Effect of substrate rigidity in tissue culture on the function of insulin-secreting INS-1E cells.

Insulin-secreting INS-1E cells are a useful tool in diabetes research. However, during permanent culture the cells tend to lose their ß cell phenotype, with resultant loss of insulin-secretory responsiveness. This can be at least partially attributed to inappropriate cell culture conditions. One of the important causative factors is the rigidity of the extracellular matrix. We have therefore systematically studied the performance of INS-1E insulin-secreting cells cultured on polyacrylamide gels of different stiffnesses and analysed changes in insulin content and secretion, glucokinase enzyme activity, gene expression of ß cell transcription factors and cell death and proliferation rates. INS-1E cells were cultured on polyacrylamide gels with a wide range of rigidities, including the one that simulates the stiffness of the pancreas. We detected changes in insulin content and the insulin-secretory response to glucose stimulation in parallel to the increasing stiffness of the polyacrylamide gels in the range 1700-111 000 Pa. On substrates with the highest and lowest rigidities, 322 and 111 000 Pa, the cells mainly formed pseudo-islets, while at rigidities of 1700-64800 Pa, including the rigidity of native pancreas tissue (3100 Pa), cells grew as a monolayer attached to the polyacrylamide gel surface. These observations provide evidence for an apparent mechanosensitivity of insulin-secreting INS-1E cells affecting morphology and cellular functions. The results can also provide practical advice regarding a selection of the materials appropriate for successful cell culture of insulin-secreting cells. Copyright © 2014 John Wiley & Sons, Ltd.

The application of impulse oscillation system for the evaluation of treatment effects in patients with COPD.

There are only a few reports of the use of impulse oscillation system (IOS) for the evaluation of COPD treatment. In this study, we applied IOS and spirometry to evaluate the effectiveness of fluticasone propionate and salmeterol (SFC) combined with tiotropium (TIO) in COPD patients. Following a 4-week run-in period with TIO (18 µg once daily) treatment, COPD patients were randomized to SFC (250/50 µg twice daily; SFC+TIO group, n=25), or TIO alone (TIO group, n=31). Pulmonary functions were recorded by IOS and spirometry before and after the study period. The SFC+TIO group showed significant improvements in inspiratory resistance at 5 Hz and resonant frequency, as well as in FVC and FEV1, after the 12-week treatment (p<0.05). Since there were no significant correlations between improvements in IOS measurements and FVC or FEV1, IOS may provide a physiological point of view that is different from spirometry and seemed to be applicable as an additional assessment tool targeting COPD patients.

Effects of free fatty acids on human salivary gland epithelial cells.

Obesity and type 2 diabetes (T2D) are characterized by decreased insulin sensitivity and higher concentrations of free fatty acids (FFAs) in plasma. Among FFAs, saturated fatty acids (SFAs), such as palmitate, have been proposed to promote inflammatory responses. Primary Sjögren's syndrome (SS) is an autoimmune disease characterized by inflammatory mononuclear cell infiltration and destruction of epithelial cells in the salivary and lacrimal glands. IL-6 production and α-fodrin degradation are increased in salivary gland epithelial cells of patients with primary SS. Although previous studies have shown a link between SS and either dyslipidemia or T2D, little is known about the clinical significance of FFAs in primary SS. Here we report that SFAs, but not unsaturated fatty acids, induced IL-6 production via NF-κB and p38 MAPK activation in human salivary gland epithelial cells. Moreover, palmitate induced apoptosis and α-fodrin degradation by caspase-3 activation. Unlike salivary gland epithelial cells, induction of IL-6 production and the degradation of α-fodrin in response to palmitate were undetectable in squamous carcinoma cells and keratinocytes. Taken together, SFAs induced IL-6 production and α-fodrin degradation in salivary gland epithelial cells, implicating a potential link between the pathogenesis of primary SS and SFAs level in plasma.

Spindle cell metaplasia arising in thyroid adenoma: characterization of its pathology and differential diagnosis.

Spindle cell metaplasia in thyroid adenoma or carcinoma is rare and its pathological features are not well characterized. Distinction of this entity from medullary or anaplastic carcinoma has an important clinical implication. We encountered a case of thyroid follicular adenoma associated with spindle cell metaplasia. It showed "tumor in tumor appearance" and neoplastic spindle cells were positive for thyroglobulin, thyroid transcription factor-1, vimentin and focally chromogranin A and somatostatin (SS). MIB-1 index was <1%. Ultrastructure of the spindle cells was reminiscent of follicular cell origin. From the findings from our case, spindle cell metaplasia appears to be a benign clinical entity, suggestive of multidirectional differentiation of follicular cells.

Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family.

Bim protein is one of the BH3-only proteins, members of the Bcl-2 family that have only one of the Bcl-2 homology regions, BH3. BH3-only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. Bim(EL), Bim(L) and Bim(S). Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimalpha1, alpha2, and beta1-beta4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C-terminal hydrophobic region. Among the novel isoforms, only Bimalpha1 and alpha2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein-fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.

Extended polyglutamine selectively interacts with caspase-8 and -10 in nuclear aggregates.

A growing number of inherited neurodegenerative disorders, including Huntington's disease, have been shown to be caused by the expansion of CAG/polyglutamine repeats. The molecular mechanism underlying these disorders, however, has yet to be clarified. We and others previously demonstrated that caspase-8 was activated by proteolysis in association with the expression of extended polyglutamine. Here, we further analyzed the selectivity of caspases in the process mediated by extended polyglutamine. Among upstream caspases, caspase-10, a close homolog of caspase-8, was also proteolytically activated, but caspase-9 was not. Caspase-8 and -10 were recruited into nuclear aggregates of extended polyglutamine, where at least a fraction of these caspases was converted to the activated forms. Caspase-8 and -10 were co-immunoprecipitated with polyglutamine only when the polyglutamine was pathologically extended, whereas caspase-2, -3, -6, -7 and -9 were not co-immunoprecipitated with polyglutamine regardless of its size. A dominant-negative form of caspase-8 with a mutation at the catalytic cysteine residue inhibited polyglutamine-mediated nuclear apoptotic phenotype. These results suggest that caspase-8 and -10 are autoactivated as a result of close proximity of the proforms of these molecules that occurs due to aggregate formation, which reveals a novel toxic gain-of-function mechanism for the pathogenesis of CAG-repeat disorders.

Comprehensive studies on subcellular localizations and cell death-inducing activities of eight GFP-tagged apoptosis-related caspases.

By using green fluorescent protein fusion, we investigated the subcellular localization of all the caspases that have been cloned from humans and implicated in the execution of apoptosis. We divided these caspases into three groups according to subcellular localization. The first group includes caspase-1, -3, -6, -7, and -9, which are expressed mainly in the cytoplasm with various levels of nuclear localization depending on the cell type. The second group has a single member, caspase-2, which is primarily localized in the nucleus. The nuclear localization was demonstrated to be mediated by a nuclear localization signal near the NH(2)-terminus of the prodomain. The third group includes caspase-8 and -10, which have a cytoplasmic distribution. These two members have potent, rapid cell death-inducing activity and are prone to make aggregates when overexpressed. Their prodomains formed marked fibrous structures in the cytoplasm whose localization seemed distinct from organelles or cytoskeletons. None of the GFP-caspases examined in this study showed a predominant mitochondrial localization as has been reported for some caspases.

Neutrophil-specific reduction in the expression of granulocyte--macrophage colony-stimulating factor receptor subunits in myelodysplastic syndromes.

The proliferative and differentiative response of neutrophils to granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be impaired in patients with myelodysplastic syndromes (MDS). To investigate the mechanisms of the defective response in MDS, we examined expression levels of GM-CSF receptor alpha (GMR alpha) and common beta (beta c) subunits on CD16(+) neutrophils, CD14(+) monocytes and CD3(+) T cells from 26 MDS patients and 10 healthy controls using flow cytometry. Expression of GMR alpha was significantly decreased on the neutrophils of five out of 26 patients and was not specific for any FAB subtype. In contrast, beta c expression on neutrophils was significantly reduced in 14 out of 26 patients with a higher proportion occurring in the advanced stages of MDS including refractory anaemia with excess of blasts (RAEB), RAEB in transformation (RAEBt) and overt leukaemia compared with refractory anaemia (RA)/RA with ringed sideroblasts (RARS) or healthy controls. Decreased beta c also correlated with the degree of hypogranular neutrophil morphology and increased infection. Expression of both subunits on T cells and monocytes in MDS was similar to normal controls. Polymerase chain reaction amplification of reverse-transcribed mRNA isolated from the affected neutrophils suggests that the reduction of beta c may result from decreased message levels. The observed reduction in GM-CSF receptor expression could account for the impaired proliferative and maturational responses in MDS.

An axonal form of Charcot-Marie-Tooth disease showing distinctive features in association with mutations in the peripheral myelin protein zero gene (Thr124Met or Asp75Val).

OBJECTIVES AND METHODS: Seven families were studied with an axonal form of Charcot-Marie-Tooth disease (CMT) associated with mutations in the peripheral myelin protein zero (MPZ) gene-Thr124Met or Asp75Val. RESULTS: Patients with these mutations commonly showed relatively late onset sensorimotor neuropathy predominantly involving the lower limbs. Sensory impairment typically was marked, and distal muscle atrophy and weakness were also present in the legs. Adie's pupil and deafness were often present, and serum creatine kinase concentrations were often raised irrespective of which MPZ mutation was present. Relatively well preserved motor and sensory nerve conduction velocities contrasted with reduced or absent compound muscle action potentials and sensory nerve action potentials. Axonal change with marked axonal sprouting was seen in sural nerve specimens. CONCLUSION: The similar associated clinical findings suggest that patients with axonal CMT with an MPZ gene mutation share distinctive clinical features.

Engagement of alpha4beta7 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro.

Asthma is characterized by an airway inflammatory infiltrate that is rich in eosinophilic leukocytes. Cellular fibronectin and VCAM-1, ligands for alpha4 integrins, are enriched in the fluid of airways of allergic patients subjected to Ag challenge. We therefore hypothesized that ligands of alpha4 integrins can promote eosinophil survival independent of cell adhesion. Cellular fibronectin and VCAM-1 increased viability of human peripheral blood eosinophil in a dose- and time-dependant manner whether the ligand was coated on the culture well or added to the medium at the beginning of the assay. Eosinophils cultured with cellular fibronectin were not adherent to the bottom of culture wells after 3 days. Treatment with mAb Fib 30 to beta7, but not mAb P4C10 or TS2/16 to beta1, increased eosinophil survival. The increased survival of eosinophils incubated with Fib 30 was blocked by Fab fragments of another anti-beta7 mAb, Fib 504. Eosinophils incubated with soluble cellular fibronectin or mAb Fib 30 for 6 h demonstrated a higher level of GM-CSF mRNA than eosinophils incubated with medium alone. Addition of neutralizing mAb to GM-CSF during incubation, but not mAbs to IL-3 or IL-5, reduced the enhancement of eosinophil survival by soluble cellular fibronectin or mAb Fib 30 to control levels. Thus, viability of eosinophils incubated with cellular fibronectin or VCAM-1 is due to engagement, probably followed by cross-linking, of alpha4beta7 by soluble ligand (or mAb) that stimulates autocrine production of GM-CSF and promotes eosinophil survival.

Regional differences in genetic subgroup frequency in hereditary cerebellar ataxia, and a morphometrical study of brain MR images in SCA1, MJD and SCA6.

Molecular genetic assessments of 69 individuals in 44 families with hereditary cerebellar ataxia (HCA) were made to determine the relative frequencies of subtypes of HCA in Yamagata, Japan. Fifteen families (34%) had SCA1, none had SCA2, nine (20%) had MJD, five (11%) had SCA6 and nine (20%) had DRPLA. These findings differ markedly from those in other regions of Japan and the rest of the world. A morphometrical study of the brain MR images also was made on 38 individuals with SCA1 (n = 14), MJD (n = 8) or SCA6 (n = 16). In SCA1, the ventral pons was atrophic in proportion to the amount of cerebellar atrophy. In MJD, both the pons and the cerebellum were atrophic, cerebellar atrophy being less pronounced than that in SCA1 and SCA6. While both the major and minor axes of the ventral pons were proportionally decreased in SCA1, the minor axis was more decreased than the major axis in MJD. In SCA6, a mild reduction in the ratio of the ventral pontine area to the posterior fossa area (Pv/PF) was observed as well as obvious cerebellar atrophy. These findings indicate that in MR images SCA1, MJD and SCA6 show different atrophic features of the cerebellum and brainstem.

The beta c component of the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3)/IL-5 receptor interacts with a hybrid GM-CSF/erythropoietin receptor to influence proliferation and beta-globin mRNA expression.

BACKGROUND: The interaction of different members of the hematopoietic growth factor receptor family may be relevant to the increased proliferation and the failure of differentiation that characterizes the myeloid leukemias. We recently demonstrated that a chimeric receptor (GMER) that is composed of the extracellular and transmembrane domains of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain (GMR alpha) and the cytoplasmic domain of the murine erythropoietin receptor mEpoR binds hGM-CSF with low affinity (3 nM) and confers both proliferative and differentiation signals to stably transfected murine Ba/F3 cells. MATERIALS AND METHODS: To investigate whether the common beta-subunit of the GM-CSF receptor (beta c) can interact with GMER, either the entire beta-subunit or a mutant, truncated beta-subunit that completely lacks the cytoplasmic domain (beta tr) was introduced into Ba/F3 cells that express GMER, and the binding of GM-CSF as well as proliferation and differentiation responses were measured. RESULTS: Scatchard analysis showed that both GMER + beta c and GMER + beta tr bound hGM-CSF with high affinity (Kd 40 pM to 65 pM). Proliferation assays showed that the maximum growth of cells expressing GMER + beta c was identical to that of cells with GMER alone. However, proliferation of the cells that expressed GMER + beta tr was reduced by 80-95% of GMER. Dose-response curves showed that the concentration of GM-CSF required for half-maximal growth was 0.5-5.0 pM for GMER + beta c and 0.5-5 nM for GMER and GMER + beta tr. The EpoR cytoplasmic domain of GMER also undergoes ligandinducible tyrosine phosphorylation. However, the tyrosine phosphorylation did not correlate with growth in cells expressing beta tr. Coexpression of beta c with GMER in Ba/F3 cells grown in hGM-CSF markedly enhanced beta-globin mRNA expression. CONCLUSIONS: These results indicate that beta c can transduce a unique signal in association with GMER to influence both proliferative and differentiation signal pathways.

Hereditary caeruloplasmin deficiency: clinicopathological study of a patient.

A 58 year old patient with dementia, oral dyskinesia, and diabetes mellitus is described. He had an undetectable concentration of serum caeruloplasmin, as an autosomal recessive trait. Brain MRI disclosed a pronounced hypointensity in the bilateral putamina, caudate, and dentate nuclei on both T1 and T2 weighted images. Pathological findings were mainly in those regions of the brain and consisted of neuronal cell loss with gliosis, heavy iron deposition, and spheroids. Visceral organs also had iron deposition, especially severe in the liver and pancreas. The present patient and other recorded cases constitute a clinicopathological entity of hereditary caeruloplasmin deficiency, different from Wilson's disease.

Localization of the gustatory pathway in the human midbrain.

The localization of the secondary gustatory pathway in the human brainstem still remains uncertain. Here we report two patients with small vascular lesions in the unilateral midbrain tegmentum who presented with taste disturbance on the ipsilateral side of the tongue. In both cases, the dorsomedial mesencephalic tegmental region lateral to the oculomotor nucleus, including the central tegmental tract and the ventral part of the periaqueductal gray, was involved commonly in the lesions. The secondary gustatory pathway arising from the nucleus of the solitary tract appears to run rostrally, without crossing, to the ipsilateral thalamic nucleus through the dorsomedial part of the tegmental region at the rostral level of the midbrain.

Effect of isolation protocol on eosinophil function: Percoll gradients versus immunomagnetic beads.

Studies of in vitro eosinophil function are dependent on efficient and reliable methods of cell isolation. Protocols using Percoll or metrizamide density gradients have been of limited use in isolating peripheral blood eosinophils in sufficient numbers and purity from subjects with normal or only slightly elevated eosinophil counts, thereby restricting comparative studies to preparations from hypereosinophilic subjects. Recently, a method utilizing negative selection by anti-CD16 coated magnetic beads has greatly improved eosinophil isolation by dramatically increased yields and purity. However, little is known as to the differential effect of various isolation methods on the functional activity of eosinophils. In this study, eosinophils were isolated by either discontinuous multiple density Percoll gradients or anti-CD16-coated magnetic beads: several functional activities were then compared using cells obtained by the two methods of isolation. Compared with Percoll isolated eosinophils, anti-CD16 bead separated eosinophils had significantly increased baseline and stimulated LTC4 production, spontaneous O2- generation, and expression of specific cell surface markers. No significant difference was observed in the cells' in vitro survival and adhesion. Such differences may be due to the isolation of eosinophils of all densities by anti-CD16 beads, or the effect of neutrophils interacting with the beads to release eosinophil agonists or primers. Alternatively, the Percoll gradient method with the eosinophils' exposure to dextran and Ficoll-Hypaque may affect subsequent cell function. Therefore, comparison of eosinophil function between cells isolated by different protocols must be considered before concluding which is the true measure of in vivo cell function.

Enzyme-linked immunosorbent assay and latex agglutination inhibition reaction test for morphine in urine.

An enzyme-linked immunosorbent assay (ELISA) and a latex agglutination inhibition reaction test (LAIRT) for morphine have been established. Rabbits were immunized with 3-carboxymethylmorphine-bovine serum albumin (BSA) conjugate to obtain anti-morphine antibody and IgG fraction of the antiserum was used as an antibody. In ELISA, 3-carboxymethylmorphine-alkaline phosphatase (ALP) conjugate and antibody adsorbed on polystyrene microtiter wells were used. The enzyme activity of antibody bound ALP was measured with the enzyme cycling method. The range of morphine measurable by ELISA was 6-600 pg/well and the analysis time for 96 wells was 90 min. In LAIRT, 3-carboxymethylmorphine-rabbit serum albumin (RSA) conjugate and the antibody were bound to latex particles covalently to prepare latex-antigen and latex-antibody reagent, respectively. The agglutination reaction with latex-antigen and latex-antibody reagents was inhibited by 0.2 microgram morphine/ml urine and the analysis time for six samples on one glass slide was 20 min. The urine samples of 47 suspected abusers were analyzed by ELISA and LAIRT and the results were compared with those of the enzyme-multiplied immunoassay technique (EMIT) and gas chromatography/mass spectrometry (GC/MS). Both ELISA and LAIRT for morphine seem to be suitable for the screening of urine samples.

A constitutively activated chimeric cytokine receptor confers factor-independent growth in hematopoietic cell lines.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GMR) comprises at least 2 distinct subunits, alpha and beta common (beta c), whereas the normal erythropoietin receptor (nEpoR) comprises only one known subunit. An arginine to cysteine (R129C) mutation of the extracytoplasmic domain of the murine EpoR leads to Epo-independent growth in transduced cells (cEpoR). To investigate the proliferative functions of the cytoplasmic regions of each GMR subunit separately and the potential of the R129C EpoR mutation to induce factor-independent growth through heterologous receptor regions, we constructed four hybrid receptors: the extracellular region of either murine nEpoR or cEpoR linked to the transmembrane and cytoplasmic regions of either the human GMR alpha or beta c subunit (nE alpha, nE beta, cE alpha, and cE beta). We then expressed them in an interleukin-3-dependent murine cell line, Ba/F3. Expression of nE beta led to Epo-dependent growth, whereas expression of cE beta conferred factor-independent growth. Surprisingly, expression of cE alpha also resulted in factor-independent cell growth, whereas nE alpha did not respond to Epo. Furthermore, the functional hybrid receptors showed Epo-dependent (nE beta) or constitutive (cE alpha and cE beta) tyrosine phosphorylation of the cytoplasmic kinases JAK1 and JAK2. We reasoned that the proliferative signal of cE alpha was transduced either through the alpha tail itself or through an accessory protein such as the endogenous murine beta common subunit (mu beta c). To distinguish these possibilities, the chimeric receptor cE alpha was expressed in the interleukin-2-dependent murine cell line, CTLL-2, that does not express mu beta c. cE alpha did not induce cell growth in CTLL-2; however, when mu beta c was coexpressed with cE alpha in CTLL-2, factor-independent growth was reconstituted. In conclusion, the cytoplasmic domain of the GMR alpha subunit requires a beta chain for transduction of a proliferative signal. Furthermore, the R129C EpoR mutation can constitutively activate heterologous receptors to mediate factor-independent proliferation.

Enzyme-linked immunosorbent assay and latex agglutination inhibition reaction test for cocaine and benzoylecgonine in urine.

An enzyme-linked immunosorbent assay (ELISA) and a latex agglutination inhibition reaction test (LAIRT) for cocaine and benzoylecgonine have been established. In ELISA with polystyrene microtiter wells coated with anti-benzoylecgonine antibody and alkaline phosphatase (ALP)-labeled benzoylecgonine, the activity of antibody-bound ALP was measured with the enzyme cycling method. The range of benzoylecgonine measurable by ELISA was 12 pg-25 ng/well; the analysis time for 96 wells was 90 min. In LAIRT, the agglutination reaction with anti-benzoylecgonine antibody-coated latex and benzoylecgonine-rabbit serum albumin (RSA) conjugate-coated latex was inhibited by 0.1 mu g benzoylecgonine/ml urine; the analysis time for six samples on one glass slide was 20 min. The urine samples of 47 abusers were analyzed by ELISA and LAIRT. From the comparison with results of the enzyme-multiplied immunoassay technique (EMIT) and gas chromatography-mass spectrometry (GC-MS), it was clarified that both ELISA and LAIRT were suitable for the screening method of urine samples.