They are useful for detecting subclinical rejection, recurrent disease, drug toxicity and polyomavirus nephropathy. Current literature mainly discusses the significance of subclinical rejection during the early post-transplant period. It has been suggested that protocol biopsies performed within the first year after kidney transplantation for the detection and treatment of subclinical rejection may be a major factor in preserving long-term graft function.[1-3] However, the benefit of long-term allograft biopsies is largely unproved, and the strategy is yet to be widely implemented. The recent progress in immunosuppressive agents has considerably

improved renal allograft survival, Sorafenib price with 5-year graft survival now exceeding 90%.[4] In addition, the prevalence of subclinical rejection has decreased over time in accordance with the development of new immunosuppressant drugs. Rush et al.[5] reported a randomized, prospective, multicentre study that used tacrolimus, mycophenolate mofetil and steroid as the baseline regimen, in which the overall prevalence of subclinical rejection between months 1 and 6 was only 4.6%. In contrast, in recent years, some reports have been published

about post-transplant recurrence of primary glomerulonephritis.[6-10] Also, calcineurin inhibitor (CNI) nephrotoxicity remains one of the most difficult issues associated with chronic allograft damage.[11-13] In this respect, the utility of long-term protocol biopsy may be of clinical significance for the detection of graft dysfunction buy Temozolomide as a result of non-immune factors, such as CNI nephrotoxicity

and recurrence of glomerulonephritis, rather than subclinical rejection. This review discusses the value of long-term protocol biopsies after kidney transplantation focusing on the issue of immunological and non-immunological factors. Early detection and treatment of subclinical rejection improves outcome. However, reported incidence rates of subclinical rejection differ widely, varying from 1% to 45% in the first 3–6 months post transplantation.[1, 2, 14-16] Some reasons for the differences in reported subclinical mafosfamide rejection rates include variation in human leukocyte antigen (HLA) matching, the incidence of delayed graft function, and the immunosuppressive protocol used.[2] Also, the difference can be explained in part by the inclusion of borderline changes, use of different inclusion criteria, and different timings of the biopsies. Comparisons between studies are complicated further by the fact that some studies include small numbers of patients and precise inclusion criteria are not reported. The treatment of subclinical rejection is a difficult problem with no easy answer. Commonly, patients with biopsies showing borderline changes or T-cell–mediated rejection were treated with corticosteroid bolus alone or thymoglobulin in combination with steroid.

“
“Maternal Selleck 5-Fluoracil immune responses during pregnancy are critical in programming the future health of a newborn. The maternal immune system is required to accommodate fetal immune tolerance as well as to provide a protective defence against infections for the immunocompromised mother and her baby during gestation and lactation. Natural immunity and antibody production by maternal B

cells play a significant role in providing such immunoprotection. However, aberrations in the B cell compartment as a consequence of maternal autoimmunity can pose serious risks to both the mother and her baby. Despite their potential implication in shaping pregnancy outcomes, the role of B cells in human pregnancy

has been poorly studied. This review focuses on the role of B cells and the implications of B cell depletion therapy in pregnancy. It highlights the evidence of an association between aberrant B cell compartment and obstetric conditions. It also alludes to the potential mechanisms that amplify these B cell aberrances and thereby contribute to exacerbation of some maternal autoimmune conditions and poor neonatal outcomes. Clinical and experimental evidence suggests strongly that maternal autoantibodies contribute directly to the pathologies of obstetric and neonatal conditions that have significant implications for the lifelong health of a newborn. The evidence for clinical benefit and safety of B cell depletion therapies in pregnancy is reviewed, and an argument Venetoclax is mounted for further clinical evaluation of B cell-targeted therapies in high-risk pregnancy, with an emphasis on improving neonatal outcomes and prevention of neonatal conditions such as congenital heart block and fetal/neonatal alloimmune thrombocytopenia. An individual’s lifetime

health is critically programmed during the gestational period. During pregnancy, the maternal immune system is required not only to accommodate the allogeneic fetus but also to maintain protection against Leukotriene-A4 hydrolase harmful infections in the otherwise immunocompromised mother and immuno-incompetent fetus [1]. The roles of innate and cell-mediated immunity, including natural killer, T helper type 1 or 2 (Th1/Th2) cells and regulatory T cells (Treg) are well documented in pregnancy [2, 3]. In contrast, there has been little focus on the role of B cells and antibody-mediated immunity. This is surprising, given the fundamental role of B cells as effectors and regulators of both innate and adaptive immune responses [4, 5]. Maternal B cells also provide a vital source of antibody-mediated protective immunity for the mother and her baby during both pregnancy and lactation [6].

The mice were exposed to bacterial aerosols generated by twin jet nebulizers (Salter Laboratories, Arvin, CA, USA) for 30 min in a whole animal exposure chamber as described 45. At each time point, mice were euthanized with intraperitoneal pentobarbital and exsanguinated by cardiac puncture. The left lung was homogenized for quantitative culture and measurement of cytokines as described 9. The right lung was lavaged for cell

counts 9. Cytospins were performed on cells from bronchoalveolar lavages and cell types were determined following a modified Wright-Giemsa selleck chemical stain (Diff-Quick, Dade Behring, Dudingen, Switzerland). For histologic preparation, the lung was inflated to 15 cm pressure with 4% paraformaldehyde, fixed in the same solution, embedded in paraffin, and 4-μm sections SRT1720 ic50 were generated. Sections stained with hemotoxylin and eosin were examined by a pathologist blinded to mouse genotype and time following infection. Inflammation was scored as a percentage of the airspaces involved derived from the examination of ten high-power fields. Human NOD1 and NOD2 constructs (gift from Gabriel Nuñez) were subcloned into the pEF6 expression vector (Promega, Madison, WI, USA). The region of the murine IFN-β promoter (−43 bp to −218 bp upstream the transcription site) containing

interferon response factor-1 and NF-κb binding sites was placed upstream a luciferase reporter construct (pGL2-IFNβ) (gift from Pierre-Yves Bochud) (Invitrogen, Carlsbad, CA, USA). pGL2–ELAM was used as previously described 46, and pRL-TK was purchased from Promega. HEK293 cells medroxyprogesterone were transfected with FUGENE HD (Roche Diagnostics, Basel, Switzerland) using the manufacturer recommended protocol. ELAM promoter-firefly luciferase and IFN-β

promoter-firefly luciferase reporter constructs were co-transfected with plasmid expression constructs containing human NOD1 and NOD2 along with Renilla luciferase expression constructs driven by the HSV thymidine kinase promoter to control for transfection efficiency. Cells were simultaneously exposed to heat-killed FlaA and WT (Corby strain) Lp and incubated overnight at 37°C in order to potentiate cytoplasmic delivery of the pathogen by the transfection reagent. Firefly luciferase activity was measured after lysis of cells using Dual Luciferase Reporter Assay System as per the manufacturer’s recommended instructions (Promega). Total luminescence over one second was measured using luciferin (to measure firefly luciferase) followed by a second reading with coelenterazine (to measure Renilla luciferase activity) with simultaneous administration of an inhibitor to firefly luciferase. Transfection efficiency of the reporter promoter was adjusted for each well by dividing the relative light units of firefly luciferase by the relative light units of Renilla luciferase.

Future studies are needed to examine the role of S100A8, S100A9 and S100A12 in other human MDSC subtypes with the aim of further characterization of these cells. This will help further our understanding of their mechanism of action and help to target them for IWR-1 purchase immunotherapeutic approaches. This research was supported (in part) by the Intramural Research Program of the National institutes of Health, National Cancer Institute, Center for Cancer Research.

This work was supported by a grant to MPM from the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer. We would like to thank the Experimental Transplantation and Immunology Branch cell sorting facility for technical assistance with cell sorting. None of the authors have any financial conflict of interest. Figure S1. PBMC were isolated by Ficoll density gradient and stained selleck chemicals llc for CD14 and HLA-DR expression. “
“DNA is immunogenic and many cells express cytosolic DNA sensors that activate the stimulator of interferon genes

(STING) adaptor to trigger interferon type I (IFN-β) release, a potent immune activator. DNA sensing to induce IFN-β triggers host immunity to pathogens but constitutive DNA sensing can induce sustained IFN-β release that incites autoimmunity. Here, we focus on cytosolic DNA sensing via the STING/IFN-β pathway that regulates immune responses. Recent studies reveal that cytosolic DNA sensing via the STING/IFN-β pathway induces indoleamine 2,3 dioxygenase (IDO), which catabolizes tryptophan to suppress effector and helper T-cell responses and activate Foxp3-lineage CD4+ regulatory T (Treg) cells. During homeostasis, and in some inflammatory settings, specialized innate immune cells in the spleen and lymph nodes may ingest and sense cytosolic DNA to reinforce tolerance that prevents autoimmunity. However, malignancies and pathogens may exploit DNA-induced regulatory responses to suppress natural and vaccine-induced immunity to malignant and infected cells. In

this review, we discuss the biologic significance of regulatory responses to DNA and novel approaches to exploit DNA-induced immune responses for therapeutic benefit. The ability of DNA to drive tolerogenic enough or immunogenic responses highlights the need to evaluate immune responses to DNA in physiologic settings relevant to disease progression or therapy. The immune adjuvant properties of DNA are well known and are exploited to enhance vaccine responses. Recent reports describe a surprisingly large array of cytosolic DNA sensors, many of which activate the stimulator of interferon genes (STING, aka MITA, ERIS, MPYS, TMEM173) to induce IFN-β in a broad range of cell types (reviewed in [1-6]. IFN-β is a potent immune cell activator, inciting host defense against many pathogens. As most mammalian cells express cytosolic DNA sensors, DNA sensing may have wider biological significance than signaling pathogen presence.

AF is associated with higher morbidity and mortality than sinus rhythm in this population. The purpose of this review is to summarize all available

evidence regarding use of warfarin in HD patients with AF for stroke prevention. The enormous heterogeneity of available studies does not allow pooling of the data in the form of meta-analysis or systematic review. Current evidence regarding use of warfarin for AF in terms of risk benefit ratio in this population is limited and conflicting. Randomized control trials evaluating the safety and efficacy of anticoagulation in this population by means of risk/benefit assessment tools are urgently needed. However, suitable HD patients with AF should be counselled PF-02341066 datasheet click here on their likelihood of reduction of stroke risk and experiencing side-effects

before initiating anticoagulant therapy. It is particularly important to incorporate the patient’s preferences and willingness to trade off benefit and risk in stroke prevention. An individualized holistic approach optimizing all potential risk factors of bleeding and ischemic stroke in HD patients with AF is recommended. Incidence rates of atrial fibrillation (AF) in haemodialysis (HD) patients (Table 1)[1-4] were higher than those of general population. The prevalence of AF in general and HD population were 1–8% and 13–23% respectively. As the prevalence of AF in chronic kidney disease (CKD) and HD is more or less similar (Table 2),[5-15] processes influencing the development of AF likely occurred early in the course of CKD. Evidence suggests inflammation associated with renal dysfunction is involved in the pathogenesis of AF. Proposed mechanisms

include decreased pro-inflammatory cytokine clearance, endotoxaemia and oxidative stress, and reduced anti-oxidant levels.[16-18] Studies have also shown that prevalence of AF is inversely correlated with glomerular filtration rate, which may mean increasing inflammation why with worsening renal function; however, age may have been a confounding factor in these studies.[19] Age was found to be an independent predictor of AF in both the general and HD populations. The prevalence of AF in HD population increased progressively with age and was much higher than in all age categories of general population.[6, 8, 9, 20] Increased prevalence of ischemic heart disease and left atrial dilatation in this population are certainly risk factors contributing to this. Compared with Caucasians, the prevalence of AF was substantially lower in blacks, Asians and Native Americans.[15] 60 (1992) 71 (2006) Atrial fibrillation was associated with higher total and cardiovascular morbidity and mortality in both general and HD populations.

2E) but were much more prevalent in the p22-phox area in the nos2−/− tissues (Fig. 2F). The CD4+

T cells were significantly increased in the sections from nos2−/− livers with an average of 321±100 CD4+ cells per section versus an average of 93±29 cells per section in the WT (p = 0.0046 by Student’s t-test). These data demonstrate that while Nos2 is not as widely expressed as p22-phox, it severely affects the ability of CD4+ and CD8+ lymphocytes to accumulate within the mycobacterial granuloma. Mycobacterium avium infected WT mice undergo a profound IFN-γ-dependent depletion of lymphocytes; however, the impact of Nos2 in this model is not to substantially deplete T cells but to reduce the level of the IFN-γ response [6, 34]. Figure 2 suggests that T cells are specifically excluded from the phagocytic areas in M. avium infected WT mice in a nitric oxide-dependent Regorafenib clinical trial manner. To determine whether the histological results in the WT lesions represented the depletion of all or a specific subset of lymphocytes from the affected organ, we compared the CD4+ T cells within infected organs by flow cytometry. We found only a modest effect of nos2 deficiency on the total frequency and number selleck of either live lymphocytes or CD4+ T cells in infected organs compared to WT mice (Supporting Information Fig.

1). This trend was seen before but had not reached statistical significance in previous studies [6, 34]. To determine whether the nos2 gene was adversely affecting activated effector cells, we compared the frequency (Fig. 3A) and number (Fig. 3B) of CD4+ T cells expressing the Th1-associated transcription factor, T-bet. We found that the CD4+ T-bet+ population was significantly and substantially increased in the nos2−/− mice relative to the WT mice in all infected

organs (Fig. 3). These data demonstrate that the presence of nos2 limits the accumulation of Th1-type T cells and that these OSBPL9 activated effector cells were either more susceptible to depletion or failed to develop in the presence of Nos2. To investigate whether all activated T-bet+ cells were equally affected by the presence of nos2, we stained CD4+ T cells from all infected organs for both T-bet and CD69, a molecule that is upregulated upon antigen exposure [35]. The pattern of staining is shown in Fig. 4A. We found that in all three organs, the frequency and number (Fig. 4B) of CD69hi T-bet+ CD4+ T cells were only modestly affected by the absence of nos2. In contrast, the CD69loT-bet+ CD4+ T-cell population failed to accumulate in the WT mice but did accumulate in the spleen, liver, and lung of the nos2−/− mice (Fig. 4C). These data demonstrate that the nos2 gene has the capacity to limit accumulation of CD69loT-bet+ CD4+ T cells.

Unlabelled forms of the biotinylated peptides were used as reference peptides to assess the validity of each experiment. Their sequences and inhibitory concentration (IC50) values were as follows: HA 306–318 (PKYVKQNTLKLAT) for DRB1*0101 (6 nM); DRB1*0401 (30 nM), DRB1*1101 (17 nM) and DRB5*0101 (8 nM), YKL (AAYAAAKAAALAA) for DRB1*0701 (42 nM); A3152–166 (EAEQLRAYLDGTGVE) for DRB1*1501 (28 nM); MT 2–16 (AKTIAYDEEARRGLE) for DRB1*0301 (660 nM); B1 21–36 (TERVRLVTRHIYNREE) for DRB1*1301 (268 nM); LOL 191–210 (ESWGAVWRIDTPDKLTGPFT) for DRB3*0101 (9 nM); and E2/E168 (AGDLLAIETDKATI)

for DRB4*0101 (3 nM). The peptide concentration that prevented binding of 50% of the labelled peptide (IC50) was evaluated. Data were expressed as relative affinity: ratios of the IC50 of the peptide by the IC50 of the reference peptide, which Ku-0059436 order binds the HLA II molecule strongly. Proliferation assays using E6 and E7 large peptides covering both whole proteins performed at entry into the study showed that blood T lymphocytes from 10 patients (nos 1, 2, 3, 4, 6, 8, 9, 11, 13, 14) proliferated in the presence of one to 10 peptides (Fig. 1). The strongest responses PD0332991 in vitro in eight patients (nos 3, 4, 6, 8, 9, 11, 13, 14) were directed against both peptides E6/2 (aa 14–34) and E6/4 (aa 45–68), whereas T cells in patient 1 proliferated against peptide E6/4 and in patient 2 against

E6/2 only, respectively (Fig. 1). SI of these strongest proliferative responses ranged from 3·1–22. Peptide E6/7 (aa 91–110) stimulated blood T lymphocytes from two patients (nos 2 and 6, SI = 3·8 and 4·3, respectively). One patient each displayed responses against peptide E6/5 (aa 61–80) (patient no. 6), peptide E6/8 (aa 105–126) (patient no. 6) and peptide E6/9 (aa 121–140) (patient no. 11). Finally, no response could be detected against peptides OSBPL9 E6/1, E6/3, E6/6 and E6/10. Only two patients (nos 2 and 6) had proliferative responses against E7 peptides. E7/7 (aa 65–87) was the better immunogenic peptide, recognized by two patients (with SI of 4 and 6), peptides E7/2 (7–27), E7/3 (21–40), E7/4 (35–55) and E7/8 (78–98) being recognized by only one patient. Peptides E7/1, E7/5 and E7/6 yielded no detectable response.

This assay was performed with E6 and E7 large peptides at entry into the study (Fig. 2). Numerous blood cells from patient 1 recognized three HPV-16 long peptides: E6/4, E7/2 and E7/3 with mean 270, 65 and 430 SFC/106 PBMCs. In patient 13 the recognized peptides were E6/7, E6/8, E7/1, E7/2, E7/3 and E7/8, with a mean of 43, 50, 38, 34, 33 and 30 SFC/106 PBMCs. These two patients both had large lesions (10 and 20 cm2, respectively). Nevertheless, their clinical outcome was different. The first patient experienced a complete and durable disappearance of the lesions 2 months after entry into the study following the electrocoagulation of less than 50% of the classic VIN lesion, whereas chronic and extensive lesions persisted in the second patient despite laser surgery.

The truncated MFG-E8 (designated as selleck chemical C2del) was abnormally glycosylated with terminal sialic acids; yet, it

bound to phosphatidylserine and enhanced the phagocytosis of apoptotic cells. When injected into mice, C2del showed greater stability than wild-type MFG-E8 and induced the production of autoantibodies, suggesting that this mutation of the MFG-E8 gene can lead to the development of SLE in humans. The human MFG-E8 gene is located on human chromosome 15q25 and is composed of eight exons (National Center for Biotechnology Information GenBank Accession Number WC_000015). To sequence the coding regions of human MFG-E8 gene in a cohort of Japanese female SLE patients (n=110), cDNA was prepared from RNA isolated from the patients’ peripheral blood mononuclear cells. Two sets of PCR primers, which amplified the cDNA corresponding to exons 1–5, and exons 4–8 of the human MFG-E8 gene, were prepared (Fig. 1A). No abnormality was found in the cDNA corresponding to the first set of exons (exons 1–5) in any of the 110 patients, but the RT-PCR of exons 4–8 from one patient yielded a longer-than-normal amplicon in addition mTOR inhibitor to the wild-type one (Fig. 1B). A sequence analysis and BLAST

search indicated that the long amplicon contained a cryptic exon of 102 bp from intron 6 of the MFG-E8 gene (Fig. 1C). This insertion caused a premature termination of the human MFG-E8 coding sequence. Exons are defined by exonic and intronic cis-regulatory elements in addition to the core splice-site motifs 17, 18. A sequence analysis of the human MFG-E8 chromosomal gene of the patient revealed a heterozygous selleck chemicals llc A-to-G point mutation located 43 bp downstream of the cryptic exon, or 937 bp from exon 5 (IVS 6-937) (Fig. 1C and D). To examine the effect of this point mutation on the pre-mRNA splicing of the human MFG-E8 gene, an MFG-E8 minigene carrying intron 6 was constructed (Fig. 2A). That is, a part of exon 6–7 of the human MFG-E8 cDNA, in pEF-BOS vector 19, was replaced by a DNA fragment of the human MFG-E8 chromosomal gene carrying exon 6, intron 6, and exon 7 from

the patient (G-allele at IVS 6-937) or a control (A-allele at IVS 6-937) individual (Fig. 2A). The splicing pattern of the MFG-E8 minigene was then assayed by expression in human HEp-2 cells. Semi-quantitative RT-PCR analysis of the RNA showed that the RNA carrying the cryptic exon was reproducibly about ten times more abundant in the cells transfected with the G-allele minigene than the cells transfected with the A-allele minigene (Fig. 2B). These results indicated that the A-to-G mutation in intron 6 (IVS 6-937 A>G) caused the aberrant inclusion of the cryptic exon in the human MFG-E8 transcript. A screening of the MFG-E8 chromosomal gene by DNA sequencing revealed the same intronic mutation (IVS 6-937 A>G) in additional one patient out of 212 Japanese female SLE patients, while none of 228 healthy female volunteers carried the mutation.

Previous study has shown that cross-linking of FcεRI activates PI3K signalling

pathway, leading to intracellular ROS production [25]. To explore whether OVA challenge–induced ROS production and subsequent activation of SOCs are related to PI3K activation, we explored the effect of PI3K inhibitor Wortmannin on ROS production and Ca2+ signalling in OVA-activated mast cells. The results demonstrated that Wortmannin (100 nm, 15 min) pretreatment significantly decreased MG 132 intracellular ROS production by ~30%. Mast cell activation–induced histamine release was similarly reduced (~30%) by inhibiting PI3K pathway. With the reduction of ROS, Ca2+ increase through SOCs in OVA-activated mast cells was diminished by ~30% (Fig. 6A,B). Consistently, the protein expressions of Orai1 and STIM1 were attenuated by ~40% and ~30%, respectively (Fig. 6C,D). We also found that inhibition of PI3K pathway reduced mast cell activation–induced histamine release (~30%) and intracellular ROS selleck production (~30%). The results indicate that PI3K-mediated ROS generation is involved in the regulation of SOCs activity and mast cell activation under food-allergic condition (Fig. 6E,F). Previous studies have demonstrated that mast cells play a critical role in allergic diseases. Using OVA-stimulated food-allergic rat model, we revealed that

mast cells were recruited and activated in the damaged intestinal tissues and peritoneal lavage, and Th2 cytokines and IgE were significantly increased, confirming

the notion that mast cells contribute to the pathogenesis of food allergy. In this study, we demonstrated that the underlying mechanism for mast cell activation Fenbendazole in the food-allergic mouse model is related to increased Ca2+ entry through SOCs. Furthermore, we found that OVA stimulation increased intracellular ROS production in mast cells through activation of phosphoinositide 3-kinase (PI3K) pathway, which results in upregulation of the expression levels of the major subunits of SOC, Orai1 and STIM1, leading to the enhancement of SOC activity and subsequent mast cell activation. Food allergy is one type of adverse reactions to non-toxic food that involves an abnormal immunological response to specific protein(s) in food. Allergens from egg seem to be one of the most frequent causes of food-allergic reaction as reported [26]. In the present study, we use OVA, which comprise 50% of the protein in egg white, to induce food allergy as previously reported [17, 27, 28]. According to our results, the food-allergic model in Brown-Norway rats has been successfully re-established. The OVA-challenged rat showed typical allergic appearances, including puffiness and redness around the eyes and mouth, diarrhoea, pilar erecti, reduced activity and/or decreased activity with increased respiratory rate and cyanosis around the mouth and tail.

All animal experiments were carried out in accordance with protocols approved by the Animal Care and Use Committee of the Kyoto University Graduate School of Medicine. Human rMFG-E8 (12 pmol in 300 μL of PBS containing 2.5% serum from C57BL/6 mice) was intravenously administered into 8-wk-old C57BL/6 female mice through the tail vein. Serum was harvested 15, 30, and 60 min after the injection, and the MFG-E8 level was measured by an indirect sandwich ELISA. In brief, a 96-well Maxisorp plate (Nalge NVP-AUY922 Nunc International) was coated with 1 μg/well of anti-FLAG mAb in 50 mM sodium bicarbonate buffer

(pH 9.6) and incubated with Reagent Diluent Concentrate 2 (R&D Systems). Triton X-100 was added to the serum samples at a final concentration of 1%, the samples were diluted ten times with TBS, and a 50-μL aliquot was applied to each well. After a 1-h incubation,

the wells were washed with wash buffer supplied with check details the Ampli Q kit (Dako), incubated with 0.8 μg/mL biotinylated hamster mAb against human MFG-E8 (clone 2–8E4A)15, washed as above, and incubated with 8000-times-diluted alkaline phosphatase-conjugated streptavidin (Dako) for 30 min. The alkaline phosphatase activity was measured using the Ampli Q kit. Human rMFG-E8 diluted with 10% normal mouse serum was used to prepare the standard curve. C57BL/6 female mice at the age of 10 wk were treated weekly with 12 pmol of hMFG-E8 for a total of four or six times, and sera were collected before, and 6 and 7 wk after the first injection. The concentration of anti-cardiolipin antibody in the sera was measured by ELISA. In brief, TCL 1 μg of cardiolipin in 100 μL of methanol was added to a 96-well plate (Immulon 1B microtiter plate; Thermo Labsystems), and the plate was air-dried. After blocking with 10% FCS, serially diluted mouse serum was added to the wells. After a 1-h incubation at room temperature, the mouse antibodies bound to the plate were detected using HRP-conjugated goat anti-mouse Ig (Dako) and peroxidase-detecting kit (Sumitomo Bakelite). The color reaction was read at 492 nm using a microplate reader (Titertek Instruments), and the titer of the antibody

was defined as the dilution that gave the absorbance of 0.1. Anti-nuclear antibody was detected by indirect immunofluorescence. In brief, human HEp-2 cells cultured on a glass slide were fixed with cold acetone and incubated with 50-times-diluted mouse serum at 37°C for 30 min. The antibodies bound to the HEp-2 cells were detected by Cy3-conjugated F(ab’)2 of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) diluted 100 times with PBS/10% normal goat serum, and observed by fluorescence microscopy (Biorevo, Keyence). The authors thank M. Fujii and M. Harayama for secretarial assistance. This work was supported in part by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports, and Culture in Japan to S. N. H. Y.