The accuracy of fine needle aspiration cytology in the clinical diagnosis of minor salivary gland tumours.

The objective of this study was to investigate the accuracy of fine needle aspiration cytology (FNAC) and biopsy for the clinical diagnosis of minor salivary gland tumours (MSGTs). This retrospective study of 32 MSGT cases was conducted over a 5-year period. Clinical features including age, sex, and location of the tumour were obtained from the patient clinical records. All cases were also assessed histologically according to the 2017 World Health Organization Classification of Head and Neck Tumours. The results of FNAC and biopsy were correlated with those of histopathology, and their sensitivity, specificity, and diagnostic efficacy were calculated using histopathology as the gold standard. Eighteen malignant MSGTs (56.3%) and 14 benign MSGTs (43.8%) were diagnosed by pathological diagnosis. The most common malignant tumour was mucoepidermoid carcinoma (seven cases, 38.9%). Most benign cases were pleomorphic adenomas (13 cases, 92.9%). FNAC was performed for 23 cases and biopsy for 13 cases. The sensitivity and specificity of FNAC were 66.7% and 91.0%, respectively, while those of biopsy were 90.0% and 100.0%, respectively. Although FNAC is a minimally invasive and cost-effective procedure, it is less accurate than biopsy in the assessment of MSGTs. Repeated FNAC or biopsy should be considered in negative and unsatisfactory FNAC cases.

Single pancreatic beta cells co-express multiple islet hormone genes in mice.

AIMS/HYPOTHESIS: It is widely accepted that production of insulin, glucagon, somatostatin and pancreatic polypeptide in islet cells is specific to beta, alpha, delta and pancreatic polypeptide cells, respectively. We examined whether beta cells express other genes encoding islet hormones. METHODS: Nested RT-PCR was performed on single beta cells of transgenic mice with green fluorescent protein (GFP) driven by mouse insulin I promoter (MIP-GFP). RESULTS: Only 55% of adult beta cells expressed the insulin gene alone, while others expressed two or more islet hormone genes; 4% expressed all four hormone genes. In embryonic and neonatal cells, 60% to 80% of GFP(+) cells co-expressed pancreatic polypeptide and insulin genes in contrast to 29% in adult. To clarify cell fate, we conducted lineage tracing using rat insulin II promoter-cre mice crossed with reporter mice Gt(ROSA)26Sor-loxP-flanked STOP-cassette-GFP. All GFP(+) cells expressed insulin I and II genes, and showed similar heterogeneity of co-expression to that seen in MIP-GFP mice. Although we report expression of other hormone genes in a significant proportion of beta cells, our lineage tracing results demonstrate that after inducing InsII (also known as Ins2) expression, beta cell progenitors do not redifferentiate to non-beta cells. CONCLUSIONS/INTERPRETATION: This study shows co-expression of multiple hormone genes in beta cells of adult mice as well as in embryos and neonates. This finding could: (1) represent residual expression from beta cell precursors; (2) result from alternative developmental pathways for beta cells; or (3) denote the differentiation potential of these cells. It may be linked to functional heterogeneity. This heterogeneity in gene expression may provide a means to characterise the functional, cellular and developmental heterogeneity seen in beta cells.

Towards better understanding of the contributions of overwork and glucotoxicity to the beta-cell inadequacy of type 2 diabetes.

Type 2 diabetes (T2D) is characterized by reduction of beta-cell mass and dysfunctional insulin secretion. Understanding beta-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when beta-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, beta-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. beta-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains beta-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human beta-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how beta-cell phenotype evolves as T2D develops and progresses.

Differentiation of COPAS-sorted non-endocrine pancreatic cells into insulin-positive cells in the mouse.

AIMS/HYPOTHESIS: The regenerative process in the pancreas is of particular interest, since insulin-producing beta cells are lost in diabetes. Differentiation of new beta cells from pancreatic non-endocrine cells has been reported in vivo and in vitro, a finding that implies the existence of pancreatic stem/progenitor cells. However, while tissue-specific stem cells are well documented in skin, intestine and testis, pancreatic stem cells have been elusive. We hypothesised that pancreatic stem/progenitor cells within the non-endocrine fraction could be a source of new islets in vitro. METHODS: To test if there were such cells within the pancreas, we generated pancreatic cell aggregates from tissue remaining after islet isolation from mouse insulin promoter 1-green fluorescent protein (MIP-GFP) mice. To eliminate any contamination of insulin-positive cells, we deleted all GFP-positive aggregates using COPAS Select and cultured with Matrigel. Immunohistochemistry, quantitative real-time PCR and single-cell nested RT-PCR were performed to confirm formation of insulin-producing cells. RESULTS: The GFP-negative cells were expanded as monolayers and then differentiated into three-dimensional cystic structures. After 1 week of culture, GFP-positive cells were found as clusters or single cells. By quantitative real-time PCR, no insulin mRNA was detected immediately after COPAS sorting, but after differentiation insulin mRNA of the whole preparation was 1.91 +/- 0.31% that of purified MIP-GFP beta cells. All GFP-positive cells expressed insulin 1; most expressed insulin 2, pancreas duodenum homeobox-1 and cytokeratin 19 by single cell nested RT-PCR. CONCLUSIONS/INTERPRETATION: Our data support the concept that within the exocrine (acinar and ductal) pancreas of the adult mouse there are cells that can give rise to insulin-positive cells in vitro.

In vivo evaluation of ocular inflammatory responses in experimental diabetes.

AIMS: Diabetic patients may have abnormal inflammatory reactions to foreign or endogenous stimuli. This study was designed to evaluate inflammatory reactions in the diabetic eye through retinal leucocyte dynamics in the inflamed eyes of diabetic rats. METHODS: Three weeks after diabetes induction in Long-Evans rats, endotoxin induced uveitis was produced by footpad injection of lipopolysaccharide (LPS). After LPS injection, leucocyte behaviour was evaluated in vivo by acridine orange digital fluorography. RESULTS: The number of rolling leucocytes increased in a biphasic manner at 12 hours and 48 hours. The number of leucocytes accumulating in the retina reached a peak at 72 hours. The maximal numbers of rolling and accumulating leucocytes in the diabetic retina decreased by 56.3% (p<0.01) and 46.7% (p<0.0001), respectively, compared with the non-diabetic retina. The levels of mRNA expression of adhesion molecules in the retina, which were upregulated after LPS injection, were also lower in diabetic rats than in non-diabetic rats. CONCLUSION: This study is the first to show that endotoxin induced inflammation is disturbed in the diabetic eye, based on evidence that the leucocyte-endothelial cell interactions stimulated by LPS were suppressed in the diabetic retina. These findings support the theory that ocular inflammatory reactions are impaired in diabetic patients.

Mastoparan stimulates GABA release from MIN6 cells: relationship between SNARE proteins and mastoparan action.

We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.

Transforming growth factor-beta 2 levels in aqueous humor of glaucomatous eyes.

PURPOSE: To determine whether clinical characteristics are correlated with increased levels of transforming growth factor-beta 2 (TGF-beta 2) in aqueous humor in glaucomatous eyes. METHODS: Aqueous humor samples were collected from 91 glaucomatous eyes. Included were samples from primary open-angle glaucoma (POAG) in 40 eyes, (pseudo)exfoliation syndrome (EXS) in 18 eyes, primary angle-closure glaucoma (PACG) in 26 eyes and uveitis-related secondary glaucoma (SG) in 7 eyes. TGF-beta 2 in aqueous humor was assessed with a specific-capture ELISA. RESULTS: The mean concentration (+/- standard error) of mature (biologically active) TGF-beta 2 in the aqueous humor of eyes with POAG was 293.6 +/- 33.6 pg/ml, significantly higher than that in eyes with PACG, EXS and SG: 147.5 +/- 28.1, 135.8 +/- 30.2 and 41.0 +/- 10.7 pg/ml, respectively (P = 0.0006, P = 0.0010 and P = 0.0003; analysis of variance). The mean concentration (+/- standard error) of total TGF-beta 2 in the aqueous humor of eyes with POAG was 1647.6 +/- 124.5 pg/ml, not significantly different from that in eyes with PACG, EXS and SG: 1482.9 +/- 148.2, 1442.7 +/- 187.8 and 1929.0 +/- 367.6 pg/ml, respectively. A multivariate analysis using logistic regression showed significant correlations between mature TGF-beta 2 concentration and history of cataract surgery (P = 0.0225) and the use of carbonic anhydrase inhibitors (P = 0.0143). CONCLUSIONS: Our results indicate that increased levels of TGF-beta 2 may play an important role in the pathogenesis of POAG.

Terson syndrome caused by ventricular hemorrhage associated with moyamoya disease--case report.

A 24-year-old female presented with Terson syndrome secondary to bilateral ventricular hemorrhage as a complication of moyamoya disease. Ophthalmoscopy and magnetic resonance imaging clearly demonstrated vitreous hemorrhage in the left eye globe. Various visual symptoms are associated with moyamoya disease, almost all of which result from ischemic lesions in the visual cortex and optic pathways. In this case, the visual disturbance was caused by Terson syndrome secondary to ventricular hemorrhage. Close ophthalmological and radiological evaluation is mandatory even in patients with moyamoya disease and hemorrhagic manifestation located in the intracerebral, subarachnoid, or intraventricular space.

Suppression of insulitis and diabetes in B cell-deficient mice treated with streptozocin: B cells are essential for the TCR clonotype spreading of islet-infiltrating T cells.

In order to clarify the role of B cells in the development of insulitis and diabetes, B cell-deficient (B(-)) mice treated with streptozocin (STZ) were studied. The extent of insulitis and the cumulative incidence of diabetes were significantly suppressed in B(-) mice (P < 0.0001), indicating that B cells are crucial for the progression of insulitis and diabetes. Accumulation of both CD4(+) T cells and B cells was observed in islets of B(+) mice, while CD4(+) T cells but not B cells were found in B(-) mice. A few CD8(+) T cells and macrophages were detectable in both types of mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B(-) mice. TCR V(beta) gene repertoire usage of islet-infiltrating T cells was restricted to some extent in the B(+) or B(-) mice, but there was no significant difference between the B(+) and B(-) mice, suggesting that the initial islet-reactive T cell response can occur in the absence of B cells. In contrast, TCR clonotype spreading of islet-infiltrating T cells was significantly suppressed in B(-) mice compared with B(+) mice (P < 0.0001). These data suggest that initial priming of T cells is not impaired and TCR V(beta) repertoire usage is not limited by the lack of B cells, while B cells are important essentially for the spreading of islet-infiltrating clonal T cells in autoimmune diabetic mice induced with STZ.

Establishment of an embryonic stem (ES) cell line derived from a non-obese diabetic (NOD) mouse: in vivo differentiation into lymphocytes and potential for germ line transmission.

A non-obese diabetic (NOD) mouse-derived embryonic stem (ES) cell line has been stably maintained in an undifferentiated state with a characteristic ES cell-like morphology, expressing the stem cell marker alkaline phosphatase, and displaying a normal diploid karyotype. After injecting the NOD-ES cells into blastocysts, chimeric mice were obtained. Small but significant numbers of lymphocytes expressed the NOD-derived MHC allele. When a chimeric mouse was mated with C57BL/6 mice, an agouti mouse was obtained, having the NOD-derived H-2 I-A(beta)g7 haplotype. Thus, an NOD-ES cell line which can differentiate into lymphocytes with potential for germ line transmission was successfully established.

Lyn-mediated down-regulation of B cell antigen receptor signaling: inhibition of protein kinase C activation by Lyn in a kinase-independent fashion.

Stimulation of the B cell Ag receptor (BCR) induces activation of tyrosine kinases such as Lyn and Syk, phosphorylation and activation of multiple signaling components, and eventually, the expression of several genes including c-myc. Syk is required for activation of phospholipase C-gamma 2 and the subsequent phosphatidylinositol hydrolysis, leading to protein kinase C (PKC) activation and intracellular Ca2+ increase. In contrast, the function of Lyn remains obscure. Here, we report that BCR-mediated induction of c-myc promoter activity and of PKC activity, but not the expression level of functional PKC, was markedly augmented in Lyn-deficient chicken B cells. This enhancement was reversed to the level of wild-type cells by the expression of exogenous Lyn of kinase-inactive form. These results indicate that Lyn inhibits BCR-mediated activation of a large portion of PKC isozymes in a kinase-independent fashion. This finding reveals a novel role of Lyn in negative regulation of BCR signaling.

Task-related coherence and task-related spectral power changes during sequential finger movements.

In order to investigate the activity of cortical regions in the control of complex movements, we studied task-related coherence (TRCoh) and task-related spectral power (TRPow) changes in 8 right-handed subjects during the execution of 4 different finger movement sequences of increasing complexity. All sequences were performed with the right hand and were paced by a metronome at 2 Hz. EEG power spectra and coherence values were computed within alpha (8-12 Hz) and beta (13-20 Hz) frequency bands for 29 scalp EEG positions during the execution of the sequences and were compared with values obtained during a rest (control) condition. Movement sequences were associated with TRPow decreases in the alpha and beta frequency bands over bilateral sensorimotor and parietal areas, with a preponderance over the contralateral hemisphere. Increases of TRCoh occurred over bilateral frontocentral regions. TRCoh decreases were present over the temporal and occipital areas. The spatial extent and the magnitude of TRPow decreases and TRCoh increases in both frequency bands were greater for sequential movements of higher complexity than for simpler ones. These results are consistent with previous findings of bilateral activation of sensorimotor areas during sequential finger movements. Moreover, the present results indicate an active intercommunication between bilateral and mesial central and prefrontal regions which becomes more intense with more complex sequential movements.

Establishment of tissue culture cell strains from normal fetal human liver and kidney.

Three cells strains of tissue culture were established from normal fetal human liver and kidney tissues. The rate of cell proliferation decreased with time after the primary culture. The cells were rarely subcultured, but the medium was renewed routinely twice a week. After 9 months of cultivation, the cells were found in both tissues to have abruptly begun to proliferate rapidly, but only in the group culture in the medium containing galactose and sodium pyruvate in place of glucose. No special treatment were given to these cultures, e.g. treatment with viruses, chemical carcinogens and others. The chromosome number was kept around diploid in the beginning but was shifted to hypotriploid after the establishment. The cell strain from liver consists of epithelial cells. The cell strains from kidney consist of mixed population of various kinds of cells. Doubling time of cells is about 33.4 hours in all of them, as determined by cinemicrography. This might be the first establishment of cell strains from untreated, normal human tissues.

An attempt in tissue culture at preventing and treating the collagen fiber formation of liver cells.

To prevent and to cure liver cirrhosis, we examined the effect of the ethanol extract of berries of Japanese ampelopsis on the collagen formation of rat liver cells in tissue culture. These cells had been transformed to produce collagen fibers very actively. When added at a time of subcultivation, the extract prevented the formation of collagen fibers. When it was added after the formation of collagen fibers, the fibers were fragmented into fine microfilaments.

Neutralization of cytotoxicity of spermine on the proliferation of rat liver cells in tissue culture.

Cytotoxicity of spermine in tissue culture was found previously. To neutralize this toxicity, the addition of various high molecular weight substances and others was attempted in this paper, e.g. lysozyme, N-acetyl-D-glucosamine, chondroitin sulfate, poly-L-glutamic acid, bovine serum fractions V and VI, fetal calf serum, methyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone and others. Into the culture of rat liver cells, strain RLC-10(2), simultaneous addition of other substances with spermine did not neutralize the toxicity. However, by the pretreatment of spermine with fetal calf serum or bovine serum albumin (fraction V) at 37 degrees C for 24 hr, the toxicity of spermine was markedly reduced. This was probably due to the denaturation of spermine caused by the pretreatment.

Tapping culture--an improved method for cell suspension culture.

A new culture vessel was designed for cell suspension culture. A silicone-covered magnet bar fixed by one end to the side wall of the bottle was held horizontally a short distance from the bottom. A standard type magnetic stirrer was used. In contrast to the conventional horizontal movement of "stirring" in cultures the bar moves vertically with a "tapping" motion. This improvement resulted in less cell injury, higher rate of cell proliferation and formation of fewer bubbles than in the conventional type. Nine cell types were simultaneously cultivated in tapping, stirring and stationary culture. All cell types proliferated more luxuriously in tapping cultures then in stirring cultures. Serial cultivation of cells in tapping cultures was also successful.