Animal models have little to teach us about type 1 diabetes: 2. In opposition to this proposal.
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Animals↗
Biomedical subjects
M von Herrath
Publications and source records attributed to M von Herrath.
Prophylaxis of autoimmune diabetes by antigen based immune modulation: are we there yet?
Autoimmune type 1 diabetes (early onset/juvenile and slow onset T1D) is a disease mediated by autoreactive T cells accompanied by autoantibodies against islet associated antigens. The genetic predisposition, the association with certain HLA alleles (DR4 and DQ8) as well as the possibility to identify high-risk subjects by autoantibody measurement, fueled hope in regards to potential prophylaxis by vaccination. Numerous preclinical studies along with accumulating evidence for the role of certain autoantigens and putative dominant epitopes created the premises of clinical trials addressing this objective. However, the disappointing outcomes of many of such trials made it clear that significant challenges to clinical application of this concept must be addressed: we do not know at this point, how to choose the most appropriate set of autoantigens, how to optimally use them as a vaccine, whether to define and enroll only a subset of recipients, how to monitor their T cell responses to the vaccine and lastly, which the end-points of clinical T1D trials from an immunologic or metabolic perspective should be? Herein, we will approach some of the opportunities and obstacles associated with this intriguing strategy to fight autoimmune diabetes based on promising novel insight gained form studies with animal models.
Autoantigens↗
About CD4pos CD25pos regulatory cells.
Autoreactive cells that escape from thymic negative selection are either anergized or counter-regulated in the peripheral lymphoid organs by regulatory T (Treg) cells. Most investigated Treg cells are CD4pos cells expressing the alpha chain of the IL-2 receptor (CD25). Absence of this particular population leads to the development of a wide range of autoimmune disorders, suggesting a critical role for CD4pos CD25pos cells in the maintenance of normal immune homeostasis. Numerous studies have aimed at unraveling the mechanisms of regulation, focusing mainly on naturally occurring Treg cells (generated in naive animals) and regulation of CD4pos autoreactive cells. In contrast, generation following antigenic priming of CD4pos CD25pos Treg cells with non-self (viral) antigen specificity and regulation of virus-activated CD4pos and CD8pos cells remain to be investigated. In this review, we describe the data collected over the past few years of intense focus on CD4pos CD25pos Treg cells and present our approach to investigate the generation of antigen-specific Treg cells during the course of an anti-viral immune response.
Animals↗
Plasmid vaccination with insulin B chain prevents autoimmune diabetes in nonobese diabetic mice.
The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-gamma-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4(-/-) NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.
Animals↗
Variable effects of transgenic c-Maf on autoimmune diabetes.
Autoimmune diabetes is associated with T helper 1 polarization, but protection from disease can be provided by the application of T helper 2 (Th2) cytokines. To test whether genetic manipulation of T-cells can provide protective Th2 responses, we developed transgenic mice in which T-cells express the interleukin-4-specific transcription factor c-Maf. When crossed with a transgenic model that combines a class II restricted T-cell receptor specific for influenza hemagglutinin with islet beta-cell expression of hemagglutinin, the c-Maf transgene provided significant protection from spontaneous autoimmunity but not from adoptively transferred diabetes. In a second transgenic model in which islet cells express the lymphocytic choriomeningitis virus nucleoprotein, the virus infection triggers autoimmune diabetes within a few weeks involving both CD4 and CD8 T-cells; here too transgenic c-Maf provided significant protection. Surprisingly, when the c-Maf transgene was backcrossed with the NOD model of spontaneous disease, no protection was evident. Thus, transgenic c-Maf can strongly influence autoimmune disease development in some models, but additional factors, such as background genetic differences, can influence the potency of its effect.
Animals↗
Report from the 1st International NOD Mouse T-Cell Workshop and the follow-up mini-workshop.
A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.
Animals↗
Neither B lymphocytes nor antibodies directed against self antigens of the islets of Langerhans are required for development of virus-induced autoimmune diabetes.
We evaluated the role of the humoral arm of the immune response in causing or contributing to virus-induced diabetes. Transgenic mice expressing the nucleoprotein (NP) or glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP) in pancreatic beta cells (RIP-LCMV) and RIP-LCMV mice with genetic dysfunction of B cells (RIP-LCMV x microMT/microMT) were compared for development of diabetes after challenge with LCMV. After inoculation with LCMV, B and T lymphocytes and macrophages infiltrated into pancreatic islets in RIP-LCMV mice, and over 50% of these mice generated Abs against host insulin or glutamate decarboxylase. However, neither B cells nor the autoantibodies played a direct role in the initiation, kinetics, or severity of the virus-induced diabetes as judged by comparing disease in RIP-LCMV mice to littermates whose functional B cells were genetically eliminated. Furthermore, the quality and quantity of T lymphocyte and macrophage infiltration was similar in the B cell-deficient and non-B cell-deficient RIP-LCMV mice. Although the development of autoantibodies to islet Ags had no direct influence on the pathogenesis of insulin-dependent (type 1) diabetes mellitus, it served as a prediabetes marker, as such autoantibodies were often elevated before the onset of disease. Hence, the RIP-LCMV model is not only useful for understanding the pathogenetic mechanisms of how islets are destroyed and spared but also for evaluating therapeutic strategies before onset of clinical diabetes.
Acute Disease↗
Local IL-4 expression in the lung reduces pulmonary influenza-virus-specific secondary cytotoxic T cell responses.
We studied the effect of lung-specific IL-4 expression on the T cell response during primary and secondary heterologous infection with influenza virus by using transgenic mice that express IL-4 under a lung-specific promoter. Subsequent to primary infection with a type A/H1N1 influenza virus these transgenic mice exhibited similar local recruitment of CD4(+) and CD8(+) T cells and only slightly decreased virus-specific CTL activity. However, during secondary challenge with a heterologous influenza virus, the local infiltration with virus-specific, MHC class I-restricted CD8(+) T cells was significantly decreased compared to that of nontransgenic littermates. The ability of IL-4 transgenic mice to clear the heterologous infection was delayed but not abrogated. This was associated with a faster virus-neutralizing antibody response in IL-4 transgenic mice and with their ability to mount significant Th1 responses even in the presence of increased local IL-4 expression. Our observations demonstrate a negative regulatory effect of IL-4 on memory Tc1/CD8(+) T cells, but are also consistent with complementary mechanisms important for virus clearance such as virus-neutralizing antibodies. The reduction of memory CTL in the presence of IL-4 might have consequences for understanding the course of influenza infection in situations where T(H)2 immunity is increased.
Animals↗
Virus-induced diabetes in a transgenic model: role of cross-reacting viruses and quantitation of effector T cells needed to cause disease.
Virus-specific cytotoxic T lymphocytes (CTL) at frequencies of >1/1, 000 are sufficient to cause insulin-dependent diabetes mellitus (IDDM) in transgenic mice whose pancreatic beta cells express as "self" antigen a protein from a virus later used to initiate infection. The inability to generate sufficient effector CTL for other cross-reacting viruses that fail to cause IDDM could be mapped to point mutations in the CTL epitope or its COO(-) flanking region. These data indicate that IDDM and likely other autoimmune diseases are caused by a quantifiable number of T cells, that neither standard epidemiologic markers nor molecular analysis with nucleic acid probes reliably distinguishes between viruses that do or do not cause diabetes, and that a single-amino-acid change flanking a CTL epitope can interfere with antigen presentation and development of autoimmune disease in vivo.
Amino Acid Sequence↗
Use of a high-affinity peptide that aborts MHC-restricted cytotoxic T lymphocyte activity against multiple viruses in vitro and virus-induced immunopathologic disease in vivo.
Binding of a specific peptide(s) from a viral protein to major histocompatibility complex (MHC) class I molecules is a critical step in the activation of CD8(+) cytotoxic T lymphocytes (CTLs). Once activated, CTLs can cause lethal disease in an infected host, for example, by killing virus-containing ependymal and ventricular cells in the central nervous system or viral protein-expressing beta cells in the pancreatic islets of Langerhans. Here we describe the usage of a designed (not natural) high-affinity peptide to compete with viral peptide(s)-MHC binding. This peptide blocks virus-induced CTL-mediated disease both in the CNS and in the pancreatic islets in vivo. Further, the blocking peptide aborts MHC-restricted killing of target cells by CTLs generated to three separate viruses: lymphocytic choriomeningitis virus, influenza virus, and simian virus 40.
Acute Disease↗
Thymic tolerance to only one viral protein reduces lymphocytic choriomeningitis virus-induced immunopathology and increases survival in perforin-deficient mice.
The outcome of viral infections is dependent on the amount of tissue destruction caused either by direct lysis of infected cells and/or by immunopathology resulting from the immune response to the virus. We investigated whether induction of tolerance to only one viral protein could reduce immunopathology caused by nonlytic lymphocytic choriomeningitis virus (LCMV) in perforin-deficient hosts. Earlier studies had shown that LCMV infection results in aplastic anemia and death in most of these mice and that this is associated with bone marrow infiltration by antiviral cytotoxic T lymphocytes (CTL) that secrete inflammatory cytokines. We report here that perforin-deficient mice exhibit severe immunopathology in multiple organs that is characterized by infiltration of anti-LCMV CTL that secrete large amounts of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Importantly, this immunopathology is significantly reduced and long-term survival of LCMV infection is increased in perforin-deficient mice expressing LCMV nucleoprotein (NP) in the thymus (and therefore deleting most of their LCMV-NP CTL) compared to the situation in thymus nonexpressors. This is due to the selective reduction of NP-specific CTL responses and their inflammatory-cytokine (IFN-gamma and TNF-alpha) secretion and to a lack of pathogenetically relevant compensatory responses to other viral proteins. Thus, "selective reduction" of the antiviral immune response to only one viral protein can significantly reduce inflammatory immunopathology and might be a therapeutic possibility for certain nonlytic infections.
Animals↗
Pathological changes in the islet milieu precede infiltration of islets and destruction of beta-cells by autoreactive lymphocytes in a transgenic model of virus-induced IDDM.
RIP-LCMV transgenic mice that express the viral glycoprotein (GP) or nucleoprotein (NP) from lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter (RIP) in pancreatic beta-cells develop autoimmune diabetes (IDDM) after infection with LCMV. Previous reports have described that the viral infection activates naive, potentially autoreactive CD8+ cytotoxic T-lymphocytes (CTL) that are present in the periphery of these mice, thus leading to the breaking of immunological unresponsiveness to the viral self-antigen expressed on beta-cells. However, we find that adoptive transfer of such CTL that were active in vitro and in vivo into uninfected RIP-LCMV recipients rarely resulted in hyperglycemia nor in insulitis, despite their ability to home to the islets and induce peri-insulitis. These observations indicated that, in addition to activated autoreactive lymphocytes, other factor(s) were required for beta-cell destruction. The present study shows that upregulation of MHC class II molecules associated with the attraction/activation of antigen presenting cells (APCs) to the islets occurs as soon as 2 days after LCMV inoculation of transgenic mice, clearly before CD4+ and CD8+ lymphocytes are found entering the islets (days 6 and 7 after LCMV inoculation). In contrast, although some MHC class II upregulation is also found in islets of non-transgenic mice 2-4 days after LCMV infection, no insulitis or IDDM develops and MHC is downregulated to normal (pre-infection) levels by day 7-10 in these mice. Associated with the activation of APCs and MHC upregulation observed in transgenic mice, viral (LCMV) infection of islets was detectable 2 days post-viral inoculation in some mice. Thus, beta-cell destruction by activated autoreactive lymphocytes is a multifactorial process that is likely to require changes within the islet milieu or dysfunction of islets.
Adoptive Transfer↗
Transforming growth factor-beta fails to inhibit allograft rejection or virus-induced autoimmune diabetes in transgenic mice.
Transgenic mice whose pancreata express transforming growth factor-beta (TGF-beta) directed by an insulin promoter (Ins-TGF-beta mice) were used to assess the effect of local TGF-beta1 on allograft rejection and on autoimmune diabetes occurring as a cross-reaction to viral antigens. Pancreatic TGF-beta1 did not delay allograft rejection, nor did it inhibit autoimmune diabetes after lymphocytic choriomeningitis infection of double transgenic mice (LCMV/TGF-beta1 mice). These results suggest that local TGF-beta1 does not serve as an immunosuppressive agent for allograft rejection or virus-mediated autoimmune diabetes.
Animals↗
Virus-induced autoimmune disease: transgenic approach to mimic insulin-dependent diabetes mellitus and multiple sclerosis.
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Amino Acid Sequence↗
Virus-induced autoimmune disease: transgenic approach to mimic insulin-dependent diabetes mellitus and other autoimmune diseases.
The technology of cloning viral genes and expressing them in vivo under cell-specific promoters allows to dissect the role of viruses, host self proteins, host genetics and immune responses in the complex etiology of autoimmune disease. Expression of a viral transgene, that is really a marker for a host "self" protein per se in beta cells of the islets of Langerhans, need not cause disease. In our model, expression of a viral gene was not associated with disease over the lifetime of the animal. However, when the host becomes infected with a virus encoding the same gene as the transgene or one closely related to it, a resultant immune response directed against the virus also recognizes the transgene leading to progressive T-cell-mediated response and destruction of the tissue expressing the viral ("self") gene, leading to autoimmune disease. This multifactorial process is influenced by whether the viral transgene is expressed in the thymus as well as in the disease-related cell or target tissue. Thymic expression influences negative selection of responder lymphocytes and thus delays the onset of the autoimmune disorder. Further, the MHC haplotype or other background genes of an individual undergoing autoimmune dysfunction play a role in the affinity of binding of the transgene products to the MHC molecule and influence the degree of negative selection that occurs, thereby influencing the vigor of the resulting immune response. The current ability to express host or viral genes in unique cell populations, and to make double- or triple-tg mice in which various cytokine genes or lymphocyte activation genes can be expressed along with the viral gene, offers a unique possibility for molecular dissection of autoimmunity. With the information on hand, approaches to the prevention and treatment of human autoimmune disease are likely to be uncovered. Finally, animal models are available in which the onset, progression and control of molecular mimicry can be evaluated. Future studies should define roles played by cytokines, bystander and immune-specific cross-reactivity to viruses and other microbes in several autoimmune diseases.
Animals↗
Simian immunodeficiency virus (SIV)-specific CTL in cerebrospinal fluid and brains of SIV-infected rhesus macaques.
Simian immunodeficiency virus (SIV)-specific CD8+ CTL were isolated from blood, cerebrospinal fluid, and brains of rhesus macaques infected i.v. with SIV. CTL were found as early as 1 wk postinfection and their appearance correlated with a decrease of viral Ag (p27) found in the blood. CTL isolated from cerebrospinal fluid and/or brain often recognized different viral proteins than CTL isolated from blood, suggesting either a unique migratory pattern to the central nervous system or a difference in activation/retention of lymphocytes in these compartments.
Amino Acid Sequence↗
Sensitization to self (virus) antigen by in situ expression of murine interferon-gamma.
Autoimmune disease results from inflammatory destruction of tissues by aberrant self-reactive lymphocytes. We studied the autoimmune potential of T lymphocytes immunologically ignorant of viral antigens acting as self antigens and whether the host defense molecule IFN-gamma could stimulate these cells to cytotoxic competency. For this purpose, we produced double transgenic mice expressing pancreatic IFN-gamma as well as lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP) or glycoprotein (GP) antigen. 100% of the NP+/IFN-gamma+ mice became diabetic before 2 mo of age, while none of the NP single transgenic littermates and only 10% of IFN-gamma single transgenic littermates did. Strikingly, NP+/IFN-gamma+ mice spontaneously developed cytotoxic T lymphocyte activity on LCMV-infected targets and vaccinia virus-NP-infected ones without prior LCMV infection but NP+/IFN-gamma- mice did not, which indicates specific sensitization to the viral antigen by IFN-gamma. These results suggest that lymphocytes ignorant of self antigens can be activated by IFN-gamma released after immunologic stimulation such as viral infection. This mechanism may account for the loss of apparent tolerance to self antigens in autoimmune diseases such as insulin-dependent diabetes mellitus.
Animals↗
Repeat determination of left ventricular wall thickness from mass and volume during one cardiac cycle for the calculation of left ventricular wall stress parameters.
Left ventricular end-diastolic wall stress, end-systolic wall stress, and systolic stress-time integral are important parameters to characterize left ventricular load and function. To obtain these parameters, left ventricular pressure, volume, and wall thickness data must be determined at short time intervals throughout one cardiac cycle. However, the measurement of wall thickness at short intervals (i.e., 20 ms) throughout a cardiac cycle is tedious. Furthermore, measurements of wall thickness are less accurate at end-systole compared with end-diastole. For these reasons we developed a computer program for calculating wall thickness at short intervals (20 ms) throughout the cardiac cycle from one single determination of left ventricular wall mass and repetitive measurements of left ventricular (LV) volume.
Computer Simulation↗