Adrian Bone - Academia.edu (original) (raw)
Papers by Adrian Bone
Biochemical Society Transactions, Jun 1, 1983
Journal of Autoimmunity, 1990
Diabetes and Viruses, 2012
There is extensive epidemiological evidence highlighting a possible aetiological role for entero... more There is extensive epidemiological evidence highlighting a possible aetiological role for enteroviral infection in type 1 diabetes. Direct evidence of the presence of enterovirus in the pancreatic islets of type 1 diabetics is, however, limited, due mainly to the paucity of samples from patients diagnosed recently with the disease. This chapter summarises the evidence implicating enteroviral infection in the human pancreas in type 1 diabetes and considers both factors indicating the presence of virus (viral capsid protein, electron microscopic visualisation of viral particles or expression of viral RNA) and the host response to a viral infection (a “viral footprint”). The relationship of these two indicators of viral infection with two apparently different forms of type 1 diabetes (autoimmune versus fulminant) is discussed. It is hypothesised that differing host responses, and perhaps different genetic variation among the viruses involved, may determine whether an enteroviral infection of beta cells causes (1) A rapid lytic cell death—characteristic of fulminant type 1 diabetes and neonatalcoxsackievirus infection, (2) A persistent infection which evokes an autoimmune reaction to beta cells, eventually resulting in their destruction—autoimmune type 1 diabetes, (3) h little host response and little damage.
Biochemical Society Transactions, Feb 1, 1994
Introduction The characteristic inability to maintain glucose homeostasis in diabetes mellitus re... more Introduction The characteristic inability to maintain glucose homeostasis in diabetes mellitus reflects an absolute or partial insulin deficiency. This lack of insulin is due to an overall reduction in the mass of properly functioning B-cells. Such a reduction occurs as a result of a combination of an increased rate of cell destruction and a decreased rate of cell repair and renewal. While considerable attention has been focused on the elucidation of the factors resulting in €3-cell loss, less information is available regarding possible common genetic control mechanisms, thus implicating alterations in islet cell repair and/or adaptive B-cell regeneration as causative factors in diabetes. There are obvious difficulties in performing studies on islet cell repair and renewal in diabetes in man. However, earlier investigations performed in models of diabetes have provided some insight into the defence and repair mechanisms activated in the islet cells following injury and onset of disease [ 11. The hypothesis that pancreatic B-cells possess defence mechanisms against cell injury gained support from observations that islets are able to survive, and even recover functional activity, after exposure to cytotoxic attack or disturbed metabolism [2, 31. The nature of the islet cell defence process is unclear but is probably due to a combination of a repair/recovery response of surviving cells and an adaptive growth response to a reduced B-cell mass. However, the magnitude and subsequent effectiveness of the islet cell defence and repair mechanisms is dependent on the extent and nature of the cytotoxic insult [4]. A short exposure of rat
Diabetes, Feb 1, 1996
Nitric oxide has been implicated as one possible mediator of interleukin-ip (IL-l)-induced inhibi... more Nitric oxide has been implicated as one possible mediator of interleukin-ip (IL-l)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-a (TNF) and interferon-7 (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14 C-labeled arginine to 14 C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with N G-monomethyl-Larginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN + TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF + IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF + IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitricoxide-mediated events predominate.
Journal of Autoimmunity, Jun 1, 1998
Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior t... more Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet-cells, but the occurrence of autoantibodies is controversial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.
Diabetologia, Mar 1, 1992
The expression of a novel regenerating (reg) gene has been reported previously in the regeneratin... more The expression of a novel regenerating (reg) gene has been reported previously in the regenerating islets of a surgical model of diabetes in rats. We exposed collagenaseisolated rat islets for three days to nutrient and non-nutrient growth factors in minimally supplemented RPMI medium (2.7 retool/1 glucose, 2 % fetal calf serum), and investigated the relationship between reg gene expression and islet cell replication. RNA was prepared from half of the islets by homogenisation in guanidinium isothiocyanate followed by phenol/chloroform extraction. Northern/dot blot analyses were used to semi-quantify reg mRNA. Islet cell replication was estimated by culturing the remaining islets in radiolabelled thymidine to determine de novo DNA synthesis. Thymidine uptake was stimulated by the following factors: 11 mmol/1 glucose (50 % increase); 10 % amino acids (126 % increase); 10 % fetal calf serum (39 % increase); 100 ng/ml insulin (45 % increase); 250ng/ml growth hormone (65% increase); 1.5nmol/1 aldosterone (29% increase); 2U/ml platelet derived growth factor (116 % increase). The results are expressed as a percentage of the thymidine incorporated into control islets cultured in minimal RPMI (1118 _+ 100 (SD) cpm/gg protein, n = 15). Increased islet cell replication was paralleled in each case by a clear rise in reg mRNA expression compared to controls. Furthermore, the rank order for reg gene expression was the same as that for thymidine uptake (r = 0.90). The present findings suggest a clear association between reg gene expression and islet cell replication in vitro, and are the first to demonstrate reg gene expression in response to individual growth factors.
Aims: Insulin producing cells are vulnerable to damage in the immediate period post islet transpl... more Aims: Insulin producing cells are vulnerable to damage in the immediate period post islet transplantation. Our aim was to study the effects of enzymatic digestion on pseudoislet extracellular matrix(ECM) and to determine if a novel three-dimensional microgravity cell culture method would restore and enhance the expression of ECM. Methods: Min6 beta cells were cultured as monolayers and then reconfigured into pseudoislet clusters (PIs) in either static dishes or a microgravity bioreactor. Specific functional markers, PDX1 and ECM proteins, were analysed by immunocytochemistry analysis (ICC). Specific ECM and insulin gene expression was analysed by qRT-PCR. PI digestion with collagenase was performed for a range of time points and cell viability was assessed (HPI assay). PIs were subsequently allowed to recover under static or bioreactor cell culture conditions. Results: PI cells cultured in both static and bioreactor conditions showed nuclear translocation of PDX1 after 1h of glucose stimulation. ECM gene and protein expression was altered in PIs maintained in static and bioreactor cultures. Fibronectin and lamininV expression was significantly increased under bioreactor compared with static conditions. CollagenIV was highly expressed in monolayers compared with PIs cultured in static and bioreactor. Insulin gene expression was markedly increased in bioreactor compared with static culture. Microscopic analysis showed an improved ECM restoration in PIs cultured in the bioreactor compared with static cultures. Conclusion: Our study has shown that microgravity cell culture has the potential to restore levels of ECM in islets following collagenase digestion.
PubMed, 1987
Several immunological responses of the spontaneously diabetic BB rat colony in Edinburgh designat... more Several immunological responses of the spontaneously diabetic BB rat colony in Edinburgh designated (BB/E) have been studied. The proliferative responses to Con A and LPS, ability to make IL-2 and to show NK activity have been studied using diabetic and non-diabetic BB/E rats and normal Wistar rats. Our data suggest that the diabetic animals in the BB/E colony do not have marked deficiencies in any of these parameters. Lymphopenia and depressed T-cell responses do not appear to be a prerequisite for the development of diabetes in the BB/E colony.
![Research paper thumbnail of Metabolic changes in rats caused by chronic feeding of ethyl 2-[5-(4-chlorophenyl)pentyl]oxiran-2-carboxylate: A new hypoglycaemic compound](https://attachments.academia-assets.com/106304651/thumbnails/1.jpg)
](Biochemical Society Transactions, Dec 1, 1981
Metabolic changes in rats caused by chronic feeding of ethyl 2-[5-(4chlorophenyl)pentyl]oxiran-2-... more Metabolic changes in rats caused by chronic feeding of ethyl 2-[5-(4chlorophenyl)pentyl]oxiran-2-carboxylate: A new hypoglycaemic compound PATRICIA P. KOUNDAKJIAN,*? KIM BARTLETT,?
Diabetologia, Nov 5, 2013
Aims/hypothesis Enteroviral infection has been implicated in the development of islet autoimmunit... more Aims/hypothesis Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes.
Journal of Endocrinology, Aug 1, 1977
The regulation of insulin biosynthesis, and insulin and glucagon secretion have been investigated... more The regulation of insulin biosynthesis, and insulin and glucagon secretion have been investigated in a human islet cell adenoma, by incubation of tumour fragments. Both biosynthesis and secretion of insulin were strongly stimulated by incubation of islet tumour cells in the presence of increasing glucose concentrations in the range 2\p=n-\8 mmol/l. However, 20 mm-glucose or 20 mm-glucose plus isobutyl methylxanthine (IBMX), both of which provide potent secretagogues for normal B cells, failed to stimulate proinsulin biosynthesis and secretion from the tumour cells. Overall rates of secretion, expressed as a proportion of total insulin content, were up to 20-fold higher than those expected for normal pancreatic tissue. Glucagon secretion from the tumour was stimulated by low glucose concentrations; normal A cells also respond in this way under these conditions. However, no stimulation of glucagon secretion occurred in the presence of IBMX. There was therefore a major alteration in the regulation both of insulin and glucagon secretion, in that release of neither hormone was stimulated by cyclic AMP. Ultrastructural examination showed the tumour to be rather heterogeneous. A and B cells with normal storage granule content and structure were seen, as well as a rather larger number of B cells containing some granules of atypical appearance. The insulin content of the tumour (13 i.u./g wet wt) was consistent with 6\ p=n-\ 8 % of the tumour cells being B cells.
European journal of endocrinology, Aug 1, 1987
A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied pros... more A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied prospectively to examine the relationship between circulating autoantibodies, islet insulin secretion, pancreatic infiltration, and islet cell replication during the pre-diabetic period. Although a higher incidence of islet cell surface (ICSA) and insulin autoantibodies (IAA) was detected in the diabetes-prone than in the low diabetic-incidence BB/E rats there was no correlation between the two antibodies in individual animals. Moreover, ICSA, but not IAA, were associated with loss
Diabetic Medicine, Sep 10, 1989
Insulin-deficient diabetes causes hypothalamic and pituitary dysfunction. The possible role of hy... more Insulin-deficient diabetes causes hypothalamic and pituitary dysfunction. The possible role of hypothalamic regulatory peptides in mediating these disturbances was investigated in spontaneously diabetic BB/E Wistar rats. Concentrations of 10 regulatory peptides were measured in the central (nucleus-rich) and lateral parts of the hypothalamus in 18 diabetic and 5 non-diabetic BB/E rats. Diabetic rats were treated with either intensified or low-dose insulin schedules to achieve moderate or severe hyperglycaemia (mean blood glucose concentrations, 8 and 20 mmol l-1 respectively). Neuropeptide Y concentration and content in the central hypothalamus were increased by 30-40% in both moderately and severely hyperglycaemic diabetic groups (p less than 0.01). Lateral hypothalamic neuropeptide Y levels did not differ significantly between the groups. The only other peptide to show any significant difference between diabetic and control rats was calcitonin gene-related peptide, whose central hypothalamic concentrations were significantly increased in the severely hyperglycaemic animals. Alterations of hypothalamic neuropeptide Y, which has potent experimental effects on hypothalamo-pituitary function, may contribute to certain neuroendocrine disturbances in insulin-deficient diabetes.
Diabetes, Nov 1, 1984
We have cultured islets from 21.5-day-old fetal rats for 1-7 days in RMPI 1640/10% fetal calf ser... more We have cultured islets from 21.5-day-old fetal rats for 1-7 days in RMPI 1640/10% fetal calf serum containing 2.8 or 11.1 mM glucose to evaluate the differential effects of age visa -vis glycemic stimulation on the maturation of selected components of stimulus-secretion coupling. After 1 day of culture in either media, acute stimulation with 3.0 mg/ml glucose during basal perifusion with 0.5 mg/ml glucose elicited only a small first phase of stimulated insulin secretion and no second phase. The acute exposure to 3.0 mg/ml glucose produced no change in the prevailing high rates of oxygen consumption (Q 02) and caused only minor increments in phosphate efflux (i.e., peak values for phosphate flush of 126 ± 16% of baseline for islets that had been cultured in 11.1 mM glucose and 162 ± 30% for islets cultured in 2.8 mM glucose). After 7 days of culture in 11.1 mM glucose, acute stimulation with 3.0 mg/ml glucose increased Q 02 (as in adult islets) and effected acute increases in the AT 32 P and GP 2 P content of prelabeled islets. The 3.0 mg/ml glucose also triggered phosphate flush to 599 ± 45% of baseline and elicited first as well as early second phases of stimulated insulin secretion that replicated the performance of adult islets. By contrast, after 7 days of culture in 2.8 mM glucose, similar acute exposures of fetal islets to 3.0 mg/ml glucose effected only a small first phase and a negligible second phase of stimulated insulin secretion despite the occurrence of the same increments in Q O2 as after culture in 11.1 mM glucose, highly significant increases in AT 32 P and GT 32 P,
Diabetologia, Jan 16, 2014
Biochemical Journal, Sep 15, 1977
![Research paper thumbnail of Causal interpretation requires appropriate study design. Reply to Priest PC [letter]](https://attachments.academia-assets.com/106304628/thumbnails/1.jpg)
](Diabetologia, Apr 29, 2009
Journal of Clinical Virology, Nov 1, 2010
Background: The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notor... more Background: The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notoriously difficult and often requires inherent knowledge about the specific virus being sought. For this reason, there is an ongoing need for reagents and methods which can identify a range of different virus types in paraffin embedded tissue. Objectives: The aim of this study was to optimise and validate the use of antisera directed against dsRNA (>50 bp in length) in paraffin-embedded formalin-fixed tissue samples. Study design: dsRNA antisera were optimised for use in a range of virally-infected tissue culture cells, Coxsackie-infected mice and human tissues. The specificity of labelling was confirmed by pre-adsorption of antisera with poly-IC and by digestion of dsRNA with RNaseIII. Results: Two different polyclonal dsRNA antisera (J2 and K1) were capable of recognising dsRNA encoded by all the multiple different viral types (including (+) ssRNA viruses, dsRNA viruses and DNA viruses) tested in paraffin-embedded formalin fixed infected cells and tissues. In contrast, the enteroviral vp1 antisera detected only a subset of the (+) ssRNA viruses tested. Staining was not seen in uninfected cells or in uninfected control tissues. Positive staining was ablated following incubation of antisera with poly-IC or by pre-treating sections with RNaseIII prior to staining. Conclusions: The dsRNA antisera J2 and K1 are useful for the detection of viral infection in formalin-fixed, paraffin-embedded, human tissue samples.
Biochemical Society Transactions, Jun 1, 1983
Journal of Autoimmunity, 1990
Diabetes and Viruses, 2012
There is extensive epidemiological evidence highlighting a possible aetiological role for entero... more There is extensive epidemiological evidence highlighting a possible aetiological role for enteroviral infection in type 1 diabetes. Direct evidence of the presence of enterovirus in the pancreatic islets of type 1 diabetics is, however, limited, due mainly to the paucity of samples from patients diagnosed recently with the disease. This chapter summarises the evidence implicating enteroviral infection in the human pancreas in type 1 diabetes and considers both factors indicating the presence of virus (viral capsid protein, electron microscopic visualisation of viral particles or expression of viral RNA) and the host response to a viral infection (a “viral footprint”). The relationship of these two indicators of viral infection with two apparently different forms of type 1 diabetes (autoimmune versus fulminant) is discussed. It is hypothesised that differing host responses, and perhaps different genetic variation among the viruses involved, may determine whether an enteroviral infection of beta cells causes (1) A rapid lytic cell death—characteristic of fulminant type 1 diabetes and neonatalcoxsackievirus infection, (2) A persistent infection which evokes an autoimmune reaction to beta cells, eventually resulting in their destruction—autoimmune type 1 diabetes, (3) h little host response and little damage.
Biochemical Society Transactions, Feb 1, 1994
Introduction The characteristic inability to maintain glucose homeostasis in diabetes mellitus re... more Introduction The characteristic inability to maintain glucose homeostasis in diabetes mellitus reflects an absolute or partial insulin deficiency. This lack of insulin is due to an overall reduction in the mass of properly functioning B-cells. Such a reduction occurs as a result of a combination of an increased rate of cell destruction and a decreased rate of cell repair and renewal. While considerable attention has been focused on the elucidation of the factors resulting in €3-cell loss, less information is available regarding possible common genetic control mechanisms, thus implicating alterations in islet cell repair and/or adaptive B-cell regeneration as causative factors in diabetes. There are obvious difficulties in performing studies on islet cell repair and renewal in diabetes in man. However, earlier investigations performed in models of diabetes have provided some insight into the defence and repair mechanisms activated in the islet cells following injury and onset of disease [ 11. The hypothesis that pancreatic B-cells possess defence mechanisms against cell injury gained support from observations that islets are able to survive, and even recover functional activity, after exposure to cytotoxic attack or disturbed metabolism [2, 31. The nature of the islet cell defence process is unclear but is probably due to a combination of a repair/recovery response of surviving cells and an adaptive growth response to a reduced B-cell mass. However, the magnitude and subsequent effectiveness of the islet cell defence and repair mechanisms is dependent on the extent and nature of the cytotoxic insult [4]. A short exposure of rat
Diabetes, Feb 1, 1996
Nitric oxide has been implicated as one possible mediator of interleukin-ip (IL-l)-induced inhibi... more Nitric oxide has been implicated as one possible mediator of interleukin-ip (IL-l)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-a (TNF) and interferon-7 (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14 C-labeled arginine to 14 C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with N G-monomethyl-Larginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN + TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF + IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF + IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitricoxide-mediated events predominate.
Journal of Autoimmunity, Jun 1, 1998
Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior t... more Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet-cells, but the occurrence of autoantibodies is controversial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.
Diabetologia, Mar 1, 1992
The expression of a novel regenerating (reg) gene has been reported previously in the regeneratin... more The expression of a novel regenerating (reg) gene has been reported previously in the regenerating islets of a surgical model of diabetes in rats. We exposed collagenaseisolated rat islets for three days to nutrient and non-nutrient growth factors in minimally supplemented RPMI medium (2.7 retool/1 glucose, 2 % fetal calf serum), and investigated the relationship between reg gene expression and islet cell replication. RNA was prepared from half of the islets by homogenisation in guanidinium isothiocyanate followed by phenol/chloroform extraction. Northern/dot blot analyses were used to semi-quantify reg mRNA. Islet cell replication was estimated by culturing the remaining islets in radiolabelled thymidine to determine de novo DNA synthesis. Thymidine uptake was stimulated by the following factors: 11 mmol/1 glucose (50 % increase); 10 % amino acids (126 % increase); 10 % fetal calf serum (39 % increase); 100 ng/ml insulin (45 % increase); 250ng/ml growth hormone (65% increase); 1.5nmol/1 aldosterone (29% increase); 2U/ml platelet derived growth factor (116 % increase). The results are expressed as a percentage of the thymidine incorporated into control islets cultured in minimal RPMI (1118 _+ 100 (SD) cpm/gg protein, n = 15). Increased islet cell replication was paralleled in each case by a clear rise in reg mRNA expression compared to controls. Furthermore, the rank order for reg gene expression was the same as that for thymidine uptake (r = 0.90). The present findings suggest a clear association between reg gene expression and islet cell replication in vitro, and are the first to demonstrate reg gene expression in response to individual growth factors.
Aims: Insulin producing cells are vulnerable to damage in the immediate period post islet transpl... more Aims: Insulin producing cells are vulnerable to damage in the immediate period post islet transplantation. Our aim was to study the effects of enzymatic digestion on pseudoislet extracellular matrix(ECM) and to determine if a novel three-dimensional microgravity cell culture method would restore and enhance the expression of ECM. Methods: Min6 beta cells were cultured as monolayers and then reconfigured into pseudoislet clusters (PIs) in either static dishes or a microgravity bioreactor. Specific functional markers, PDX1 and ECM proteins, were analysed by immunocytochemistry analysis (ICC). Specific ECM and insulin gene expression was analysed by qRT-PCR. PI digestion with collagenase was performed for a range of time points and cell viability was assessed (HPI assay). PIs were subsequently allowed to recover under static or bioreactor cell culture conditions. Results: PI cells cultured in both static and bioreactor conditions showed nuclear translocation of PDX1 after 1h of glucose stimulation. ECM gene and protein expression was altered in PIs maintained in static and bioreactor cultures. Fibronectin and lamininV expression was significantly increased under bioreactor compared with static conditions. CollagenIV was highly expressed in monolayers compared with PIs cultured in static and bioreactor. Insulin gene expression was markedly increased in bioreactor compared with static culture. Microscopic analysis showed an improved ECM restoration in PIs cultured in the bioreactor compared with static cultures. Conclusion: Our study has shown that microgravity cell culture has the potential to restore levels of ECM in islets following collagenase digestion.
PubMed, 1987
Several immunological responses of the spontaneously diabetic BB rat colony in Edinburgh designat... more Several immunological responses of the spontaneously diabetic BB rat colony in Edinburgh designated (BB/E) have been studied. The proliferative responses to Con A and LPS, ability to make IL-2 and to show NK activity have been studied using diabetic and non-diabetic BB/E rats and normal Wistar rats. Our data suggest that the diabetic animals in the BB/E colony do not have marked deficiencies in any of these parameters. Lymphopenia and depressed T-cell responses do not appear to be a prerequisite for the development of diabetes in the BB/E colony.
Biochemical Society Transactions, Dec 1, 1981
Metabolic changes in rats caused by chronic feeding of ethyl 2-[5-(4chlorophenyl)pentyl]oxiran-2-... more Metabolic changes in rats caused by chronic feeding of ethyl 2-[5-(4chlorophenyl)pentyl]oxiran-2-carboxylate: A new hypoglycaemic compound PATRICIA P. KOUNDAKJIAN,*? KIM BARTLETT,?
Diabetologia, Nov 5, 2013
Aims/hypothesis Enteroviral infection has been implicated in the development of islet autoimmunit... more Aims/hypothesis Enteroviral infection has been implicated in the development of islet autoimmunity in type 1 diabetes and enteroviral antigen expression has been detected by immunohistochemistry in the pancreatic beta cells of patients with recent-onset type 1 diabetes. However, the immunohistochemical evidence relies heavily on the use of a monoclonal antibody, clone 5D8/1, raised against an enteroviral capsid protein, VP1. Recent data suggest that the clone 5D8/1 may also recognise non-viral antigens; in particular, a component of the mitochondrial ATP synthase (ATP5B) and an isoform of creatine kinase (CKB). Therefore, we evaluated the fidelity of immunolabelling by clone 5D8/1 in the islets of patients with type 1 diabetes.
Journal of Endocrinology, Aug 1, 1977
The regulation of insulin biosynthesis, and insulin and glucagon secretion have been investigated... more The regulation of insulin biosynthesis, and insulin and glucagon secretion have been investigated in a human islet cell adenoma, by incubation of tumour fragments. Both biosynthesis and secretion of insulin were strongly stimulated by incubation of islet tumour cells in the presence of increasing glucose concentrations in the range 2\p=n-\8 mmol/l. However, 20 mm-glucose or 20 mm-glucose plus isobutyl methylxanthine (IBMX), both of which provide potent secretagogues for normal B cells, failed to stimulate proinsulin biosynthesis and secretion from the tumour cells. Overall rates of secretion, expressed as a proportion of total insulin content, were up to 20-fold higher than those expected for normal pancreatic tissue. Glucagon secretion from the tumour was stimulated by low glucose concentrations; normal A cells also respond in this way under these conditions. However, no stimulation of glucagon secretion occurred in the presence of IBMX. There was therefore a major alteration in the regulation both of insulin and glucagon secretion, in that release of neither hormone was stimulated by cyclic AMP. Ultrastructural examination showed the tumour to be rather heterogeneous. A and B cells with normal storage granule content and structure were seen, as well as a rather larger number of B cells containing some granules of atypical appearance. The insulin content of the tumour (13 i.u./g wet wt) was consistent with 6\ p=n-\ 8 % of the tumour cells being B cells.
European journal of endocrinology, Aug 1, 1987
A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied pros... more A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied prospectively to examine the relationship between circulating autoantibodies, islet insulin secretion, pancreatic infiltration, and islet cell replication during the pre-diabetic period. Although a higher incidence of islet cell surface (ICSA) and insulin autoantibodies (IAA) was detected in the diabetes-prone than in the low diabetic-incidence BB/E rats there was no correlation between the two antibodies in individual animals. Moreover, ICSA, but not IAA, were associated with loss
Diabetic Medicine, Sep 10, 1989
Insulin-deficient diabetes causes hypothalamic and pituitary dysfunction. The possible role of hy... more Insulin-deficient diabetes causes hypothalamic and pituitary dysfunction. The possible role of hypothalamic regulatory peptides in mediating these disturbances was investigated in spontaneously diabetic BB/E Wistar rats. Concentrations of 10 regulatory peptides were measured in the central (nucleus-rich) and lateral parts of the hypothalamus in 18 diabetic and 5 non-diabetic BB/E rats. Diabetic rats were treated with either intensified or low-dose insulin schedules to achieve moderate or severe hyperglycaemia (mean blood glucose concentrations, 8 and 20 mmol l-1 respectively). Neuropeptide Y concentration and content in the central hypothalamus were increased by 30-40% in both moderately and severely hyperglycaemic diabetic groups (p less than 0.01). Lateral hypothalamic neuropeptide Y levels did not differ significantly between the groups. The only other peptide to show any significant difference between diabetic and control rats was calcitonin gene-related peptide, whose central hypothalamic concentrations were significantly increased in the severely hyperglycaemic animals. Alterations of hypothalamic neuropeptide Y, which has potent experimental effects on hypothalamo-pituitary function, may contribute to certain neuroendocrine disturbances in insulin-deficient diabetes.
Diabetes, Nov 1, 1984
We have cultured islets from 21.5-day-old fetal rats for 1-7 days in RMPI 1640/10% fetal calf ser... more We have cultured islets from 21.5-day-old fetal rats for 1-7 days in RMPI 1640/10% fetal calf serum containing 2.8 or 11.1 mM glucose to evaluate the differential effects of age visa -vis glycemic stimulation on the maturation of selected components of stimulus-secretion coupling. After 1 day of culture in either media, acute stimulation with 3.0 mg/ml glucose during basal perifusion with 0.5 mg/ml glucose elicited only a small first phase of stimulated insulin secretion and no second phase. The acute exposure to 3.0 mg/ml glucose produced no change in the prevailing high rates of oxygen consumption (Q 02) and caused only minor increments in phosphate efflux (i.e., peak values for phosphate flush of 126 ± 16% of baseline for islets that had been cultured in 11.1 mM glucose and 162 ± 30% for islets cultured in 2.8 mM glucose). After 7 days of culture in 11.1 mM glucose, acute stimulation with 3.0 mg/ml glucose increased Q 02 (as in adult islets) and effected acute increases in the AT 32 P and GP 2 P content of prelabeled islets. The 3.0 mg/ml glucose also triggered phosphate flush to 599 ± 45% of baseline and elicited first as well as early second phases of stimulated insulin secretion that replicated the performance of adult islets. By contrast, after 7 days of culture in 2.8 mM glucose, similar acute exposures of fetal islets to 3.0 mg/ml glucose effected only a small first phase and a negligible second phase of stimulated insulin secretion despite the occurrence of the same increments in Q O2 as after culture in 11.1 mM glucose, highly significant increases in AT 32 P and GT 32 P,
Diabetologia, Jan 16, 2014
Biochemical Journal, Sep 15, 1977
Diabetologia, Apr 29, 2009
Journal of Clinical Virology, Nov 1, 2010
Background: The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notor... more Background: The detection of viral infection in paraffin-embedded, formalin-fixed tissue is notoriously difficult and often requires inherent knowledge about the specific virus being sought. For this reason, there is an ongoing need for reagents and methods which can identify a range of different virus types in paraffin embedded tissue. Objectives: The aim of this study was to optimise and validate the use of antisera directed against dsRNA (>50 bp in length) in paraffin-embedded formalin-fixed tissue samples. Study design: dsRNA antisera were optimised for use in a range of virally-infected tissue culture cells, Coxsackie-infected mice and human tissues. The specificity of labelling was confirmed by pre-adsorption of antisera with poly-IC and by digestion of dsRNA with RNaseIII. Results: Two different polyclonal dsRNA antisera (J2 and K1) were capable of recognising dsRNA encoded by all the multiple different viral types (including (+) ssRNA viruses, dsRNA viruses and DNA viruses) tested in paraffin-embedded formalin fixed infected cells and tissues. In contrast, the enteroviral vp1 antisera detected only a subset of the (+) ssRNA viruses tested. Staining was not seen in uninfected cells or in uninfected control tissues. Positive staining was ablated following incubation of antisera with poly-IC or by pre-treating sections with RNaseIII prior to staining. Conclusions: The dsRNA antisera J2 and K1 are useful for the detection of viral infection in formalin-fixed, paraffin-embedded, human tissue samples.