Hideyuki Murata

Laser microdissection-based analysis of cytokine balance in the kidneys of patients with lupus nephritis

To determine the cytokine balance in patients with lupus nephritis (LN), we analysed kidney‐infiltrating T cells. Renal biopsy samples from 15 systemic lupus erythematosus (SLE) patients were used. In accordance with the classification of International Society of Nephrology/Renal Pathology Society, they were categorized into Class III, Class III+V (Class III‐predominant group, n = 4), Class IV, Class IV+V (Class IV‐predominant group, n = 7) and Class V (n = 4) groups. The single‐cell samples of both the glomelular and interstitial infiltrating cells were captured by laser‐microdissection. The glomerular and interstitial infiltrating T cells produced interleukin (IL)‐2, IL‐4, IL‐10, IL‐13 and IL‐17 cytokines in the Class III‐predominant, Class IV‐predominant and Class V groups. Interferon‐gamma was detected only in the glomeruli of the Class III‐predominant and Class V group samples. The expression level of IL‐17 was correlated closely with clinical parameters such as haematuria, blood urea nitrogen level, SLE Disease Activity Index scores in both glomeruli and interstitium, urine protein level in glomeruli and serum creatinine and creatinine clearance levels in interstitium. This suggests that the glomerular infiltrating T cells might act as T helper type 1 (Th1), Th2 and Th17 cells while the interstitial infiltrating T cells, act as Th2 and Th17 cells in the Class III‐predominant and Class V groups. In contrast, both the glomerular and interstitial infiltrating T cells might act as Th2 and Th17 cells in the Class IV‐predominant group. The cytokine balances may be dependent upon the classification of renal pathology, and IL‐17 might play a critical role in SLE development.

Microchimerism in Japanese women patients with systemic sclerosis

To examine whether microchimerism occurs in Japanese women patients with systemic sclerosis, we analysed the Y-chromosome in DNA by PCR. There was no significant difference between patients and healthy people, suggesting that microchimerism may not be the sole pathogenesis in Japanese women with systemic sclerosis.

Common T cell receptor clonotype in lacrimal glands and labial salivary glands from patients with Sjögren's syndrome.

Sjogrens syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS.

Mannose binding lectin gene polymorphism in patients with type I diabetes

Our purpose was to investigate a possible relationship between occurrence of type I diabetes and polymorphism of the mannose binding lectin gene. Polymorphism of codon 54 of the mannose binding lectin (MBL) gene, whose presence of the minority allele leads to significant reduction of serum MBL concentration, was investigated in 128 Japanese patients with type I diabetes and 78 healthy volunteers by restriction fragment length polymorphism method. Frequencies of the minority allele were compared between the patient group and the control group. Frequency of the minority allele was 24.2% in the patient group and 19.9% in the control group. The probability of being heterozygous or homozygous for the minority allele was 41.4% in the patient group and 33.3% in the control group. Patients with DRB1*0405-DQB1*0401 and/or DRB1*0901-DQB1*0303 haplotypes, the two major type I diabetes-prone human leukocyte antigen haplotypes, showed a slightly higher probability of being heterozygous or homozygous for allele B of the MBL gene. Possession of the minority allele of the MBL gene may be a minor risk factor for having type I diabetes.

Conserved CDR 3 region of T cell receptor BV gene in lymphocytes from bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an inflammatory lung disease characterized by the accumulation of inflammatory cells and deposition of collagen, resulting in lung remodelling. High numbers of T cells are present in bronchoalveolar lavage fluid (BALF) of IPF patients, although the characteristics of these cells are yet to be determined. To elucidate the pathogenic mechanisms of IPF, we analysed the T cell receptor (TCR) of BALF lymphocytes in three patients with IPF and three healthy subjects as control. TCR repertoire of BALF lymphocytes and T cell clonality were examined by family PCR and Southern blot analysis, and single‐strand conformation polymorphism (SSCP), respectively. We observed that the TCR repertoire in the lung was heterogeneous, both in the control subjects and three patients with IPF. SSCP analysis demonstrated an increase in the number of accumulated T cell clones in BALF of two of the three patients, but not in the healthy subject. Furthermore, junctional sequence analysis showed the presence of conserved amino acid motifs (ETGRSG, LAxG, QGQ, GxQP, GRxG, VAR, PGT, GTI, GGT, TGR, LxLxQ, SGQ) in the TCR‐CDR 3 region of BAL lymphocytes in patients with IPF, whereas only two amino acid motifs (VTTG, GGE) were found in the control. Our findings suggest that T cells in BALF of patients with IPF expand oligoclonally in the lung, suggesting antigen stimulation of these cells.

Analysis of T Cell Receptor Vβ Gene Expression and Clonality in Bronchoalveolar Fluid Lymphocytes from a Patient with Chronic Eosinophilic Pneumonitis

Abstract. Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. T lymphocytes in bronchoalveolar lavage (BAL) from patients with chronic eosinophilic pneumonitis are considered to recognize unknown antigens. To analyze the pathogenesis of CEP, we examined the T cell receptor (TCR) repertoire and T cell clonotype of BAL lymphocytes and peripheral blood lymphocytes (PBLs) in a 66-year-old woman patient with CEP. The expression of TCR BV gene was analyzed by the family PCR method using specific primers for 20 TCR BV genes and BC gene. The clonotype of BAL and peripheral T cells was examined by the PCR-single-strand conformation polymorphism (SSCP) method. Functional sequences of some T cell clones were also carried out. A TCR repertoire of BAL T cells was heterogeneous as well as PBLs. However, SSCP analysis showed that distinct T cell clonotypes were detected in BAL T cells, TCR BV3, BV4, BV6, BV8, BV9, BV14, and BV18-positive T cell clones especially, expanded clonally in BAL from the patient. Sequencing analysis showed that GVD, LGG, RDXS, and SSG amino acid sequence motif were found in the CDR3 in lung-specific T cells. BAL-specific T cell clones accumulated in the patient with CEP. Thus, we can conclude that BAL T cells are induced by the antigen-driven stimulation and these cells might play a crucial role in the generation of CEP.

T cell epitopes of prothrombin in patients with antiphospholipid syndrome

Antiphospholipid syndrome (APS) is characterised by arterial and/or venous thrombosis and recurrent fetal loss in association with the presence of antiphospholipid antibodies (aPL). In addition to β2-glycoprotein I, prothrombin (PT) is an important autoantigen recognised by aPL. PT is a coagulation proenzyme abundantly present in blood (70–100 μg/ml), and binds to negatively charged phospholipids such as phosphatidylserine. PT comprises two major domains, fragment-1 (F-1) and prethrombin-1 (Pre-1). We previously reported that antiprothrombin antibody (aPT) is a mixture of antibodies against both F-1 and Pre-1, and that there are significant clinical differences between anti-F-1 and anti-Pre-1.1,2 aPL, including aPT, are not mere serological markers of the disease, but are important players in the pathogenesis of APS. Therefore, antigen-specific immunosuppressive treatments, if developed, will help to prevent …

An HLA-B27-positive patient diagnosed with ulcerative colitis 15 years after the onset of arthropathy

Abstract A 28-year-old woman had persistent pain of both hip joints since the age of 13 years. X-ray analysis showed destructive changes in both hip joints and ossification of sacroiliitic joints. The patient had mild diarrhea and slight abdominal pain for 8 years. Blood-stained stool was not noticed. Barium enema showed changes consistent with the diagnosis of ulcerative colitis (UC). Inflammatory bowel syndrome should be considered in patients with persistent coxitis, even in the absence of severe abdominal symptoms.

Quantitative analysis of fetal microchimerism in Japanese women patients with systemic sclerosis

To the Editor: J. Lee Nelson et al. have shown a quantitative difference in the amount of fetal DNA in the peripheral blood of women with systemic sclerosis (SSc), and have put forward the hypothesis that SSc may be caused by a graft-versus-hostlike reaction induced by fetal cells in the mother. This was supported by the findings of Carol M. Artlett. In contrast, our previous study (in Japanese) demonstrated that there was no significant difference between the frequency of microchimerism in SSc patients and healthy subjects. In this study, to examine whether the number of fetal progenitor cells is related to the pathogenesis, the amount of malespecific DNA in peripheral blood leukocytes (PBL) was estimated by quantitative polymerase chain reaction (PCR) using the TaqMan probe method (Fig. 1). The primers specific for DYZ1 (positions 78–100, 3511–3573) and the TaqMan probe (positions 12–40) were used for quantification. We analysed the amounts of male-specific genes in DNA extracted from peripheral blood lymphocytes from seven Japanese women patients with SSc bearing the Ychromosome, six healthy Japanese subjects with the Ychromosome, and eight healthy Japanese women without children as controls. The quantities of Y-chromosomespecific DNA were described in arbitrary units. These units represent the amount of Y-chromosome-specific DNA in women, taking the amount of Y-chromosome-specific DNA in 10ng of genomic DNA from the blood of one male as 10. The results showed that the amount of Y-chromosomespecific DNA in patients with SSc (18.3 30.8, mean SD) was significantly higher than those in healthy subjects with a Y-chromosome sequence (2.11 2.78) (P 0.03) and in controls without children (0.23 0.19) (P 0.03). These amounts were compared using the Mann–Whitney U-test. These findings support the notion that a large number of fetal cells might induce clinical features of SSc, and indicate that a degree of microchimerism is one of the essential factors in the generation of SSc.