Increase of viral hemorrhagic septicemia virus growth by knockout of IRF9 gene in Epithelioma papulosum cyprini cells.

Viral hemorrhagic septicemia virus (VHSV) has been a notorious pathogen in freshwater and marine fish. Due to the lack of effective treatment measures against VHSV disease, the development of prophylactic vaccines has been required, and methods that can produce high-titered viruses would be advantageous in producing cost-effective vaccines. Type I interferon (IFN) responses are the key elements of vertebrates' antiviral activities, and IFN-stimulated gene factor 3 (ISGF3) complex formed through type I IFNs up-regulates the expression of IFN-stimulated genes (ISGs). IFN regulatory factor 9 (IRF9) is a key component of ISGF3, so the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity. In this study, to increase the replication of VHSV, we generated IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contains an EPC cell's U6 promoter-driven guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette. In the clones of IRF9 knockout EPC cells, there were no increase in ISG15 gene by poly I:C, and in Mx1 gene by both poly I:C and VHSV. Interestingly, although the increased folds were conspicuously lower than control EPC cells, the expression of ISG 15 gene in all the IRF9 knockout clones was significantly increased by VHSV infection. Control EPC cells pre-treated with poly I:C did not show any CPE when infected with VHSV, however, IRF9 knockout EPC cells showed CPE by VHSV infection in spite of being pretreated with poly I:C. The replication of VHSV in IRF9 knockout EPC cells was significantly faster and higher than that in control EPC cells indicating that the IRF9 knockout-mediated decrease of type I IFN responses allowed VHSV to replicate efficiently. Considering an economical aspect for the production of fish vaccines, the present IRF9 knockout EPC cells can be used to get higher-titered VHSV.

Inflammation-Targeting Mesenchymal Stem Cells Combined with Photothermal Treatment Attenuate Severe Joint Inflammation.

Current clinical therapeutic efficacy for the treatment of osteo- and rheumatoid-arthritis is obviously limited. Although mesenchymal stem cells (MSCs) are considered as a source of promising regenerative therapy, un-modified or genetically engineered MSCs injected in vivo restrict their clinical utility because of the low drug efficacy and unpredicted side effect, respectively. Herein, a strategy to enhance the migration efficacy of MSCs to inflamed joints via an inflammation-mediated education process is demonstrated. To reinforce the limited anti-inflammatory activity of MSCs, gold nanostar loaded with triamcinolone is conjugated to MSC. Furthermore, near-infrared laser-assisted photothermal therapy (PTT) induced by gold nanostar significantly elevates the anti-inflammatory efficacy of the developed drugs, even in advanced stage arthritis model. An immunological regulation mechanism study of PTT is first suggested in this study; the expression of the interleukin 22 receptor, implicated in the pathogenesis of arthritis, is downregulated in T lymphocytes by PTT, and Th17 differentiation from naïve CD4 T cell is inhibited. Collectively, inflammation-targeting MSCs conjugated with triamcinolone-loaded gold nanostar (Edu-MSCs-AuS-TA) promote the repolarization of macrophages and decrease neutrophil recruitment in joints. In addition, Edu-MSCs-AuS-TA significantly alleviate arthritis-associated pain, improve general locomotor activity, and more importantly, induce cartilage regeneration even for severe stages of arthritis model.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Greater Plasma Protein Adsorption on Mesoporous Silica Nanoparticles Aggravates Atopic Dermatitis.

Purpose: The protein corona surrounding nanoparticles has attracted considerable attention as it induces subsequent inflammatory responses. Although mesoporous silica nanoparticles (MSN) are commonly used in medicines, cosmetics, and packaging, the inflammatory effects of the MSN protein corona on the cutaneous system have not been investigated till date. Methods: We examined the greater plasma protein adsorption on MSN leads to serious inflammatory reactions in Dermatophagoides farinae extract (DFE)-induced mouse atopic dermatitis (AD)-like skin inflammation because of increased uptake by keratinocytes. Results: We compare the AD lesions induced by MSN and colloidal (non-porous) silica nanoparticles (CSN), which exhibit different pore architectures but similar dimensions and surface chemistry. MSN-corona treatment of severe skin inflammation in a DFE-induced in vivo AD model greatly increases mouse ear epidermal thickness and infiltration of immune cells compared with the CSN-corona treatment. Moreover, MSN-corona significantly increase AD-specific immunoglobulins, serum histamine, and Th1/Th2/Th17 cytokines in the ear and lymph nodes. MSN-corona induce more severe cutaneous inflammation than CSN by significantly decreasing claudin-1 expression. Conclusion: This study demonstrates the novel impact of the MSN protein corona in inducing inflammatory responses through claudin-1 downregulation and suggests useful clinical guidelines for MSN application in cosmetics and drug delivery systems.

Stem Cell Mimicking Nanoencapsulation for Targeting Arthritis.

Mesenchymal stem cells (MSCs) are considered a promising regenerative therapy due to their ability to migrate toward damaged tissues. The homing ability of MSCs is unique compared with that of non-migrating cells and MSCs are considered promising therapeutic vectors for targeting major cells in many pathophysiological sites. MSCs have many advantages in the treatment of malignant diseases, particularly rheumatoid arthritis (RA). RA is a representative autoimmune disease that primarily affects joints, and secreted chemokines in the joints are well recognized by MSCs following their migration to the joints. Furthermore, MSCs can regulate the inflammatory process and repair damaged cells in the joints. However, the functionality and migration ability of MSCs injected in vivo still show insufficient. The targeting ability and migration efficiency of MSCs can be enhanced by genetic engineering or modification, eg, overexpressing chemokine receptors or migration-related genes, thus maximizing their therapeutic effect. However, there are concerns about genetic changes due to the increased probability of oncogenesis resulting from genome integration of the viral vector, and thus, clinical application is limited. Furthermore, it is suspected that administering MSCs can promote tumor growth and metastasis in xenograft and orthotopic models. For this reason, MSC mimicking nanoencapsulations are an alternative strategy that does not involve using MSCs or bioengineered MSCs. MSC mimicking nanoencapsulations consist of MSC membrane-coated nanoparticles, MSC-derived exosomes and artificial ectosomes, and MSC membrane-fused liposomes with natural or genetically engineered MSC membranes. MSC mimicking nanoencapsulations not only retain the targeting ability of MSCs but also have many advantages in terms of targeted drug delivery. Specifically, MSC mimicking nanoencapsulations are capable of encapsulating drugs with various components, including chemotherapeutic agents, nucleic acids, and proteins. Furthermore, there are fewer concerns over safety issues on MSC mimicking nanoencapsulations associated with mutagenesis even when using genetically engineered MSCs, because MSC mimicking nanoencapsulations use only the membrane fraction of MSCs. Genetic engineering is a promising route in clinical settings, where nano-encapsulated technology strategies are combined. In this review, the mechanism underlying MSC homing and the advantages of MSC mimicking nanoencapsulations are discussed. In addition, genetic engineering of MSCs and MSC mimicking nanoencapsulation is described as a promising strategy for the treatment of immune-related diseases.