Protection from type 1 diabetes in the face of high levels of activated autoaggressive lymphocytes in a viral transgenic mouse model crossed to the SV129 strain.

In comparing the incidence of virally induced type 1 diabetes in F(1) crosses of RIP-LCMV mice to three different mouse strains identical at the major histocompatibility complex H-2D(b) locus, we surprisingly found that disease development was reduced by 80% in F(1) crosses to the SV129 genetic background and by 60% after eight backcrosses to the original C57BL/6 RIP-LCMV mice. In this model, diabetes is strongly dependent on a virally induced H-2D(b)-restricted cytotoxic T-cell (CTL) response. Importantly, numbers and effector functions of autoaggressive CD4 and CD8 lymphocytes were not decreased in the protected mice, and CTLs were still able to kill syngeneic islet cells in vitro with equal efficacy compared with CTLs from the original RIP-LCMV strain. Furthermore, CTLs were able to extravasate into islets in vivo, and no evidence for induction of regulatory cells was observed. However, regeneration of beta-cells in islets under "attack" occurred only in the protected SV129-crossed animals, whereas it was not evident at any time in any mice that developed diabetes. Thus, genetic factors can "override" the diabetogenic potential of high numbers of autoaggressive lymphocytes through, for example, increased islet regeneration. This finding has important implications for interpreting numbers and pathogenicity of autoreactive lymphocytes in prediabetic patients of genetically diverse backgrounds.

Tolerance induction with agonist peptides recognized by autoaggressive lymphocytes is transient: therapeutic potential for type 1 diabetes is limited and depends on time-point of administration, choice of epitope and adjuvant.

Immunization with agonist peptides recognized by autoaggressive lymphocytes has been used successfully in several animal models for type 1 diabetes (T1D) or multiple sclerosis (MS) to prevent disease. Depending on the timing of immunization, use of adjuvant and route of administration either elimination of autoaggressive T cells or induction of regulation reflected by cytokine shifts were described. Since it was also reported that such agonist peptides could enhance autoimmunity by activating aggressive lymphocytes, our goal was to re-evaluate their efficacy in an antigen-specific model of virally-induced T1D that allowed us to precisely track the autoaggressive response. We find that rather than the route of administration (oral versus sc) the precise timing is important for inducing tolerance to self-antigens. Tolerance is transient and only immunization during a susceptible phase 10 to 20 days prior to the induction of disease but not in prediabetic mice resulted in protection. Further, use of a stronger adjuvant (CFA) compared to IFA enhanced the protective effect. Mechanistically, a transient loss of autoaggressive T cells was responsible for preventing disease, the effect was quantitative and no regulatory lymphocytes or cytokine shifts were induced by any of our treatments. Thus, MHC class I-restricted agonist peptides might only find a limited use in treating autoimmune disorders, because tolerance induction is transient and treatment has to be given very early, ideally prior to activation of the aggressive response.

Frequency with which surgeons undertake pancreaticoduodenectomy determines length of stay, hospital charges, and in-hospital mortality.

Others have suggested that in certain technically challenging operations, outcome and experience are related. Because pancreaticoduodenectomy is a technically complex procedure, this study was undertaken to evaluate mortality, length of hospital stay, and hospital charges when compared to volume of experience. The database of the State of Florida Agency for Health Care Administration was queried for pancreaticoduodenectomies undertaken during a recent 33-month period. Length of stay, hospital charges, and in-hospital mortality were stratified by the frequency of pancreaticoduodenectomy. A total of 282 surgeons performed 698 pancreaticoduodenectomies over 33 months. Eighty-nine percent of surgeons performed one pancreaticoduodenectomy per year or less and accounted for 52% of the procedures. Overall mortality rate was 5.1%. Average hospital charges were $72,171.64. The more frequently pancreaticoduodenectomy was undertaken, the shorter the hospital stay (P = 0.025, regression analysis) and the lower the hospital charges (P = 0.008, regression analysis) and in-hospital mortality (P = 0.036, log likelihood ratio test). Surgeons who undertake pancreaticoduodenectomy more frequently have patients with shorter hospital stays, lower hospital charges, and lower in-hospital mortality rates, independent of hospital volume. Variations exist among surgeons and among different areas of the state. Data regarding cost and mortality are available for use in programs of cost and quality improvement.

Virus-induced autoimmune diabetes: most beta-cells die through inflammatory cytokines and not perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes.

Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic beta-cells. The main factors directly implicated in beta-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse beta-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the beta-cells. Unexpectedly, we found that the resulting perforin-mediated killing of beta-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of beta-cells also requires a direct effect of gamma-interferon (IFN-gamma), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-gamma receptor on their beta-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most beta-cells occurs as cytokine-mediated death and requires IFN-gama in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes.

Disruption of the STAT4 signaling pathway protects from autoimmune diabetes while retaining antiviral immune competence.

The role of the STAT4 signaling pathway in autoimmune diabetes was investigated using the rat insulin promoter lymphocytic choriomeningitis virus model of virally induced autoimmune diabetes. Abrogation of STAT4 signaling significantly reduced the development of CD4+-T cell-dependent but not CD4+-T cell-independent diabetes, illustrating the fine-tuned kinetics involved in the pathogenesis of autoimmunity. However, the absence of STAT4 did not prevent the generation of autoreactive Th1/Tc1 T cell responses, as well as protective antiviral immunity. Protection from insulin-dependent diabetes mellitus was associated with decreased numbers of autoreactive CTL precursors in the pancreas and the spleen and a general as well as Ag-specific reduction of IFN-gamma secretion by T lymphocytes. A shift from Th1 to Th2 T cell immunity was not observed. Hence, our results implicate both CTL and cytokines in beta cell destruction. Selective inhibition of the STAT4 signal transduction pathway might constitute a novel and attractive approach to prevent clinical insulin-dependent diabetes mellitus in prediabetic individuals at risk.

Autoreactive CD4+ T cells protect from autoimmune diabetes via bystander suppression using the IL-4/Stat6 pathway.

Targeted immune regulation can be achieved by use of tissue-specific T cells and offers the potential for organ-specific suppression of destructive autoimmune processes. Here, we report the generation and characterization of insulin B chain-specific "autoreactive" CD4+ regulatory T cells that locally suppress diabetogenic T cell responses against an unrelated self-antigen (viral transgene) in a virus-induced model for type 1 diabetes. Interleukin 4 (IL-4) is essential for prevention of diabetes since regulatory T cells cannot be induced in the absence of IL-4 or stat6 (IL-4 signaling pathway). Our observations demonstrate that autoreactive regulatory T cells can suppress autoreactive destructive T cell activity of differential antigenic specificity locally in the pancreatic draining lymph node, probably via cytokine-mediated modulation of antigen-presenting cells.

DNA immunization to prevent autoimmune diabetes.

Mice expressing lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP) as a transgene in their beta cells develop insulin-dependent diabetes mellitus (IDDM) only after LCMV infection. Inoculation of plasmid DNA encoding the insulin B chain reduced the incidence of IDDM by 50% in this model. The insulin B-chain DNA vaccination was effective through induction of regulatory CD4 lymphocytes that react with the insulin B chain, secrete IL-4, and locally reduce activity of LCMV-NP-autoreactive cytotoxic T lymphocytes in the pancreatic draining lymph node. In contrast, similar vaccination with plasmids expressing the LCMV viral ("self") protein did not prevent IDDM, because no such regulatory cells were induced. Thus, DNA immunization with plasmids expressing self-antigens might constitute a novel and attractive therapeutic approach to prevent autoimmune diseases, if the antigens are carefully preelected for an ability to induce regulatory lymphocytes in vivo.

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.

In vivo treatment with a MHC class I-restricted blocking peptide can prevent virus-induced autoimmune diabetes.

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-gamma secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-gamma and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.

Low-affinity cytotoxic T-lymphocytes require IFN-gamma to clear an acute viral infection.

The majority of the response of cytotoxic T-lymphocytes (CTL) to lymphocytic choriomeningitis virus (LCMV) in H-2d mice is directed toward one epitope located on the nucleoprotein (NP, aa 118-126), and usually no primary responses to other epitopes are detectable. Previous studies have shown that thymic expression of lymphocytic choriomeningitis virus-nucleoprotein (LCMV-NP) in H-2d transgenic mice (Thy-NP mice) leads to deletion of high-affinity anti-LCMV-NP CTL by negative selection. Selection is incomplete, so that low-affinity NP-specific CTL pass through the thymus and are detectable in the periphery. To analyze the importance of interferon-gamma (IFN-gamma) in the ability of low-affinity antiviral CTL to clear an acute viral infection, double transgenic mice were generated that are IFN-gamma deficient and express the NP of LCMV in the thymus (Thy-NP x IFN-gamma -/- mice). When infected with LCMV, these bigenic mice were unable to clear the infection despite generating low-affinity primary antiviral CTL, and they became persistently infected. In contrast, IFN-gamma competent Thy-NP mice cleared LCMV within 7-8 days and IFN-gamma deficient mice that did not express NP in their thymus generated high-affinity CTL that terminated an acute LCMV infection within 10-12 days post-viral challenge. Persistently infected IFN-gamma deficient mice selectively depleted LCMV-specific CTL and displayed reduced levels of antigen-presenting cells in the spleen, and 60% of these mice died at 2-3 months postinfection. Thus, IFN-gamma is required for clearing an acute viral infection in the absence of a high-affinity CTL response. In the absence of IFN-gamma persistent viral infection results despite the presence of low-affinity CTL.

The extinction of naturally occurring conditioned reactions in psychoactive substance users: analog studies.

In the present series of studies we develop an analog approach for the study of conditioned reactions to drug stimuli. The analog we study is the naturally occurring conditioned reaction of salivation at the sight of a lemon. We show that this conditioned reaction can be extinguished, that spontaneous recovery occurs, and that the conditioned reaction increases after "relapse." Further, we show that massed extinction trials lead to greater extinction than do spaced trials. This analog provides an approach that can be used to develop cue-exposure treatments that minimize spontaneous recovery from extinction and reduce the likelihood of relapse.