Tetsufumi Inoue

Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids

Background: Anti-citrullinated protein antibodies (ACPA) are specifically and frequently detected in sera of patients with rheumatoid arthritis (RA). Citrullinated fibrin or fibrinogen is a candidate autoantigen of such antibodies. Objective: To investigate the presence of citrullinated fibrinogen (cFBG) in the plasma or synovial fluid of patients with RA and control patients, and to determine cFBG levels and their relationship with serum markers for RA if it is present. Methods: A sandwich enzyme linked immunosorbent assay (ELISA) to measure cFBG was established using monoclonal antibodies cF16.1 and cF252.1, generated by immunising mice with R16Cit and R252Cit, the fibrinogen Aα chain derived sequences with citrulline at position 16 and 252, respectively, and the presence of cFBG was further investigated with immunoprecipitation-western blotting. Results: Positive signals were detected in 11/15 RA synovial fluids (RASFs), but not in osteoarthritis synovial fluids or RA plasma with sandwich ELISA for cFBG using cF16.1 and an anti-modified citrulline (AMC) antibody. The presence of cFBG in RASFs was confirmed by immunoprecipitation-western blotting. Furthermore, most RA sera strongly reacted against R16Cit. No relationship was seen between RASF cFBG levels and C reactive protein or anti-cyclic citrullinated peptide antibody levels of the paired sera. Conclusion: cFBG is detected as a soluble citrullinated autoantigen in RASFs and may therefore be a genuine candidate antigen for ACPA in patients with RA.

Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome

Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient.

Peptidylarginine deiminase 4 (PADI4) identified as a conformation‐dependent autoantigen in rheumatoid arthritis

Objective: Peptidylarginine deiminase (PADI) catalyses the post‐translational modification of arginine to citrulline, which is specifically recognized by sera from rheumatoid arthritis (RA) patients. The PADI4 gene has recently been identified as a risk factor for RA. We aimed to determine whether PADI4 constitutes an autoantigen in RA. Methods: Serum samples were obtained from 42 patients with RA, 19 patients with systemic lupus erythematosus (SLE), 23 patients with other rheumatic diseases, and 40 normal individuals. The presence of antibodies against recombinant human PADI4 (anti‐PADI4) was examined using enzyme‐linked immunosorbent assay (ELISA) and Western blotting. Results: For ELISA, the prevalence of anti‐PADI4 among RA patients (50%) was significantly higher than that of normal individuals (2.5%), SLE (10.5%), and other rheumatic diseases (4.3%), while for Western blot analysis, PADI4 was recognized only by a portion of the ELISA‐positive serum samples. Conclusions: PADI4 is an autoantigen in some RA patients, and its conformational epitope(s) may be important.

A sensitive and simple method for determination of IgM in cerebrospinal fluid by a solid-phase enzyme-immunoassay: Comparison of two different methods

A sensitive and simple solid-phase enzyme-immunoassay for quantitative determination of IgM in cerebrospinal fluid has been developed. This method is based on a sandwich technique and is much more sensitive than the competition enzyme-immunoassay which we described previously. The IgM values obtained by this sandwich technique were well correlated with those by the competition technique. This new method is considered to be more suitable for the estimation of IgM in cerebrospinal fluid because of its simplicity and good sensitivity.

AN ALTERNATIVELY SPLICED FORM OF THE HUMAN CD94 GENE

Human CD94 is one of the lectin-type natural killer (NK) receptors which are type II transmembrane proteins expressed on NK cells and subsets of T cells that possess a C-type lectin domain (Chang et al. 1995). CD94 recognizes HLA class I molecules on target cells by heterodimerization with NKG2-A or -C, other lectin-type NK receptors (Lazetic et al. 1996), and inhibits or activates the NK cytotoxicity (Houchins et al. 1997). cDNA variants of several NK receptors have been produced by alternative splicing (Dohring et al. 1996; Plougastelet et al. 1996; Yabe et al. 1993). We investigated an alternatively spliced cDNA variant of CD94 using reverse transcription-polymerase chain reaction (PCR) methods. RNA was extracted from the peripheral blood lymphocytes of four different individuals. The cDNA was synthesized and amplified with a set of primers specific for CD94 (59-ACACATCGTGCCTTCTCTACTTC-39, 59-TCTTTACTCTCCACCTTCTCTGCCC39) for 35 cycles. Two fragments, of 620 and 527 base pairs, were amplified (data not shown). The size of the fragment with the shorter length was at least one-fifth that of the longer fragment, in all individuals tested (data not shown). The sequence of the longer fragment was identical to that of the CD94 cDNA, whereas that of the shorter fragment was a 93 base-deleted form (DDBJ accession number AB009597). These data suggest that the shorter fragment may be the alternatively spliced form of CD94. It is necessary to know the genomic structure of CD94 to confirm the alternative splicing. Since the sequences of exons 4, 5, and 6 of the CD94 gene have already been reported to GenBank by Rodrõ Âguez and co-workers (1998) (accession number AJ000673), we investigated exons 1, 2, and 3. Genomic DNA was also extracted from peripheral blood lymphocytes and amplified using the same set of primers. Only one fragment, of approximately 7 kilobase pairs, was amplified. Exons 1, 2, and 3 and introns 1 and 2 of the PCR product were completely sequenced (Fig. 1 A) (DDBJ accession number AB010084). All acceptor and donor sites matched with the conventional splice consensus sequences (Shapiro et al. 1987). The genomic sequence was compared with the two cDNA sequences, and it was determined that exon 2 is completely identical to the missing portion in the shorter fragment (Fig. 1 B). These data indicate that the difference between the two fragments could be attributed to alternative splicing. It was also determined that the shorter fragment is a result of fusion between exon 1 and exon 3 without interruption of the established frame (Fig. 1 C). Exon 2 encodes the transmembrane portion of CD94. Our data suggest that some amounts of the alternatively spliced form of CD94 mRNA exist in peripheral blood lymphocytes. The translated protein probably resides in the cytosol, because the signal peptide/membrane anchor domain of the type II transmembrane protein is necessary for its expression on the lumen of the endoplasmic reticulum (ER). The function of the protein has yet to be clarified. One possibility is that the protein may form a heterodimer with NKG2-A or C in the cytosol, which cannot enter the ER lumen and consequently prevent surface expression of the receptors.

Inhibition of l-leucine methyl ester mediated killing of THP-1, a human monocytic cell line, by a new anti-inflammatory drug, T614

T614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) is a member of the family of methanesulfonanilide non-steroidal anti-inflammatory drugs (mNSAIDs), most of which act as cyclooxygenase (COX)-2 inhibitors. L-leucine methyl ester (Leu-OME) is a reagent which has been shown to kill phagocytes following interaction with intracellular proteases. There are two pathways whereby Leu-OME becomes cytotoxic to phagocytes. Within lysosomes, Leu-OME is converted into free Leu, which causes disruption of the lysosomes and subsequent cell necrosis. The other is the conversion of Leu-OME into (Leu-Leu)(n)-OME, which is associated with the induction of apoptosis. In the present study, we examined the action of T614 on Leu-OME mediated killing of THP-1, a human monocytic cell line. We revealed that T614 and phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, inhibited Leu-OME mediated killing of THP-1 cells. All the other mNSAIDs, including nimesulide (NIM-03), fluosulide (CGP28238), FK3311 and NS398, also rescued THP-1 from Leu-OME mediated killing, although to a lesser degree. Of the classical NSAIDs tested, a protective effect was observed with diclofenac at high concentration, but not with naproxen or indomethacin. Unlike conventional lysosomal inhibitors, such as chloroquine and ammonium chloride (NH(4)Cl), T614 and PMSF did not raise lysosomal pH, as measured by flow cytometry using fluorescein isothiocyanate dextran (FITC-dextran). Therefore, the mechanism whereby T614 and PMSF inhibit Leu-OME killing is distinct from that of chloroquine or NH(4)Cl. Based on the similarity of T614 and PMSF, we suggest that, besides their roles as COX-2 inhibitors, T614 and other mNSAIDs may act as lysosomal protease inhibitors.

Generation of reactive oxygen species is required for bucillamine, a novel anti-rheumatic drug, to induce apoptosis in concert with copper

Rheumatoid arthritis (RA) is considered to be a proliferative disorder of synovial tissue, which is composed of macrophage-like, fibroblast-like and dendritic cells. Bucillamine (BUC) is a novel disease-modifying anti-rheumatic drug, which is a structural analogue of cysteine. Some of the pharmacological actions of BUC have been shown to depend on the generation of reactive oxygen species (ROS) in the presence of copper. In this study, we examined whether BUC in concert with copper can induce apoptosis via generation of ROS. THP.1, a human monocytic cell line, was used as surrogate for synovial cells. We observed that BUC plus copper can induce THP.1 to undergo apoptosis, as evidenced by the presence of DNA degradation, which is preceded by ROS generation and increase in membrane permeability. Moreover, catalase rescued THP.1 from BUC-mediated cell death, indicating that generation of ROS is essential for the induction of apoptosis Red blood cells (RBC), probably acting as a scavenger of ROS, also rescued THP.1 from cell death mediated by BUC plus copper. Collectively, we suggest that ROS derived from BUC in the presence of copper may suppress the outgrowth of rheumatoid arthritis synovial cells in vivo through the induction of apoptosis.

A genome-wide association study identifies two novel susceptibility loci and trans population polygenicity associated with bipolar disorder

Genome-wide association studies (GWASs) have identified several susceptibility loci for bipolar disorder (BD) and shown that the genetic architecture of BD can be explained by polygenicity, with numerous variants contributing to BD. In the present GWAS (Phase I/II), which included 2964 BD and 61 887 control subjects from the Japanese population, we detected a novel susceptibility locus at 11q12.2 (rs28456, P=6.4 × 10−9), a region known to contain regulatory genes for plasma lipid levels (FADS1/2/3). A subsequent meta-analysis of Phase I/II and the Psychiatric GWAS Consortium for BD (PGC-BD) identified another novel BD gene, NFIX (Pbest=5.8 × 10−10), and supported three regions previously implicated in BD susceptibility: MAD1L1 (Pbest=1.9 × 10−9), TRANK1 (Pbest=2.1 × 10−9) and ODZ4 (Pbest=3.3 × 10−9). Polygenicity of BD within Japanese and trans-European-Japanese populations was assessed with risk profile score analysis. We detected higher scores in BD cases both within (Phase I/II) and across populations (Phase I/II and PGC-BD). These were defined by (1) Phase II as discovery and Phase I as target, or vice versa (for ‘within Japanese comparisons’, Pbest~10−29, R2~2%), and (2) European PGC-BD as discovery and Japanese BD (Phase I/II) as target (for ‘trans-European-Japanese comparison,’ Pbest~10−13, R2~0.27%). This ‘trans population’ effect was supported by estimation of the genetic correlation using the effect size based on each population (liability estimates~0.7). These results indicate that (1) two novel and three previously implicated loci are significantly associated with BD and that (2) BD ‘risk’ effect are shared between Japanese and European populations.

Quantitation of IgG, IgA and IgM in the cerebrospinal fluid by a solid-phase enzyme-immunoassay

A sensitive, specific and simple solid-phase enzyme-immunoassay for quantitative determination of IgG, IgA and IgM in the cerebrospinal fluid (CSF) has been developed. Using this technique, we have established the control values for CSF immunoglobulins from 30 neurologically normal subjects. The value of IgG determined by our method was closely correlated with that found by single radial immunodiffusion although the latter was found 1.5 times higher. The values of IgG and IgA showed significant positive correlations with CSF total protein values, while the value of IgM did not. This finding suggested that the concentration of CSF IgM might be less affected by transudation from the serum than those of IgG and IgA in neurologically normal subjects. Our method needs no complicated technique nor special apparatus other than microplate photometer, and offers more facility and convenience for routine laboratory use.

Cholinergic-drug induced sicca syndrome in Parkinson's disease: a case report and a review of the literature.

A 67-year-old woman developed severe sicca manifestations after initial treatment of Parkinsons disease with an anti-cholinergic drug, which prompted us to look for the presence of Sjögrens syndrome. The results of sialography, labial salivary gland biopsy, Rose-Bengal test as well as the presence of antinuclear antibody were consistent with the diagnosis of Sjögrens syndrome. The sicca symptoms diminished by cessation of the anti-cholinergic drug, and the parkinsonian features were controlled by levodopa. We suggest that Sjögrens syndrome should be considered, if patients with Parkinsons disease complain severe xerostomia.