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Background Undifferentiated carcinoma of the esophagus with rhabdoid features is a very rare histologic finding that is occasionally associated with the loss of SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1); however, until now, few survey reports of this type of tumor have been published. In this study, we describe a case of esophageal carcinoma with undifferentiated components and rhabdoid features that was exclusively positive for vimentin and SMARCB1 in a patient with prolonged survival. Case presentation A 67-year-old man complained of a stomachache and loss of appetite persisting for 1 month. He was then admitted to the hospital. Diagnostic imaging studies revealed a transdiaphragmatic circular ulcerative tumor of the esophagogastric region. Biopsy specimens showed undifferentiated round cell carcinoma. The patient underwent lower esophageal resection and total gastrectomy with lymph node dissection. Microscopic analysis revealed that most of the primary tumor consisted of large undifferentiated round cells and scattered rhabdoid cells. The tumor invaded the muscular layer in the esophagus and the subserosal layer in the stomach, and metastasis was noted in only one lymph node. Immunohistochemical analysis revealed that the round and rhabdoid cells found in the primary tumor were diffusely positive for SMARCB1 and vimentin. The tumor displayed focal positivity for the anti-pan-cytokeratin antibody AE1/AE3. In the positive lymph node, round undifferentiated carcinoma cells were admixed with squamous carcinoma cells that were positive for cytokeratin 5/6 and 34βE12. The MIB-1 index was 19.7% and 0.5% for the round cells from the primary tumor and epithelial cells from the metastatic lymph node lesion, respectively, and 70.1% for the round cells from the metastatic lymph node lesion. The patient has been alive for 10 years after surgery without tumor recurrence. Conclusions We reported a rare case of esophageal carcinoma with undifferentiated components, rhabdoid features, and a good prognosis.

In order to investigate the involvement of the IER3/ PP2A-B56γ/ERK-positive feedback loop, which leads to sustained phosphorylation/activation of ERK in carcinogenesis, we immunohistochemically examined the expression of IER3 and phosphorylated ERK in lung tumor tissues. IER3 was overexpressed in all cases of adenocarcinomas examined, but was not overexpressed in squamous cell carcinomas. Phosphorylated ERK (pERK) was also overexpressed in almost all adenocarcinomas. EGFR and RAS, whose gene product is located upstream of ERK, were sequenced. Activating mutation of EGFR, which is a possible cause of overexpression of IER3 and pERK, was found only in 5 adenocarcinomas (42%). No mutation of RAS was found. We further examined the sequences of all exons of B56γ gene (PPP2R5C) and IER3, but no mutation was found. Using a single nucleotide insertion in intron 1 of PPP2R5C, which was found in the process of sequencing, allelic deletion of PPP2R5C was examined. Eight cases were informative (67%), and the deletion was found in 4 of them (50%). Three cases having deletion of PPP2R5C did not have EGFR mutation. Finally, PPP2R5C deletion or EGFR mutation that could be responsible for IER3/pERK overexpression was found in at least 8 cases (67% or more). This is the first report of a high incidence of deletion of PPP2R5C in human carcinomas., Embargo Period 12 months

We report four cases of adenoma of the nipple, a rare benign epithelial benign tumor occurring under the nipple and areola. Clinically, erosion findings of the nipple require discrimination from Paget's disease. In addition, a mass found in the nipple warrants discrimination from ductal carcinoma. Two cases underwent aspiration biopsy cytology and the other underwent tumor imprint cytology, respectively, revealing a large number of epithelial cell populations in the necrotic background material. These large cell clusters had a papillary or sheet structure and exhibited decreased cell cohesiveness at the cluster part. In addition, small clusters and solitary epithelial cells were also present. Furthermore, a two-cell pattern comprising both duct epithelial and myoepithelial cells was observed in the cell clusters. Two other cases underwent nipple brush cytology, revealing a few small papillary clusters with isolated epithelial cells in the hemorrhagic background. The clusters appeared as benign duct proliferative lesions such as papilloma, papillomatosis, and adenosis. An accurate diagnosis warrants the recognition of regular nuclei with bland chromatin and myoepithelial cells and the identification of the lesion location.

Background To compare lung cancer detection rate by sputum cytology, we need some assurance that the estimates do not vary widely if different observers evaluate the same specimens. The aim of this study was to determine inter-rater agreement of sputum cytology diagnoses. Methods Slides of sputum cytology from 150 subjects were selected from a pool of slides held by six of the laboratories that had participated in a population-based lung cancer screening program over the last ten years in Japan. The cytotechnologists in these laboratories had considerable experience with sputum cytology. Each case was re-evaluated six times. Cases that were diagnosed as the same category by all six laboratories were selected as consensus cases to serve as standardized sputum cytology cases. Thirty-seven cytotechnologists with various levels of experience in sputum cytology then re-evaluated these consensus cases. Inter-rater agreement was calculated by kappa statistics including Fleiss' kappa. Results All pairs of interlaboratory agreement for the 150 cases showed statistically significant kappa values, most pairs showing substantial agreement. Fleiss' kappa value across the six laboratories was 0.5. Fourteen cases were identified as the consensus cases, and the agreement among observers with less experience of sputum cytology showed significantly lower than the agreement among those with considerable experience (Fleiss' kappa value 0.27 vs. 0.45, P

Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species.

The insulin-like growth factor 2 gene (IGF2) is an imprinting gene, which mediates cell growth and apoptosis. The loss of imprinting (LOI) of IGF2 has been associated with the development of cancer. In the present study, loss LOI of IGF2 in lung cancer was analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in combination with DNA sequencing of samples collected by laser capture microdissection. The status of each sample was assigned as imprinting when PCR-RFLP revealed only one band or sequence with a single peak; otherwise, the case was classified as LOI. LOI was identified in eight out of 13 adenocarcinoma cases (62%), but was not detected in any of the nine squamous cell carcinoma cases (0%). These results suggest that IGF2 LOI is involved in the molecular pathogenesis of lung adenocarcinoma, but not squamous cell carcinoma, and that LOI may be detected through increased IGF2 expression levels.

[Purpose] The present study examined satellite cell and hepatocyte growth factor (HGF) responses to exercise intensity in the rat soleus muscle. [Methods] HGF levels assessed during postnatal growth of the gastrocnemius muscle. Depression of HGF levels occurred up to postnatal week 4, so 4-week-old rats were used the exercise training experiment. Rats walked or ran at speeds of 16 or 24 m/min, at -16% grade, 30 min/trial. Soleus muscles were removed after 72 h. Animals were injected with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) 1 h before sampling. The right soleus muscle was used for immunofluorescence, and the left soleus muscle was used to measure HGF protein levels. [Result] HGF levels were unchanged, although numbers of BrdU-positive nuclei increased 2.4-fold in rats with exercise training. [Conclusion] The relationship between activation of satellite cells and HGF production after exercise training remains unclear. However, this study indicates the exercise intensity necessary to activate satellite cells. In the future, this result may facilitate the creation of exercise training intensity as an index of satellite cell activity for muscle strength training.

An immunohistochemical study was made on an autopsy case of papillary carcinoma of the thyroid with anaplastic transformation in the metastatic foci occurring in a 72-year-old woman. The anaplastic carcinoma cells were sarcomatous in appearance, and they were vimentin-positive and cytokeratin-negative. Whereas, papillary tumor cells which were intermingled in the anaplastic carcinoma contained both cytokeratin and vimentin. The close correlation between tumor cell anaplasia and the expression of the different intermediate filament proteins in thyroid carcinoma was briefly discussed.

[Purpose] The present study aimed to determine the effects of short muscle strength exercise on hepatocyte growth factor expression and satellite cell activation. [Subjects] The study included 72 2–12-week-old male Sprague-Dawley rats. [Methods] The rat plantaris muscle was contracted with a 5-min electrical stimulation of the sciatic nerve, and then, the mRNA expressions of hepatocyte growth factor and myogenic regulatory factors in the plantaris muscle were determined, and the phosphorylation of the hepatocyte growth factor receptor (c-Met) was examined. [Results] The mRNA expressions of hepatocyte growth factor and myogenic regulatory factors increased after a short muscle contraction compared to that un-contraction. Immunofluorescence analysis showed the expression of hepatocyte growth factor protein and the possibility that downstream biological changes occurred in the hepatocyte growth factor-bound c-Met. [Conclusion] Our results demonstrated that activation of satellite cells induced hepatocyte growth factor expression during muscle contraction with a short 5-min electrical stimulation, which simulates short muscle strength exercise in physical therapy. The present study provides evidence for the use of short muscle strength exercise in physical therapy.

Little is known about the functions of two different β5 integrins: repeated-FNK (FNKFNK764–769) and single-FNK (FNK764–766) amino acid sequences in the cytoplasmic domain. We examined whether they occurred as germ line mutations or somatic mutations associated with neoplastic transformation, and whether there were functional alterations. Out of six cultured cell lines, only KATO-III cells had the single-FNK β5 sequence. The single-FNK β5 was found in 9 out of 79 patients with colon carcinoma, but no somatic mutations were detected in cancerous tissues. CHO cells were transformed with expression vectors containing single-FNK or repeated-FNK β5 cDNA, which were derived from KATO-III cells. CHO cells transfected with single-FNK and repeated-FNK showed similar adhesiveness to, and proliferative activity on, vitronectin substrates.

The present study examined the localization of hepatocyte growth factor in rat skeletal muscle, and investigated whether levels of hepatocyte growth factor differ between skeletal muscles. Levels of hepatocyte growth factor in soleus and tibialis anterior muscles were measured using enzyme-linked immunosorbent assay. Localization of hepatocyte growth factor and proliferating cell nuclear antigen in the soleus muscle was visualized using immunofluorescence analysis. Level of hepatocyte growth factor was 3.2 ± 1.4 ng/g tissue in the soleus muscle and 3.4 ± 0.4 ng/g tissue in the tibialis anterior muscle. No significant differences were identified between muscles with differential contractile characteristics. Existence of hepatocyte growth factor was observed in cytoplasm of small cells conterminous to muscle fibers. Cells in a similar position displayed reactivity to proliferating cell nuclear antigen, suggesting that they represented activated skeletal muscle satellite cells. Hepatocyte growth factor is produced in normal rat skeletal muscle by activated skeletal muscle satellite cells.

金沢大学大学院医学系研究科保健学専攻, Purpose E2F1 plays a critical role in cell proliferation, and its function is controlled by the retinoblastoma (RB) protein. We examined the expression of E2F1 and the aberration of RB gene and protein to elucidate what factors contribute to the overexpression of E2F1 in non-small cell lung carcinomas. Methods The expression level of E2F1 in tissues of non-small cell lung carcinomas was measured by means of quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. For RB, we examined loss of heterozygosity (LOH) by PCR-restriction fragment length polymorphism and a variable number of tandem repeats, and protein expression by immunohistochemistry. Results Fifteen cases of carcinoma (46%) showed high transcription levels of E2F1 gene. Immunohistochemically, almost all (14 of 15) cases overexpressing E2F1 mRNA were positive for E2F1 protein. LOH at the RB locus was found in 13 of 30 informative cases. In 13 cases with LOH, ten showed overexpression of E2F1 mRNA and protein. Immunohistochemical positivity for phosphorylated RB protein was also closely correlated with overexpression of E2F1. Conclusions Our results suggest that overexpression of E2F1, induced both by LOH at the RB locus and anomalous phosphorylation of the RB protein, is involved in the development of non-small cell lung carcinoma.

Background Tumors of the so-called intrathyroidal epithelial thymoma type are a rare group of thyroid neoplasm tumors. Of this type of tumor, spindle epithelial tumor with thymuslike differentiation (SETTLE) has been reported only 17 times in English literature. Methods An 18-year-old woman was initially seen with a 6-cm left thyroid mass that was resected with a left thyroidectomy. Histopathologic features of the excised left thyroid tumor together with an immunohistochemistry and electron microscopy led us to a diagnosis of SETTLE. Results Most of the specimen was composed of highly cellular spindle cells that formed intersecting and streaming fascicles. The spindle cells showed both vimentin immunoreactivity and cytokeratin immunoreactivity but no immunoreactivity for thyroglobulin or calcitonin. Electron microscopic examination of the spindle cells demonstrated prominent cytoplasmic tonofilaments, desmosomes, and basal lamina consistent with epithelial cell origin. DNA content analysis by flow cytometry revealed DNA diploidy. Conclusions SETTLE of thyroid gland is an extremely rare entity. A review of the literature reveals that SETTLE has distinctive morphologic features and an immunohistochemical profile. © 2002 Wiley Periodicals, Inc. Head Neck 24: 888–893, 2002

We report four cases of adenoma of the nipple, a rare benign epithelial benign tumor occurring under the nipple and areola. Clinically, erosion findings of the nipple require discrimination from Paget's disease. In addition, a mass found in the nipple warrants discrimination from ductal carcinoma. Two cases underwent aspiration biopsy cytology and the other underwent tumor imprint cytology, respectively, revealing a large number of epithelial cell populations in the necrotic background material. These large cell clusters had a papillary or sheet structure and exhibited decreased cell cohesiveness at the cluster part. In addition, small clusters and solitary epithelial cells were also present. Furthermore, a two-cell pattern comprising both duct epithelial and myoepithelial cells was observed in the cell clusters. Two other cases underwent nipple brush cytology, revealing a few small papillary clusters with isolated epithelial cells in the hemorrhagic background. The clusters appeared as benign duct proliferative lesions such as papilloma, papillomatosis, and adenosis. An accurate diagnosis warrants the recognition of regular nuclei with bland chromatin and myoepithelial cells and the identification of the lesion location.

To compare lung cancer detection rate by sputum cytology, we need some assurance that the estimates do not vary widely if different observers evaluate the same specimens. The aim of this study was to determine inter-rater agreement of sputum cytology diagnoses.Slides of sputum cytology from 150 subjects were selected from a pool of slides held by six of the laboratories that had participated in a population-based lung cancer screening program over the last ten years in Japan. The cytotechnologists in these laboratories had considerable experience with sputum cytology. Each case was re-evaluated six times. Cases that were diagnosed as the same category by all six laboratories were selected as consensus cases to serve as standardized sputum cytology cases. Thirty-seven cytotechnologists with various levels of experience in sputum cytology then re-evaluated these consensus cases. Inter-rater agreement was calculated by kappa statistics including Fleiss' kappa.All pairs of interlaboratory agreement for the 150 cases showed statistically significant kappa values, most pairs showing substantial agreement. Fleiss' kappa value across the six laboratories was 0.5. Fourteen cases were identified as the consensus cases, and the agreement among observers with less experience of sputum cytology showed significantly lower than the agreement among those with considerable experience (Fleiss' kappa value 0.27 vs. 0.45, P0.05). Moreover, cytotechnologists with less experience under-diagnosed the slides significantly more often than those with considerable experience.When the observers have considerable experience with sputum cytology, inter-observer agreement is good.

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.

OST cells enhance the induction of matrix metalloproteinase (MMP)-9 by tumour necrosis factor (TNF)-α and the corresponding metastasis to lungs in vivo (Kawashima et al., 1994). We focused on the adhesive and migratory properties of OST cells, and investigated the expression of integrins in OST cells stimulated by TNFαin vitro. OST cells potentiated not only adhesion to the extracellular matrix (ECM) but also the migration on ECM. On competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses, the amounts of α2 (4.9-fold), α5 (1.2-fold) and αv (4.9-fold) were upregulated by TNFα at the transcriptional level. Alpha-5 showed a slight increase by flow cytometry; however, α2 and αv integrins remained unchanged at the protein level. Immunofluorescence study disclosed integrins of α2β1 and α5β1 were much clustered at cell processes by TNFα stimulation, probably related to increased cell adhesion and migration. Therefore, the upregulation of α2β1 and α5β1 integrins seems to contribute to tumour invasion and metastatic potential.

耳下腺および口蓋小唾液腺より発生した腺房細胞癌2例を報告する. 症例1は25歳女性. 耳下腺再発腫瘍にて術中迅運組織診と同時に捺印細胞診が行われた. 症例2は67歳女性軟口蓋再発腫瘍にて生検材料採取時に捺印細胞診を施行した. 両症例で乳頭状細胞集塊が認められ, 特に症例1では, 毛細血管周囲の葡萄の房状細胞集塊が特徴的だった. 腫瘍細胞の核は偏在性で, 小空胞状または泡沫状の淡明な細胞質を有していた. 両症例とも核異型は弱かった. これらの所見はこの腫瘍の組織学的特徴と一致し, 細胞診で腺房細胞癌を推定できる所見と考えられた.

BACKGROUND: Chordoma that arises along the spinal axis metastasizes to the lung on occasion. Chordoma has been reported to have characteristic cytologic features in fine needle aspiration cytology in primary and metastatic foci. CASE: A 76-year-old female had a tumor on the buttock and tumors in the lung. Exfoliative cytology of the sputum showed cohesive, epithelioid clusters composed of pale-stained, broad cytoplasm with a lacelike pattern, minimal nuclear atypism with anisonucleosis and characteristic mucoid substances. CONCLUSION: Cytopathologists should be aware of the possibility of metastatic chordoma when epithelioid tumor cell clusters with benign-looking features embedded in a mucoid substance are detected in aspiration cytology or sputum.

Breast cancer treatment with trastuzumab, a monoclonal antibody that targets human epidermal growth factor receptor type 2 (HER2), has largely been successful in improving the prognosis of HER2-positive disease. However, a critical issue associated with trastuzumab treatment is its cardiotoxic adverse effects, including cardiac insufficiency. We measured levels of cardiac troponin I, a marker of myocardial damage, with a highly sensitive method (hs-cTnI) using a fully automated chemiluminescent immunoassay system (ADVIA Centaur® XP) in breast cancer patients and examined the relationship between administration of trastuzumab and epirubicin and concentrations of hs-cTnI. The coefficient of variation for within-run repeatability was 1.34–5.93 %, using plasma pools and controls of 3 concentrations, and that for between-run reproducibility was 3.99–8.79 %, indicating high precision of the assay. In a dilutional linearity test with highly concentrated specimens, hs-cTnI values could be measured up to 50 ng/mL with linearity. No influence from coexisting substances was observed. The concentration of hs-cTnI was at or above the reference range (0.04 ng/mL) in 9 of 214 total breast cancer cases (4.2 %). The hs-cTnI concentration was at or above the reference range in 4 of 49 cases (8.2 %) that were administered trastuzumab, and in 5 of 165 cases (3.0 %) that were not. Trastuzumab did not cause elevation of hs-cTnI when not administered in combination with epirubicin. These results suggest that epirubicin and trastuzumab cause cardiotoxicity through different mechanisms. Epirubicin can cause myocardial necrosis, while trastuzumab can cause cardiomyopathy without myocardial necrosis.

Immunolocalization of a variety of integrins using monoclonal antibodies against beta 3, beta 5, alpha 3 beta 1 and alpha 6, and polyclonal antibodies against vitronectin receptor (alpha v beta 3) and beta 1 were investigated on PLP-fixed paraffin sections of 19 cases of advanced gastric carcinomas. The beta 5 integrin, which pairs only with the alpha v subunit, was positive in seven cases (37%), and was associated closely with scirrhous invasion (P < 0.05). beta 5 was positive chiefly in the cytoplasm of carcinoma cells and infrequently in cell membranes. beta 3, which is another subunit pairing with alpha v, was positive in six cases (32%), and tended to be associated with scirrhous invasion (P < 0.01). beta 3 was also located chiefly in the cytoplasm. Five of the seven beta 5-positive cases showed coexpression of beta 3. Polyclonal antibodies to alpha v beta 3 also showed a significant difference among the amounts of stroma (P < 0.05). Anti-beta 1 antibodies showed clear positivity in many cases (89%). Of the beta 1 integrins, alpha 3 beta 1 was positive in a few cases (26%) without any preferential pattern, and laminin receptor subunit alpha 6 stained on cell membranes of neoplastic epithelia in many cases of carcinoma (89%) except for mucinous carcinoma These distinctive patterns of integrin positivity indicate a close association of beta 5 and beta 3 expression with scirrhous invasion in gastric carcinoma.

金沢大学大学院医学系研究科保健学専攻, We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3, poorly differentiated type; OSC-19, well-differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-αv antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-β1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-β5 and anti-αv antibodies, and only HSC-β3 cells were positive for antiβ-3 antibody. α5β1 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects αvβ3-and/or αvβ5-mediated carcinoma cell invasion.

Immunohistochemical study of major salivary gland tumours was performed on 60 pleomorphic adenomas, five basal cell adenomas and 10 adenoid cystic carcinomas to determine the diagnostic value of each antigen. Immunoreactivity examined were intermediate filaments (keratin, vimentin, desmin and glial fibrillary acidic protein [GFAP]) and related substances (actin, S-100 protein and secretory component). In pleomorphic adenomas, there was positive immunoreactivity for GFAP which was not observed in normal tissue or other neoplastic tissues. Immunoreactivity of GFAP was closely related to myxomatous and early chondromatous differentiation in pleomorphic adenoma. It is considered that GFAP immunoreactivity should be assessed in the occasional differential diagnostic dilemma of pleomorphic adenoma versus adenoid cystic carcinoma and basal cell adenoma, because of its ability to show potential and definite myxochondromatous differentiation.

Thickened ligamenta flava obtained from 14 patients with spinal canal stenosis were examined with special reference to type VI collagen. The characteristic histological finding in the thickened area was rupture or normal elastic fibre meshwork with resultant fibrosis which usually appeared hyaline. Using an immunohistological method, collagen types VI, I and III were found to be present in the hyaline matrix. Ultrastructural study revealed many microfilamentous structures of type VI collagen admixed in loosely packed, banded collagen fibres. With differential salt precipitation of pepsin-extracted collagen the existence of type VI collagen was confirmed by SDS-polyacrylamide gel electrophoresis analysis and Western blotting analysis using anti-type VI collagen antibody. Quantification of type VI collagen in pepsin-extracted crude collagen samples by an inhibition enzyme-linked immunosorbent assay showed an increasing amount of type VI collagen in the thickened ligamenta flava compared to the normal ligaments. Thus, increase of type VI collagen is the main contribution to the thickening of the ligamentum flavum. This may represent an adaptational and reparative process associated with disruption of elastic fibres.

Autopsy findings of primary malignant melanoma arising in an ovarian dermoid cyst in an 86-year-old woman are presented. The right ovary was replaced by a dermoid cyst, 14 x 9 x 9 cm in size, in which several nodular tumors with diameters less than 3.2 cm were localized. They comprised diffusely proliferating anaplastic cells with prominent nucleoli. Some of them contained melanin pigments in the cytoplasm. The tumor cells were positive for S-100 protein and ultrastructurally showed melanosomes. In addition, several benign pigmented lesions resembling dermal nevus, pigmented schwannoma, or cellular blue nevus were present in the dermoid cyst, one of which contained a malignant melanomatous component. Histologic transition between benign and malignant components and the presence of another small focus of atypical melanocytes in the benign lesion suggested that the malignant melanoma arose in close association with the previously existing benign pigmented lesions in the dermoid cyst.

A rare case of malignant melanocytic schwannoma in the nasopharynx is presented. Light microscopy showed proliferation of spindle-shaped tumor cells with mitoses. The essential histological hallmark was melanin deposition. Electron-microscopic examination confirmed the schwannian differentiation of the tumor cells. The patient has remained well without evidence of metastasis or local recurrence of the tumor 18 months after the excision of the neoplasm.

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.

To ascertain the implications of loss of imprinting (LOI) of the insulin-like growth factor II gene (IGF2) for carcinogenesis, the precise frequency of LOI in colorectal carcinoma was examined using a laser capture microdissection method, and compared to the matched normal colorectal mucosa. LOI was examined by PCR-restriction fragment length polymorphism in combination with direct sequencing. The status was assigned as imprinting when PCR-RFLP showed only one band or sequence with a single peak, otherwise cases were classified as LOI. LOI was found in 13 of 24 informative cases of carcinoma (54%), which was higher than the ratios reported previously. LOI was also found in the normal colorectal mucosae in 14 cases (58%). The LOIs in carcinomas and in the normal mucosae were closely correlated: 10 of 13 LOI-positive carcinomas showed LOI in the matched normal mucosae. These results suggest that LOI of IGF2 in colorectal carcinoma and LOI in the background mucosa play important roles in carcinogenesis.

A rare case of berry splenic artery aneurysm (SAA) rupture associated with segmental arterial mediolysis (SAM) and portal hypertension is reported. A 66-year-old woman, diagnosed as having liver cirrhosis and portal hypertension 6 years earlier, suddenly developed a lancinating pain in the upper abdomen and lost consciousness. She recovered consciousness while being transferred to hospital by ambulance. During the investigations, her level of consciousness suddenly deteriorated. Ultrasonography showed a massive intraperitoneal hemorrhage, and she died 5(1/2) h after admission. On gross examination at autopsy it was not possible to find the rupture point of the vessel because the pancreas was embedded in a massive hematoma. However, careful dissection of the pancreatic tail after fixation revealed a berry aneurysm measuring 0.8 cm in diameter in a branch adjacent to the bifurcation in the distal third of the main splenic artery. Microscopic examination detected a rupture of the aneurysm. The histology of the arterial wall proximal to the aneurysm showed typical SAM. In general, berry SAA caused by SAM is rare and unlikely to rupture. The SAA in the present case likely occurred and ruptured due to the combination of SAM and portal hypertension.

金沢大学大学院医学系研究科保健学専攻, Background/Aims: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. Methods: An antisense sequence corresponding to B cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. Results: Antisense-transfected cells showed less B protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-B also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. Conclusion: These results suggested that PP2A-B promotes cell migration through the MAPK cascade.

Little is known about the functions of two different beta5 integrins: repeated-FNK (FNKFNK764-769) and single-FNK (FNK764-766) amino acid sequences in the cytoplasmic domain. We examined whether they occurred as germ line mutations or somatic mutations associated with neoplastic transformation, and whether there were functional alterations. Out of six cultured cell lines, only KATO-III cells had the single-FNK beta5 sequence. The single-FNK beta5 was found in 9 out of 79 patients with colon carcinoma, but no somatic mutations were detected in cancerous tissues. CHO cells were transformed with expression vectors containing single-FNK or repeated-FNK beta5 cDNA, which were derived from KATO-III cells. CHO cells transfected with single-FNK and repeated-FNK showed similar adhesiveness to, and proliferative activity on, vitronectin substrates.

金沢大学大学院医学系研究科保健学専攻, Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and β1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes.

Objective Recent experimental evidence indicates that angiogenesis affects tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is considered to be an important regulator of tumor angiogenesis. The present study was designed to examine the role of VEGF on angiogenesis and lymph node metastasis in primary nasopharyngeal carcinomas (NPCs). Study design Formalin-fixed paraffin-embedded biopsy specimens were obtained from 29 primary NPCs that consisted of 22 differentiated nonkeratinizing carcinomas and seven undifferentiated carcinomas. Methods Microvessels were highlighted by staining endothelial cells with von Willebrand factor (VWF) using immunohistochemical techniques, and were counted (per x 400 field) in the most active area of angiogenesis on light microscopy. The expression of VEGF was also studied with immunohistochemistry. Positive ratio for VEGF was graded on a scale of 1 and 2. Scale 1 represents patients with less than the mean value of the positive ratio, and scale 2 represents patients with more than the corresponding value. Results There was a significant correlation between increased microvessel count and the progression of regional lymph node involvement. The microvessel counts and the progression of N factor were significantly higher in scale 2 patients than in scale 1 patients. Conclusion These results suggest that VEGF plays an important role in lymph node metastasis through induction of angiogenesis in NPCs.

Extracellular signal-regulated kinase (ERK) signalling plays a central role in various biological processes, including cell migration, but it remains unknown what factors directly regulate the strength and duration of ERK activation. We found that, among the B56 family of protein phosphatase 2A (PP2A) regulatory subunits, B56γ1 suppressed EGF-induced cell migration on collagen, bound to phosphorylated-ERK, and dephosphorylated ERK, whereas B56α1 and B56β1 did not. B56γ1 was immunolocalized in nuclei. The IER3 protein was immediately highly expressed in response to costimulation of cells with EGF and collagen. Knockdown of IER3 inhibited cell migration and enhanced dephosphorylation of ERK. Analysis of the time course of PP2A-B56γ1 activity following the costimulation showed an immediate loss of phosphatase activity, followed by a rapid increase in activity, and this activity then remained at a stable level that was lower than the original level. Our results indicate that the strength and duration of the nuclear ERK activation signal that is initially induced by ERK kinase (MEK) are determined at least in part by modulation of the phosphatase activity of PP2A-B56γ1 through two independent pathways. © 2013 Kawahara et al.