An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.     

Systemic sclerosis biomarkers discovered using mass-spectrometry-based proteomics: a systematic review

Abstract

Context: Systemic sclerosis (SSc) is an autoimmune disease with incompletely known physiopathology. There is a great challenge to predict its course and therapeutic response using biomarkers.

Objective: To critically review proteomic biomarkers discovered from biological specimens from systemic sclerosis patients using mass spectrometry technologies.

Methods: Medline and Embase databases were searched in February 2014.

Results: Out of the 199 records retrieved, a total of 20 records were included, identifying 116 candidate proteomic biomarkers.

Conclusion: Research in SSc proteomic biomarkers should focus on biomarker validation, as there are valuable mass-spectrometry proteomics studies in the literature.     

Novel procedures for high-throughput analysis of a frequent insertion-deletion polymorphism in the human T-cell receptor beta locus

The human T-cell receptor beta locus (TRB) contains two frequent insertion-deletion polymorphisms. In one, the insertion comprises two functional variable beta genes, TRBV6-2/TRBV6-3 and TRBV4-3, and the pseudogene TRBV3-2. Deletion of these TRBV genes may confer resistance and/or susceptibility to autoimmunity, analogously to findings in rodent models. Curiously, the TRBV domains in the insertion react with the HERV-K18 superantigen associated with type 1 diabetes. While this region has been extensively characterized before, typing methods compatible with high-throughput analysis are not yet available. Here, two novel procedures are reported that are suitable for large-scale association analysis of this polymorphism. One features a duplex TaqMan 5-exonuclease assay that quantifies the gene dosage of TRBV3-2 present at 0, 1 or 2 copies, with its closely related diploid relative TRBV3-1 as an internal reference, using the 2^-C_T method. The other technique consists of two complementary long PCRs with primers specific for unique regions in the locus. The first discriminates individuals heterozygous or homozygous for the deletion, and the second, individuals heterozygous or homozygous for the insertion from other genotypes. These simple, solid, and cross-validated procedures can now be used in conjunction with flanking single-nucleotide polymorphisms for large-scale linkage studies.     

Granulocyte-dendritic cell unbalance in the non-obese diabetic mice

Several investigators, including ourselves, have reported lower yield of GM-CSF bone marrow-derived dendritic cells (DC) with altered MHC class II and co-stimulatory molecules expression in the non-obese diabetic (NOD) mice. However, whether this defect was intrinsic to the DC lineage and/or related to abnormal expansion of other cell types responding to GM-CSF remained an opened issue. We performed phenotypical and morphological analysis of cells from GM-CSF-supplemented-bone marrow-cultures and of freshly isolated bone marrow and blood cells from unmanipulated prediabetic NOD mice. The results show a heretofore undescribed bias towards generation of granulocytes in NOD mice, concomitant with quantitative and qualitative alterations of the DC lineage in both the bone marrow and the blood of this mouse strain. We propose that increased generation of granulocytes in NOD mice might contribute to autoimmunity. First, high numbers of granulocytes per se might favor inflammatory environment. Second, granulocytes, by interfering with DC development, might favor unbalanced antigen presenting cell function leading to T cell autoimmunity.     

Suppression of antibody production by TNF-related apoptosis-inducing ligand (TRAIL)

TNF-related apoptosis-inducing ligand (TRAIL), a type II membrane protein belonging to the TNF family, induces apoptotic cell death in various types of tumor cells. However, little is known about its pathological and physiological functions in the immune system. In this study, we showed that administration of neutralizing anti-TRAIL mAb markedly increased serum auto-Ab levels, particularly of IgG1 subclass, in autoimmune-prone C3H/HeJ gld/gld mice without affecting lymphocytosis and lymphocytes populations. In an experimental system where TNP-specific Ab production was induced by immunization with TNP-modified syngeneic B lymphoma cells, expression of TRAIL on these cells significantly reduced TNP-specific Ab production, especially of IgG1 subclass, without affecting T cell priming. These results suggest a new role for TRAIL in the suppression of Ab production.     

A high incidence of Shigella-induced arthritis in a primate species: major histocompatibility complex class I molecules associated with resistance and susceptiblity, and their relationship to HLA-B27

 The human major histocompatibility complex (MHC) class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies. Rodents that express HLA-B27 develop spondyloarthropathies, implicating HLA-B27 in the etiology of these disorders. To determine whether an HLA-B27-like molecule was associated with spondyloarthropathies in nonhuman primates, we analyzed the MHC class I cDNAs expressed in a cohort of rhesus macaques that developed reactive arthritis after an outbreak of shigellosis. We identified several cDNAs with only limited sequence similarity to HLA-B27. Interestingly, one of these MHC molecules had a B pocket identical to that of HLA-B39. Pool sequencing of radiolabeled peptides bound by this molecule demonstrated that, like HLA-B27 and HLA-B39, it could bind peptides with arginine at the second position. However, extensive analysis of the MHC class I molecules in this cohort revealed no statistically significant association between any particular MHC class I allele and susceptibility to reactive arthritis. Furthermore, none of the rhesus MHC class I molecules bore a strong resemblance to HLA-B27, indicating that reactive arthritis can develop in this animal model in the absence of an HLA-B27-like molecule. Surprisingly, there was a statistically significant association between the rhesus macaque MHC A locus allele, Mamu-A*12, and the absence of reactive arthritis following Shigella infection. Received: 26 July 1999 / Revised: 28 December 1999     

A common and recurrent 13-bp deletion in the autoimmune regulator gene in British kindreds with autoimmune polyendocrinopathy type 1.

Autoimmune polyendocrinopathy type 1 (APS1) is an autosomal recessive disorder characterized by autoimmune hypoparathyroidism, autoimmune adrenocortical failure, and mucocutaneous candidiasis. Recently, an autoimmune regulator gene (AIRE-1), which is located on chromosome 21q22.3, has been identified, and mutations in European kindreds with APS1 have been described. We used SSCP analysis and direct DNA sequencing to screen the entire 1,635-bp coding region of AIRE-1 in 12 British families with APS1. A 13-bp deletion (964del13) was found to account for 17 of the 24 possible mutant AIRE-1 alleles, in our kindreds. This mutation was found to occur de novo in one affected subject. A common haplotype spanning the AIRE-1 locus was found in chromosomes that carried the 964del13 mutation, suggesting a founder effect in our population. One of 576 normal subjects was also a heterozygous carrier of the 964del13 mutation. Six other point mutations were found in AIRE-1, including two 1-bp deletions, three missense mutations (R15L, L28P, and Y90C), and a nonsense mutation (R257*). The high frequency of the 964del13 allele and the clustering of the other AIRE-1 mutations may allow rapid molecular screening for APS1 in British kindreds. Furthermore, the prevalence of the 964del13 AIRE-1 mutation may have implications in the pathogenesis of the more common autoimmune endocrinopathies in our population.     

Mechanism of regulation of autoimmunity by iNKT cells

iNKT cells, CD1d dependent natural killer T cells are a unique population of T cells. The capacity of iNKT cells to produce regulatory cytokines first provided an indication of their regulatory potential. Later on, in experimental models as well as in patients afflicted with an auto-immune disease, such as Type 1 diabetes mellitus, multiple sclerosis, and systemic lupus erythematosus along with others, a deficit in iNKT cell number was observed, suggesting the role these cells may possibly have in the prevention of auto-immune diseases. More importantly, experimental strategies which focused on increasing the volume or stimulation of iNKT cells in laboratory animals, demonstrated an improved level of protection against the development of auto-immune diseases. This article reviews the mechanism of protection against autoimmunity by iNKT cells, discusses the obstacles against and indications for the potential use of iNKT cell manipulation in the treatment of human auto-immune diseases.     

Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"

Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori-specific Th1 response, characterized by high IFN-gamma, TNF-alpha, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas-Fas ligand-mediated killing of B cells. In H. pylori-infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori-induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry.     

The mouse immune response to human fibrinogen reveals an autoimmune component against mouse fibrinogen

The present experiments were carried out to analyze whether immunization of mice with human fibrinogen would induce autoimmunity like other heterologous proteins such as collagen type II, thyroglobulin or myelin basic protein. Our results demonstrate that human fibrinogen induces very strong immune responses in all mouse strains analyzed. Autoimmune responses with short-term memory to mouse fibrinogen are induced in genetically susceptible mice. These autoimmune Th2-type responses induce splenomegaly, enhanced coagulation times, and production of rheumatoid factors. The short-lived autoimmune memory was not regulated by either suppressor T cells or exhaustion of immune cells; rather this potentially dangerous autoimmune response was regulated by unknown, antigen-specific feedback mechanisms (they do not influence immune responses to proteins like HSA and OA in the same mice). Such feedback mechanisms were not found in the immune responses to other heterologous proteins inducing significant cross-reactive autoimmunity such as collagen type II, thyroglobulin, or myelin basic protein.