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Biomedical subjects

J H Scarpello

Publications and source records attributed to J H Scarpello.

At least 19 recordsLinked to original sources
PubMed2001

Effects of tyrosine kinase inhibitors on cell death induced by sodium fluoride and pertussis toxin in the pancreatic beta-cell line, RINm5F.

1. Sodium fluoride causes apoptosis of pancreatic beta-cells and this response is enhanced by pre-treatment with pertussis toxin. In the present study, tyrosine kinase inhibitors were used to investigate the mechanisms of action of NaF and pertussis toxin in the beta-cell line, RINm5F. 2. Exposure of RINm5F cells to low concentrations of genistein or tyrphostin A25 resulted in significant inhibition of cell death induced by 5 mM NaF. Higher concentrations (>25 microM) were cytotoxic in the absence of NaF but, paradoxically, the combination of genistein and NaF induced less cell death than when each agent was used alone. 3. The increase in cell death induced by 100 microM genistein was markedly inhibited by ciprofloxacin, a drug which binds to topoisomerase II. Etoposide (which inhibits topoisomerase II but has no effect on tyrosine kinase activity) also caused an increase in RINm5F cell death. Neither etoposide nor ciprofloxacin altered the response to 5 mM NaF. 4. Pertussis toxin markedly enhanced the extent of RINm5F cell death induced by NaF and this effect was completely prevented by 25 microM genistein. The inhibition caused by genistein was not affected by ciprofloxacin but was reproduced by a structurally dissimilar tyrosine kinase inhibitor, herbimycin A. 5. The results demonstrate that RINm5F beta-cells express a pertussis toxin sensitive pathway that is anti-apoptotic. The activity of this pathway is most evident in cells exposed to pro-apoptotic stimuli where the effects of pertussis toxin can be blocked by inhibitors of tyrosine kinase enzymes. A genistein-sensitive tyrosine kinase does not appear to be involved in RINm5F cell survival under basal conditions.

Animals↗
PubMed2000

Dissociation between Fas expression and induction of apoptosis in human islets of Langerhans.

There is increasing evidence that inappropriate induction of apoptosis in pancreatic beta-cells may precede the development of type 1 diabetes in animal models and in man. One mechanism by which this has been proposed to occur involves up-regulation of the death receptor Fas on beta-cells, resulting in apoptosis of the Fas-bearing beta-cells upon ligation of the receptor. We have examined this hypothesis in isolated human islets of Langerhans and show that--in contrast to data obtained with rodent beta-cells--expression of Fas per se is not sufficient to allow induction of apoptosis upon addition of agonistic anti-Fas serum.

Animals↗
PubMed1998

Effect of metformin on bile salt circulation and intestinal motility in type 2 diabetes mellitus.

Gastrointestinal symptoms can be a limiting factor in optimizing metformin therapy, particularly at the onset of treatment. The underlying cause remains unclear. We have investigated whether metformin changes oral-caecal transit and if it causes bile salt malabsorption using the lactulose breath test and orally administered 14C-glycocholate followed by breath 14CO2 measurement over 6 h and stool collection for 72 h, respectively. Twenty-four diet and/or sulphonylurea treated patients underwent 7 days of baseline investigations before entering a randomized double-blind crossover study of 21 days duration with either metformin (850 mg bd) or placebo. No difference was observed in the oral-caecal transit time but a change in fasting plasma glucose was observed of 2.6 mmol l-1 (95% CI 1.3, 3.8). Significant increases in percentage 14CO2 breath elimination were observed during treatment with metformin (9.7 +/- 6.3) compared with placebo (3.1 +/- 1.9) p = 0.020. In addition, percentage faecal 14C bile salt excretion was increased with metformin (17.2 +/- 9.9 vs 10.1 +/- 6.9) p = 0.037. A significant association (p = 0.002) emerged for stool bile salt content and liquidity of the stool. We conclude that metformin may cause gastrointestinal disturbances by reducing ileal bile salt reabsorption leading to elevated colonic bile salt concentrations.

Adult↗
PubMed1998

An audit of the management of thyroid cancer in a district general hospital.

OBJECTIVE: Thyroid cancer is the commonest endocrine malignancy yet it appeared to present infrequently to the endocrinologists at this large District General Hospital. The management of well-differentiated thyroid cancer remains controversial with a wide variation in clinical practice. The aim of this survey was to determine the characteristics of the patients diagnosed with thyroid cancer and whether any deficiencies existed in the management of subjects diagnosed with thyroid cancer over a five-year period using standards of care based upon long-term outcome data and recently published USA guidelines. DESIGN AND PATIENTS: Retrospective case-note survey of all patients newly registered with thyroid cancer from 1990 to 1994 in North Staffordshire (estimated total population 450,000). RESULTS: The annual incidence of all thyroid cancer was two per 100,000 of which well-differentiated tumours comprised 70%. Medical records were obtained in 48 new cases (91% of total) identified. Fifteen subjects who presented as surgical emergencies received only palliative treatment and had a poor outcome. Two patients presented with metastatic medullary thyroid carcinoma (3% of total). Thirty-one patients (97% of whom presented with a thyroid nodule) were referred electively to either surgical (n = 22), ENT (n = 2) or endocrinology (n = 7) outpatients with well-differentiated papillary (n = 17) and follicular (n = 14) tumours. Thirteen patients (42%) had fine-needle aspiration cytology performed preoperatively. Of the 22 tumours (71%) greater than 1.5 cm, five (27%) had a total thyroidectomy and two (9%) also had radioiodine ablation. There was inadequate serum thyrotrophin suppression postoperatively in 12 patients (39%) and only five (16%) were being monitored for recurrence with serum thyroglobulin measurements. CONCLUSIONS: Deficiencies in the optimum management of small, well-differentiated thyroid cancers were identified. Improved communication between specialties has led to the development of an agreed management protocol to increase the quality of care offered to patients with thyroid cancer and for auditing the coordinated service in the future.

Adult↗
PubMed1998

Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation.

IDDM results from a progressive loss of pancreatic beta-cells that, in humans, may be triggered by a combination of genetic and environmental factors. Recently, attention has been focused on the hypothesis that the loss of beta-cells is initiated by inappropriate induction of apoptosis. We now demonstrate that human islets of Langerhans undergo apoptosis upon exposure to interleukin-1beta. The cytokine also sharply increases the number of cells that enter apoptosis on treatment with a stimulatory anti-Fas antibody. Western blotting and immunocytochemistry clearly show for the first time that human pancreatic beta-cells normally express Fas ligand. The results suggest that human islet cells are primed to undergo apoptosis by interleukin-1beta and that this involves the close association between cell-surface Fas and its ligand.

Apoptosis↗
PubMed1997

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15.

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell line HIT-T15 in a dose- and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 microM) and dibutyryl cGMP (300 microM) were able to fully relieve this inhibition. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10-250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.

Alkaloids↗
PubMed1996

Heterotrimeric G-proteins are implicated in the regulation of apoptosis in pancreatic beta-cells.

Recent studies have provided evidence that apoptosis of pancreatic beta-cells is important in the early etiology of both type I and type II diabetes mellitus. The mechanisms responsible for induction of apoptosis are unknown, but we present evidence that the signal transduction pathway controlling the process in pancreatic beta-cells is regulated by G-proteins. We have employed the global G-protein activator fluoride and show that this agent induces apoptosis in clonal RINm5F pancreatic beta-cells and also in the cells of normal rat islets of Langerhans. The process is time and concentration dependent and may reflect the formation of AIF4- since it was inhibited by the aluminum chelator deferoxamine. Induction of apoptosis by fluoride was confirmed by acridine orange staining of cell nuclei, by electron-microscopic examination of chromatin condensation, and by oligonucleosomal degradation of DNA. The involvement of G-proteins was confirmed by culture of beta-cells in the presence of pertussis toxin (PTX) prior to exposure to fluoride. PTX did not affect the extent of cell death under control conditions but it consistently, and markedly, enhanced the response to fluoride. The results demonstrate that apoptosis can be induced in pancreatic beta-cells by sustained activation of a G-protein-dependent signaling pathway(s) and they further suggest that a pertussis toxin-sensitive G-protein is involved in attenuation of the response. Treatment of RINm5F pancreatic beta-cells with dibutyrylcAMP resulted in a dose-dependent, saturable increase in cell death, suggesting that a sustained rise in intracellular cAMP may form part of the effector system controlling apoptosis.

Animals↗
PubMed1996

A specific inhibitor of cytosolic phospholipase A2 activity, AACOCF3, inhibits glucose-induced insulin secretion from isolated rat islets.

In pancreatic beta-cells, arachidonic acid accumulation results primarily from phospholipid hydrolysis by phospholipase A2, and activation of this enzyme has been shown to accompany glucose-induced insulin secretion. Inhibitors of phospholipase A2 attenuate the secretory response, although the compounds used to date have not discriminated between cytosolic and secretory isoforms of phospholipase A2. In this work, the specific cytosolic phospholipase A2 inhibitor, AACOCF3, caused a dose-dependent inhibition of glucose-induced insulin secretion from isolated rat islets and this response was significantly relieved by exogenous arachidonic acid. The results suggest that, despite the low levels of expression of cytosolic phospholipase A2 in rat islets, this enzyme contributes to the control of glucose-induced insulin secretion in rat pancreatic beta-cells.

Animals↗
PubMed1996

Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans.

Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas. All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15. Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells. Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.

Base Sequence↗
PubMed1995

Phospholipase A2 expression in human and rodent insulin-secreting cells.

The expression of different isoforms of phospholipase A2 in human and rat islets of Langerhans and in the clonal B-cell lines, HIT-T15 and RINm5F has been investigated, using polyclonal antisera specific to human cytosolic (cPLA2) or human Groups I and II secretory (sPLA2) isoforms. Abundant levels of a 100-kDa protein corresponding to cPLA2 were detected in cytosolic extracts of human islets. A 100-kDa cPLA2 was not detectable in rat islets, RINm5F or HIT-T15 cells using an anti-cPLA2 serum raised against cloned human cPLA2 cDNA, despite the antiserum being cross-reactive with cPLA2 from rat kidney. Human and rat islets were found to express a 21-kDa protein immunoreactive with Group I sPLA2 antiserum. Group II sPLA2 was not detected in human or rat islets. RIN cells did not express detectable levels of either Group I or Group II sPLA2, but HIT cells expressed variable quantities of Group II sPLA2. These differences in PLA2 expression suggest that caution should be exercised when extrapolating conclusions about lipid-derived signalling molecules from insulin-secreting cell lines to normal islets of Langerhans.

Animals↗
PubMed1995

Comparison of colour discrimination and electroretinography in evaluation of visual pathway dysfunction in aretinopathic IDDM patients.

The slow progression of diabetic retinopathy makes it difficult to assess the effects of intervention therapy. There is thus a need for surrogate markers of visual change in diabetes. Colour vision tests and electroretinography (ERG) may be useful in this regard; yet little is known of their relative performance in the assessment of visual dysfunction in diabetes. The aim of the present study was to compare colour discrimination (100 hue test) and ERG indices (oscillatory potentials (OP) and pattern ERG (PERG)) in the evaluation of aretinopathic IDDM patients. Colour discrimination was abnormal in 10 aretinopathic IDDM patients when compared with nine age matched controls; mean square root 100 hue error scores were 10.38 (SD 2.89) versus 4.77 (1.87) respectively, p < 0.01. OP implicit times of the ERG were also abnormal; for example, for right eye, mean OP1 implicit time for diabetics versus OP1 implicit time for controls was 20.1 (2.0) versus 18.6 (1.4) ms, p = 0.03. Comparison of the two techniques suggested that the 100 hue test was more sensitive and more specific than ERG OP implicit times in the detection of diabetic visual dysfunction in these patients.

Adult↗
PubMed1995

Effect of short term changes in blood glucose on visual pathway function in insulin dependent diabetes.

BACKGROUND: Visual pathway function is abnormal in patients with insulin dependent diabetes mellitus (IDDM) without retinopathy, yet the mechanism underlying this abnormality is unknown. It is hypothesised that short term changes in blood glucose level affect visual pathway function in IDDM. METHODS: Colour discrimination was measured in 10 uncomplicated aretinopathic IDDM patients during hyperinsulinaemic clamp, with the Farnsworth Munsell 100 hue test (100 hue test). After stable euglycaemia, patients were made hyperglycaemic (14 mmol/l), maintained euglycaemic (5 mmol/l), and rendered hypoglycaemic (2.5 mmol/l), in random order, on separate occasions at least 1 week apart. RESULTS: Short term (1-2 hours) changes in blood glucose did not affect colour discrimination: mean (SD) 100 hue error score at 2.5 mmol/l was 34 (22) compared with 35 (33) at 5 mmol/l, and 39 (28) at 14 mmol/l. CONCLUSION: These data suggest that short term (1-2 hours) changes in blood glucose are not the mechanism for visual pathway dysfunction in aretinopathic IDDM patients.

Adult↗
PubMed1994

Extent and duration of practice effects on performance with the Farnsworth-Munsell 100-Hue test.

The extent and persistence of practice effects on serial performance in the Farnsworth-Munsell 100-Hue test (100-Hue test) were evaluated in an experiment in which six subjects performed the 100-Hue test up to 17 times over six weeks, and then once more after 7 months. A practice effect occurred which was highly statistically significant for the group as a whole (P < 0.001) and for three individual subjects (P < 0.05). A practice effect was still evident 7 months after the last test performance. Error scores were reduced almost to zero after 5-10 retests so that it was unclear whether the effects of practice had disappeared or whether failure to improve further was a 'floor effect'. As a control against a floor effect, a second experiment was performed in which subjects' performance was impaired by placing neutral density filters in front of their eyes (thus artificially raising their 100-Hue error score). Under these conditions, error scores continued to fall and were halved after 17 tests (P < 0.03). It is concluded that practice has a large effect on 100-Hue test performance which continues over many retests and for many months after testing.

Adult↗
PubMed1994

Effect of diabetes associated increases in lens optical density on colour discrimination in insulin dependent diabetes.

Optical density (OD) of the crystalline lens has been shown in non-diabetics to increase linearly with age over the first five decades and at an increased rate thereafter; in insulin dependent diabetic (IDDM) patients, lens OD increases with age and with duration of diabetes at a rate similar to that in non-diabetics over the age of 60 years. Recently, it has been established that colour discrimination is abnormal in a majority of young patients with uncomplicated IDDM and angiographically normal retinas. Colour discrimination loss was attributed to functional abnormalities in the retina or neural pathways; yet the possibility exists that increases in lens OD may account for part or all of the colour discrimination loss in IDDM. In the present study, colour discrimination was compared in aretinopathic IDDM patients and age-matched controls, and then in a group of aretinopathic IDDM patients individually matched to controls with respect to lens OD. Colour discrimination was significantly worse in diabetic patients than in age-matched controls, and was significantly worse when diabetic patients were compared with controls matched for OD. The magnitude of the difference in 100 hue error score between diabetic patients and OD matched controls was, however, considerably less than the difference between diabetic patients and age-matched controls. These data suggest that colour discrimination loss in aretinopathic IDDM patients cannot be explained solely on the basis of diabetes induced increases in lens OD, but must involve abnormalities of the retina or its neural connections.

Adolescent↗