Enhanced efficacy of the novel recombinant clone VasSF in a mouse model of antineutrophil cytoplasmic antibody-associated vasculitis.

Based on the efficacy of intravenous immunoglobulin (IVIg) for the treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), we developed a recombinant single-chain-fragment variable clone, VasSF, therapeutic against AAV in a mouse model (SCG/Kj mice). VasSF is thought to bind to vasculitis-associated apolipoprotein A-II (APOA2) as a target molecule. VasSF is a promising new drug against AAV, but difficulties in the yield and purification of VasSF remain unresolved. We produced monomers of new VasSF molecules by modifying the plasmid structure for VasSF expression and simplifying the purification method using high-performance liquid chromatography. We compared the therapeutic effects between 5-day continuous administration of the monomers, as in IVIg treatment, and single shots of 5-day-equivalent doses. We also evaluated the life-prolonging effect of the single-shot treatment. Two-dimensional western blots were used to examine the binding of VasSF to APOA2. Our improved manufacturing method resulted in a 100-fold higher yield of VasSF than in our previous study. Monomerization of VasSF stabilized its efficacy. Single shots of a small amount (1/80 000 of IVIg) produced sufficient therapeutic effects, including decreased glomerular crescent formation, a decreasing trend of serum ANCA against myeloperoxidase (MPO-ANCA), decreases in multiple proinflammatory cytokines, and a trend toward prolonged survival. Two-dimensional western blots confirmed the binding of VasSF to APOA2. The newly produced pure VasSF monomers are stable and therapeutic for AAV with a single low-dose injection, possibly by removing vasculitis-associated APOA2. Thus, the new VasSF described herein is a promising drug against AAV.

Characteristics of Japanese physician influencers on Twitter during the COVID-19 pandemic and fact-checking their tweets on COVID-19-related drugs.

BACKGROUND: During the coronavirus disease-2019 (COVID-19) pandemic, segments of the public relied on social media platforms such as Twitter for medical information shared by medical personnel. Although physicians are likely to disseminate more accurate information on Twitter than non-medical individuals, it cannot be taken for granted. As such, tweets written by physicians in Japan should also be scrutinized for accuracy. PURPOSE: The purpose of this study was to create a profile of the most popular physician influencers on Twitter in Japan, and to do a fact-check of their tweets regarding COVID-19-related drugs. DESIGN: This is a retrospective observational study. METHODS: We purchased Twitter data for Japan for the initial 9 months of the COVID-19 pandemic (from January 2020 to September 2020), and extracted tweets with keywords related to COVID-19 at a sampling rate of 3%. The most popular physicians were identified and selected consecutively by searching for the top 1000 accounts using Twitter's search function. These top accounts were considered influencers and their tweets and retweets concerning COVID-19-related drugs were fact-checked against scientific literature. RESULTS: We identified 21 physician influencers with real names: most were male in their 40s and 50s working at private medical facilities. The contents of their tweets were mainly sourced from scientific publications that were current at that time. The fact-check revealed that only one of 50 tweets was not correct while the others had no identifiable inaccuracies. CONCLUSIONS: Except for one tweet, tweets written and retweeted by Japanese physician influencers concerning the COVID-19-related drugs contained predominantly accurate information.

Cutaneous T-cell-attracting chemokine as a novel biomarker for predicting prognosis of idiopathic pulmonary fibrosis: a prospective observational study.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease that leads to respiratory failure and death. Although there is a greater understanding of the etiology of this disease, accurately predicting the disease course in individual patients is still not possible. This study aimed to evaluate serum cytokines/chemokines as potential biomarkers that can predict outcomes in IPF patients. METHODS: A multi-institutional prospective two-stage discovery and validation design using two independent cohorts was adopted. For the discovery analysis, serum samples from 100 IPF patients and 32 healthy controls were examined using an unbiased, multiplex immunoassay of 48 cytokines/chemokines. The serum cytokine/chemokine values were compared between IPF patients and controls; the association between multiplex measurements and survival time was evaluated in IPF patients. In the validation analysis, the cytokines/chemokines identified in the discovery analysis were examined in serum samples from another 81 IPF patients to verify the ability of these cytokines/chemokines to predict survival. Immunohistochemical assessment of IPF-derived lung samples was also performed to determine where this novel biomarker is expressed. RESULTS: In the discovery cohort, 18 cytokines/chemokines were significantly elevated in sera from IPF patients compared with those from controls. Interleukin-1 receptor alpha (IL-1Rα), interleukin-8 (IL-8), macrophage inflammatory protein 1 alpha (MIP-1α), and cutaneous T-cell-attracting chemokine (CTACK) were associated with survival: IL-1Rα, hazard ratio (HR) = 1.04 per 10 units, 95% confidence interval (95% CI) 1.01-1.07; IL-8, HR = 1.04, 95% CI 1.01-1.08; MIP-1α, HR = 1.19, 95% CI 1.00-1.36; and CTACK, HR = 1.12 per 100 units, 95% CI 1.02-1.21. A replication analysis was performed only for CTACK because others were previously reported to be potential biomarkers of interstitial lung diseases. In the validation cohort, CTACK was associated with survival: HR = 1.14 per 100 units, 95% CI 1.01-1.28. Immunohistochemistry revealed the expression of CTACK and CC chemokine receptor 10 (a ligand of CTACK) in airway and type II alveolar epithelial cells of IPF patients but not in those of controls. CONCLUSIONS: CTACK is a novel prognostic biomarker of IPF. Trial registration None (because of no healthcare intervention).

Role of interleukin 5-induced eosinophils in interleukin 33-triggered airway inflammation in mice.

BACKGROUND: Interleukin (IL)-5 is essential for allergen induced eosinophilic airway inflammation, but not activation of T helper type 2 (Th2) cells in the lung. Although an excessive Th2 reaction is observed without IL-5 signaling, the mechanisms have remained unknown. OBJECTIVE: To evaluate the negative-feedback mechanism in eosinophilic airway inflammation, we examined IL-33 triggered eosinophilic airway inflammation in IL-5Rα-/- mice. METHODS: Mice were administered intranasal IL-33 for 3 days. Airway hyperresponsiveness (AHR) was evaluated and bronchoalveolar lavage (BAL) was performed 24 h after the last IL-33 treatment. The number of inflammatory cells and cytokine levels in the BAL fluid (BALF) were analyzed, and histologic examination was performed. RESULTS: Compared with IL-33 treated wild-type (WT) mice, intranasal administration of IL-33 in IL-5Rα-/- mice reduced eosinophilic airway inflammation, AHR, and basement membrane thickening, while we found excessive IL-33 induced IL-5 and IL-13 production in the airway without IL-5 signaling. The numbers of eosinophils with a ringshaped nucleus (resident) and segmented nucleus (inflammatory) were increased in WT mice, but not in IL-5Rα-/- mice following intranasal administration of IL-33, and the numbers of SiglecF-positive eosinophils with (resident) or without (inflammatory) expression of CD62L were also significantly increased by IL-33 treatment in WT mice, but not in IL5Rα-/- mice. The number of ILC2 cells in the BALF was significantly higher in IL-33 treated IL-5Rα-/- mice than in IL-33 treated WT mice. CONCLUSIONS: These findings suggest the possibility that IL-5 induced eosinophils contribute to the negative-feedback mechanisms in IL-33 induced ILC2 mediated airway inflammation.

Impaired HVJ-stimulated Interferon producing capacity in MPO-ANCA-associated vasculitis with rapidly progressive glomerulonephritis lead to susceptibility to infection.

ANCA-associated RPGN leads to renal failure through systemic vasculitis and diffuse crescentic glomerulonephritis. MPO-ANCA-RPGN patients are highly susceptible to infections. Our aim in this study was to uncover reasons why these patients were susceptible to infections. We analyzed various aspects of type I interferon system including HVJ-stimulated IFN-α producing capacity and plasmacytoid dendritic cell (pDC) number in whole blood in MPO-ANCA-RPGN patients. Compared with healthy subjects, MPO-ANCA-RPGN patients showed impaired HVJ-stimulated IFN-α producing capacity and lower pDC number with or without glucocorticoid treatment. Immuno-histological staining of MPO-ANCA-RPGN kidney samples revealed a few but apparent pDC in T cell infiltrating regions even in patients with low pDC number in their peripheral blood. Patients' low HVJ-stimulated IFN-α producing capacity and pDC numbers persisted even after patients underwent several years of treatment. Former infection was determined using patients' serum BPI, Lamp-2 and Calprotectin, since they are reflective of a history of infection. These markers were higher in MPO-ANCA-RPGN patients than in healthy subjects. These results indicate that impaired HVJ-stimulated IFN-α production as well as dysfunction of the IFN system might have resulted from a previous bout of infection and can be partially implicated in patients' long-term susceptibility and vulnerability to infection.

Serum matrix metalloproteinase levels in polymyositis/dermatomyositis patients with interstitial lung disease.

OBJECTIVE: We aimed to clarify the clinical significance of serum levels of MMPs in interstitial lung disease (ILD) complicated with PM/DM (PM/DM-ILD). METHODS: We retrospectively analysed serum levels of seven subsets of MMPs in 52 PM/DM-ILD patients diagnosed at Kyoto University Hospital or Tenri Hospital from January 2005 to December 2014. The patients were sub-grouped based on the presence of anti-amimoacyl-tRNA synthetase antibody (anti-ARS antibody), anti-melanoma differentiation-associated protein 5 antibody (anti-MDA5 antibody) or lack of the antibodies (ARS-ILD, MDA5-ILD and other-ILD groups, respectively) and independently analysed. Eighteen PM/DM patients without ILD and 55 healthy control were also analysed. Associations between serum levels of MMPs and clinical findings including mortality were analysed. RESULTS: Among the MMPs analysed, MMP-7 serum levels in the ARS-ILD group were significantly higher compared with those in any of the other groups of PM/DM patients or in healthy controls. On the other hand, in the MDA5-ILD group, serum MMP-7 levels >5.08 ng/ml were associated with worse overall survival both in univariate (P = 0.017; odds ratio 18.0; 95% CI 1.69, 192.00) and multivariate (P = 0.027; odds ratio 14.60; 95% CI 1.11, 192.00) analyses. Immunohistochemical analysis suggested that MMP-7 was expressed in type II alveolar epithelial cells adjacent to the fibrotic lesions. CONCLUSION: Serum MMP-7 levels were higher in anti-ARS antibody-positive PM/DM-ILD patients, while higher serum MMP-7 levels among anti-MDA5 antibody-positive PM/DM-ILD patients were associated with a worse prognosis. Fibrotic processes may be associated with the elevation of serum MMP-7 levels.

Phase I clinical trial of adoptive transfer of expanded natural killer cells in combination with IgG1 antibody in patients with gastric or colorectal cancer.

Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells without prior sensitization, and have the potential to exert antibody-dependent cellular cytotoxicity (ADCC). In this clinical trial, we examined the safety and efficacy of the use of NK cells, generated using a novel expansion system, in combination with IgG1 antibodies for the treatment of advanced gastric or colorectal cancers. Treatment consisted of trastuzumab- or cetuximab-based chemotherapy, plus adoptive NK cell therapy. For administration of expanded NK cells, dose escalation with a sequential 3 + 3 design was performed in three steps, at doses of 0.5 × 109 , 1.0 × 109 , and 2.0 × 109 cells/injection (N = 9). After 3 days of IgG1 antibody administration, patients were infused with expanded NK cells three times at triweekly intervals. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D (97.6%) and CD16 (69.6%). The combination therapy was very well tolerated with no severe adverse events. Among six evaluable patients, four presented stable disease (SD) and two presented progressive disease. Of the four SD patients, three showed an overall decrease in tumor size after combination therapy. Immune monitoring suggested that combination therapy enhanced whole blood IFN-γ production and reduced peripheral regulatory T cells (Tregs). In conclusion, this phase I trial provides evidence of good tolerability, induction of Th1 immune responses, and preliminary anti-tumor activity for this combination therapy, in patients with advanced gastric and colorectal cancer that have received previous therapy.

Tentative diagnostic criteria and disease severity classification for Castleman disease: A report of the research group on Castleman disease in Japan.

OBJECTIVES: To determine the tentative diagnostic criteria and disease severity classification for Castleman disease (CD) and describe the clinical and pathologic features among human herpesvirus 8 (HHV-8) negative idiopathic multicentric CD (iMCD) in the Japanese population. METHODS: We established the working groups for the research of CD in Japan and had meetings to discuss and define the tentative diagnostic criteria and disease severity classification for CD. We subsequently analyzed 142 patients classified into iMCD by using the nationwide Japanese patient registry. RESULTS: We proposed the preliminary diagnostic criteria and disease severity classification for CD based on our discussion. In addition, we made a proposal for the disease activity score. We identified clinical and pathological features of patients with iMCD diagnosed by these diagnostic criteria. In the disease severity classification, 37, 33 and 30% patients were categorized into mild, moderate and severe diseases, respectively. CONCLUSION: This is the first proposal for diagnosis and classification of CD by the Japanese group. Further studies are required to validate whether they can distinguish CD from other inflammatory diseases and to determine their sensitivity and specificity.

Matrix metalloproteinase-10: a novel biomarker for idiopathic pulmonary fibrosis.

BACKGROUND: Matrix metalloproteinases (MMPs) are believed to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), and MMP-7 has been described as a useful biomarker for IPF. However, little is known regarding the significance of MMP-10 as a biomarker for IPF. METHODS: This observational cohort study included 57 patients with IPF. Serum MMPs were comprehensively measured in all patients, and the relationships between these markers and both disease severity and prognosis were evaluated. Bronchoalveolar lavage fluid (BALF) MMP-7 and -10 levels were measured in 19 patients to investigate the correlation between these markers and their corresponding serum values. Immunohistochemical staining for MMP-10 was also performed in IPF lung tissue. RESULTS: Serum MMP-7 and -10 levels correlated significantly with both the percentage of predicted forced vital capacity (ρ = -0.31, p = 0.02 and ρ = -0.34, p < 0.01, respectively) and the percentage of predicted diffusing capacity of the lung for carbon monoxide (ρ = -0.32, p = 0.02 and ρ = -0.43, p < 0.01, respectively). BALF MMP-7 and -10 levels correlated with their corresponding serum concentrations. Only serum MMP-10 predicted clinical deterioration within 6 months and overall survival. In IPF lungs, the expression of MMP-10 was enhanced and localized to the alveolar epithelial cells, macrophages, and peripheral bronchiolar epithelial cells. CONCLUSIONS: MMP-10 may be a novel biomarker reflecting both disease severity and prognosis in patients with IPF.

Renal redox dysregulation in AKI: application for oxidative stress marker of AKI.

Oxidative stress is a major determinant of acute kidney injury (AKI); however, the effects of an AKI on renal redox system are unclear, and few existing AKI markers are suitable for evaluating oxidative stress. We measured urinary levels of the redox-regulatory protein thioredoxin 1 (TRX1) in patients with various kinds of kidney disease and in mice with renal ischemia-reperfusion injury. Urinary TRX1 levels were markedly higher in patients with AKI than in those with chronic kidney disease or in healthy subjects. In a receiver operating characteristic curve analysis to differentiate between AKI and other renal diseases, the area under the curve for urinary TRX1 was 0.94 (95% confidence interval, 0.90-0.98), and the sensitivity and specificity were 0.88 and 0.88, respectively, at the optimal cutoff value of 43.0 µg/g creatinine. Immunostaining revealed TRX1 to be diffusely distributed in the tubules of normal kidneys, but to be shifted to the brush borders or urinary lumen in injured tubules in both mice and humans with AKI. Urinary TRX1 in AKI was predominantly in the oxidized form. In cultured human proximal tubular epithelial cells, hydrogen peroxide specifically and dose dependently increased TRX1 levels in the culture supernatant, while reducing intracellular levels. These findings suggest that urinary TRX1 is an oxidative stress-specific biomarker useful for distinguishing AKI from chronic kidney disease and healthy kidneys.

Predictive biomarkers of COVID-19 prognosis identified in Bangladesh patients and validated in Japanese cohorts.

Despite high vaccination rates globally, countries are still grappling with new COVID infections, and patients diagnosed as mild dying at home during outpatient treatment. Hence, this study aim to identify, then validate, biomarkers that could predict if newly infected COVID-19 patients would subsequently require hospitalization or could recover safely with medication as outpatients. Serum cytokine/chemokine data from 129 COVID-19 patients within 7 days after the onset of symptoms in Bangladesh were used as training data. The majority of patients were infected with the Omicron variant and over 88% were vaccinated. Patients were divided into those with mild symptoms who recovered, and those who deteriorated to moderate or severe illness. Using the Lasso method, 15 predictive markers were identified and used to classify patients into these two groups. The biomarkers were then validated in a cohort of 194 Covid patients in Japan with a predictive accuracy that exceeded 80% for patients infected with Delta and Omicron variants, and 70% for Wuhan and Alpha variants. In an environment of widespread vaccination, these biomarkers could help medical practitioners determine if newly infected COVID-19 patients will improve and can be managed on an out-patient basis, or if they will deteriorate and require hospitalization.

Whole blood interferon-γ levels predict the therapeutic effects of adoptive T-cell therapy in patients with advanced pancreatic cancer.

A core challenge in administering immune-based treatments for cancer is the establishment of easily accessible immunological assays that can predict patients' clinical responses to immunotherapy. In this study, our aim was to predict the therapeutic effects of adoptive T-cell therapy in patients with advanced pancreatic cancer. To do this, we evaluated whole blood cytokine levels and peripheral regulatory T cells (Tregs) in 46 patients with unresectable or recurrent pancreatic cancer who received adoptive T-cell therapy at 2-week intervals. To test immune function, venous blood was obtained from patients before the start of therapy and 2 weeks after the 4th treatment. Whole blood interferon (IFN)-α levels (after stimulation with the Sendai virus) were evaluated, as well as the levels of 9 cytokines stimulated with phytohemagglutinin [interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12(p70), IL-13, tumor necrosis factor-α, IFN-γ, and granulocyte-monocyte colony-stimulating factor]. Peripheral Tregs were analyzed by flow cytometry. Using the obtained data, we then observed the relationship between these immunological parameters and clinical outcome of patients. We found that the whole blood production of IFN-γ, IL-2, IL-4, IL-5 and IL-13 significantly increased after adoptive T-cell therapy, whereas the number of peripheral Tregs did not change. Multivariate Cox proportional hazards analyses indicated that the number of peripheral Tregs before receiving adoptive T-cell therapy and the change in IFN-γ levels after adoptive T-cell therapy were independent variables predicting overall survival. The findings of this study indicate that the assay of whole blood IFN-γ production offers promise for evaluating the clinical response of patients to cancer immunotherapy.

Analysis of systemic and airway inflammation in obstructive sleep apnea.

PURPOSE: The presence of both systemic and airway inflammation has been suggested in obstructive sleep apnea (OSA) by increased levels of inflammatory biomarkers in the circulation and respiratory specimens. We aimed to investigate the relationship between systemic and airway inflammation in OSA. METHODS: This study was conducted by simultaneously measuring various biomarkers both in serum and induced sputum of 43 patients. We compared the relationships of these biomarker levels with polysomnographic data and obesity measurements and also investigated their interrelationships between systemic and local compartments. We also assessed the relation of inflammatory markers with proximal airway resistance measured by impulse oscillometry. RESULTS: In multiple regression analyses, each measured serum biomarker [leptin, interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF)] significantly correlated with waist circumference or fat area determined by computed tomography. In contrast, regarding airway inflammation, sputum IL-6, IL-8, TNF-α, and VEGF significantly correlated with OSA severity as indicated by the respiratory disturbance index or oxygen desaturation indices. Sputum IL-6, IL-8, TNF-α, and VEGF were significantly related to sputum neutrophil number, and sputum IL-8 and TNF-α were related to proximal airway resistance independently of body mass index. There were no significant interrelationships between the same biomarkers in serum and induced sputum. CONCLUSIONS: Systemic and airway inflammation in OSA might be differently regulated by OSA itself and comorbid obesity, depending on the type of cytokine. Although we did not find apparent interrelationships between systemic and local compartments, further studies are needed to clarify this concept.

Clinical features of patients with anti-neutrophil cytoplasmic autoantibodies targeting native myeloperoxidase antigen.

OBJECTIVES: Anti-neutrophil cytoplasmic autoantibodies (ANCA) are useful diagnostic markers in systemic vasculitic disorders with small-vessel involvement, but depending on the particular test used, the myeloperoxidase (MPO)-ANCA results are variable. In the present study, we performed a comparative analysis between our originally developed nMPO-ANCA assay that targets the native MPO antigen and other commercially available assays using sera of patients with clinical features of ANCA-associated vasculitis (AAV). METHODS: Sera of 24 patients strongly suspected of having AAV were examined for the presence of MPO-ANCAs by our nMPO-ANCA assay and by other commercial-based MPO-ANCA assays. These results were correlated to indirect immunofluorescence microscopy staining patterns and patient clinical parameters. RESULTS: Eighteen out of 24 patients (75 %) were positive for nMPO-ANCA, compared with 13 out of 24 patients (54 %) by one of the most frequently used commercial-based MPO-ANCA enzyme-linked immunosorbent assays (ELISAs) in Japan. Interestingly, the patients who tested positive with our nMPO-ANCA assay alone showed clinical features of AAV marked by continuous fever, polyarthritis, and mild nephritis. The titers of nMPO-ANCA decreased in association with clinical improvement after treatment. CONCLUSIONS: Our data suggest that a positive nMPO-ANCA result, which identifies antibodies to human native MPO antigen, correlates with AAV disease activity. Moreover, the nMPO-ANCA test has clinical utility in detecting AAV-affected patients who have tested negative using commercially available assays.

The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction.

Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we use immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2'-5'-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5'UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s lead to a modest induction of IFN-α expression, which is abrogated upon HELZ2 overexpression. Notably, IFN-α expression is enhanced upon overexpression of an ORF1p RNA binding mutant, suggesting ORF1p binding might protect L1 RNA from "triggering" IFN-α induction. Thus, ISG proteins can inhibit retrotransposition by different mechanisms.

Enhancement of IL-6 Production Induced by SARS-CoV-2 Nucleocapsid Protein and Bangladeshi COVID-19 Patients' Sera.

Coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by severe acute respiratory syndrome coronavirus 2 that can have detrimental effects on multiple organs and accelerate patient mortality. This study, which encompassed 130 confirmed COVID-19 patients who were assessed at three different time points (i.e., 3, 7, and 12 days) after the onset of symptoms, investigated interleukin-6 (IL-6) enhancement induced by a viral nucleocapsid (N) protein from a myeloid cell line. Disease severity was categorized as mild, moderate, or severe. The severe cases were characterized as having significant elevations in serum IL-6, C-reactive protein, D-dimer, ferritin, creatinine, leukocytes, and neutrophil-to-lymphocyte ratio and decreased hemoglobin, hematocrit, and albumin levels compared with mild and moderate cases. To evaluate IL-6-inducing activity, heat-inactivated sera from these patients were incubated with and without the N protein. The findings showed a progressive increase in IL-6 production in severe cases upon N protein stimulation. There was a strong correlation between anti-N antibodies and levels of IL-6 secreted by myeloid cells in the presence of N protein and sera, indicating the crucial role that the anti-N antibody plays in inducing IL-6 production. Uncontrolled IL-6 production played a pivotal role in disease pathogenesis, exacerbating both disease severity and mortality. Efficiently targeting the N protein could potentially be employed as a therapeutic strategy for regulating the immune response and alleviating inflammation in severe cases.

Peripheral helper-T-cell-derived CXCL13 is a crucial pathogenic factor in idiopathic multicentric Castleman disease.

Castleman disease (CD) is a rare lymphoproliferative disorder. Among subtypes of CD, idiopathic multicentric CD-not otherwise specified (iMCD-NOS) has a poor prognosis and its pathogenesis is largely unknown. Here we present a xenotransplantation model of iMCD-NOS pathogenesis. Immunodeficient mice, transplanted with lymph node (LN) cells from iMCD-NOS patients, develop iMCD-like lethal inflammation, while mice transplanted with LN cells from non-iMCD patients without inflammation serve as negative control. Grafts depleted of human CD3+ T cells fail to induce inflammation in vivo. Upon engraftment, peripheral helper T (Tph) cells expand and levels of human CXCL13 substantially increase in the sera of mice. A neutralizing antibody against human CXCL13 blocks development of inflammation and improves survival in the recipient mice. Our study thus indicates that Tph cells, producing CXCL13 play a critical role in the pathogenesis of iMCD-NOS, and establishes iMCD-NOS as an immunoregulatory disorder.

Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

Beneficial effects of anti-apolipoprotein A-2 on an animal model for coronary arteritis in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is usually treated with high-dose intravenous immunoglobulin (IVIg) as severe infectious and other diseases. Due to issues that are associated with immunoglobulin preparation, such as the risk of possible contamination by infectious agents and limited blood banking resources, recombinant immunoglobulins are required. We developed a novel recombinant antibody drug candidate, "VasSF," based on the therapeutic effects it exerted on a mouse spontaneous crescentic glomerulonephritis model (SCG/Kj). Apolipoprotein A-2 (ApoA2) has been identified as one of VasSF's target molecules. METHODS: Here, we tested the potential of anti-apolipoprotein A-2 antibodies (anti-ApoA2) as a new therapeutic drug against KD by examining its effect on a mouse model, in which KD was induced via Candida albicans water-soluble fraction (CAWS). CAWS (2 mg/mouse) was injected intraperitoneally into C57BL/6NCrSlc mice for five consecutive days. The incidence and histological severity of vasculitis in CAWS-induced coronary arteritis in mice administered anti-ApoA2 was examined. The following experimental groups were tested: solvent (only PBS (-) injection); anti-ApoA2 antibodies at dosages of 0.05 mg, 0.1 mg, and 0.5 mg/kg/day; human IgG at 0.1 mg/kg/day. RESULTS: The group treated with anti-ApoA2 0.5 mg/kg/day showed a lower incidence of panvasculitis induced by CAWS, less inflammation of the coronary arteries and aortic roots, and lower levels of serum IL-6, M-CSF, and MIP-1α and 32 cytokines/chemokines compared with those in the solvent group. CONCLUSIONS: The anti-ApoA2 treatment suppressed the development of coronary arteritis in an animal KD model and anti-ApoA2 shows potential as an effective therapeutic candidate for the treatment of KD vasculitis. The use of specific antibodies that display higher vasculitis-suppressing effects, such as anti-ApoA2, may attenuate KD as well as other infectious diseases, with less severe adverse side effects than treatment with IVIg.

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells.

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.