Search

The influenza virus surface glycoprotein hemagglutinin (HA) is responsible for viral attachment to sialic acid-containing host cell receptors and it facilitates the initial stage of viral infection. In the present study, we isolated an RNA aptamer specific to the glycosylated receptor-binding domain of the HA protein (gHA1) after 12 cycles of the systematic evolution of ligands by exponential enrichment procedure (SELEX), and we then investigated if the selected aptamer suppresses viral infection in host cells. Nitrocellulose filter binding and enzyme-linked immunosorbent assay (ELISA) experiments revealed that 1 RNA aptamer, HA12-16, bound specifically to the gHA1 protein. Cell viability assay showed that the HA12-16 RNA aptamer suppressed viral infection in host cells by enhancing cell viability. Immunofluorescence microscopic analysis further demonstrated that the HA12-16 RNA aptamer suppresses viral attachment to host cells by neutralizing the receptor-binding site of influenza virus HA. These results indicate that the isolated RNA aptamer can be developed as an antiviral reagent against influenza through appropriate therapeutic formulation.

To investigate the impact of dose reduction of tumor necrosis factor inhibitor (TNFi) on radiographic progression in ankylosing spondylitis (AS).One hundred and sixty-five patients treated with etanercept or adalimumab were selected from a consecutive single-center observational cohort based on the availability of radiographs at baseline and after two- and/or four-years of follow up. Radiographs were assessed by two blinded readers using the modified Stokes AS Spinal Score (mSASSS). Radiographic progression in patients treated with standard-dose TNFi (standard-dose group, n = 49) was compared with patients whose dosage was tapered during the treatment (tapering group, n = 116) using linear mixed models.Baseline characteristics between two groups were comparable except for higher BASDAI (7.1 vs. 6.3, p = 0.003) in the standard-dose group. At two years after the treatment, mean dose quotient (S.D.) of the tapering group was 0.59 (0.17). During follow up, rate of radiographic progression in overall patients was 0.90 mSASSS units/year. Radiographic progression over time between the two groups was similar at the entire group level. However, in the subgroup of patients with baseline syndesmophytes, progression occurred significantly faster in the tapering group after the adjustment for baseline status (1.23 vs. 1.72 mSASSS units/year, p = 0.023). Results were consistent when radiographic progression was assessed by the number of newly developed syndesmophytes (0.52 vs. 0.73/year, p = 0.047). Sensitivity analysis after multiple imputation of missing radiographs also showed similar results.A dose tapering strategy of TNFi is associated with more rapid radiographic progression in AS patients who have syndesmophytes at baseline.

Objective. To develop a decision model to identify SSc-associated interstitial lung disease (ILD) patients who are eligible for watchful waiting at ILD diagnosis. Methods. One hundred and fifty-one SSc-ILD patients who received medical care at Seoul National University Hospital from 1986 to 2013 were enrolled in this retrospective cohort study. ILD was diagnosed by chest CT. Patients with and without immunosuppressive treatment were compared in terms of char- acteristics at ILD diagnosis to identify distinguishing variables. After multivariate analysis, a decision model for watchful waiting was formulated. Its validity was assessed by comparing the survival of patients whose management in real practice did and did not accord with the management recommended by the model. Results. The untreated group had better survival than the immunosuppressive treatment group (P = 0.0316, by log-rank test). The untreated group was less likely to have gastrointestinal involvement (P = 0.008) and pulmonary arterial hypertension (PAH), as determined by echocardiography) (P = 0.015) and more likely to have favourable initial forced vital capacity (P = 0.0004), favourable initial lung diffusion capacity for carbon monoxide (P = 0.0002) and a low CT grade (P<0.001). The final watchful waiting decision model included lack of PAH and limited ILD extent on CT. Application of the model to the cohort revealed that patients who were eligible for watchful waiting (as determined by the model) and underwent this management strategy had better survival than eligible patients who underwent immuno-suppressive treatment (P = 0.048, by log-rank test). Conclusion. Watchful waiting may be effective for SSc-ILD patients who have minimal pulmonary involvement on CT and lack PAH on echocardiography at baseline. [ABSTRACT FROM AUTHOR]

The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. Here, we demonstrate the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by Gram-positive bacteria and fungi. Our analysis shows that ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spatzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions. [ABSTRACT FROM AUTHOR]

Background/Aims The aim of this study was to investigate long-term post-discontinuation outcomes in patients with rheumatoid arthritis (RA) who had been treated with tumor necrosis factor-α inhibitors (TNF-αi) which was then discontinued. Methods Sixty Korean patients with RA who participated in a 5-year GO-BEFORE and GO-FORWARD extension trials were included in this retrospective study. Golimumab was deliberately discontinued after the extension study (baseline). Patients were then followed by their rheumatologists. We reviewed their medical records for 2 years (max 28 months) following golimumab discontinuation. Patients were divided into a maintained benefit (MB) group and a loss-of-benefit (LB) group based on treatment pattern after golimumab discontinuation. The LB group included patients whose conventional disease-modifying antirheumatic drug(s) were stepped-up or added/switched (SC) and those who restarted biologic therapy (RB). Results The mean age of patients at baseline was 56.5 years and 55 (91.7%) were females. At the end of follow-up, 23 (38.3%) patients remained in the MB group. In the LB group, 75.7% and 24.3% were assigned into SC and RB subgroups, respectively. Fifty percent of patients lost MB after 23.3 months. Demographics and clinical variables at baseline were comparable between MB and LB groups except for age, C-reactive protein level, and corticosteroid use. Restarting biologic therapy was associated with swollen joint count (adjusted hazard ratio [HR], 1.90; 95% confidence interval [CI], 1.01 to 3.55) and disease duration (adjusted HR, 1.12; 95% CI, 1.02 to 1.23) at baseline. Conclusions Treatment strategies after discontinuing TNF-αi are needed to better maintain disease control and quality of life of patients with RA.

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses. [ABSTRACT FROM AUTHOR]

The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates. [ABSTRACT FROM AUTHOR]

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-KB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of 3-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation. [ABSTRACT FROM AUTHOR]

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway. [ABSTRACT FROM AUTHOR]