Myra A. Lipes

Recessive tolerance to preproinsulin 2 reduces but does not abolish type 1 diabetes

Although autoimmune diseases can be initiated by immunization with a single antigen, it is not clear whether a single self antigen is essential for the initiation and, perhaps, the perpetuation of spontaneous autoimmunity. Some studies have suggested that insulin may represent an essential autoantigen in type 1 diabetes. Here we show that unlike tolerance to glutamic acid decarboxylase, tolerance to transgenically overexpressed preproinsulin 2 substantially reduced the onset and severity of type 1 diabetes in nonobese diabetic mice. However, some mice still developed type 1 diabetes, suggesting that insulin is a key, but not absolutely essential, autoantigen. The results are consistent with the idea that the human IDDM2 locus controls susceptibility to type 1 diabetes by regulating intrathymic preproinsulin expression.

MRI of insulitis in autoimmune diabetes

Development of imaging techniques that would allow the mapping of immune cells in vivo could greatly aid our understanding of a number of inflammatory and autoimmune diseases. The current study focused on imaging of autoimmune destruction of the insulin‐producing pancreatic beta‐cells by cytotoxic lymphocytes, the cause of insulin‐dependent diabetes mellitus (IDDM; Type 1 diabetes). Using high‐resolution MR microscopy and a conventional clinical MR imaging system, it was possible to visualize the infiltration of immune cells in the diabetic mouse pancreas. Mouse lymphocytes were visualized by magnetically labeling them with recently developed magnetic nanoparticles (CLIO‐Tat). The results from this study could potentially lead to detection of immune infiltration during diabetes formation in vivo, which would be one of the earliest parameters of disease development. Magn Reson Med 47:751–758, 2002.

Impaired thymic tolerance to α-myosin directs autoimmunity to the heart in mice and humans

Autoimmunity has long been linked to myocarditis and its sequela, dilated cardiomyopathy, the leading causes of heart failure in young patients. However, the underlying mechanisms are poorly defined, with most clinical investigations focused on humoral autoimmunity as the target for intervention. Here, we show that the α-isoform of myosin heavy chain (α-MyHC, which is encoded by the gene Myh6) is the pathogenic autoantigen for CD4+ T cells in a spontaneous mouse model of myocarditis. Further, we found that Myh6 transcripts were absent in mouse medullary thymic epithelial cells (mTECs) and peripheral lymphoid stromal cells, which have been implicated in mediating central and peripheral T cell tolerance, respectively. Transgenic expression of α-MyHC in thymic epithelium conferred tolerance to cardiac myosin and prevented myocarditis, demonstrating that α-MyHC is a primary autoantigen in this disease process. Remarkably, we found that humans also lacked α-MyHC in mTECs and had high frequencies of α-MyHC-specific T cells in peripheral blood, with markedly augmented T cell responses to α-MyHC in patients with myocarditis. Since α-MyHC constitutes a small fraction of MyHC in human heart, these findings challenge the longstanding notion that autoimmune targeting of MyHC is due to its cardiac abundance and instead suggest that it is targeted as a result of impaired T cell tolerance mechanisms. These results thus support a role for T cell-specific therapies for myocarditis.

Transgenic Insulin (B:9-23) T-Cell Receptor Mice Develop Autoimmune Diabetes Dependent Upon RAG Genotype, H-2g7 Homozygosity, and Insulin 2 Gene Knockout

A series of recent studies in humans and the NOD mouse model have highlighted the central role that autoimmunity directed against insulin, in particular the insulin B chain 9-23 peptide, may play in the pathogenesis of type 1 diabetes. Both pathogenic and protective T-cell clones recognizing the B:9-23 peptide have been produced. This report describes the successful creation of BDC12-4.1 T-cell receptor (TCR) transgenic mice with spontaneous insulitis in F1 mice (FVB × NOD) and spontaneous diabetes in NOD.RAG−/− (backcross 1 generation). Disease progression is heterogeneous and is modified by a series of genetic factors including heterozygosity (H-2g7/H-2q) versus homozygosity for H-2g7, the presence of additional T-/B-cell receptor–rearranged genes (RAG+ versus RAG−/−), and the insulin 2 gene knockout (the insulin gene expressed in the NOD thymus). Despite lymphopenia, 40% of H-2g7/g7 BDC12-4.1 TCR+ RAG−/− Ins2−/− mice are diabetic by 10 weeks of age. As few as 13,500 transgenic T-cells from a diabetic TCR+ RAG−/− mouse can transfer diabetes to an NOD.scid mouse. The current study demonstrates that the BDC12-4.1 TCR is sufficient to cause diabetes at NOD backcross 1, bypassing polygenic inhibition of insulitis and diabetogenesis.

A Spontaneous Model for Autoimmune Myocarditis Using the Human MHC Molecule HLA-DQ8

Genome-wide analyses have shown that the MHC class II region is the principal locus that confers susceptibility to a number of human autoimmune diseases. Due to the high degree of linkage disequilibrium across the MHC, it has been difficult to dissect the contribution of individual genes to disease susceptibility. As a result, intensive efforts have been made to generate mice transgenic for human class II molecules as models of autoimmune disease. However, in every case, additional manipulations—such as immunization with Ag in adjuvant, expression of immunostimulants on target tissues, or coexpression of TCR transgenes—have been required to induce disease. In this study, we show that expression of the human HLA-DQ8 (DQA1*0301/DQB1*0302) molecule alone in three lines of transgenic nonobese diabetic murine class II-deficient (mII−/−) mice results in the spontaneous development of autoimmune myocarditis. The disease shares key features of human myocarditis and was characterized by lymphocytic infiltrates in the myocardium and cardiac myocyte destruction, circulating IgG autoantibodies against cardiac myosin heavy chain, and premature death due to heart failure. We demonstrate that myocarditis could be transferred into healthy HLA-DQ8+RAG-1−/−mII−/− nonobese diabetic recipients with lymphocytes, but not sera. It has been widely thought that autoimmune myocarditis is of infectious etiology, with the immune responses arising secondary to cardiac damage from pathogens. These studies provide direct experimental evidence that spontaneous autoimmune myocarditis can occur in the absence of infection and that expression of HLA-DQ8 confers susceptibility to this organ-specific autoimmune disease.

Myocardial Parvovirus B19 Persistence: Lack of Association with Clinicopathologic Phenotype in Adults with Heart Failure

Background—Multiple viruses have been isolated from the heart, but their significance remains controversial. We sought to determine the prevalence of cardiotropic viruses in endomyocardial biopsy (EMB) samples from adult patients with heart failure (HF) and to define the clinicopathologic profile of patients exhibiting viral positivity. Methods and Results—EMB from 100 patients (median ejection fraction, 30%; interquartile range [IQR], 20% to 45%) presenting for cardiomyopathy evaluation (median symptom duration, 5 months; IQR, 1 to 13 months) were analyzed by polymerase chain reaction for adenovirus, cytomegalovirus, enteroviruses, Epstein-Barr virus, and parvovirus B19. Each isolate was sequenced, and viral load was determined. Parvovirus B19 was the only virus detected in EMB samples (12% of subjects). No patient had antiparvovirus IgM antibodies, but all had IgG antibodies, suggesting viral persistence. The clinical presentation of parvovirus-positive patients was markedly heterogeneous with both acute and chronic HF, variable ventricular function, and ischemic cardiomyopathy. No patient met Dallas histopathologic criteria for active or borderline myocarditis. Two patients with a positive cardiac MRI and presumed “parvomyocarditis” had similar viral loads to autopsy controls without heart disease. The oldest parvovirus-positive patients were positive for genotype 2, suggesting lifelong persistence in the myocardium. Conclusions—Parvovirus B19 was the only virus isolated from EMB samples in this series of adult patients with HF from the United States. Positivity was associated with a wide array of clinical presentations and HF phenotypes. Our studies do not support a causative role for parvovirus B19 persistence in HF and, therefore, advocate against the use of antiviral therapy for these patients.Background— Multiple viruses have been isolated from the heart, but their significance remains controversial. We sought to determine the prevalence of cardiotropic viruses in endomyocardial biopsy (EMB) samples from adult patients with heart failure (HF) and to define the clinicopathologic profile of patients exhibiting viral positivity. Methods and Results— EMB from 100 patients (median ejection fraction, 30%; interquartile range [IQR], 20% to 45%) presenting for cardiomyopathy evaluation (median symptom duration, 5 months; IQR, 1 to 13 months) were analyzed by polymerase chain reaction for adenovirus, cytomegalovirus, enteroviruses, Epstein-Barr virus, and parvovirus B19. Each isolate was sequenced, and viral load was determined. Parvovirus B19 was the only virus detected in EMB samples (12% of subjects). No patient had antiparvovirus IgM antibodies, but all had IgG antibodies, suggesting viral persistence. The clinical presentation of parvovirus-positive patients was markedly heterogeneous with both acute and chronic HF, variable ventricular function, and ischemic cardiomyopathy. No patient met Dallas histopathologic criteria for active or borderline myocarditis. Two patients with a positive cardiac MRI and presumed “parvomyocarditis” had similar viral loads to autopsy controls without heart disease. The oldest parvovirus-positive patients were positive for genotype 2, suggesting lifelong persistence in the myocardium. Conclusions— Parvovirus B19 was the only virus isolated from EMB samples in this series of adult patients with HF from the United States. Positivity was associated with a wide array of clinical presentations and HF phenotypes. Our studies do not support a causative role for parvovirus B19 persistence in HF and, therefore, advocate against the use of antiviral therapy for these patients.

Metabolic decompensation in pump users due to lispro insulin precipitation

Small, short term studies show that lispro insulin (Humalog; Eli Lilly & Co, Indianapolis, IN), commonly used in pump therapy, is stable in insulin pumps.1 However, in agreement with reports by others,2 we have noted several patients who have developed erratic and unpredictable glucose fluctuations with lispro insulin that have resolved when the treatment was changed to buffered regular insulin (Velosulin; Novo Nordisk, Princeton, NJ) and aspart insulin (Novolog; Novo Nordisk, Princeton, NJ). We have …

Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice

Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP‐GlcNAc: Galβ 1–3GalNAcαRβ 1–6‐N‐acetylglucosaminyltransferase (core 2 GlcNAc‐T), a developmentally regulated enzyme of O‐linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc‐T with severe increase in cardiac core 2 GlcNAc‐T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O‐glycan and increases of c‐fos gene expression and AP‐1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc‐T also induced c‐fos promoter activation, mitogen activated‐protein kinase (MAPK) phosphorylation, Trk receptor glyco‐sylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc‐T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.—Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N‐acetylglyco‐saminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice. FASEB J. 13, 2329–2337 (1999)

Myocardial Infarction Triggers Chronic Cardiac Autoimmunity in Type 1 Diabetes

Acute myocardial infarction triggers an autoimmune attack on the heart in a mouse model of type 1 diabetes and in human type 1 diabetic patients. After a Heart Attack, Diabetics Skip a Beat There’s no way to sugarcoat it: Patients with type 1 (autoimmune) diabetes experience more severe downstream symptoms after a myocardial infarction (MI)—heart attack—than do nondiabetic individuals. MI triggers an inflammatory response, which promotes cardiac repair in healthy patients but has unclear effects in those with preexisting autoimmunity. Gottumukkala et al. now show that the increased morbidity and mortality of type 1 diabetics after MI may be caused by the immune system attacking the damaged heart. In a humanized mouse model of diabetes, the authors found that MI gives rise to post-infarction autoimmune (PIA) syndrome, which is characterized by lymphocytic infiltration and both cardiac autoantibody and autoimmune T cell responses, which are intriguingly restricted to cardiac myosin. Gottumukkala et al. then detected cardiac tissue–directed autoantibodies after MI in 83% of human type 1 diabetic patients tested, but these autoantibodies were absent from type 2 diabetic patients who had suffered MI. These data suggest that an autoimmune disease such as type 1 diabetes may predispose individuals to further autoimmunity (PIA) in the context of inflammation and tissue damage. The antigen-restricted autoimmune response suggests that antigen-specific tolerance induction may be used to improve the health of diabetics after MI. Patients with type 1 diabetes (T1D) suffer excessive morbidity and mortality after myocardial infarction (MI) that is not fully explained by the metabolic effects of diabetes. Acute MI is known to trigger a profound innate inflammatory response with influx of mononuclear cells and production of proinflammatory cytokines that are crucial for cardiac repair. We hypothesized that these same pathways might exert “adjuvant effects” and induce pathological responses in autoimmune-prone T1D hosts. Here, we show that experimental MI in nonobese diabetic mice, but not in control C57BL/6 mice, results in a severe post-infarction autoimmune (PIA) syndrome characterized by destructive lymphocytic infiltrates in the myocardium, infarct expansion, sustained cardiac autoantibody production, and T helper type 1 effector cell responses against cardiac (α-)myosin. PIA was prevented by inducing tolerance to α-myosin, demonstrating that immune responses to cardiac myosin are essential for this disease process. Extending these findings to humans, we developed a panel of immunoassays for cardiac autoantibody detection and found autoantibody positivity in 83% post-MI T1D patients. We further identified shared cardiac myosin autoantibody signatures between post-MI T1D patients and nondiabetic patients with myocarditis, which were absent in post-MI type 2 diabetic patients, and confirmed the presence of myocarditis in T1D by cardiac magnetic resonance imaging techniques. These data provide experimental and clinical evidence for a distinct post-MI autoimmune syndrome in T1D. Our findings suggest that PIA may contribute to worsened post-MI outcomes in T1D and highlight a role for antigen-specific immunointervention to selectively block this pathway.

Genetic engineering of insulin expression in nonislet cells: implications for beta-cell replacement therapy for insulin-dependent diabetes mellitus.

Abstract Insulin-dependent diabetes mellitus (IDDM) occurs as a consequence of autoimmune destruction of the insulin-producing pancreatic β-cells. Although progress has been made in the field of islet transplantation, an appealing alternative strategy for β-cell replacement therapy for IDDM is to target insulin expression to non-islet cells. We have recently generated transgenic nonobese diabetic (NOD) mice in which insulin gene expression was targeted to proopiomelanocortin (POMC)-expressing cells of the intermediate lobe (IL) of the pituitary. We have shown that POMC-expressing IL pituitary cells secreted large amounts of mature insulin, similar to islet β-cells. However, in contrast to the insulin-producing islet β-cells, the insulin-producing IL pituitary cells were not attacked by the immune system. Remarkably, transplantation of small amounts of the transgenic IL tissues into diabetic NOD mice resulted in the restoration of near-normogylcemia and the complete reversal of diabetic symptoms. In separate experiments, IL allografts showed enhanced viability and were highly vascularized, compared with similarly transplanted islet allografts. These features are highly advantageous in the transplantation setting and demonstrate the considerable potential of these non-islet cell types for insulin-gene delivery in IDDM.