Use of synthetic peptides for the detection and quantification of autoantibodies

Summary and conclusions The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins).Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity.Synthetic peptide antigens have beeen used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications. It is unlikely that short synthetic peptides will substitute for native proteins in all instances since some autoantibodies show a striking preference for conformational epitopes.Abbreviations r recombinant - SLE systemic lupus erythematosus     

B-cell epitopes of scleroderma-specific autoantigens

Conclusions It can be concluded that the precise localization of the epitopes on autoantigens associated with scleroderma has not been determined yet, and further subcloning experiments will be required to map the epitopes more precisely. However, the fact that the antigenicities of the C-terminal ends of topo I as well as of CENP-B are highly affected by the length of the fusion segments suggest that most, if not all, antigenic determinants on these parts of the autoantigens are conformational epitopes. Studies based upon molecular modelling of antibodies reacting with antigens suggest that over 90% of B-cell epitopes are conformational [50]. This implies that the most successful approach to allocate B-cell epitopes on autoantigens in the near future may be the use of techniques for mapping conformational epitopes. Such techniques are currently being developed [reviewed in 51]. Until now, the limited data available indicate that the B-cell epitopes on the scleroderma-associated autoantigens are distributed over the entire proteins. The C-terminal parts of the antigens seem to be good candidates for harboring the major autoimmune epitopes, but more experimental data will be necessary to confirm this suggestion.     

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Cloning and expression of mouse 60 kDa ribonucleoprotein SS-A/Ro

Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5′ RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kindney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.     

Immunoreactivity between a monoclonal lupus autoantibody and the arginine/aspartic acid repeats within the U1-snRNP 70K autoantigen is conformationally restricted

Immunoreactivity of the arginine/aspartic acid (RD) repeats of the 70K protein of U1 small nuclear ribonucleoprotein (snRNP) was determined to be conformationally dependent. The monoclonal autoantibody 2.73, isolated from a lupus-prone MRL/n mouse model, is reactive with the RD repeat regions of U1 snRNP 70K protein. Immunochemical analysis of the antigenic determinants with use of chemically synthesized peptides characterized the 2.73 epitope as the RD repeat [Pelsue, S.,et al. (1993)Autoimmunity,15, 231–236] Analysis by circular dichroism (CD) and nuclear magnetic resonance spectroscopy indicates conformational preferences in the immunoreactive peptides. Computer analyses of CD spectra obtained on the RD-containing peptides predict-turns and-sheet to be the preferred conformations of the RD repeats. This structure was also predicted by the Chou-Fasman algorithm. The RD repeat is believed to be a conserved structural motif; however, the biological function is still unclear. Immunological and biochemical analysis of autoimmune antibodies and their respective antigenic determinants has helped to characterize the possible mechanisms that lead to autoimmune diseases. This is the first report of a conformationally dependent, linear epitope of an autoantibody.     

Autoantibody microarrays for biomarker discovery

Identification of autoantigens and the detection of autoantibody reactivity are useful in biomarker discovery and for explaining the role of important biochemical pathways in disease. Despite all of their potential advantages, the main challenge to working with autoantibodies is their sensitivity. Nevertheless, proteomics may hold the key to overcoming this limitation by providing the means to multiplex. Clearly, the ability to detect multiple autoantigens using a platform such as a high-density antigen microarray would improve sensitivity and specificity of detection for autoantibody profiling. The aims of this review are to: briefly describe the current status of antigen–autoantibody microarrays; provide examples of their use in biomarker discoveries; address current limitations; and provide examples and strategies to facilitate their implementation in the clinical setting.     

Expression of the Centrosomal Colon Cancer Autoantigen Gene during Spermatogenesis in the Maturing Rat Testis

We analyzed the gene and protein expression of serologically defined colon cancer antigen 8. Gene expression was upregulated in the maturing rat testis, and was localized to the spermatocytes. Protein was detected in the spermatids and at the sites of mRNA expression. Specific expression of colon cancer antigen 8 was observed in the maturing rat testis.     

The NOTCH4-GATA4-IRG1 axis as a novel target in early-onset colorectal cancer

The early onset of colorectal cancer (CRC) in individuals younger than 50 years is an emerging phenomenon, and obesity is a strong risk factor. Inflammatory mechanisms are mediated by immune cells, with macrophages and their phenotypical changes playing a significant role in CRC. Obesity-related hormones, such as leptin and adiponectin, affect macrophage polarization and cytokine expression. Macrophage metabolism, and therefore polarization, directly affects tumor progression and survival in patients with CRC. Altered obesity-related hormone levels induce phosphoinositide kinase-3 (PI3K)/serine-threonine-protein kinase (AKT) activation in colon cancer, causing increased cell survival, hyperplasia, and proliferation. Investigating the effects of obesity-related mechanisms on PI3K/Akt signaling can provide new insights for targeting mechanisms in CRC and obesity among the young. Central molecules for the control of cell proliferation, differentiation, and tumorigenesis within the gastrointestinal tract include downstream targets of the PI3K/AKT pathway, such as Neurogenic locus notch homolog 4 (Notch4) and GATA binding proteins (GATA). Leptin and adiponectin both alter gene expression within this pathway, thereby affecting TAM-mediated CRC progression. Our goal is to introduce the NOTCH4-GATA4-IRG1 axis as a link between inflammation and sporadic CRC and to discuss this pathway as a new potential immunotherapeutic target in individuals affected with obesity and early-onset CRC.     

综述与专论：免疫吸附治疗自身免疫疾病的研究进展

自身免疫疾病治疗的常规方法 (如免疫抑制剂和血浆置换等)缺乏足够的安全性和有效性,因此,寻找新的治疗方法十分必要。免疫吸附(immunoadsorption,IA)是一种通过选择性或非选择性去除自身致病抗体,从而实现对自身免疫疾病治疗的方法。本文介绍了免疫吸附在扩张型心肌病、特发性膜性肾病、系统性红斑狼疮、重症肌无力4种自身免疫疾病中的研究和临床应用,讨论了该治疗方法的有效性和安全性;同时指出,免疫吸附要在更多的疾病中实现临床应用,还需要在可信度、地域性、样本量等方面做更加深入的临床前试验。