Telomere length determined by the fluorescence in situ hybridisation distinguishes malignant and benign cells in cytological specimens.

BACKGROUND: Telomeres are tandem repeats of TTAGGG at the end of eukaryotic chromosomes that play a key role in preventing chromosomal instability. The aim of the present study is to determine telomere length using fluorescence in situ hybridisation (FISH) on cytological specimens. METHODS: Aspiration samples (n = 41) were smeared on glass slides and used for FISH. RESULTS: Telomere signal intensity was significantly lower in positive cases (cases with malignancy, n = 25) as compared to negative cases (cases without malignancy, n = 16), and the same was observed for centromere intensity. The difference in DAPI intensity was not statistically significant. The ratio of telomere to centromere intensity did not show a significant difference between positive and negative cases. There was no statistical difference in the signal intensities of aspiration samples from ascites or pleural effusion (n = 23) and endoscopic ultrasound-guided FNA samples from the pancreas (n = 18). CONCLUSIONS: The present study revealed that telomere length can be used as an indicator to distinguish malignant and benign cells in cytological specimens. This novel approach may help improve diagnosis for cancer patients.

Characterization of Spontaneous Mammary Tumors in Domestic Djungarian Hamsters (Phodopus sungorus).

Mammary tumors that spontaneously occurred in domestic Djungarian hamsters (Phodopus sungorus) were histologically examined. Forty-five mammary tumors included 14 adenomas, 18 adenocarcinomas, 1 lipid-rich carcinoma, 2 adenoacanthomas, 2 malignant adenomyoepitheliomas, 1 benign mixed tumor, and 7 "balloon cell" carcinosarcomas. The latter 4 types were newly recognized neoplasms in Djungarian hamsters. The relatively high incidence of spontaneous mammary carcinosarcomas in domestic Djungarian hamsters is intriguing. Carcinosarcomas exhibited anomalous histological features made up of a mixture of glandular cells, polygonal cells (including "balloon cells"), and sarcomatous spindle cells in varying proportions. Transitional features from glandular cells to polygonal cells and subsequently to sarcomatous spindle cells were observed. Using immunohistochemistry, we observed that glandular cells exhibited an epithelial phenotype (cytokeratin(+)/vimentin(-)), spindle cells exhibited a mesenchymal phenotype (cytokeratin(-)/vimentin(+)), and polygonal cells exhibited an intermediate phenotype (cytokeratin(+)/vimentin(+)). Reduction or loss of ß-catenin expression and gain of S100A4 expression were observed in polygonal and spindle cells. The polygonal cell population included a varying number of characteristic cells that were expanded by large intracytoplasmic vacuoles. Electron microscopy revealed that these "balloon cells" had large cytoplasmic lumens lined by microvilli. These observations suggest that epithelial-mesenchymal transition may account for the pathogenesis of mammary carcinosarcomas in Djungarian hamsters.

Cellular sources of tenascin-C in canine mammary carcinomas.

Tenascin-C (Tn-C) is an extracellular matrix glycoprotein implicated in the progression of several human cancers. In canine mammary carcinomas, accumulation of Tn-C has been recognized in 3 different areas: regions of proliferating myoepithelial cells in complex carcinoma, basement membrane zone in low-grade simple carcinoma, and reactive stroma in high-grade simple carcinoma. To identify the Tn-C synthesizing cells in these areas, we utilized double-labeling immunohistochemistry, branched DNA in situ hybridization, and in situ hybridization-immunohistochemistry double-labeling techniques. In complex carcinomas, Tn-C was generated by proliferating myoepithelial cells. Tn-C in low-grade simple carcinomas was also derived from myoepithelial cells existing as a basal monolayer. However, stromal Tn-C in high-grade carcinomas was mainly synthesized by fibroblasts/myofibroblasts, similar to human breast cancer. Thus, the origin of Tn-C in canine mammary carcinomas differs between low- and high-grade malignancies. The role of myoepithelial cell-generated Tn-C is not yet understood.

Novel method of screening the oxidation and reduction abilities of photocatalytic materials.

Two analytical methods for the evaluation of photocatalytic oxidation and reduction abilities were developed using a photocatalytic microreactor; one is product analysis and the other is reaction rate analysis. Two simple organic conversion reactions were selected for the oxidation and reduction. Since the reactions were one-to-one conversions from the reactant species to the product species, the product analysis was simply performed using gas chromatography, and the reactions were monitored in situ in the photocatalytic microreactor using the UV absorption spectra. The partial oxidation and reduction abilities for each functional group can be judged from the yield and selectivity, and the corresponding reaction rate, while the total oxidation ability can be judged from the conversion. We demonstrated the application of these methods for several kinds of visible light photocatalysts.

Improvement in skin color achieved by smoking cessation.

It has been well known that habitual smoking accelerates premature skin ageing recognized as 'smoker's face'. However, the effect of smoking cessation on the appearance of skin has not been elucidated. The aim of this study was to evaluate objectively the effect of smoking cessation on the skin's appearance. The stratum corneum carbonyl protein level and skin colour of the cheek and the hand were measured. The change before and during the smoking cessation treatment (0, 2, 4, 8 and 12 weeks), and the success or failure in smoking cessation, was compared and examined. Eighty-four cases who had smoking cessation treatment were examined. The level of the stratum corneum carbonyl protein did not show any difference comparing before and after treatment for the smoking cessation success group and the failure group. The lightness of skin colour showed an upward tendency 4-12 weeks after starting the treatment in the success group and increased significantly compared with the failure group. The redness showed a significant decrease in comparison with before the treatment, and it also showed a significant decrease compared with the failure group. The yellowness did not show any clear tendency. Also, the haemoglobin showed a decreased tendency. Furthermore, multivariate statistical analysis showed a possibility that the lightness and haemoglobin could be changed by smoking cessation treatment. In conclusion, our study showed that an upward tendency of skin lightness was seen to correspond with a haemoglobin decrease accompanied by smoking cessation. If we can easily measure skin improvement as an effect of smoking cessation, it is thought to be a useful aid for smoking cessation support.

Characteristics of paediatric patients with 2009 pandemic influenza A(H1N1) and severe, oxygen-requiring pneumonia in the Tokyo region, 1 September-31 October 2009.

Few reports describe the features of 2009 pandemic influenza A(H1N1) pneumonia in children. We retrospectively reviewed 21 consecutive children admitted to hospital from September to October 2009 in the Tokyo region. The diagnosis of 2009 pandemic influenza A(H1N1) virus infection was based on positive results of real-time RT-PCR or rapid influenza antigen test. All patients were hospitalised for pneumonia with respiratory failure and severe hypoxia. The median interval from onset of influenza symptoms to admission was 14 hours (range: 5-72 hours) and the median interval from the onset of fever (≥38 degrees C) to hospitalisation was 8.5 hours (range: 0-36 hours). All patients required oxygen inhalation. Four patients required mechanical ventilation. Chest radiography revealed patchy infiltration or atelectasis in all patients. Antiviral agents and antibiotics were administrated to all patients. Antiviral agents were administered to 20 patients within 48 hours of influenza symptom onset. No deaths occurred during the study period. Paediatric patients with this pneumonia showed rapid aggravation of dyspnoea and hypoxia after the onset of influenza symptoms.

Chronic Basophilic Leukaemia in a Dog.

A 13-year-old neutered female mixed-breed dog with a clinical history of emaciation, inappetence and vomiting for 2 months was presented. Blood tests showed marked leucocytosis with increased neutrophil and basophil count, mild thrombocytosis and anaemia. Seven days after the initial visit, the dog died and was submitted for necropsy examination. Grossly, the bone marrow was red in colour and hepatomegaly and splenomegaly with discolouration were observed. A bone marrow smear showed an increased proportion of basophilic lineage cells. Histologically, the bone marrow showed high cellular density and numerous basophilic lineage cells with a round or segmented nucleus. The cytoplasm contained basophilic granules exhibiting metachromasia on toluidine blue staining. Immunohistochemically, the neoplastic basophils were diffusely positive for vimentin and myeloperoxidase, but negative for CD3, BLA36, CD163, CD204 and c-kit. The immunohistochemical features of neoplastic basophils that had invaded the liver and spleen were similar to those of the basophils in the bone marrow. Based on the clinicopathological and histopathological findings, chronic basophilic leukaemia was diagnosed. The present case study provides insights into the pathological features of chronic basophilic leukaemia in dogs.

Diffuse Pulmonary Meningotheliomatosis with Sarcomatous Transformation in a Shiba Dog.

A 2-year-old neutered female Shiba dog exhibited laboured breathing for 1 month. Computed tomography of the thoracic cavity revealed multiple nodules (2-5 mm diameter) in the lungs. Grossly, the lungs were firm and normal in shape. The nodules were grey-white in colour. Microscopically, the nodules were non-encapsulated and exhibited an irregular shape. They were composed of polygonal or spindle cells with indistinct cell borders arranged in sheets. The cells had large, round, hyperchromatic nuclei and abundant pale eosinophilic cytoplasm with no atypia. Intrapulmonary arterial emboli and infiltration into the bronchioles were observed. Immunohistochemically, the cells were positive for vimentin and negative for cytokeratin, glial fibrillary acidic protein and α-smooth muscle actin. Ultrastructurally, the cells displayed cytoplasmic processes, desmosomes and intermediate filaments. These findings led to a diagnosis of diffuse pulmonary meningotheliomatosis with sarcomatous transformation. To the best of our knowledge, this is the first report of diffuse pulmonary meningotheliomatosis in a dog.

The cell-surface heparan sulfate proteoglycan glypican-1 regulates growth factor action in pancreatic carcinoma cells and is overexpressed in human pancreatic cancer.

Heparan sulfate proteoglycans (HSPGs) play diverse roles in cell recognition, growth, and adhesion. In vitro studies suggest that cell-surface HSPGs act as coreceptors for heparin-binding mitogenic growth factors. Here we show that the glycosylphosphatidylinositol- (GPI-) anchored HSPG glypican-1 is strongly expressed in human pancreatic cancer, both by the cancer cells and the adjacent fibroblasts, whereas expression of glypican-1 is low in the normal pancreas and in chronic pancreatitis. Treatment of two pancreatic cancer cell lines, which express glypican-1, with the enzyme phosphoinositide-specific phospholipase-C (PI-PLC) abrogated their mitogenic responses to two heparin-binding growth factors that are commonly overexpressed in pancreatic cancer: fibroblast growth factor 2 (FGF2) and heparin-binding EGF-like growth factor (HB-EGF). PI-PLC did not alter the response to the non-heparin-binding growth factors EGF and IGF-1. Stable expression of a form of glypican-1 engineered to possess a transmembrane domain instead of a GPI anchor conferred resistance to the inhibitory effects of PI-PLC on growth factor responsiveness. Furthermore, transfection of a glypican-1 antisense construct attenuated glypican-1 protein levels and the mitogenic response to FGF2 and HB-EGF. We propose that glypican-1 plays an essential role in the responses of pancreatic cancer cells to certain mitogenic stimuli, that it is relatively unique in relation to other HSPGs, and that its expression by pancreatic cancer cells may be of importance in the pathobiology of this disorder.

Pancreatic Colloid Carcinoma in an Elderly Cat.

A 21-year-old neutered female domestic shorthaired cat was presented with a history of inappetence, vomiting and haematuria. The cat was humanely destroyed at the owner's request and a necropsy examination was performed. A 0.8 × 0.5 × 0.5 cm mass was located in the left lobe of the pancreas. The mass was gelatinous in nature and the external and cut surfaces were pale yellow in colour. Microscopically, the mass was non-capsulated and comprised an accumulation of extracellular stromal mucin containing suspended neoplastic columnar epithelial cells forming tubular structures. Immunohistochemically, these cells diffusely expressed cytokeratin (CK) AE1/AE3, CK7 and carcinoembryonic antigen and were partially positive for CK19 and trypsin, but negative for vimentin. The tumour was diagnosed as a colloid carcinoma. The clinical presentation in this case was caused by chronic renal failure complicated by secondary renal hyperparathyroidism and associated metastatic calcinosis. To the best of our knowledge, this is the first report of colloid carcinoma arising from the pancreas in a cat.

Cutaneous Angiolymphoid Hyperplasia in a Dog.

A 5-year-old male miniature dachshund was presented with a dermal nodule on the left forelimb that increased to 5 mm in diameter over a 2-month period. Grossly, the nodule was firm, and both the external and cut surfaces were homogeneously pale pink in colour. Microscopically, the nodule was comprised of mainly plump endothelial cells and inflammatory cells; among the latter, lymphocytes were predominant, with few scattered plasma cells, mast cells and macrophages. Lymphoid follicles with germinal centres were often observed. Mitotic figures were not observed amongst the endothelial cells. Immunohistochemically, the endothelial cells were positive for vimentin, factor VIII-related antigen and CD31, and the surrounding cells were positive for smooth muscle actin. Lymphocytes expressed CD3 or BLA36. These findings led to a diagnosis of cutaneous angiolymphoid hyperplasia. To the best of our knowledge, this is the first report of a cutaneous proliferative disorder comprising an admixture of proliferating vascular endothelial cells and lymphocytic infiltration with follicle formation in a dog.

Expression of nestin in rat and human glomerular podocytes.

Nestin is a neuroepithelial precursor cell marker expressed in a variety of human cell types during development. However, no information exists on the expression of nestin in mature glomeruli as well as during the glomerular development. Here, we examined nestin expression in rat and human glomerular tissues in quiescent states using RT-PCR and immunohistochemical methods. Nestin mRNA was detected in the rat glomeruli in parallel with its expression in developing rat brains. In the normal mature rat glomeruli, WT-1 positive cells expressed nestin. Co-expression of nestin and vimentin was observed in mature rat podocytes. Immunoelectron microscopy revealed nestin localization in the cell bodies and primary processes of podocytes. A similar expression pattern was observed for vimentin. In matured glomeruli, nestin was not expressed by mesangial and endothelial cells. In the newborn rat, early developing glomeruli (metanephric cap, metanephric vesicle, comma-shaped vesicle and S-shaped body phases) expressed nestin. In the capillary loop stage, Bowman's capsules also expressed nestin. Immunoelectron microscopy demonstrated that developing podocytes and endothelial cells in S-shaped phase glomeruli expressed nestin. Additionally, in immature glomeruli, the mesangial cells in capillary stage of glomerulus also expressed nexin. As in the rat, WT-1 positive cells in human glomeruli also expressed nestin and immunoelectron microscopy confirmed nestin expression in human glomerular podocytes. These results reveal that in normal condition nestin is expressed in several glomerular cell types at early stage of development and becomes confined to podocytes in mature glomeruli, thus implicating nestin in podocyte functions.

The helix-loop-helix protein Id2 is overexpressed in human pancreatic cancer.

Id2 belongs to the Id family of helix-loop-helix (HLH) proteins, which upon heterodimerization with basic HLH proteins prevent basic HLH proteins from DNA binding. Proteins of the Id family act as negative regulatory transcriptional factors, and their expression correlates with cell proliferation and arrested differentiation in many cell lineages. In this study, we characterized the expression of Id2 in normal and cancerous pancreatic tissues. Pancreatic cancers markedly overexpressed Id2 mRNA in comparison to the normal pancreas. Furthermore, there was abundant Id2 immunoreactivity in the cancer cells within the pancreatic tumor mass. In PANC-1 human pancreatic cancer cells, steady-state Id2 mRNA levels increased upon serum addition and decreased after induction of differentiation with either sodium butyrate or 12-O-tetradecanoylphorbol-13-acetate. Inhibition of Id2 expression with Id2 antisense oligonucleotides inhibited the growth of these cells, whereas random and sense oligonucleotides were without effect. These findings suggest that Id2 may have a role in human pancreatic cancer.

Fibroblast growth factor-5 stimulates mitogenic signaling and is overexpressed in human pancreatic cancer: evidence for autocrine and paracrine actions.

Fibroblast growth factor (FGF)-1 and -2 are overexpressed in human pancreatic cancer. In this study the role of FGF-5 in human pancreatic cancer was investigated, as FGF-5 has a classical signal sequence for secretion not found in FGF-1 or -2. Northern blot analysis with a 306 bp FGF-5 cDNA revealed the presence of 4.0 kb and 1.6 kb FGF-5 mRNA transcripts in both normal and cancerous pancreatic tissues. Densitometric analysis indicated that 4.0 kb and 1.6 kb FGF-5 mRNA transcripts levels were increased 2.4- and 2.7-fold in the cancers by comparison with normal tissues, respectively (P < 0.002, P < 0.0001). Immunohistochemistry and in situ hybridization demonstrated that FGF-5 localized in the cancer cells, stromal fibroblast and inflitrating macrophages. FGF-5 mRNA was also detected in COLO-357 human pancreatic cancer cells. Furthermore, secreted FGF-5 protein was present in conditioned medium of COLO-357 cells. Exogeneous FGF-5 (0.37 nM) increased the growth of COLO-357 cells by 48% (P < 0.0001) and increased mitogen-activated protein kinase activity. COLO-357 cells expressed the IIIc isoform of the type I FGF receptor, the preferred FGF receptor for FGF-5. These observations suggest that FGF-5 may participate in autocrine and paracrine pathways promoting pancreatic cancer cell growth in vivo.

The TGF-beta signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer.

Transforming growth factor-beta (TGF-beta) signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaRII) with the type I TGF-beta receptor (TbetaRI). Activated TbetaRI then mediates TGF-beta signals by inducing the phosphorylation of Smad2 and/or Smad3, which separately hetetorodimerize with Smad4 and translocate to the nucleus. Phosphorylation of Smad2/Smad3 by activated TbetaRI is inhibited by two newly discovered members of the Smad family, Smad6 and Smad7. We now report that Smad7 mRNA levels are increased in human pancreatic cancer by comparison with the normal pancreas, and that by in situ hybridization, Smad7 is over-expressed in the cancer cells within the tumor mass. Stable transfection of COLO-357 human pancreatic cancer cells with a full-length Smad7 construct leads to complete loss of the growth inhibitory response to TGF-beta1, without altering TGF-beta1-mediated induction of PAI-I. Furthermore, Smad7 transfected COLO-357 cells display enhanced anchorage-independent growth and accelerated growth in nude mice. These findings point to a previously unrecognized mechanism for selective suppression of TGF-beta-mediated growth inhibition in cancer cells that allows for continued activation of the PAI-I promoter by TGF-beta1, which may act to enhance the tumorigenicity of certain cancer cells.

Enhanced expression of vascular endothelial growth factor in human pancreatic cancer correlates with local disease progression.

Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been implicated in cancer growth. In the present study, we characterized VEGF expression in cultured human pancreatic cancer cell lines and determined whether the presence VEGF in human pancreatic cancers is associated with enhanced neovascularization or altered clinicopathological characteristics. VEGF mRNA transcripts were present in all six tested cell lines (ASPC-1, CAPAN-1, MIA-PaCa-2, PANC-1, COLO-357, and T3M4). Immunoblotting with a highly specific anti-VEGF antibody revealed the presence of VEGF protein in all of the cell lines. Northern blot analysis of total RNA revealed a 5.2-fold increase in VEGF mRNA transcript in the cancer samples in comparison with the normal pancreas. Immunohistochemical and in situ hybridization analysis confirmed the expression of VEGF in the cancer cells within the tumor mass. Immunohistochemical analysis of 75 pancreatic cancer tissues revealed the presence of strong VEGF immunoreactivity in the cancer cells in 64% of the cancer tissues. The presence of VEGF in these cells was associated with increased blood vessel number, larger tumor size, and enhanced local spread but not with decreased patient survival. These findings indicate that VEGF is commonly overexpressed in human pancreatic cancers and that this factor may contribute to the angiogenic process and tumor growth in this disorder.

Insulin-like growth factor I improves cardiovascular function and suppresses apoptosis of cardiomyocytes in dilated cardiomyopathy.

To investigate how insulin-like growth factor I (IGF-I) modulates cardiovascular function and myocardial apoptosis in heart failure, the therapeutic effects of IGF-I were determined in a canine model of dilated cardiomyopathy. The animals were paced at 220 beats/min, and the left ventricular (LV) chamber became dilated after 2 weeks. A subset of paced dogs was treated with s.c. injections of IGF-I from week 3 to week 4. After 4 weeks of pacing, untreated paced dogs developed significant ventricular dysfunction. IGF-I-treated paced dogs showed better cardiac output, stroke volume, LV end-systolic pressure, and LV end-diastolic pressure. Moreover, pulmonary wedge pressure and systemic vascular resistance were increased in the untreated group and decreased in the IGF-I-treated group. IGF-I treatment was associated with less thinning of the ventricular wall. Compared with the controls, untreated paced dogs showed increased apoptosis of cardiac muscle cells, which was partially suppressed by IGF-I treatment. The myocardial apoptotic index was negatively related to the thickness of the ventricular wall and to cardiac output, suggesting that ventricular remodeling/dysfunction involves the occurrence of myocardial apoptosis. Due to the close resemblance between this experimental model of dilated cardiomyopathy and human heart failure, the results of this study provide evidence that IGF-I may be a potential therapeutic agent for the failing human heart.

Expression of hsp90 and cyclin D1 in human breast cancer.

The integral roles of heat shock proteins (hsps) in the cell cycle and in multistep processes leading to tumorigenesis have been implied. We examined the expression of hsp90alpha, hsp90beta and cyclin D1 in human breast cancer. Levels of mRNAs coding for hsp90alpha and cyclin D1 were significantly higher in cancer tissues than in non-cancer tissues. Moreover, there was a close relationship between the extent of the two mRNA levels, suggesting that increased expression of hsp90alpha, an isoform of the hsp90 family, is associated with the proliferation of human breast cancer. Hsp90beta was expressed in cancer cells, but not associated with cell proliferation.

Insulin-like growth factor II activates mitogenic signaling in pancreatic cancer cells via IRS-1.

Insulin-like growth factor-II (IGF-II) action and expression were examined in 3 human pancreatic cancer cell lines. IGF-II expression was also studied in 17 normal and 12 malignant pancreatic tissues. IGF-II enhanced the growth of all 3 cell lines. In COLO-357 and PANC-1 cells, one-half maximal stimulation occurred at 0.3 and 0.4 nM IGF-II, respectively. In ASPC-1 cells, one-half maximal stimulation occurred at 0.9 nM IGF-II. A monoclonal antibody (alpha IR3) that blocks ligand binding to the insulin-like growth factor I (IGF-I) receptor (IGF-IR) inhibited IGF-II-mediated growth stimulation, and IGF-II enhanced insulin-receptor substrate 1 (IRS-1) phosphorylation. IGF-II mRNA transcripts were present in COLO-357 cells, in 4 of 17 normal human pancreatic tissues, and in 8 of the 12 cancer samples. By immunohistochemistry, IGF-II was present in the islets of both normal and malignant pancreatic tissues, and occasionally in the cancer cells within the tumor mass. These findings indicate that IGF-II acts via IGF-IR to enhance mitogenic signaling in pancreatic cancer cells and suggest that islet-derived IGF-II may contribute to pancreatic cancer cell growth in vivo.

Purification and characterization of LasR as a DNA-binding protein.

In Pseudomonas aeruginosa, the activator protein LasR and a cognate autoinducer (AI) are required for expression of the elastase gene (lasB). In the present study, we investigated the binding properties of the P. aeruginosa lasR gene product. The LasR protein was overexpressed and purified as a glutathione S-transferase (GST) fusion protein. Using gel retardation and UV cross-linking analysis, we demonstrated that the GST-LasR could bind to a separate site in the lasB upstream operator regions 1 and 3 in the presence of the autoinducer. Regions 1 and 3 are located at 105 and 42 base pairs upstream, respectively, from the lasB transcriptional start site. Our present results clearly demonstrate that LasR is a specific DNA-binding protein that regulates the transcription of the lasB gene in the presence of an autoinducer.