and a region at 9p21 is thought to be involved in the tumorigenesis. Solitary TE occurs more commonly than multiple TE and is not inherited. Histologically, TE tumors contain horn cysts and abortive hair papillae. A basal cell carcinoma appearance in some or all regions of a TE tumor can happen. In sporadic basal cell carcinoma, frequent deletions at 9q22.3 (Drosophila patched gene) have occurred. The objective of this study is to test whether loss of heterozygosity (LOH) on either 9p21 or on chromosome 9q22.3 could be detected in archival sporadic TE. OBSERVATIONS: We studied 29 randomly selected cases of sporadic TE by microdissection and polymerase chain reaction using paraffin-embedded, formalin-fixed tissue specimens on glass slides. Analysis was performed with the polymorphic markers IFNA and D9S171 (9p21) as well as D9S15, D9S303, D9S287, and D9S252 (9q22.3). RESULTS: The LOH at 9q22.3 was identified in 14 (48%) of 29 cases with at least 1 marker, while LOH could not be demonstrated using the markers IFNA and D9S171 (9p21). CONCLUSIONS: The results show that the Drosophila patched gene LOH can be frequently identified in paraffin-embedded sporadic TE after routine processing and indicates a common gatekeeper mechanism for both TE and basal cell carcinoma.

Chromosomes, Human, Pair 9↗

PubMed2001

Laryngeal mucosa elasticity and viscosity in high and low relative air humidity.

In earlier work we showed that low relative humidity (RH) of inhaled air causes acoustic voice parameters such as jitter and shimmer to deteriorate. Other authors have shown negative effects on vocal efficiency. To explain these changes in the mechanical properties of the vocal folds, the effects of changes in RH of the air passing over microdissected mucosa of sheep larynges were studied. The dissected surface of the tissue specimen just touched Ringer solution and air of varying RH was blown over the specimen. The mucosa specimen was subjected to sinusoidal oscillations of length (strain) and the resulting force (stress) was measured. The gain and phase angle between the imposed strain and resulting stress were measured, and elasticity and viscosity were calculated. Two different air conditions were tested: air with high RH (100%) vs air with low RH (0%). Viscosity and stiffness increased significantly in both ambient conditions (P < 0.01). Dry dehydrating air resulted in a stiffer and more viscous cover than humid air (P < 0.001). These changes in mechanical characteristics may contribute to the effects on voice parameters described in earlier work.

Animals↗

PubMed1992

Epithelial injury and interstitial fibrosis in the proximal alveolar regions of rats chronically exposed to a simulated pattern of urban ambient ozone.

Electron microscopic morphometry was used to study the development of lung injury during and after chronic (78 weeks) exposure to a pattern of ozone (O3) designed to simulate high urban ambient concentrations that occur in some environments. The daily exposure regimen consisted of a 13-hr background of 0.06 ppm, an exposure peak that rose from 0.06 to 0.25 ppm, and returned to the background level over a 9-hr period, and 2-hr downtime for maintenance. Rats were exposed for 1, 3, 13, and 78 weeks. Additional groups of rats exposed for 13 or 78 weeks were allowed to recover in filtered clean air for 6 or 17 weeks, respectively. Rats exposed to filtered air for the same lengths of time were used as controls. Samples from proximal alveolar regions and terminal bronchioles were obtained by microdissection. Analysis of the proximal alveolar region revealed a biphasic response. Acute tissue reactions after 1 week of exposure included epithelial inflammation, interstitial edema, interstitial cell hypertrophy, and influx of macrophages. These responses subsided after 3 weeks of exposure. Progressive epithelial and interstitial tissue responses developed with prolonged exposure and included epithelial hyperplasia, fibroblast proliferation, and interstitial matrix accumulation. The epithelial responses involved both type I and type II epithelial cells. Alveolar type I cells increased in number, became thicker, and covered a smaller average surface area. These changes persisted throughout the entire exposure and did not change during the recovery period, indicating the sensitivity of these cells to injury. The main response of type II epithelial cells was cell proliferation. The accumulation of interstitial matrix after chronic exposure consisted of deposition of both increased amounts of basement membrane and collagen fibers. Interstitial matrix accumulation underwent partial recovery during follow-up periods in air; however, the thickening of the basement membrane did not resolve. Analysis of terminal bronchioles showed that short-term exposure to O3 caused a loss of ciliated cells and differentiation of preciliated and Clara cells. The bronchiolar cell population stabilized on continued exposure; however, chronic exposure resulted in structural changes, suggesting injury to both ciliated and Clara cells. We conclude that chronic exposure to low levels of O3 causes epithelial inflammation and interstitial fibrosis in the proximal alveolar region and bronchiolar epithelial cell injury.

Administration, Inhalation↗

PubMed2004

alpha(2)-Macroglobulin: a novel cytochemical marker characterizing preneoplastic and neoplastic rat liver lesions negative for hitherto established cytochemical markers.

We tried to identify a novel marker characteristic for rat hepatocellular preneoplastic and neoplastic lesions, undetectable by well established cytochemical markers. Glutathione S-transferase placental (GST-P)-negative hepatocellular altered foci (HAF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) were generated by two initiation-promotion models with N-nitrosodiethylamine (NDEN) and peroxisome proliferators, Wy-14,643 and clofibrate. Total RNAs isolated from laser-microdissected GST-P-negative HAF (amphophilic cell foci) and adjacent normal tissues were applied to microarray analysis. As a result, five up-regulated genes were identified, and further detailed examinations of the gene demonstrating most fluctuation, ie, that for alpha(2)-macroglobulin (alpha(2)M) were performed. In reverse transcriptase-polymerase chain reaction, alpha(2)M mRNA was overexpressed not only in amphophilic GST-P-negative HAF but also in amphophilic GST-P-negative HCA and HCC. In situ hybridization showed accumulation of alpha(2)M mRNA to be evenly distributed within GST-P-negative HAF (predominantly amphophilic cell foci). Distinctive immunohistochemical staining for alpha(2)M could be consistently demonstrated in GST-P-negative HAF, HCA, and HCC induced not only by peroxisome proliferators but also N-nitrosodiethylamine alone. Thus our findings suggest that alpha(2)M is an important novel cytochemical marker to identify hepatocellular preneoplastic and neoplastic lesions, particularly amphophilic cell foci, undetectable by established cytochemical markers and is tightly linked to rat hepatocarcinogenesis.

Adenoma↗

PubMed1998

Quantitative biochemical analysis of samples from squamous cell carcinoma.

Samples of tumor, tumor stroma, and mixed tumor and stroma were microdissected from three squamous cell carcinomas, as well as from adjacent nontumor tissue and assayed for malate dehydrogenase or lactate dehydrogenase activity as indicators of the relative contribution of aerobic and anaerobic processes, respectively, to energy metabolism. Malate and lactate dehydrogenase activities were greater in tumor tissue than in tumor stroma in all three tumors. However, the magnitudes of malate and lactate dehydrogenase activities were different among the tumors, suggesting that tumors vary in the degree to which aerobic and anaerobic reactions contribute to energy production. These results indicate that a quantitative assessment of enzymes involved in energy production may be a useful probe in the understanding of tumor metabolism.

Carcinoma, Squamous Cell↗

PubMed2004

Prostate cancer is characterized by epigenetic silencing of 14-3-3sigma expression.

In order to identify tumor suppressive genes silenced by CpG methylation in prostate carcinoma (PCa), we determined genome-wide expression changes after pharmacological reversal of CpG methylation-mediated transcriptional repression in three PCa cell lines using microarray analysis. Thereby, epigenetic silencing of the 14-3-3sigma gene was detected in the cell line LNCaP. 14-3-3sigma encodes a p53-regulated inhibitor of cell cycle progression. Laser microdissection was used to isolate different cell types present in diseased prostatic tissue. Subsequent methylation-specific PCR analysis showed CpG methylation of 14-3-3sigma in all 41 primary PCa samples analysed, which was accompanied by a decrease or loss of 14-3-3sigma protein expression. In contrast, normal prostate epithelial and benign prostate hyperplasia cells showed high levels of 14-3-3sigma expression. PCa-precursor lesions (prostatic intraepithelial neoplasia) also displayed decreased levels of 14-3-3sigma expression in luminal cells, which are known to contain shortened telomeres. RNA interference-mediated inactivation of 14-3-3sigma compromised a DNA damage-induced G(2)/M arrest in the PCa cell line PC3. The generality of CpG methylation and downregulation of 14-3-3sigma expression in PCa suggests that it significantly contributes to the formation of PCa, potentially by allowing the escape from a DNA damage-induced arrest elicited by telomere shortening.

14-3-3 Proteins↗

PubMed2004

NOX3, a superoxide-generating NADPH oxidase of the inner ear.

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.

Adaptor Proteins, Signal Transducing↗

PubMed1992

Localization of occult persisting medullary thyroid carcinoma before microsurgical reoperation: high sensitivity of selective venous catheterization.

In 14 patients with occult persisting medullary thyroid carcinoma, tumor tissue was removed by microsurgical reoperation in 13 of 14 patients. This resulted in biochemical improvement in all but 1 patient and biochemical cure in 3 patients (21%). The lateral compartment of the neck or the upper mediastinum was involved in all but 1 patient. Before microsurgical reoperation, selective venous catheterization (SVC) for serum sampling along with serum calcitonin (CT) determination was done and compared to other localization methods. Tumor tissue could be localized correctly by SVC in 89% (CT gradient 1.21-2.02), computed tomography in 38%, and ultrasound in 28%. In patients with an elevated CT level after initial surgery and clinically occult disease, SVC is recommended for localization of tumor tissue. The affected side of the neck should be reoperated on with microdissection of the central and lateral compartment of the neck and the upper mediastinum. With this procedure, the cure rate of reoperation in patients with persistent occult MTC can be improved.

Calcitonin↗