Hidetaka Shiraiwa

Epstein-Barr Virus Induces Erosive Arthritis in Humanized Mice

Epstein-Barr virus (EBV) has been implicated in the pathogenesis of rheumatoid arthritis (RA) on the basis of indirect evidence, such as its presence in affected joint tissues, antigenic cross reactions between EBV and human proteins, and elevated humoral and cellular anti-EBV immune responses in patients. Here we report development of erosive arthritis closely resembling RA in humanized mice inoculated with EBV. Human immune system components were reconstituted in mice of the NOD/Shi-scid/IL-2Rγnull (NOG) strain by transplantation with CD34+ hematopoietic stem cells isolated from cord blood. These humanized mice were then inoculated with EBV and examined pathologically for the signs of arthritis. Erosive arthritis accompanied by synovial membrane proliferation, pannus formation, and bone marrow edema developed in fifteen of twenty-three NOG mice transplanted with human HSC and inoculated with EBV, but not in the nine NOG mice that were transplanted with HSC but not inoculated with EBV. This is the first report of an animal model of EBV-induced arthritis and strongly suggest a causative role of the virus in RA.

Identification of candidate genes for Sjögren's syndrome using MRL/lpr mouse model of Sjögren's syndrome and cDNA microarray analysis

Sjögrens syndrome is a chronic autoimmune disease characterized by focal lymphocytic infiltration of lacrimal and salivary glands, but the precise mechanism of this syndrome is poorly understood. To clarify the mechanism of onset and progression of Sjögrens syndrome, it is necessary to identify Sjögrens syndrome-related genes. For this purpose, we used MLR/MpJ-lpr/lpr (MRL/lpr) mouse as a model of human secondary Sjögrens syndrome and analyzed specific mRNA expression pattern in MRL/lpr mouse salivary glands by in-house cDNA microarray. Among arrayed 2304 genes, 13 genes were isolated as highly expressed genes in MRL/lpr mouse salivary gland in comparison with MRL/MpJ-+/+ (MRL/+) mouse tissue. Subsequently, we performed RT-PCR analysis and confirmed the high expression level of nine genes; caspase3, Ly-6C.2, vimentin, Mel-14 antigen, cathepsin B, mpt1, Laptm5, Gnai2 and UCP2. Five of the nine genes have already been identified in patients with Sjögrens syndrome or mice models of the syndrome, but the remaining four genes; mpt1, Laptm5, Gnai2, and UCP2 have not been reported previously as Sjögrens syndrome-related genes. Although, further experiments are necessary to examine the relationship between these four genes and Sjögrens syndrome, our system of mouse model of Sjögrens syndrome combined with in-house cDNA microarray is suitable for the isolation of Sjögrens syndrome-related genes.

A New Tactile Skin Sensor for Measuring Skin Hardness in Patients with Systemic Sclerosis and Autoimmune Raynaud's Phenomenon

We used a new tactile sensor to measure the elastic properties of skin in patients with systemic sclerosis or Raynauds phenomenon. The sensor consists of a piezoelectric vibrator with vibration pickup to measure frequency changes when the sensor is placed on the skin. The mean frequency change at the skin surface of the proximal third phalanx in patients with systemic sclerosis was significantly lower than in age- and sex-matched controls. The results in systemic sclerosis patients were statistically correlated to the Modified Rodnan Skin Thickness Score. This technique was also used to measure the therapeutic efficacy of salpogrelate, a new specific serotonin receptor antagonist. A greater mean frequency change was seen after treatment. We conclude that this new tactile sensor is useful for quantitatively measuring skin sclerosis and may help determine the efficacy of therapeutic treatments.

SLAM-Associated Protein Solves a Mystery of Autoimmunity

Abstract:  SLAM‐associated protein (SAP) is essential for viral protection, lifelong immune memory (vaccination), and lifelong autoantibody production. We discuss how SAP is a key player in the development of autoimmune disease.

Increased soluble CD4 molecules and the role of soluble CD4 production in patients with rheumatoid arthritis.

We investigated the concentration of soluble CD4 molecules (sCD4) in serum, and the mechanism of sCD4 production from T lymphocytes, in patients with rheumatoid arthritis (RA). The concentration of sCD4 molecules was determined using a solid-phase enzyme-linked immunosorbent assay method. Using reverse transcription polymerase chain reaction (RT-PCR) techniques, we studied the presence of alternatively spliced mRNA encoding the transmembrane site of CD4, and the mRNA encoding a conservative region of the CD4 binding site of the human immunodeficiency virus (HIV), in the serum of RA patients. Levels of sCD4 found in RA patients were higher than in normal controls (199 U/ml compared with 8.4 U/ml, respectively), and correlated with additional medical parameters. The results of RT-PCR suggested that the higher sCD4 levels may be due to shedding from the cell membrane after protease digestion, not to alternative splicing or a reaction to viral binding to sCD4.

Epstein-Barr-Virus-Infected CD15 (Lewis X)-Positive Hodgkin-Lymphoma-Like B Cells in Patients with Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA), especially those who are treated with methotrexate (MTX), might have an increased risk of Hodgkin lymphoma (HL), a malignancy that is associated with Epstein-Barr virus (EBV). Here we describe a monoclonal EBV-infected B-lymphoblastoid cell line (LCL) called TKS-1 that was established from cells that spontaneously converted from an MTX-treated RA patient. TKS-1 has properties similar to HL cells and it is distinctly different from control LCLs established from normal individuals. TKS-1 cells express the HL -associated surface markers CD15 and CD30 (Takei et al. 1989). Like Hodgkin Reed-Sternberg (H-RS) cells of EBV-positive HL, TKS-1 cells express EBNA1 mRNA transcribed from the Qp promoter of the virus, whereas control LCLs use the Cp or Wp promoter to transcribe mRNA. TKS-1 cells can proliferate in an anchorage-independent manner and possess a cloning efficiency comparable to that of the Burkitt lymphoma (BL) line Raji. In addition, two EBV-positive LCLs established by cocultivated CD34+ cells isolated from the bone marrow of patients with RA and peripheral blood B lymphocytes from a healthy EBV-seronegative individual also expressed CD15. These results indicate that EBV-infected B-lymphoblastoid cells from patients with RA tend to acquire properties similar to HL cells.

Effect of L -Asparaginase Combined with Vincristine and Prednisolone on Acute Myeloblastic Leukemia (M0) Associated with Non-Hodgkin Lymphoma

A 66-year-old Japanese woman was referred to us because of severe anemia and fever and presented at our hospital. She was eventually diagnosed as having acute myeloblastic leukemia (AML; M0) with non-Hodgkin lymphoma (NHL). We investigated the therapeutic efficacy of L-asparaginase (L-Asp), vincristine and prednisolone for both her AML and NHL. Asparagine synthetase (AS) activity in her AML blast cells was undetectable. A lymph node biopsy specimen revealed NHL of the marginal zone B cell type. Complete remission (CR) of AML and NHL was achieved. CR of the AML lasted for 18 months without further consolidation therapy. We conclude that L-Asp can be an effective drug for the treatment of AML in which blasts are negative for AS.

Detection of Grb-2-related adaptor protein gene (GRAP) and peptide molecule in salivary glands of MRL/lpr mice and patients with Sjögren's syndrome.

The pathogenesis of Sjögrens syndrome (SS) is poorly understood. In this study we used an in-house mouse spleen cDNA microarray to analyse genes in spleens from MRL/lpr (an SS mouse model) mice. We have previously demonstrated that GRAP genes were up-regulated in salivary glands of the same mice. The microarray analysis showed that seven out of 2304 genes were highly expressed in spleens from the MRL/lpr mice, one of which was the GRAP gene. In other words, the GRAP gene is highly expressed in the salivary glands and spleen of MRL/lpr mice. We also carried out immunohistochemical studies. Mouse and human Grb-2-related adaptor protein (GRAP) antigens were expressed on ductal cells and infiltrating lymphocytes in salivary glands of MRL/lpr mice and SS patients, but only weakly in controls (MRL/+ mice and individuals with salivary cysts). These results suggest that the GRAP gene might have a role in the pathogenesis of SS.

Efficacy and safety of minodronic acid hydrate in patients with steroid-induced osteoporosis

Minodronic acid hydrate, an oral bisphosphonate, has a greater inhibitory effect on bone resorption than do other approved drugs; however, this has been studied only in patients with primary osteoporosis. Here, we administered minodronic acid hydrate to patients with steroid‐induced osteoporosis who have been treated with steroids for rheumatoid arthritis or other collagen diseases, and the efficacy and safety of minodronic acid hydrate were prospectively investigated.

SAT0104 Subclinical Myocardial Inflammation and Fibrosis are Common in Active Rheumatoid Arthritis, Assessed by Cardiac Magnetic Resonance Imaging

Background Rheumatoid arthritis (RA) is a multi-organ inflammatory disorder associated with high cardiovascular morbidity and mortality. Importantly, cardiac involvements are typically clinically silent, only manifesting as myocardial abnormalities after an extended subclinical phase. Myocardial abnormalities may arise from a number of distinct processes, including myocardial inflammation (myocarditis), and/or myocardial fibrosis, any of which may be active in RA. Objectives We sought to assess cardiac involvement using a cardiac magnetic resonance imaging (CMR) approach and to determine its association with disease characteristics in RA patients without cardiac symptoms. Methods Consecutive RA patients and controls without cardiac symptoms were enrolled. RA patients and control subjects with no history and/or clinical findings of systemic and pulmonary hypertension, coronary artery disease, valvular heart disease, atrial fibrillation, diabetes mellitus, and dyslipidemia underwent CMR. RA patients received conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) or biologic DMARDs (bDMARDs). Late gadolinium enhancement (LGE) was obtained for the assessment of myocardial fibrosis. Using a black-blood T2-weighted image (T2-WI), myocardial inflammation could be assessed. Left ventricular geometry was classified into four categories: concentric remodeling, concentric or eccentric hypertrophy, or normal. We compared the patients and controls in terms of prevalence of CMR abnormalities, and explored possible associations between CMR abnormalities and RA disease characteristics. Results Sixty patients (mean age, 55.2±1.3 years; 85% female) and 20 controls (mean age 52.6±2.3 years; 86% female) were enrolled. Thirty RA patients received csDMARDs [25, methotrexate (MTX) (8.7±2.1mg); 5, other drugs)] and 30 RA patients received bDMARDs [(15, infliximab 3 mg/kg; 15, tocilizumab 8 mg/kg plus methotrexate (8.6±1.4 mg) mg/kg]. The control group showed no myocardial abnormalities. Twenty RA patients (33%) demonstrated myocardial abnormalities. T2-WI was seen in seven RA patients (11%). LGE was found in 19 RA patients (32%), six of whom also demonstrated T2-WI. Simplified Disease Activity Index (SDAI) scores were significantly higher in LGE-positive compared to LGE-negative (p=0.011). LGE was significantly associated with high NT-proBNP and eccentric hypertrophy (p=0.005, p=0.018, respectively). The use of bDMARDs was significantly associated with LGE-negative findings (p=0.0017). Other RA characteristics such as disease duration, autoantibody status, and cardiovascular risk factors were not significantly associated with myocardial abnormalities. After adjusting for confounding by age, RA duration, rheumatoid factor, and bDMARDs, the association of LGE with SDAI remained significant (p=0.023), in which the SDAI scores were, on average, 10.9 units higher in LGE-positive than in LGE-negative. Conclusions Subclinical myocardial inflammation and fibrosis are common in active RA patients without cardiac manifestation. Abnormal CMR findings were associated with higher RA disease activity, suggesting a role for inflammation in the pathogenesis of myocardial involvement in RA. Disclosure of Interest None declared