Analysis of PLA2R1 and HLA-DQA1 sequence variants in Japanese patients with idiopathic and secondary membranous nephropathy.

BACKGROUND: Several recent studies in patients with idiopathic membranous nephropathy (iMN) from Western and Asian counties showed that some single nucleotide polymorphisms (SNPs) within the PLA2R1 and HLA-DQA1 genes are significantly associated with iMN. However, there is only 1 report on analysis of PLA2R1 and HLA regions in Japanese patients with iMN. METHODS: A total of 58 patients with iMN, 26 patients with secondary MN (sMN), and 50 patients with other diseases were enrolled. All patients were Japanese. We selected 6 SNPs within PLA2R1 and 1 SNP within HLA-DQA1, which were significantly associated with iMN in reported white European cohorts, and sequenced these exons using genomic DNA prepared from peripheral mononuclear cells from each patient. We then analyzed differences in PLA2R1 and HLA-DQA1 sequence variants among the 3 groups. RESULTS: Genotypic and allelic frequency distributions for 3 out of 6 SNPs within PLA2R1, rs3749117, rs35771982, and rs2715918 were significantly different between the iMN and control groups. Allelic frequency distributions for SNP rs2187668 within HLA-DQA1 were significantly different between the iMN and control groups. There were no correlations between PLA2R1 and HLA-DQA1 sequence variants and clinical parameters in patients with iMN. There were no significant differences in genotypic or allelic frequency distributions for examined SNPs between the sMN and control groups. CONCLUSIONS: There are some differences in PLA2R1 SNP distributions between previously reported cohorts from other countries and our Japanese cohort of patients with iMN, while there is a significant association between SNP rs35771982 and iMN in most of reported cohorts.

The effect of hepatocyte growth factor on secretory functions in human eosinophils.

Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is now recognized as a humoral mediator in inflammatory and immune responses. Previous studies indicated that HGF negatively regulated allergic airway inflammation. In view of eosinophils playing a role in the pathogenesis of asthma, especially in airway remodeling as a rich source of pro-fibrogenic mediators, the effects of HGF on the different types of eosinophil secretory functions were examined in this study. We found that HGF significantly inhibited IL-5-induced secretion of TGF-ß and VEGF from human eosinophils. The inhibitory effect is not associated with TGF-ß transcription; rather, it is associated with ultrastructural granule emptying and loss of intracellular TGF-ß contents, indicating HGF inhibits the process of piecemeal degranulation. The effect of HGF on extracellular trap cell death (ETosis) that mediates cytolytic degranulation was also investigated; however, immobilized IgG- or phorbol myristate acetate-induced ETosis was only minimally attenuated by HGF. These results reveal the effect of HGF on the distinct pathways of eosinophil secretory functions and also provide novel insights into the role of HGF in the pathogenesis of allergic inflammation.

Decline of CSF orexin (hypocretin) levels in Prader-Willi syndrome.

Prader-Willi syndrome is a congenital neurodevelopmental disorder resulting from deletion of the paternal copies of genes within the chromosome region 15q11-q13. Patients with Prader-Willi syndrome often exhibit excessive daytime sleepiness, excessive appetite, and obesity. As is the case in narcolepsy, orexin (hypocretin) may be responsible for these symptoms. However, reports showing cerebrospinal fluid orexin levels in Prader-Willi syndrome patients have been limited. The aim of this study was to examine the relationship between the characteristic symptoms of Prader-Willi syndrome and cerebrospinal fluid orexin levels. We clinically identified 14 Prader-Willi syndrome patients and examined their cerebrospinal fluid orexin levels. A total of 12 patients with a 15q11-q13 deletion and two patients with maternal uniparental disomy of chromosome 15 were identified. A total of 37 narcoleptic patients and 14 idiopathic hypersomnia patients were recruited for comparison. Cerebrospinal fluid orexin levels (median [25-75 percentiles]) in the 14 Prader-Willi syndrome patients were intermediate (192 [161-234.5] pg/ml), higher than in the narcoleptic patients, but lower than in the idiopathic hypersomnia patients. Body mass index of the Prader-Willi syndrome patients was higher than in the narcoleptic and idiopathic hypersomnia patients. There was also a negative correlation between Epworth sleepiness scale scores and orexin levels in Prader-Willi syndrome patients. Decreased cerebrospinal fluid orexin levels in Prader-Willi syndrome may play an important role in severity of obesity and excessive daytime sleepiness.

[Viewpoint: Skill Certifications for Japanese Medical Technologists].

With the development of medicine, the field of clinical laboratory medicine evolves rapidly, and it will be more specialized in the near future. Medical technologists are required to hone their skills and knowledge, in order to keep up with the evolution. In recent years, board certifications by several medical societies are considered to indicate the skills of medical technologists. The number of board-certified medical technologists in populated areas such as Tokyo, Kanagawa, Osaka, and Fukuoka is greater than in less populated areas such as Kyusyu and Tohoku. The rate of certified medical technologists among prefectures is the highest in Mie (10.1%), followed by Nagasaki (8.8%). Tokyo, Ishikawa, Kyoto, and Osaka have acquisition rates greater than 7%. In contrast, prefectures of Miyazaki, Kumamoto, Yamanashi, and Akita have low acquisition rates of less than 4%. Being certified is not only an opportunity for personal career advancement, but also a chance to improve the laboratory. More technologists are being certified in our laboratory, and we are encouraging a future increase in their number. However, there are some problems to be overcome. Assignment of competent staff and long-term and premeditated rotation are considered to be important for staff to find the work rewarding, and the laboratory to be trusted by physicians.

Effect of CYP3A5 and ABCB1 polymorphisms on the interaction between tacrolimus and itraconazole in patients with connective tissue disease.

PURPOSE: The aim of this study was to investigate the effect of itraconazole (ITCZ), a potent inhibitor of CYP3A4 and P-glycoprotein, on the blood concentration 12 h after tacrolimus administration (C 12h) in relation to CYP3A5 6986A>G and ABCB1 3435C>T genotype status in patients with connective tissue disease (CTD). METHODS: Eighty-one CTD patients taking tacrolimus (Prograf®) once daily at night (2100 hours) were enrolled in this study. Whole blood samples were collected 12 h after tacrolimus administration at steady state. RESULTS: The dose-adjusted tacrolimus C 12h with or without ITCZ co-administration was significantly higher in patients with CYP3A5*3/*3 than in those with the CYP3A5*1 allele [CYP3A5 *1/*1 vs. *1/*3 vs. *3/*3 = 1.67 vs. 2.70 vs. 4.83 ng/mL/mg (P = 0.003) and 0.68 vs. 0.97 vs. 2.20 ng/mL/mg (P < 0.001), respectively], but differences were not observed for ABCB1 genotypes. However, there was no difference in the increase rate of the dose-adjusted C 12h of tacrolimus between CYP3A5 or ABCB1 genotypes (P = 0.378 and 0.259). On the other hand, reduction of the estimated glomerular filtration rate exhibited a correlation with the C 12h of tacrolimus after ITCZ co-administration (r = -0.482, P = 0.009). CONCLUSIONS: In CYP3A5*3/*3 patients, because the metabolic pathway for tacrolimus occurs only through CYP3A4, the combination with ITCZ seems to lead to a higher risk of acute renal dysfunction. Therefore, we suggest that the target blood tacrolimus concentration be set as low as possible through dose-adjustment for patients with the CYP3A5*3/*3 allele.

Effects of CYP3A5 polymorphism and the tacrolimus 12 h concentration on tacrolimus-induced acute renal dysfunction in patients with lupus nephritis.

1. The purpose of the present study was to investigate the effect of tacrolimus concentration in blood 12 h after administration (C12h) on acute renal dysfunction in patients with lupus nephritis (LN) taking tacrolimus once daily. 2. Five of the 35 LN patients experienced tacrolimus-induced acute renal dysfunction. 3. The average annual C12h of tacrolimus was higher for patients with events of elevated serum creatinine level than for patients not experiencing these events [6.4 (5.6-8.8) versus 2.8 (2.2-4.6) ng/mL, respectively, p=0.001]. 4. The average annual tacrolimus C12h was higher for patients with CYP3A5*3/*3 (PM) than for patients with the CYP3A5*1 allele (EM) [4.6 (3.2-6.6) versus 2.5 (2.0-3.1) ng/mL, respectively, p=0.002]. 5. The area under the receiver operating characteristic was 0.887, which gave the best sensitivity (80.0%) and specificity (86.7%) at a tacrolimus C12h (average annual C12h or C12h at events) threshold of 5.2 ng/mL. 6. C12h measurements, CYP3A5 genotyping and dose adjustments of tacrolimus should be performed to prevent acute renal dysfunction in LN patients taking tacrolimus once daily.

Validation of the Japanese histologic classification 2013 of immunoglobulin A nephropathy for prediction of long-term prognosis in a Japanese single-center cohort.

BACKGROUND: A new Japanese histologic classification (JHC) of immunoglobulin A nephropathy (IgAN) for prediction of long-term prognosis was proposed in 2013. The goal of this study was to validate the JHC system in a Japanese single-center cohort. METHODS: A retrospective study was conducted in 198 Japanese adult patients with IgAN. Clinical findings including blood pressure, urinary protein, estimated glomerular filtration rate (eGFR), and outcomes were evaluated in these patients. The glomerular lesion percentage score (GLPS) [number of glomeruli with cellular crescents, fibrocellular crescents, global sclerosis, segmental sclerosis, or fibrous crescents/number of total obtained glomeruli × 100 (%)] was assessed in each patient and categorized into histologic grades (HGs) of HG1 (<25 %), HG2 (25-49 %), and HG3/4 (≥50 %). Associations of GLPS (HG) with disease progression (50 % eGFR decline or end-stage renal disease requiring dialysis) within 10 years after biopsy and the rate of annual eGFR decline were examined. RESULTS: During a median follow-up period of 12.0 years after biopsy, disease progression occurred in 12.8 % (12/94) of HG1 patients, 32.3 % (21/65) of HG2 patients, and 46.2 % (18/39) of HG3/4 patients. The risk of disease progression was significantly higher in the HG2 and HG3/4 groups than in the HG1 group (odds ratios: 3.3 and 5.9 vs. 1). A higher GLPS was significantly associated with a higher risk of disease progression and a greater annual eGFR decline. CONCLUSION: The newly proposed JHC system 2013 based on GLPS (HG) was well correlated with long-term prognosis in our cohort of Japanese adult patients with IgAN.

Downregulated expression of miR-155, miR-17, and miR-181b, and upregulated expression of activation-induced cytidine deaminase and interferon-α in PBMCs from patients with SLE.

OBJECTIVE: Recent studies on systemic lupus erythematosus (SLE) revealed that microRNAs (miRNAs or miRs) were involved in its pathogenesis. However, only a limited number of miRNAs have been examined. METHODS: We performed quantitative real-time reverse transcription-polymerase chain reaction analyses of peripheral blood mononuclear cells (PBMCs) obtained from 31 untreated SLE patients and 31 healthy subjects to examine the expression levels of miR-155, miR-17, and miR-181b, as well as those of activation-induced cytidine deaminase (AID) and interferon-α (IFN-α) messenger RNAs (mRNAs). We examined the relationship between miR-181b, AID, and IFN-α with a luciferase reporter assay. RESULTS: The expression levels of miR-155, miR-17, and miR-181b were significantly lower in SLE patients than those in healthy controls, whereas those of AID and IFN-α mRNAs were significantly higher in SLE patients than those in healthy controls. The expression levels of miR-155, miR-17, and miR-181b inversely correlated with those of AID and IFN-α mRNAs in SLE patients. The results of the luciferase reporter assay revealed that miR-181b negatively regulated AID and IFN-α. CONCLUSIONS: The results of the present study demonstrated for the first time that there is a differential expression and inverse correlation between the levels the miR-155, miR-17, and miR-181b and target molecules, AID and IFN-α mRNAs, in PBMCs of untreated SLE patients. These alterations may contribute to the pathogenesis of SLE.

Validation of the 2010 histopathological classification of ANCA-associated glomerulonephritis in a Japanese single-center cohort.

OBJECTIVES: To validate the 2010 histopathological classification system of anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (GN) in a Japanese single-center cohort. METHODS: We retrospectively studied 54 patients (28 renally limited pauci-immune GN, 25 microscopic polyangiitis, and one Churg-Strauss syndrome). RESULTS: There were 17 patients with focal GN, eight patients with crescentic GN, 19 patients with mixed GN, and 10 patients with sclerotic GN. Detailed information regarding treatment was available in 39 patients. All these patients were treated with steroids with or without immunosuppressive agents. Hemodialysis was introduced in two patients with crescentic GN and three patients with sclerotic GN. During the follow-up period, 27 of 54 patients died. The major cause of death was pneumonia. Significant differences were observed in estimated glomerular filtration rate among patients with focal, crescentic, mixed, and sclerotic GN at entry and 1- and 5-year follow-up. Patients with focal GN had preserved renal function and favorable outcome. CONCLUSIONS: Our validation study suggests that the 2010 histopathological classification of ANCA-associated GN might aid in prognostication of patients at the time of diagnosis and in therapy selection.

Serum interleukin 6 levels as a useful prognostic predictor of clinically amyopathic dermatomyositis with rapidly progressive interstitial lung disease.

OBJECTIVES: Rapidly progressive interstitial lung disease (RP-ILD) is life-threatening in patients with clinically amyopathic dermatomyositis (CADM). Useful prognostic markers are necessary for treatment selection. This study aimed to investigate differences in clinical and laboratory characteristics between surviving and non-surviving patients. METHODS: Twelve CADM patients with RP-ILD were enrolled. Six patients lived (Group A) and six patients died (Group B) after immunosuppressive treatment for RP-ILD. Clinical manifestations and laboratory data before treatment were compared between the two groups. RESULTS: Among the clinical manifestations and laboratory data examined, serum interleukin 6 (IL-6) levels in Group B were significantly higher than those in Group A (mean ± SD 28.5 ± 21.0 vs. 7.2 ± 1.6 pg/mL; p = 0.009). Simple regression analysis showed that serum IL-6 was the only significant prognostic factor (p = 0.032). Kaplan-Meier estimates showed that the cumulative survival rate was significantly lower in patients with serum IL-6 levels of ≥ 9 pg/mL than in patients with those of < 9 pg/mL (p = 0.04). CONCLUSIONS: Serum IL-6 levels may predict the prognosis of CADM patients with RP-ILD. The intensity of immunosuppressive treatment can be decided according to serum IL-6 levels at an early phase of the disease.

Validation of the 2010 histopathological classification of ANCA-associated glomerulonephritis in a Japanese single-center cohort.

OBJECTIVES: To validate the 2010 histopathological classification system of anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (GN) in a Japanese single-center cohort. METHODS: We retrospectively studied 54 patients (28 renally limited pauci-immune GN, 25 microscopic polyangiitis, and one Churg-Strauss syndrome). RESULTS: There were 17 patients with focal GN, eight patients with crescentic GN, 19 patients with mixed GN, and 10 patients with sclerotic GN. Detailed information regarding treatment was available in 39 patients. All these patients were treated with steroids with or without immunosuppressive agents. Hemodialysis was introduced in two patients with crescentic GN and three patients with sclerotic GN. During the follow-up period, 27 of 54 patients died. The major cause of death was pneumonia. Significant differences were observed in estimated glomerular filtration rate among patients with focal, crescentic, mixed, and sclerotic GN at entry and 1- and 5-year follow-up. Patients with focal GN had preserved renal function and favorable outcome. CONCLUSIONS: Our validation study suggests that the 2010 histopathological classification of ANCA-associated GN might aid in prognostication of patients at the time of diagnosis and in therapy selection.

Distribution of glomerular IgG subclass deposits in patients with membranous nephropathy and anti-U1 ribonucleoprotein antibody.

BACKGROUND: It is known that a predominant glomerular deposition of IgG4 is characteristic of idiopathic membranous nephropathy (MN) and that significant deposition of other IgG subclasses is also observed in lupus MN. However, there is no report focusing on the distribution of glomerular IgG subclass deposits in MN patients with anti-U1 ribonucleoprotein (RNP) antibody. METHODS: We evaluated clinicopathological features and the distribution patterns of glomerular IgG subclass deposits in seven MN patients with positive anti-RNP antibody and negative antibodies to double-stranded DNA (dsDNA) and Smith antigen (Sm) (RNP-MN group) and in seven age- and sex-matched lupus MN patients with positive anti-dsDNA antibody and negative antibodies to RNP and Sm (L-MN group). RESULTS: Mixed connective tissue disease was diagnosed in four patients in the RNP-MN group. Two patients in the RNP-MN group and three patients in the L-MN group developed nephrotic syndrome. Renal insufficiency was not present in all patients in both groups. Hypocomplementemia was found in two patients in the RNP-MN group and six patients in the L-MN group. In the RNP-MN group, positive stainings for glomerular IgG1, IgG2, IgG3 and IgG4 were observed in one, seven, zero and five patients, respectively. On the contrary, in the L-MN group, positive stainings for glomerular IgG1, IgG2, IgG3 and IgG4 were observed in seven, seven, seven, and six patients, respectively. CONCLUSIONS: This is the first study showing striking differences in the distribution of glomerular IgG subclass deposits between RNP-MN and L-MN groups. RNP-MN and L-MN may result from different immunological mechanisms.

Renal biopsy in patients aged 80 years and older: a single-center experience in Japan.

BACKGROUND: There is a paucity of data on renal biopsy in a large number of the very elderly (age ≥ 80 years) worldwide. METHODS: Clinicopathological features in 73 patients aged ≥ 80 years were evaluated and compared with control groups of 172 patients aged 60 - 61 years and 128 patients aged 70 - 71 years. RESULTS: The common indications for biopsy in the very elderly were nephrotic syndrome (NS), followed by proteinuria without NS and/or hematuria, and acute kidney injury (AKI). Histological diagnoses were considered to potentially modify treatment in 57 cases (78.1%): the most frequent diagnosis was membranous nephropathy, followed by minimal change disease, and various other diseases. There were no biopsy procedure-related serious complications. Clinical assessment of treatments was evaluated in 38 of 54 patients with AKI and/or NS. Improvement in renal dysfunction or NS was observed in 24 of 30 (80%) patients who received immunosuppressive therapy. There were statistically significant differences in the disease spectrum between the very elderly and control groups. CONCLUSIONS: This is the first report of renal biopsy findings in a relatively large number of Japanese very elderly patients. Histological observations are useful aids in estimating the prognosis and therapy selection for renal disorders, even in the very elderly.

Clonal hematopoiesis in adult pure red cell aplasia.

Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.

Simultaneous herpes simplex virus esophagitis and lupus enteritis in a patient with systemic lupus erythematosus.

A 52-year-old woman with a 6-year history of systemic lupus erythematosus (SLE) developed acute abdominal pain, nausea, vomiting, and diarrhea accompanied by hypocomplementemia. Herpes simplex virus (HSV) esophagitis and lupus enteritis were diagnosed on the basis of the results of endoscopic and histological examinations and abdominal computed tomography (CT) findings. Treatment with acyclovir followed by high-dose intravenous steroids improved her symptoms. To our knowledge, this is the first case of simultaneous HSV esophagitis and lupus enteritis.

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4-28/IGHJ4 gene in primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4-28 in ITP patients. One hundred eighty-six distinct IGHV4-28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4-28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4-28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.

Urine Xanthine Crystals in Tumor Lysis Syndrome.

Urine xanthine crystals are remarkably rare but can be observed by routine urine microscopy. We report the results of a 67-year-old man with T-cell-prolymphocytic leukemia whose urine contained xanthine crystals after chemotherapy and prophylactic administration of febuxostat. Accumulation of xanthine was due to tumor lysis syndrome causing a massive release of DNA. The metabolized DNA caused an increase of xanthine, which was not readily converted to uric acid by xanthine oxidase because of febuxostat inhibition of this enzyme.