Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

Antitumor activity, pharmacokinetics, tumor-homing effect, and hepatotoxicity of a species cross-reactive c-Met antibody.

The receptor tyrosine kinase c-Met plays critical roles in promoting tumor growth, invasion, metastasis, and angiogenesis in various types of cancer and is a promising therapeutic target. The development of a species cross-reactive therapeutic antibody could provide useful to comprehensive preclinical assessment in animal models. Towards this goal, we developed human/mouse cross-reactive c-Met antibodies using an antibody phage library. IRCR201, a c-Met antibody with species cross-reactivity, successfully inhibited the HGF/c-Met signaling pathway via degradation of c-Met and disruption of the binding with its partners, and demonstrated strong in vivo antitumor activity. In pharmacokinetic analysis, IRCR201 exhibited a nonlinear pharmacokinetic profile and showed rapid serum clearance at low dosage. Ex vivo fluorescence imaging and immunohistochemistry demonstrated strong tumor accumulation of IRCR201. Hepatotoxicity analysis revealed that IRCR201 does not significantly affect primary human and mouse hepatocytes. Serum chemistry analysis demonstrated that the alanine aminotransferase serum level was elevated in mice treated with 30 mg/kg IRCR201 than in PBS-treated mice, whereas the levels of aspartate aminotransferase and blood urea nitrogen did not significantly differ. Thus, IRCR201 is a potent therapeutic antibody that can disrupt the HGF/c-Met signaling axis and its species cross-reactivity would enable to evaluate precise biological activity in animal models.

Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain.

Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.

Vaccinia-based influenza vaccine overcomes previously induced immunodominance hierarchy for heterosubtypic protection.

Growing concerns about unpredictable influenza pandemics require a broadly protective vaccine against diverse influenza strains. One of the promising approaches was a T cell-based vaccine, but the narrow breadth of T-cell immunity due to the immunodominance hierarchy established by previous influenza infection and efficacy against only mild challenge condition are important hurdles to overcome. To model T-cell immunodominance hierarchy in humans in an experimental setting, influenza-primed C57BL/6 mice were chosen and boosted with a mixture of vaccinia recombinants, individually expressing consensus sequences from avian, swine, and human isolates of influenza internal proteins. As determined by IFN-γ ELISPOT and polyfunctional cytokine secretion, the vaccinia recombinants of influenza expanded the breadth of T-cell responses to include subdominant and even minor epitopes. Vaccine groups were successfully protected against 100 LD50 challenges with PR/8/34 and highly pathogenic avian influenza H5N1, which contained the identical dominant NP366 epitope. Interestingly, in challenge with pandemic A/Cal/04/2009 containing mutations in the dominant epitope, only the group vaccinated with rVV-NP + PA showed improved protection. Taken together, a vaccinia-based influenza vaccine expressing conserved internal proteins improved the breadth of influenza-specific T-cell immunity and provided heterosubtypic protection against immunologically close as well as distant influenza strains.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. METHODS: Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. RESULTS: Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. CONCLUSIONS: Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Transgenic viral expression of PH-20, IL-12, and sPD1-Fc enhances immune cell infiltration and anti-tumor efficacy of an oncolytic virus.

Oncolytic viruses are of significant clinical interest due to their ability to directly infect and kill tumors and enhance the anti-tumor immune response. Previously, we developed KLS-3010, a novel oncolytic virus derived from the International Health Department-White (IHD-W) strain vaccinia virus, which has robust tumoricidal effects. In the present study, we generated a recombinant oncolytic virus, KLS-3020, by inserting three transgenes (hyaluronidase [PH-20], interleukin-12 [IL-12], and soluble programmed cell death 1 fused to the Fc domain [sPD1-Fc]) into KLS-3010 and investigated its anti-tumor efficacy and ability to induce anti-tumor immune responses in CT26.WT and B16F10 mouse tumor models. A single injection of KLS-3020 significantly decreased tumor growth. The roles of the transgenes were investigated using viruses expressing each single transgene alone and KLS-3020. PH-20 promoted virus spread and tumor immune cell infiltration, IL-12 activated and reprogrammed T cells to inflammatory phenotypes, and sPD1-Fc increased intra-tumoral populations of activated T cells. The tumor-specific systemic immune response and the abscopal tumor control elicited by KLS-3020 were demonstrated in the CT26.WT tumor model. The insertion of transgenes into KLS-3020 increased its anti-tumor efficacy, supporting further clinical investigation of KLS-3020 as a novel oncolytic immunotherapy.

The semaphorin 3A/neuropilin-1 pathway promotes clonogenic growth of glioblastoma via activation of TGF-ß signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-ß receptor 1 (TGF-ßR1), which in turn leads to activation of canonical TGF-ß signaling in both GSCs and NPCs. TGF-ß signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-ß pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-ßR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Pharmacogenomic profiling reveals molecular features of chemotherapy resistance in IDH wild-type primary glioblastoma.

BACKGROUND: Although temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application. METHODS: We stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n = 29) and sensitive (n = 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy. RESULTS: In vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P = 1.12e-4 for progression-free survival (PFS) and 3.63e-4 for overall survival (OS)). Moreover, we found that elevated gene expression of EGR4, PAPPA, LRRC3, and ANXA3 was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration in PTEN, EGFR, and CDKN2A/B was more frequent in TMZ-sensitive samples (Fisher's exact P = 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P = 4.58e-4 for PFS and 3.66e-4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage ( http://www.wang-lab-hkust.com:3838/TMZEP ). CONCLUSIONS: We identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.

Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site.

Many proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effects in vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV(9)-IV(10)-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic process in vitro and analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical significance of these findings will require further validation in vivo.

Optimization of linear double-stranded RNA for the production of multiple siRNAs targeting hepatitis C virus.

RNA interference (RNAi)-based gene silencing possesses great therapeutic potential for inhibiting replication of human viruses such as hepatitis C virus (HCV). However, one of the putative limitations for its use as a therapy is the rapid emergence of escape variants. These contain deletions or mutations within the viral genome sequences complementary to the small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) being used for treatment. As a potential solution to this problem, we constructed an expression system for duplex RNAs harboring two siRNA units using convergent H1 and U6 Pol III promoters. Here, the length and orientation of the transcript, tandem siRNA (tsiRNA), were optimized to be processed by the intracellular ribonuclease Dicer into functional siRNAs targeting different sequences. Assessment in transfected cells indicates that the length of the tsiRNA duplex (40-42 base pairs) is more critical for both siRNA-producing capacity and gene silencing activity than the orientation of each siRNA unit. In Huh7 cells replicating full-length HCV RNA, expression of length-optimized tsiRNA inhibited viral protein levels as efficiently as a single 21-nucleotide siRNA-expression construct, without affecting miRNA maturation or induction of an interferon response. We verified that the anti-viral activity of tsiRNA was achieved by precise cleavage of two target sites. A distinct advantage of this strategy is that each side of the optimized linear duplex RNA could enter into the Dicer-mediated processing machinery, thus likely providing more equal and efficient production of multiple siRNAs required for reducing the chance of viral escape.

Sequencing and characterization of Varicella-zoster virus vaccine strain SuduVax.

BACKGROUND: Varicella-zoster virus (VZV) causes chickenpox in children and shingles in older people. Currently, live attenuated vaccines based on the Oka strain are available worldwide. In Korea, an attenuated VZV vaccine has been developed from a Korean isolate and has been commercially available since 1994. Despite this long history of use, the mechanism for the attenuation of the vaccine strain is still elusive. We attempted to understand the molecular basis of attenuation mechanism by full genome sequencing and comparative genomic analyses of the Korean vaccine strain SuduVax. RESULTS: SuduVax was found to contain a genome that was 124,759 bp and possessed 74 open reading frames (ORFs). SuduVax was genetically most close to Oka strains and these Korean-Japanese strains formed a strong clade in phylogenetic trees. SuduVax, similar to the Oka vaccine strains, underwent T- > C substitution at the stop codon of ORF0, resulting in a read-through mutation to code for an extended form of ORF0 protein. SuduVax also shared certain deletion and insertion mutations in ORFs 17, 29, 56 and 60 with Oka vaccine strains and some clinical strains. CONCLUSIONS: The Korean VZV vaccine strain SuduVax is genetically similar to the Oka vaccine strains. Further comparative genomic and bioinformatics analyses will help to elucidate the molecular basis of the attenuation of the VZV vaccine strains.

Development of a new advanced animal cradle for small animal multiple imaging modalities: acquisition and evaluation of high-throughput multiple-mouse imaging.

The physiological conditions of small animals are an essential component to be considered when acquiring images for pre-clinical studies, and they play a vital role in the overall results of a study. However, several previous studies did not consider these conditions. In this study, a new animal cradle that can be modified and adjusted to suit multiple imaging modalities such as positron emission tomography (PET)/computed tomography (CT) and magnetic resonance imaging (MRI) was developed. Unlike previous cradles where only one mouse can be imaged at a time, a total of four mice can be imaged simultaneously using this new cradle. Additionally, fusion images with high-throughput multiple-mouse imaging (MMI) of PET/MRI and PET/CT images can be acquired using this newly developed cradle. The dynamic brain images were also acquired simultaneously by applying PET dynamic imaging technology to high-throughput MMI methods. The results of this study suggest that the newly developed small animal cradle can be widely used in pre-clinical studies.

Characterization of Gut Microbiome in Korean Patients with Metabolic Associated Fatty Liver Disease.

Metabolic associated fatty liver disease (MAFLD) is a new concept where the presence of both fatty liver and metabolic abnormality are necessary for diagnosis. Several studies have reported that altered gut microbiome is closely associated with metabolic diseases and non-alcoholic fatty liver disease. However, the studies on MAFLD population are scarce. This prospective study aimed to identify differences in gut microbiome between patients with MAFLD and healthy controls in Korean population. In this study, patients with MAFLD and age, sex-matched healthy controls were included, and their stool samples were collected. Taxonomic composition of gut microbiota was analyzed using 16S ribosomal ribonucleic acid pyrosequencing. Twenty-two MAFLD patients and 44 healthy controls were included. Taxonomic diversity was lower in patients with MAFLD in the aspect of alpha and beta diversity. The differences were also found at phylum, class, family, and genus levels between the two groups. Phylum Proteobacteria, family Enterobactereriaceae, genus Citrobacter abundance was significantly increased and genus Faecalibacterium was significantly decreased in patients with MAFLD. In addition, butyrate-producing bacteria were decreased and ethanol-producing bacteria were increased in patients with MAFLD. The composition of gut microbiome was different between MAFLD and healthy controls in Korean population. This could offer potential targets for therapeutic intervention in MAFLD.

Membrane-based hybridization capture of intracellular peptide nucleic acid.

Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized, mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here we present a PNA detection method: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer to a Western blotting membrane and then hybridization with a radiolabeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization.

cIRCR201-dPBD, a Novel Pyrrolobenzodiazepine Dimer-Containing Site-Specific Antibody-Drug Conjugate Targeting c-Met Overexpression Tumors.

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody-drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug-conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC-MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

Double-filtered leukoreduction as a method for risk reduction of transfusion-associated graft-versus-host disease.

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GvHD) is caused by leukocytes, specifically T cells within a transfused blood product. Currently, the prevention of transfusion-associated graft-versus-host disease is performed by irradiation of blood products. With a sufficient reduction of leukocytes, the risk for TA-GvHD can be decreased. With consistent advances in current state-of-the-art blood filters, we herein propose that double filtration can sufficiently reduce leukocytes to reduce the risk for TA-GvHD. MATERIALS: Thirty RBC concentrates were filtered with leukocyte filters, followed by storage at 1-6 oC for 72 hours, and then a second filtration was performed. Residual leukocytes in the double-filtered RBC units (n = 30) were assessed with flow cytometric methods, and an additional assay with isolated peripheral blood mononuclear cells (PBMCs) (n = 6) was done by both flow cytometric methods and an automated hematology analyzer. Quality of the RBCs after filtration was evaluated by hematological and biochemical tests. In vitro T cell expansion was performed using anti-CD3/CD28-coated Dynabeads or anti-CD3 (OKT3). In vivo experiment for GvHD was performed by using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. RESULTS: Double-filtered blood products showed residual leukocyte levels below detection limits, which calculated to be below 1200-2500 cells per blood unit. In vitro expansion rate of T cells showed that 6x103 and 1x103 cell-seeded specimens showed 60.8±10.6 fold and 10.2±9.7-fold expansion, respectively. Cell expansion was not sufficiently observed in wells planted with 1x102 or 10 cells. In vivo experiments showed that mice injected with 1x105 or more cells cause fatal GvHD. GvHD induced inflammation was observed in mice injected with 1x104 or more cells. No evidence of GvHD was found in mice injected with 103 cells. CONCLUSIONS: Our study suggests that additional removal of contaminating lymphocytes by a second leukodepletion step may further reduce the risk for TA-GvHD.

Targeted delivery of siRNA against hepatitis C virus by apolipoprotein A-I-bound cationic liposomes.

BACKGROUND/AIMS: Hepatitis C virus (HCV) is one of the major human hepatic RNA viruses. Recently, we developed a liver-specific siRNA delivery technology using DTC-Apo composed of cationic liposomes (DTC) and apolipoprotein A-I (apo A-I). Here, we investigated whether DTC-Apo nanoparticles can systemically deliver siRNA into mouse hepatocytes expressing HCV proteins and inhibit their expression efficiently. METHODS: A transient HCV model was constructed by hydrodynamic injection of plasmid DNA expressing viral structural proteins under hepatic control region and alpha1-antitrypsin promoter elements. Using this model, DTC-Apo containing HCV-core-specific siRNA was intravenously injected to assess antiviral activity as well as the duration of silencing. RESULTS: Post-administration of DTC-Apo/HCV-specific siRNA at a dose of 2mg siRNA/kg inhibited viral gene expression by 65-75% in the liver on day 2. Improved activity (95% knockdown on day 2) without immunotoxicity was obtained by 2'-OMe-modification at two U sequences on its sense strand. Notably, the gene silencing effect of the modified siRNA was still maintained at day 6, while the unmodified one lost RNAi activity after day 4. CONCLUSIONS: Our results suggest that DTC-Apo liposome is a highly potential delivery vehicle to transfer therapeutic siRNA especially targeting HCV to the liver.

Hepatic siRNA delivery using recombinant human apolipoprotein A-I in mice.

Apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein (HDL), plays a key role in reverse cholesterol transport from peripheral tissues to liver or steroidogenic organs. Class B, type 1 scavenger receptor (SR-BI) is abundantly expressed in these target tissues and recognizes apo A-I of HDL for selective cholesteryl ester uptake. Recently, we reported the liver-targeting potential of plasma-derived apo A-I and the efficient delivery of therapeutic small interfering RNAs (siRNA) assembled with cationic liposome and apo A-I. In this study, we expressed and purified recombinant human apo A-I (rhapo A-I), low endotoxin grade, from an Escherichia coli expression system. The liver-targeting property of rhapo A-I was compared to that of plasma-derived apo A-I. Using a hepatitis C virus mouse model, intravenous administration of virus-specific siRNA with liposome and rhapo A-I significantly inhibited viral protein expression, demonstrating great promise for its use in clinical applications.

Oral administration of 1,4-aryl-2-mercaptoimidazole inhibits T-cell proliferation and reduces clinical severity in the murine experimental autoimmune encephalomyelitis model.

T cells play a pivotal role in the initiation and progression of multiple sclerosis. We have found that 1,4-aryl-2-mercaptoimidazole (KRM-III) inhibited T-cell antigen receptor- and phorbol myristate acetate/ionomycin-induced activation of nuclear factor of activated T cells (NFAT) and T-cell proliferation with an IC(50) of 5 microM. The KRM-III-mediated inhibitory effect was specific for NFAT activation but not for nuclear factor kappaB. Oral administration of 90 mg/kg KRM-III resulted in complete abrogation of anti-CD3 antibody-induced T-cell activation and a 45.8% reduction in footpad swelling in bovine serum albumin-induced delayed-type hypersensitivity. In the murine experimental autoimmune encephalomyelitis (EAE) model, oral administration of KRM-III significantly attenuated the severity of disease when given before or after disease onset. Draining lymph node cells from KRM-III-treated mice showed markedly reduced proliferation in response to myelin oligodendrocyte glycoprotein peptide. Histological analysis indicated that KRM-III reduced the infiltration of inflammatory cells to the white matter of spinal lumbar cords. These results demonstrate that KRM-III efficiently inhibits T-cell activation and inflammatory responses and lessens EAE clinical signs, which suggest KRM-III as a potential lead compound for the treatment of T-cell-driven autoimmune diseases.

Innovative Distribution Priorities for the Medical Devices Industry in the Fourth Industrial Revolution.

PURPOSE: This study aimed to set priorities for improving the medical device distribution structure and to suggest an innovative improvement plan for the distribution structure using the analytic hierarchy process (AHP) method, focusing on stakeholders in the medical device industry. METHODS: This study conducted a survey with 35 specialists using the AHP method, which is a multiple-criteria decisionmaking methodology, in order to set priorities for improvement plans to address the problems faced by the medical device distribution structure. RESULTS: The AHP analysis showed that supply stability was the most important factor, followed by greater transparency, efficiency, smart supply, and cost reduction. CONCLUSIONS: It is necessary to establish a stable supply system and manage crises through supply stability, as well as to provide opportunities for fair trade through greater transparency. As steps towards those goals, we propose establishing a unique device identification system, an information disclosure system, online distribution, and a group purchasing organization system in Korea.