Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.
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MG Baroni, MG Cavallo, M Mark, L Monetini, B Stoehrer, and P Pozzilli
P Pozzilli, R Buzzetti, C Giovannini, C Gragnoli, and R D G Leslie
Autoimmune diseases affect up to 10% of the population. These diseases may be organ specific (e.g. thyroiditis) or non-organ specific (e.g. rheumatoid arthritis). Immunemediated damage to endocrine cells represents a major fraction of the organ-specific autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM), Hashimoto's thyroiditis and Addison's disease due to adrenalitis. These endocrinopathies are probably due to environmental factors operating in genetically susceptible individuals to induce a destructive process (Leshin et al. 1985). The destructive process is believed to be mediated by T-lymphocytes (Birk & Cohen 1993) although other immune effector systems such as macrophages and cytokines may also be important (Brennan & Feldman 1992). Intervention with immunotherapy has been considered either to ameliorate the clinical disease in its early stage or even to prevent the disease developing (Pozzilli et al. 1992).
Immunotherapy could be specifically targeted at immune effector systems or it could involve non-specific immunomodulation (Adorini et al. 1990).
J. Krug, A. J. K. Williams, P. E. Beales, I. Doniach, E. A. M. Gale, and P. Pozzilli
Autoimmune reactions to parathyroid cells have been observed in human autoimmune polyendocrinopathy, but such findings have not been described in animal models of polyendocrine autoimmunity. We report here three cases of lymphocytic infiltrations in 12 parathyroid glands identified in a total of 18 thyroid glands studied in the non-obese diabetic (NOD) mouse.
The majority of parathyroid-infiltrating lymphocytes possessed the helper/inducer phenotype as defined by the L3T4 monoclonal antibody. Parathyroiditis was accompanied by lymphocytic thyroiditis only on one occasion, whereas in other cases of thyroiditis, lymphocytic infiltration of the parathyroid was undetectable. We conclude that parathyroiditis in the NOD mouse is part of the wide spectrum of autoimmunity observed in this animal model of diabetes.
Journal of Endocrinology (1991) 131, 193–196
M G Cavallo, F Dotta, L Monetini, S Dionisi, M Previti, L Valente, A Toto, U Di Mario, and P Pozzilli
In the present study we have evaluated the expression of different beta-cell markers, islet molecules and autoantigens relevant in diabetes autoimmunity by a human insulinoma cell line (CM) in order to define its similarities with native beta cells and to discover whether it could be considered as a model for studies on immunological aspects of Type 1 diabetes.
First, the positivity of the CM cell line for known markers of neuroendocrine derivation was determined by means of immunocytochemical analysis using different anti-islet monoclonal antibodies including A2B5 and 3G5 reacting with islet gangliosides, and HISL19 binding to an islet glycoprotein. Secondly, the expression and characteristics of glutamic acid decarboxylase (GAD) and of GM2-1 ganglioside, both known to be islet autoantigens in diabetes autoimmunity and expressed by human native beta cells, were investigated in the CM cell line. The pattern of ganglioside expression in comparison to that of native beta cells was also evaluated. Thirdly, the binding of diabetic sera to CM cells reacting with islet cytoplasmic antigens (ICA) was studied by immunohistochemistry. The results of this study showed that beta cell markers identified by anti-islet monoclonal antibodies A2B5, 3G5 and HISL-19 are expressed by CM cells; similarly, islet molecules such as GAD and GM2-1 ganglioside are present and possess similar characteristics to those found in native beta cells; the pattern of expression of other gangliosides by CM cells is also identical to human pancreatic islets; beta cell autoantigen(s) reacting with antibodies present in islet cell antibodies (ICA) positive diabetic sera identified by ICA binding are also detectable in this insulinoma cell line.
We conclude that CM cells show close similarities to native beta cells with respect to the expression of neuroendocrine markers, relevant beta cell autoantigens in Type 1 diabetes (GAD, GM2-1, ICA antigen), and other gangliosides. Therefore, this insulinoma cell line may be considered as an ideal model for studies aimed at investigating autoimmune phenomena occurring in Type 1 diabetes.
Journal of Endocrinology (1996) 150, 113–120
L Monetini, F Barone, L Stefanini, A Petrone, T Walk, G Jung, R Thorpe, P Pozzilli, and MG Cavallo
Enhanced cellular immune response to bovine beta-casein has been reported in patients with type 1 diabetes. In this study we aimed to establish beta-casein-specific T cell lines from newly diagnosed type 1 diabetic patients and to characterise these cell lines in terms of phenotype and epitope specificity. Furthermore, since sequence homologies exist between beta-casein and putative beta-cell autoantigens, reactivity to the latter was also investigated. T cell lines were generated from the peripheral blood of nine recent onset type 1 diabetic patients with different HLA-DQ and -DR genotypes, after stimulation with antigen pulsed autologous irradiated antigen presenting cells (APCs) and recombinant human interleukin-2 (rhIL-2). T cell line reactivity was evaluated in response to bovine beta-casein, to 18 overlapping peptides encompassing the whole sequence of beta-casein and to beta-cell antigens, including the human insulinoma cell line, CM, and a peptide from the beta-cell glucose transporter, GLUT-2. T cell lines specific to beta-casein could not be isolated from HLA-matched and -unmatched control subjects. beta-Casein T cell lines reacted to different sequences of the protein, however a higher frequency of T cell reactivity was observed towards the C-terminal portion (peptides B05-14, and B05-17 in 5/9 and 4/9 T cell lines respectively). Furthermore, we found that 1 out of 9 beta-casein-specific T cell lines reacted also to the homologous peptide from GLUT-2, and that 3 out of 4 of tested cell lines reacted also to extracts of the human insulinoma cell line, CM. We conclude that T cell lines specific to bovine beta-casein can be isolated from the peripheral blood of patients with type 1 diabetes; these cell lines react with multiple and different sequences of the protein particularly towards the C-terminal portion. In addition, reactivity of beta-casein T cell lines to human insulinoma extracts and GLUT-2 peptide was detected, suggesting that the potential cross-reactivity with beta-cell antigens deserves further investigation.