Synthetic peptides in the diagnosis of systemic autoimmune diseases

Proteins recognized by antibodies from patients with autoimmune diseases have been intensively studied over the two past decades since cDNAs encoding autoantigens have become available. Identity of many of them has been defined, and specific structural motifs or post-translational modifications, which may be important to explain the generation of such antibodies during the autoimmune process, have been pointed out. Immunological analysis of sera from autoimmune patients with recombinant fragments and with short peptides has revealed the presence of dominant epitopes along proteins; some of them are targeted by antibodies from patients with specific diseases or disease subsets. Innovative technologies such as peptide arrays and biosensors as well as the exploitation of large peptides libraries have recently open up new perspectives. Peptides bearing natural modifications, peptide analogues, as well as mimotopes of protein or non-protein antigens (DNA, RNA, sugar) have been developed and might advantageously replace native antigens in routine immunoassays. Although numerous conformational epitopes have not yet been identified, and cannot be identified by the approaches classically used in epitope mapping studies, such peptides and peptide analogues may represent efficient probes to detect the presence of circulating autoantibodies in the serum of autoimmune patients and help for establishing specific and sensitive early diagnostic tests. They may also lead to the design of high-affinity ligands for purifying autoantibodies. These different aspects are discussed and epitope mapping studies of a number of autoantigens (e.g. histones, sn and hnRNP proteins and Ro proteins) are summarized.     

Molecular technology and the recombinant TSH have changed diagnostics of thyroid carcinoma with positive I-131 whole body scan but low serum thyroglobulin

The early detection of recurrent differentiated thyroid carcinoma (DTC) cells in the post surgery DTC patients relies on the sensitivity of measuring both the level of thyroglobulin (Tg) and 131-Iodine distribution by Whole Body Scan (WBS). Undetectable level of Tg associated with negative WBS or elevated levels of Tg associated with positive WBS ("concordant") is ordinarily indicative of either absence or presence of disease. At times, elevated level of Tg with negative WBS or low levels of Tg with positive WBS ("discordant") could also occur. In the present study, we retrospectively reviewed series of 573 patients with DTC followed in the Diagnostic Imaging and Radiotherapy of the University "Federico II" of Naples between 1993 and 1997. We focused on 9 out of 573 patients (1.56%) who had a discordant pattern with low level of Tg/positive WBS in the post-surgical follow-up. Four patients were metastatic at presentation while 5 patients with metastasis during follow-up still remained in persistently low levels of Tg (<5 ng/mL). This result does point to some flaw in the evaluation of "discordant" cases. Reviewing data previously described series by resetting cut-off values of Tg <1 ng/ml as undetectable changed the apparent "discordant" subgroup of patients into "concordant". Recent introduction of recombinant human TSH (rhTSH) to enhance the expression level of Tg brought significant increase in the sensitivity of diagnostic evaluation of thyroid cancer patients. The role of burdensome WBS in the follow up evaluation of DTC patients is significantly reduced over time especially in low-risk patients while the relevance of Tg assay is steadily increased. Sensitive Tg assays, significantly improved our ability to assess disease status in follow-up of DTC. Given the possibility of late disease relapses, the need for long-term follow-up, and reduced delay in treatment of persistent disease, there is still need for greater sensitive diagnostic tools for DTC.     

Streptavidin-enhanced assay for sensitive and specific detection of single nucleotide polymorphism in TP53

This paper reports an approach to detection of single nucleotide polymorphism based on special amplification assay and surface plasmon resonance biosensor technology. In this assay, a part of the target DNA is recognized by a probe (probe A) coupled with streptavidin–oligonucleotide (SON) complexes ex situ, and when the mixture is injected in the sensor, another part of the target DNA is recognized by a DNA probe (probe B) immobilized on the sensor surface. To achieve high sensitivity and specificity, the assay is optimized in terms of composition of SON complexes, probe design, and assay temperature. It is demonstrated that this approach provides high specificity (no response to targets containing single-mismatched bases) and sensitivity (improves sensor response to perfectly matched oligonucleotides by one order of magnitude compared to the direct detection method). The assay is applied to detection of a short synthetic analogue of TP53 containing a “hot spot”—single nucleotide mismatch frequently mutated in germ line cancer—at levels down to 40 pM.     

Effect of selenium supplementation on thyroid antibodies

Selenium is an essential component of selenoproteins, enzymes with extensive regulatory and protective effect in organism. Immunological effects of Se are documented and are distinct even above concentrations necessary for maximal activity of selenoenzymes. Therefore, we investigated effect of supplementation by 100 μg of yeast-bound Se on concentrations of thyroid autoantibodies TPOAb and TgAb in the group of 253 seniors living in the Asylum Houses of South Bohemia. Increase of serum selenium from 59 to 150 μg Se/L serum in supplemented group and from 59 to 72 μg Se/L serum in group with placebo were detected by Instrumental Neutron Activation Analysis (INAA) and proved increased Se intake during the trial. Autoantibodies were analyzed by ELISA at the beginning of the trial and after 1 year. Statistical evaluation of results in whole groups (regardless of increased autoantibodies) by ANOVA manifested significant decrease of TPOAb and TgAb in non-supplemented group while supplementation did not effect serum autoantibodies concentrations. Evaluation of groups of seniors created from those with increased autoantibodies, ANOVA demonstrated decrease of TPOAb in both groups but Se supplementation did not affect the decrease. In opposite, TgAb increased significantly and Se supplementation led to higher increase of TgAb. Recent results of possibility to decrease serum concentration of TPOAb proved this effect only for high TPOAb concentrations and for higher Se supplements. From this point of view, it is necessary to conduct subsequent trials with the patients with autoimmune thyreoiditis with different levels of autoantibodies and detect also serum Se levels.     

Epitope promiscuity of human monoclonal autoantibodies to T-cell receptor-combining site determinants

To characterize the binding specificity and light- and heavy-chain variable region usage in monoclonal human autoantibodies (mAAbs) to T-cell receptors, we constructed heterohybridomas from peripheral blood B cells of three rheumatoid arthritis (RA) patients. From a panel of more than 200 heterohybridomas secreting IgM autoantibodies binding to T-cell receptor Vbeta chain first complementarity determining segments (CDR1), we characterized two IgM/lambda molecules from a single patient in detail. These bound to both CDR1 peptide epitopes and intact TCR of recombinant single-chain T-cell receptor constructs, and to T-cell surface TCR. Spectratype analysis using epitopes mimicking a set of 24 Vbeta genes indicated that one molecule bound only a few members of the set, whereas the second showed considerable epitope promiscuity by binding to more than half of the tested CDR1 peptides. Both mAAbs used variants of a Vlambda3 gene that were very similar to one another and to the germline gene. The epitope-promiscuous autoantibody used a V(H)4 gene identical to a germline prototype, while the other incorporated a V(H)3 sequence differing in only a single residue from its germline prototype. The CDR3s of both were large and distinct from each other as well as from the corresponding segments of rheumatoid factors and "cold agglutinins" using the same or related V(H) germline genes. These mAAbs offer models for deciphering the basis of epitope promiscuity, and serve as candidates for direct use in immunomodulation because they are of intrinsic human origin and do not require molecular engineering to adapt them for use in therapy.     

Modification of β2 I by glutardialdehydeI by glutardialdehyde

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on β₂glycoprotein I (β₂GPI) only when native β₂GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene, β₂GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-β₂GPI epitope at GDA: β₂GPI mol ratios in the range of 500–2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of β₂GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of β₂GPI, but does not expose the epitope. The circular dichroism spectra of native and modified β₂GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that β₂GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of β₂GPI, conditions were defined that are necessary for binding of human autoantibodies. The authors also suggest that treatment of phospholipid-binding proteins with chemical agents might provide a strategy to modify their structure and permit exposure of epitopes, resulting in synthetic antigens for therapeutic and diagnostic use.     

Photoassisted Synthesis of Luminescent Mannose–Au Nanodots for the Detection of Thyroglobulin in Serum

We have employed mannose‐modified gold nanodots (Man–Au NDs) as a luminescence sensor for the detection of the thyroid‐cancer marker thyroglobulin (Tg) in homogeneous solutions. The luminescent Man–Au NDs are prepared through the reaction of 2.9 nm‐diameter gold nanoparticles (Au NPs) with 11‐mercapto‐3,6,9‐trioxaundecyl‐α‐D ‐mannopyranoside (Man‐RSH) under the irradiation of a light‐emitting diode (LED). We have found that the irradiation enhances the quantum yield (～11 %), alters the emission wavelength and lifetimes, and shortens the preparation time. A luminescence assay has been developed for Tg based on the competition between Tg and Man–Au NDs for the interaction with the concanavalin A (Con A). Because luminescence quenching of the Man–Au NDs by Con A is inhibited by Tg selectivity, we have obtained a highly sensitive and selective assay for Tg.     

桥本氏甲状腺炎合并甲状腺乳头状微小癌患者的超声特点与临床参数的相关性分析

本文探讨了桥本氏甲状腺炎合并甲状腺乳头状微小癌患者的超声特点与临床参数的相关性。选择2010年1月~2018年12月我院收治的桥本氏甲状腺炎合并甲状腺乳头状微小癌患者75例作为研究组,单纯桥本甲状腺炎患者696例作为对照组,分析了两组患者的临床病理特征、二维超声图像特征及内部血流图像特征。结果显示,两组平均年龄、肿块直径、病灶数量、甲状腺结构、形态、晕圈比较无统计学差异(P>0.05);研究组的性别构成、病灶钙化情况、内部回声情况、内部血流情况与对照组有统计学差异(P<0.05)。多因素Logistic回归分析显示,女性、微钙化、边缘回声不清晰、内部极低回声、内部无血流是桥本氏甲状腺炎合并甲状腺乳头状微小癌的危险因素(P<0.05)。说明超声能够对桥本氏甲状腺炎合并甲状腺乳头状微小癌进行有效诊断。     

Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody

In this study we have applied epitope excision and epitope extraction strategies, combined with matrix assisted laser desorption/ionization mass spectrometry, to determine the fine structure of epitopes recognized by a polyclonal antibody to human immunodeficiency virus envelope glycoprotein gp120. This is the first application of this approach to epitope mapping on a large, heavily glycosylated protein. In the epitope excision method, gp120 in the native form is first bound to the antibody immobilized on sepharose beads and cleaved with endoproteinase enzymes. In the epitope extraction method, the gp120 was first proteolytically cleaved and then allowed to react with the immobilized antibody. The fragments that remain bound to the antibody, after repeated washing to remove the unbound peptides, contain the antigenic region that is recognized by the antibody, and the bound peptides in both methods can be characterized by direct analysis of the immobilized antibody by matrix assisted laser desorption ionization/mass spectrometry. In this study we have carried out epitope excision and extraction experiments with three different enzymes and have identified residues 472–478 as a major epitope. In addition, antigenic regions containing minor epitopes have also been identified.     

Natural catalytic immunity is not restricted to autoantigenic substrates

The autoimmune repertoire is well known from previous studies to be capable of producing catalytic antibodies directed to self-antigens. In the present study, we explored the ability of 26 monoclonal light chains (Lchains) from multiple myeloma patients to cleave radiolabeled gp 120, a foreign protein. One L chain with this activity was identified. ¹²⁵I-gp120 and unlabeled gp 120 were cleaved at several sites by the L chain, as shown by SDS-polyacrylamide gel electrophoresis, autoradiography, and immunoblotting, respectively. The apparent dissociation constant of the L chain was 130–145 nM, indicating high-affinity gp 120 recognition. ¹²⁵I-albumin was not cleaved by the L chain, and various proteins and peptides did not inhibit gp 120 cleavage by the L chain, suggesting that the activity is not a nonspecific phenomenon. The substrate recognition determinants may be conserved in different HIV-1 strains, because gp 120 isolated from strains SF2, MN, and IIIB was found to be cleaved by the L chain. Micromolar concentrations of a synthetic peptide corresponding to residues 23–30 of gp 120 inhibited the cleavage of ¹²⁵I-gp 120, suggesting that these residues are components of the epitope recognized by the L chain. The toxic effect of gp120 in neuronal cultures was reduced by about 100-fold by pretreatment of the protein with the L chain. These observations open the possibility of utilizing gp120-cleaving antibodies in the treatment of AIDS.     

T-cell epitopes of the La/SSB autoantigen: prediction based on the homology modeling of HLA-DQ2/DQ7 with the insulin-B peptide/HLA-DQ8 complex

T-cell epitopes are important components of the inappropriate response of the immune system to self-proteins in autoimmune diseases. In this study, the candidate T-cell epitopes of the La/SSB autoantigen, the main target of the autoimmune response in patients with Sjogren's Syndrome (SS), and Systemic Lupus Erythematosus (SLE) were predicted using as a template the HLA-DQ2 and DQ7 molecules, which are genetically linked to patients with SS and SLE. Modeling of DQ2 and DQ7 was based on the crystal structure of HLA-DQ8, an HLA molecule of high risk factor of type I diabetes, which is also an autoimmune disease. The quality and reliability of the modeled DQ2 and DQ7 was confirmed by the Ramachandran plot and the TINKER molecular modeling software. Common and/or similar candidate T-cell epitopes, obtained by comparing three different approaches the Taylor's sequence pattern, the TEPITOPE quantitative matrices, and the MULTIPRED artificial neural network, were subjected to homology modeling with the crystal structure of the insulin-B peptide complexed with HLA-DQ8, and the best superposed candidate epitopes were placed into the modeled HLA-DQ2 and DQ7 binding grooves to perform energy minimization calculations. Six T-cell epitopes were predicted for HLA-DQ7 and nine for HLA-DQ2 covering parts of the amino-terminal and the central regions of the La/SSB autoantigen. Residues corresponding to the P1, P4, and P9 pockets of the HLA-DQ2 and DQ7 binding grooves experience very low SASA because they are less exposed to the microenvironment of the groove. The proposed T-cell epitopes complexed with HLA-DQ2/DQ7 were further evaluated for their binding efficiency according to their potential interaction energy, binding affinity, and IC50 values. Our approach constitutes the ground work for a rapid and reliable experimentation concerning the T-cell epitope mapping of autoantigens, and could lead to the development of T-cell inhibitors as immunotherapeutics in autoimmune diseases.     

Idiotypic network dysregulation

The pathogenesis of autoimmune disease is still an enigma. Whereas the diverse clinical manifestations of many autoimmune diseases cannot be explained by the existence of autoantibodies, idiotypic dysregulation may provide an alternative explanation. Experimental models, serum level changes of pathogenic idiotypes during exacerbation and remission, and the increased expression of pathogenic idiotypes following common infections all support this notion. In this article we review experimental models of autoimmune disease induction (systemic lupus erythematosus, antiphospholipidsyn drome, Goodpasture's syndrome, autoimmune thyroiditis, and vasculitis) by manipulation of the idiotypic network and discuss the utilization of idiotypes for the immunotherapy of autoimmune diseases and other conditions that involve the immune system (e.g., atherosclerosis).     

Chemical synthesis and enzymatic assembly of fragments of the DNA coding immunodominant epitopes of human immunodeficiency virus

More than twenty 13- to 55-membered oligo(poly)nucleotides have been synthesized by the H-phosphonate solid-phase method in the manual variant using an original procedure. From the oligonucleotides obtained, seven DNA duplexes coding immunodominant epitopes of HIV-1 proteins have been formed: 598–609 and 737–748 gp41, 91–115 and 105–115 gag, and 940–951 pol and a number of its mutants. The ligase assembly and polymerization of the DNA duplexes obtained has been carried out. The efficiency of ligation amounted to from 60 to 90%. The possibility of their directed polymerization was determined by the flanking of the duplexes by partially completed half-sites of restrictases BamHI and XhoI. Abbreviations adopted: HIV-1 — human immunodeficiency virus, type 1; (+)-chain — the coding chain — and (−)-chain the chain complementary to the coding chain of the DNA duplex; CPG — controlled-pore glass; PAAG — polyacrylamide gel; Py — pyridine.     

Prediction of T-cell epitopes based on least squares support vector machines and amino acid properties

T-lymphocyte (T-cell) is a very important component in human immune system. It possesses a receptor (TCR) that is specific for the foreign epitopes which are in a form of short peptides bound to the major histocompatibility complex (MHC). When T-cell receives the message about the peptides bound to MHC, it makes the immune system active and results in the disposal of the immunogen. The antigenic determinants recognized and bound by the T-cell receptor is known as T-cell epitope. The accurate prediction of T-cell epitopes is crucial for vaccine development and clinical immunology. For the first time we developed new models using least squares support vector machine (LSSVM) and amino acid properties for T-cell epitopes prediction. A dataset including 203 short peptides (167 non-epitopes and 36 epitopes) was used as the input dataset and it was randomly divided into a training set and a test set. The models based on LSSVM and amino acid properties were evaluated using leave-one-out cross-validation method and the predictive ability of the test set, and obtained the results of 0.9875 and 0.9734 under the ROC curves, respectively. This result is more satisfactory than that were reported before. Especially, the accuracy of true positive gets a marked enhancement.     

Multiplex real-time PCR using SYBR® GreenER™ for the detection of DNA allergens in food

Thyroid hormones are essential hormones for regulating growth and development in humans and wildlife. Methods to monitor precise and low levels of these hormones in serum and tissues are needed to assess overall health, whether from disease considerations or possibly from environmental contaminant exposures. Common and routine methods typically rely upon radioimmunoassays, which can be expensive, and typically only measure thyroxine and 3,3′,5-triidothyronine, which can be a limitation in fully evaluating impacts on thyroid regulation. In this study we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of five thyroid hormones—thyroxine, 3,3′,5-triidothyronine, 3,3′,5′-triiodothyronine, 3,3′-diiodothyronine, and 3,5-diiodothyronine—in serum samples. The LC-MS/MS parameters were optimized and calibrated over a wide concentration range (1.0–500 ng/mL) with on-column detection limits of 1.5–7.0 pg. With use of spiked bovine serum samples, the mean method recoveries were calculated to be 81.3–111.9% with relative standard deviations of 1.2–9.6% at spiking levels ranging from 10 to 100 ng/mL. This method was compared with measurements made by standard radioimmunoassays and with measurements made in a serum Standard Reference Material (SRM 1951b). Development of this method expands the capacity to measure thyroid hormones by including a larger suite of thyroid hormones, and has promising applications for measuring catabolism of thyroid hormones in vitro.     

Idiotypic network dysregulation: a common etiopathogenesis of diverse autoimmune diseases

The pathogenesis of autoimmune disease is still an enigma. Whereas the diverse clinical manifestations of many autoimmune diseases cannot be explained by the existence of autoantibodies, idiotypic dysregulation may provide an alternative explanation. Experimental models, serum level changes of pathogenic idiotypes during exacerbation and remission, and the increased expression of pathogenic idiotypes following common infections all support this notion. In this article we review experimental models of autoimmune disease induction (systemic lupus erythematosus, antiphospholipid syndrome, Goodpasture's syndrome, autoimmune thyroiditis, and vasculitis) by manipulation of the idiotypic network, and discuss the utilization of idiotypes for the immunotherapy of autoimmune diseases and other conditions that involve the immune system (e.g., atherosclerosis).     

Has immunoblotting replaced electroimmunoprecipitation? Examples from the analysis of autoantigens and transglutaminase-induced polymers of the human erythrocyte membrane

The virtues and drawbacks of immunoblotting and electroimmunoprecipitation in the characterization of macromolecules in crude mixtures are presented. Interactions between autoantibodies and human erythrocyte membrane proteins were studied by means of crossed-affinoimmunoelectrophoresis with autologous immunoglobulins incorporated into the first dimension gel and by immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis separated erythrocyte membrane proteins with autologous immunoglobulins as primary antibodies. Substrates for transglutaminase in calcium-activated human erythrocyte membranes were examined by immunoelectrophoretic and immunoblotting methods. The experiments concerning autoantibodies complemented each other and showed that epitopes on Band 3 protein, spectrin and ankyrin are recognized by circulating immunoglobulin autoantibodies in normal individuals. The polymer experiments showed the presence of spectrin, ankyrin, Band 3, Band 4.1, glucose transporter, actin and haemoglobin epitopes in the polymer (M_r 3 · 10⁶-5 · 10⁶). It is concluded that the two techniques complement each other. The most evident advantage of immunoblotting is its sensitivity and applicability while electroimmunoprecipitation in some instances allows an easier identification of distinct protein species and still has a rôle for quantification and certain monitoring purposes.     

Diagnosis of cold thyroid nodules by 201T1 scientigraphy

Fifty two patients with cold thyroid nodule demonstrated on the thyroid scan were imaged with 201Tl which were given intravenously as thallium chloride in dose of 2 mCi. Thirty nine of 52 patients were confirmed and investigated whether 201Tl concentrated or not. Fourteen of 15 (93 percent) thyroid carcinomas, 5 of 17 (29 percent) thyroid adenomas, 1 of 2 adenomatous goiters and all of 5 of chronic thyroiditis were visualized as positive with 201 Tl. One thyroid carcinoma did not concentrate 201Tl which was confirmed to have cystic degeneration. Of the 19 benign cold thyroid nodules except chronic thyroiditis 6 were positively visualized with 201Tl. However, 201Tl did not accumulate in the other 13 benign nodules, 11 out of which were confirmed to have cystic degeneration. The data suggests that if the thyroid nodule is found to have negative accumulation of 201Tl, malignancy can be ruled out except a small microscopical lesion.     

Autoantibody detection by direct counting of antigen-displayed yeast cells

In this study, we report a new immunoassay platform based on yeast surface display technology for detection of autoantibodies involved in autoimmune diseases, e.g., systemic lupus erythematosus (SLE) and Sj?gren's syndrome (SS). The autoantigens of Ro52/SSA epitope and SmD were chosen to be displayed on the yeast surface with their respective antibodies as the analytes. By using magnetic beads modified with protein G, yeast cells bound with specific target antibody can be captured. The amount of analytes could be determined by counting the number of fluorescent yeast cells captured in a magnetic field. The platform showed promising results in the detection of SLE autoantibodies with high sensitivity and multiplex detection capability over the traditional approaches.     

Biosensor analyses of serum autoantibodies: application to antiphospholipid syndrome and systemic lupus erythematosus

Autoimmune disorders are rare human diseases characterized by the presence of circulating autoantibodies that bind the body’s own structural compounds as target antigens. The detection of autoantibodies is important for the diagnostic process. Immunofluorescence and immunoassay methods do not allow a reliable characterization of binding characteristics. Therefore, novel analytical techniques should be considered. This review describes the application of surface plasmon resonance biosensor systems for the diagnosis of autoimmune disorders. The covalent attachment of native antigens to the sensor chip is a suitable method for obtaining highly reproducible analyses of autoantibodies, allowing the evaluation of kinetic rate and affinity constants, and it may enable the identification of disease-relevant autoantibodies linked to disease progression. The autoantibody microarray is another future-oriented technique. Patterns of differential antigen recognition should allow early diagnosis. This is due to the fact that a broad range of autoreactive B cell responses in autoimmune disorders can only be mirrored by including a sufficient number of antigens in a microarray format.