Abnormal glycosylation of α2-macroglobulin,a non-acute-phase protein,in patients with autoimmune diseases

Previous studies from this and other laboratories have shown that abnormal glycosylation of several acute-phase proteins can be detected in various pathological conditions including autoimmune diseases. In the present study, we have investigated if abnormal glycosylation is limited to acute-phase proteins. We used the concanavalin A (Con A) blots in conjunction with the peptide mapping techniques to analyze serum samples and cerebrospinal fluids (CSF) obtained from patients with autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD), scleroderma (SCL), Sjögren's syndrome (SS), and polymyositis (PM); diseases of probable autoimmune origin: hepatopathies (HP); diseases of suspected autoimmune origin: schizophrenia and Alzheimer's disease (AZ); and conditions not related to autoimmunity: pregnancy (PG) and elevation of the carcinoembryonic antigen (CEA), in comparison to normal donors (NHS). We have micropurified two human proteins; ₂-macroglobulin, a non-acute-phase protein, and-chain of haptoglobin, a known acute-phase protein, from serum samples of individual patients with SLE, RA, MCTD, SCL and SS, and from PG and NHS for analysis. The identity of the purified proteins was confirmed by immunoblots using either monospecific polyclonal or monoclonal antibodies, and by directN-terminal amino acid sequencing. Peptide maps for each of these proteins were generated usingStaphylococcus aureus protease V₈, a Glu-C endopeptidase. When the peptide fragments of ₂-macroglobulin were resolved by SDS-PAGE and visualized using silver staining, no differences were noted between patient samples and controls. However, when they were examined by lectin blots using Con A, the Con A-reactive fragments increased specifically and significantly in samples derived from patients of SLE, SCL, MCTD, and RA. Similarly when the peptide fragments of the-chain of haptoglobin were visualized by silver staining, no differences were noted; however, the Con A reactivity of specific fragments increased in SLE, RA, SCL, and SS patients. Analysis of these results indicated that there has been a selective increase in Con A-reactive fragments in both acute-phase and non-acute-phase proteins in autoimmune conditions. Thus, the study of changes in glycosylation patterns in selected serum proteins may be a valuable diagnostic approach to define the pathophysiology of inflammatory and autoimmune disorders.     

Pulmonary epithelial expression of human α1-antitrypsin in transgenic mice results in delivery of α1-antitrypsin protein to the interstitium

α₁-Antitrypsin (α₁AT) therapy is used as a treatment for α₁AT deficiency. It has also been proposed as a therapy for cigarette smoke-induced emphysema, although the efficacy of such therapy is as yet unproven. Moreover, the optimal route of delivery of α₁AT to the lung interstitium, the crucial locus of action, is unknown. We created transgenic mice with expression of the human α₁AT gene directed by a human surfactant protein C (SpC) promoter fragment or a rat Clara cell 10-kDa protein (CC10) promoter fragment in order to examine the ability of pulmonary epithelial cell expression of α₁AT to deliver protein to the interstitium, and to produce a model that would allow studies on the efficacy of α₁AT in preventing lung damage after cigarette smoke exposure. Four transgenic lines were studied. In situ hybridization and light microscopic immunohistochemistry showed that two CC10 driven lines expressed human α₁AT in type II alveolar cells and airway epithelial cells; α₁AT expression was seen in the alveolar parenchyma in two SpC driven lines, and in small airway epithelium in one of the SpC lines. Electron microscopic immunochemistry showed the presence of the human α₁AT protein in the interstitium in all lines. Mean levels of human protein varied from 0.37 to 2.9 μg/g lung protein and serum levels from 0.72 to 1.3 μg/ml, compared to normal human serum α₁AT levels of 2–5 mg/ml. We conclude that transgene-mediated expression of α₁AT in pulmonary epithelial cells results in diffuse expression of the transgene in the alveolar parenchyma and reproducibly leads to transfer of protein to the interstitium. The present model is, however, limited by low levels of protein production; limited protein production may be a problem in other forms of gene therapy in which relatively large amounts of extracellular protein are needed in the lung for a therapeutic effect. Received: 5 August 1998 / Accepted: 25 January 1999     

Changes in thymosin-α1 content in patients with nonspecific gynecologic diseases depending on inflammation type and efficacy of antiinflammatory and immunomodulating therapy

Plasma content of thymosin-α₁ and its circadian variations in patients with inflammatory gynecologic diseases differ from those in healthy donors and depend on the type of inflammation and efficacy of treatment. It is concluded that not only the absolute content of thymic hormones, but also their biorhythmic variations are important for immune regulation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 327–329, September, 2000     

Changes in thymosin-α1 content in patients with nonspecific gynecologic diseases depending on inflammation type and efficacy of antiinflammatory and immunomodulating therapy

Plasma content of thymosin-₁ and its circadian variations in patients with inflammatory gynecologic diseases differ from those in healthy donors and depend on the type of inflammation and efficacy of treatment. It is concluded that not only the absolute content of thymic hormones, but also their biorhythmic variations are important for immune regulation.     

Alterations in the expression of signal-transducing CD3ζ chain in T cells from patients with chronic inflammatory/autoimmune diseases

The CD3ζ chain, a component of the T cell receptor (TCR)/CD3 complex, is considered to be a limiting factor in the assembly and transport of the TCR/CD3 complex to the cell surface and is crucial to receptor signaling function. Recent studies have demonstrated altered expression and function of this signal transduction molecule in T and natural killer cells in patients with chronic inflammatory/autoimmune diseases. In this review, current knowledge concerning the expression of CD3ζ chain as well as the mechanisms responsible for abnormal expression of this molecule in systemic lupus erythematosus, rheumatoid arthritis, and childhood idiopathic nephrotic syndrome are summarized.     

Direct selection of hepatoma cell variants deficient in α1-antitrypsin gene expression

Expression plasmids containing the human ₁-antitrypsin (₁ AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT^– HPRT^– rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes incis, as only three of 20 clones tested were affected for expression of ₁AT mRNA. In contrast, double selection yielded predominantlytrans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal ₁AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.     

Serum α1-antitrypsin and α2-macroglobulin in Alzheimer's and Binswanger's disease

The deposition of _A4-amyloid in senile plaques and small cerebral vessels is one of the pathological hallmarks of Alzheimer's disease. Recent data suggest that protease inhibitors such as ₂-macroglobulin may be involved in the process of forming _A4 amyloid deposits. Compared to 34 persons without neurological diseases, the serum content of al-antitrypsin was increased in 16 patients with Alzheimer's disease and 15 with Binswanger's disease. In the latter a2 macroglobulin was also elevated in serum. Our results show no evidence of a blood-borne origin of the protein or peptid deposited in the walls of small vessels in Alzheimer's or Binswanger's disease. Nevertheless, the elevated proteinase inhibitor concentrations may play a role in the pathogenesis of these diseases.Abbreviations AD Alzheimer's disease - APP -amyloid peptide precursor peptide - BD Binswanger's disease - IMCT Information-Memory-Concentration Test - MID multi-infarct dementia - MMSE Mini-Mental State Examination Correspondence to: T. Wetterling     

Characterization of the autoimmune response against the nerve tissue S100β in patients with type 1 diabetes

Type 1 diabetes results from destruction of insulin-producing beta cells in pancreatic islets and is characterized by islet cell autoimmunity. Autoreactivity against non-beta cell-specific antigens has also been reported, including targeting of the calcium-binding protein S100β. In preclinical models, reactivity of this type is a key component of the early development of insulitis. To examine the nature of this response in type 1 diabetes, we identified naturally processed and presented peptide epitopes derived from S100β, determined their affinity for the human leucocyte antigen (HLA)-DRB1*04:01 molecule and studied T cell responses in patients, together with healthy donors. We found that S100β reactivity, characterized by interferon (IFN)-γ secretion, is a characteristic of type 1 diabetes of varying duration. Our results confirm S100β as a target of the cellular autoimmune response in type 1 diabetes with the identification of new peptide epitopes targeted during the development of the disease, and support the preclinical findings that autoreactivity against non-beta cell-specific autoantigens may have a role in type 1 diabetes pathogenesis.     

Are immunoconjugates useful for therapy with autoimmune diseases?

Immunoconjugates or immunotoxins (ITs) are targeting molecules which consist of a monoclonal antibody together with a toxin-thereby they can selectively kill target cells in a highly efficient manner. The use of ITs as a drug targeting approach is one of the most attractive research fields for tumor therapy; however, the study of ITs for the treatment of autoimmune diseases has been given little attention until recently. It has been shown that ITs could help alleviate the symptoms of myasthenia gravis and rheumatoid arthritis in animal models. In the last 3 yr ITs have been used in clinical trials (phase I and phase II) for the treatment of various autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus and psoriasis. This article reviews the main progress on the application of ITs for the therapies of autoimmune diseases. The preliminary results suggest the future may hold some promise, but side effects, in addition to there being no convincing efficacy, remain unresolved.     

The absence of immunoreactivity to donkey’s milk in patients with recurrent aphthous ulcers and immunoreactivity to cow’s milk

Despite the numerous benefits of milk constituents for human health a considerable number of the general population follow a milk-restricted diet due to clinically confirmed or self-assessed adverse reactions to cow’s milk consumption. Recurrent aphthous ulcers (RAU) are currently one of the most common oral disorders, with a worldwide distribution and insufficiently defined etiology, which, among other factors, implies the immunological reaction to food proteins. The aim of this study was to determine the immune-reactivity to donkey’s milk proteins in patients with RAU and compare it to the reactivity towards the proteins from cow’s and goat’s milks, in a set of simultaneous experiments. Levels of serum IgA, IgG and IgE antibodies to the same quantity of the examined antigens were determined by enzyme-linked immunosorbent assay. The results indicate that patients with RAU with increased immunity to cow’s milk proteins could consider the use of donkey’s milk as the best protein source.     

Immunoregulatory effects of α-GalCer in a murine model of autoimmune myocarditis

This study was designed to investigate the role of α-galactosylceramide (α-GalCer) on experimental autoimmune myocarditis (EAM), and to explore the underlying mechanisms. Balb/c mice were immunized with porcine cardiac myosin to establish the EAM model. All the immunized mice were divided into two groups, the α-GalCer group and the EAM group. α-GalCer or vehicle was given intraperitoneally at the time of immunization. Then α-GalCer or PBS was injected on alternate days for 6 weeks. Myocardial inflammation was evaluated by H & E staining and the expression levels of C/EBPβ and α-SMA were determined by immunohistochemistry. CD4⁺CD25⁺Foxp3⁺ Tregs and iNKT cells were analyzed and sorted by flow cytometry. Western blot analysis was performed to detect MMP-2 and MMP-9 protein expression. Following α-GalCer treatment for 6 weeks, myocardial inflammation improved significantly in the α-GalCer treated group compared to the EAM group. The proportions of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and NK1.1⁺ iNKT cells were statistically increased in the α-GalCer treated group compared to the EAM and normal control groups. In contrast to the EAM group, α-GalCer treatment significantly increased myocardial MMP-2 and MMP-9 expression. Expression of C/EBPβ increased significantly in the EAM group compared to the other two groups. In contrast, the expression of α-SMA did not differ significantly among the three groups. This study demonstrated that α-GalCer alleviates EAM. Thus, α-GalCer represents a potential therapeutic target for autoimmune-inflammation mediated cardiac damage. α-GalCer protects EAM through upregulation of the proportion of iNKT and Tregs and increased expression of myocardial MMP-2 and MMP-9.     

Is it time for biosimilars in autoimmune diseases?

The last two decades have witnessed a revolution in the treatment of autoimmune diseases due to the introduction of biological agents which, although now included as standard treatment in patients with autoimmune rheumatological, dermatological and gastrointestinal diseases. The use of biological agents is associated with greater costs compared with the mainly anti-inflammatory and immunosuppressant drugs used in the pre-biological era. Biosimilars are highly similar copies of biological drugs, but not identical to approved ‘reference’ agents. Biological agents are complex proteins involved in the immune response and their exact replicas are extremely difficult, if not impossible, to obtain. Three scenarios have converged to provide a specific opportunity for biosimilars in autoimmune diseases: growing demand for biologics due to successful clinical use; the nearing of patent expiry for the four top-selling biological brands; and the search to reduce health costs due to the financial crisis. We aimed to review the crucial topics of efficacy, safety and regulatory approach of upcoming biosimilars.     

In silico mapping of polymorphic miRNA–mRNA interactions in autoimmune thyroid diseases

Abstract

MicroRNAs (miRNAs) are important regulators of gene expression and translation. The genetic variants altering miRNA targets have been associated with many diseases. Here we systematically mapped the human genetic polymorphisms that may affect miRNA–mRNA interactions in the autoimmune thyroid disease (AITD) pathway. We also mapped the polymorphic miRNA target sites in the genes that have been linked to AITDs or other thyroid-related diseases/phenotypes in genome-wide association studies (GWAS). These genetic polymorphisms may potentially contribute to the pathogenesis of AITDs and other thyroid diseases. The polymorphic miRNA–mRNA interactions we mapped in the AITD pathway and the GWAS-informed thyroid disease loci may provide insights into the possible miRNA-mediated molecular mechanisms through which genetic variants assert their influences on thyroid diseases and phenotypes.     

Negative regulation of TLR signaling in myeloid cells—implications for autoimmune diseases

Toll-like receptors (TLR) are transmembrane pattern recognition receptors that recognize microbial ligands and signal for production of inflammatory cytokines and type I interferon in macrophages and dendritic cells (DC). Whereas TLR-induced inflammatory mediators are required for pathogen clearance, many are toxic to the host and can cause pathological inflammation when over-produced. This is demonstrated by the role of TLR-induced cytokines in autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. Because of the potent effects of TLR-induced cytokines, we have diverse mechanisms to dampen TLR signaling. Here, we highlight three pathways that participate in inhibition of TLR responses in macrophages and DC, and their implications in autoimmunity; A20, encoded by the TNFAIP3 gene, Lyp encoded by the PTPN22 gene, and the BCAP/PI3K pathway. We present new findings that Lyp promotes TLR responses in primary human monocytes and that the autoimmunity risk Lyp620W variant is more effective at promoting TLR-induced interleukin-6 than the non-risk Lyp620R protein. This suggests that Lyp serves to downregulate a TLR inhibitory pathway in monocytes, and we propose that Lyp inhibits the TREM2/DAP12 inhibitory pathway. Overall, these pathways demonstrate distinct mechanisms of negative regulation of TLR responses, and all impact autoimmune disease pathogenesis and treatment.     

Concentrations of α- and β-defensins in plasma of patients with inflammatory bowel disease

Background:  Impaired production/release of defensins, representative endogenous antimicrobial peptides, is associated with the pathogenesis of inflammatory bowel disease (IBD). Material and methods:  Employing in house radioimmunoassay, we examined concentrations of the major forms α-defensins, human neutrophil peptides (HNP) 1–3 and human β-defensin (HBD)-2 in plasma of 55 IBD patients consisting of 29 patients with ulcerative colitis (UC) and 26 with Crohn’s disease (CD) and 57 controls. Results:  The circulating HNP 1–3, but not HBD-2, levels in IBD patients were significantly higher than those in controls. Plasma HNP 1–3 concentrations in CD patients significantly correlated with Crohn’s disease activity index, peripheral white blood cell counts, serum CRP values and TNF-α levels. Conclusions:  Elevation of circulating α-defensins levels is suggestive of their physiopathological roles in IBD. Plasma HNP 1–3 concentrations may be an indicator for CD activity and their association with CRP and TNF-α supports a possible association with the inflammatory process. Received 2 June 2008; returned for revision 20 June 2008; received from final revision 25 June 2008; accepted by C. Kasserra 19 August 2008     

Anticytokine therapy,particularly anti-IFN-γ, in Th1-mediated autoimmune diseases

Anticytokine therapy was proposed in 1974 in Nature, in which it was stated that hyperproduced interferon can cause autoimmune disease and anti-interferon can be therapeutic. In 1989, the use of antibodies to tumor necrosis factor-α in combination with antibodies to certain types of interferon was proposed to treat various autoimmune diseases, including AIDS. The first anticytokine therapy was conducted in 1975. Anti-interferon-γ has brought improved and often striking results in the treatment of various T-helper 1-mediated autoimmune diseases, including inflammatory skin diseases. Anti-interferon-γ may be a universal treatment for these conditions. In AIDS and other virus-induced autoimmune diseases, the virus may stimulate cytokines (interferons), which increase, rather than halt, viral replication. Tumor necrosis factor-α inhibitors have also shown good clinical results, however, they may result in complications and are ineffective in some autoimmune diseases.