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PubMed1996

Immunohistochemistry of transmissible gastroenteritis virus antigens in fixed paraffin-embedded tissues.

An immunohistochemistry technique was developed using fixed tissues to study the presence and location of transmissible gastroenteritis virus (TGEV) antigens in situ. Experimentally infected gnotobiotic and conventional pigs as well as pigs with natural TGEV infection were examined. The staining technique was based on detection of the major structural protein of TGEV, the nucleocapsid, by using a pool of 3 monoclonal antibodies. Formalin and periodate-lysine-paraformaldehyde (PLP)-fixed intestinal tissues from a gnotobiotic pig inoculated with virulent TGEV were used to determine optimal antibody concentrations and incubation times. The intestinal tissues remained in their respective fixatives for 6 months, and serial sections were removed at sequential times and embedded in paraffin blocks. PLP and 10% neutral buffered formalin were acceptable fixatives and preserved TGEV nucleocapsid antigenicity for up to 6 months. Formalin, in comparison with PLP as a fixative, was better for preserving original tissue morphology and provided better antigen detection. Conventional crossbred pigs were inoculated with virulent TGEV, and animals were euthanized on various postexposure days. Intestinal tissues were positive for TGEV nucleocapsid antigens on postexposure days 2, 4, and 8. The immunohistochemistry technique detected TGEV antigen in stored paraffin-embedded tissues from 14 naturally infected pigs previously confirmed as positive for TGEV using a direct immunofluorescence assay on intestinal mucosal smears, whereas 9 naturally infected pigs confirmed negative for TGEV antigen by the same immunofluorescence assay showed no staining consistent with the presence of TGEV antigen. Immunohistochemistry provides a method to detect TGEV and possibly other closely related coronaviruses such as porcine respiratory coronavirus in situ. A diagnostic test using the same fixed tissues processed for histopathology provides veterinary practitioners an alternative to delivering live pigs or refrigerated fresh intestinal samples containing infectious virus to a diagnostic laboratory. Investigators can utilize this technique to retrospectively screen fixed tissues for TGEV antigen.

Animals↗
PubMed1994

Selective elimination of hypothalamic neurons by grafted hypertension-inducing neural tissue.

Embryonic hypothalamic tissue originating from spontaneously hypertensive rats (SHR) was implanted in young normotensive Wistar Kyoto rats in an attempt to localize hypothalamic regions directly responsible for the induction of hypertension. A 25% increase in host systolic blood pressure as compared with the controls was recorded 3 months after implantation in the animals receiving rostral hypothalamic tissue (R-SHR), whereas blood pressure was not affected in the animals grafted with caudal hypothalamic tissue (C-SHR). The hypertension in the R-SHR group was accompanied by hypertrophy of the heart and kidneys. The number of vasopressin-immunopositive (VPi) parvocellular cells in the hypothalamic paraventricular nucleus (PVN) of the R-SHR group was massively reduced (by 72%), while that of the tyrosine hydroxylase-immunopositive cells displayed no change. In the suprachiasmatic nucleus of these animals the VPi cell number was unaltered. In the C-SHR, the amount of parvocellular VPi cells was also unaltered. Likewise, oxytocin-containing cells were the same in all groups. DNA nick-end labeling of the tissue revealed that PVN cells are undergoing programmed cell death. These results implicate a selective degeneration by hypothalamic PVN cells in the pathogenesis of hypertension.

Animals↗
PubMed1981

Restriction endonuclease analysis of mitochondrial DNA from virus-transformed, tumor and control cells of human, hamster and avian origin. Sequence conservation and intraspecific variation.

This study compares over 70 recognition sites for restriction endonucleases on mtDNAs from various control versus malignant cells, derived from Syrian hamster, chick embryo, viper and human cells, exhibiting a wide spectrum of cellular transformation and tumor histories. Agents for transformation in vitro and in vivo include Rous sarcoma viruses, simian virus 40, polyoma virus and adenovirus. The results show a striking intraspecific sequence homogeneity of different mtDNAs regardless of tissue origin and oncogenic history. mtDNA from human biopsy specimens of tumor versus pathologically normal areas yielded indistinguishable restriction cleavage patterns reflecting either the "wild-type' form (with seven restriction endonucleases) or, in one individual, a variant pattern detected with HpaI. The precise position of the HpaI variant site was determined on the physical map of human mtDNA. Additional cleavage sites in the previously reported restriction map of Syrian hamster mtDNA are also presented. It is concluded that (1) mtDNA sequence in higher animal cells are highly conserved in malignant transformation; (2) no evidence for integration of viral sequences in mtDNA is apparent; (3) variant patterns in mtDNA are likely to be intraspecific polymorphisms that pre-exist neoplastic transformation. The possibility is discussed that altered regulatory interaction with the mitochondrial genome, rather than evident changes in mtDNA primary structure, determine anomalous mitochondrial functions in malignant transformation.

Adenoviruses, Human↗
PubMed2001

Tumour metastasis: is tissue an issue?

Single tumour cells, or multicellular aggregates, escape into blood and lymphatic vessels from a primary solid tumour as it progresses. However, the ability of such cells to develop into metastatic outgrowths is very limited, as shown by the poor prognostic power of the presence of circulating tumour cells in cancer patients. An explanation for this low efficiency may be a temporary absence of a suitable microenvironment once the tumour cells escape from their original tissue compartment. On the basis of histopathological observations, experimental studies, and the generally accepted poor prognosis of histopathologically confirmed intravascular tumour location, we propose that the structural and functional organisation of intravascular tumour cells as a tissue has a key role in providing the optimum microenvironment for sustained malignant dissemination. Such a tissue may be a fixed or a mobile intravascular microsatellite, or an intravascular micrometastasis, which locally develops into an overt in-transit metastasis.

Humans↗
PubMed1985

Pathogenesis of heterogeneity in human multinodular goiter. A study on growth and function of thyroid tissue transplanted onto nude mice.

Functional and morphologic heterogeneity of human multinodular goiters was investigated in 300 samples from "cold" and "hot" regions of 20 goiters transplanted onto nude mice. Transplants were labeled with [3H]thymidine and radioiodine, while the host's thyroid-stimulating hormone (TSH) secretion was either stimulated or suppressed. Proliferation and function of follicular cells were assessed in whole follicles reconstructed from autoradiographs of serial sections. Hot transplants had a higher autonomous iodine uptake than those of cold tissue in TSH-suppressed hosts. Functional autonomy widely varied among the follicles, but even more so among individual cells. Hot grafts differed from cold ones only by a comparatively larger fraction of autonomous cells. Intercellular differences of iodinating activity were not abolished by TSH. Grafts faithfully reproduced the individual growth pattern of the original tissue. Between 0.5% and 7% of all follicular cells replicated despite suppression of TSH. Up to 70% of these cells were clustered, forming scattered foci of autonomously growing tissue. Other cells only started replicating after long-term TSH stimulation. Thus, goiters contained subsets of cells with high and others with low growth response. Progenies of replicating cells remained clustered, sometimes budding outwards to form new follicles. Autonomy of growth and autonomy of function are independent traits of epithelial cells. Epithelial cells have their individual growth pattern, replication rate, and functional capacity. These traits are passed on from a mother cell to its progeny during follicle neogenesis. To this main mechanism accounting for the morphologic and functional heterogeneity of human goiters, inheritable modifications of gene expression must probably be added.

Animals↗
PubMed1988

Characterization of the pore transport properties and tissue alteration of excised human skin during iontophoresis.

Pores through which charged carriers move during iontophoresis were demonstrated by the use of the cathodic (-) iontophoretic transport of fluorescein from the epidermis to the dermis. Both dermatomed (0.8-mm) human cadaver skin and full-thickness female human breast skin were investigated. The density of pores, as visualized by fluorescein transport, was approximately 2-5 cm-2. A set of microelectrodes rastered across the visualized pore gave a maximal response when directly above the pore, demonstrating that the pore was a locus of charge transport. Fluorescein was also sometimes observed at the diffusion cell-tissue interface. This indicates that edge damage had occurred as the result of clamping the tissue in a diffusion cell. Studies were conducted to determine if tissue damage occurred during iontophoretic transport. The electrical resistance across excised skin was measured at 0.2 Hz and found to decrease initially by approximately an order of magnitude after the application of an iontophoretic current of 0.16 mA/cm2 for 1 h. The electrical resistance then increased, reaching a plateau value which was lower than the original tissue resistance before application of an iontophoretic current. Controls were carried out to demonstrate that the observed electrical resistance changes were not just due to tissue hydration effects. These results imply that the passage of current through excised human skin at clinically acceptable current densities can lead to tissue damage which is not fully reversible.

Adult↗
PubMed2005

Localization of ferruginol, a diterpene phenol, in Cryptomeria japonica heartwood by time-of-flight secondary ion mass spectrometry.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was applied to the investigation of heartwood extractives in Sugi (Cryptomeria japonica). Sugi heartwood tissue generated secondary ions that were not produced from sapwood tissue by TOF-SIMS. Among the peculiar ions generated from heartwood, two positive ions of m/z 285 and 301 were remarkable due to their appearance in a larger mass range and with a high intensity. These two ions were not generated from heartwood tissue preextracted with n-hexane, and the n-hexane extract of Sugi heartwood produced both ions. Gas chromatography-mass spectrometry of the n-hexane extract demonstrated that ferruginol, a diterpene phenol, the molecular weight of which is 286, constituted one of the predominant constituents of the extract. Authentic ferruginol also generated both ions by TOF-SIMS. The molecular formula of the m/z 285 ion generated from Sugi heartwood tissue was estimated to be C(20)H(29)O, which corresponds well with that of ferruginol, i.e. C(20)H(30)O, by peak identification. All these results strongly suggest that the m/z 285 ion generated from Sugi heartwood tissue originated significantly from ferruginol in Sugi heartwood. By TOF-SIMS imaging, the m/z 285 ion was detected uniformly in the tracheid cell walls, in the cell walls of the axial parenchyma cells and ray parenchyma cells, and also inside these parenchyma cells. These results indicate that ferruginol was distributed almost evenly in Sugi heartwood tissue.

Abietanes↗
PubMed1998

A comparative study of epithelialization of subcutaneous fascial flaps and muscle-only flaps in the oral cavity. A rabbit model.

Epithelialization of subcutaneous fascial flaps and muscle-only flaps used for reconstruction of the oral mucosa were studied in a rabbit model. Angiography was performed to help to design a subcutaneous cervical fascial flap and a cleidomastoid muscle-only flap. These flaps were transferred into a mucoperiosteal defect on the mandibular alveolus. The flaps were gradually infiltrated by acute inflammatory cells from the periphery and then replaced by granulation tissue originating from the oral mucosa. The epithelialization was by secondary intention. An epithelial tongue from the surrounding epithelium migrated onto the granulating flaps with eventual coverage after three weeks. After two months, the muscle-only flap was covered by a poorly organized epithelium, different from the highly uniform epithelium in the fascial flap. The granulation tissue which replaced the muscle-only flap matured to fibrous tissue, associated with severe contraction. This was characterized by dense regular collagen fibers, no elastic fibers, and few capillaries, while the fibrous tissue which replaced the fascial flap was characterized by random collagen and elastic fiber morphology and numerous dilated blood vessels, and was associated with mild contraction. This experiment supports the view that the different extracellular matrices of these flaps may play a role in epithelial configuration and contraction.

Animals↗
PubMed2000

Is mechanism-based pain treatment attainable? Clinical trial issues.

Woolf et al have recently called for the development of a mechanism-based pain taxonomy to guide the individualization of treatment based on each patient's pain mechanisms. Although any scientific physician could endorse this ideal, small academic clinical trials so far have failed to identify obvious differences in the response of different pain symptoms in the same condition to various drugs. In contrast, there are clear differences in the analgesic responses of patient groups distinguished on the basis of etiology or tissue origin of pain, factors which tend to be associated with groups of mechanisms. The few tests to diagnose pain mechanisms remain too delicate, time-consuming, or uncomfortable for general clinical use. To understand how best to exploit new mechanistic insights to assign treatments, one must scrutinize the relative value of diagnostic classifications based on etiology, tissue, and individual patients' pain characteristics in large clinical trials. Research priorities should include developing simple methods for assessing pain mechanisms in the clinic and increasing the efficiency of pain assessment methods in clinical trials. I describe a collaborative research agenda for academic pain researchers and funding agencies, the pharmaceutical industry, and regulatory bodies.

Journal Article↗
PubMed1990

Karyometric marker features in fine needle aspirates of microinvasive follicular carcinoma of the thyroid.

Karyometric measurements were performed on fine needle aspirates of clearly identifiable tumor areas and adjacent normal-appearing areas in the surgical specimens from ten patients with microinvasive follicular carcinoma of the thyroid. Similar measurements were performed on aspirates from nine patients free of thyroid disease (controls). A total of 95 karyometric features were evaluated for each nucleus. As compared with the control nuclei, the normal-appearing nuclei showed a 6% increase in total nuclear optical density (OD) while the tumor nuclei showed a 14% increase. Analysis of variance indicated a significant difference between the normal-appearing nuclei and the control nuclei, with most of the difference due to the differences of tissue origin. Discriminant analysis selected nine features that produced a statistically highly significant separation of tumor nuclei from control nuclei. A similar analysis selected five features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei; the validity of those karyometric features as markers of malignancy in normal-appearing nuclei from tissues adjacent to microinvasive follicular carcinomas of the thyroid was demonstrated by analysis in further training and test sets of nuclei.

Adenocarcinoma↗
PubMed1994

No tumor-specific expression levels of protein kinase C isoenzymes and of c-fos in human breast cancer cell cultures.

Epithelial cells derived from 46 human breast tissue samples of patients suffering from breast cancer have been cultivated. Twenty-five of these cell cultures stemmed from normal and 21 from tumor tissues. Moderate to large variations of protein levels of three protein kinase C (PKC) isoenzymes (alpha, delta and epsilon) were found among the various cell cultures. The cell cultures also exhibited very heterogeneous basal as well as inducible levels of c-fos mRNA. However, none of these variations could be correlated with the character of the original tissue nor with any clinical parameter of the respective patient. Our results suggest that altered levels of PKC isoenzymes or of the protooncogene c-fos per se cannot serve as an indication for a transformed behavior of the epithelial cell fraction of human breast tissue.

Adult↗
PubMed1998

Artificial skin equivalent differentiation depends on fibroblast donor site: use of eyelid fibroblasts.

Our objective was to construct and explore human skin equivalents from several normal and pathologic donor skin sites to determine if the fibroblast origin influences epidermal differentiation. Also, we wanted to find out if fibroblasts from some donor sites produced epidermis of superior quality for plastic surgery repairs. Skin equivalents were constructed from 15 normal skin specimens, 9 normal eyelid specimens, 15 lesional skin specimens taken from patients with psoriasis, and 4 specimens from keloid scars. Results show that the tissue origin of the donor fibroblasts determines epidermal differentiation and the time period for regeneration. Eyelid fibroblasts were very dependable in establishing well-differentiated skin equivalents in all nine specimens. Our findings contribute to the accumulated knowledge of wound healing and should also be of value in skin grafting, especially when large areas are denuded as in burns, severe trauma, or cancer ablation.

Adolescent↗
PubMed1975

A variant of hexokinase isoenzymes detected in ascites tumor cells.

In the hexokinase isoenzyme pattern on the cellulose acetate membrane electropherogram of the supernatant fraction of Ehrlich ascites tumor cells, a band was detected moving slower than of common Type I isoenzyme. This band was assumed to be due to the presence of a variant of the hexokinase isoenzymes in the cells, since such a band was not detected in the normal tissues, and a change in the intensity of the band in response to a sulfhydryl agent, etc., differed from that of the common Type I and II isoenzymes. The band was also found in AH-7974 and MH-134 ascites tumor cells, in spite of the difference in the species of the tumor-bearing animals and originating tissue.

Animals↗
PubMed2000

The hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue.

Hormone-sensitive lipase (HSL) mediates triglyceride hydrolysis in adipocytes, in which its expression varies with physiological stress and is controlled posttranslationally and transcriptionally. We sequenced the mouse HSL gene for 8.2 kb upstream of the translation start codon and studied the steady-state HSL mRNA levels in mouse adipose tissue. In 50 clones derived from primer extension and PCR of mouse adipose cDNA, we found five distinct 5' extremities that correspond to distinct exons in genomic DNA. Exon A is located approximately 7 kb 5' to the HSL translation start site. Exons B. C, and D are clustered 1.5-2 kb upstream, and the previously described exon 1 is immediately upstream and contiguous with the previously described HSL translation start site. Exon A is located -7 kb upstream and contains an in-frame methionine codon that could potentially generate another HSL isoform with 43 additional N-terminal residues. cDNA clones containing the newly described exons suggested that each exon has several transcription start sites but that all splice to an acceptor site located 20 nt upstream of the translation initiation codon in exon 1. HSL transcription in mouse adipose tissue originates from multiple sites in the 7-kb region between exon A and exon 1, with peaks at exon C (50-70% of HSL transcripts), exon 1 (5-30%), and exon A (approximately 10%). There are multiple potential transcription factor-binding elements upstream of each exon, suggesting the possibility of differential transcriptional regulation of HSL in different tissues and under various physiologic conditions.

Adipose Tissue↗
PubMed2006

Low meprin alpha expression differentiates primary ovarian mucinous carcinoma from gastrointestinal cancers that commonly metastasise to the ovaries.

BACKGROUND: Currently, no specific immunohistochemical markers are available to differentiate primary mucinous epithelial ovarian cancer (MOC) from adenocarcinomas originating at other sites that have metastasised to the ovary, which may have an impact on patient management and prognosis. AIM: To investigate the expression of two intestinal markers, galectin 4 and meprin alpha, in mucinous carcinomas of the ovary and gastrointestinal tract. METHODS: Using immunohistochemical analysis, the expression of galectin 4 and meprin alpha was investigated in 10 MOCs and in 38 mucinous adenocarcinomas of colon, pancreas, stomach and appendix, the most common sites of origin of ovarian metastases. RESULTS: Total cytoplasmic galectin 4 expression was relatively consistent between the different carcinomas. Membranous meprin alpha expression was significantly lower in MOCs compared with gastrointestinal carcinomas. Moreover, meprin alpha expression showed greater discrimination between the ovarian and gastrointestinal carcinomas than the cytokeratins CK7 and CK20, the current standard immunohistochemical markers used to determine the tissue origin of mucinous carcinomas involving the ovaries. CONCLUSIONS: Meprin alpha is a useful additional marker in differentiating primary from secondary mucinous adenocarcinomas of the ovary.

Adenocarcinoma↗
PubMed1990

Karyometric marker features in fine needle aspirates of invasive follicular carcinoma of the thyroid.

Karyometric measurements were performed on fine needle aspirates of clearly identifiable tumor areas and adjacent normal-appearing areas in the surgical specimens from ten patients with invasive follicular carcinoma of the thyroid. Similar measurements were performed on aspirates from nine patients free of thyroid disease (controls). A total of 95 karyometric features were evaluated for each nucleus. Analysis of variance of optical density values indicated (1) a similarity between tumor and normal-appearing nuclei from carcinoma cases, (2) a significant difference between those nuclei and control nuclei and (3) that most of the differences were due to the differences of tissue origin. Stepwise linear discriminant analysis selected ten features that produced a statistically highly significant separation of tumor nuclei from control nuclei. A similar analysis selected six features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei; the validity of those karyometric features as markers of malignancy in normal-appearing nuclei from tissues adjacent to invasive follicular carcinomas of the thyroid was demonstrated by analysis in further training and control sets of nuclei. This analysis in thyroid aspirates identified more marker features than did a previous similar analysis using tissue sections.

Adenocarcinoma↗
PubMed1995

Human cell lines in pharmacotoxicology. An introduction to a panel discussion.

Various types of cells lines are used in pharmacotoxicology. Established cell lines are easily available, with few ethical restrictions. Some specific properties are preserved, although they have kept the phenotype of the original tissue, which is frequently a tumor phenotype. They are usually more resistant to toxic compounds than freshly isolated cells. Some drug-metabolizing enzymes are expressed and regulated in these cells. Immortalized cell lines are also of interest in toxicology. They are mainly examined for their potential in mutagenicity testing. These cells and numerous others of animal or human origin can be transfected with cDNA coding for human enzymes. They are used for determination of the individual enzyme involved in a particular metabolic pathway, or, when multiple transfections are successfully achieved, for mutagenicity testing. Regulation studies are also possible in such cells after transfection of DNA elements regulating gene transcription.

Cell Line↗
PubMed1995

Quantitative differences in neoglycoprotein binding for vascular endothelial cells from porcine brain, ovary, and testis in vitro.

Carrier-immobilized carbohydrates are valuable tools for assessing the glycoligand-binding capacity of cell surfaces. A panel of 10 types of fluorescent neoglycoproteins has been synthesized to determine the extent of their specific binding to endothelial cells in vitro that have been obtained from porcine brain, ovary, and testis. Different sugar moieties revealed a nonuniform capacity to bind to the endothelial cells as determined by flow cytometric analysis, the histogenetic origin of the preparations being an important factor. Binding of mannose, xylose, and glucuronic acid was especially pronounced, but the extent of cell-associated fluorescence was significantly different dependent on the source of the endothelial cells. These results clearly reveal that endothelial cells in vitro display the capacity to specifically recognize defined carbohydrate moieties. Moreover, endothelial cells of different tissue origin can exhibit variability in this property, potentially endowing this cell type with site-dependent molecular properties.

Animals↗