Biomedical subjects

R B Nagle

Publications and source records attributed to R B Nagle.

At least 73 records · Page 4Linked to original sources

PubMed1990

Assessment of S-(1,2-dichlorovinyl)-L-cysteine induced toxic events in rabbit renal cortical slices. Biochemical and histological evaluation of uptake, covalent binding, and toxicity.

A renal cortical slice system was utilized to investigate the events leading to site-specific nephrotoxicity induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). DCVC uptake into renal cortical slices was shown to be rapid (5-15 min) as well as time- and concentration-dependent. Of the total amount taken up at 1 h, 40% was subsequently covalently bound. These observations were confirmed by autoradiography, illustrating uptake and binding in the proximal tubule cells. Following these events, toxicity was evidenced by alterations in ATP content and O2 consumption between 4 and 8 h as well as leakage of the brush border enzymes (gamma glutamyl transpeptidase and alkaline phosphatase) as early as 4 h. Light microscopy provided a sequence of histopathological changes from an initial S3 lesion between 4 and 8 h to a lesion encompassing all proximal tubule segments (by 12 h). Electron microscopy demonstrated not only the specificity of DCVC toxicity (at 6 h) but also illustrated mitochondrial damage and loss of brush borders. A comparison of continuous versus short-term exposure to DCVC indicated that an irreversible sequence of events was initiated as early as 30 min. By utilizing an in vitro model which allows correlation of biochemical and histological changes, a sequence of events leading to DCVC induced toxicity was established.

Animals↗

PubMed1990

Improved methods for separation of human cytokeratins.

The cytokeratins from human bladder and esophageal epithelia were separated using chromatographic techniques. The cytokeratins were first extracted from fresh autopsy tissue using high and low salt buffers. Urea, 8.0-9.5 M, was used to solubilize the resulting cytokeratin pellet. Imidazole was found to increase the solubility of the pellet but reducing agents such as 2-mercaptoethanol were not beneficial. DEAE ion exchange chromatography produced three fractions which were analyzed by using one and two-dimensional electrophoresis. The third fraction was shown to contain the acidic cytokeratins and was further fractionated on a moderately polar reverse phase HPLC column using an acetonitrile elution gradient. Tetramethylammonium tetrafluoroborate was added to the mobile phase to react with any unreacted silanol groups on the stationary phase, and trifluoroacetic acid was added to ion pair with the protein. HPLC fractions of the acidic proteins from human esophagus revealed seven reproducible peaks. All seven peaks were shown by Western blotting to contain an epitope found on cytokeratin 13. The results suggest that the isolation and separation procedures have produced a series of peptide products which all retain a similar epitope but which vary significantly in their hydrophobic characteristics.

Chromatography, High Pressure Liquid↗

PubMed1990

Anomalous cytokeratin expression in malignant melanoma: one- and two-dimensional western blot analysis and immunohistochemical survey of 100 melanomas.

Immunohistochemical characterization is an accepted method of human cell typing and tumor diagnosis. The differentiation of undifferentiated carcinoma from amelanotic melanoma is usually achieved by demonstration of cytokeratin (CK) intermediate filaments in carcinoma but not in melanoma. In examination of 100 melanomas fixed in formalin or methacarn and frozen tissue sections, we have found CK-immunoreactivity in 2, 8, and 21% of cases, respectively, with multiple anticytokeratin antibodies displaying overlapping antigenic specificities. In addition, we have confirmed the anomalous expression of low molecular weight CK proteins by one- and two-dimensional gel electrophoresis and immunoblotting of extracts of an immunohistochemically positive case. This latter finding indicates that CK staining in melanomas reflects the presence of authentic CK peptides and is not an artefact induced by fixation or cross-reacting antibodies. These observations have direct implications for the application of immunohistochemistry to the present practice of diagnostic surgical pathology. The anomalous CK expression by melanoma limits the diagnostic reliability of immunohistochemically demonstrated CK alone to indicate a diagnosis of carcinoma, without the concomitant detection of additional tumor-associated antigens. The finding of anomalous CK expression only in metastatic or recurrent melanomas raises an interesting question of possible association with tumor progression.

Blotting, Western↗

PubMed1990

Coexpression patterns of vimentin and glial filament protein with cytokeratins in the normal, hyperplastic, and neoplastic breast.

The authors studied by immunohistochemistry the intermediate filament (IF) protein profile of 66 frozen samples of breast tissue, including normal parenchyma, all variants of fibrocystic disease (FCD), fibroadenomas, cystosarcoma phylloides, and ductal and lobular carcinomas. Monoclonal antibodies (MAbs) to cytokeratins included MAb KA 1, which binds to polypeptide 5 in a complex with polypeptide 14 and recognizes preferentially myoepithelial cells; MAb KA4, which binds to polypeptides 14, 15, 16 and 19; individual MAbs to polypeptides 7, 13, and 16, 17, 18, and 19, and the MAb mixture AE1/AE3. The authors also applied three MAbs to vimentin (Vim), and three MAbs to glial filament protein (GFP). Selected samples were studied by double-label immunofluorescence microscopy and by staining sequential sections with some of the said MAbs, an MAb to alpha-smooth muscle actin, and well-characterized polyclonal antibodies for the possible coexpression of diverse types of cytoskeletal proteins. Gel electrophoresis and immunoblot analysis also were performed. All samples reacted for cytokeratins with MAbs AE1/AE3, although the reaction did not involve all cells. Monoclonal antibody KA4 stained preferentially the luminal-secretory cells in the normal breast and in FCD, whereas it stained the vast majority of cells in all carcinomas. Monoclonal antibody KA1 stained preferentially the basal-myoepithelial cells of the normal breast and FCD while staining tumor cell subpopulations in 4 of 31 carcinomas. Vimentin-positive cells were found in 8 of 12 normal breasts and in 12 of 20 FCD; in most cases, Vim-reactive cells appeared to be myoepithelial, but occasional luminal cells were also stained. Variable subpopulations of Vim-positive cells were noted in 9 of 20 ductal and in 1 of 7 lobular carcinomas. Glial filament protein-reactive cells were found in normal breast lobules and ducts and in 15 of 20 cases of FCD; with rare exceptions, GFP-reactivity was restricted to basally located, myoepithelial-appearing cells. Occasional GFP-reactive cells were found in 3 of 31 carcinomas. Evaluation of sequential sections and double-label immunofluorescence microscopy showed the coexpression of certain cytokeratins (possibly including polypeptides 14 and 17) with vimentin and alpha-smooth muscle actin together with GFP in some myoepithelial cells. The presence of GFP in myoepithelial cells was confirmed by gel electrophoresis and immunoblotting. Our results indicate that coexpression of cytokeratin with vimentin and/or GFP is comparatively frequent in normal basal-myoepithelial cells of the breast.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenofibroma↗

PubMed1989

Keratin immunoreactivity as an aid to the diagnosis of persistent adenocarcinoma in irradiated human prostates.

Postirradiation prostatic biopsy is believed by many to be the best measure of radiation effectiveness in prostatic cancer. Therapeutic irradiation may induce prostatic glandular atypia, which in its severe form can be confused with persistent adenocarcinoma on prostatic biopsies. In the current study, 37 postirradiation prostate biopsy specimens were evaluated by immunohistochemistry using a specific monoclonal anticytokeratin antibody (KA1) that reacts with the basal cells of normal or hyperplastic glands, but is nonreactive with the lumenal cells or with prostatic carcinoma cells. Persistent carcinoma was observed in 19 cases in which antibody staining was absent. The noncarcinomatous glands retained reactivity, but this reactivity appeared in a new and previously undescribed pattern. The irradiated lesion was characterized by cellular pleomorphisism, with enlargement of nuclei and loss of polarity. The immunoreactivity was seen in the enlarged basal cells and was seen to focally extend to involve the lumenal cell layer. In five of 37 cases, glands were seen that were so atypical on the routinely stained sections that a distinction from cancer could not be made. These same glands in the adjacent section reacted with KA1 in each case allowing us to conclude that the changes were benign. We conclude that the interpretation of postirradiation prostatic biopsy specimens may be aided by immunohistochemistry with this anticytokeratin antibody.

Adenocarcinoma↗

PubMed1989

Prostatic intraepithelial neoplasia: a lesion that may be confused with cancer on prostatic ultrasound.

Most prostatic adenocarcinomas reveal a hypoechoic peripheral zone lesion on transrectal ultrasonography. Numerous benign processes can have similar transrectal ultrasound findings. We report 8 cases of transrectal ultrasound-guided prostatic biopsies of peripheral zone hypoechoic lesions that demonstrated prostatic intraepithelial neoplasia, a presumed premalignant lesion. Immunohistochemistry using an antibody directed against cytokeratins 5 and 14 was performed to exclude carcinoma. Of the men 2 had invasive carcinoma on repeat biopsy and 1 had carcinoma diagnosed on subsequent transurethral resection. Patients with prostatic intraepithelial neoplasia on biopsy of hypoechoic peripheral zone lesions merit careful monitoring.

Adenocarcinoma↗

PubMed1989

Serum prostate-specific antigen and prostate pathology in men having simple prostatectomy.

Prostate-specific antigen (PSA) is a sensitive and specific serum marker for monitoring disease activity in men with prostatic carcinoma. Despite reports of elevation of levels of this analyte in men with benign prostatic hyperplasia, no information is available correlating the serum levels with the actual prostatic abnormalities in men having prostatectomy for presumed benign disease. In the present investigation, the authors compared preoperative serum PSA levels with prostate disease in 81 men with bladder outlet obstruction. Five pathologic groups were found: incidental high-grade carcinoma (n = 3), low-grade carcinoma (n = 11), acute inflammation (n = 16) with or without chronic inflammation, Prostatic intraepithelial neoplasia (PIN) (n = 25), and benign hyperplasia (n = 26). Serum PSA levels were significantly elevated in both low- and high-grade carcinoma, acute inflammation, and PIN when compared with the patients with benign hyperplasia with and without chronic inflammation. Within the four groups with elevated levels, use of PSA levels could separate only the high-grade cancer patients who were subsequently shown to have metastatic disease. Only one patient with simple hyperplasia had PSA levels in the abnormal range.

Adenocarcinoma↗

PubMed1989

Expression of beta-actin during progression of mouse skin tumors.

Recombinant DNA techniques have been employed to isolate sequences that are over-expressed during multi-stage carcinogenesis in the mouse skin. One of these six cDNA sequences, mal-4, detected 1.9-kb transcripts that were expressed at levels 10-fold higher in squamous cell carcinomas (SCCs) in comparison to normal epidermis. A full-length cDNA for mal-4 was obtained from a lambda gt10 cDNA library made from an SCC-producing cell line, PDVC57. DNA sequencing of 1300 out of 1900 bp of the mal-4 cDNA and searching of Genbank showed greater than 95% DNA sequence similarity to mouse beta-actin over the entire cDNA and greater than 98% similarity over the 3' untranslated region, which is unique to the various isoforms of actins. Southern analysis showed no amplification or rearrangement of the beta-actin gene had occurred during tumor progression. The RNA:RNA hybrid protection assay was utilized to screen for the expression of mutated beta-actin(s) in mouse skin tumors. No evidence for mutation was obtained in any of the tumors examined. Fluorescence microscopy of tumor sections stained with rhodamine-conjugated phalloidin showed a peripheral pattern of F-actin localization with no gross differences between papillomas and carcinomas. Approximately equal amounts of beta-actin were observed following two-dimensional gel electrophoresis of proteins extracted from normal epidermis, papillomas or SCCs, indicating that the over-expression of beta-actin RNA in SCCs did not result in an increased steady-state level of beta-actin protein. These results suggest that alterations in actin metabolism accompany tumor progression.

Actins↗

PubMed1989

Intermediate filaments. Efficacy in surgical pathologic diagnosis.

The use of antibodies specific for the various types of intermediate filament proteins has become an important part of the immunohistochemical approach to surgical pathology. These proteins are expressed in a cell lineage-specific pattern, which is reproduced with great fidelity even in poorly differentiated neoplasms. The historic discovery, embryology, and structure of these proteins are reviewed. A brief discussion of the major applications and precautions in the choice and use of these antibodies is presented.

Amino Acid Sequence↗

PubMed1989

The relationship of basement membrane to histologic grade of human prostatic carcinoma.

The presence or absence of basement membrane (BM) was examined in normal and neoplastic adult prostatic tissue as well as prostate cell lines using immunohistochemistry and electron microscopy. Immunohistochemical studies using antibodies directed against laminin and type IV collagen were done on 55 samples of human prostate representing various grades of prostate carcinoma. The percentage of glandular structures surrounded by BM was determined. Benign prostates (n = 15) had BM around 99% of the acini. Gleason Grade II adenocarcinoma (n = 9) had 65%, Grade III (n = 5) had 23%, Grade IV (n = 12) had 15%, and Grade V (n = 7) had 0% BM around glandular structures, respectively. None of the metastases (n = 7) had visible BM. By transmission electron microscopy, 32 prostates were examined for the glandular profile of BM. One hundred percent of the acini in the benign prostates (n = 17) had BM. Of the low grade carcinomas, Gleason I and II (n = 4), 44% of the acini had BM, and of the high grade carcinomas, Gleason IV and V (n = 7), 34% had BM. None of the metastases (n = 4) had BM by electron microscopy. We conclude that, in prostatic carcinoma, there is a progressive loss of BM with decreasing differentiation, and that in prostate carcinoma metastases there is a complete loss of epithelial BM.

Adenocarcinoma↗

PubMed1989

Ultrastructural morphometry distinguishes Burkitt's-like lymphomas from neuroendocrine neoplasms: useful criteria applied to the evaluation of a poorly differentiated neuroendocrine neoplasm of the nasal cavity masquerading as Burkitt's-like lymphoma.

The present study describes the potential usefulness of ultrastructural morphometry in diagnosis. Ultrastructural morphometric criteria were applied to the evaluation of a poorly differentiated neuroendocrine neoplasm of the nasal cavity that was initially thought to be a Burkitt's-like lymphoma (BLL). Although the nasal lesion in question failed to stain with over 50 cell lineage-relevant antibodies, it did stain for vimentin (an intermediate filament protein) and Ki-67 (a nuclear antigen associated with cell proliferation). Routine electron microscopy revealed a primitive neoplasm with abundant cytoplasmic lipid droplets and sparse dense granules with no intercellular junctions. Treatment options, which included extensive facial surgery, prompted more study. Tissue processed for the uranaffin reaction revealed sparse uranaffin-positive granules indicating the presence of true neurosecretory granules. An ultrastructural morphometric analysis of the neoplastic nuclei of this patient placed the tumor outside the morphometric domains for BLLs (18 cases) and neuroblastomas (11 cases) and within the morphometric domain of neuroendocrine carcinomas (9 cases). A greater mean standard deviation (P less than 0.05) and mean coefficient of variation (P less than 0.02) of nuclear perimeter in the neuroendocrine (NE) group related to the BLL group indicated greater nuclear pleomorphism within the NE group as illustrated in bivariate graphic displays. The possible origin of the neoplasm within the nasal mucosa is discussed.

Burkitt Lymphoma↗

PubMed1988

Sequential structural changes in the fat body of the tobacco hornworm, Manduca sexta, during the fifth larval stadium.

Light and electron microscopy revealed a series of structural changes that occur in the fat body of the tobacco hornworm, Manduca sexta, during the fifth, i.e. the final, larval stadium. At each developmental stage studied, the cells of the fat body were homogeneous in structure. We found no evidence suggesting the presence of more than one type of fat body cell. Our structural data are consistent with published observations on biochemical activities of M. sexta fat body at particular developmental stages. Specific points of agreement include: (a) acquisition of Golgi complex (GC) and rough endoplasmic reticulum (RER) concomitant with the time of major protein production; (b) loss of many cellular organelles (such as GC and RER) as protein production drastically decreases; (c) accumulation of protein granules and urate granules after the onset of wandering (i.e. during the pre-pupal period); (d) accumulation of lipid and glycogen throughout the feeding period. In addition we found that (a) the plasma membrane reticular system (PMRS) developed during the period when protein secretion was great; (b) the PMRS was lost abruptly at the onset of wandering; and (c) the nucleus changed in shape from being roughly spherical to elliptoid in the pre-pupal stage. We found that the structure of M. sexta fat body is similar to that published for other Lepidoptera. However, it differs from that of Heliothis zea in that regional differences are not obviously apparent.

Animals↗

PubMed1988

Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins.

The immunohistochemical reactivity on frozen sections of diverse benign and malignant epithelial proliferations of human breast tissue from 156 patients was examined using antibodies to different cytokeratins. Antibodies recognizing cytokeratins 18 and 19 reacted with luminal epithelial cells but not with myoepithelial cells of normal mammary gland, cystic disease, adenosis, papilloma, and fibroadenoma or with a subpopulation of proliferating cells in sclerosing adenosis and epitheliosis. These antibodies reacted with the tumor cells of all in situ and invasive carcinomas. KA1 antibody, which by one- and two-dimensional gel electrophoresis and immunoblotting was shown to bind preferentially to cytokeratin 14 in a complex with cytokeratin 5, reacted with the nonproliferating myoepithelium of normal gland, cystic disease, adenosis, papilloma, fibroadenoma, and in situ carcinoma; it also reacted with a subpopulation of proliferating cells in sclerosing adenosis and epitheliosis (papillomatosis) but was negative with the tumor cells of all preinvasive and most invasive carcinomas. In adenotic and epitheliotic proliferations, groups of cells were identified that reacted strongly with KA1 antibody in addition to antibodies to cytokeratins 18 and 19. The data are discussed with respect to epithelial cell heterogeneity in the breast. We show that by using such antibodies, benign epithelial proliferations are clearly distinguished from carcinomas.

Adenofibroma↗

PubMed1988

A comprehensive immunotopographic map of human thymus.

We have achieved a comprehensive immunotopographic mapping of human thymus by using a large battery of monoclonal antibodies and the methodological refinement of comparative serial tissue section immunohistochemistry, allowing analysis of multiple phenotypes in the same tissue site. Previous immunohistochemical studies of thymus have concentrated on the majority T-cell and epithelial cell populations. Besides demonstrating the complexity of T-cell antigenic expression (e.g., simultaneous cortical expression of Leu 2, Leu 3, CALLA, Tdt, and Leu 6), we delineate surprisingly complex B-cell zones (e.g., septal B-follicles with DRC+C3d+ dendritic cells and zonal maturation of B-cells). Whereas septal B-follicles were found in 25% of cases, medullary B-cells were universally present as a substantial minority component. This expanded immunotopographic knowledge of the complex T-, B-, epithelial, and reticulum cell neighborhoods suggests that the thymus is an organ capable of a broad repertoire of immunological responses, not limited to T-cell development.

Adolescent↗

PubMed1988

Enhancement of DMN-induced kidney tumors by 1,2-dichlorovinylcysteine in Swiss-Webster mice.

1,2-Dichlorovinylcysteine (DCVC) is known to cause enlarged nuclei in renal proximal tubule epithelium. This study further characterized the cellular changes induced by DCVC. Also a preliminary investigation of the initiator and promoter potential of DCVC was conducted. When given for 4 weeks in drinking water to Swiss-Webster mice, DCVC (50 or 100 micrograms/ml) caused a progressive change in renal epithelia which was persistent at 23 weeks. Enlarged nuclei with uneven chromatin dispersal, multiple nucleoli, and irregular nuclear membranes resided in enlarged, abnormally-shaped cells. These changes were resolved by week 50. Mice initiated with dimethylnitrosamine developed renal tumors by week 27. Mice fed DCVC (10 or 50 micrograms/ml) for 14 weeks following dimethylnitrosamine initiation, had a slightly higher tumor incidence, a higher incidence of invasive tumors, and had multiple renal tumors unlike animals given dimethylnitrosamine alone.

Animals↗

PubMed1988

Intermediate filaments: a review of the basic biology.

This review presents the current knowledge of the basic biology of intermediate filaments, including their phylogenetic distribution and their distribution within mammalian cells. The current understanding of their structure, recently described using recombinant DNA, peptide sequencing, and immunologic methods, is discussed in terms of a revised chemical classification that not only includes the acidic and neutral basic keratins, vimentin, glial fibrillary protein, desmin, and neurofilament, but also includes the more recently related nuclear lamins. This new structural knowledge allows a more rational approach to the diagnostic use and development of polyclonal and monoclonal anti-intermediate filament antibodies. Many of the problems encountered with fixation, cross-reactivity, and epitope masking can be related to these structural concepts.

Animals↗

PubMed1987

Differential patterns of injury to the proximal tubule of renal cortical slices following in vitro exposure to mercuric chloride, potassium dichromate, or hypoxic conditions.

The innate susceptibility of renal cell types to these agents was investigated using precision-cut rabbit renal cortical slices made perpendicular to the cortical-papillary axis. Slices were incubated in DME/F12 medium containing 10 microM, 100 microM, or 1 mM concentrations of either metal for 12 hr or in Krebs-Hepes buffer gassed with nitrogen (100%) for 0.75 to 5 hr of hypoxic exposure. To simulate postischemic reperfusion, some slices were transferred to vessels gassed with oxygen after an initial hypoxic period. Mercuric chloride (100 microM) exposure resulted in damage to the straight regions of proximal tubules by 12 hr leaving convoluted regions unaffected. Hypoxia (2.25 hr) and potassium dichromate (100 microM for 12 hr) both caused injury to the convoluted proximal tubules without affecting straight proximal tubular regions. Mercury concentrations of 10 microM and 1 mM had no effect or injured all cell types within the slice, respectively. Similar results were observed for hypoxic periods less than 1.5 hr or greater than 3 hr of exposure. Potassium dichromate had no measurable affect at 10 microM, but at 1 mM focal lesions were observed after 4 hr of exposure, and by 12 hr all cell types within the slice were affected. Intracellular potassium content normalized to DNA correlated well, but always preceded the pathological lesions observed. These results demonstrate that injury to specific regions of the proximal tubule by these agents relates to an innate susceptibility of the intoxicated cell type independent of physiologic feedback or blood delivery patterns proposed as mechanisms of selective injury from in vivo studies.

Animals↗

PubMed1987

Cytokeratin characterization of human prostatic carcinoma and its derived cell lines.

Two murine monoclonal anti-cytokeratin antibodies with defined specificity were shown to distinguish between basal cells and luminal cells in human prostate tissue. Forty-one biopsies or transurethral resection specimens were characterized using these two antibodies. In cases of benign prostatic hyperplasia, focal loss of the basal cell layer was noted in areas of glandular proliferation. Ten cases of adenocarcinoma of the prostate, varying in Gleason's histological grade from 2 to 4, were also studied. In each case the carcinoma was shown to represent the luminal cell phenotype with no evidence of involvement of the basal cell phenotype. An analysis of three established metastatic prostatic carcinoma cell lines (DU-145, PC-3, and LNCaP) using two-dimensional electrophoresis showed that the cytokeratin complement of each cell line was slightly different but retained the phenotype of the luminal cell. It was concluded that during both hyperplasia and neoplastic transformation of the prostate, the luminal cell phenotype is primarily involved and that the basal cell phenotype does not appear to contribute to either intraluminal proliferation or invasive cell populations.

Antibodies, Monoclonal↗