Composition of the von Willebrand factor storage organelle (Weibel-Palade body) isolated from cultured human umbilical vein endothelial cells.

von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by centrifugation on a self-generated Percoll gradient. Stimulation of EC by 4-phorbol 12-myristate 13-acetate (PMA) resulted in the disappearance of this organelle fraction and the synchronous loss of Weibel-Palade bodies as judged by immunoelectron microscopy. Electrophoretic and serologic analyses of biosynthetically labeled dense organelle fraction revealed that it is comprised almost exclusively of VWF and its cleaved pro sequence. These two polypeptides were similarly localized exclusively to Weibel-Palade bodies by ultrastructural immunocytochemistry. The identity of the dense organelle as the Weibel-Palade body was further established by direct morphological examination of the dense organelle fraction. The VWF derived from this organelle is distributed among unusually high molecular weight multimers composed of fully processed monomeric subunits and is rapidly and quantitatively secreted in unmodified form after PMA stimulation. These studies: establish that the Weibel-Palade body is the endothelial-specific storage organelle for regulated VWF secretion; demonstrate that in cultured EC, the VWF concentrated in secretory organelles is of unusually high molecular weight and that this material may be rapidly mobilized in unmodified form; imply that proteolytic processing of VWF involved in regulated secretion takes place after translocation to the secretory organelle; provide a basis for further studies of intracellular protein trafficking in EC.

Ultrastructural localization of keratin proteins in human skin using low-temperature embedding and the protein A-gold technique.

Human skin was embedded in Lowicryl K4M and keratin proteins were localized by incubation with antikeratin antisera, followed by protein A-gold. The antikeratin antisera labeled all intermediate filament (tonofilament) structures in all layers of the epidermis. The association of keratin filaments with hemidesmosomes, desmosomes, and keratohyaline granules was clearly visualized. Desmosomes and keratohyaline granules were not labeled by the antikeratin antisera. No nonfilamentous structures were labeled. The technique described is suitable for studying the distribution of keratin filaments in normal and diseased tissue.

Malignant mesothelioma following radiation therapy.

BACKGROUND: Studies of the growing population of long-term survivors of cancer have led to increased recognition of the neoplastic complications of therapy. The causes of secondary malignancies are probably multifactorial, but radiation therapy and chemotherapy have certainly been implicated in the development of posttherapy neoplasia. PATIENTS AND METHODS: A case of pleural mesothelioma after successful radiation therapy for Hodgkin's disease is described with a review of radiation-associated mesotheliomas reported in the literature. In Hodgkin's disease, patients may receive radiation, chemotherapy, or combined treatment; the most common secondary malignancy is acute nonlymphocytic leukemia while sarcomas are the second most common solid tumors. CONCLUSIONS: Although mesothelioma is an uncommon sarcoma, its occurrence has been documented numerous times after exposure to diagnostic or therapeutic radiation.

Malignant mesothelioma: ultrastructural distinction from adenocarcinoma.

Mesotheliomas and metastatic adenocarcinomas involving the pleura are frequently difficult to distinguish by light-microscopic and histochemical methods. In a double-blind study, we have compared ultrastructural features of 10 mesotheliomas of epithelial type and 10 adenocarcinomas from the lung, breast, and upper GI tract, i.e., sites known to give rise to metastases which mimic mesothelioma. Mesotheliomas were observed to have a significantly greater microvillus length/diameter ratio (LDR) than adenocarcinomas (p less than 0.01) and more abundant intermediate filaments (p less than 0.001). Mesotheliomas had more complex microvilli than adenocarcinomas, whereas adenocarcinomas had rootlets (2/10 cases) and lamellar inclusion bodies (2/10 cases), both of which were absent in the mesotheliomas. This study provides quantitative and qualitative ultrastructural features of potential utility in the differential diagnosis of pleural mesotheliomas and adenocarcinomas.

Use of anti-horseradish peroxidase antibody-gold complex in the ABC technique.

We report a modification of the avidin-biotin-peroxidase complex (ABC) technique for the light and electron microscopic detection of antigens in tissue sections. An immunological approach was used instead of the DAB reaction to reveal ABC bound to antigen-antibody complexes. Affinity-purified polyclonal antibodies against horseradish peroxidase were complexed to particles of colloidal gold and applied for reaction with the horseradish peroxidase molecules of the ABC. For light microscopic immunolabeling, the signal produced by the anti-horseradish peroxidase antibody-gold complex required silver intensification. The ABC immunogold reaction as compared with the standard ABC technique, in particular with silver intensification of the DAB reaction product, provided superior resolution in paraffin sections. Furthermore, section pre-treatment to block endogenous peroxidase activity could be omitted and no potentially hazardous substrate was used. The ABC immunogold reaction was successfully applied for electron microscopic immunolabeling on Lowicryl K4M thin sections. We propose that the ABC immunogold reaction is a useful alternative to the standard ABC technique and can be equally well applied to light and electron microscopy.

Localization of keratin proteins in the human epidermis by a postembedding immunoperoxidase technique.

Individual keratin species were localized ultrastructurally to the tonofilaments and tonofibrils of the human epider-misusing a postembedding immunoperoxidase method. Low molecular weight keratin (45 kD) was localized primarily to the stratum basale and displayed a fine cytoplasmic staining of individual tonofilaments and some tonofibrillar staining. Higher molecular weight keratins (55 and 63 kD) were found predominantly in the suprabasal, differentiated squamous epithelial cells, staining tonofibrils with a web-like pattern. The technique utilized is suitable for studying the distribution of keratin proteins in normal and disease states.

Cytokeratin 20 immunoreactivity in renal oncocytomas.

Cytokeratins (CKs) are a group of 20 antigenically distinct intermediate filaments, generally confined to epithelia and their neoplasms. Immunostaining for CKs, in particular coordinate staining for CK7 and CK20, has become a useful tool in diagnostic pathology. Although studies defining CK distribution in neoplasms identify 0--7.7% of renal cell carcinomas (RCCs) positive for CK20, none has described the incidence of CK20 immunopositivity in renal oncocytomas (ROs). Distinction between RCC and RO may be difficult but this distinction is clinically significant, prompting us to establish the incidence of CK20 positivity in RO. We selected fifteen surgical cases of RO from our archives and studied their immunoreactivity for CKs including CK7 and CK20; 12/15 (80%) were positive for CK20, with variation in the number of cells staining. There was also variation in the distribution of CKs within the cells, including diffuse cytoplasmic, perinuclear, and a punctate or dot-like pattern. Such punctate staining corresponds to cytoplasmic balls of intermediate filaments and has been described with CAM 5.2 in RO and CK20 in Merkel cell carcinomas. Our findings suggest that CK20 immunohistochemistry is a useful tool for distinguishing RCCs from ROs. (J Histochem Cytochem 49:919-920, 2001)

Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes.

Involucrin immunoreactivity was localized ultrastructurally with protein A--gold in epidermis and cultured keratinocytes embedded in Lowicryl K4M. In the skin, immunoreactivity was found predominantly in cells of the granular layer and inner stratum corneum. The label was associated primarily with amorphous cytoplasmic material and especially keratohyaline granules. Some labeling was observed at the cell periphery, but little with keratin filaments. Tissue samples examined without aldehyde fixation showed relatively greater labeling in the outer stratum corneum than fixed tissue. In cultured cells, the labeling was also associated primarily with cytoplasmic granular material and to a lesser extent with the cell periphery. Upon treatment with the ionophore X537A, keratin filaments were found in aggregated arrays and the plasma membranes became convoluted. That involucrin immunoreactivity persisted in the cytoplasm in cultured cells and in vivo after cross-linking occurs could account for considerable isopeptide bonding detected in epidermal keratin fractions and indicates that not all the involucrin participates in envelope formation.

Plasmacytoma with aberrant expression of myeloid markers, T-cell markers, and cytokeratin.

Plasmacytomas are localized neoplastic proliferations of monoclonal plasma cells. When multifocal, the process is referred to as multiple myeloma. These lesions exhibit a pattern of antigen expression and cytomorphology that usually leads to a ready diagnosis. However, potentially troublesome variations in immunophenotype occur. We describe a case of a plasmacytoma from a patient who presented with sudden onset of pain and a lytic lesion of the left proximal humerus. Hematoxylin and eosin-stained sections showed a lymphoproliferative lesion composed of large lymphoid cells, some with plasmacytoid and immunoblastic features. The lesion also showed significant mitotic activity. Immunohistochemical staining was positive for CD45 (LCA), CD56 (N-CAM), CD43 (MT1), and cytokeratin CAM5.2. There was also clonal staining for lambda light chains. In addition, flow cytometric analysis showed positivity for myeloid markers such as CD13, CD33, CD38, and CD138. Significant negative markers include CD20 (L26), CD45RO (UCHL-1), and CD79alpha. The unusual phenotypic features of this plasmacytoma illustrate potential diagnostic pitfalls. It is important to fully study such lesions to correctly classify them, because this has significant impact on prognosis and management.

An ultrastructural comparison of mesotheliomas with adenocarcinomas of the lung and breast.

The ultrastructural features of 15 mesotheliomas were compared with those of equal numbers of adenocarcinomas of the lung and of the breast in a double-blind study. Combined quantitative and qualitative features were evaluated to provide criteria for distinguishing among these three tumors, which may present as either primary or metastatic pleural tumors. mesotheliomas could be distinguished from adenocarcinomas of the lung by length of microvilli (mean ratios of length to diameter [LDR], 15.7 and 8.7, respectively; P less than 0.01) and content of tonofilaments. Length of microvilli was also useful in distinguishing mesotheliomas from breast adenocarcinomas (mean LDR, 15.7 and 6.9, respectively; P less than 0.001). Adenocarcinomas of the lung could be distinguished from adenocarcinomas of the breast by tonofilament content and the presence of intracytoplasmic lumina. Combined quantitative and qualitative criteria are essential for maximal ultrastructural discrimination among these tumors.

Ultrastructural distinctions between adult pleomorphic rhabdomyosarcomas, pleomorphic liposarcomas, and pleomorphic malignant fibrous histiocytomas.

The ultrastructural features of five pleomorphic rhabdomyosarcomas, five high-grade malignant fibrous histiocytomas, and five pleomorphic liposarcomas were studied. Electron microscopy was found to be consistently useful in distinguishing between these tumors. The rhabdomyosarcomas showed thick and thin filaments in complexes and consistently contained glycogen. The malignant fibrous histiocytomas had numerous lysosomes, often in cells with ruffled borders, and contained cells showing "myofibroblastic" differentiation. The liposarcomas showed abundant and coalescing lipid droplets, sparse stroma with condensation of amorphous granular materials surrounding plasma membranes, and prominent vascularity. Fourteen of the 15 tumors could be identified on the basis of ultrastructure; thus, electron microscopic examination is an important diagnostic tool for pleomorphic tumors.

Ultrastructural localization of Leu M1 in Reed-Sternberg cells and normal myeloid cells.

An antigen Leu M1 has been localized to myelomonocytic cells and Reed-Sternberg cells by light microscopic immunocytochemical studies. We used both pre- and post-embedding immunoelectron microscopy to define the ultrastructural distribution of this antigen. Post-embedding techniques heavily labeled the granules of polymorphonuclear leukocytes and the nonspecific granules of eosinophils. At high concentrations there was labeling of the specific granules of the eosinophil. The antibody consistently labeled the perinuclear granules and vesicles of Reed-Sternberg cells. Some Reed-Sternberg cells also exhibited labeling of the endoplasmic reticulum, suggesting that these cells have the capacity to synthesize this antigen. Although plasma membranes were labeled with the post-embedding technique, these structures were most heavily labeled with the pre-embedding method. These results indicate that Leu M1 is synthesized and packaged by Reed-Sternberg cells and represents an integral structural component of these cells.

Detection of human papillomavirus in laryngeal lesions by in situ hybridization.

Human papillomavirus (HPV) is associated with human neoplasms of squamous epithelium. Squamous papillomas and verrucous carcinomas are two types of squamous neoplasms of the larynx that present difficult problems in differential diagnosis. Using in situ hybridization with biotinylated DNA probes, we examined benign squamous papillomas and verrucous squamous carcinomas of the larynx for the presence of HPV. Forty-two biopsy specimens from 18 patients with laryngeal papillomas and 11 biopsy specimens from seven patients with verrucous carcinomas were obtained from the files of Pennsylvania Hospital, Philadelphia, PA. Tissue sections were hybridized with an HPV DNA cocktail. The HPV-positive cases then were subtyped further with DNA probes specific for HPV subtypes 6/11, 16/18, and 31/33/35. All benign squamous papillomas (42 of 42) were positive for HPV subtype 6/11. None of the verrucous carcinomas contained demonstrable HPV (none of 11). Some of the squamous papillomas were recurrences, which shows the persistence of the virus. These results indicate that laryngeal papillomas may be related to HPV, but verrucous carcinomas are not.

Giant cell myocarditis after mitral valve replacement: case report and studies of the nature of giant cells.

A 22-year-old man with Marfan's syndrome and a history of antinuclear antibody-positive hepatitis died 25 days after undergoing cardiac valve replacement surgery for mitral valve prolapse. Giant cell myocarditis was found at autopsy. The multinucleated giant cells were shown by immunoperoxidase techniques to contain lysozyme, but not myosin or creatine phosphokinase, suggesting that they were derived from macrophage, rather than myocyte, precursors.

Immunoperoxidase staining for involucrin: a potential diagnostic aid in cervicovaginal pathology.

Involucrin, a protein subunit of keratinocyte cross-linked envelopes, is a distinctive marker for suprabasal differentiation in stratified squamous epithelium. Immunoperoxidase staining for involucrin was used to evaluate paraffin sections of tissue obtained by colposcopically directed biopsies of infectious, metaplastic, and dysplastic lesions of the cervix and vagina. Areas of normal squamous epithelium, papillary and flat condyloma acuminatum, and mature and immature squamous metaplasia showed positive staining in 99 per cent of samples lacking significant inflammation and in 60 per cent of those with moderate or severe inflammation. In contrast, only 19 per cent of the squamous cell dysplasias, even those without much inflammation, showed positive staining, and no area with moderate or severe inflammation showed positive staining. These findings indicate that expression of involucrin is modulated by cellular pathologic features and microenvironment. We suggest that immunoperoxidase staining for involucrin may be useful in distinguishing mild dysplasia from immature metaplasia and flat condyloma in some biopsy specimens in which routine histologic examination yields an indeterminate diagnosis.

Medullary carcinoma arising in a thyroid with Hashimoto's disease.

A 51-year-old woman presented with a large goiter and on pathologic examination was found to have both Hashimoto's thyroiditis and medullary carcinoma of the thyroid. To our knowledge, this is the first well-documented case of the coexistence of these two entities in the same patient in the English literature. The association of Hashimoto's disease and carcinoma of the thyroid is reviewed and its relevance discussed.

Ampullary somatostatinoma: psammomatous variant of gastrointestinal carcinoid tumor--an immunohistochemical and ultrastructural study. Report of a case and review of the literature.

A 28-year-old man presented with epigastric pain and obstructive jaundice associated with a histologically and immunologically unusual variant of carcinoid tumor involving the ampulla of Vater. The tumor contained abundant psammoma bodies and exhibited immunoreactivity only for somatostatin. Immunoperoxidase studies for insulin, glucagon, vasoactive intestinal peptide, calcitonin, serotonin, and ACTH had negative results. In contrast to most somatostatinomas of pancreatic origin, clinically this ampullary somatostatinoma was not accompanied by features of the somatostatinoma syndrome. A literature review of the clinical and hormonal features in reported cases of gastrointestinal and pancreatic somatostatinomas is presented.

Eccentric exercise-induced muscle damage impairs muscle glycogen repletion.

Five healthy untrained young male subjects were studied before, immediately after, and 10 days after a 45-min bout of eccentric exercise on a cycle ergometer (201 W). The subjects were sedentary at all other times and consumed a eucaloric meat-free diet. Needle biopsies of the vastus lateralis muscle were examined for intracellular damage and glycogen content. Immediately after exercise, muscle samples showed myofibrillar tearing and edema. At 10 days, there was myofibrillar necrosis, inflammatory cell infiltration, and no evidence of myofibrillar regeneration. Glycogen utilization during the exercise bout was 33 mmol glycosyl units/kg muscle, consistent with the metabolic intensity of 44% of maximal O2 uptake; however, the significant glycogen use by type II fibers contrasted with concentric exercise performed at this intensity. At 10 days after exercise, muscle glycogen was still depleted, in both type I and II fibers. It is possible that the alterations in muscle ultrastructures were related to the lack of repletion of muscle glycogen. Damage produced by eccentric exercise was more persistent than previously reported, indicating that more than 10 days may be necessary for recovery of muscle ultrastructure and carbohydrate reserves.

Fluoroquinolone's effect on growth of human chondrocytes and chondrosarcomas. In vitro and in vivo correlation.

Clinical and in vitro studies have demonstrated that fluoroquinolones are toxic to chondrocytes; however, the exact mechanism of fluoroquinolone arthropathy is unknown. We investigated the toxicity of ciprofloxacin on normal cartilage and on cartilaginous tumors. Normal human cartilage, enchondroma, and chondrosarcoma explants were cultured either alone or with the addition of ciprofloxacin at 1, 10, or 20 mg/L of medium. Samples were collected up to twenty-one days after treatment and were processed for electron microscopy and conventional light microscopy. The specimens were characterized morphologically with use of conventional light microscopy, electron microscopy, and immunohistochemistry to identify extracellular matrix, cell proliferation, and apoptosis. Cultures of normal chondrocytes expressed type-II collagen. Electron microscopy revealed a large amount of glycogen in the cells; the presence of fat droplets, rough endoplasmic reticulum, and prominent Golgi apparatus; and a proteoglycan layer surrounding the cells. With prolonged ciprofloxacin treatment and with increased doses, there was an increase in dilated rough endoplasmic reticulum, the appearance of phagosomes, and disintegrated bundles of vimentin filaments. The treated chondrocytes showed a decrease in cell proliferation, but there was no induction of apoptosis or effect on the expression of extracellular matrix proteins. Ciprofloxacin-treated chondrosarcoma cultures and tissue samples showed changes in cartilage matrix composition. Ultrastructural analysis demonstrated clumped glycogen, dilation of endoplasmic reticulum, numerous abnormal lysosomes containing degeneration products, and a decreased proteoglycan deposit surrounding the tumor cells. Treated chondrosarcoma cells and tissue specimens did not proliferate, and apoptosis was induced. In contrast, the in vitro growth of other noncartilaginous malignant tumors like osteosarcoma and liposarcoma was unaffected by ciprofloxacin. Our results indicate that ciprofloxacin is toxic to chondrocytes. In vitro and in vivo treated chondrosarcomas are the most affected.

Comparison of glycoprotein expression between ovarian and colon adenocarcinomas.

OBJECTIVE: Tumor-associated antigens may be expressed as surface glycoproteins. These molecules undergo qualitative and quantitative modifications during cell differentiation and malignant transformation. During malignant transformation, incomplete glycosylation is common, and certain glycosylation pathways are preferred. These antigens might help distinguish between ovarian and colonic adenocarcinomas in the primary and metastatic lesions. Different cytokeratins have been proposed as relatively organ-specific antigens. DESIGN: We used monoclonal antibodies against T1, Tn, sialosyl-Tn, B72.3, CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use in distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot be distinguished from colonic adenocarcinomas using immunohistochemistry.