In-depth proteomic analysis of juvenile dermatomyositis serum reveals protein expression associated with muscle-specific autoantibodies.

OBJECTIVES: The clinical symptoms and complications of JDM differ depending on the type of muscle-specific autoantibodies (MSAs) present. We aimed to identify protein expression profiles specific for MSAs that characterize various clinical features by comprehensively analyzing the proteins present in the serum of patients with JDM. METHODS: We analysed sera from patients with JDM that were positive for anti-melanoma differentiation-associated protein 5 (MDA5) antibodies (n = 5), anti-nuclear matrix protein 2 (NXP2) antibodies (n = 5) and anti-transcriptional intermediary factor 1 alpha or gamma subunit (TIF1-γ) antibodies (n = 5), and evaluated healthy controls (n = 5) via single-shot liquid chromatography-tandem mass spectrometry (MS) in data-independent acquisition mode, which is superior for comparative quantitative analysis. We identified different protein groups based on MSAs and performed pathway analysis to understand their characteristics. RESULTS: We detected 2413 proteins from serum MS analysis; 508 proteins were commonly altered in MSAs, including many myogenic enzymes and IFN-regulated proteins. Pathway analysis using the top 50 proteins that were upregulated in each MSA group revealed that the type I IFN and proteasome pathways were significantly upregulated in the anti-MDA5 antibody group alone. CONCLUSION: Although JDM serum contains many proteins commonly altered in MSAs, the pathways associated with clinical features of MSAs differ based on protein accumulation. In-depth serum protein profiles associated with MSAs may be useful for developing therapeutic target molecules and biomarkers.

Clinical impact of myositis-specific autoantibodies on long-term prognosis of juvenile idiopathic inflammatory myopathies: multicentre study.

OBJECTIVES: This study aimed to investigate the clinical characteristics, treatment and prognosis of juvenile idiopathic inflammatory myopathies (JIIM) in Japan for each myositis-specific autoantibody (MSA) profile. METHODS: A multicentre, retrospective study was conducted using data of patients with JIIM at nine paediatric rheumatology centres in Japan. Patients with MSA profiles, determined by immunoprecipitation using stored serum from the active stage, were included. RESULTS: MSA were detected in 85 of 96 cases eligible for the analyses. Over 90% of the patients in this study had one of the following three MSA types: anti-melanoma differentiation-associated protein 5 (MDA5) (n = 31), anti-transcriptional intermediary factor 1 alpha and/or gamma subunits (TIF1γ) (n = 25) and anti-nuclear matrix protein 2 (NXP2) (n = 25) antibodies. Gottron papules and periungual capillary abnormalities were the most common signs of every MSA group in the initial phase. The presence of interstitial lung disease (ILD) was the highest risk factor for patients with anti-MDA5 antibodies. Most patients were administered multiple drug therapies: glucocorticoids and MTX were administered to patients with anti-TIF1γ or anti-NXP2 antibodies. Half of the patients with anti-MDA5 antibodies received more than three medications including i.v. CYC, especially patients with ILD. Patients with anti-MDA5 antibodies were more likely to achieve drug-free remission (29 vs 21%) and less likely to relapse (26 vs 44%) than others. CONCLUSION: Anti-MDA5 antibodies are the most common MSA type in Japan, and patients with this antibody are characterized by ILD at onset, multiple medications including i.v. CYC, drug-free remission, and a lower frequency of relapse. New therapeutic strategies are required for other MSA types.

External validation of the EULAR/ACR idiopathic inflammatory myopathies classification criteria with a Japanese paediatric cohort.

OBJECTIVES: To assess the performance of the EULAR/ACR idiopathic inflammatory myopathies (IIMs) classification criteria to classify juvenile IIMs (JIIMs) in an Asian paediatric population. METHODS: Sixty-eight JIIM patients and 49 non-JIIM patients diagnosed at seven major paediatric rheumatology centres in Japan between 2008 and 2015 were enrolled. Retrospective data were collected, and each patient's data form was submitted. The expert group reviewed the forms and re-examined the diagnoses. The EULAR/ACR criteria were then applied and the probability of having JIIM was determined for each case. The sensitivity and specificity of the EULAR/ACR criteria were compared with those of other existing criteria. RESULTS: The sensitivity/specificity of the EULAR/ACR classification criteria were 92.1/100% with muscle biopsy data (n = 38); 86.7/100% without muscle biopsy data (n = 30) and 89.7/100% in our total cohort (n = 68). The sensitivity of Bohan and Peter's criteria and Tanimoto's criteria were 80.9 and 64.7% in our total cohort, respectively. Among 68 physician-diagnosed JIIM patients, seven cases (three JDM and four overlap myositis) were not classified as JIIM because the probability did not reach the cut-off point (55%). The three JDM patients all presented with only one of the three skin manifestations that are listed in the criteria: Gottron's sign. CONCLUSION: Our validation study with Japanese JIIM cases indicates that the EULAR/ACR classification criteria for IIM generally perform better than existing diagnostic criteria for myositis.

Primaquine plus clindamycin as a promising salvage therapy for Pneumocystis jirovecii pneumonia: A retrospective analysis in Japanese patients.

Treatment of intractable Pneumocystis jirovecii pneumonia (PCP) patients with primaquine (PQ) in combination with clindamycin (CLDM) was conducted by the Research Group on Chemotherapy of Tropical Diseases (RG-CTD), as a kind of compassionate use. Primaquine was not nationally licensed at the time but imported by RG-CTD for the use in a clinical research to investigate safety and efficacy in malaria treatment. Eighteen Japanese adult patients thus treated were analyzed. Prior to the treatment with PQ-CLDM, most of the patients had been treated with trimethoprim-sulfamethoxazole first, all of which being followed by pentamidine and/or atovaquone treatment. This combination regimen of PQ-CLDM was effective in 16 (89%) patients and developed adverse events (AEs) in five (28%) patients. AEs included skin lesions, methemoglobinemia, and hepatic dysfunction, though none of them were serious. As a second-line or salvage treatment for PCP, PQ-CLDM appears to be a better option than pentamidine or atovaquone. Currently in Japan, both PQ and CLDM are licensed drugs but neither of them is approved for treatment of PCP. Considering the potentially fatal nature of PCP, approval of PQ-CLDM for treating this illness should be urged.

Clinical practice guidance for juvenile dermatomyositis (JDM) 2018-Update.

Juvenile dermatomyositis is the most common type of juvenile idiopathic inflammatory myopathy mainly affecting the skin and proximal muscles. We have published the Japanese version of 'Clinical practice guidance for juvenile dermatomyositis (JDM) 2018 'consisting of a review of articles in the field and evidence-informed consensus-based experts' opinion on the treatment strategy in collaboration with The Pediatric Rheumatology Association of Japan and The Japan College of Rheumatology under the financial support by 'Research on rare and intractable diseases, Health and Labor Sciences Research Grants'. This article is a digest version of the Japanese guidance.

Evaluation of systemic activity of pediatric primary Sjögren's syndrome by EULAR Sjögren's syndrome disease activity index (ESSDAI).

OBJECTIVES: The purpose of this study is to evaluate systemic disease activity of pediatric Sjögren's syndrome (SS) using European League Against Rheumatism (EULAR) Sjögren's syndrome disease activity index (ESSDAI). METHODS: We retrospectively reviewed medical records of patients with SS who have been diagnosed according to 1999 Japanese diagnostic criteria for SS before 16 years old at KKR Sapporo Medical Center, Hokkaido University Hospital, and affiliated hospitals. We analyzed clinical and laboratory data and calculated ESSDAI at both diagnosis and peak activity. RESULTS: Twenty-five patients (2 boys and 23 girls) were enrolled. Only 4 patients had sicca symptoms at diagnosis. Mean ESSDAI scores at diagnosis and peak activity were 12.68 (2-31) and 15.08 (2-38), respectively. Only 3 patients were inactive (ESSDAI score <5) at diagnosis. Frequently involved domains at diagnosis were the biological (96%) followed by the constitutional (68%), glandular (44%), articular (44%), cutaneous domains (28%), renal (16%), and central nervous system (12%). At peak activity, biological domain (96%) was followed by the constitutional (72%), glandular (60%), articular (44%), cutaneous (28%), central nervous system (20%), and renal domains (16%). CONCLUSION: Pediatric SS is suspected from active systemic manifestations. The items of ESSDAI are useful clues to the diagnosis of pediatric SS.

Myositis-specific autoantibodies in Japanese patients with juvenile idiopathic inflammatory myopathies.

OBJECTIVES: The aim of our study is to clarify the association of myositis-specific autoantibodies (MSAs) with clinical and laboratory features in Japanese patients with juvenile idiopathic inflammatory myopathies (JIIMs). METHODS: We retrospectively analyzed the frequency of MSAs and their association with clinical or laboratory findings in 25 Japanese patients with JIIMs in Hokkaido district. RESULTS: Eighteen of the 25 patients (72%) were positive for MSAs; seven with anti-melanoma differentiation associated gene (MDA) 5 (28%), five with anti-transcriptional intermediary factor (TIF)-1γ (20%), four with anti-MJ/nuclear matrix protein (NXP)-2 (16%), two with anti-Jo-1 (8%), one with anti- HMG-CoA reductase, one with anti-signal recognition peptide (SRP) antibodies (4% each), including co-existence and transition of MSAs in one patient each. Anti-MDA5 antibodies were related to interstitial lung disease (ILD) and arthritis but not to amyopathic juvenile dermatomyositis. Drug-free remission was achieved, once ILD was overcome in this group. Anti-TIF-1γ antibodies were associated with typical rashes and mild myositis. Anti-MJ/NXP2 and anti-SRP antibodies were associated with severe muscle weakness. No patient was complicated with malignancy. CONCLUSION: Anti-MDA5 antibodies are prevalent and closely associated with ILD in our series compared with other countries. There was no apparent difference in clinical features associated with other MSAs among races.

Progression of palindromic rheumatism to juvenile idiopathic arthritis in a Japanese girl carrying heterozygous L110P-E148Q substitutions of MEFV gene.

Palindromic rheumatism (PR), a rare disease in children, is characterized by recurrent arthritis or periarthritis and asymptomatic interval. We report evolution of PR to juvenile idiopathic arthritis in a Japanese girl with heterozygous complex L110P-E148Q allele of MEFV gene. Poor response to colchicine alone suggests that the MEFV substitution could increase the susceptibility to arthritis rather than caused arthritis associated with atypical Familial Mediterranean Fever. Weekly methotrexate is a choice for such cases.

Difficulty in the diagnosis of bone and joint pain associated with pediatric acute leukemia; comparison with juvenile idiopathic arthritis.

OBJECTIVES: Acute leukemia often causes osteoarthralgia. The aim of this study is characterization of leukemia-associated osteoarthralgia in comparison with juvenile idiopathic arthritis (JIA). METHODS: We retrospectively reviewed clinical records of 31 patients with acute leukemia and 13 patients with articular JIA diagnosed between January 2008 and March 2013. Clinical and laboratory findings at the initial examination were compared among the three groups; 10 leukemia with and 21 leukemia without osteoarthralgia and 13 JIA groups. RESULTS: Eleven of the 31 leukemic patients (35%) had osteoarthralgia before the diagnosis of leukemia. Peripheral leukemic cells were initially absent in 10 of the 31 leukemia patients including three with osteoarthralgia. Platelet counts over 300 × 109/L were common in JIA, but not in osteoarthralgia group. Mean serum lactate dehydrogenase levels were higher in both of the leukemia groups than JIA group but often within normal or near-normal levels in the leukemia groups. Magnetic resonance imaging was examined in three leukemic patients and demonstrated osteomyelitis-like bone marrow edema in two and periarticular infiltration similar to synovitis in one patient. Three leukemic patients with osteoarthralgia showed partial and transient responses to antibiotic therapy. CONCLUSIONS: Leukemia-associated osteoarthralgia is often indistinguishable from rheumatic diseases by imaging and laboratory findings and should be confirmed by bone marrow examination.

A heterozygous dominant-negative mutation in the coiled-coil domain of STAT1 is the cause of autosomal-dominant Mendelian susceptibility to mycobacterial diseases.

Heterozygous dominant-negative mutations of STAT1 are responsible for autosomal-dominant Mendelian susceptibility to mycobacterial diseases (AD-MSMD). So far, only 7 mutations have been previously described and are localized to 3 domains: the DNA-binding domain, the SH2 domain, and the tail segment. In this study, we demonstrated the first coiled-coil domain (CCD) mutation of c.749G>C, p.G250A (G250A) in STAT1 as a genetic cause of AD-MSMD in a patient with mycobacterial multiple osteomyelitis. This de novo heterozygous mutation was shown to have a dominant-negative effect on the gamma-activated sequence (GAS) transcriptional activity following IFN-γ stimulation, which could be attributable to the abolished phosphorylation of STAT1 from the wild-type (WT) allele. The three-dimensional structure of STAT1 revealed the G250 residue was located distant from a cluster of residues affected by gain-of-function mutations responsible for chronic mucocutaneous candidiasis.

Two novel gain-of-function mutations of STAT1 responsible for chronic mucocutaneous candidiasis disease: impaired production of IL-17A and IL-22, and the presence of anti-IL-17F autoantibody.

Heterozygous gain-of-function (GOF) mutations of STAT1 are responsible for chronic mucocutaneous candidiasis disease (CMCD), one of the primary immunodeficiency diseases characterized by susceptibility to mucocutaneous Candida infection. To date, 30 aa changes have been reported: 21 in the coiled-coil domain and 9 in the DNA-binding domain. In this study, we report two novel STAT1 GOF mutations of p.K278E in coiled-coil domain and p.G384D in DNA-binding domain in Japanese CMCD patients. Ectopic expression of these STAT1 mutants in HeLa cells was associated with increased phosphorylation of the mutant and the endogenous wild-type STAT1 due to impaired dephosphorylation, indicating heterodimers of the wild-type and mutant STAT1 cause impaired dephosphorylation, as did homodimers of the mutants. Because IL-17A production was not significantly reduced at least in one of the patients following PMA plus ionomycin stimulation, we further studied Th17-associated cytokines IL-17A, IL-17F, and IL-22 in response to more physiologically relevant stimulations. IL-17A and IL-22 production from PBMCs and CD4(+) cells was significantly reduced in four patients with STAT1 GOF mutations, including the previously reported R274Q in response to anti-CD3 plus anti-CD28 Abs or Candida stimulations. In contrast, IL-17F production was comparable to healthy controls in response to anti-CD3 plus anti-CD28 Abs stimulation. These results indicate impaired production of IL-17A and IL-22 rather than IL-17F was associated with the development of CMCD in these patients. Additionally, only the anti-IL-17F autoantibody was detected in sera from 11 of 17 patients with STAT1 GOF mutations, which may be useful as a marker for this disease.

Disease specificity of anti-tryptophan hydroxylase-1 and anti-AIE-75 autoantibodies in APECED and IPEX syndrome.

Autoantibodies to autoimmune enteropathy-related 75 kDa antigen (AIE-75) and villin are disease markers of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome which is characterized by a peripheral tolerance defect. On the other hand, anti-tryptophan hydroxylase-1 (TPH-1) antibodies are detected in autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy (APECED), a central tolerance defect, especially when complicated with gastrointestinal dysfunction. However, to date, anti-AIE-75 and anti-villin antibodies or anti-TPH-1 antibodies have not been tested in APECED or IPEX syndrome, respectively. In the present study, we confirmed the disease specificity of both anti-AIE-75 and anti-TPH-1, although anti-villin antibodies were detected in some patients with APECED. Our observation suggests that immunotolerance to AIE-75 depends on the peripheral mechanism, whereas the tolerance to TPH-1 depends on the central mechanisms.

Outcomes in two Japanese adenosine deaminase-deficiency patients treated by stem cell gene therapy with no cytoreductive conditioning.

OBJECTIVE: We here describe treatment outcomes in two adenosine deaminase (ADA)-deficiency patients (pt) who received stem cell gene therapy (SCGT) with no cytoreductive conditioning. As this protocol has features distinct from those of other clinical trials, its results provide insights into SCGT for ADA deficiency. PATIENTS AND METHODS: Pt 1 was treated at age 4.7 years, whereas pt 2, who had previously received T-cell gene therapy, was treated at age 13 years. Bone marrow CD34(+) cells were harvested after enzyme replacement therapy (ERT) was withdrawn; following transduction of ADA cDNA by the γ-retroviral vector GCsapM-ADA, they were administered intravenously. No cytoreductive conditioning, at present considered critical for therapeutic benefit, was given before cell infusion. Hematological/immunological reconstitution kinetics, levels of systemic detoxification, gene-marking levels, and proviral insertion sites in hematopoietic cells were assessed. RESULTS: Treatment was well tolerated, and no serious adverse events were observed. Engraftment of gene-modified repopulating cells was evidenced by the appearance and maintenance of peripheral lymphocytes expressing functional ADA. Systemic detoxification was moderately achieved, allowing temporary discontinuation of ERT for 6 and 10 years in pt 1 and pt 2, respectively. Recovery of immunity remained partial, with lymphocyte counts in pts 1 and 2, peaked at 408/mm(3) and 1248/mm(3), approximately 2 and 5 years after SCGT. Vector integration site analyses confirmed that hematopoiesis was reconstituted with a limited number of clones, some of which were shown to have myelo-lymphoid potential. CONCLUSIONS: Outcomes in SCGT for ADA-SCID are described in the context of a unique protocol, which used neither ERT nor cytoreductive conditioning. Although proven safe, immune reconstitution was partial and temporary. Our results reiterate the importance of cytoreductive conditioning to ensure greater benefits from SCGT.

Clinical and laboratory features of fatal rapidly progressive interstitial lung disease associated with juvenile dermatomyositis.

OBJECTIVE: Rapidly progressive interstitial lung disease (RP-ILD) is a rare but potentially fatal complication of JDM. The aim of this study was to establish markers for the prediction and early diagnosis of RP-ILD associated with JDM. METHODS: The clinical records of 54 patients with JDM were retrospectively reviewed: 10 had RP-ILD (7 died, 3 survived), 19 had chronic ILD and 24 were without ILD. Routine tests included a high-resolution CT (HRCT) scan of the chest and measurement of serum levels of creatine phosphokinase, ferritin and Krebs von den Lungen-6 (KL-6). Anti-melanoma differentiation-associated gene 5 (MDA5) antibodies and IL-18 levels were measured by ELISA. RESULTS: No differences were found in the ratio of juvenile clinically amyopathic DM between the three groups. Initial chest HRCT scan findings were variable and could not distinguish between RP-ILD and chronic ILD. Anti-MDA5 antibodies were positive in all 8 patients with RP-ILD and 10 of 14 with chronic ILD, but none of the patients without ILD. Serum levels of anti-MDA5 antibody, ferritin, KL-6 and IL-18 were significantly higher in the RP-ILD group than in the chronic ILD and non-ILD groups. Serum levels of IL-18 positively correlated with serum KL-6 (R = 0.66, P < 0.001). CONCLUSION: High serum levels of IL-18, KL-6, ferritin and anti-MDA5 antibodies (e.g. >200 units by ELISA) are associated with RP-ILD. These can be used as an indication for early intensive treatment. Both alveolar macrophages and autoimmunity to MDA5 are possibly involved in the development of RP-ILD associated with JDM.

Fusarium falciforme infection in a patient with chronic granulomatous disease: Unique long-term course of epidural abscess.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease characterized by recurrent life-threatening bacterial and fungal infections with granuloma formation. Species of the genus Fusarium are opportunistic environmental microorganisms that are rarely pathogenic in humans. We report here the first case of X-linked CGD complicated with epidural abscess caused by Fusarium falciforme infection. The abscesses extended along the dura mater for >7 years and finally resulted in fatal meningitis and cervical myelitis. Early intervention with hematopoietic stem cell transplantation should be considered, especially in patients with severe CGD, before the development of serious infectious complication.

Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.

Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of primary immunodeficiency diseases characterized by chronic and recurrent Candida infections of the skin, nails, and oropharynx. Gain-of-function mutations in STAT1 were very recently shown to be responsible for autosomal-dominant or sporadic cases of CMC. The reported mutations have been exclusively localized in the coiled-coil domain, resulting in impaired dephosphorylation of STAT1. However, recent crystallographic analysis and direct mutagenesis experiments indicate that mutations affecting the DNA-binding domain of STAT1 could also lead to persistent phosphorylation of STAT1. To our knowledge, this study shows for the first time that a DNA-binding domain mutation of c.1153C>T in exon 14 (p.T385M) is the genetic cause of sporadic CMC in two unrelated Japanese patients. The underlying mechanisms involve a gain of STAT1 function due to impaired dephosphorylation as observed in the coiled-coil domain mutations.

Orofacial Granulomatosis among Pediatric Patients Well Controlled by Corticosteroid Treatment: A Rare Case Series.

Orofacial granulomatosis (OFG) is a rare disease entity characterized by nonnecrotizing granulomatous inflammation in the oral and maxillofacial regions, typically characterized by recurrent or persistent edema, primarily in the lips and occasionally in the gingiva. OFG is often associated with Crohn's disease and sarcoidosis, and an accurate diagnosis requires systemic examination of patients. Pediatric patients possess unique oral conditions where dental plaque rapidly forms, especially during tooth replacement due to tooth crowding. Moreover, controlling oral hygiene can be challenging, rendering it difficult to distinguish plaque-induced gingivitis from nonplaque-induced gingivitis. We elucidate the reports of pediatric patients who developed OFG in the lips and/or gingiva alone, which was well controlled through corticosteroid treatment. The patients demonstrated recurrent lips and/or gingival swelling with redness, which failed to improve despite oral health care and treatment with antibiotics and/or corticosteroid ointment. Incision biopsy was performed, which demonstrated granulomatous inflammation. Further systemic examination ruled out Crohn's disease and sarcoidosis and confirmed OFG diagnosis. Corticosteroid treatment orally or through gargling was administered to the patients, which provided improvement of symptoms after 1 month. As OFG may be associated with intractable diseases, monitoring the patient regularly is crucial. Pediatric patients with OFG require a collaborative approach with pediatricians and pediatric dentists to manage their oral and overall health.

A de novo dominant-negative variant is associated with OTULIN-related autoinflammatory syndrome.

OTULIN-related autoinflammatory syndrome (ORAS), a severe autoinflammatory disease, is caused by biallelic pathogenic variants of OTULIN, a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here, we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells, which confirms that the patient has ORAS. However, although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN, the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis.

Rapid progression to pulmonary arterial hypertension crisis associated with mixed connective tissue disease in an 11-year-old girl.

Mixed connective tissue disease (MCTD) is rare in pediatric rheumatic diseases. Pulmonary arterial hypertension (PAH) associated with MCTD usually progresses gradually and is difficult to note at the asymptomatic phase. We report a 11-year-old girl with MCTD complicated with rapidly progressive PAH. Although PAH was not detected by echocardiogram or chest CT scan at the initial examination, it became clear in 1 year and suddenly came to cardiac arrest during an invasive procedure. She was successfully treated with extracorporeal assist and both vasodilative and immunosuppressive medication. A combination of echocardiogram and plasma BNP levels could be a useful marker for the follow-up of such cases. PAH could develop early in the course of pediatric MCTD and needs attention to unexpected acute exacerbation, especially under emotional stress.