Kimiko Yurugi

Progressive graft fibrosis and donor‐specific human leukocyte antigen antibodies in pediatric late liver allografts

The role of donor‐specific anti‐human leukocyte antigen antibodies (DSAs) that develop late after living donor liver transplantation is unknown. Seventy‐nine pediatric recipients who had good graft function and underwent protocol liver biopsy more than 5 years after transplantation (median = 11 years, range = 5‐20 years) were reviewed. DSAs were determined with the Luminex single‐antigen bead assay at the time of the last biopsy, and complement component 4d (C4d) immunostaining was assessed at the times of the last biopsy and the previous biopsy. The donor specificity of antibodies could be identified in 67 patients: DSAs were detected in 32 patients (48%), and they were usually against human leukocyte antigen class II (30 cases) but were rarely against class I (2 cases). These patients had a higher frequency of bridging fibrosis or cirrhosis (28/32 or 88%) than DSA‐negative patients (6/35 or 17%, P < 0.001). Fibrosis was likely to be centrilobular‐based. DSA‐positive patients, in comparison with DSA‐negative patients, had higher frequencies of diffuse/focal endothelial C4d staining (P < 0.001) and mild/indeterminate acute rejection [15/32 (47%) versus 5/35 (14%), P = 0.004]. Four DSA‐negative patients were off immunosuppression, whereas no patients in the DSA‐positive group were (P = 0.048). In conclusion, the high prevalence of graft fibrosis and anti–class II DSAs in late protocol biopsy samples suggests that humoral alloreactivity may contribute to the process of unexplained graft fibrosis late after liver transplantation. Liver Transpl 18:1333–1342, 2012.

B‐cell surface marker analysis for improvement of rituximab prophylaxis in ABO‐incompatible adult living donor liver transplantation

Although the effectiveness of rituximab has been reported in ABO blood group (ABO)‐incompatible (ABO‐I) organ transplantation, the protocol is not yet established. We studied the impact of the timing of rituximab prophylaxis and the humoral immune response of patients undergoing ABO‐I living donor liver transplantation (LDLT), focusing on clinicopathological findings and the B‐cell subset. From July 2003 to December 2005, 30 adult patients were treated with hepatic artery infusion (HAI) protocol without splenectomy for ABO‐I LDLT. A total of 17 patients were treated only with HAI (no prophylaxis), and the other 13 were treated with rituximab prophylaxis at various times prior to transplantation. For B‐cell study of the spleen, another 4 patients undergoing ABO‐I LDLT both with HAI after prophylaxis and eventual splenectomy, and 3 patients with ABO‐compatible LDLT with splenectomy were enrolled. The mortality of the 30 patients with HAI, without splenectomy, and with/without rituximab prophylaxis was 33% and the main cause of death was sepsis. Peripheral blood B cells were completely depleted, anti‐donor blood‐type antibody titer was lower, and clinical and pathological antibody‐mediated rejection was not observed in patients with prophylaxis earlier than 7 days before transplantation (early prophylaxis). Early rituximab prophylaxis significantly depleted B cells and memory B cells in the spleen but not in lymph nodes. On the other hand, B cells and memory B cells increased and memory B cells became dominant during antibody‐mediated rejection. In conclusion, early prophylaxis with rituximab depletes B cells, including memory B cells, in the spleen and is associated with a trend toward lower humoral rejection rates and lower peak immunoglobulin (Ig)G titers in ABO‐I LDLT patients. Liver Transpl 13:579–588, 2007.

Two Susceptibility Loci to Takayasu Arteritis Reveal a Synergistic Role of the IL12B and HLA-B Regions in a Japanese Population

Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor.

A Clinical, Pathological, and Genetic Characterization of Methotrexate-associated Lymphoproliferative Disorders

Objective. Methotrexate-associated lymphoproliferative disorders (MTX-LPD) often regress spontaneously during MTX withdrawal, but the prognostic factors remain unclear. The aim of our study was to clarify the clinical, histological, and genetic factors that predict outcomes in patients with MTX-LPD. Methods. Patients with MTX-LPD diagnosed between 2000 and 2012 were analyzed retrospectively regarding their clinical course, site of biopsy, histological typing, Epstein-Barr virus (EBV) in situ hybridization and immunostaining, and HLA type. Results. Twenty-one patients, including 20 with rheumatoid arthritis (RA) and 1 with polymyositis, were analyzed. The mean dose of MTX was 6.1 mg/week and the mean duration of treatment was 71.1 months. Clinically, 5 patients were diagnosed with EBV-positive mucocutaneous ulcer (EBVMCU) and had polymorphic histological findings. The proportion of those patients successfully treated solely by withdrawal of MTX was significantly greater than that of those without EBVMCU (75% vs 7.7%, p = 0.015). The HLA-B15:11 haplotype was more frequent in patients with EBV+ RA with MTX-LPD than in healthy Japanese controls (p = 0.0079, Bonferroni’s method). EBV latency classification and HLA typing were not associated with the prognosis of MTX-LPD in our cohort. Conclusion. Our data demonstrate that patients in the EBVMCU, a specific clinical subgroup of MTX-LPD, had a better clinical outcome when MTX was withdrawn than did other patients with MTX-LPD.

Intraoperative blood loss during living donor liver transplantation: an analysis of 635 recipients at a single center.

BACKGROUND: Living‐donor liver transplantation (LDLT) has been an important option in the treatment of patients with end‐stage liver disease. Massive intraoperative blood loss can occur during LDLT, necessitating blood transfusion. The purpose of this study was to present blood loss data from the recipients of LDLT, to assess the effect of massive intraoperative blood loss on prognosis, and to assess the reliability of preoperative information in predicting intraoperative blood transfusion requirements in LDLT.

Does a positive lymphocyte cross-match contraindicate living-donor liver transplantation?

BACKGROUND There is still no consensus on the importance of lymphocyte cross-matching (LCM) in the field of living-donor liver transplantation (LDLT). METHODS LCM examinations are routinely performed before LDLT, and the results of complement-dependent cytotoxicity were used in this study. A total of 1157 LDLT cases were evaluated. The recipients were divided into four groups based on the LCM and ABO compatibilities: (1) negative LCM and identical/compatible ABO; (2) negative LCM and incompatible ABO; (3) positive LCM and identical/compatible ABO; and (4) positive LCM and incompatible ABO. The diagnosis of antibody-mediated rejection (AMR) was made based on the clinical course, immunological assays and histopathological findings. C4d immunostaining was added if AMR was suspected. RESULTS The LCM-positive LDLT recipients showed significantly poorer outcomes than the LCM-negative recipients. Among the LCM-positive recipients, 44.1% of recipients eventually died and 85.2% of recipients revealed positive C4d findings. The survival rate of LCM-positive and ABO-incompatible group was 0.50. The survival days were compared with the LCM-negative and ABO-identical/compatible group, and the LCM-positive and ABO-identical/compatible group clearly showed early death after LDLT, although the ABO-incompatible groups did not show significant. The factors of age, disease, pre-transplant scores, LCM, ABO compatibility and graft-recipient weight ratio showed statistical significance in multivariate analysis for important factors of LDLT outcomes. However, the LCM and ABO compatibilities had no synergetic effects on the LDLT survival. CONCLUSION HLA antigens are more widely expressed than ABO antigens, and advanced immunological strategies must be established for LCM-positive LDLT as well as for ABO-incompatible LDLT.

Rapid quantitation of immunoglobulin G antibodies specific for blood group antigens A and B by surface plasmon resonance

BACKGROUND:  The measurement of immunoglobulin (Ig) G blood group A/B antibody(anti‐A/B) levels is important for ABO‐unmatched organ recipients because the effective removal of the antibodies improves their prognosis. Currently existing methods to detect IgG anti‐A/B suffer limitations owing to high costs, low throughput, and poor adaptability to automation.

ACPA-Negative RA Consists of Two Genetically Distinct Subsets Based on RF Positivity in Japanese

HLA-DRB1, especially the shared epitope (SE), is strongly associated with rheumatoid arthritis (RA). However, recent studies have shown that SE is at most weakly associated with RA without anti-citrullinated peptide/protein antibody (ACPA). We have recently reported that ACPA-negative RA is associated with specific HLA-DRB1 alleles and diplotypes. Here, we attempted to detect genetically different subsets of ACPA-negative RA by classifying ACPA-negative RA patients into two groups based on their positivity for rheumatoid factor (RF). HLA-DRB1 genotyping data for totally 954 ACPA-negative RA patients and 2,008 healthy individuals in two independent sets were used. HLA-DRB1 allele and diplotype frequencies were compared among the ACPA-negative RF-positive RA patients, ACPA-negative RF-negative RA patients, and controls in each set. Combined results were also analyzed. A similar analysis was performed in 685 ACPA-positive RA patients classified according to their RF positivity. As a result, HLA-DRB1*04:05 and *09:01 showed strong associations with ACPA-negative RF-positive RA in the combined analysis (p = 8.8×10−6 and 0.0011, OR: 1.57 (1.28–1.91) and 1.37 (1.13–1.65), respectively). We also found that HLA-DR14 and the HLA-DR8 homozygote were associated with ACPA-negative RF-negative RA (p = 0.00022 and 0.00013, OR: 1.52 (1.21–1.89) and 3.08 (1.68–5.64), respectively). These association tendencies were found in each set. On the contrary, we could not detect any significant differences between ACPA-positive RA subsets. As a conclusion, ACPA-negative RA includes two genetically distinct subsets according to RF positivity in Japan, which display different associations with HLA-DRB1. ACPA-negative RF-positive RA is strongly associated with HLA-DRB1*04:05 and *09:01. ACPA-negative RF-negative RA is associated with DR14 and the HLA-DR8 homozygote.

Association of Takayasu arteritis with HLA-B*67:01 and two amino acids in HLA-B protein

OBJECTIVE Takayasu arteritis (TAK) is a rare autoimmune arteritis that affects large arteries. Although the association between TAK and HLA-B 52:01 is established, the other susceptibility HLA-B alleles are not fully known. We performed genetic association studies to determine independent HLA-B susceptibility alleles other than HLA-B 52:01 and to identify important amino acids of HLA-B protein in TAK susceptibility. METHODS One hundred patients with TAK and 1000 unrelated healthy controls were genotyped for HLA-B alleles in the first set, followed by a replication set containing 73 patients with TAK and 1000 controls to compare the frequencies of HLA-B alleles. Step-up logistic regression analysis was performed to identify susceptibility amino acids of HLA-B protein. RESULTS Strong associations of susceptibility to TAK with HLA-B 52:01 and HLA-B 67:01 were observed (P = 1.0 × 10(-16) and 9.5 × 10(-6), respectively). An independent susceptibility effect of HLA-B 67:01 from HLA-B 52:01 was also detected (P = 1.8 × 10(-7)). Amino acid residues of histidine at position 171 and phenylalanine at position 67, both of which are located in antigen binding grooves of the HLA-B protein, were associated with TAK susceptibility (P ≤ 3.8 × 10(-5)) with a significant difference from other amino acid variations (ΔAIC ≥ 9.65). CONCLUSION HLA-B 67:01 is associated with TAK independently from HLA-B 52:01. Two amino acids in HLA-B protein are strongly associated with TAK susceptibility.

Association of anti-human leukocyte antigen and anti-angiotensin II type 1 receptor antibodies with liver allograft fibrosis after immunosuppression withdrawal.

Background Many pediatric patients who receive a living-donor liver transplant undergo withdrawal of immunosuppression (IS). For them, the high incidence of long-term progressive graft fibrosis is of particular concern. Methods We conducted a cross-sectional study including 81 pediatric patients who underwent IS withdrawal after living-donor liver transplant at Kyoto University Hospital and whose serum samples and pathological data could be obtained during the analysis period. We examined the association of donor-specific anti-human leukocyte antigen (HLA) antibody (DSA) and angiotensin II type 1 receptor antibody (anti-AT1R Ab) with posttransplant graft fibrosis. Normalized mean fluorescence intensity (MFI) 5,000 or higher and anti-AT1R Ab concentrations 17 U/mL or higher were both considered high level. The patients were classified into an advanced fibrosis group (AFG) (Ishak score≥3) and a control group (CG) (Ishak score⩽2). Results Only one patient demonstrated DSA class I. Among those who demonstrated DSA class II, more AFG patients than CG patients demonstrated high-level mean fluorescence intensity, although the difference was not significant (64% vs. 39%; P=0.053). The incidence of high-level DSA-DRB1, however, was significantly higher in the AFG than that in the CG (40% vs. 4%; P<0.001), but there was no significant difference in DSA-DQB1 or DSA-DRB345. High-level anti-AT1R Ab was significantly more frequent in the AFG than in the CG (65% vs. 36%; P=0.02). All patients with both high-level DSA-DRB1 and high-level anti-AT1R Ab were found to have advanced fibrosis (P<0.001). Conclusion Anti-AT1R Ab and DSA-DRB1 may be candidates as biomarkers of graft fibrosis; both HLA and non-HLA immunity may be involved in graft fibrosis after IS withdrawal.