Blake Middleton

Infectious Th1 and Th2 autoimmunity in diabetes-prone mice

Summary: In the non‐obese diabetic (NOD) mouse, a Thl‐biased autoimmune response arises spontaneously against glutamic acid decarboxylase, concurrent with the onset of insulitis. Subsequently, Th1‐type autore‐activity spreads intra‐ and intermolecularly to other β‐cell autoantigens (βCAAs), suggesting that a spontaneous Th1 cascade underlies disease progression. Induction of Th2 immunity to a single βCAA results in the spreading of Th2–type T‐cell and humoral responses to other βCAAs in an infectious manner. Thus, both Th1 and Th2 autoimmunity can evolve in amplificatory cascades defined by site‐specific, but not antigen‐specific, positive feedback circuits. Despite the continued presence of Th1 auto‐immunity, the induction of Th2 spreading is associated with active tolerance to βCAAs and reduced disease incidence. With disease progression there is an attenuation of βCAA‐inducible Th2 spreading, presumably because of a reduced availability of uncommitted βCAA‐reactive precursor T cells. We discuss the implications of these findings for the rational design of antigen‐based immunotherapeutics.

Modulating the potency of an activator in a yeast in vitro transcription system.

The intrinsic stimulatory potential or potency of a eukaryotic gene activator is controlled by the interaction between the activation domain and the transcriptional machinery. To further understand this interaction, we undertook a biochemical study to identify parameters that could be used to modulate activator potency. We considered how varying the number of activation domains, their flexibility, and the number of promoter sites affects potency in a yeast nuclear extract. The effects of GAL4 derivatives bearing either one, two, or four herpes simplex virus VP16 activation domains (amino acids 413 to 454) were measured on DNA templates containing one or two GAL4 sites in a Saccharomyces cerevisiae nuclear extract. We found that multimerized VP16 activation domains acted synergistically to increase the potency of the activators. The spacing between the activation domains was critical, such that the increased flexibility imparted by a protein linker contributed to increased activator potency. With highly potent activators, the levels of transcription stimulated on a single site were saturating, whereas the stimulatory effect of weaker activators increased with the number of sites. We discuss how these biochemical studies relate to the mechanism of gene activation and synergy in a yeast in vitro system.

Compensatory Energetic Relationships between Upstream Activators and the RNA Polymerase II General Transcription Machinery

Activation of RNA polymerase II transcriptionin vivo and in vitro is synergistic with respect to increasing numbers of activator binding sites or increasing concentrations of activator. The Epstein-Barr virus ZEBRA protein manifests both forms of synergy during activation of genes involved in the viral lytic cycle. The synergy has an underlying mechanistic basis that we and others have proposed is founded largely on the energetic contributions of (i) upstream ZEBRA binding to its sites, (ii) the general pol II machinery binding to the core promoter, and (iii) interactions between ZEBRA and the general machinery. We hypothesize that these interactions form a network for which a minimum stability must be attained to activate transcription. One prediction of this model is that the energetic contributions should be reciprocal, such that a strong core promoter linked to a weak upstream promoter would be functionally analogous to a weak core linked to a strong upstream promoter. We tested this view by measuring the transcriptional response after systematically altering the upstream and core promoters. Our data provide strong qualitative support for this hypothesis and provide a theoretical basis for analyzing Epstein-Barr virus gene regulation.

BCG Vaccine-Induced Neuroprotection in a Mouse Model of Parkinson's Disease

There is a growing interest in using vaccination with CNS antigens to induce autoreactive T cell responses that home to damaged areas in the CNS and ameliorate neurodegenerative disease. Neuroprotective vaccine studies have focused on administering oligodendrocyte antigens or Copaxone® in complete Freunds adjuvant (CFA). Theoretical considerations, however, suggest that vaccination with a neuronal antigen may induce more robust neuroprotective immune responses. We assessed the neuroprotective potential of vaccines containing tyrosine hydroxylase (a neuronal protein involved in dopamine synthesis) or Copaxone® in CFA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinsons disease. Surprisingly, we observed that the main beneficial factor in these vaccines was the CFA. Since the major immunogenic component in CFA is Mycobacterium tuberculosis, which closely related to the bacille Calmette-Guérin (BCG) that is used in human vaccines, we tested BCG vaccination in the MPTP mouse model. We observed that BCG vaccination partially preserved markers of striatal dopamine system integrity and prevented an increase in activated microglia in the substantia nigra of MPTP-treated mice. These results support a new neuroprotective vaccine paradigm in which general (nonself-reactive) immune stimulation in the periphery can limit potentially deleterious microglial responses to a neuronal insult and exert a neurorestorative effect in the CNS. Accordingly, BCG vaccination may provide a new strategy to augment current treatments for a wide range of neuropathological conditions.

Antigen-Based Therapies Using Ignored Determinants of β Cell Antigens Can More Effectively Inhibit Late-Stage Autoimmune Disease in Diabetes-Prone Mice

As organ-specific autoimmune diseases do not become manifest until well-advanced, interventive therapies must inhibit late-stage disease processes. Using a panel of immunogenic peptides from various β cell Ags, we evaluated the factors influencing the efficacy of Ag-based therapies in diabetes-prone NOD mice with advanced disease. The ability of the major β cell autoantigen target determinants (TDs) to prime Th2 responses declined sharply between 6 and 12 wk of age, whereas the ability of immunogenic ignored determinants (IDs) of β cell Ags to prime Th2 responses was unaffected by the disease process. The different patterns of TD and ID immunogenicity (even from the same β cell Ag) may be due to the exhaustion of uncommitted TD-reactive, but not ID-reactive, T cell pools by recruitment into the autoimmune cascade. Therapeutic efficacy was associated with a peptide’s immunogenicity and ability to promote Th2 spreading late in the disease process but not its affinity for I-Ag7 or its expression pattern (β cell specific/nonspecific or rare/abundant). Characterization of some IDs revealed them to be “absolute” cryptic determinants. Such determinants have little impact on T cell selection, leaving large precursor T cell pools available for priming by synthetic peptides. Traditional Ag-based therapeutics using whole autoantigens or their TDs cannot prime responses to such determinants. These findings suggest a new strategy for designing more efficacious Ag-based therapeutics for late-stage autoimmune diseases.

Bacillus Calmette‐Guerin vaccine‐mediated neuroprotection is associated with regulatory T‐cell induction in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mouse model of Parkinson's disease

We previously showed that, in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of Parkinsons disease (PD), vaccination with bacillus Calmette‐Guerin (BCG) prior to MPTP exposure limited the loss of striatal dopamine (DA) and dopamine transporter (DAT) and prevented the activation of nigral microglia. Here, we conducted BCG dose studies and investigated the mechanisms underlying BCG vaccinations neuroprotective effects in this model. We found that a dose of 1 × 106 cfu BCG led to higher levels of striatal DA and DAT ligand binding (28% and 42%, respectively) in BCG‐vaccinated vs. unvaccinated MPTP‐treated mice, but without a significant increase in substantia nigra tyrosine hydroxylase‐staining neurons. Previous studies showed that BCG can induce regulatory T cells (Tregs) and that Tregs are neuroprotective in models of neurodegenerative diseases. However, MPTP is lymphotoxic, so it was unclear whether Tregs were maintained after MPTP treatment and whether a relationship existed between Tregs and the preservation of striatal DA system integrity. We found that, 21 days post‐MPTP treatment, Treg levels in mice that had received BCG prior to MPTP were threefold greater than those in MPTP‐only‐treated mice and elevated above those in saline‐only‐treated mice, suggesting that the persistent BCG infection continually promoted Treg responses. Notably, the magnitude of the Treg response correlated positively with both striatal DA levels and DAT ligand binding. Therefore, BCG vaccine‐mediated neuroprotection is associated with Treg levels in this mouse model. Our results suggest that BCG‐induced Tregs could provide a new adjunctive therapeutic approach to ameliorating pathology associated with PD and other neurodegenerative diseases.

Major histocompatibility complex class I molecules modulate embryonic neuritogenesis and neuronal polarization

We studied cultured hippocampal neurons from embryonic wildtype, major histocompatibility complex class I (MHCI) heavy chain-deficient (K(b)D(b)-/-) and NSE-D(b) (which have elevated neuronal MHCI expression) C57BL/6 mice. K(b)D(b)-/- neurons displayed slower neuritogenesis and establishment of polarity, while NSE-D(b) neurons had faster neurite outgrowth, more primary neurites, and tended to have accelerated polarization. Additional studies with ß2M-/- neurons, exogenous ß2M, and a self-MHCI monomer suggest that free heavy chain cis interactions with other surface molecules can promote neuritogenesis while tripartite MHCI interactions with classical MHCI receptors can inhibit axon outgrowth. Together with the results of others, MHCI appears to differentially modulate neuritogenesis and synaptogenesis.

Multimodality Imaging of β-Cells in Mouse Models of Type 1 and 2 Diabetes

OBJECTIVE β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 “MIP-TF” mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy. RESEARCH DESIGN AND METHODS MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes. RESULTS MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve–intraperitoneal glucose tolerance test (AUC-IPGTT). CONCLUSIONS MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.

A potential role for shed soluble major histocompatibility class I molecules as modulators of neurite outgrowth.

The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCIs actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed “NSE-Db”) whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off its transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptors signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCIs biological activities in the nervous system.

Multimodality Imaging of β-Cells in Mouse Models of Type I and II Diabetes

OBJECTIVE β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 “MIP-TF” mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy. RESEARCH DESIGN AND METHODS MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes. RESULTS MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve–intraperitoneal glucose tolerance test (AUC-IPGTT). CONCLUSIONS MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.