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M Osborn

Publications and source records attributed to M Osborn.

At least 145 records · Page 8Linked to original sources
PubMed1986

Reactivity of a panel of neurofilament antibodies on phosphorylated and dephosphorylated neurofilaments.

The work of the Sternbergers and their colleagues has shown that monoclonal antibodies reactive with neurofilament subunit proteins may be sensitive to the state of phosphorylation of these proteins. We therefore examined the ability of our previously described panel of monoclonal and polyclonal neurofilament antibodies to bind to normal and to enzymatically dephosphorylated neurofilament subunits. All the monospecific antibodies, both mono- and polyclonal, which we had previously documented as reactive with neurofilament H protein proved to bind only to the phosphorylated form of this protein, and H antibody staining of neurofilamentous profiles in frozen sections could be abolished by appropriate pretreatment of sections with alkaline phosphatase. In contrast, all monospecific antibodies, both mono- and polyclonal, reactive with native M and L proved to bind with apparently undiminished affinity following enzymatic dephosphorylation of the appropriate antigen, either in frozen sections or on Western blots. The class of monoclonal antibodies which react with both H and M were variable in their response to dephosphorylated neurofilaments; some completely lost their reactivity whilst others were partially or wholly unaffected. We stained frozen sections of nervous tissues from various mammalian species with the panel of antibodies, and observed filamentous staining of the perikarya and dendrites of a variety of different types of neuron with all antibodies, both mono- and polyclonal, directed against L and M. Antibodies with strong reactivity for phosphorylated H always failed to stain neurofilamentous dendritic and perikaryal profiles. We further describe the isolation and characterization of a new monoclonal antibody, which recognizes both phosphorylated and enzymatically dephosphorylated forms of H.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Monoclonal antibodies to titin in conjunction with antibodies to desmin separate rhabdomyosarcomas from other tumor types.

Titin is a major constituent protein of sarcomeric muscles and is thought to give rise to an elastic filament component underlying the myofibrillar organization. Monoclonal antibodies to titin have been characterized on normal and pathological human material and on human cell lines in culture. A positive immunocytochemical reaction was restricted to sarcomeric muscles and did not occur on visceral or vascular smooth muscles or on various nonmuscle tissues. When different tumor types were examined titin antibodies reacted solely with rhabdomyosarcomas and did not react with leiomyosarcoma or leiomyoma, or with the nonmuscle tumor types tested. In rhabdomyosarcomas a noticeably smaller population of cells were positive with antibodies to titin than with antibodies to desmin showing that individual cells within a rhabdomyosarcoma achieve different degrees of myogenic differentiation. The results reinforce the use of desmin as a marker for muscle sarcomas and show that a positive identification of rhabdomyosarcoma can be achieved by immunocytochemistry with the parallel use of desmin and titin antibodies.

Animals↗
PubMed1986

Villin--a marker of brush border differentiation and cellular origin in human renal cell carcinoma.

Expression of villin, a 95-kd F-actin bundling and severing protein, is restricted in animal tissues to epithelial cells with a brush border. Thus, the enterocytes of the intestine and epithelial cells of proximal but not distal tubules of the kidney are strongly positive. Here we report a similar staining pattern for human intestine and kidney. In four human colon adenocarcinomas villin expression was seen in tubular and glandular structures but not in the undifferentiated parts. Fourteen human renal carcinomas (9-Grade I and 5 Grade II) were villin-positive, and 2 sarcomatous renal carcinomas (Grade III) were villin-negative. The percentage of tumor cells that were villin-positive varied from 10-90% for the Grade I and II types. Our results indicate that villin may be a grading marker that deserves further study in renal carcinoma. They also raise the question whether the majority of renal carcinomas are derived from the proximal tubular epithelium rather than from the distal epithelium.

Adenocarcinoma↗
PubMed1985

Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

Amino Acids↗
PubMed1985

A monoclonal antibody recognizing desmosomes: use in human pathology.

A mouse monoclonal IgG1 antibody that recognizes desmosomes in human tissues has been isolated and characterized. DP1 recognizes desmoplakin I (250 kD) in immunoblots of partially purified desmosome preparations. In immunofluorescence microscopy DP1 reacts with desmosomes in epithelial cells from a variety of human tissues including skin, esophagus, uterus, intestine, and in liver hepatocytes. In axilla skin, the epithelial cells in epidermis, sweat glands, and in the outer hair follicles are strongly stained whereas relatively little staining is seen in the epithelial cells of the apocrine glands. Nonepithelial cells are not stained, although decoration of myocardial desmosomes is seen. In cells in culture DP1 is also specific for desmosomes in epithelial cells. Thus the mouse epidermal line HEL, bovine MBCK cells, as well as the human lines A431, HT29, and HeLa are positively stained by DP1. Desmosomes are retained in tumors of epithelial origin such as adenocarcinoma and squamous cell carcinoma. Thus DP1 should be a useful diagnostic marker in human pathology.

Animals↗
PubMed1985

Detection of metastatic tumour cells in routine bone marrow smears by immuno-alkaline phosphatase labelling with monoclonal antibodies.

The present study describes 11 cases (10 carcinomas, one rhabdomyosarcoma) in which immuno-alkaline phosphatase labelling with monoclonal antibodies was used to demonstrate metastatic cells in routine smears of aspirated bone marrow. Carcinoma cells were detected using antibodies against epithelial cytokeratins, milk fat globule membrane antigen and carcinoembryonic antigen, and rhabdomyosarcoma cells with monoclonal anti-desmin. In four of the carcinoma cases it had not been possible to identify malignant cells in routinely stained marrow smears, whilst the case of disseminated rhabdomyosarcoma had initially been diagnosed (and treated) as a case of acute lymphoblastic leukaemia. The anti-cytokeratin antibody was found to be the most valuable of the anti-epithelial reagents used, since it labelled malignant cells in all of the 10 cases of carcinoma and gave the strongest reactions. These results suggest that immunocytochemical labelling should be used in cases of suspected carcinoma whenever conventional examination of marrow smears yields negative results, and furthermore (as illustrated by the case of rhabdomyosarcoma) that the technique is of value for identifying the true nature of poorly differentiated neoplasms in bone marrow.

Aged↗
PubMed1985

An ultrastructural and immunohistological study of the rat olfactory epithelium: unique properties of olfactory sensory cells.

The olfactory epithelium contains three cell types: basal cells, supporting cells and sensory neurons. Electron microscopy as well as immunofluorescence microscopy with intermediate-filament antibodies were used to study the rat olfactory epithelium in order to obtain more information about these different cell types and to try to investigate their histogenetic origins. We found mitoses in the basal-cell layer, as well as multiple centrioles and tonofilaments in some basal cells. As revealed by electron microscopy, the supporting cells contained tonofilaments and reacted strongly with antibodies to keratin, in line with their known epithelial nature. When antibodies to other intermediate-filament types were used, i.e. glial fibrillary acidic protein, vimentin, desmin and neurofilaments, no reaction was seen in the cells of the olfactory epithelium, with the exception of occasional staining of a few axons in the subepithelial layer by neurofilament antibodies. In particular, the cell bodies, dendrites and most axons of the sensory neurons were negative for a variety of antibodies against neurofilaments. Olfactory sensory neurons therefore belong to the very few cells in adult animals which seem to lack intermediate filaments. We discuss whether this finding is related to the fact that these cells are also unique among neurons in that they are not permanent cells but constantly turn over.

Animals↗
PubMed1985

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments.

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.

Animals↗
PubMed1985

Desmin is a specific marker for rhabdomyosarcomas of human and rat origin.

Putative human rhabdomyosarcoma (RMS) has been divided into two groups according to desmin content. Twenty-five tumors with histologic features consistent with but not necessarily sufficient to prove a diagnosis of RMS were desmin-positive. More than 95% of the tumor cells were desmin-positive, suggesting a muscle origin and supporting the diagnosis of RMS. Nine tumors for which the preferred first histologic diagnosis was also RMS were desmin-negative. Reexamination of the original histologic slides together with results from intermediate filament typing resulted in a diagnosis other than RMS for all tumors in this second group, and in several instances other tests were used to prove the correctness of the final diagnosis. The results on human material were extended to a rat model system in which RMS was induced by nickel sulfide. Again, all 24 tumors tested were desmin-positive. Vimentin was coexpressed in a varying percentage of tumor cells in RMS of human and rat origin. The results show that desmin is an excellent marker for rhabdomyosarcoma, yielding few if any false-positive or false-negative results in frozen or alcohol-fixed material.

Adolescent↗
PubMed1985

Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments, and tubulin in Torpedo electric organ.

Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.

Actins↗
PubMed1984

Diagnostic value of intermediate filament antibodies in clinical cytology.

Antibodies to intermediate filament (IF) proteins can distinguish the major tumour groups as shown by results with sectioned human material. In this study we evaluate the use of similar methods in the cytology of human tumors. Smears obtained from fine needle aspiration biopsies were investigated using well characterized antibodies, each specific for only one of the five types of intermediate filaments. Tumor cells of different carcinomas, thymomas, and the epithelial part of pulmonary blastomas were positive with antibodies recognizing cytokeratins. Tumor cells in non-muscle sarcomas, including lymphoma and Ewing's sarcoma, could be specifically identified with antibodies to vimentin. Tumor cells of muscle sarcomas were desmin-positive. Finally, tumor cells in pheochromocytoma and bronchus carcinoid were positive with antibodies specific for neurofilaments. Specimens were also examined in parallel using conventional cytochemical stains, such as May-Grünwald-Giemsa. In addition, in most cases sections of the tumor were examined both by histology and IF typing of frozen sections to confirm the diagnosis made on the cytologic specimens. The results show that IF typing is a valuable diagnostic aid in clinical cytology.

Adolescent↗
PubMed1984

Tissue polypeptide antigen (TPA) is related to the non-epidermal keratins 8, 18 and 19 typical of simple and non-squamous epithelia: re-evaluation of a human tumor marker.

Because of the broad clinical interest which tissue polypeptide antigen (TPA) has attracted as a tumor marker, human cell lines and human tissues have been analyzed for TPA expression using immunofluorescence microscopy. Epithelial cell lines including HeLa, MCF-7, and A-431 are recognized by TPA antibodies whereas human lines of non-epithelial origin are not. The positive staining patterns coincide with keratin-type intermediate filaments of the cytoskeleton. On tissue sections a subset of epithelial cells including uterine epithelium, bile duct cells in liver and tumor cells in breast carcinoma are strongly positive; cells of the squamous epithelia of skin and tongue as well as cells of non-epithelial origin are negative. In immunoblots of human epidermis, human tongue mucosa, human hair follicles, Detroit 562 cells, HeLa cells, MCF-7 and RT-4 cells, only keratins 8, 18 and 19 show TPA antigenicity. Conversely a TPA preparation is recognized by various antibodies known to react with keratins, including alpha-IFA, KG 8.13.2 and two antibodies which recognize keratins 18 (CK2) and 19, respectively. Our results thus relate TPA to human keratins 8, 18 and 19 which are known cytoskeletal components in both normal and malignant epithelial cells of simple and non-squamous origin. We speculate that the elevated levels of circulating TPA antigenicity present in the sera of patients with carcinoma, which are often used to monitor tumor progression, correspond to soluble proteolytic fragments originating from this particular keratin subgroup.

Animals↗
PubMed1984

Typing of intermediate filaments in malignant fibrous pleural tumors.

We report two cases of a malignant fibrous pleural tumor, one of the localized type and one of the diffuse type. In both cases, typing of intermediate filaments by immunofluorescence microscopy showed that the tumor cells were positive for vimentin and negative for (cyto)keratin and desmin. This result supports the concept that malignant fibrous pleural tumors do not arise from the (cyto)keratin-positive pleural mesothelium but from the submesothelial fibrous tissue. Thus, instead of the usual term "malignant fibrous mesothelioma", the term "malignant submesothelial fibrosarcoma" should be preferred.

Adolescent↗
PubMed1984

Esthesioneuroblastoma: ultrastructural, immunohistological and biochemical investigation of one case.

A case of esthesioneuroblastoma, the pathological diagnosis of which almost always causes great difficulties, was investigated ultrastructurally, biochemically, and immunohistologically, using antibodies against the five known types of intermediate filaments [keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and neurofilaments]. The tumour cells did not react with antibodies against any of the five intermediate filament proteins. Ultrastructural investigations showed dense cored secretory granules in the cytoplasm and cell processes. Thus, immunohistology offers by "exclusion" a differential diagnosis to avoid often misdiagnosed tumours (undifferentiated carcinomas, embryonal rhabdomyosarcomas, and malignant lymphomas), since carcinomas react with antikeratin, embryonal rhabdomyosarcomas with antibodies to desmin and malignant lymphomas show immunofluorescence with antibodies to vimentin. The biological behaviour (age distribution, tendency to metastasize), the normal values of biochemical parameters, homovanillic acid and vanilmandelic acid (HVA, VMA), and the absence of neurofilaments distinguish this type of tumour from the peripheral sympathetic neuroblastoma.

Cytoplasmic Granules↗