Survey of diversity awareness and environment among members of the Pediatric Rheumatology Association of Japan.

BACKGROUND: Diversity management has gained traction in Japan. The Pediatric Rheumatology Association of Japan (PRAJ) has an Advisory Committee for Diversity Promotion with a broader focus on promoting diversity. The objectives of this study were to better understand the problems faced by PRAJ members regarding the work environment, childcare and nursing care, and work-life balance. METHODS: A web-based questionnaire was administered to members of the PRAJ and 79 responses were evaluated. RESULTS: Of the respondents, 73% were male and 27% were female. A total of 14% worked for more than 12 h on weekdays, and 22% worked for more than 60 h per week and 38% had fewer than 4 days off per month. Regarding childcare, 54% of the respondents were raising preschool children and 83% had taken parental leave for less than 1 year. A total of 17% of participants had family members in need of care. For both childcare and caregiving, the burden was greater for women. Only 18% of the respondents reported a well-balanced work-life balance, and the most common reasons for a lack of balance were not having enough time, heavy workload, and heavy housework load. CONCLUSIONS: The working hours of the respondents were long, and female members had a greater burden of childcare and caregiving, which was considered a barrier to the career development of women. In the future, there will be a need to promote a sense of equality in diverse human resources, develop support for family life, and shorten working hours.

Kinetics of Enzymatic Reactions at the Solid/Liquid Interface in Nanofluidic Channels.

Nanostructures can realize highly efficient reactions due to their structural advantages. However, the mechanism of accelerating enzyme reactions in a nanospace is still unknown from a kinetic perspective because it is difficult to control a well-defined nanospace, enzyme density, and reaction time. Here, we investigated kinetic parameters of an immobilized enzyme in micro- and nanochannels using nanofabrication, partial enzyme patterning, fluidic control, and a high sensitivity detection system. Devices with channel depths of 300 nm, 4.4 µm, and 13.6 µm were fabricated. Kinetic parameters were determined by the Michaelis-Menten model. Compared to the bulk reaction, all kcats for immobilized enzyme reactors were decreased, although the kcats were approximately the same for the immobilized enzyme reactors of different depths. An ultrafast enzyme reaction could overcome the drawback due to immobilization by an increase of the apparent [E]0 due to the decreased channel depth.

The Use of a Combination of a Sugar and Surfactant to Stabilize Au Nanoparticle Dispersion against Aggregation during Freeze-Drying.

Drying a suspension of nanoparticles typically results in the irreversible aggregation of nanoparticles; however, solutions that contain unstable ingredients are often converted into dried powders to prolong their shelf lives. In this study, the use of a combination of a surface-active agent and sugar was investigated with regard to avoiding the aggregation of nanoparticles during drying. Suspensions of Au nanoparticles (∼60 nm diameter, AuNPs) were freeze-dried in the presence of different combinations of various sugars with a surfactant. Sucrose monopalmitate (SEC16) was mainly used as the surfactant, based on a comparison of antiaggregation effects conferred by various surfactants. The freeze-dried AuNP suspension was then reconstituted, and the avoidance of AuNP aggregation was then examined. The results demonstrated that the use of a combination of a small amount of SEC16 and sugar resulted in a greater redispersibility of AuNPs after freeze-drying than when the individual components were used. Repetition tests of freeze-drying and reconstitution were conducted. The sucrose/SEC16 mixture was freeze-dried on an electroless-plated Au film and then analyzed by infrared spectroscopy. Strong interactions between SEC16 and the Au surface were detected, and these interactions appear to play a crucial role in the antiaggregation of AuNPs during freeze-drying.

Picoliter enzyme reactor on a nanofluidic device exceeding the bulk reaction rate.

Single-cell analyses have recently become important to understand cell heterogeneity, the mechanism of cell function, and diseases. In contrast to single-cell analyses that target nucleic acids, single-cell protein analyses still pose challenges. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale with femtoliter-picoliter volumes which are smaller than the volume of a single cell exploring ultimate analytical performances (e.g. single-cell target proteomics). However, single-cell shotgun proteomics, which is used to analyze even unknown proteins, is still challenging because there is no digestion column with picoliter volume. The issues were long reaction time (overnight) and much larger reaction volume (microliter) in the conventional bulk method. In this study, an ultra-fast picoliter enzyme reactor using a nanochannel was developed. A device with a channel depth of 300 nm and a volume of 32.4 pL was fabricated. To prevent the self-digestion of trypsin (enzyme), the picoliter enzyme reactor was prepared by immobilizing trypsinogen which was activated to trypsin by enterokinase. The enzyme density obtained by the trypsinogen immobilization process was 2.5 times higher than that obtained by the conventional trypsin immobilization process. Furthermore, the apparent enzyme concentration was 36 times higher due to an extremely high surface-to-volume ratio of the nanochannel, compared to the limit concentration in the bulk. Finally, the enzyme reaction in the picoliter enzyme reactor was accelerated 25 times compared to that in the bulk. Using the picoliter enzyme reactor, protein solution with picoliter volume will be digested without self-digestion and artificial modification, which will greatly contribute to single-cell shotgun proteomics.

Surfactant-Free Solid Dispersions of Hydrophobic Drugs in an Amorphous Sugar Matrix Dried from an Organic Solvent.

The technique for homogeneously dispersing hydrophobic drugs in a water-soluble solid matrix (solid dispersion) is a subject that has been extensively investigated in the pharmaceutical industry. Herein, a novel technique for dispersing a solid, without the need to use a surfactant, is reported. A freeze-dried amorphous sugar sample was dissolved in an organic solvent, which contained a soluble model hydrophobic component. The suspension of the sugar and the model hydrophobic component was vacuum foam dried to give a solid powder. Four types of sugars and methanol were used as representative sugars and the organic medium. Four model drugs (indomethacin, ibuprofen, gliclazide, and nifedipine) were employed. Differential scanning calorimetry analyses indicated that the sugar and model drug (100:1) did not undergo segregation during the drying process. The dissolution of the hydrophobic drugs in water from the solid dispersion was then evaluated, and the results indicated that the Cmax and AUC0-60 min of the hydrophobic drug in water were increased when the surfactant-free solid dispersion was used. Palatinose and/or α-maltose were superior to the other tested carbohydrates in increasing Cmax and AUC0-60 min for all tested model drugs, and the model drug with a lower water solubility tended to exhibit a greater extent of over-dissolution.

Characteristics of proteinaceous additives in stabilizing enzymes during freeze-thawing and -drying.

Protein-stabilizing characteristics of sixteen proteins during freeze-thawing and freeze-drying were investigated. Five enzymes, each with different instabilities against freezing and dehydration, were employed as the protein to be stabilized. Proteinaceous additives generally resulted in greater enzyme stabilization during freeze-thawing than sugars while the degree of stabilization for basic lysozyme and protamine were inferior to that of neutral and acidic proteins. Freeze-drying-induced inactivation of enzyme was also reduced by the presence of a proteinaceous additive, the extent of which was lower than that for a sugar. In both freeze thawing and freeze drying, the enzymes stabilization by the proteinaceous additive increased with increasing additive concentration. The enhancement of enzyme inactivation caused by pH change was also reduced in the presence of proteinaceous additives. The combined use of a sugar such as sucrose and dextran tended to increase the stabilizing effect of the proteinaceous additive.

Prediction of long-term remission of oligo/polyarticular juvenile idiopathic arthritis with S100A12 and vascular endothelial growth factor.

OBJECTIVES: This study aimed to evaluate the usefulness of S100A12 and vascular endothelial growth factor (VEGF) for predicting the stability of remission for discontinuing methotrexate (MTX) and/or biological agents in Japanese patients with oligo/polyarticular juvenile idiopathic arthritis (JIA). METHODS: Forty-four patients with oligo/polyarticular JIA who received MTX with or without biological agents were enrolled. Serum concentration of both S100A12 and VEGF were simultaneously evaluated by ELISA in active and in remission phase determined by activity markers including DAS-28. RESULTS: S100A12 and VEGF were correlated with DAS-28. Of the 22 patients with oligo/polyarticular JIA in clinical remission, 13 patients with low S100A12 and VEGF concentrations could discontinue treatment without relapse over 2 years. However, nine patients without low S100A12 and VEGF concentrations relapsed afterwards, even though they had been in clinical remission. The cut-off levels of S100A12 and VEGF for division into two groups of the maintenance remission and relapse groups were 177 ng/ml and 158 pg/ml, respectively. CONCLUSIONS: S100A12 and VEGF are useful markers for assessing disease activity of oligo/polyarticular JIA in remission phase. These markers should be kept low when clinicians consider tapering or discontinuing treatments in oligo/polyarticular JIA patients.

The safety and effectiveness of HBV vaccination in patients with juvenile idiopathic arthritis controlled by treatment.

OBJECTIVES: To evaluate the safety and effectiveness of hepatitis B virus (HBV) vaccination in patients with juvenile idiopathic arthritis (JIA) controlled by treatment. METHODS: Among 49 patients with juvenile idiopathic arthritis (JIA) at the outpatient clinic of Kagoshima University Hospital, we enrolled 25 who were controlled by treatment. All children were unimmunized and were vaccinated against HBV according to the schedule. Their responses to the vaccine and vaccine adverse events were examined during their visits. RESULTS: Nineteen of the 25 patients with JIA controlled by treatment developed effective antibody responses (76%). All eight patients with JIA below 10 years of age achieved seroconversion. The seroconversion was not influenced by biologics. Five adverse events were observed (6.7%). The rate of all adverse events did not surpass that of a previous report, and all adverse events were immediately resolved. None of the patients with JIA experienced a flare-up or clinical deterioration related to the vaccination. CONCLUSIONS: HBV vaccination is safe and effective. Pediatric rheumatologists should consider HBV vaccination for unimmunized patients with JIA, because the response to HBV vaccine might be influenced by age, and children have a higher risk for potential HBV infection than adults.

Disease activity score in 28 joints at 3 months after the initiation of biologic agent can be a predictive target for switching to the second biologic agent in patients with polyarticular juvenile idiopathic arthritis.

OBJECTIVE: To clarify polyarticular juvenile idiopathic arthritis (pJIA) patients who failed to maintain prolonged remission with the first biologic agent. METHODS: Fourteen pJIA patients were observed for 47.5 months (median) after initiating the first biologic agent. RESULTS: Eight maintained sustained clinical remission (median 47 months) with the first biologic agents, while the six switched to the second one due to lack of efficacy, thereafter. Receiver operating characteristic (ROC) analysis revealed that disease activity score in 28 joints (DAS28) of 2.37 at 3 months could distinguish between the two patient groups (p = 0.001). CONCLUSION: pJIA patients with DAS28 >2.37 at 3 months of the initial biologic therapy may be considered to switch to the second biologics.

Polymorphobacter multimanifer gen. nov., sp. nov., a polymorphic bacterium isolated from Antarctic white rock.

A Gram-stain-negative, non-spore-forming, aerobic, oligotrophic bacterium (strain 262-7(T)) was isolated from a crack of white rock collected in the Skallen region of Antarctica. Strain 262-7(T) grew at temperatures between -4 and 30 °C, with optimal growth at 25 °C. The pH range for growth was between pH 6.0 and 9.0, with optimal growth at approximately pH 7.0. The NaCl concentration range allowing growth was between 0.0 and 1.0%, with an optimum of 0.5%. Strain 262-7(T) showed an unprecedented range of morphological diversity in response to growth conditions. Cells grown in liquid medium were circular or ovoid with smooth surfaces in the lag phase. In the exponential phase, ovoid cells with short projections were observed. Cells in the stationary phase possessed long tentacle-like projections intertwined intricately. By contrast, cells grown on agar plate medium or in liquid media containing organic compounds at low concentration exhibited short- and long-rod-shaped morphology. These projections and morphological variations clearly differ from those of previously described bacteria. Ubiquinone 10 was the major respiratory quinone. The major fatty acids were C(17 : 1)ω6c (28.2%), C(16 : 1)ω7c (22.6%), C(18 : 1)ω7c (12.9%) and C(15 : 0) 2-OH (12.3%). The G+C content of genomic DNA was 68.0 mol%. Carotenoids were detected from the cells. Comparative analyses of 16S rRNA gene sequences indicated that strain 262-7(T) belongs to the family Sphingomonadaceae, and that 262-7(T) should be distinguished from known genera in the family Sphingomonadaceae. According to the phylogenetic position, physiological characteristics and unique morphology variations, strain 262-7(T) should be classified as a representative of a novel genus of the family Sphingomonadaceae. Here, a novel genus and species with the name Polymorphobacter multimanifer gen. nov., sp. nov. is proposed (type strain 262-7(T) = JCM 18140(T) = ATCC BAA-2413(T)). The novel species was named after its morphological diversity and formation of unique projections.

The discoidin domain of Bacillus circulans ß-galactosidase plays an essential role in repressing galactooligosaccharide production.

The recently cloned ß-galactosidase from Bacillus circulans ATCC 31382, designated BgaD, contains a multiple domain architecture including a F5/8 type C domain or a discoidin (DS) domain in the C-terminal peptide region. Here we report that the DS domain plays an essential role in repressing the production of galactooligosaccharides (GOSs). We prepared deletion mutants and point-mutated forms of rBgaD-A (deletion of the BgaD signal peptide) to compare their reaction behaviors. The yields of GOSs for all of the point-mutated forms as well as the deletion mutants of rBgaD-As increased as compared to rBgaD-A. In particular, W1540A mutant BgaD-A (rBgaD-A_W1540A) produced much more GOSs than rBgaD-A. Surface plasmon resonance experiments indicated that both the wild-type and the W1540A mutant DS domains showed high affinity for galactosyllactose. rBgaD-A, which has a wild-type DS domain, showed high hydrolytic activity toward galactosyllactose, while the hydrolytic activities of rBgaD-D, without a DS domain, and rBgaD-A_W1540A, with a mutant DS domain were extremely low. The findings obtained in this study indicate that the wild-type DS domain of rBgaD-A has a function that aids galactosyllactose molecules to be properly oriented within the active site, so that they can be hydrolyzed efficiently to produce galactose/glucose by inhibiting the accumulation of GOSs.

Safety and efficacy of tocilizumab, an anti-IL-6-receptor monoclonal antibody, in patients with polyarticular-course juvenile idiopathic arthritis.

We evaluated the safety and efficacy of tocilizumab in polyarticular-course juvenile idiopathic arthritis (pJIA) with polyarticular or oligoarticular onset. Patients received 8 mg/kg tocilizumab every 4 weeks in the open-label studies: initial study (to week 12) and then an extension study (at least 48 weeks). Nineteen patients intractable to conventional methotrexate therapy were enrolled. Seventeen patients had polyarticular-onset pJIA; two had oligoarticular-onset pJIA. Mean age was 11.6 years; mean disease duration 5.3 years. American College of Rheumatology Pediatric (ACR Pedi) 30, 50, 70, and 90 response rates, respectively, were 94.7%, 94.7%, 57.9%, and 10.5% at week 12, and 100%, 94.1%, 88.2%, and 64.7% at week 48. Mean disease activity score (DAS28) remained below the remission level (2.6) from week 24. Administration was discontinued in two patients during the extension study because the ACR Pedi 50 response was judged insufficient (one patient) and antitocilizumab antibodies developed (one patient). Adverse events were generally mild, and the four serious adverse events resolved spontaneously or with treatment. In conclusion, tocilizumab showed early and sustained efficacy and tolerability for treating intractable pJIA, which suggests that it is a promising new treatment for this disease.

Safety and efficacy of long-term etanercept in the treatment of methotrexate-refractory polyarticular-course juvenile idiopathic arthritis in Japan.

OBJECTIVES: Previous short-term trials found etanercept (0.2 or 0.4 mg/kg) to be effective and well tolerated in Japanese children with juvenile idiopathic arthritis (JIA) who were intolerant/resistant to methotrexate. The aim of this study was to evaluate the long-term safety and efficacy of etanercept in Japanese children with JIA. METHODS: Patients (4-19 years) who received etanercept in one of three short-term studies continued onto this long-term open-label study. RESULTS: Of the 32 patients enrolled, 18 (56.3%) completed 192 weeks of the study and 14 (43.8%) were discontinued; 7 (21.9%) for patient refusal, 2 (6.3%) for adverse events (AEs), and 5 (15.6%) for lack of efficacy. All patients reported AEs; 31 (96.9%) reported infections and 6 (18.8%) reported serious AEs. Main efficacy assessments included change from baseline in the American College of Rheumatology Pediatric core components, including mean improvements from baseline in the physician global assessment (90.7%), patient/guardian global assessments (54.1%), Childhood Health Assessment Questionnaire (84.6%), and median improvements in C-reactive protein levels (92.7%). No unexpected safety results were reported, and early efficacy responses were sustained in the long term. CONCLUSIONS: This study provides further evidence that etanercept is an effective therapeutic option for Japanese children with polyarticular-course JIA.

Causes of the production of multiple forms of ß-galactosidase by Bacillus circulans.

The presence of multiple types of ß-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four ß-galactosidases, ß-Gal-A, ß-Gal-B, ß-Gal-C, and ß-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. ß-Gal-B, ß-Gal-C, and ß-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while ß-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for ß-Gal-A, showed biphasic behavior. ß-Gal-A was truncated to yield multiple ß-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of ß-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for ß-Gal-D.

Cloning and expression of a ß-galactosidase gene of Bacillus circulans.

A gene of ß-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(®). Using the cloned gene, recombinant ß-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.

Etanercept in the treatment of disease-modifying anti-rheumatic drug (DMARD)-refractory polyarticular course juvenile idiopathic arthritis: experience from Japanese clinical trials.

Efficacy, safety, and pharmacokinetics results from 4 studies-3 open-label (OL) and 1 randomized double-blind (DB)-have provided data for approval of etanercept for treatment of disease-modifying anti-rheumatic drug (DMARD)-refractory juvenile idiopathic arthritis (JIA) in Japan. Results from the 3 shorter-term (2 OL and 1 DB) studies are reported here. Subjects (4-17 years) enrolled in the OL studies had active JIA, i.e. ≥5 swollen joints and ≥3 joints with limitation of motion and pain or tenderness. Subjects enrolled in the primary OL study received etanercept 0.4 mg/kg subcutaneously twice weekly; in the lower-dose OL study subjects received etanercept 0.2 mg/kg. Subjects in the primary OL study who completed ≥48 weeks could continue into a 12-week DB dose-down extension study in which subjects received etanercept 0.4 or 0.2 mg/kg twice weekly. The primary endpoint in all 3 studies, i.e. 30% improvement in the American College of Rheumatology criteria for JIA (ACR Pedi 30) at 12 weeks, was achieved by ≥80% of subjects by week 2 and sustained to week 12. Common adverse events reported were injection site reactions, nasopharyngitis, and gastroenteritis. These results provide further evidence that etanercept is effective therapy for DMARD-refractory polyarticular JIA patients.

A case of cryopyrin-associated periodic fever syndrome during canakinumab administration complicated by inflammatory bowel disease.

Cryopyrin-associated periodic fever syndrome (CAPS) is a highly debilitating disorder, which is characterized by unregulated interleukin-1ß production driven by autosomal dominantly inherited mutations in the NLRP3 gene. Patients with CAPS often present with early-onset episodes of fever and rash. These patients also present with variable systemic signs and symptoms, such as arthritis, sensorineural hearing loss, chronic aseptic meningitis, and skeletal abnormalities, but minimal gastrointestinal symptoms. Recently, effective therapies for CAPS targeted against interleukin-1 have become available. We report a case of a young Japanese woman with CAPS who developed inflammatory bowel disease during canakinumab therapy. The patient had colostomy after intestinal perforation and changed canakinumab to infliximab. To the best of our knowledge, this is the first report of a case of inflammatory bowel disease secondary to CAPS complicated by gastrointestinal symptoms and arthritis which canakinumab could not control. Patients with CAPS who have symptoms that cannot be controlled by canakinumab should be considered for possible co-morbidities.

Immobilization of surface non-affinitive protein onto a metal surface by an external electric field.

We investigated an alternate technique to coat the surface with a protein having no surface affinity, without the use of any exotic chemical agents. An external electric field was utilized to prepare the protein coating on a metal substrate. Stainless steel (St) substrate and lysozyme (LSZ) were used as the surface to be coated and the model non-adsorptive protein, respectively. Dynamics of the adsorption of LSZ on the St surface in the presence and absence of an external electric potential (EEP) were monitored by in-situ ellipsometry. Applying negative surface potential (-0.4 V vs Ag/AgCl) forced the adsorption of LSZ onto the St surface where LSZ did not adsorb without applying any EEP. The repetition of the EEP-application and -cut-off indicated the controllability of the LSZ coating amount depending on the total duration of the EEP-application. The coated LSZ largely remained bound to the surface even by the cut-off of the external electric field, the ratio of which to the detached amount was roughly constant (approximately 7:3). Furthermore, the LSZ coated surface on the St substrate was found to be reversibly switched between being affinitive and non-affinitive to a typical model protein adsorbate (bovine serum albumin) by the EEP-application and cut-off.

Purification, characterization, molecular cloning, and expression of a new aminoacylase from Streptomyces mobaraensis that can hydrolyze N-(middle/long)-chain-fatty-acyl-L-amino acids as well as N-short-chain-acyl-L-amino acids.

We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees Celsius (at pH 7.5).

S100A12 and vascular endothelial growth factor can differentiate Blau syndrome and familial Mediterranean fever from systemic juvenile idiopathic arthritis.

OBJECTIVES: Systemic juvenile idiopathic arthritis (sJIA) has recently become regarded as one of the autoinflammatory syndromes (AIS). However, other AIS, such as familial Mediterranean fever (FMF) and Blau syndrome, have been initially misdiagnosed as sJIA because of the clinical similarities. Making the correct diagnosis in the early stage of these AIS is desirable. Therefore, we evaluated serum S100A12 and vascular endothelial growth factor (VEGF) levels to determine if they could be biomarkers for differentiating these AIS. METHOD: Serum S100A12 and VEGF levels were examined in patients with Blau syndrome (n = 4), FMF (n = 4), and sJIA (n = 11) in the active and inactive phases. RESULTS: In the active phase, S100A12 levels were significantly higher in patients with sJIA and FMF compared with those with Blau syndrome (p < 0.001). VEGF levels of patients with sJIA were significantly higher than those of patients with others (p = 0.001). In the inactive phase, there was no significant difference in VEGF levels. However, colchicine-resistant patients or patients without treatment with FMF showed high levels of S100A12 compared with others. CONCLUSIONS: Measuring both serum S100A12 and VEGF levels may be useful for differentiating patients with Blau syndrome and FMF from those with sJIA at the early stage.