BacMam Expressing Highly Glycosylated Porcine Interferon Alpha Induces Robust Antiviral and Adjuvant Effects against Foot-and-Mouth Disease Virus in Pigs.

Foot-and-mouth disease (FMD) is an acute contagious disease that affects cloven-hoofed animals and has severe global economic consequences. FMD is most commonly controlled by vaccination. Currently available commercial FMD vaccines contain chemically inactivated whole viruses, which are thought to be slow acting as they are effective only 4 to 7 days following vaccination. Hence, the development of a novel rapid vaccine or alternative measures, such as antiviral agents or the combination of vaccines and antiviral agents for prompt FMD virus (FMDV) outbreak containment, is desirable. Here, we constructed a recombinant baculovirus (BacMam) expressing consensus porcine interferon alpha (IFN-α) that has three additional N-glycosylation sites driven by a cytomegalovirus immediate early (CMV-IE) promoter (Bac-Con3N IFN-α) for protein expression in mammalian cells. Bac-Con3N IFN-α expressing highly glycosylated porcine IFN-α protein increased the duration of antiviral effects. We evaluated the antiviral effects of Bac-Con3N IFN-α in swine cells and mice and observed sustained antiviral effects in pig serum; additionally, Bac-Con3N IFN-α exhibited sustained antiviral effects in vivo as well as adjuvant effects in combination with an inactivated FMD vaccine. Pigs injected with a combination of Bac-Con3N IFN-α and the inactivated FMD vaccine were protected against FMDV at 1, 3, and 7 days postvaccination. Furthermore, we observed that in combination with the inactivated FMD vaccine, Bac-Con3N IFN-α increased neutralizing antibody levels in mice and pigs. Therefore, we suggest that Bac-Con3N IFN-α is a strong potential antiviral and adjuvant candidate for use in combination with inactivated FMD vaccines to protect pigs against FMDV. IMPORTANCE Early inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in pigs is highly desirable as FMDV transmission and shedding rates are higher in pigs than in cattle. However, commercial FMD vaccines require at least 4 to 7 days postvaccination (dpv) for protection, and animals are vulnerable to heterologous viruses before acquiring high antibody levels after the second vaccination. Therefore, the development of antiviral agents for use in combination with FMD vaccines is essential. We developed a novel antiviral and immunostimulant, Bac-Con3N IFN-α, which is a modified porcine IFN-α-expressing recombinant baculovirus, to improve IFN stability and allow its direct delivery to animals. We present a promising candidate for use in combination with inactivated FMD vaccines as pigs applied to the strategy had early protection against FMDV at 1 to 7 dpv, and their neutralizing antibody levels were higher than those in pigs administered the vaccine only.

Midbody plays an active role in fibroblast-myofibroblast transition by mediating TGF-ß signaling.

The transition of fibroblasts into myofibroblasts is a crucial step in kidney fibrosis. However, the biological processes involved in this transdifferentiation are incompletely understood. In this study, we discovered that the midbody plays a role in the fibroblast-myofibroblast transition by mediating TGF-ß/Smad signaling. Combining bulk RNA-seq, histology, and the western blot of unilateral ureteral obstruction kidneys, we demonstrated that the pathway related to microtubules is implicated in kidney fibrosis, and the blocking of microtubule dynamics by colchicine improved kidney fibrosis. Subsequently, to explore microtubule-based organelles in detail, we cultured NRK-49F (rat kidney fibroblast cell line) and HKC-8 (human proximal tubule cell line) under transforming growth factor-ß1 (TGF-ß1) stimulation, which caused deciliation in both cell lines during epithelial-mesenchymal and fibroblast-myofibroblast transition. We identified another microtubule-based organelle, the midbody, whose formation is promoted by TGF-ß1 in fibroblasts as a result of proliferation in contrast to tubular cells. Notably, TGF-ß receptors were present in the midbody of both cell lines. In TGF-ß1-treated fibroblasts, colchicine or Hedgehog pathway inhibitor 4 impaired the midbody formation, and attenuated the upregulation of canonical TGF-ß/Smad signaling and α-SMA expression. These findings offer novel insight into the midbody as an active organelle involved in fibroblast-myofibroblast transition by mediating TGF-ß/Smad signaling, which could be a potential therapeutic target.

Workplace Violence Experienced by Community Mental Health Workers.

A cross-sectional online survey was conducted to 1) investigate the prevalence of workplace violence and workers' emotional distress, 2) explore factors associated with workplace violence, and 3) assess workers' needs for preventive measures. A total of 763 community mental health workers participated in Korea. Among them, 85.85% of workers experienced workplace violence, including verbal (74.31%), emotional (66.45%), infectious (47.44%), informational (42.60%), sexual (32.50%), and physical (23.72%) abuse. Binary logistic regression analysis indicated that sex, occupation, certification, and working institution were significantly associated with workplace violence. Workplace violence affects workers' depression, anger, and anxiety negatively. The most-needed preventive measure is a two-person home visit.

TDAG51 is a crucial regulator of maternal care and depressive-like behavior after parturition.

Postpartum depression is a severe emotional and mental disorder that involves maternal care defects and psychiatric illness. Postpartum depression is closely associated with a combination of physical changes and physiological stress during pregnancy or after parturition in stress-sensitive women. Although postpartum depression is relatively well known to have deleterious effects on the developing fetus, the influence of genetic risk factors on the development of postpartum depression remains unclear. In this study, we discovered a novel function of T cell death-associated gene 51 (TDAG51/PHLDA1) in the regulation of maternal and depressive-like behavior. After parturition, TDAG51-deficient dams showed impaired maternal behavior in pup retrieving, nursing and nest building tests. In contrast to the normal dams, the TDAG51-deficient dams also exhibited more sensitive depressive-like behaviors after parturition. Furthermore, changes in the expression levels of various maternal and depressive-like behavior-associated genes regulating neuroendocrine factor and monoamine neurotransmitter levels were observed in TDAG51-deficient postpartum brain tissues. These findings indicate that TDAG51 plays a protective role against maternal care defects and depressive-like behavior after parturition. Thus, TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition.

Nanoelectrical characterization of individual exosomes secreted by Aß42-ingested cells using electrostatic force microscopy.

Quantifying the physical properties of individual exosomes containing amyloid-ß42 (Aß42) is crucial for a better understanding of an underpinning mechanism of Alzheimer's disease expression which is associated with the Aß42 transfer. Because of the lack of proper tools, however, there have been very few studies on how the amount of Aß42 affects the physical properties of exosomes. To answer the question, we investigated the physical properties of exosomes secreted by neuroblastoma by probing individual exosomes using electrostatic force microscopy. Interestingly, we observed that when the higher concentration of Aß42 oligomers was fed to cells, the higher surface charge of the exosomes appeared. This result indicates that the exosomes contain more Aß42 with the increase in Aß42 concentration in cell media, implying that they serve as transport vesicles for Aß42. Our approach could help to better understand how the neuronal exosomes are related to the propagation of neurodegenerative diseases and to seek how to make an early diagnosis of those diseases.

Regulation of Osteoblast Differentiation by Cytokine Networks.

Osteoblasts, which are bone-forming cells, play pivotal roles in bone modeling and remodeling. Osteoblast differentiation, also known as osteoblastogenesis, is orchestrated by transcription factors, such as runt-related transcription factor 1/2, osterix, activating transcription factor 4, special AT-rich sequence-binding protein 2 and activator protein-1. Osteoblastogenesis is regulated by a network of cytokines under physiological and pathophysiological conditions. Osteoblastogenic cytokines, such as interleukin-10 (IL-10), IL-11, IL-18, interferon-γ (IFN-γ), cardiotrophin-1 and oncostatin M, promote osteoblastogenesis, whereas anti-osteoblastogenic cytokines, such as tumor necrosis factor-α (TNF-α), TNF-ß, IL-1α, IL-4, IL-7, IL-12, IL-13, IL-23, IFN-α, IFN-ß, leukemia inhibitory factor, cardiotrophin-like cytokine, and ciliary neurotrophic factor, downregulate osteoblastogenesis. Although there are gaps in the body of knowledge regarding the interplay of cytokine networks in osteoblastogenesis, cytokines appear to be potential therapeutic targets in bone-related diseases. Thus, in this study, we review and discuss our osteoblast, osteoblast differentiation, osteoblastogenesis, cytokines, signaling pathway of cytokine networks in osteoblastogenesis.

Pax5 Negatively Regulates Osteoclastogenesis through Downregulation of Blimp1.

Paired box protein 5 (Pax5) is a crucial transcription factor responsible for B-cell lineage specification and commitment. In this study, we identified a negative role of Pax5 in osteoclastogenesis. The expression of Pax5 was time-dependently downregulated by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulation in osteoclastogenesis. Osteoclast (OC) differentiation and bone resorption were inhibited (68.9% and 48% reductions, respectively) by forced expression of Pax5 in OC lineage cells. Pax5 led to the induction of antiosteoclastogenic factors through downregulation of B lymphocyte-induced maturation protein 1 (Blimp1). To examine the negative role of Pax5 in vivo, we generated Pax5 transgenic (Pax5Tg) mice expressing the human Pax5 transgene under the control of the tartrate-resistant acid phosphatase (TRAP) promoter, which is expressed mainly in OC lineage cells. OC differentiation and bone resorption were inhibited (54.2-76.9% and 24.0-26.2% reductions, respectively) in Pax5Tg mice, thereby contributing to the osteopetrotic-like bone phenotype characterized by increased bone mineral density (13.0-13.6% higher), trabecular bone volume fraction (32.5-38.1% higher), trabecular thickness (8.4-9.0% higher), and trabecular number (25.5-26.7% higher) and decreased trabecular spacing (9.3-10.4% lower) compared to wild-type control mice. Furthermore, the number of OCs was decreased (48.8-65.3% reduction) in Pax5Tg mice. These findings indicate that Pax5 plays a negative role in OC lineage specification and commitment through Blimp1 downregulation. Thus, our data suggest that the Pax5-Blimp1 axis is crucial for the regulation of RANKL-induced osteoclastogenesis.

Uric acid and inflammation in kidney disease.

Asymptomatic hyperuricemia is frequently observed in patients with kidney disease. Although a substantial number of epidemiologic studies have suggested that an elevated uric acid level plays a causative role in the development and progression of kidney disease, whether hyperuricemia is simply a result of decreased renal excretion of uric acid or is a contributor to kidney disease remains a matter of debate. Over the last two decades, multiple experimental studies have expanded the knowledge of the biological effects of uric acid beyond its role in gout. In particular, uric acid induces immune system activation and alters the characteristics of resident kidney cells, such as tubular epithelial cells, endothelial cells, and vascular smooth muscle cells, toward a proinflammatory and profibrotic state. These findings have led to an increased awareness of uric acid as a potential and modifiable risk factor in kidney disease. Here, we discuss the effects of uric acid on the immune system and subsequently review the effects of uric acid on the kidneys mainly in the context of inflammation.

Rhabdomyolysis-Induced AKI Was Ameliorated in NLRP3 KO Mice via Alleviation of Mitochondrial Lipid Peroxidation in Renal Tubular Cells.

INTRODUCTION: A recent study showed that early renal tubular injury is ameliorated in Nod-like receptor pyrin domain-containing protein 3 (NLRP3) KO mice with rhabdomyolysis-induced acute kidney injury (RIAKI). However, the precise mechanism has not been determined. Therefore, we investigated the role of NLRP3 in renal tubular cells in RIAKI. METHODS: Glycerol-mediated RIAKI was induced in NLRP3 KO and wild-type (WT) mice. The mice were euthanized 24 h after glycerol injection, and both kidneys and plasma were collected. HKC-8 cells were treated with ferrous myoglobin to mimic a rhabdomyolytic environment. RESULTS: Glycerol injection led to increase serum creatinine, aspartate aminotransferase (AST), and renal kidney injury molecule-1 (KIM-1) level; renal tubular necrosis; and apoptosis. Renal injury was attenuated in NLRP3 KO mice, while muscle damage and renal neutrophil recruitment did not differ between NLRP3 KO mice and WT mice. Following glycerin injection, increases in cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), and a decrease in the glutathione peroxidase 4 (GPX-4) level were observed in the kidneys of mice with RIAKI, and these changes were alleviated in the kidneys of NLRP3 KO mice. NLRP3 was upregulated, and cell viability was suppressed in HKC-8 cells treated with ferrous myoglobin. Myoglobin-induced apoptosis and lipid peroxidation were significantly decreased in siNLRP3-treated HKC-8 cells compared to ferrous myoglobin-treated HKC-8 cells. Myoglobin reduced the mitochondrial membrane potential and increased mitochondrial fission and reactive oxygen species (ROS) and lipid peroxidation levels, which were restored to normal levels in NLRP3-depleted HKC-8 cells. CONCLUSIONS: NLRP3 depletion ameliorated renal tubular injury in a murine glycerol-induced acute kidney injury (AKI) model. A lack of NLRP3 improved tubular cell viability via attenuation of myoglobin-induced mitochondrial injury and lipid peroxidation, which might be the critical factor in protecting the kidney.

Alterations in Lipid Profile of the Aging Kidney Identified by MALDI Imaging Mass Spectrometry.

During aging, the kidney undergoes functional and physiological changes that are closely affiliated with chronic kidney disease (CKD). There is increasing evidence supporting the role of lipid or lipid-derived mediators in the pathogenesis of CKD and other aging-related diseases. To understand the role of lipids in various metabolic processes during kidney aging, we conducted matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) analysis in kidneys harvested from young (2 months old, n = 3) and old mice (24 months old, n = 3). MALDI-IMS analysis showed an increase in ceramide level and a decrease in sphingomyelin (SM) and phosphatidylcholine (PC) levels in kidneys of old mice. The increased expression of cPLA2 and SMPD1 protein in aged kidney was confirmed by immunohistochemistry and Western blot analysis. Our MALDI-IMS data showed the altered distribution of lipids in aged kidney as indicative of aging-related functional changes of the kidney. Combined analysis of MALDI-IMS and IHC confirmed lipidomic changes and expression levels of responsible enzymes as well as morphological changes.

Evaluation of novel inactivated vaccines for the SAT 1, SAT 2 and SAT 3 serotypes of foot-and-mouth disease in pigs.

BACKGROUND: The foot-and-mouth disease (FMD) virus is classified into seven serotypes, of which the South African types have South African Territories (SAT)1, SAT2, and SAT3 that are prevalent in Africa. Especially SAT2 have spread to Arabian Peninsula and the Palestinian Autonomous Territories. Of these viruses, the incidence of SAT2 is the highest. It is important to prepare for the spread of the virus to other continents, even though most FMD viruses are bovine-derived. In particular, due to the high breeding density of pigs in Asia, more attention is usually paid to the immunity and protection of pigs than cattle. For this reason, this study investigated the immunity and protection of pigs against the SAT viruses. METHODS: Specific vaccines were developed for SAT1, SAT2, and SAT3 serotypes. These vaccine viruses were designed to be distinguished from the wild-type strain. An immunogenicity test was conducted using these vaccines in both cattle (n = 5/group) and pigs (n = 20/group). RESULTS: High virus-neutralizing titer of antibodies (> 1:100) was induced in only 2 weeks after the immunization of cattle with the individual vaccine for SAT1, SAT2 or SAT3, and a clear immune response was induced after the second immunization in pigs. When the vaccinated pigs (n = 4-5/group) were challenged by the homologous wild-type virus strain 4 weeks after immunization, all the pigs were protected from the challenge. CONCLUSIONS: This study confirmed that these vaccines can be used against SAT1, SAT2, and SAT3 viruses in cattle and pigs. The vaccine strains developed in this study are expected to be used as vaccines that can protect against FMD in the event of a future FMD outbreak in pigs in consideration of the situation in Asia.

Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer.

BACKGROUND: Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC. METHODS: Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel. RESULTS: We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93). CONCLUSIONS: We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

Impending extrusion of Ex-PRESS shunt treated by shunt-position adjustment: a case report.

BACKGROUND: To report a case of impending extrusion of Ex-PRESS shunt treated by shunt-position adjustment. CASE PRESENTATION: A 56-year-old Asian woman presented with impending extrusion and malposition of Ex-PRESS shunt in her left eye. The bleb of the left eye was shallow and diffuse. In the past, the patient was treated by Ex-PRESS shunt implantation under the scleral flap in both eyes. There had been no Ex-PRESS shunt-related complication in her right eye, and she reported no history of left-eye trauma. Based on these findings, we hypothesized that the source of the left-eye problem was a loosely fixed Ex-PRESS shunt spur. It was thought, furthermore, that this inadequate scleral resistance during the Ex-PRESS shunt implantation was due to the low scleral rigidity resulting from high myopia and insufficient maintenance of the anterior chamber. We proceeded to make an incision in the area adjacent to the Ex-PRESS shunt using a super sharp blade. The shunt was then pushed into the anterior chamber with forceps, and the spur was fixed firmly. Pushing the shunt to the anterior chamber was found to have been sufficient to fix it firmly. In fact, when the sclera was palpated with a sponge, aqueous outflow was observed with no shunt displacement. Postoperative intraocular pressure (IOP) was managed well, and the bleb had formed with diffuse, prominent shapes. The Ex-PRESS shunt was well sustained with good positioning. CONCLUSIONS: When an Ex-PRESS shunt operation is performed on a patient who shows a tendency for low scleral rigidity, shunt implantation should be accomplished carefully and with force adequate for firm spur fixation.

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis.

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.

Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.

Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 106 M-1 min-1) and paraoxon hydrolysis (0.015 × 106 M-1 min-1) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP.

Robust Real-Time Reverse Transcription-PCR for Detection of Foot-and-Mouth Disease Virus Neutralizing Carryover Contamination.

During an outbreak of foot-and-mouth disease (FMD), real-time reverse transcription-PCR (rRT-PCR) is the most commonly used diagnostic method to detect viral RNA. However, while this assay is often conducted during the outbreak period, there is an inevitable risk of carryover contamination. This study shows that the carryover contamination can be prevented by the use of target-specific restriction endonuclease in that assay.

Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs.

UNLABELLED: Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE: The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the virus has associated mechanisms of resistance to type I interferons and siRNAs. We have developed recombinant adenoviruses for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) to enhance the inhibitory effects of these antiviral agents observed in previous studies. Here, we show enhanced antiviral effects against FMDV by combination treatment with Ad-porcine IFN-αγ and Ad-3siRNA to overcome the mechanisms of resistance of FMDV in swine.

Hyperuricemia-induced NLRP3 activation of macrophages contributes to the progression of diabetic nephropathy.

IL-1ß-secreting nucleotide-binding oligomerization domain protein 3 (NLRP3) inflammasomes play a pivotal role in triggering innate immune responses in metabolic disease. We investigated the role of soluble uric acid in NLRP3 inflammasome activation in macrophages to demonstrate the effect of systemic hyperuricemia on progressive kidney damage in type 2 diabetes. THP-1 cells, human acute monocytic leukemia cells, were cultured to obtain macrophages, and HK-2 cells, human renal proximal tubule cells, were cultured and stimulated with uric acid. In vivo, we designed four rat groups as follows: 1) Long-Evans Tokushima Otsuka (LETO); 2) Otsuka Long-Evans Tokushima Fatty (OLETF); 3) OLETF+high-fructose diet (HFD) for 16 wk; and 4) OLETF+HFD+allopurinol (10 mg/dl administered in the drinking water). Soluble uric acid stimulated NLRP3 inflammasomes to produce IL-1ß in macrophages. Uric acid-induced MitoSOX mediates NLRP3 activation and IL-1ß secretion. IL-1ß from macrophages activates NF-κB in cocultured proximal tubular cells. In vivo, intrarenal IL-1ß expression and macrophage infiltration increased in HFD-fed OLETF rats. Lowering the serum uric acid level resulted in improving the albuminuria, tubular injury, macrophage infiltration, and renal IL-1ß (60% of HFD-fed OLETF) independently of glycemic control. Direct activation of proximal tubular cells by uric acid resulted in (C-X-C motif) ligand 12 and high mobility group box-1 release and accelerated macrophage recruitment and the M1 phenotype. Taken together, these data support direct roles of hyperuricemia in activating NLRP3 inflammasomes in macrophages, promoting chemokine signaling in the proximal tubule and contributing to the progression of diabetic nephropathy through cross talk between macrophages and proximal tubular cells.

Inactivation of foot-and-mouth disease virus by citric acid and sodium carbonate with deicers.

Three out of five outbreaks of foot-and-mouth disease (FMD) since 2010 in the Republic of Korea have occurred in the winter. At the freezing temperatures, it was impossible to spray disinfectant on the surfaces of vehicles, roads, and farm premises because the disinfectant would be frozen shortly after discharge and the surfaces of the roads or machines would become slippery in cold weather. In this study, we added chemical deicers (ethylene glycol, propylene glycol, sodium chloride, calcium chloride, ethyl alcohol, and commercial windshield washer fluid) to keep disinfectants (0.2% citric acid and 4% sodium carbonate) from freezing, and we tested their virucidal efficacies under simulated cold temperatures in a tube. The 0.2% citric acid could reduce the virus titer 4 logs at -20°C with all the deicers. On the other hand, 4% sodium carbonate showed little virucidal activity at -20°C within 30 min, although it resisted being frozen with the function of the deicers. In conclusion, for the winter season, we may recommend the use of citric acid (>0.2%) diluted in 30% ethyl alcohol or 25% sodium chloride solvent, depending on its purpose.