Benefits of BCG-induced metabolic switch from oxidative phosphorylation to aerobic glycolysis in autoimmune and nervous system diseases.

The most commonly used vaccine worldwide, bacillus Calmette-Guerin (BCG), appears to have the ability to restore blood sugar control in humans with early-onset but long-duration type 1 diabetes when a repeat vaccination strategy is used. This is a process that may be driven by a metabolic switch from overactive oxidative phosphorylation to accelerated aerobic glycolysis and a reset of the immune system. BCG is a live, attenuated strain of Mycobacteria bovis, a cousin of M. tuberculosis. Humans and Mycobacteria, which are found in the environment and in warm-blooded hosts, share a long coevolutionary history. In recent times, humans have had fewer exposures to these and other microorganisms that historically helped shape the immune response. By 're-introducing' an attenuated form of Mycobacteria via BCG vaccination, humans might benefit from an immunological perspective, a concept supported by a growing body of data in autoimmunity and robust data on the nonspecific immune effects of BCG related to protection from diverse infections and early mortality. New findings of immune and metabolic defects in type 1 diabetes that can be corrected with repeat BCG vaccination suggest that this therapeutic strategy may be applicable in other diseases with inadequate aerobic glycolysis, including Parkinson's disease, dementia, depression and other disorders affecting the nervous system.

Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic antibodies in advanced Sézary syndrome.

Sézary syndrome (SS) is a rare form of cutaneous T-cell lymphoma often refractory to treatment. SS is defined as adenopathy, erythroderma with high numbers of atypical T cells. This offers an opportunity for new interventions and perhaps antibody-based therapeutic by virtue of its high expression of the TNFR2 oncogene on the tumor cells and on T-regulatory cells (Tregs). Potent human-directed TNFR2 antagonistic antibodies have been created that preferentially target the TNFR2 oncogene and tumor-infiltrating TNFR2+ Tregs. Here we test the therapeutic potential of TNFR2 antagonists on freshly isolated lymphocytes from patients with Stage IVA SS and from healthy controls. SS patients were on a variety of end-stage multi-drug therapies. Baseline burden Treg/T effector (Teff) ratios and the responsiveness of tumor and infiltrating Tregs to TNFR2 antibody killing was studied. We show dose-escalating concentrations of a dominant TNFR2 antagonistic antibody killed TNFR2+ SS tumor cells and thus restored CD26- subpopulations of lymphocyte cell numbers to normal. The abundant TNFR2+ Tregs of SS subjects are also killed with TNFR2 antagonism. Beneficial and rapid expansion of Teff was observed. The combination of Treg inhibition and Teff expansion brought the high Treg/Teff ratio to normal. Our findings suggest a marked responsiveness of SS tumor cells and Tregs, to targeting with TNFR2 antagonistic antibodies. These results show TNFR2 antibodies are potent and efficacious in vitro.

Low levels of C-peptide have clinical significance for established Type 1 diabetes.

AIM: To determine whether the low C-peptide levels (< 50 pmol/l) produced by the pancreas for decades after onset of Type 1 diabetes have clinical significance. METHODS: We evaluated fasting C-peptide levels, duration of disease and age of onset in a large cross-sectional series (n = 1272) of people with Type 1 diabetes. We then expanded the scope of the study to include the relationship between C-peptide and HbA1c control (n = 1273), as well as diabetic complications (n = 324) and presence of hypoglycaemia (n = 323). The full range of C-peptide levels was also compared with 1,5-Anhydroglucitol, a glucose responsive marker. RESULTS: C-peptide levels declined for decades after diagnosis, and the rate of decline was significantly related to age of onset (P < 0.0001), after adjusting for disease duration. C-peptide levels > 10 pmol/l were associated with protection from complications (e.g. nephropathy, neuropathy, foot ulcers and retinopathy; P = 0.03). Low C-peptide levels were associated with poor metabolic control measured by HbA1c (P < 0.0001). Severe hypoglycaemia was associated with the lowest C-peptide levels compared with mild (P = 0.049) or moderate (P = 0.04) hypoglycaemia. All levels of measurable C-peptide were responsive to acute fluctuations in blood glucose levels as assessed by 1,5-Anhydroglucitol (P < 0.0001). CONCLUSIONS: Low C-peptide levels have clinical significance and appear helpful in characterizing groups at-risk for faster C-peptide decline, complications, poorer metabolic control and severe hypoglycaemia. Low C-peptide levels may be a biomarker for characterizing at-risk patients with Type 1 diabetes.

The promise of Hox11+ stem cells of the spleen for treating autoimmune diseases.

The spleen of human adults uniquely possesses a reservoir of multilineage adult stem cells that express the developmental transcription factor HOX11. In contrast to hematopoietic stem cells, HOX11+ stem cells hold potentially broader therapeutic applications because they are less lineage restricted. HOX11/TLX1 is part of a homeodomain gene family essential for organogenesis of the spleen and for contributions to development of hindbrain, cochlea, pancreas, salivary glands, among other organs and tissues. While HOX11/TLX1 displays widespread patterns of expression during embryogenesis, its expression was thought to cease after birth. Recent findings in human post-mortem tissue have shattered this dogma, finding that HOX11/TLX1 stem cells are uniquely and abundantly expressed throughout adulthood in the human spleen. While their role in humans is not yet understood, HOX11/TLX1 stem cells from the spleen of normal mice have been harvested to assist in both the treatment and cure at least two autoimmune diseases: type 1 diabetes, Sjogren's syndrome, and possibly their comorbid hearing loss. The splenic stem cells are infused, with an immune therapy, into diseased NOD mice, where they can home to the diseased organ, differentiate into the appropriate cell type, and assume normal functioning with the endogenous regeneration of the animal due to disease removal. This review covers HOX11/TLX1+ stem cells' success in an animal model and their potential for treating autoimmune diseases in organs that mirror their extensive expression patterns during embryogenesis.

The therapeutic potential of tumor necrosis factor for autoimmune disease: a mechanistically based hypothesis.

Excess levels of tumor necrosis factor-alpha (TNF-alpha) have been associated with certain autoimmune diseases. Under the rationale that elevated TNF-alpha levels are deleterious, several anti-TNF-alpha therapies are now available to block the action of TNF-alpha in patients with autoimmune diseases with a chronic inflammatory component to the destructive process. TNF-alpha antagonists have provided clinical benefit to many patients, but their use also is accompanied by new or aggravated forms of autoimmunity. Here we propose a mechanistically based hypothesis for the adverse events observed with TNF-alpha antagonists, and argue for the opposite therapeutic strategy: to boost or restore TNF-alpha activity as a treatment for some forms of autoimmunity. Activation defects in the transcription factor nuclear factor kappaB leave autoreactive T cells sensitive to TNF-alpha-induced apoptosis. Treatment with TNF-alpha, by destroying autoreactive T cells, appears to be a highly targeted strategy to interrupt the pathogenesis of type 1 diabetes, lupus and certain forms of autoimmunity.

Central role of defective apoptosis in autoimmunity.

Lymphocyte development, selection and education represent tightly controlled immune processes that normally prevent autoimmunity. Lymphocyte development likely induces cellular selection through apoptosis to remove potentially autoreactive cells. Dysregulated apoptosis, both interrupted as well as accelerated apoptosis, are now demonstrated as central defects in diverse murine autoimmune disease. In murine models of autoimmune lupus, mutations in cell death receptor Fas (CD95) and its ligand, FasL (CD95 L), have been identified. These errors create a lymphoid system resistant to apoptosis. In contrast, select lymphoid subpopulations of maturing autoimmune prone non-obese diabetic mice have identifiable and pathogenic T cells with both in vivo and in vitro heightened apoptosis after drug interventions. In part, these defects are due to faulty activation of transcription factors such as nuclear factor-kappaB (NF-kappaB) that normally protect against apoptotic death. The genetic basis of interrupted NF-kappaB in pathogenic memory T cells in diabetes is attributable to a developmentally controlled gene defect in an essential subunit of the proteasome. No specific gene in most common forms of human autoimmune disease has yet been identified. Functional assays from diverse laboratories repeatedly demonstrate heightened apoptosis in multiple cellular signaling pathways for cell death, suggesting a common theme in disease causality.

Selected contribution: Association of gender-related LMP2 inactivation with autoimmune pathogenesis.

Recent results in an animal model of autoimmune diabetes, the nonobese diabetic (NOD) mouse, suggest a hypothesis to explain the role of major histocompatibility complex (MHC) in autoimmunity. The genome MHC region contains immune response genes that are important for T cell education and antigen presentation by MHC molecules. Two such genes encoding the LMP2 and LMP7 proteasome subunits are located in this high-risk MHC genomic region. Proteasome containing the LMP2 subunit is essential for T cell education and proteolytically activates transcription factor nuclear factor-kappaB. Splenocytes of NOD mouse with marked female specificity for disease expression are defective in LMP2 expression. The spontaneous defective LMP2 expression in NOD mice, which is gender biased toward female cohorts, is restricted to select lymphoid and myeloid cells and is developmentally controlled with lowered LMP2 protein and heightened tumor necrosis factor-alpha-induced apoptosis. These defects are apparent only after approximately 7 wk of age. These data suggest a proteasome role in autoimmune progression, and a gender developmental and lineage restriction of LMP2 expression may contribute to the diverse autoimmune characteristics preferentially observed in female NOD mice.

Reversal of established autoimmune diabetes by restoration of endogenous beta cell function.

In NOD (nonobese diabetic) mice, a model of autoimmune diabetes, various immunomodulatory interventions prevent progression to diabetes. However, after hyperglycemia is established, such interventions rarely alter the course of disease or allow sustained engraftment of islet transplants. A proteasome defect in lymphoid cells of NOD mice impairs the presentation of self antigens and increases the susceptibility of these cells to TNF-alpha-induced apoptosis. Here, we examine the hypothesis that induction of TNF-alpha expression combined with reeducation of newly emerging T cells with self antigens can interrupt autoimmunity. Hyperglycemic NOD mice were treated with CFA to induce TNF-alpha expression and were exposed to functional complexes of MHC class I molecules and antigenic peptides either by repeated injection of MHC class I matched splenocytes or by transplantation of islets from nonautoimmune donors. Hyperglycemia was controlled in animals injected with splenocytes by administration of insulin or, more effectively, by implantation of encapsulated islets. These interventions reversed the established beta cell-directed autoimmunity and restored endogenous pancreatic islet function to such an extent that normoglycemia was maintained in up to 75% of animals after discontinuation of treatment and removal of islet transplants. A therapy aimed at the selective elimination of autoreactive cells and the reeducation of T cells, when combined with control of glycemia, is thus able to effect an apparent cure of established type 1 diabetes in the NOD mouse.

Implications of altered apoptosis in diabetes mellitus and autoimmune disease.

Lymphocyte development, selection and education represent tightly controlled immune processes that normally prevent autoimmunity. Lymphocyte development requires cellular selection through apoptosis to remove potentially autoreactive cells. Dysregulated apoptosis, both interrupted as well as accetuated apoptosis, are now demonstrated as central defects in diverse human and murine autoimmune disease. In murine models of autoimmune lupus, mutations in cell death receptor CD95 (Fas) and its ligand CD95L (FasL) have been identified; these errors create a lymphoid system resistant to apoptosis. In contract, select lymphoid subpopulations of auto immune diabetic mice have accelerated apoptosis due to faulty activation of transcription factor NF-kappaB that normally protects against apoptotic death. The genetic basis of interrupted NF-kappaB in diabetes is a gene defect in an essential subunit of the proteasome. Although no specific gene in most common forms of human autoimmune disease has been identified, functional assays repeatedly demonstrate apoptotic defects in multiple cellular signaling pathways for cell death.

Bayesian analysis of case control polygenic etiology studies with missing data.

Many genetic studies are based on analysing multiple DNA regions of cases and controls. Usually each is tested separately for association with disease. However, some diseases may require interacting polymorphisms at several regions, and most disease susceptibility is polygenic. In this paper, we develop new methods for determining combinations of polymorphisms that affect the risk of disease. For example, two different genes might produce normal proteins, but these proteins improperly function when they occur together. We consider a Bayesian approach to analyse studies where DNA data from cases and controls have been analysed for polymorphisms at multiple regions and a polygenic etiology is suspected. The method of Gibbs sampling is used to incorporate data from individuals who have not had every region analysed at the DNA sequence or amino acid level. The Gibbs sampling algorithm alternatively generates a sample from the posterior distribution of the sequence of combinations of polymorphisms in cases and controls and then uses this sample to impute the data that are missing. After convergence the algorithm is used to generate a sample from the posterior distribution for the probability of each combination in order to identify groups of polymorphisms that best discriminate cases from controls. We apply the methods to a genetic study of type I diabetes. The protein encoded by the TAP2 gene is important in T cell function, and thus may affect the development of autoimmune diseases such as insulin dependent diabetes mellitus (IDDM). We determine pairs of polymorphisms of genetic fragments in the coding regions of linked HLA genes that may impact the risk of IDDM.

The role of the proteasome in autoimmunity.

Type 1 diabetes is believed to be caused by T cell-mediated autoimmunity, with a prediabetic state characterized by the production of autoantibodies specific for proteins expressed by pancreatic beta cells. The non-obese diabetic (NOD) mouse is a spontaneous model of Type 1 diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) region of the genome. A specific proteasome defect has now been identified in NOD mouse lymphocytes that results from down-regulation of expression of the proteasome subunit LMP2, which is encoded by a gene in the MHC genomic region. This defect both prevents the proteolytic processing required for the production and activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which plays an important role in immune and inflammatory responses, in addition to increasing the susceptibility of the affected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). The proteasome dysfunction is both tissue- and developmental stage-specific and likely contributes to disease pathogenesis and tissue targeting.

Identification of premature ovarian failure patients with underlying autoimmunity.

Although known causes of premature ovarian failure (POF) include X chromosome deletions, radiation and chemotherapy, and genetic defects of the gonadotropin hormones or receptors, at least one third to one half of cases remain idiopathic. A significant proportion of patients with apparently idiopathic POF have some evidence for an autoimmune etiology. However, the only gold standard for detecting autoimmune causes of immune ovarian destruction has been invasive ovarian biopsy. Serum antibodies to ovarian and other self-tissue have been described in up to one third of women with POF, but the tests are not well standardized, not well correlated with ovarian histology, and highly variable. Recently, specific defects of expression of cell surface markers on peripheral blood lymphocytes have been shown to identify, in population-based studies, individuals destined to develop autoimmune pancreatic destruction and type I diabetes mellitus, even before any other evidence of autoimmunity. We, therefore, sought to test the ability of cell surface marker expression in women with POF to identify autoimmune defects. Seventeen women with POF, 11 of whom had positive antibody titers to ovary, thyroid, or antinuclear antibody, were studied on at least two occasions and compared in blinded fashion with normal controls and patients with autoimmune type I diabetes mellitus. The most useful marker for identifying autoimmunity was the surface density of conformationally correct HLA class I molecules on macrophages, a structure essential for T cell education. Using this marker, 7 of the 9 patients with autoantibodies and 3 of the 8 patients without autoantibodies were identified as having evidence of a defect in self-antigen presentation similar to that of type I diabetics (chi-square, p = 0.03). Subsequent testing identified antismooth muscle antibodies in 1 of the women with a defect of HLA class I molecules but no previously identified autoimmunity. In addition, there were increased numbers of CD8 T cells in both autoimmune POF and insulin-dependent diabetes mellitus (IDDM) patients. Exclusive to POF patients was a statistically significant increase in CD8 density on T cells. This was most prominent in POF patients with an underlying autoimmune etiology. These data further support a role for autoimmunity in POF patients and suggest that the further development of cell surface markers in combination with other diagnostic tests could result in diagnosis before the development of complete ovarian failure. The possibility for disease-specific therapy to prevent further autoimmune ovarian damage in selected POF patients is also envisioned.

Essential role of human leukocyte antigen-encoded proteasome subunits in NF-kappaB activation and prevention of tumor necrosis factor-alpha-induced apoptosis.

The multisubunit proteasome complex is the principal mediator of nonlysosomal protein degradation. The proteasome subunit varies minimally between cells with the exception of LMP2, LMP7, and LMP10 subunits in rodent and human cells. LMP2 and LMP7 subunits are encoded by the human lymphocyte antigen region, and they optimize proteolytic mediated antigen presentation. The proteasome is also important for the function of transcription factor nuclear factor-kappaB (NF-kappaB). It is required for NF-kappaB subunits p50 and p52 generation and catalyzes degradation of phosphorylated IkappaBalpha. These proteasome-mediated reactions have now been shown to be defective in T2 cells, a human lymphocyte cell line that lacks both LMP2 and LMP7. Although T2 cells contain normal expression of p100 and p105, the abundance of p50 and p52 was greatly reduced. Tumor necrosis factor-alpha (TNF-alpha) induced normal phosphorylation of IkappaBalpha but failed to induce degradation of phosphorylated IkappaBalpha. Both DNA binding assays and luciferase assays revealed that TNF-alpha-induced NF-kappaB activation is defective in T2 cells. Unlike parental cells, T2 cells were susceptible to TNF-alpha-induced apoptosis. These data indicate human leukocyte antigen-linked proteasome subunits are essential for NF-kappaB activation and protection of cells from TNF-alpha-induced apoptosis.

A role for NF-kappaB and the proteasome in autoimmunity.

Type 1 diabetes (also known as insulin-dependent diabetes mellitus or juvenile-onset diabetes) is usually caused by T cell-mediated autoimmunity, with a prediabetic state characterized by the production of autoantibodies specific for proteins expressed by pancreatic beta cells. The non-obese diabetic (NOD) mouse is a spontaneous model of type 1 diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) region of the genome. A specific proteasome defect has been identified in NOD mouse lymphocytes that results from down-regulation of expression of the proteasome subunit LMP2, which is encoded by a gene in the MHC genomic region. This defect both prevents the proteolytic processing required for the production and activation of the transcription factor nuclear factor kappaB (NF-kappaB), which plays important roles in immune and inflammatory responses, as well as increases the susceptibility of the affected cells to apoptosis induced by tumor necrosis factor alpha (TNF-alpha). The proteasome dysfunction is both tissue and developmental stage specific and likely contributes to disease pathogenesis and tissue targeting.

Defective function of the proteasome in autoimmunity: involvement of impaired NF-kappaB activation.

Type 1 diabetes (also known as insulin-dependent diabetes mellitus or juvenile-onset diabetes) is usually caused by T cell-mediated autoimmunity, with a prediabetic state characterized by the production of autoantibodies specific for proteins expressed by pancreatic beta cells. The nonobese patient with diabetes (NOD) mouse is a spontaneous model of type 1 diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) region of the genome. A specific proteasome defect has been identified in NOD mouse in select lymphocytic and monocytic lineages that results from down-regulation of expression of the proteasome subunit LMP2, which is encoded by a gene in the MHC genomic region. This defect prevents the proteolytic processing required for the production and activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which plays important roles in immune and inflammatory responses, as well as increases the susceptibility of the affected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). The novel role of the proteasome in dysfunction in autoimmunity is presented and documented to be both tissue and developmental stage specific. We propose a role of the proteasome as a step in disease pathogenesis and tissue targeting.

Antibodies for transplantation.

The use of antibodies in transplantation has become a clinical reality. Antibodies have been used to both dampen the recipient's immune response and to obscure the immunogenicity of the donor graft. Traditionally, antibodies have been administered to the transplant recipient to transiently inactivate the host's T cells, the lymphocytes responsible for recognizing and attacking foreign proteins, cells, and tissues. Antibodies can also be used to eliminate any highly immunogenic passenger cells from a donor graft prior to transplantation, and antibodies can mask or conceal antigens present on donor cells that might trigger rejection.

NOD mice are defective in proteasome production and activation of NF-kappaB.

The nonobese diabetic (NOD) mouse is an animal model of human type I diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) of the genome. We have identified in NOD lymphocytes a specific proteasome defect that results from the lack of the LMP2 subunit. The pronounced proteasome defect results in defective production and activation of the transcription factor NF-kappaB, which plays an important role in immune and inflammatory responses as well as in preventing apoptosis induced by tumor necrosis factor alpha. The defect in proteasome function in NOD mouse splenocytes was evident from impaired NF-kappaB subunit p50 and p52 generation by proteolytic processing and impaired degradation of the NF-kappaB-inhibitory protein IkappaBalpha. An obligatory role of MHC-linked proteasome subunits in transcription factor processing and activation has been established in a spontaneous-disease model and mutant cells similarly lacking the MHC-encoded subunit. These data suggest that NOD proteasome dysfunction is due to a tissue- and developmental-stage-specific defect in expression of the MHC-linked Lmp2 gene, resulting in altered transcription factor NF-kappaB activity, and that this defect contributes to pathogenesis in NOD mice. These observations are consistent with the diverse symptomatology of type I diabetes and demonstrate clear sex-, tissue-, and age-specific differences in the expression of this error which parallel the initiation and disease course of insulin-dependent (type I) diabetes mellitus.

Novel splicing of the human MHC-encoded peptide transporter confers unique properties.

Presentation of intracellularly derived antigenic peptides to T cells requires their assembly together with MHC class I molecules in the endoplasmic reticulum (ER). Such peptides are delivered to the ER by an MHC-encoded transporter composed of TAP1 and TAP2 protein delivery. Here, the first alternative splicing of Tap2 is described. The human splice variant, termed Tap2iso, lacks exon 11 and original 3' untranslated region and contains a newly identified exon 12 and 3' untranslated region. The full-length Tap2iso cDNA (2496 bp) predicts a protein of 653 amino acids. Tap2iso mRNA was normally coexpressed with Tap2 mRNA in all human lymphocyte cell lines examined. Function of TAP2iso was evaluated at multilevel in TAP1/2iso and TAP1/2 cotransfected T2 cells, a mutant cell line deplete of endogenous Tap gene products. The TAP1-TAP2iso transporter facilitated the maturation of MHC class I molecules in the ER and restored surface expression of class I. Importantly, TAP1-TAP2iso transporters expressed in T2 cells exhibited distinct and opposing influences on peptide selectivities, at times exceeding 30-fold differences in competition experiments and attributable to diversity in the 3'-COOH tail. The common coexpression of an alternative splice product of the Tap2 gene may contribute to broaden immune diversity, a mechanism previously described to occur predominantly at the level of the TCR and MHC class I gene products.