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PubMed1987

Electron microscopy procedure influences detection of rotaviruses.

Technical parameters of electron microscope staining procedures (type of stain, pH of stain, and time of staining) influence particle integrity for three groups of rotaviruses. Simian rotavirus SA11 (group A), Chinese adult diarrhea rotavirus and porcine rotavirus-like agent (group B), and porcine pararotavirus (group C) were tested. All rotavirus strains were quite stable in uranyl acetate and phosphotungstic acid at pH 4.5 and relatively stable in ammonium molybdate. However, staining with phosphotungstic acid at higher pH values with increased staining time yielded a reduction in the number of particles and particles that were broken or degraded to single-shelled particles or core particles. The different staining procedures were also tested in immunoelectron microscopy experiments. Antibody molecules bound to rotavirus particles were observed clearly only with phosphotungstic acid staining and not with uranyl acetate. We therefore recommend that uranyl acetate and phosphotungstic acid at pH 4.5 be used for negative staining of rotaviruses; phosphotungstic acid at pH 4.5 is optimal for immunoelectron microscopy. These technical points may be critical for rotavirus detection and are important for studies pertaining to the epidemiology and clinical importance of the non-group A rotaviruses.

Animals↗
PubMed1992

[The phenotype of mast cells in primary adenoid liver tumours of rats and its relation to tumor cells].

Mast cells in adenoid liver tumours of 32 rats induced with nitrosomorpholine were observed ultrastructurally, and among them, some were studied immunocytochemically via immunogold techniques. Data indicated that mast cells which located in tumour tissues presented positive expression of rat mast cell protein (RMCP) II, indicating origination from the mucosal mast cells, while those in the connective tissues around tumours were largely stained negatively with either RMCP II or RMCP I antisera, with the exception of only a few cells showing positive RMCP II staining. Ultrastructural observation showed that mast cells in tumors contacted closely with the tumor cells. Membranes of the intracellular granules in these mast cells were fusing together. The content inside the granules were discharged and spread along the intercellular space between the tumor cells. There was not any lesion obtained ultrastructurally at the contacting point between the tumor cells and the mast cells. The significance of mucosal mast cells in adenoid liver tumors is briefly discussed.

Animals↗
PubMed1996

[Detection of micrometastases in axillary lymph nodes of node-negative breast cancer patients and its clinical significance].

Monoclonal antibodies against cytokeratin (AE1/AE3) were applied as a prob, using APAAP immunohistochemistry technique to detect 635 lymph nodes from 45 breast cancer patients with negative lymph nodes. Micrometastases were identified in 14 lymph nodes of 9 cases (20%). A significant difference was found between cytokeratin staining positive group and cytokeratin staining negative group in disease-free and over-all Kaplan-Meier survival curves. The detection of micrometastases had more clinical value for T1 and T2 patients. One of 2 T1 cytokeratin positive cases relapsed while only 1 of 19 T1 negative cases relapsed within 5 years, three of 5 T2 cytokeratin positive cases relapsed while 1 of 17 negative cases did. The presence of micrometastases had the same value in predicting local recurrence and distant metastases.

Adenocarcinoma↗
PubMed1998

Immunocytochemistry on cytologic specimens of limited quantity.

Some cytologic specimens may be limited in quantity, and this may hamper or preclude the performance of immunocytochemistry (ICC) in cases where more than one antibody (ab) is required by ICC to arrive at a definitive diagnosis. There is very little information in the cytology literature regarding the use of ICC for specimens that are limited in quantity. In this study, we describe a method, derived from the principles of double immunolabelling, whereby more than one ab test can be repeatedly used on the same Papanicolaou stained slide. Multiple cytologic scrape preparations fixed in 95% ethanol were obtained from fresh surgical specimens including carcinomas of the breast, endometrium, stomach, ovary and colon. Nonneoplastic tissues included tonsil (2), lymph node (2) and myometrium. Papanicolaou stained slides or unstained slides were subjected to two sequential ICC procedures, the first in which the ab was known to be nonreactive with the cells (insulin, glucagon, or somatostatin) and the second in which the ab was known to be positive in the cells. Positive controls for the known positive abs included a single-step ICC procedure as well as the tissue section. The test abs included CAM 5.2, AE1/3, K903, LCA, L26, UCHL-1, s-100, mCEA, GCDFP-15, vimentin, muscle specific actin and desmin. Identical two-step ab procedures were carried out on the tissues from the same surgical specimens. For Papanicolaou stained cytologic specimens, abs were reactive and gave excellent results for the repeat second-step ICC method. There was no false positive or false negative staining. This "repeat ICC" method also gave excellent results on the tissue sections. Immunocytochemistry can be performed more than once on the very same cytologic specimen if the initial ICC antibody attempt is negative. This method may be especially useful in situations where more than one antibody is needed on a very limited cytologic sample size.

Antibodies, Monoclonal↗
PubMed1975

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.

Bacteria↗
PubMed2002

Reduced aquaporin 4 expression in the muscle plasma membrane of patients with Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), previous freeze-fracture electron microscopic studies demonstrated that muscle plasma membrane contained markedly decreased numbers of orthogonal arrays. Recent investigations showed that orthogonal arrays were composed of aquaporin 4 (AQP4) molecules, a member of the water channel protein family. OBJECTIVES: To study whether the immunostainability of anti-AQP4 antibody is reduced in muscles of patients with DMD and whether, if it is reduced, the problem is at the genomic DNA, messenger RNA (mRNA), or posttranscriptional level. PATIENTS AND METHODS: We analyzed the muscle and blood samples from 6 boys with DMD, 6 normal control subjects, and 12 patients with neuromuscular diseases at the protein, genomic DNA, and mRNA levels. At the protein level, immunohistochemical staining and immunoblot analysis were performed. At the genomic DNA and mRNA levels, the polymerase chain reaction and reverse transcription polymerase chain reaction, respectively, were used to screen for mutations in the AQP4 gene. RESULTS: At the protein level, immunohistochemical staining of our originally generated rabbit anti-AQP4 antibody in DMD muscles was markedly reduced. Most of the DMD myofibers showed negative staining with sporadic partially positive fibers at their myofiber surface, whereas the control muscles displayed continuous myofiber surface staining. Immunoblot analysis showed that the content of AQP4 in DMD muscles was remarkably decreased. Amplification of leukocyte genomic DNA by polymerase chain reaction showed that the patients with DMD had genomic DNA of the AQP4 molecule. Quantitative reverse transcription polymerase chain reaction demonstrated that DMD skeletal muscles contained markedly decreased AQP4 mRNA compared with controls. CONCLUSION: The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.

Aquaporin 4↗
PubMed1992

Large cell lymphoma of the mediastinum with marked tropism for germinal centers.

The cases are presented of three patients with large cell malignant lymphoma of the mediastinum with a distinctive growth pattern characterized by marked tropism of tumor cells for germinal centers. The patients' ages ranged from 35 to 70 years; one was a woman, and two were men. Histologically, the lesions were characterized by a proliferation of large atypical cells arranged in clusters that encroached on mantle zones and invaded germinal centers. Immunohistochemical studies showed positive staining of the tumor cells for leukocyte common antigen, L-26 (pan-B-cell antigen), and lambda light chain restriction and negative staining with Leu-M1, UCHL-1 (pan-T-cell antigen), Ber-H2 (Ki-1 antigen), S-100 protein, HMB-45, low-molecular weight keratins, epithelial membrane antigen, carcinoembryonic antigen, and placental alkaline phosphatase. Electron microscopy done on one specimen showed cells with abundant cytoplasm, some of them containing surface microvilli, scant organelles, and scattered polyribosomes consistent with lymphoid cells. Because of their unusual growth pattern and morphology, these tumors can be mistaken for primary or metastatic carcinomas, germ cell tumors (particularly seminomas), and metastatic malignant melanomas. It is proposed that the "germinotropic" features observed in these lesions are the result of active colonization of germinal centers by the tumor cells. The possible pathogenetic implications of this phenomenon are discussed.

Adult↗
PubMed1994

Immunohistochemical localization of syndecan-1 in normal and pathological human uterine cervix.

Expression of syndecan-1, a cell surface proteoglycan that binds growth factors and extracellular matrix components, was studied in normal and pathological human uterine cervix using immunohistochemical methods. Normal cervical squamous epithelium showed positive staining for syndecan-1 in all cell layers, except the basal cell layer, whereas endocervical columnar epithelium stained weakly. In non-neoplastic reactive lesions, metaplastic squamous cells were positive for syndecan-1, whereas columnar cells showed weak or negative staining. In cervical condylomas, cells showing koilocytotic atypia were positive for syndecan-1. The progression of cervical intraepithelial neoplasia (CIN) grade I to grade III was associated with reduced syndecan-1 expression and localization of syndecan-1 to more superficial cell layers. In squamous cell carcinomas (SCCs), syndecan-1 expression correlated with histological differentiation, being absent from most poorly differentiated tumours. The results suggest that loss of syndecan-1 from atypical cells is an early event during cervical carcinogenesis and show a close association of syndecan-1 expression with preserved epithelial morphology and differentiation.

Carcinoma, Squamous Cell↗
PubMed2002

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.

Cell Line↗
PubMed1990

Keratin filaments of epithelial and taste-bud cells in the circumvallate papillae of adult and developing mice.

Keratin filaments of epithelial- and taste-bud cells in the circumvallate papillae of adult and developing mice were studied by immunocytochemistry using monoclonal antikeratin antibodies (PKK2 and PKK3) and by conventional electron microscopy. Elongated cells (type-I, -II, and -III cells) of the taste buds were stained by PKK3 antibody, which reacts with 45-kdalton keratin, whereas basal cells of the taste buds and surrounding epithelial cells showed negative staining with PKK3. Such PKK3-reactive cells occurred at 0 day after birth, when taste-buds first appeared in the dorsal surface epithelium of the papillae. Thus 45-kdalton keratin seems to be an excellent immunocytochemical marker for identifying taste-bud cells. Epithelial cells in all layers of the trench wall and basal layer cells of the dorsal surface contained densely aggregated bundles of keratin filaments that reacted with PKK2 antibody, but not with PKK3. In contrast, taste-bud cells and spinous and granular layer cells of the dorsal surface possessed loose aggregated bundles of filaments that reacted with PKK3, but not with PKK2. These results suggest that the aggregation and distribution pattern of keratin filaments may reflect differences in the keratin subtypes that comprise these filaments.

Actin Cytoskeleton↗
PubMedDate not supplied

Small-cell neuroendocrine carcinoma as a variant form of prostate cancer recurrence: a case report and short literature review.

BACKGROUND: Small-cell neuroendocrine carcinoma has been recognized as a rare histologic variant occurring in only 0.5% to 2% of prostatic primary tumors. However, recent autopsy studies suggest development to this phenotype in up to 10% to 20% of the cases with hormone-refractory disease. CASE PRESENTATION: A case of conventional adenocarcinoma before androgen-ablation therapy but showing progression to small-cell neuroendocrine carcinoma at the recurrence. The immunohistochemistry of the tumor showed strong positive staining for progastrin-releasing peptide (ProGRP), a carboxy terminal region common to 3 precursors for gastrin-releasing peptide, but almost negative staining for chromogranin-A and prostate-specific antigen. Combination chemotherapy based on cisplatin and etoposide was effective for controlling the tumor progression for 7 months, and the serum ProGRP level correlated well to the clinical course. Neither objective nor subjective responses were observed to somatostatin analogue therapy performed in the late stage of disease. CONCLUSIONS: The present case reminds the urologist that small-cell neuroendocrine carcinoma may be a variant form of disease recurrence during androgen ablation in advanced prostate cancer. A strategic approach for this phenotype evaluating serum neuroendocrine markers, such as ProGRP, should be taken when serum prostate-specific antigen does not reflect the disease state. This approach would allow one to choose alternative therapies targeting neuroendocrine cells other than androgen ablation.

Aged↗
PubMed2000

Expression of cytokeratins in human enamel organ.

Cytokeratin (CK) is a filament which plays a central role in epithelial tissue and, like the polypeptides of intermediate filaments in general, shows a high degree of tissue specificity. The CK expression patterns of odontogenic epithelia are still poorly described. We studied the distribution of individual CK polypeptides in the human enamel organ at bell stage and in remnants of the dental lamina. Our immunohistochemical study showed that epithelial cells stained for CKs 7, 13, 14 and 19 with slight changes in their pattern during the differentiation phase of odontogenesis. There was negative staining for all other CK polypeptides tested (CKs 8, 10, 16, 17 and 18). Most of the CKs in the enamel organ epithelia did not show differences related to the stage-specific state of differentiation, except for CKs 14 and 19 at the inner enamel epithelium. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, and this was substituted by CK 19 at the late bell stage when the ameloblasts were fully differentiated.

Ameloblasts↗
PubMed1982

Interaction of Mycoplasma hyopneumoniae with the porcine respiratory epithelium as observed by electron microscopy.

An in vivo-passaged strain of Mycoplasma hyopneumoniae attained viability titers of 10(6) to 10(8) color-changing units per mg of tissue in pig lungs and caused gross and histological pulmonic lesions. Mycoplasmas were readily located in the lumina of the respiratory tract by electron microscopy. In sections of tissue fixed in glutaraldehyde-osmium, the organisms were found to possess many radial fibrils on the outer surface of the limiting membrane. These fibrils appeared to interconnect adjacent mycoplasmas and to extend between the organism and epithelial cell. Ruthenium red staining demonstrated a thick, dark layer of capsular material enveloping the entire mycoplasma cell. The capsular material was seen to bridge the space between the mycoplasma and host cell. The general morphology of the in vitro-passaged strains grown in broth medium was essentially similar to that of the in vivo-passaged strain. In these organisms, however, no long fibrils were seen, although a fuzzy layer was present outside the cell membrane. The ruthenium red-positive capsule was stained less intensely, and its width was only about one-half that observed for the in vivo-passaged strain. In negatively stained preparations, the cells had an outer fringe of amorphous material apparently corresponding to the fuzzy layer seen in thin sections. The in vitro-passaged strain grew poorly in pig lungs and lost its ability to produce gross pulmonic lesions. The organisms in the respiratory tract had a capsule much thinner than that of the in vivo-passaged strain.

Animals↗
PubMed1972

Arrangement of herpesvirus deoxyribonucleic acid in the core.

The core of the herpes simplex virion consists of an electron-dense toroidal structure 50 nm high, with an inside diameter of 18 nm and an outside diameter of 70 nm, penetrated by a less dense cylindrical mass. The toroid contains deoxyribonucleic acid (DNA), as evident from the observation that uranyl ion staining is removed by ethylenediaminetetraacetic acid under conditions which result in the extraction of uranyl ions bound to nuclear DNA. Studies on negatively stained preparations of purified capsids suggest that the toroid consists of DNA arranged as if it were spooled around the cylindrical mass.

Carcinoma↗
PubMedDate not supplied

Lectin expression during wound healing of the rabbit sinus mucosa: a study of regenerating epithelium and early polyp formation.

Lectin expression during wound healing of the rabbit sinus mucosa was examined. Positive UEA-I staining was evident on squamous or cuboidal as well as columnar regenerating epithelial cells (RE cells). PNA staining of columnar RE cells first became evident after neuraminidase treatment, while squamous or cuboidal RE cells stained positively with PNA alone. Fucosylation within RE cells thus occurred from a relatively early period, and sialylation followed at later stages. Ingrowing epithelial cells of early polyp formation stained negatively with UEA-I, indicating that unfucosylated RE cells may represent aberrant cellular behavior. We concluded that these patterns of lectin staining indicate a functional maturation as well as an integration of regenerating mucosa.

Animals↗
PubMed1996

Increase in lectin binding sites on epithelial cells by chronic bladder infection in rats.

Using lectin histochemistry we assessed whether chronic bladder infection modifies carbohydrate residues of glycoconjugates on uroepithelial cells in rats. The bladder infection was produced by implanting a knotted silk thread with Escherichia coli into the bladder. One or 4 weeks after the implantation the bladder was excised, incubated with sixteen biotinylated lectins and stained. The bladder epithelia as a whole stained more strongly positive for eight lectins in the infected rats than in the control rats having a sterile silk thread in the bladder. In the infected rats, the superficial epithelial layer that stained negative for Arachis hypogaea (PNA) in the controls became strongly positive for PNA, whereas the middle and deep epithelial layers increased in staining for Canavalia ensiformis and six other lectins. These results indicate that chronic bladder infection increases carbohydrate residues on uroepithelial cells and may facilitate bacterial adherence to uroepithelial cells.

Animals↗
PubMed1989

Isolation of Fusobacterium nucleatum and electron microscopic observations of spirochetes from tropical skin ulcers in Papua New Guinea.

Tropical ulcer is a disabling condition of the lower leg affecting mainly young adults and older children. Microscopic observations of lesion material have shown fusiform bacilli and spirochetes. We used anaerobic culture techniques to isolate and identify these fusiform bacilli. Electron microscopic (EM) studies were performed to characterize the spirochetes. Material collected on swabs was used to inoculate pre-reduced media and to prepare smears for gram staining; the swabs were placed in fixative for EM study. After incubation, colonies containing fusiform bacilli were subcultured. The anaerobic gram-negative fusiform isolates were identified as Fusobacterium nucleatum using biochemical reactions, hemagglutination testing, and reaction of antigen preparations of the isolates and ATCC strains in serological tests with rabbit antisera. EM observations of negatively stained spirochetes revealed an 8-16-8 periplasmic flagellar arrangement. F. nucleatum and spirochetes may participate in the pathogenesis of this polymicrobic infection.

Adolescent↗
PubMed1993

[A case of sarcoidosis presenting as pulmonary nodules associated with pleural indentation fifteen years after onset].

A 65-year-old woman was admitted to our hospital because of pulmonary nodules, about 3 cm in diameter in the right upper lobe accompanied by pleural indentation, and 5 mm in diameter in the left middle lung field, revealed by chest X-ray. She had no clinical signs or symptoms except achalasia-like discomfort swallowing. Results of physical examinations were within normal limits. A specimen obtained from the nodule in right S2 by transbronchial biopsy showed noncaseating epithelioid granuloma containing asteroid bodies, and negative staining for acid-fast bacilli. No malignant cells were found in the specimen. Bacteriological examination of a bronchial lavage specimen was negative for pyogenic bacteria, mycobacteria and fungi. A PPD skin test was negative. BALF revealed an increase of lymphocytes and elevation of CD4/CD8 ratio. Fifteen years previously, the patient had been diagnosed as having sarcoidosis by scalene node biopsy, and had been treated with steroid therapy. Pulmonary nodules associated with pleural indentation in sarcoidosis is relatively rare, and is usually due to malignancy such as lung cancer. This possibility was ruled out clinically in the present case. Therefore, it was concluded that the pulmonary nodules in this case were due to the sarcoidosis itself.

Age of Onset↗