Glycemic Control, Cardiac Autoimmunity, and Long-Term Risk of Cardiovascular Disease in Type 1 Diabetes Mellitus.

BACKGROUND: Poor glycemic control is associated with increased risk of cardiovascular disease (CVD) in type 1 diabetes mellitus (T1DM); however, little is known about mechanisms specific to T1DM. In T1DM, myocardial injury can induce persistent cardiac autoimmunity. Chronic hyperglycemia causes myocardial injury, raising the possibility that hyperglycemia-induced cardiac autoimmunity could contribute to long-term CVD complications in T1DM. METHODS: We measured the prevalence and profiles of cardiac autoantibodies (AAbs) in longitudinal samples from the DCCT (Diabetes Control and Complications Trial) in participants with mean hemoglobin A1c (HbA1c) ≥9.0% (n=83) and ≤7.0% (n=83) during DCCT. We assessed subsequent coronary artery calcification (measured once during years 7-9 in the post-DCCT EDIC [Epidemiology of Diabetes Interventions and Complications] observational study), high-sensitivity C-reactive protein (measured during EDIC years 4-6), and CVD events (defined as nonfatal myocardial infarction, stroke, death resulting from CVD, heart failure, or coronary artery bypass graft) over a 26-year median follow-up. Cardiac AAbs were also measured in matched patients with type 2 diabetes mellitus with HbA1c ≥9.0% (n=70) and ≤7.0% (n=140) and, as a control for cardiac autoimmunity, patients with Chagas cardiomyopathy (n=51). RESULTS: Apart from HbA1c levels, the DCCT groups shared similar CVD risk factors at the beginning and end of DCCT. The DCCT HbA1c ≥9.0% group showed markedly higher cardiac AAb levels than the HbA1c ≤7.0% group during DCCT, with a progressive increase and decrease in AAb levels over time in the 2 groups, respectively ( P<0.001). In the HbA1c ≥9.0% group, 46%, 22%, and 11% tested positive for ≥1, ≥2, and ≥3 different cardiac AAb types, respectively, similar to patients with Chagas cardiomyopathy, compared with 2%, 1%, and 0% in the HbA1c ≤7.0% group. Glycemic control was not associated with AAb prevalence in type 2 diabetes mellitus. Positivity for ≥2 AAbs during DCCT was associated with increased risk of CVD events (4 of 6; hazard ratio, 16.1; 95% CI, 3.0-88.2) and, in multivariable analyses, with detectable coronary artery calcification (13 of 31; odds ratio, 60.1; 95% CI, 8.4-410.0). Patients with ≥2 AAbs subsequently also showed elevated high-sensitivity C-reactive protein levels (6.0 mg/L versus 1.4 mg/L in patients with ≤1 AAbs; P=0.003). CONCLUSIONS: Poor glycemic control is associated with cardiac autoimmunity in T1DM. Furthermore, cardiac AAb positivity is associated with an increased risk of CVD decades later, suggesting a role for autoimmune mechanisms in the development of CVD in T1DM, possibly through inflammatory pathways.

Cardiac autoimmunity as a novel biomarker, mediator, and therapeutic target of heart disease in type 1 diabetes.

Patients with type 1 diabetes (T1D) suffer excess mortality from cardiovascular disease (CVD) that has persisted despite substantial reductions in microvascular complications. Although T1D and type 2 diabetes (T2D) are etiologically distinct, it has generally been assumed that CVD in T1D is "the same disease" as that found in T2D. Here, we review the most recent epidemiological and clinical studies on heart disease in T1D, highlighting differences between CVD in T1D and T2D. In addition, we discuss experimental and clinical evidence for a post-myocardial infarction (MI) autoimmune heart syndrome in T1D, including the development of diagnostic assays which we believe can, for the first time, differentiate between heart disease in T1D and T2D. We postulate that a clinically unrecognized form of chronic myocardial inflammation ("myocarditis") triggered by MI contributes to the poor CVD outcomes in T1D. These findings provide a conceptual shift in our understanding of CVD in T1D and have important diagnostic and therapeutic implications.

Luminex-Coupled EliFACS: A Multiparametric Method to Enumerate and Functionally Characterize Antigen-Specific T cells in Human Peripheral Blood.

We describe a Luminex-coupled EliFACS assay that integrates multiplexing technology, enzyme-linked immunospot (ELISPOT), and intracellular cytokine FACS staining for the detection of multiple parameters of antigen-specific T-cell activation in human peripheral blood. Although our protocol is for measuring T-cell responses against cardiac myosin heavy chain and myelin basic protein, the major autoantigens in myocarditis and multiple sclerosis, respectively, these methods could be used for the detection of T-cell responses to other antigens, including foreign antigens.

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 x 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-2(g7)/H-2(q)) versus homozygosity for H-2(g7), 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-2(g7/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.

Role of impaired central tolerance to α-myosin in inflammatory heart disease.

For more than a half century, autoimmunity has been linked to a diverse array of heart diseases, including rheumatic carditis, myocarditis, Chagas' cardiomyopathy, post-myocardial infarction (Dressler's) syndrome, and idiopathic dilated cardiomyopathy. Why the heart is targeted by autoimmunity in these seemingly unrelated conditions has remained enigmatic. Here, we discuss our recent studies indicating that this susceptibility is mediated by impaired negative selection of autoreactive α-myosin heavy-chain-specific CD4(+) T cells in the thymus of both mice and humans. We describe how this process may place the heart at increased risk for autoimmune attack following ischemic or infectious injury, providing a rationale for the development of antigen-specific tolerogenic therapies.

Myocardial infarction triggers chronic cardiac autoimmunity in type 1 diabetes.

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.

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.

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.

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.

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.

Expression of the B7.1 costimulatory molecule on pancreatic beta cells abrogates the requirement for CD4 T cells in the development of type 1 diabetes.

Although HLA-DQ8 has been implicated as a key determinant of genetic susceptibility to human type 1 diabetes, spontaneous diabetes has been observed in HLA-DQ8 transgenic mice that lack expression of murine MHC class II molecules (mII(-/-)) only when the potent costimulatory molecule, B7.1, is transgenically expressed on pancreatic beta cells. To study the contribution of HLA-DQ8 to the development of diabetes in this model, we crossed RIP-B7.1mII(-/-) mice with a set of transgenic mouse lines that differed in their HLA-DQ8 expression patterns on APC subpopulations, in particular dendritic cells and cortical thymic epithelial cells. Surprisingly, we found that even in the absence of HLA-DQ8 and CD4 T cells, a substantial fraction of the RIP-B7.1mII(-/-) mice developed diabetes. This disease process was remarkable for not only showing insulitis, but also inflammatory destruction of the exocrine pancreas with diffusely up-regulated expression of MHC class I and ICAM-1 molecules. Expression of HLA-DQ8 markedly increased the kinetics and frequency of diabetes, with the most severe disease in the lines with the highest levels of HLA-DQ8 on cortical thymic epithelial cells and the largest numbers of CD4 T cells. However, the adoptive transfer of diabetes was not HLA-DQ8-dependent and disease could be rapidly induced with purified CD8 T cells alone. Expression of B7.1 in the target tissue can thus dramatically alter the cellular and molecular requirements for the development of autoimmunity.