Establishment of a lethal mouse model of emerging tick-borne orthonairovirus infections.

Emerging and reemerging tick-borne virus infections caused by orthonairoviruses (family Nairoviridae), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129 mice) both for a better understanding of the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel orthonairovirus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.

Lipidomic Analysis Reveals Differences in the Extent of Remyelination in the Brain and Spinal Cord.

During demyelination, lipid-rich myelin debris is released in the central nervous system (CNS) and must be phagocytosed and processed before new myelin can form. Although myelin comprises over 70% lipids, relatively little is known about how the CNS lipidome changes during demyelination and remyelination. In this study, we obtained a longitudinal lipidomic profile of the brain, spinal cord, and serum using a genetic mouse model of demyelination, known as Plp1-iCKO-Myrf. The mass spectrometry data is available at the Metabolomics Workbench, where it has been assigned Study ID ST002958. This model has distinct phases of demyelination and remyelination over the course of 24 weeks, in which loss of motor function peaks during demyelination. Using principal component analysis (PCA) and volcano plots, we have demonstrated that the brain and spinal cord have different remyelination capabilities and that this is reflected in different lipidomic profiles over time. We observed that plasmalogens (ether-linked phosphatidylserine and ether-linked phosphatidylcholine) were elevated specifically during the early stages of active demyelination. In addition, we identified lipids in the brain that were altered when mice were treated with a remyelinating drug, which may be CNS biomarkers of remyelination. The results of this study provide new insights into how the lipidome changes in response to demyelination, which will enable future studies to elucidate mechanisms of lipid regulation during demyelination and remyelination.

Development of flavivirus subviral particles with low cross-reactivity by mutations of a distinct antigenic domain.

The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. KEY POINTS: • The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. • Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. • Inoculation of mutated SVPs induces antibodies with low cross-reactivity.

Identification of the Acidification Mechanism of the Optimal pH for RNase He1.

Ribonuclease (RNase) He1 is a small ribonuclease belonging to the RNase T1 family. Most of the RNase T1 family members are active at neutral pH, except for RNase Ms, U2, and He1, which function at an acidic pH. We crystallized and analyzed the structure of RNase He1 and elucidated how the acidic amino residues of the α1ß3- (He1:26-33) and ß67-loops (He1:87-95) affect their optimal pH. In He1, Ms, and U2, the hydrogen bonding network formed by the acidic amino acids in the ß67-loop suggested that the differences in the acidification mechanism of the optimum pH specified the function of these RNases. We found that the amino acid sequence of the ß67-loop was not conserved and contributed to acidification of the optimum pH in different ways. Mutations in the acidic residues in He1 promoted anti-tumor growth activity, which clarified the role of these acidic amino residues in the binding pocket. These findings will enable the identification of additional targets for modifying pH-mediated enzymatic activities.

Quantitative evaluation of skin barrier function using water evaporation time related to transepidermal water loss.

BACKGROUND: Transepidermal water loss (TEWL) is often used as an index for skin barrier function. The skin barrier tester, SBT-100 (Rousette Strategy Inc), measures the TEWL, water evaporation time, and time constant by contacting the skin and diffusing water into the closing measurement chamber. However, the relationship between the TEWL and time constant has not been sufficiently investigated. This study involved analyzing the underlying measurement principle and obtaining data through two experiments. MATERIALS AND METHODS: The TEWL and time constant were measured using SBT-100. Experiment 1 produced a simple simulation model for continuous water evaporation from the skin using a moisture-permeable film. In experiment 2, four skin sites of 43 healthy volunteers were examined from May to September 2018. RESULTS: In experiment 1, the TEWL increased and time constant decreased, following an increase in humidity in the external environment. Both parameters demonstrated significant negative correlation (drying: ρ = -0.832, p < 0.001). For the 43 healthy volunteers who participated in experiment 2, their TEWL increased and time constant decreased in summer. For all skin measurement sites, both data demonstrated significant negative correlation (forehead: ρ = -0.909, p < 0.001; back of the left hand: ρ = -0.829, p < 0.001; left lateral elbow: ρ = -0.896, p < 0.001; left lateral malleolus: ρ = -0.865, p < 0.001). CONCLUSION: Results indicated that the time constant is significantly correlated with TEWL. Furthermore, the time constant can be used as a parameter for evaluating skin barrier function.

Outcome of rituximab treatment in Japanese patients with ANCA-associated vasculitis in daily clinical practice: A two-centre study in Fukushima, Japan.

OBJECTIVES: Rituximab (RTX) efficacy for anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) has been reported in large randomized studies; however, the efficacy of RTX in Japanese AAV patients, especially the elderly, is not well known. We aimed to determine the clinical efficacy of RTX in Japanese AAV patients including elderly patients. METHODS: This study included 78 AAV patients newly diagnosed with AAV and treated in Fukushima Medical University Hospital or Ohta-Nishinouchi Hospital from April 2004 to September 2019. Clinical records were retrospectively reviewed, and clinical efficacy and outcome (1-year survival) between the RTX treatment group (23 cases) and the conventional therapy group (immunosuppressive therapy other than RTX, 55 cases) were compared. We also analysed the clinical efficacy and outcome in elderly-onset (>75 years) AAV patients. RESULTS: The RTX group showed similar efficacy and 1-year survival compared to the conventional therapy group. Conversely, after 6 months of treatment, prednisolone doses significantly decreased in the RTX group compared to the conventional therapy group (p < 0.01). In the elderly-onset AAV patients, clinical efficacy and outcome were not significantly different. CONCLUSIONS: RTX was effective in Japanese AAV patients and may be useful for prompt tapering of prednisolone doses, even in elderly-onset AAV patients.

A Human Endogenous Bornavirus-Like Nucleoprotein Encodes a Mitochondrial Protein Associated with Cell Viability.

Endogenous retroviruses (ERVs) are sequences in animal genomes that originated from ancient retrovirus infections; they provide genetic novelty in hosts by being coopted as functional genes or elements during evolution. Recently, we demonstrated that endogenous elements from not only from retroviruses but also nonretroviral RNA viruses are a possible source of functional genes in host animals. The remnants of ancient bornavirus infections, called endogenous bornavirus-like elements (EBLs), are present in the genomes of a wide variety of vertebrate species, and some express functional products in host cells. Previous studies have predicted that the human EBL locus derived from bornavirus nucleoprotein, termed hsEBLN-2, expresses mRNA encoding a protein, suggesting that hsEBLN-2 has acquired a cellular function during evolution. However, the detailed function of the hsEBLN-2-derived product remains to be elucidated. In this study, we show that the hsEBLN-2-derived protein E2 acts as a mitochondrial protein that interacts with mitochondrial host factors associated with apoptosis, such as HAX-1. We also demonstrate that knockdown of hsEBLN-2-derived RNA increased the levels of PARP and caspase-3 cleavage and markedly decreased cell viability. In contrast, overexpression of E2 enhanced cell viability, as well as the intracellular stability of HAX-1, under stress conditions. Our results suggest that hsEBLN-2 has been coopted as a host gene, the product of which is involved in cell viability by interacting with mitochondrial proteins. IMPORTANCE Our genomes contain molecular fossils of ancient viruses, called endogenous virus elements (EVEs). Mounting evidence suggests that EVEs derived from nonretroviral RNA viruses have acquired functions in host cells during evolution. Previous studies have revealed that a locus encoding a bornavirus-derived EVE, hsEBLN-2, which was generated approximately 43 million years ago in a human ancestor, may be linked to the development of some tumors. However, the function of hsEBLN-2 has not been determined. In this study, we found that the E2 protein, an expression product of hsEBLN-2, interacts with apoptosis-related host proteins as a mitochondrial protein and affects cell viability. This study suggests that nonretroviral RNA viral EVEs have been coopted by hosts with more diverse functions than previously thought, showing a pivotal role for RNA virus infection in evolution.

Isolation and whole-genome sequencing of a novel aviadenovirus from owls in Japan.

Adenoviruses have been reported to infect a variety of birds. Here, we isolated a novel adenovirus from the liver of a dead owl chick (Bengal eagle owl; Bubo bengalensis) at a raptor-breeding facility in Japan and determined the complete genome sequence of the virus. We performed necropsies on the dead owl chicks and found that they had enlarged livers, pericardial edema, and focal necrosis of the liver tissue. Transmission electron microscopy of the liver tissue revealed a virus-like structure, appearing as paracrystalline arrays in the nucleus, and immunohistochemical staining with anti-adenovirus antibodies showed positive reactions in hepatocytes and other cells. Attempts to isolate the virus from homogenized liver tissue of a dead owl chick showed a cytopathic effect on chicken-derived cultured cells after multiple blind passages. Further, we determined the complete genome sequence of this virus and performed phylogenetic analysis, revealing that this adenovirus belongs to the genus Aviadenovirus, forming a cluster with fowl and turkey aviadenoviruses. The amino acid sequence divergence between the DNA polymerase of this virus and its closest known adenovirus relative is approximately 29%, implying that this virus can be assigned to a new species in the genus Aviadenovirus. Based on our data, this novel owl adenovirus is a likely cause of fatal infections in owls, which may threaten wild and captive owl populations. Further, this virus is unique among raptor adenoviruses in that it infects chicken-derived cultured cells, raising the importance of further investigations to evaluate interspecies transmission of this virus.

Effect of tongue-hold swallow on posterior pharyngeal wall using dynamic area detector computed tomography.

PURPOSE: The purpose of this study was to elucidate the effects of the tongue-hold swallow (THS) on the pharyngeal wall by quantifying posterior pharyngeal wall (PPW) anterior bulge during the THS. In addition, the effect of tongue protrusion length on the extent of pharyngeal wall anterior bulge was analysed. METHODS: Thirteen healthy subjects (6 males and 7 females, 23-43 years) underwent 320-row area detector CT during saliva swallow (SS) and THS at two tongue protrusion lengths (THS1 protrude the tongue as much as 1/3 of premeasured maximum tongue protrusion length (MTP-L) and THS2 protrude the tongue as much as 2/3 of MTP-L). To acquire images of the pharynx at rest, single-phase volume scanning was performed three times during usual breathing with no tongue protrusion (rest), protrusion of the tongue at 1/3 of MTP-L (rTHS1) and protrusion of the tongue at 2/3 of MTP-L (rTHS2). Length from cervical spine to PPW (PPW-AP) and the volume of pharyngeal cavity was measured and was compared between rest, rTHS1 and rTHS2 and between SS, THS1 and THS2. Correlation between MTP-L and PPW-AP was calculated in three conditions, SS, THS1 and THS2. RESULTS: PPW-AP at rest, rTHS1 and rTHS2 was 2.9 ± 0.6 mm, 3.0 ± 0.5 mm and 3.0 ± 0.5 mm, respectively, showing no significant differences across swallows. PPW-AP at the maximum pharyngeal constriction was 8.1 ± 2.0 mm, 9.1 ± 2.4 mm and 8.7 ± 2.0 mm in SS, THS1 and THS2, respectively. Compared to SS, PPW-AP in THS1 was significantly larger (p = 0.04) and PPW-AP in THS2 was not significantly different (p = 0.09). Pharyngeal volume at rest, rTHS1 and rTHS2 was 16.4 ± 5.2 mm3 , 18.4 ± 4.5 mm3 and 21.3 ± 6.2 mm3 , respectively. It was significantly larger during rTHS2 compared with rest or rTHS1 (rTHS2-rest p = 0.007, rTHS2-rTHS1 p = 0.007). Pharyngeal volume was completely obliterated (zero volume) at maximum pharyngeal contraction in all except one subject. There was no correlation between MTP-L and PPW-AP in any of the three conditions (SS, THS1 and THS2). DISCUSSION: This study demonstrated that the expanded pharyngeal cavity due to the tongue protrusion was completely obliterated by the increase in anterior motion of pharyngeal wall during THS. It also became clear that the degree of tongue protrusion did not linearly correlate with the movement of PPW during THS. There was no relationship between PPW motion and the MTP-L, suggesting that the effect of tongue protrusion is better determined in each subject by analysing the motion of PPW using imaging tools.

Clinical characteristics and treatment of 50 cases of Blau syndrome in Japan confirmed by genetic analysis of the NOD2 mutation.

OBJECTIVES: To collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis. METHODS: Fifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians. RESULTS: The study population comprised 26 males and 24 females aged 0-61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment. CONCLUSIONS: In patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

IgG4-Related Disease Complicated by Brain Parenchymal Lesions Successfully Treated with Corticosteroid Therapy: A Case Report.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is distinguished by the infiltration of IgG4-positive plasma cells in a variety of tissues and organs including the pancreas, salivary glands, retroperitoneal lesions, kidney, and lymph nodes with elevated serum IgG4 levels. Even so, central nervous system (CNS) lesions such as brain parenchymal lesions associated with IgG4-RD are scarce. So far, only six cases of IgG4-RD in relation with brain parenchymal lesions have been described, with its characteristics still being not clear. Here we have detailed a case of IgG4-RD with brain parenchymal lesions and reviewed previously-reported cases of IgG4-RD with brain parenchymal lesions. A 62-year-old Japanese male suffering from lung silicosis was admitted to our hospital for abdominal discomfort and altered consciousness. He has shown no major neurologic abnormalities except for drowsiness, urinary retention, and fecal incontinence. Brain magnetic resonance imaging has shown scattered hyperintense signals in the brain parenchyma. The serum IgG4 levels were elevated and systemic lymph nodes were enlarged. Biopsy from inguinal lymph nodes has shown massive infiltration of IgG4-positive plasma cells: the ratio of IgG4-positive/IgG-positive plasma cells was nearly 100%. Based on clinical courses, images, laboratory data, and pathological findings, a diagnosis of IgG4-RD that was complicated by brain parenchymal lesions and sacral nerve disturbance was confirmed. The patient was then given methylprednisolone pulse therapy (1g for 3 days) succeeding oral prednisolone (1 mg per body weight). The clinical and radiological improvements together with steroid therapy proposed IgG4-RD to be the cause of the lesions.

Crystal Structure of Catechol O-Methyltransferase Complexed with Nitecapone.

Catechol O-methyltransferase (COMT) is known as an important drug-target protein in the field of Parkinson's disease. All clinically approved COMT inhibitors bring a 5-substituted-3-nitrocatechol ring as a pharmacophore, and they bind to COMT with S-adenosylmethionine (SAM) and an Mg2+ ion to form a quaternary complex (COMT/SAM/Mg2+/inhibitor). However, structural information about such quaternary complexes is only available for a few inhibitors. Here, a new crystal structure of COMT complexed with nitecapone (5), SAM and Mg2+ is revealed. Comparison of the structures of these complexes indicates that conformation of the catechol binding pocket is almost constant regardless of structure of the inhibitors. The only restriction of the side chain of inhibitors (i.e., the substituent at the 5-position of 3-nitrocatechol) seems to be that it does not make steric repulsion with COMT. However, recent crystallographic and biochemical studies suggest that COMT is a flexible protein, and its conformational flexibility seems crucial for its catalytic process. Based on this information, implications of these quaternary inhibitor complexes were investigated. Met 40 in the α2α3-loop makes atomic contacts with SAM or S-adenosylhomocysteine and the 3-position of the catechol inhibitor. This interaction seems to play a critical role in the affinity of the inhibitor and to stabilize the COMT/SAM/Mg2+/nitrocatechol inhibitor complex by fixing the flexible α2α3-loop.

X-Ray Crystallographic Structure of Hericium erinaceus Ribonuclease, RNase He1 in Complex with Zinc.

RNase He1 is a guanylic acid-specific ribonuclease of the RNase T1 family from Hericium erinaceus (Japanese name: Yamabushitake). Its RNA degrading activity is strongly inhibited by Zn2+, similar to other T1 family RNases. However, RNase He1 shows little inhibition of human tumor cell proliferation, unlike RNase Po1, another T1 family RNase from Pleurotus ostreatus (Japanese name: Hiratake). Here, we determined the three-dimensional X-ray crystal structure of RNase He1 in complex with Zn, which revealed that Zn binding most likely prevents substrate entry into the active site due to steric hindrance. This could explain why RNase He1 and other T1 family RNases are inhibited by Zn. The X-ray crystal structures revealed that RNase He1 and RNase Po1 are almost identical in their catalytic sites and in the cysteine residues involved in disulfide bonds that increase their stability. However, our comparison of the electrostatic potentials of their molecular surfaces revealed that RNase He1 is negative whereas RNase Po1 is positive; thus, RNase He1 may not be able to electrostatically bind to the plasma membrane, potentially explaining why it does not exhibit antitumor activity. Hence, we suggest that the cationic characteristics of RNase Po1 are critical to the anti-tumor properties of the protein.

Factors Affecting and Adjustments for Sex Differences in Current Perception Threshold With Transcutaneous Electrical Stimulation in Healthy Subjects.

OBJECTIVE: Current perception threshold (CPT) measurement is a noninvasive, easy, and semi-objective method for determining sensory function using transcutaneous electrical stimulation. Previous studies have shown that CPT is determined by physical characteristics, such as sex, age, physical sites, and presence of neuropathy. Although the CPT reported in males is clearly higher than that in females, the reason for this difference remains unclear. This study investigates the cause of sex-based differences in CPT and suggests an adjustment method, which may suppress the sex difference in CPT. MATERIALS AND METHODS: Electrical stimulation was applied with PainVision® via five sizes of circular surface electrodes. Seventy healthy participants were examined thrice under each electrode. The relationship among body water percentage, body fat percentage, and CPT was then analyzed. RESULTS: CPT values are higher in males than that in females, with statistically significant sex differences with each electrode pairs (EL 1: p < 0.001; EL 2: p = 0.006; EL 3: p < 0.001; EL 4: p < 0.001; EL 5: p < 0.001). By adjusting for body fat percentage or body water percentage, the log-transformation values (CPT values) no longer exhibit sex differences with any electrode pairs (body fat: p = 0.09; body water: p = 0.08). CONCLUSION: We conclude that sensitivity for perceiving electrical stimulation can be influenced by the subjects' characteristics, such as body fat or body water percentages.

HLA-DQB1 DPB1 alleles in Japanese patients with adult-onset Still's disease.

Objective: HLA class II alleles are major determinants of genetic predisposition to rheumatic diseases. Predisposing effects of HLA had been suggested in AOSD, however, ethnic differences may account for variations in AOSD association with HLA. We determined the contribution of HLA-DQB1, DPB1 alleles to susceptibility to Adult-onset Still's disease (AOSD) in the Japanese population. Methods: HLA-DQB1 and DPB1 alleles were analyzed in 87 Japanese patients with AOSD and 413 Japanese healthy subjects. Results: We found significant association between HLA-DQB1*06:02 (Pc = 0.010, odds ratio: 2.54) and AOSD, whereas there was no association between the DQB1*06:02 allele and disease phenotypes of AOSD. Moreover, we did not find a predisposing effect of the HLA-DPB1 allele to AOSD. Haplotype analysis showed that presence of DRB1*15:01-DQB1*06:02 was associated with Japanese patients with AOSD. However, conditional logistic regression tests were unable to demonstrate independent association between DRB1*1501 or DQB1*0602 and AOSD. Conclusions: Our results show significant association between AOSD and the HLA DQB1*06:02 allele, and between the DRB1*1501-DQB1*06:02 haplotype and AOSD susceptibility. These findings suggest that genetic susceptibility to AOSD depends on the genotype combinations of HLA DRB1 and DQB1 alleles.

Fatty Acids as Useful Serological Markers for Crohn's Disease.

BACKGROUND: We have previously reported that patients with Crohn's disease (CD) have a very specific erythrocyte membrane phospholipid fatty acid profile. The findings of this study suggest that the activities of enzymes involved in the metabolism of linoleic acid (LA), that is, delta-6 desaturase, are higher in CD patients than in healthy individuals. METHODS: We evaluated the utilities of various fatty acid compositions of the plasma (p-) as new serological markers for CD compared to those of erythrocyte membranes (e-). RESULTS: Fifty CD patients and 50 healthy individuals were enrolled. In both plasma and erythrocyte membranes, the weight percentages of palmitic acid (PA) were significantly higher, while those of LA were significantly lower in CD patients than in controls. Fatty acids with high sensitivity and specificity were p-PA (0.86 and 0.74) and e-PA (0.80 and 0.74). With PA and LA as a CD fatty acid index (CDFAi), that is, CDFAi = (PA/LA), the sensitivity and specificity of plasma CDFAi (p-CDFAi) and e-CDFAi were 0.80 and 0.80; and 0.82 and 0.88 respectively. CONCLUSION: In CD patients, various fatty acids were specifically altered in both plasma and erythrocytes, and p-PA and p-CDFAi are potentially useful as new serological markers for CD.

TNF-α potentiates uric acid-induced interleukin-1ß (IL-1ß) secretion in human neutrophils.

OBJECTIVE: Monosodium urate (MSU) has been shown to promote interleukin-1ß (IL-1ß) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1ß induction in human neutrophils. METHODS: Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1ß, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1ß mRNA expressions in human neutrophils were analyzed by real-time PCR method. RESULTS: TNF-α stimulation induced pro-IL-1ß mRNA expression; however, MSU stimulation did not induce pro-IL-1ß mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1ß secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1ß and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation. CONCLUSION: Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1ß secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

Successful Immunosuppressive Treatment of Mixed Connective Tissue Disease Complicated by Microscopic Polyangiitis.

Mixed connective tissue disease (MCTD) is characterized by a combination of clinical features of systemic lupus erythematosus, systemic sclerosis, and polymyositis with elevated antibodies to U1 small nuclear ribonucleoprotein (U1-RNP). MCTD is often accompanied by interstitial lung disease as pulmonary involvement. On the other hand, microscopic polyangiitis (MPA) is a systemic autoimmune disease characterized by the inflammation of small vessels (arterioles, capillaries, and venules) mainly affecting the lung and kidney. MPA is associated with elevated serum anti-neutrophil cytoplasmic antibody (ANCA). Complication of MPA in patients with MCTD is rare. So far, only nine case reports of MCTD complicated by MPA with serum myeloperoxidase-specific ANCA (MPO-ANCA) are available. Here, we describe a 64-year-old male suffering from MCTD with MPA. The patient developed interstitial pneumonia with alveolar hemorrhage accompanied by myositis, scleroderma, and elevated anti-U1-RNP antibody and MPO-ANCA levels with substantial systemic inflammation. Strong immunosuppressive therapy (corticosteroid, intravenous immunoglobulin, and cyclosporine A) ameliorated the myositis, interstitial lung disease, and inflammation, with the decrease of MPO-ANCA levels, despite that severe lung complications are often associated with poor outcomes. In conclusion, MCTD may be accompanied by MPA with alveolar hemorrhage. Severe lung complications may indicate a poor outcome, and therefore prompt immunosuppressive treatment should be performed in such patients.

Possible participation of IgG4 in the activation of complement in IgG4-related disease with hypocomplementemia.

OBJECTIVE: To investigate which IgG subclasses contribute to the activation of the complement pathway in IgG4-related disease (IgG4RD) patients with hypocomplementemia. METHODS: Sera of IgG4RD patients were analyzed for the binding ability of IgG subclasses to complement component 1q (C1q). Polyethylene glycol (PEG) precipitates containing immune complexes (ICs) in sera of IgG4RD patients were analyzed for IgG subclass composition by Western blotting. PEG precipitates containing ICs (PEG-ICs) in sera of patients were also analyzed for their ability to consume complement in normal human serum (NHS) using a total complement hemolytic (CH50) assay and a commercial kit to measure the complement capacity of all three individual complement pathways. RESULTS: The C1q binding assay revealed high serum levels of C1q-binding IgG4 in IgG4RD patients with hypocomplementemia. ICs in PEG precipitates were formed with IgG4 in IgG4RD patients, regardless of the presence or absence of hypocomplementemia. We observed a marked reduction of CH50 and reduced complement activity in the classical complement pathway as well as the mannan-binding lectin complement pathway in NHS incubated with PEG-IC isolated from IgG4RD patients with hypocomplementemia. CONCLUSION: Our results suggest that IgG4 may participate in the activation of complement in IgG4RD patients with hypocomplementemia.

Clinical and structural remission rates increased annually and radiographic progression was continuously inhibited during a 3-year administration of tocilizumab in patients with rheumatoid arthritis: A multi-center, prospective cohort study by the Michinoku Tocilizumab Study Group.

OBJECTIVE: To evaluate the clinical and structural efficacy of tocilizumab (TCZ) during its long-term administration in patients with rheumatoid arthritis (RA). METHODS: In total, 693 patients with RA who started TCZ therapy were followed for 3 years. Clinical efficacy was evaluated by DAS28-ESR and Boolean remission rates in 544 patients. Joint damage was assessed by calculating the modified total Sharp score (mTSS) in 50 patients. RESULTS: When the reason for discontinuation was limited to inadequate response or adverse events, the 1-, 2-, and 3-year continuation rates were 84.0%, 76.8%, and 72.2%, respectively. The mean DAS28-ESR was initially 5.1 and decreased to 2.5 at 6 months and to 2.2 at 36 months. The Boolean remission rate was initially 0.9% and increased to 21.7% at 6 months and to 32.2% at 36 months. The structural remission rates (ΔmTSS/year ≤ 0.5) were 68.8%, 78.6%, and 88.9% within the first, second, and third years, respectively. The structural remission rate at 3 years (ΔmTSS ≤ 1.5) was 66.0%, and earlier achievement of swollen joint count (SJC) of 1 or less resulted in better outcomes. CONCLUSIONS: TCZ was highly efficacious, and bone destruction was strongly prevented. SJC was an easy-to-use indicator of joint destruction.