Identification and characterization of a novel adenovirus in the cloacal bursa of gulls.

Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species.

Expression of E-cadherin, alpha- & beta-catenin, and CD44V6 and the subcellular localization of E-cadherin and CD44V6 in normal epidermis and basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin is a locally invasive, rarely metastasizing epithelial tumor. In the current study, the expression of E-cadherin, alpha- and beta-catenin and CD44V6 in normal epidermis and on BCC cells were investigated. A significantly reduced expression of alpha-catenin and CD44V6 and a slightly reduced expression of E-cadherin on BCC cells were observed compared with the overlying epidermis. Immunoelectron microscopy was used to investigate whether the decreased expression of E-cadherin and CD44V6 was due to either an absence or downregulation of specific membrane structures or due to an overall downregulation of these adhesion molecules in all membrane structures in BCC. E-cadherin and CD44V6 were expressed in adherens junctions, desmosomes, and complex interdigitating membrane structures both in normal epidermis and in BCC. A quantitative analysis showed that only a percentage of desmosomes was stained. In addition, the effect of pro-inflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), was investigated in biopsy specimens of normal skin and BCC, using a biopsy culture system and immunohistochemistry. The expression of E-cadherin and CD44V6 was not significantly decreased after culturing BCC or normal skin biopsy specimens for 48 hours with or without recombinant human (rHu)IFN-gamma or rHuTNF-alpha. It may be concluded that the decreased expression of both E-cadherin and CD44V6, observed in light microscopy, was not attributable to the absence of specific specialized structures in BCC and most likely also not caused by downregulation by local cytokines, but rather by generic downregulation of both of these adhesion molecules during malignant transformation.

[Reading children's temperatures with the tympanic infrared thermometer and the rectal mercury thermometer: equally good results in the emergency room]. / Temperatuurmeting bij kinderen: met de trommelvliesinfraroodmeter en de rectale kwikthermometer even goede resultaten op de spoedeisendehulpafdeling.

OBJECTIVE: To compare the results of reading body temperatures with a tympanic infrared thermometer and a rectal mercury thermometer in children in an emergency department. DESIGN: Prospective comparative study. SETTING: St. Elisabeth Hospital, Tilburg, the Netherlands. METHOD: In children up to 11 years of age seen in the emergency room between 1 January 1994 and 1 April 1994, the body temperature was measured with a rectal mercury thermometer as well as with a tympanic infrared thermometer. Data were collected on temperature read, clinical picture on arrival (not ill, ill, seriously ill) and appearance of the tympanic membrane (signs of acute otitis media, presence of cerumen). For the statistical comparison, the differences between the findings of the two methods were plotted against the means. The sensitivity and specificity of the results of tympanic measurement in relation to the values read rectally were determined. RESULTS: Data were collected on 213 children, of whom 19 were younger than 3 months, 46 between 3 and 12 months, and 148 between 1 and 11 years. The mean temperatures measured with the rectal and tympanic thermometers were 38.01 and 38.03 degrees C, respectively. The mean difference between the rectal and tympanic temperatures was -0.013 degree C. The correlation between the rectal and tympanic temperatures was high (r = 0.86; P = 0.0001). The results were the same in groups differing in age, severity of disease and appearance of the tympanic membrane. The sensitivity of the tympanic measurement for fever (rectal temperature > 38.0 degrees C) was 80.6% with a specificity of 92.5%. The sensitivity was 83.8% when a rectal temperature > 38.5 degrees C was taken as the criterion, with a specificity of 95.9%. CONCLUSION: The tympanic infrared measurement in children in an emergency department gave the same results as rectal measurement using a mercury thermometer.

A new monoclonal antibody for specific immunocytochemical staining of nucleoli.

We have isolated a hybridoma cell line (clone 1E10) producing a monoclonal antibody which specifically recognizes nucleoli. The antibody (IgM, k isotype) was found to react in a nucleolar pattern with a variety of cell types. Specific staining was only obtained on cryostat sections of unfixed tissues. Paraffin embedding destroyed the epitope. Tissue specificity or species specificity was not observed. Nucleoli in neoplastic cells were highly reactive, presumably due to the larger size of nucleoli in these cells. Immunoelectron-microscopy (using a pre-embedding as well as a post-embedding technique) confirmed the specific nucleolar localization of the immunoreactivity. Immunoreactivity was confined to the granular component of the nucleolus. The intensity of the immunoreactivity increased after cell or tissue pretreatment with DNase, pronase or trypsin, indicating that the target epitope is not DNA or a protein. On Western blots of immunoreactive cells no specific signal was obtained, which supports the non-protein nature of the epitope. Acid hydrolysis and RNase digestion abolished the immunoreactivity. Parallel staining experiments with methylgreen pyronin and acridin orange confirmed the RNA nature of the epitope. In spot blots, immunoreactivity was not found with tRNA or mRNA. These observations indicate that 1E10 recognizes a conformational RNA epitope which occurs only in the nucleolus.

Characterization of distinct functions for growth factors in murine transitional epithelial cells in primary organotypic culture.

Although previous studies indicate that growth factors can affect several physiological processes in epithelia, their role in the biological dynamics of transitional epithelium of the bladder is not yet established. This study investigates the functional consequences of a direct action of EGF, TGF beta, FGF-1, FGF-2, PDGF-AA, and PDGF-BB on mouse urothelium in organoid-like primary cultures. Confluent and nonconfluent cultures served as a model for intact and regenerating urothelium, respectively. EGF and FGF-1 stimulated in both models under serum-free conditions the BrdU and [3H]-thymidine incorporation. This resulted in an increase in the number of cell layers, but the cultures assumed a less organoid-like morphology. In addition, EGF and FGF-1 stimulated the expansion of nonconfluent cultures. TGF beta inhibited proliferation, caused a decrease in the number of cell layers, and blocked expansion. Moreover, TGF beta induced the terminal differentiation and apoptosis of urothelial cells. In nonconfluent cultures PDGF-BB slightly stimulated the increase in the outgrowth area, but no other effect on the parameters for proliferation and differentiation was observed. FGF-2 and PDGF-AA did not affect any of the studied parameters. These data are consistent with the hypothesis that EGF and FGF-1 can promote wound healing and/or hyperplasia through direct action on the epithelial cells, while TGF beta promotes the development of a normal, differentiated transitional epithelium.

Ultrastructural characterization of transitional cells in oligodendrogliomas.

In oligodendroglial tumors the expression of glial fibrillary acidic protein (GFAP) is found in cells with an astrocytic morphology representing preexistent or neoplastic astrocytes. In addition, a proportion of the GFAP-positive cells has the morphology of miniature gemistocytes (minigemistocytes) or oligodendrocytes (gliofibrillary oligodendrocytes or GFOC). Both minigemistocytes and GFOC are considered as cells transitional between astrocytic and oligodendroglial lineage. Though minigemistocytes can readily be distinguished in routinely stained histological sections, GFAP immunostaining is obligatory for the identification of the GFOC. In the present study, the GFOC is characterized at the ultrastructural level using an immunogold-silver stain on semithin (1 micron) slides for identification of GFAP immunoreactivity and subsequent processing of the adjacent slide for immunoelectron microscopy. In analogy with the minigemistocytes, the glial filaments in the GFOC are arranged in parallel bundles. The finding of cells with ultrastructural features intermediate between those of GFOC and minigemistocytes suggests a close relationship and a possible interconvertibility between the two transitional cell types in oligodendrogliomas.

Ultrastructural and immunohistochemical segregation of gemistocytic subsets.

Gemistocytes are frequently encountered in cases of reactive gliosis as well as in glial tumors. Recently, miniature forms of gemistocytes (minigemistocytes) were recognized as cellular constituents of oligodendrogliomas. Antibodies specific for the intermediate filaments glial fibrillary acidic protein and vimentin are reactive with gemistocytic cells, but do not react specifically with these cells. In a study of 23 glial tumors we found the monoclonal antibody Pm43 selectively reactive with the classical gemistocytes as well as with the minigemistocytes. Nevertheless, at the ultrastructural level a striking difference in the arrangement of the glial filaments between both gemistocytic cell types was found. Immunoelectron microscopy showed that the reactivity for the newly discovered gemistocytic marker Pm43 was confined to identical intermediate filaments. Despite immunohistochemical homology, a clearly different ultrastructure divides classic gemistocytes and minigemistocytes into two subsets.

Phrenic nerve paralysis following neck dissection.

One of the complications of neck dissection to control regional metastatic disease in cancer of the head and neck is phrenic nerve paralysis. The resulting elevation of the ipsilateral diaphragm can be diagnosed on a postoperative chest X-ray and confirmed by fluoroscopy. Symptoms can be respiratory, cardiac or gastrointestinal. In a retrospective study, unilateral phrenic nerve paralysis was observed in 14 (8%) of 176 consecutive neck dissections. None of the patients with postoperative phrenic nerve paralysis displayed severe symptoms, although a significantly higher number sustained atelectasis with or without pulmonary infiltrates to complicate the postoperative course.

Quantitative energy-filtered image analysis in cytochemistry. I. Morphometric analysis of contrast-related images.

A combination of energy-filtered electron microscopy (EFEM) and an image-analyzing system (IBAS/2000) is used for morphometric analyses of cells and (reaction) products. Image contrast is objectively established and segmentation is based upon intrinsic contrasts, in ultrathin sections. Cross-sectioned platinum-stained erythrocytes are used as a model to determine optimal conditions for constant measuring results for contrast, area and perimeter. Results are related to changes in: (1) the objective-lens diaphragm diameter, (2) three most frequently used contrast modes obtainable by electron spectroscopical imaging (ESI) in a Zeiss EM 902 transmission electron microscope (e.g., global, zero loss (or deltaE - 0 eV) and deltaE = 250 eV), and (3) the number of image integrations (1-250X) acquired by real-time video. A thresholding procedure is proposed for objective segmentation of such contrast-related images and applied to measure the area fraction of nuclear chromatin and the diameter of nominal 1 nm colloidal gold particles.

Cytopathology of malignant mesothelioma. Reappraisal of the diagnostic value of collagen cores.

The identification of malignant mesothelial cells in cytological smears prepared from serous effusions is still hampered by the lack of features specific for mesothelial differentiation. We examined the diagnostic value of collagen cores within clusters of tumour cells in cytological smears prepared from effusions from 43 patients with malignant mesothelioma and of 62 cases of metastatic adenocarcinoma. In Giemsa-stained smears collagen cores were detected in 51% of the cases of malignant mesothelioma and in none of the smears with metastatic adenocarcinoma. Using the Azan stain, collagen cores were detected in 64% of the malignant mesotheliomas and 4% of the adenocarcinomas. Immunoelectron microscopy demonstrated that the collagen cores are largely composed of collagen type III fibrils and some elastin embedded in a homogenous extracellular matrix. It can be concluded that the presence of collagen cores within clusters of tumour cells is highly suggestive of mesothelial differentiation and a common finding in malignant mesothelioma.

Effect of phlebotomy on the ferritin iron content in the rat liver as determined morphometrically with the use of electron energy loss spectroscopy.

Phlebotomy of untreated and iron-loaded rats results in a significant decrease in total liver iron. In iron-loaded rats a marked decrease in iron-containing particles is observed ultrastructurally in lysosomes and cytoplasm of hepatic sinusoidal cells but not in parenchymal cells. This remarkable phenomenon was further investigated in a morphometric study, based on element-specific (iron) distribution images made in situ in the parenchymal cell by means of electron energy loss spectroscopy. With the use of this technique it could be shown that in spite of phlebotomy the ferritin iron content of the iron-loaded liver parenchymal cell is not decreased.