Islet-cell autoantigen 69 mediates the antihyperalgesic effects of electroacupuncture on inflammatory pain by regulating spinal glutamate receptor subunit 2 phosphorylation through protein interacting with C-kinase 1 in mice.

Electroacupuncture (EA) is widely used in clinical settings to reduce inflammatory pain. Islet-cell autoantigen 69 (ICA69) has been reported to regulate long-lasting hyperalgesia in mice. ICA69 knockout led to reduced protein interacting with C-kinase 1 (PICK1) expression and increased glutamate receptor subunit 2 (GluR2) phosphorylation at Ser880 in spinal dorsal horn. In this study, we evaluated the role of ICA69 in the antihyperalgesic effects of EA and the underlying mechanism through regulation of GluR2 and PICK1 in spinal dorsal horn. Hyperalgesia was induced in mice with subcutaneous plantar injection of complete Freund adjuvant (CFA) to cause inflammatory pain. Electroacupuncture was then applied for 30 minutes every other day after CFA injection. When compared with CFA group, paw withdrawal frequency of CFA+EA group was significantly decreased. Remarkable increases in Ica1 mRNA expression and ICA69 protein levels on the ipsilateral side were detected in the CFA+EA group. ICA69 expression reached the peak value around day 3. More importantly, ICA69 deletion impaired the antihyperalgesic effects of EA on GluR2-p, but PICK1 deletion could not. Injecting ICA69 peptide into the intrathecal space of ICA69-knockout mice mimicked the effects of EA analgesic and inhibited GluR2-p. Electroacupuncture had no effects on the total protein of PICK1 and GluR2. And, EA could increase the formation of ICA69-PICK1 complexes and decrease the amount of PICK1-GluR2 complexes. Our findings indicate that ICA69 mediates the antihyperalgesic effects of EA on CFA-induced inflammatory pain by regulating spinal GluR2 through PICK1 in mice.

A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome.

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3-α5 chains (α3-α5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of α3/α4/α5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular α(IV) chain and the absence of high-throughput screening (HTS) platforms to assess α345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion α345(IV) proteins to monitor α345(IV) trimerization of wild-type and clinically associated mutant α5(IV). The α345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular- and secretion-dependent defects of mutant α5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote α345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting α345(IV) trimers to treat Alport syndrome.

Cutting Edge: Processing of Oxidized Peptides in Macrophages Regulates T Cell Activation and Development of Autoimmune Arthritis.

APCs are known to produce NADPH oxidase (NOX) 2-derived reactive oxygen species; however, whether and how NOX2-mediated oxidation affects redox-sensitive immunogenic peptides remains elusive. In this study, we investigated a major immunogenic peptide in glucose-6-phosphate isomerase (G6PI), a potential autoantigen in rheumatoid arthritis, which can form internal disulfide bonds. Ag presentation assays showed that presentation of this G6PI peptide was more efficient in NOX2-deficient (Ncf1m1J/m1J mutant) mice, compared with wild-type controls. IFN-γ-inducible lysosomal thiol reductase (GILT), which facilitates disulfide bond-containing Ag processing, was found to be upregulated in macrophages from Ncf1 mutant mice. Ncf1 mutant mice exhibited more severe G6PI peptide-induced arthritis, which was accompanied by the increased GILT expression in macrophages and enhanced Ag-specific T cell responses. Our results show that NOX2-dependent processing of the redox-sensitive autoantigens by APCs modify T cell activity and development of autoimmune arthritis.

Proteolysis by Granzyme B Enhances Presentation of Autoantigenic Peptidylarginine Deiminase 4 Epitopes in Rheumatoid Arthritis.

Proteolysis of autoantigens can alter normal MHC class II antigen processing and has been implicated in the induction of autoimmune diseases. Many autoantigens are substrates for the protease granzyme B (GrB), but the mechanistic significance of this association is unknown. Peptidylarginine deiminase 4 (PAD4) is a frequent target of autoantibodies in patients with rheumatoid arthritis (RA) and a substrate for GrB. RA is strongly associated with specific MHC class II alleles, and elevated levels of GrB and PAD4 are found in the joints of RA patients, suggesting that GrB may alter the presentation of PAD4 by RA-associated class II alleles. In this study, complementary proteomic and immunologic approaches were utilized to define the effects of GrB cleavage on the structure, processing, and immunogenicity of PAD4. Hydrogen-deuterium exchange and a cell-free MHC class II antigen processing system revealed that proteolysis of PAD4 by GrB induced discrete structural changes in PAD4 that promoted enhanced presentation of several immunogenic peptides capable of stimulating PAD4-specific CD4+ T cells from patients with RA. This work demonstrates the existence of PAD4-specific T cells in patients with RA and supports a mechanistic role for GrB in enhancing the presentation of autoantigenic CD4+ T cell epitopes.

The role of T-cell receptor recognition of peptide:MHC complexes in the formation and activity of Foxp3⁺ regulatory T cells.

Foxp3(+) regulatory T (Treg) cells are required to prevent the immune system from spontaneously mounting a severe autoaggressive lymphoproliferative disease and can modulate immune responses in a variety of settings, including infections. In this review, we describe studies that use transgenic mice to determine how signals through the T-cell receptor (TCR) contribute to the development, differentiation, and activity of Treg cells in in vivo settings. By varying the amount and quality of the self-peptide recognized by an autoreactive TCR, we have shown that the interplay between autoreactive thymocyte deletion and Treg cell formation leads to a Treg cell repertoire that is biased toward low abundance agonist self-peptides. In an autoimmune disease setting, we have demonstrated that diverse TCR specificities can be required in order for Treg cells to prevent disease in a mouse model of autoimmune inflammatory arthritis. Lastly, we have shown that Treg cells initially selected based on specificity for a self-peptide can be activated by TCR recognition of a viral peptide, and that they can acquire a specialized phenotype and suppress antiviral effector cell activity at the site of infection. These studies provide insights into the pivotal role that TCR specificity plays in the formation and activity of Treg cells.

GAMETOPHYTE DEFECTIVE 1, a putative subunit of RNases P/MRP, is essential for female gametogenesis and male competence in Arabidopsis.

RNA biogenesis, including biosynthesis and maturation of rRNA, tRNA and mRNA, is a fundamental process that is critical for cell growth, division and differentiation. Previous studies showed that mutations in components involved in RNA biogenesis resulted in abnormalities in gametophyte and leaf development in Arabidopsis. In eukaryotes, RNases P/MRP (RNase mitochondrial RNA processing) are important ribonucleases that are responsible for processing of tRNA, and transcription of small non-coding RNAs. Here we report that Gametophyte Defective 1 (GAF1), a gene encoding a predicted protein subunit of RNases P/MRP, AtRPP30, plays a role in female gametophyte development and male competence. Embryo sacs were arrested at stages ranging from FG1 to FG7 in gaf1 mutant, suggesting that the progression of the gametophytic division during female gametogenesis was impaired in gaf1 mutant. In contrast, pollen development was not affected in gaf1. However, the fitness of the mutant pollen tube was weaker than that of the wild-type, leading to reduced transmission through the male gametes. GAF1 is featured as a typical RPP30 domain protein and interacts physically with AtPOP5, a homologue of RNases P/MRP subunit POP5 of yeast. Together, our data suggest that components of the RNases P/MRP family, such as RPP30, play important roles in gametophyte development and function in plants.

Peptide-based immunotherapy of experimental autoimmune encephalomyelitis without anaphylaxis.

Administration of peptide antigens in tolerogenic form holds promise as a specific treatment for autoimmune and allergic disorders. However, experiments in rodent autoimmune models have highlighted the risk of anaphylaxis in response to systemic peptide application once the aberrant immune response is underway. Thus, mice with clinical signs of experimental autoimmune encephalomyelitis (EAE) or diabetes have been reported to suffer fatal anaphylaxis upon administration of native autoantigenic peptides. Clearly, this might represent a significant barrier to the use of synthetic peptides in the treatment of ongoing human autoimmune conditions. Here we describe the development of an altered peptide ligand (APL) engineered to prevent anaphylaxis (no antibody binding) whilst retaining the ability to silence pathogenic myelin-reactive T lymphocytes. Administration of the APL to mice with an ongoing anti-myelin immune response did not cause anaphylaxis, but led to complete protection from the subsequent induction of EAE and, when given during ongoing EAE, led to a rapid remission of clinical signs. The approach of removing antibody recognition whilst maintaining the desired functional effect (in this case T cell tolerance) may be of value in other situations in which there is a risk of triggering anaphylaxis with peptide-based drugs.

Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate.

Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell-mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)-specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor-kappaB (NF-kappaB), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor alpha and interleukin-1beta were suppressed in eyes of PDTC-treated EAU mice. However, when T cells from PDTC-treated EAU mice, Ag-presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell-proliferative response and cytokine production. Pretreatment of Ag-primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector-phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF-kappaB pathway in the lesion could be a novel target for the successful control of uveoretinitis.

Endoproteolytic cleavage of human thyroperoxidase: role of the propeptide in the protein folding process.

Human thyroperoxidase (hTPO), the key enzyme involved in thyroid hormone synthesis, is synthesized in the form of a 933-amino acid polypeptide that subsequently undergoes posttranslational modifications such as N- and O-glycosylation and heme fixation. In the present study, it was established that the N-terminal part of hTPO is cleaved during the maturation of the enzyme. In the first set of experiments performed in this study, Chines hamster ovary (CHO) cells transfected with hTPO cDNA generated four different species after deglycosylation, namely a 98-kDa species, which corresponds to the full-length deglycosylated hTPO, and two 94-kDa and one 92-kDa species, which were truncated in the N-terminal parts. The three latter forms were detected only at the cell surface. A proprotein convertase inhibitor prevented these cleavages, and experiments using monensin and brefeldin A showed that they occurred in a post-endoplasmic reticulum compartment. Site-directed mutagenesis studies were performed in which Arg65 was identified as one of the cleavage sites. In the second part of the study, hTPO from human thyroid glands was purified using a monoclonal antibody recognizing the folded form of hTPO. Amino acid determination showed that the N-terminal part of this protein begins at Thr109. This cleavage process differs from that observed in CHO cells. The fact that this hTPO was endoglucosaminidase H-sensitive indicated that the cleavage of the propeptide occurs in the endoplasmic reticulum. To analyze the role of the hTPO prosequence, cDNAs with and without prosequence (Cys15-Lys108) were transfected into CHO cells. hTPO propeptide deletion drastically decreased the proportion of the folded hTPO form, and under these conditions the cell surface activity disappeared completely. These results strongly suggest that the prosequence plays a crucial role as an intramolecular chaperone, facilitating the folding of hTPO.

SET-related cell division autoantigen-1 (CDA1) arrests cell growth.

We used an autoimmune serum from a patient with discoid lupus erythematosus to clone a cDNA of 2808 base pairs. Its open reading frame of 2079 base pairs encodes a predicted polypeptide of 693 amino acids named CDA1 (cell division autoantigen-1). CDA1 has a predicted molecular mass of 79,430 Daltons and a pI of 4.26. The size of the cDNA is consistent with its estimated mRNA size. CDA1 comprises an N-terminal proline-rich domain, a central basic domain, and a C-terminal bipartite acidic domain. It has four putative nuclear localization signals and potential sites for phosphorylation by cAMP and cGMP-dependent kinases, protein kinase C, thymidine kinase, casein kinase II, and cyclin-dependent kinases (CDKs). CDA1 is phosphorylated in HeLa cells and by cyclin D1/CDK4, cyclin A/CDK2, and cyclin B/CDK1 in vitro. Its basic and acidic domains contain regions homologous to almost the entire human leukemia-associated SET protein. The same basic region is also homologous to nucleosome assembly proteins, testis TSPY protein, and an uncharacterized brain protein. CDA1 is present in the nuclear fraction of HeLa cells and localizes to the nucleus and nucleolus in HeLa cells transfected with CDA1 or its N terminus containing all four nuclear localization signals. Its acidic C terminus localizes mainly to the cytoplasm. CDA1 levels are low in serum-starved cells, increasing dramatically with serum stimulation. Expression of the CDA1 transgene, but not its N terminus, arrests HeLa cell growth, colony numbers, cell density, and bromodeoxyuridine uptake in a dose-dependent manner. The ability of CDA1 to arrest cell growth is abolished by mutation of the two CDK consensus phosphorylation sites. We propose that CDA1 is a negative regulator of cell growth and that its activity is regulated by its expression level and phosphorylation.

Identification of two rodent genes encoding homologues to seminal vesicle autoantigen: a gene family including the gene for prolactin-inducible protein.

We cloned two new paralogous genes that encode proteins homologous to seminal vesicle autoantigen (SVA) in rodents. The open reading frame of one mouse gene encodes a polypeptide consisting of 151 amino acid residues which has 43% identity to SVA. RT-PCR analysis showed selective expression in the colon, and thus the protein was tentatively named "SVA-like protein in the colon (SLP-C)". The other mouse gene has an open reading frame encoding 144 amino acid residues with 46 and 65% identity to SVA and SLP-C, respectively. Expression of this gene was detected in the mammary, submaxillary, parotid, and lacrimal glands, and this protein was named "SLP in the mammary gland (SLP-M)". Orthologs of both genes were also found in rats. The three homologous genes coding for SVA, SLP-C, and SLP-M may have been generated by gene duplication with divergence of tissue expression in the course of evolution. They comprise a unique structurally-related gene family. Moreover, these genes share significant sequence homology with that of another secretory glycoprotein, prolactin-inducible protein.

Molecular properties of the Goodpasture epitope.

Goodpasture disease fulfils all criteria for a classical autoimmune disease, where autoantibodies targeted against the non-collagenous domain of the alpha3-chain of collagen IV initiates an inflammatory destruction of the basement membrane in kidney glomeruli and lung alveoli. This leads to a rapidly progressive glomerulonephritis and severe pulmonary hemorrhage. Previous studies have indicated a limited epitope for the toxic antibodies in the N-terminal part of the non-collagenous domain. The epitope has been partially characterized by recreating the epitope in the non-reactive alpha1-chain by exchanging nine residues to the corresponding ones of alpha3. In this study we have investigated to what extent each of these amino acids contribute to the antibody binding in different patient sera. The results show that seven of the nine substitutions are enough to get an epitope that is recognized equally well as the native alpha3-chain by all sera from 20 clinically verified Goodpasture patients. Furthermore, the patient sera reactivity against the different recombinant chains used in the study are very similar, with some minor exceptions, strongly supporting a highly defined and restricted epitope. We are convinced that the restriction of the epitope is of significant importance for the understanding of the etiology of the disease. Thereby also making every step on the way to characterization of the epitope a crucial step on the way to specific therapy for the disease.

Identification of an apoptotic cleavage product of BARD1 as an autoantigen: a potential factor in the antitumoral response mediated by apoptotic bodies.

We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.

Experimental autoimmune uveitis as animal model for human posterior uveitis.

Uveitis is an intraocular inflammatory disease that mostly affects children and young adults. It is one of the major causes of blindness in young individuals in India and the world. It is responsible for about 10 per cent of total visual impairment. Unfortunately, etiological diagnosis is not evident in a majority of these patients. It is generally felt that autoimmune mechanism may be involved in so called 'idiopathic' cases which has led to search for the putative autoantigens in experimental animal models. It has been demonstrated that experimental autoimmune uveitis (EAU) can be elicited against several retinal proteins in rats, mice and sub-human primates. These include the S-antigen, a major protein on retinal photoreceptor cell, interphotoreceptor retinoid binding protein (IRBP) and several others. There are many similarities between clinical entities and the EAU, but the EAU differs from the clinical conditions in being self-limited, and requiring complete Freund's adjuvant for induction of the disease. The disease can be induced only in susceptible strains. Nevertheless, use of the EAU model has allowed for identification of disease causing epitopes of antigens and evaluation of disease modifying strategies which could be applied in clinical situations. There has been significant progress in this field, but still a lot more is required to be learnt to translate it into clinical practice.

The 56K autoantigen is identical to human annexin XI.

Anti-56K autoantibodies are present in sera from patients with various autoimmune diseases, predominantly in sera from patients with rheumatoid arthritis, systemic lupus erythematosus, or Sjögren's syndrome. To clarify the molecular structure of this autoantigen, we isolated a 2.0-kilobase pair cDNA clone considered to encode the full-length 56K autoantigen. The longest open reading frame encodes a 505-amino acid polypeptide, with a predicted molecular mass of 54.4 kDa. The in vitro translated protein is recognized by all anti-56K positive patient sera tested. Antibodies affinity-purified using the bacterially expressed recombinant protein recognized the 56K autoantigen in a HeLa cell extract. cDNA sequencing revealed that the 56K cDNA shares a high degree of homology in both nucleotide (87%) and amino acid sequence (92.5%) with bovine annexin XI, indicating that the 56K cDNA encodes the human homologue of annexin XI, a member of the Ca(2+)-dependent phospholipid binding protein family. Anti-56K autoantibody exhibits both a cytoplasmic and a nuclear staining in immunofluorescence experiments. Patients' sera recognize preferentially the N-terminal region of the protein, which is specific for 56K/annexin XI and not shared by other annexins, indicating that the autoimmune response to 56K/annexin XI in these patients is specific for this annexin family member.

T cell-B cell interaction: autoreactive T cells recognize B cells through a terminal mannose-containing superantigen-like glycoprotein.

Rat autoreactive T cells (ATs) recognize a membrane component(s) on syngeneic B cells in association with class II MHC antigens resulting in proliferation of ATs as well activation and differentiation of B cells. Results presented herein indicate that ATs recognize a stimulating antigen(s) SA, in association with class II MHC antigens, on the B cell surface. Our studies using inhibitors of carbohydrate and protein synthesis suggest that SA is a glycoprotein(s) with a high turnover rate but is not an immunoglobulin. Treatment of B cells with mannosidase abrogates their ability to stimulate AT proliferation. Furthermore, pretreatment of B cells with GNA (a lectin from Galanthus nivalis that reacts with free terminal-mannose residues on glycoconjugates) also inhibits their ability to stimulate ATs. However, these treatments do not affect the competence of B cells to stimulate an allogeneic MLR or present a conventional antigen to T cells. The frequency of CD4+ T cells proliferating in response to syngeneic B cells is very high (0.2-0.5%) and is in line with frequencies seen in "superantigen"-type responses. Moreover, T cell receptors expressed on ATs use mainly V beta 6, V beta 11, and V beta 8 regions. Based on these data, SA appears to be a fast turnover, terminal mannose-containing, superantigen-like glycoprotein on the B cell surface.

Iodine content of rat thyroglobulin affects its antigenicity in inducing lymphocytic thyroiditis in the BB/Wor rat.

The BB/Wor rat develops spontaneous insulin dependent diabetes mellitus (DM) and lymphocytic thyroiditis (LT). We have recently demonstrated that immunization of BB/Wor rats with allogeneic thyroglobulin (Tg) induces LT at an early age. The incidence of spontaneous and Tg induced LT is extremely variable among different BB/Wor sublines. It has been shown that high iodine diet significantly increases the incidence of spontaneous lymphocytic thyroiditis (LT) and low iodine diet significantly decreases the incidence of LT in genetically predisposed BB/Wor rats. Recent studies on thyroglobulin (Tg) induced LT in chicken and mouse have shown that iodine rich Tg is far more antigenic than Tg with a low iodine content, suggesting that a high iodine diet increases the immunogenicity of Tg molecule. In order to determine whether the extent of Tg iodination would affect its immunogenicity in the BB/Wor rats, the current study was carried out. Normal iodine Tg (NTg) or low iodine Tg (LTg) was obtained from thyroids of rats that were placed on regular diet or regular diet plus 0.5% methimazole, respectively. 120 rats from the NB (highly susceptible) and BB (low susceptible) sublines were randomized in three groups. Immunization was carried out with a 1:1 emulsion of complete Freund's adjuvant (CFA) and LTg, NTg (0.6 mg/rat) or saline at 30 and 37 days of age. Since spontaneous LT rarely occurs before age 75 days, rats were sacrificed at age 65 days to specifically study Tg induced LT. Immunization with NTg induced LT in 31% of the NB rats, but not in the BB subline. LTg did not induce LT in either subline.(ABSTRACT TRUNCATED AT 250 WORDS)