Humoral link of autoimmune reactions to neuron-specific enolase in post-radiation encephalopathy patients]

The purpose of this research was to study the humoral link of autoimmune response to the brain antigen i.e. neurospecific enolase (NSE) in liquidators of the Chernobyl accident aftereffects diagnosed as having "postradiation encephalopathy" (PREP). Determined in the PREP patients' serum were: NSE content, level of autoantibodies to NSE, concentration and size of the immune complexes (IC) as well as NSE content in the IC. The majority of PREP patients revealed high serum level of NSE and high level of autoantibodies to NSE. Concentration of small and middle-size IC was 2-3 times higher than normal. So the presence of circulating NSE and autoantibodies thereto in the liquidators' blood serum evidences of an organic lesion of the brain neurons, this causing the development of neurological syndromes. It is likely that at certain stages of the PREP pathogenesis there appear autoimmune responses to neuroantigens which prove the destructive effects of radiation on the CNS. Probably, in patients with PREP 2-3 degrees the autoimmune processes become chronic due to pathogenic action of free autoantibodies to NSE and of small NSE-anti-NSE complexes which are known to be the most pathogenic and capable of being fixed on complementary brain structures promoting their damage.     

Autoantibodies to the thymus and skin epithelium in NZB/N mice and (NZBxNZW)F1 hybrids

Antibodies reacting with thymus and skin epithelial cells were revealed by indirect immunofluorescence in sera of NZB/N mice and (NZB X NZW)F1 hybrids (B/W) 1-2 and 4-5 months of age. Similar antibodies were not found in sera of BALB/c mice. The inhibition experiments with DNA have shown that antibodies reacting with the thymus and skin epithelium differ from those reacting with the cellular nucleus. Positive reactions with the epithelium were obtained in all thymus and skin tissue samples of humans, guinea-pigs and NZB/N, B/W and BALB/c mice, including autologous tissues of NZB/N and B/W mice. Thus, antibodies reacting with thymus and skin epithelial tissues belong to autoantibodies. These autoantibodies are revealed during the first month of life before the onset of autoimmune processes. The role of these autoantibodies in the damage of thymus epithelium and the development of immunoregulatory disturbances, typical of autoimmune processes, needs further study.     

Cycloferon efficacy in viral and bacterial diseases of children (clinical review)

The authors' findings and literature data on the pharmacotherapeut efficacy of cycloferon, an interferon inductor (immunomodulators) are described. The drug effect in the treatment of various socially significant children' diseases, including acute respiratory tract viral infection, bronchial asthma, allergic conditions with infection protection disturbances, mycoplasmic infection, bronchopulmonary complications of acute respiratory tract viral infection with low intensity of free radical oxidation is indicated. The use of cycloferon at the background of vaccination was shown to provide inhibition of the autoimmune processes causing postvaccinal complications in frequently ill children. The results of the use of cycloferon in the treatment of gastrointestinal tract and intestinal infections of both the viral and bacterial genesis are discussed. Cycferon is recommended to be used for correction of the intestine dysbiosis (the microflora level came to normal in 95% of the children). The use of the drug in surgical pathology and in particular in appendicular peritonitis for decreasing the postoperative complications and correction of the immune disturbances due to chronic viral hepatitis C and B in children under the complex therapy is described. The cycloferon safety and efficacy were confirmed by the postmarketing randomized trials.     

Detection of autoantibodies against phenylalanyl-, tyrosyl-, and tryptophanyl-tRNA-synthetase and anti-idiotypic antibodies to it in serum from patients with autoimmune diseases]

Sera of patients bearing autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) and sera of clinically healthy donors were examined by ELISA for the presence of autoantibodies against tryptophanyl-, tyrosyl- and phenylalanyl-tRNA synthetases. Pure bovine synthetases served as antigens. It was shown that in patients with both autoimmune diseases all three enzyme autoantibodies were revealed at serum dilution 1/1600-1/3200. Moreover, by means of monoclonal antibodies against the same enzymes used for immunoaffinity sorption, antiidiotypic antibodies of IgG type against autoantibodies were detected. A conclusion has been made that autoimmune diseases are characterized by autoimmune response for many aminoacyl-tRNA synthetases irrespectively of their quaternary structure, intracellular location etc both at the level of primary and secondary antibodies.     

Allotype-specific immunoregulation of autoantibody production by host B cells in chronic graft-versus host disease

A chronic graft-vs-host (GVH) reaction induced in nonautoimmune mice by the transfer of Ia-incompatible spleen cells results in a syndrome that closely resembles SLE in the spectrum of autoantibodies and immunopathology. We have utilized Ia- and Igh-congenic strains to study the immunoregulation of autoantibody-producing B cells in this model. We have found that the autoantibodies are produced almost entirely by the host B cells. The transferred donor B cells contributed neither to the autoimmune response nor to the total serum Ig, with rare exceptions. The donor cell population did, however, exert an Igh allotype-specific immunoregulatory effect on the host B cells. For example, in allotype-heterozygous recipients, the autoantibodies were preferentially made by those host cells that expressed the donor allotype, whereas those host B cells that expressed nondonor allotype were relatively suppressed. In allotype-homozygous recipients, the donor cells frequently suppressed the host IgG2a allotype completely. This suppression sometimes prevented the IgG antichromatin response, although in other cases the response occurred with the use of a different isotype. In a final set of experiments, a chronic GVH reaction was induced in which both the donors and the recipients were Igh allotype heterozygous and yet differed at Ia. In this case, no donor influence on allotype should be expected; yet the IgG2a autoantibodies were clearly skewed toward the b allotype. These results show that host B cells play a unique role in the GVH autoimmune syndrome. In addition, they are immunoregulated in allotype-specific manners, some of which presumably involve interaction with donor T cells.     

Hormonal correction of the resident microflora of the vagina and uterus cervix in women with chronic cervicitis

Clinical, microbiological and hormonal examination of women with chronic cervicitis revealed lesions in the upper section of the reproductive tract in a high proportion of those examined, hormonal disturbances being registered in 96.7% of women. Dysbiotic manifestations (suppression of lacto- and bifidoflora and the excessive growth of opportunistic microorganisms) in the uterus cervix and vagina observed in patients with chronic cervititis were not associated with the etiology of the inflammatory process. The degree of dysmicrobiocenosis in the lower section of the genital tract in women with chronic cervicitis depends on the character of hormonal disturbances. The most significant inhibition of the resident flora was observed when ovarian dysfunction occurred and less significant--in cases of hyperprolactinemia and changes in the level of hypophysial hormones. Hormonal disturbances led to contamination of vagina and cervical canal with opportunistic microorganisms that was inversely proportional to the presence of lactic acid bacteria and bifidobacteria in these organs. Complex therapy of women with chronic cervicitis with the use of preparations for the correction of hormonal disturbances made it possible to restore the normal microflora of the genital tract and to improve the results of treatment.     

Identification of monoclonal insulin autoantibodies in insulin autoimmune syndrome associated with HLA-DRB1*0401

We have characterized HLA and insulin autoantibodies in a Japanese female patient with insulin autoimmune syndrome. Serological HLA typing demonstrated the patient had HLA-DR4, and DNA typing showed she had HLA-DRB1*0401 which has not been reported in patients with insulin autoimmune syndrome in Japan. A single binding affinity of insulin autoantibodies was demonstrated by Scatchard analysis and immunoglobulin class of insulin autoantibodies was exclusively IgG-kappa. HLA-DRB1*0406 is strikingly associated with patients with insulin autoimmune syndrome who have polyclonal insulin autoantibodies. The present report demonstrated the first Japanese patient with insulin autoimmune syndrome carrying HLA-DRB1*0401 who was revealed to have monoclonal insulin autoantibodies. The present results indicate that HLA molecules are the major determinants of polyclonal insulin autoantibodies and monoclonal insulin autoantibodies in insulin autoimmune syndrome.     

Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry

Normal sera contain a large number of naturally occurring autoantibodies which can mask important disease-associated ones. Western blotting has evolved as the most important tool to demonstrate autoantibodies in autoimmune diseases, because of its ability to simultaneous screening for a wide spectrum of different antigens. In previous studies we have shown the diagnostic potential of the analysis of autoantibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western blotting procedure remains very time-consuming and is also limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF MS (surface-enhanced laser desorption/ionization-time of flight mass spectrometry) technology for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The microscale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biologically activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of patients (n = 12). After washing, the chips were incubated with a complex solution of autoantigens and subsequently washed again. If the Protein-A bound autoantibodies recognized their antigens, these proteins could be separated by their molecular masses and were to be detected by mass spectrometry. Previous studies using monoclonal antibodies have demonstrated that the detection limit is in the attomole level. Furthermore, all sera were analyzed by conventional Western blotting for direct comparison. In the present study, we have shown complex on-chip antibody-antigen reactions. At higher molecular weights (> 30 kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular mass, the Western blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time-consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of autoantibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex autoantibody distributions for diagnostic purposes and early detection of autoimmune diseases might be possible.     

Dysbacteriosis in patients with colon polyposis

The study revealed the most profound changes in the composition of intestinal microflora in patients with polyposis of the large intestine. In these patients anaerobic microflora (bifidobacteria, lactic acid bacteria) was more often suppressed than in other examined groups, in particular, patients with cholelithic disease. The associations of hemolytic Escherichia with Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter freundii were often observed as well as the increased content of enterococci and fungi of the genus Candida. The determination of frequency and degree of manifestations showed that dysbacteriosis was registered in the absolute majority of patients (97.4%) with polyposis of the large intestine. Among patients with cholelithic disease disturbances in microbiocenosis were detected in 60.0% of cases, the profundity and character of the microflora composition changes being less pronounced.     

High Levels of Soluble Ctla-4 Are Present in Anti-Mitochondrial Antibody Positive,but Not in Antibody Negative Patients with Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a chronic autoimmune cholestatic liver disease frequently characterized by anti-mitochondrial autoantibodies (AMA). A minority of patients are AMA-negative. Cytotoxic-T-Lymphocyte-Antigen-4 (CTLA-4) is a surface molecule expressed on activated T-cells delivering a critical negative immunoregulatory signal. A soluble form of CTLA-4 (sCTLA-4) has been detected at high concentrations in several autoimmune diseases, and its possible functional meaning has been suggested. We aimed to evaluate sCTLA-4 concentration in sera of patients with PBC and to correlate it to immunological abnormalities associated with the disease. Blood samples were collected from 82 PBC-patients diagnosed according to international criteria (44 AMA-positive/MIT3-positive and 38 AMA-negative-MIT3-negative), and 65 controls. sCTLA-4 levels were evaluated by ELISA and Western blot. Increased sCTLA-4 concentrations were found in all AMA-positive PBC-patients, but in none of the AMA-negative ones, nor in normal controls or in controls with unrelated liver diseases. sCTLA-4 presence was associated with autoantibodies against MIT3, but not with nuclear autoantibodies (sp100, gp210). This is the first study to demonstrate that levels of sCTLA-4 are elevated in sera of PBC patients. However, they are clearly restricted to patients with AMA positivity, suggesting an immunological difference with respect to AMA-negative ones.     

Fcgamma receptors in the initiation and progression of systemic lupus erythematosus

Systemic lupus erythematosus, a systemic autoimmune disorder, is characterized by the production of autoantibodies to nuclear constituents and inflammatory lesions in multiple organ systems. Although the pathogenesis of the disease is largely unknown, recent studies have suggested that disturbances in apoptosis and/or clearance of apoptotic cells may play an important role in the induction and perpetuation of autoantibody production. When autoantibodies subsequently complex to autoantigens present on apoptotic cells, ligation of Fcgamma receptor will result in inflammation and disease development. Indeed, mice deficient in activating Fcgamma receptors were protected against inflammation in models of immune complex-mediated autoimmune disease, whereas deletion of the inhibitory Fcgamma receptors increased autoantibody production and susceptibility to immune complex-induced inflammation. Additionally, functional polymorphisms in Fcgamma receptors were shown to be associated with development of human systemic lupus erythematosus. This review focuses on the role of Fcgamma receptors in the initiation of autoantibody production, inflammatory handling of immune complexes, and disease development in systemic lupus erythematosus.     

Potential influence of nicotine on inflammation and induction of autoantibodies in rats with adjuvant arthritis

The influence of chronic administration of nicotine diluted in the drinking water on the parameters of systemic inflammation and autoimmune processes in rats (August line) with adjuvant-induced arthritis, were studied. The experiments have shown that nicotine acts as an antiphlogistic means (the amount of C-reactive protein rises in the blood) and activates autoimmune processes: induction of rheumatic factor, of autoantibodies to serotonin, and glial fibrillar acid protein. It was supposed that nicotine has a potential impact on immunocompetent cells.     

Cardiac myosin-induced myocarditis. Heart autoantibodies are not involved in the induction of the disease

We recently demonstrated that cardiac myosin is capable of inducing autoimmune myocarditis in genetically predisposed mice. This disease parallels coxsackievirus B3-induced autoimmune myocarditis in many respects and is associated with high-titer autoantibodies specific for cardiac myosin. The following lines of evidence suggest that these autoantibodies are not involved in the induction of autoimmune myocarditis: 1) immunoperoxidase staining of heart sections from cardiac myosin-immunized A/J and A.SW mice revealed IgG depositions only along damaged muscle fibres in infiltrated areas, but not in intact tissue; 2) myosin autoantibodies did not bind to the surface of viable cardiac myocytes isolated from mice, but only reacted with myocytes permeabilized with detergent; 3) mice treated with a single high dose of cyclophosphamide, which reduces the humoral immune response, still developed severe myocarditis, despite the fact that their autoantibody titers were reduced to the level of adjuvant-injected controls; and 4) passive transfer of high-titer myosin autoantibodies failed to induce myocarditis, although the titers in the recipients were comparable to those found in mice with cardiac myosin-induced disease. Together, the results suggest that high-titer myosin autoantibodies are secondary rather than primary to the disease.     

肾血管性高血压大鼠发病过程中心血管AT1-受体自身抗体的变化

实验观察了肾血管性高血压（RVH）大鼠血浆中抗AT1－受体自身抗体在发病过程中的作用及其变化规律。采用两肾一夹Goldblatt RVH模型,以合成的大鼠AT1－受体细胞外第二环165－191位氨基酸序列作为特慢性抗原,用SA－ELISA法检测大鼠血清中抗AT1-受体自身抗体。结果表明,RVH模型组术后2周时大鼠血清中抗AT1－受体自身抗体的阳性率和平均滴度与术前相比明显增高,较高的阳性率和平均滴度持续几周后逐渐下降,12周时下降到正常水平。结果提示,自身免疫机制参与了高血压的形成,抗AT1－受体自身抗体可能与心肌肥厚有关。     

Screening for associated autoimmunity in type 1 diabetes mellitus with respect to diabetes control

As an autoimmune disease, type 1 diabetes mellitus (DM) can be associated with other autoimmune disorders. The aim of this study was to detect subclinically associated autoimmune thyroid disease, coeliac disease, and Addison's disease. The presence of autoantibodies was evaluated with special regard to the control of diabetes and to the clinical status of the patient. Fifty-one type 1 diabetic patients (22 men, 29 women, mean age 37+/-11 years, mean duration of diabetes 16+/-13 years) were included into this study. Specific antibodies to islet antigens--glutamic acid decarboxylase (GAD65), protein thyrosine phosphatase IA-2alpha, and to thyroid autoantigens--thyroid microsomal peroxidase (TPO) and thyroglobulin (TG) and also thyroid stimulating hormone (TSH) were measured by RIA. Autoantigens of the small intestine--tissue transglutaminase autoantibodies (ATTG), IgA and IgG antibodies to gliadin (AGA-IgA, AGA-IgG) were evaluated by ELISA. Endomysial autoantibodies (EMA) and adrenal cortex antibodies (ACA) were detected by indirect immunofluorescence microscopy. Eleven new cases of thyreopathy (22 % of patients) were detected by the assessment of thyroid autoantibodies and TSH. Two new cases of thyreotoxicosis were diagnosed during the study. Coeliac disease was diagnosed in at least two cases. Addison's disease was not diagnosed, although the ACA were positive in two patients. No influence of single or combined autoantibody positivity on the control of diabetes was found if normal organ function was preserved. In both patients with thyreotoxicosis the control of diabetes was worsened and improved after treatment. The screening of autoantibodies in type 1 diabetic patients could reveal subclinical cases of AITD or coeliac disease. Subclinical forms of these disorders have no influence on diabetes control. However, impaired organ function may be associated with the worsened control of diabetes as we demonstrated on two newly diagnosed cases of thyreotoxicosis. We suggest the need for the follow-up of patients with positive autoantibodies because further deterioration of the respective organs can be expected.     

Protracted diarrhoea of infancy: evidence in support of an autoimmune variant.

Circulating autoantibodies to enterocytes were detected by indirect immunofluorescence in 14 out of 25 patients with idiopathic protracted diarrhoea of infancy. Similar specificities were not found in 50 control children with nongastroenterological disorders. The immunofluorescence pattern was more accentuated on the apical border of mature enterocytes. Enterocyte autoantibodies were mainly of IgG class (13/14), but 11 sera were positive for IgM and IgA classes, and five out of 14 positive sera also had the ability to fix complement. Absorption of sera positive for autoantibodies with an IgA coupled immunoabsorbent did not modify the intensity of the staining, indicating that these antibodies were not directed against secretory IgA. High titres and the complement fixing ability of enterocyte autoantibodies indicated a poorer prognosis despite the use of immunosuppressive drugs. Organ specific and non-organ specific autoimmune diseases or corresponding autoantibodies or both were often found in children with enterocyte autoantibodies and their family. These data show the existence of an autoimmune variant of protracted diarrhoea of infancy, despite the rare occurrence of autoimmune diseases in childhood.     

Radioimmunologic determination of pituitary autoantibodies in patients with Addison's disease]

The occurrence of pituitary autoantibodies was investigated in 34 patients, 25 females and 9 males, with Addison's disease of autoimmune origin and in 10 patients with tuberculous etiology of the disease. In both groups adrenal autoantibodies were also determined. Autoantibodies were determined by radioimmunoassay using solid phase technique in tubes coated with proteins of human adrenal and pituitary microsomal fractions. Pituitary autoantibodies were detected in 28 of 34 patients (20 females and 8 males) with autoimmune Addison's disease but only in 3 patients (2 females and one male) with nonimmune etiology of the disease. Both antipituitary and antiadrenal antibodies remain present in the circulation for many years as they were detected in autoimmune Addison's disease even after 10 or more years after the onset of the disease. There was a close relationship between the occurrence of pituitary antibodies and adrenal autoantibodies: they both appeared in the same patients and high titres of pituitary autoantibodies were associated with high titres of adrenal autoantibodies. This suggests a close similarity or identity of pituitary and adrenal autoantigens.     

Cytochrome P450 enzymes and UDP-Glucuronosyltransferases as hepatocellular autoantigens

Cytochromes P450 and UDP-Glucuronosyltransferases (UGT) are targets of microsomal autoantibodies in liver and kidney (LKM). LKM autoantibodies are observed in autoimmune hepatitis, in some patients with viral hepatitis, drug-induced hepatitis and autoimmune hepatitis as disease component of the autoimmune polyglandulars syndrome type 1 (APS-1). In autoimmune hepatitis LKM antibodies are markers of autoimmune hepatitis type 2. The major target of LKM-1 antibodies is cytochrome P450 2D6; a second less frequent target was the described UGTs of family 1. In autoimmune hepatitis LKM-1 autoantibodies are usually directed against small linear epitopes. LKM autoantibodies are also associated with infection with hepatitis viruses C and D. In hepatitis C about 1–2% of patients develop LKM-1 autoantibodies. About 60% of these autoantibodies are conformation dependent. The presence of LKM autoantibodies in hepatitis C may be associated with an increased risk in interferon treatment. LKM-3 autoantibodies are found in about 8% of patients with hepatitis D and are directed against conformational epitopes. Patients treated with certain drugs may develop drug induced hepatitis. In hepatitis induced by tienilic acid, tienilic acid is activated by and covalently bound to cytochrome P450 2C9. Activation of the immune system results in the formation of autoantibodies against cytochrome P450 2C9 (LKM-2) and infiltration of the liver with immune cells. A similar mechanism has been described for dihydralazine induced hepatitis, where autoantibodies are directed against P450 1A2 (LM). Autoantibodies directed against cytochrome P450 1A2 also are found in patients suffering from hepatitis as a disease component of APS-1.Abbreviations AIH autoimmune hepatitis - APS1 autoimmune polyendocrine syndrome type 1 - APS-1 autoimmune polyglandular syndrome type 1 - LKM microsomal autoantibodies in liver and kidney - HSV-1 herpes simplex virus type 1 - UGT UDP-glucuronosyltransferases     

Circulating and synovial antibody profiling of juvenile arthritis patients by nucleic acid programmable protein arrays

Introduction

Juvenile idiopathic arthritis (JIA) is a heterogeneous disease characterized by chronic joint inflammation of unknown cause in children. JIA is an autoimmune disease and small numbers of autoantibodies have been reported in JIA patients. The identification of antibody markers could improve the existing clinical management of patients.

Methods

A pilot study was performed on the application of a high-throughput platform, the nucleic acid programmable protein array (NAPPA), to assess the levels of antibodies present in the systemic circulation and synovial joint of a small cohort of juvenile arthritis patients. Plasma and synovial fluid from 10 JIA patients was screened for antibodies against 768 proteins on NAPPAs.

Results

Quantitative reproducibility of NAPPAs was demonstrated with > 0.95 intra-array and inter-array correlations. A strong correlation was also observed for the levels of antibodies between plasma and synovial fluid across the study cohort (r = 0.96). Differences in the levels of 18 antibodies were revealed between sample types across all patients. Patients were segregated into two clinical subtypes with distinct antibody signatures by unsupervised hierarchical cluster analysis.

Conclusion

The NAPPAs provide a high-throughput quantitatively reproducible platform to screen for disease-specific autoantibodies at the proteome level on a microscope slide. The strong correlation between the circulating antibody levels and those of the inflamed joint represents a novel finding and provides confidence to use plasma for discovery of autoantibodies in JIA, thus circumventing the challenges associated with joint aspiration. We expect that autoantibody profiling of JIA patients on NAPPAs could yield antibody markers that can act as criteria to stratify patients, predict outcomes and understand disease etiology at the molecular level.