Birefringent (calcium oxalate) crystals in thyroid diseases. A clinicopathological study with possible implications for differential diagnosis.

To elucidate the nature and significance of calcium oxalate crystals in the pathologic thyroid, we used polarized light microscopy to review 357 thyroid lesions. Under polarized light, calcium oxalate crystals had brilliant birefringence, and they were invariably identified within the colloid of follicles. The highest prevalence of crystals (87.9%) was in nodular goiters; they were also found in 60.0% of follicular adenomas and 33.3% of follicular carcinomas. The prevalence of crystals in papillary carcinomas was very low (5.4%). Therefore, the overall prevalence was 69.4% in benign nodules and 7.6% in malignant nodules. A heavy deposit of crystals was seen only in benign nodules except for one case of follicular carcinoma. Graves' disease, focal thyroiditis, and subacute thyroiditis showed low prevalence: 25.0, 46.9, and 40.0%, respectively. In cases of toxic nodules, the crystals were sparsely identified within nodules, but abundantly observed in surrounding inactive tissues. Immunohistochemistry for thyroid hormones confirmed that the crystals tended to appear in inactive follicles. On tissue x-ray film, the crystals appeared as microcalcifications. As a result of these findings, we suggest that examinations of crystals are likely to be useful in the differential diagnosis of thyroid diseases and in possible estimations of the functional state of lesions.

Confocal laser scanning microscopic observation of angioarchitectures in human thyroid neoplasms.

Confocal laser scanning microscopy (CLSM) was employed to study the blood vascular system of human thyroid tumors. CLSM observation combined with immunohistochemistry for type IV collagen clearly visualized 3-dimensional images of the microvascular structures. CLSM observation showed that normal thyroid follicles were tightly covered by branching microvessels, whereas microvessels in follicular adenomas were more prominent and more irregular in shape. Microfollicular adenomas showed that neoplastic small follicles were attached to the capillaries and had a "grape-like" appearance. Strikingly well-developed vascular networks were seen in neoplastic follicles of papillary carcinomas. Interestingly, papillae of papillary carcinoma occasionally contained contained aggregated vascular complexes (glomeruloid structure) composed of tortuous, densely packed, and irregularly arranged small vessels. Such aggregated vascular complexes were seen in 5 of 7 papillary carcinoma tissues but not in other histological thyroid tumors. Our findings indicate that the fundamental vascular pattern correlates well with the growth pattern, suggesting an interdependence between parenchyma and stroma characteristic for thyroid tumors. CLSM observation combined with immunohistochemistry may contribute to a better understanding of morphological characteristics of angioarchitecture in human surgical materials.

Expression of vascular endothelial growth factor (VEGF) in human thyroid neoplasms.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is an angiogenic factor that plays important roles in tumor growth. Angiogenesis studies on VEGF deal with various types of malignant tumors, but very little is known about the role or significance of VEGF in human thyroid neoplasms. Therefore, in the current study, we determined whether VEGF is found in normal and neoplastic thyroids and whether its expression is altered in different histological types of thyroid neoplasms. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that all specimens of thyroid tumors expressed bands corresponding to 121-, 165-, and 189-amino acid forms of VEGF. Northern blot analysis showed an increase in VEGF mRNA levels in neoplastic tissues in comparison with normal thyroid samples. By nonisotopic in situ hybridization, most of the tumor cells in follicular adenomas expressed VEGF mRNA, whereas VEGF mRNA expression was identified only in epithelium of isolated follicles in normal thyroid tissues. In papillary thyroid carcinomas, an intense labeling with VEGF probe was often found in overlying tumor cells of neoplastic papillae. VEGF expression was distinctly intensified in undifferentiated carcinoma cells that were immediately adjacent to necrotic foci. The immunohistochemical localizations of VEGF protein were comparable to the localization of VEGF mRNA. In conclusion, our results suggest that the histological types of thyroid tumor may determine the vascular pattern through a paracrine mechanism involving VEGF.

Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure using monoclonal antibody MIB-1.

To increase our understanding of the basic biological mechanisms of thyroid diseases, growth activity (GA) in 232 thyroid lesions was determined by immunohistochemistry using monoclonal antibody MIB-1. The GA tended to be higher in hyperplastic lesions, adenomatous goiter (MIB-1-positive cell rate, 0.73%), and Graves' disease (1.68%) than in normal tissue (0.19%). The GA for differentiated thyroid carcinomas (2.00%) was much lower than for adenocarcinomas of other organs, such as breast, lung, stomach and colon (44.67%). Of the thyroid carcinomas, the highest GA was observed in undifferentiated carcinomas (32.67%), and follicular carcinomas (3.18%) showed a higher GA than papillary carcinomas (1.83%). There was no significant difference between the GA of follicular carcinomas and solid/trabecular adenomas, although widely invasive follicular carcinomas showed a higher GA than minimally invasive carcinomas. No significant correlations between GA and patient age, sex, and tumor diameter, metastasis, or histological features were observed in papillary carcinomas. Familial medullary carcinomas showed a higher GA than sporadic tumors. All latent papillary carcinomas had a very low GA. Our findings suggest that immunohistochemical investigation using the antibody MIB-1 contributes to the understanding of growth characteristics and biological activities in thyroid diseases.

Different organization of intermediate filaments in columnar cells of rat large intestinal mucosa as revealed by confocal laser scanning microscopy and quick-freezing and deep-etching method.

The relationship between cell differentiation and ultrastructural changes of intermediate filaments (IF) was studied in columnar cells of large intestinal mucosa of rats by confocal laser scanning microscopy and quick-freezing and deep-etching method. A feature of the IF in immature columnar cells was minibundle formation with prominent branching, which organized the meshwork structures. The minibundles, which appeared to be formed by the attachment of two or more IF in side-to-side fashion, were loosely distributed throughout the cytoplasm. In contrast, in mature columnar cells, the IF were densely distributed under the terminal web in the cytoplasm and beneath the upper part of the lateral membrane regions, whereas the other areas of the cytoplasm contained only a small number of IF. Minibundle formation was not observed, and the branching was rarely identified. The changes in the distribution and density of IF, which are expressed in specific areas of mature columnar cells, apparently represent a characteristic of intracellular differentiation. It is suggested that the dissociation of minibundled IF, which was often observed in the immature columnar cells, is an important step in the acquisition of functional polarity in cells of this type.

Autonomic nerve tumour with skeinoid fibres: ultrastructure of skeinoid fibres examined by quick-freezing and deep-etching method.

A case of gastrointestinal autonomic nerve tumour with skeinoid fibres (SFs) of the jejunum in a 79-year-old Japanese man, was examined by the quick-freezing and deep-etching (QF-DE) method. The tumour consisted of spindle cells with immunohistochemical reactions for vimentin, NSE and CD34. Electron microscopically, features of the neural cells of the myenteric plexus were observed. The QF-DE method demonstrated intercellular meshwork structures, consisting of thin filaments (7-15 nm), with granular deposits. Fully developed parts of the deposits formed nodular aggregates composed of irregularly surfaced thick fibrils (30-48 nm) with a tendency to linear arrangement (SFs). We detected many interconnecting thin filaments (ICTFs) between the SFs, which were pre-existing components in the meshwork, avoiding the granular deposits. The focal thickening formed by the connection between SFs and ICTFs revealed a periodicity typical of SFs (33-45 nm). We conclude that SFs are formed by decoration of the granular deposits along pre-existing intercellular meshwork structures.

Poorly differentiated desmin-negative and vimentin-positive leiomyosarcoma of the stomach examined by the immunohistochemical and quick-freezing and deep-etching methods.

A poorly differentiated leiomyosarcoma of the stomach in a 41-year-old woman is reported. The diagnosis was confirmed by the diffuse immunohistochemical reaction to HHF35, and the presence of focal density and caveolas in some of the tumour cells by conventional electron microscopy. Immunohistochemically, most tumour cells had an undifferentiated nature, in which negative immunostaining for desmin, alpha-smooth muscle actin, and type IV collagen, and positive immunostaining for vimentin were observed. By the quick-freezing and deep-etching (QF-DE) method, these tumour cells revealed the loss of bundled actin and myosin filaments, which constitute desmin associated structures (focal densities and dense patchy areas). Their cytoplasm had many mitochondria and other cell organelles. The intermediate filaments (IFs), which were determined to be vimentin by immunohistochemistry, were observed in the inter-organellar spaces, and connected with these cell organelles. Actin filaments formed a meshwork structure and were distributed mainly in subplasmalemmal regions. Although a basal lamina was not detected by conventional electron microscopy, basal lamina-like structures, an association between the extracellular matrices and the cell membrane, were observed. Using the QF-DE method, three dimensional ultrastructural alterations of the cytoskeleton and extracellular matrix of the leiomyosarcoma were observed.

Development of a conductivity-based immunosensor for sensitive detection of methamphetamine (stimulant drug) in human urine.

A simple immunosensor based on a conductivity method was developed for determination of methamphetamine (MA, a stimulant drug) in urine. Anti-MA antibody was immobilized onto the surface of a pair of platinum electrodes. The reaction of MA with the antibody causes a decrease in the conductivity of the anti-MA immobilized layer between the electrodes. A linear relationship was obtained between the conductivity and MA concentration in the range of 1-10 micrograms/ml. The method requires the sample to be rinsed with water on the electrodes after the immunoreaction. This detection system was applied to the determination of MA in urine and proved to be a useful and a simple detection technique of MA in forensic science in comparison with a gas chromatography-mass spectrometry method.

Flow injection analysis for residual chlorine using Pb(II) ion-selective electrode detector.

A simple flow injection analysis (FIA) system for residual chlorine in tap water has been developed by using a Pb(II) ion-selective electrode (ISE) detector. The method is based on a specific response of the Pb(II)-ISE to residual chlorine. The FIA system consists of a millivolt meter, a peristaltic pump, a Pb(II)-ISE detector and a recorder. A linear working curve between peak height and concentration of residual chlorine was obtained from 0.1 to 1 mg l(-1) for the developed FIA system. The relative standard deviation for repeated injections of a 0.2 mg l(-1) residual chlorine sample was 2%. The regression line and its correlation factor between the conventional o-tolidine colorimetric method and the present method were Y=0.75X+0.17 and 0.967, respectively, for this determination.

Leualacin, a novel calcium blocker from Hapsidospora irregularis. I. Taxonomy, fermentation, isolation, physico-chemical and biological properties.

A new calcium blocker, designated leualacin, has been isolated from Hapsidospora irregularis. The compound inhibits the binding of 3H-nitrendipine, a well known synthetic calcium blocker, to cardiac Ca channel in a competitive manner, although its structure is completely different from dihydropyridines.

Aquastatin A, an inhibitor of mammalian adenosine triphosphatases from Fusarium aquaeductuum. Taxonomy, fermentation, isolation, structure determination and biological properties.

A new inhibitor of mammalian adenosine triphosphatases, designated aquastatin A, has been isolated from a fungus identified as Fusarium aquaeductuum. The structure of this compound has been determined by MS and NMR analyses. It inhibits Na+/K(+)-ATPase with an IC50 value of 7.1 microM, and H+/K(+)-ATPase with an apparent IC50 value of 6.2 microM.

Folipastatin, a new depsidone compound from Aspergillus unguis as an inhibitor of phospholipase A2. Taxonomy, fermentation, isolation, structure determination and biological properties.

A new inhibitor of phospholipase A2 was isolated from the fermentation broth of Aspergillus unguis. The structure, with a depsidone carbon skeleton, was assigned by spectroscopic experiments.

Tc-99m-MIBI scintigraphy for detecting parathyroid adenoma and hyperplasia.

We performed scintigraphy with technetium-99m-methoxyisobutylisonitrile (MIBI) in 10 patients with parathyroid adenoma (7 lesions) or hyperplasia (9 lesions). Correlation between an amount of accumulation of MIBI and histological types of the lesions were evaluated with special reference to an amount of oxyphilic cell in the lesions. Selected lesions were also evaluated for mitochondrial density by electromicroscopy and showed increased mitochondrial density in the oxyphilic cells. All lesions equal to or above 220 mg showed positive scintigraphic results despite differences in cell types. Undetected lesions were all equal to or below 100 mg. The scintigraphic results for 2 lesions with abundant oxyphilic cells were both positive although those for 11 lesions with abundant chief cells only 6 were positive, probably because these lesions were smaller in the hyperplasia group. In conclusion, MIBI uptake in parathyroid lesions was not dependent on the cell type but either on the size or functional state of the lesions.

Clinical and immunohistochemical characteristics of benign giant cell tumour of bone with pulmonary metastases: case series.

PURPOSE: Giant cell tumour of bone with pulmonary metastases is rare. However, some patients die of pulmonary metastases, and histological examination cannot distinguish between benign tumour and malignant metastases. In this study, we present clinical and immunohistochemical findings associated with giant cell tumour of bone with pulmonary metastases. METHODS: Five patients with benign giant cell tumour of bone with pulmonary metastases (one man and 4 women) were studied. Patients' ages ranged between 20 and 23 years (mean age, 21.8 years). Tumours were in the distal femur in 2 cases, and in the proximal tibia, distal tibia, and lumbar spine in one case each. The tissue specimens from primary tumours, recurrent tumours, and pulmonary metastases were studied using immunohistochemical techniques. RESULTS: Three of the 5 primary tumours were of the spontaneous regression or growth cessation type, or the continuously slow-growing type, showing 4.2% to 6.2% of positive cells for Ki-67 after immunohistochemical staining. However, 2 patients with the rapid-growing type of disease died of pulmonary metastases; their primary, recurrent, and metastatic tumour specimens contained 9.0% to 11.5% of positive cells for Ki-67. CONCLUSION: Three of the 5 primary tumours had a benign clinical pattern and immunohistochemistry. Two of the 5 patients died of pulmonary metastases, which had an aggressive clinical pattern and a high prevalence of positive cells in Ki-67. Examination of Ki-67 should be carried out for aggressive type of giant cell tumour.

[Study of the microwave fixation using the immunohistochemical method].

We evaluated microwave fixation of formalin immersed and microwave irradiated kidney and liver tissues by keratin immunostain using the paraffin embedded section. In the microwave irradiated tissues, the formalin fixed areas and the alcohol fixed areas were clearly detected because; 1) Alcohol fixed tissues are easily digested by pepsinization but formalin fixed tissues are not, and 2) Formalin fixed tissues revealed intense keratin staining after pepsinization, whereas intense keratin staining were noted in alcohol fixed tissues without preliminary pepsinization. The microwave irradiation fixed completely the tissues in about 20 sec. A thin, formalin-fixed layer was observed in the periphery of the tissues. A thin, alcohol-fixed layer was observed beneath the peripheral formalin fixed layer in some specimens. This area was thought to have been fixed during the dehydration of the paraffin blocks. However with microwave fixation, most of the central areas differed from those of the formalin or alcohol fixed areas. They showed sharp contrast on hematoxylin-eosin staining, conspicuous cellular membrane and only slight cellular shrinkage and swelling. Unlike the formalin and alcohol fixed tissues, the intensity of keratin staining was independent of preliminary pepsinization and keratin staining was observed. Therefore, the central areas were thought to be fixed by the microwave irradiation. This fixation method seems to be useful for immunostaining.

High quality single atomic layer deposition of hexagonal boron nitride on single crystalline Rh(111) four-inch wafers.

The setup of an apparatus for chemical vapor deposition (CVD) of hexagonal boron nitride (h-BN) and its characterization on four-inch wafers in ultra high vacuum (UHV) environment is reported. It provides well-controlled preparation conditions, such as oxygen and argon plasma assisted cleaning and high temperature annealing. In situ characterization of a wafer is accomplished with target current spectroscopy. A piezo motor driven x-y stage allows measurements with a step size of 1 nm on the complete wafer. To benchmark the system performance, we investigated the growth of single layer h-BN on epitaxial Rh(111) thin films. A thorough analysis of the wafer was performed after cutting in atmosphere by low energy electron diffraction, scanning tunneling microscopy, and ultraviolet and X-ray photoelectron spectroscopies. The apparatus is located in a clean room environment and delivers high quality single layers of h-BN and thus grants access to large area UHV processed surfaces, which had been hitherto restricted to expensive, small area single crystal substrates. The facility is versatile enough for customization to other UHV-CVD processes, e.g., graphene on four-inch wafers.

Note: An ion source for alkali metal implantation beneath graphene and hexagonal boron nitride monolayers on transition metals.

The construction of an alkali-metal ion source is presented. It allows the acceleration of rubidium ions to an energy that enables the penetration through monolayers of graphene and hexagonal boron nitride. Rb atoms are sublimated from an alkali-metal dispenser. The ionization is obtained by surface ionization and desorption from a hot high work function surface. The ion current is easily controlled by the temperature of ionizer. Scanning Tunneling Microscopy measurements confirm ion implantation.

Immunohistochemical study of cytokeratin distribution in the collecting duct of the human kidney.

In order to assess the expression of different cytokeratins in the collecting duct cells (CDCs) of the human kidney, three consecutive sections were stained with periodic acid-Schiff, CAM 5.2 and AE-1 (CAM 5.2 recognizes cytokeratins #19,18,8 and AE-1 #19,16,15,14,10 of Moll's catalog.), respectively. By comparing these sections, it was found that most CDCs in the inner medulla were both CAM 5.2- and AE-1-positive, whereas in the outer medulla and cortex, 77% of the CDCs were both CAM 5.2- and AE-1-positive, 15% CAM 5.2-positive and AE-1-negative, 8% both CAM 5.2- and AE-1-negative, and 0.4% CAM 5.2-negative and AE-1-positive. Recent studies have shown that most CDCs express low-molecular-weight cytokeratins #7,8,18 and 19 (17, 18, 19, 20). Of these cytokeratins, CAM 5.2 recognizes cytokeratins #8,18,19 and AE-1 recognizes cytokeratin #19. Therefore, most CDCs belong to one of the following three major types; 1. Those positive for cytokeratins #8,18 and 19 (CAM 5.2- and AE-1-positive), 2. Those positive for cytokeratins #8 and 18 and negative for #19 (CAM 5.2-positive and AE-1-negative) and 3. Those negative for cytokeratins #8,18 and 19 (CAM 5.2- and AE-1-negative). A few CAM 5.2-negative and AE-1-positive cells were thought to express high-molecular-weight cytokeratins. The significance of these various cytokeratin expressions is discussed.