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Recent strategies in cancer immunotherapy based on interleukin-2 (IL-2) are generally focused on reducing regulatory T cell (Treg) development by modifying IL-2 receptor alpha (IL-2Rα) domain. However, the clinical utility of high-dose IL-2 treatment is mainly limited by severe systemic toxicity. We find that peritumorally injectable 'BALLkine-2', recombinant human IL-2 (rIL-2) loaded porous nanoparticle, dramatically reduces systemic side effects of rIL-2 by minimizing systemic IL-2 exposure. Notably, in cynomolgus monkeys, subcutaneous (SC)-injection of BALLkine-2 not only dramatically reduces systemic circulation of rIL-2 in the blood, but also increases half-life of IL-2 compared to IV- or SC-injection of free rIL-2. Peritumorally-injected BALLkine-2 enhances intratumoral lymphocyte infiltration without inducing Treg development and more effectively synergizes with PD-1 blockade than high-dose rIL-2 administration in B16F10 melanoma model. BALLkine-2 could be a highly potent therapeutic option due to higher anti-tumor efficacy with lower and fewer doses and reduced systemic toxicity compared to systemic rIL-2.

Artificial, synthetic chaperones have attracted much attention in biomedical research due to their ability to control the folding of proteins and peptides. Here, we report bio-inspired multifunctional porous nanoparticles to modulate proper folding and intracellular delivery of therapeutic α-helical peptide. The Synthetic Nano-Chaperone for Peptide (SNCP) based on porous nanoparticles provides an internal hydrophobic environment which contributes in stabilizing secondary structure of encapsulated α-helical peptides due to the hydrophobic internal environments. In addition, SNCP with optimized inner surface modification not only improves thermal stability for α-helical peptide but also supports the peptide stapling methods in situ, serving as a nanoreactor. Then, SNCP subsequently delivers the stabilized therapeutic α-helical peptides into cancer cells, resulting in high therapeutic efficacy. SNCP improves cellular uptake and bioavailability of the anti-cancer peptide, so the cancer growth is effectively inhibited in vivo. These data indicate that the bio-inspired SNCP system combining nanoreactor and delivery carrier could provide a strategy to expedite the development of peptide therapeutics by overcoming existing drawbacks of α-helical peptides as drug candidates.

Among various strategies to treat neurodegenerative disorders, cell replacement therapies have drawn much attention recently. Such a trend led to the increase in demand for the rare and specialized cells, followed by the outburst development of various cell reprogramming strategies. However, several limitations on these conventional methods remain to be solved, including the genetic instability of the viral vectors and the high cytotoxicity or poor performance of the non-viral carriers. Therefore, non-viral methods need to be developed to ensure safe and efficient cell reprogramming. Here, we introduce a polymer-modified nano-reagent (Polymer-functionalized Nanodot, PolyN) for the safe and efficient, non-viral direct cell reprogramming. PolyN facilitated the highly efficient contemporary overexpression of the transgene compared to the conventional reagent. With our nano-reagent, we demonstrated the SOX2-mediated cell reprogramming and successfully generated the neuron-like cell from the human fibroblast.

We report a novel high-throughput screening tool for drug discovery of direct-acting antiviral agents against RNA viruses., Direct-acting agents against viral components are considered as the most promising candidates for the successful antiviral therapeutics. To date, no direct-acting drugs exist for the treatment against dengue virus (DV) infection, which can develop into life-threatening diseases. RNA-dependent RNA polymerase (RdRp), an RNA virus–specific enzyme highly conserved among various viral families, has been known as the broad-range antiviral drug target. Here, we developed an RNA-based graphene biosensor system [RNA nano-graphene oxide system (RANGO)] to enable the fluorescence-based quantitative analysis of the RdRp enzyme activity. We used the RANGO system to a high-throughput chemical screening to identify novel direct-acting antiviral drug candidates targeting DV RdRp from the FDA-approved small-molecule library. RANGO accelerated the massive selection of drug candidates. We found that one of the selected hit compounds, montelukast, showed antiviral activity in vitro and in vivo by directly inhibiting replication of DV and thus relieved related symptoms.

Skin fibrosis occurs in a variety of human diseases but the current anti-fibrosis treatments are not sufficient. One major cause of fibrotic diseases shared across diverse organ fibrosis is uncontrolled overexpression of the connective tissue growth factor (CTGF, also known as CCN2). Here, we examine the anti-fibrotic activity of RNAi therapy utilizing siRNA against CTGF with a new drug delivery system (DDS), 'DegradaBALL', which is based on porous nanoparticles, for durable CTGF gene silencing. DegradaBALL is a modular DDS having many favorable properties for RNA delivery such as effective intracellular uptake, convenient drug loading, biocompatibility, sustained release profile and biodegradability. DegradaBALL loaded with siCTGF, named 'LEM-S401', showed highly durable and effective CTGF gene-silencing in TGF-β induced lung fibrosis and skin fibrosis model cells, A549 and HaCaT, respectively. In addition, LEM-S401 induced knockdown of collagen types I and III, which are excess extracellular matrix components in fibrotic skin in addition to CTGF in the mouse wound healing model. Most importantly, we showed that LEM-S401 effectively inhibited the formation of hypertrophic scars in wound-associated dermal fibrosis mouse models, during both the epidermis recovery and tissue remodeling process. Our findings suggest that LEM-S401 could be a highly potent therapeutic option for skin fibrotic diseases.

Small interfering RNA (siRNA) is an attractive therapeutic candidate for sequence-specific gene silencing to treat incurable diseases using small molecule drugs. However, its efficient intracellular delivery has remained a challenge. Here, we have developed a highly biocompatible fluorescent carbon dot (CD), and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo. We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA, via endocytosis in tumor cells, with low cytotoxicity and unexpected immune responses. Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h. Moreover, we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy. We have developed a multifunctional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression, that shows excellent potential for gene therapy.

The mesoporous silica nanoparticle (MSN) has been utilized for various drug delivery systems. For the efficient loading and functional delivery of bioactive molecules, the pore size should be finely controlled. Here, we synthesize MSNs having different pore sizes and investigate the relationship between the pore size of the MSN and the efficacy and kinetics of gene silencing induced by siRNA loaded MSNs. We found that MSNs with various pore sizes present great differences in gene down-regulation of green fluorescence protein (GFP), vascular endothelial growth factor (VEGF) and hepatitis C virus non-structural protein 3 (HCV NS3) as well as the endocytosis pathways of MSNs in cancer cells. In specific, MSNs with a 7 and 10 nm pore size show the most significant gene knockdown with a long-lasting effect and great protective effect of the cargo from nuclease-mediated degradation compared to MSNs with a 2, 4 and 23 nm pore size. In addition, although similar amounts of siRNA was delivered inside cells, the degree of gene knockdown was significantly different depending on pore size of the employed MSN. The present study indicates that the porosity in nanoparticles acts as a crucial factor in a potent biomolecule cargo delivery system and the optimized MSN based system should provide better opportunities in the application of porous nanoparticles in biomedical research and clinical applications in the future.

MicroRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes. Therefore, control over the disease-related miRNA expression is important both for basic research and for a new class of therapeutic modality to treat serious diseases such as cancer. Here, we present a high-throughput screening strategy to identify small molecules that modulate miRNA expression in living cells. The screen enables simultaneous monitoring of the phenotypic cellular changes associated with the miRNA expression by measuring quantitative fluorescent signals corresponding to target miRNA level in living cells based on a novel biosensor composed of peptide nucleic acid and nano-sized graphene oxide. In this study, the biosensor based cellular screening of 967 compounds (including FDA-approved drugs, enzyme inhibitors, agonists, and antagonists) in cells identified four different classes of small molecules consisting of (i) 70 compounds that suppress both miRNA-21 (miR-21) expression and cell proliferation, (ii) 65 compounds that enhance miR-21 expression and reduce cell proliferation, (iii) 2 compounds that suppress miR-21 expression and increase cell proliferation, and (iv) 21 compounds that enhance both miR-21 expression and cell proliferation. We further investigated the hit compounds to correlate cell morphology changes and cell migration ability with decreased expression of miR-21.

Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin‐6/signal transducer and activator of transcription 3 (IL‐6/STAT3) signaling up‐regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL‐6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL‐6, with a concomitant decrease of hypoxia‐inducible factor 1 alpha (HIF‐1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF‐κB) p65 subunit to positively regulate the transcription of HIF‐1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF‐1α and CD133 expression were not observed in Toll‐like receptor 4/IL‐6 double‐knockout mice. Long‐term silencing of CD133 by a lentiviral‐based approach inhibited cancer cell‐cycle progression and suppressed in vivo tumorigenicity by down‐regulating expression of cytokinesis‐related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF‐1α proteins. Conclusion: IL‐6/STAT3 signaling induces expression of CD133 through functional cooperation with NF‐κB and HIF‐1α during liver carcinogenesis. Targeting STAT3‐mediated CD133 up‐regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment. (Hepatology 2015;62:1160‐1173)

An improved cell conversion strategy for neural differentiation of mouse embryonic stem (mES) cells is developed by incorporating functionalized mesoporous silica nanoparticle (MSN) as an efficient delivery carrier of retinoic acid (RA), which is a pleiotropic factor required for initiation of neural differentiation. Traditional RA-mediated neural differentiation methods required either preactivation of the cells to the differentiating state by embryoid body (EB) formation or repetitive treatment of the differentiation factor. Our modified cell conversion system involves only singular treatment of the RA/MSN complex, which simplified the whole process and accelerated neural induction to be finished within 6 days with high quality. With our new method, neural cells were successfully derived from mES cells with stable expression of neurite marker gene.

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.

Aberrantly activated signal transducer and activator of transcription (STAT) proteins are implicated with human cancers and represent essential roles for cancer cell survival and proliferation. Therefore, the development of small-molecule inhibitors of STAT signaling bearing pharmacological activity has therapeutic potential for the treatment of human cancers. In this study, we identified sophoraflavanone G as a novel small-molecule inhibitor of STAT signaling in human cancer cells. Sophoraflavanone G inhibited tyrosine phosphorylation of STAT proteins in Hodgkin's lymphoma and tyrosine phosphorylation of STAT3 in solid cancer cells by inhibiting phosphorylation of the Janus kinase (JAK) proteins, Src family tyrosine kinases, such as Lyn and Src, Akt, and ERK1/2. In addition, sophoraflavanone G inhibited STAT5 phosphorylation in murine-bone-marrow-derived pro-B cells transfected with translocated Ets Leukemia (TEL)-JAKs and cytokine-induced rat pre-T lymphoma cells, as well as STAT5b reporter activity in TEL-JAKs and STAT5b reporter systems. Sophoraflavanone G also inhibited nuclear factor-κB (NF-κB) signaling in multiple myeloma cells. Furthermore, sophoraflavanone G inhibited cancer cell proliferation and induced apoptosis by regulating the expression of apoptotic and anti-apoptotic proteins. Our data suggest that sophoraflavanone G is a novel small-molecule inhibitor of STAT signaling by targeting upstream signals of STATs that may have therapeutic potential for cancers caused by persistently activated STAT proteins.

Bone marrow stromal cell antigen 2 (BST-2) is a type II transmembrane protein that is known to be a therapeutic target in several types of cancer. However, despite its clinical importance, the roles of BST-2 expression have remained elusive. Here, we found that BST-2 expression is up-regulated in tamoxifen-resistant MCF-7 human breast cancer (TRM-7) cells, resulting in enhanced invasiveness and migration. Matrigel and wound healing assays also showed that overexpression of BST-2 increased invasion and migration in MCF-7 cells, whereas invasion and migration were decreased by the silencing of BST-2 in TRM-7 cells. In addition, B16F10 cells expressing BST-2 showed increased metastatic melanoma nodule growth in a lung metastasis mouse model. Furthermore, BST-2 expression and promoter activity were regulated by activated signal transducer and activator of transcription 3 (STAT3). Taken together, our results indicate that BST-2 is an important factor in the invasiveness and motility of tamoxifen-resistant breast cancer cells, and that its expression and activity are regulated by activated STAT3. Therefore, regulation of BST-2 is a potential therapeutic target for tamoxifen-resistant breast cancer.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by the oxidative burst in activated macrophages and neutrophils cause oxidative stress-implicated diseases. Quercetin is flavonoid that occurs naturally in plants and is widely used as a nutritional supplement due to its antioxidant and anti-inflammatory properties. In this study, we investigated antioxidant activities and mechanisms of action in zymosan-induced macrophages of quercetin and quercetin-related flavonoids such as quercitrin, isoquercitrin, quercetin 3-O-β-(2″-galloyl)-rhamnopyranoside (QGR) and quercetin 3-O-β-(2″-galloyl)-glucopyranoside (QGG) as well as gallic acid, a building moiety of QGR and QGG. QGR and QGG exhibited stronger antioxidant activities compared with quercetin, whereas quercitrin, isoquercitrin and gallic acid exhibited weak-to-no antioxidant activities, assessed by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging, superoxide production, superoxide scavenging, nitric oxide (NO) production, peroxynitrite (ONOO−) scavenging and myeloperoxidase (MPO) activity. Regarding mechanisms, the quercetin-containing flavonoids QGR and QGG differentially targeted compared with quercetin in the NF-κB signaling pathway that inhibited the DNA binding activity of the NF-κB complex without affecting the degradation and phosphorylation of IκBα and NF-κB phosphorylation. In addition, QGR and QGG inhibited CRE and activator protein (AP-1) transcriptional activity and JNK phosphorylation by inhibiting the cAMP/protein kinase A (PKA) and protein kinase C (PKC) signaling in a different manner than quercetin. Our results showed that although QGR and QGG exhibited stronger antioxidant activities than quercetin in macrophages, their mechanisms of action in terms of the NF-κB, PKA and PKC signaling pathways were different.

α-Enolase (ENO1) is a multifunctional glycolytic enzyme expressed abundantly in the cytosol. It has been implicated in autoimmune and inflammatory diseases. Serum Abs against ENO1 were reported in rheumatoid arthritis (RA). Cell-surface expression of ENO1 has been found to be increased rapidly in response to inflammatory stimuli, but its expression and function has not been reported in RA. In this study, we show that cell-surface expression of ENO1 is increased on monocytes and macrophages isolated from RA patients but not on those from osteoarthritis patients, and Ab against ENO1 can stimulate these cells to produce higher amounts of proinflammatory mediators, such as TNF-α, IL-1 α/β, IFN-γ, and PGE2 via p38 MAPK and NF-κB pathway. The frequency of ENO1-positive cells in synovial fluid mononuclear cells was higher than PBMCs. ENO1-positive cells were also found in the inflamed synovium from RA patients and arthritic ankle tissues of mice with collagen-induced arthritis. Taken together, these findings suggest that Abs against ENO1 present in RA sera may stimulate monocytes and macrophages expressing cell-surface ENO1 and contribute to production of proinflammatory mediators during the effector phase of synovial inflammation.

Cyclooxygenase-2 (COX-2) is a key inflammatory response molecule, and associated with many immune functions of monocytes/macrophages. Particularly, interferon gamma (IFNgamma)-induced COX-2 expression appears in inflammatory conditions such as viral infection and autoimmune diseases. Recently, statins have been reported to show variable effects on COX-2 expression, and on their cell and species type dependences. Based on the above description, we compared the effect of simvastatin on IFNgamma-induced COX-2 expression in human monocytes versus murine macrophages. In a result, we found that simvastatin suppresses IFNgamma-induced COX-2 expression in human THP-1 monocytes, but rather, potentiates IFNgamma-induced COX-2 expression in murine RAW264.7 macrophages. However, signal transducer and activator of transcription 1/3 (STAT1/3), known as a transcription factor on COX-2 expression, is inactivated by simvastatin in both cells. Our findings showed that simvastatin is likely to suppress IFNgamma-induced COX-2 expression by inhibiting STAT1/3 activation in human THP-1 cells, but not in murine RAW264.7 cells. Thus, we concluded that IFNgamma-induced COX-2 expression is differently regulated by simvastatin depending on species specific mechanism.

The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins.

The naive T-cell pool in peripheral lymphoid tissues is fairly stable in terms of number, diversity and functional capabilities in spite of the absence of prominent stimuli. This stability is attributed to continuous tuning of the composition of the T-cell pool by various homeostatic signals. Despite extensive research into the link between signal transducer and activator of transcription 3 (Stat3) and T-cell survival, little is known about how Stat3 regulates homeostasis by maintaining the required naive T-cell population in peripheral lymphoid organs. We assessed whether the elimination of Stat3 in T cells limits T-cell survival. We demonstrated that the proportion and number of single-positive thymocytes as well as T cells in the spleen and lymph nodes were significantly decreased in the Stat3-deficient group as a result of the enhanced susceptibility of Stat3-deleted T lymphocytes to apoptosis. Importantly, expression of the anti-apoptotic Bcl-2 and Bcl-xL was markedly decreased in Stat3-deleted single-positive thymocytes and T lymphocytes, suggesting that Stat3 helps to maintain the T-cell pool in the resting condition by promoting the expression of Bcl-2 family genes. These findings suggest the importance of Stat3 in the integration of homeostatic cues for the maintenance and functional tuning of the T-cell pool.

Recently, statins have been shown to have anti-inflammatory effects on lung inflammatory diseases. However, the mechanisms of action of simvastatin in viral pneumonia have yet to be elucidated, although viral infection remains a considerable health threat. In this study, we hypothesised that simvastatin inhibits polyinosinic-polycytidylic acid (poly I:C)-induced airway inflammation, such as RANTES (regulated on activation, normal T-cell expressed and secreted) expression and inflammatory cell recruitment. In bronchial cells, the effect of simvastatin on poly I:C-induced RANTES expression and signal transducer and activator of transcription (STAT)3-mediated signal transduction was determined using an ELISA and short hairpin (sh)RNA system. In a poly I:C-induced pneumonia mouse model, immunological changes in the lungs after simvastatin inhalation, such as inflammatory cell recruitment and cytokine/chemokine release, were examined. In poly I:C-stimulated bronchial cells, RANTES secretion was increased by STAT3 activation, and simvastatin suppressed poly I:C-induced STAT3 activation, resulting in inhibition of RANTES expression. In BALB/c mice stimulated with inhaled poly I:C, RANTES expression and neutrophil infiltration into the airway were elevated. However, simvastatin treatment attenuated STAT3 activation, RANTES release and subsequent neutrophilia in the lungs. These findings suggest that simvastatin inhibits airway inflammation, but there are other mechanisms that need to be fully elucidated.

The initiation and growth of hepatocellular carcinoma (HCC) are closely linked to chronic inflammation. Not only is cyclin D1 overexpressed, but it is also related to aggressive progression in HCC. However, the mechanism of expression cyclin D1, a cell-cycle regulator of paramount importance, in the tumor microenvironment remains unknown. Here, we investigated the mechanism of cyclin D1 expression induced by interleukin-6 (IL-6) and whether 3-[3,4-dihydroxy-phenyl]-acrylic acid 2-[3,4-dihydroxy-phenyl]-ethyl ester (CADPE), a derivate of caffeic acid, suppresses cyclin D1 expression. CADPE significantly inhibited IL-6-induced signal transducer and activator of transcription 3 (STAT3) activity in the Huh7 HCC cell line and attenuated IL-6-induced cyclin D1 transcription. Moreover, overexpression of constitutively active STAT3 increased cyclin D1 transcriptional activity and protein expression, whereas overexpression of a dominant-negative STAT3 deletion mutant (STAT3 (1-588)) reduced cyclin D1 transcriptional activity. In addition, CADPE effectively deacetylated histone 4 and prevented STAT3 recruitment to the cyclin D1 promoter, consistent with a role for the CADPE target, STAT3, in the regulation of cyclin D1 transcription. Collectively, these results indicate that CADPE suppresses cyclin D1 expression in HCC cells by blocking both IL-6-mediated STAT3 activation and recruitment of STAT3 to the cyclin D1 promoter.

Fraxinellone and sauchinone, isolated from natural substance, are known to have an anti-inflammatory effect in inflammatory conditions. However, the anti-inflammatory actions of these compounds have been insufficiently demonstrated in viral-induced neuroinflammation. A viral component (double-stranded (ds)RNA) triggers a toll-like receptor 3-dependent inflammatory response that stimulates pro-inflammatory mediators in the brain. In present study, we initially examined the biological effects of fraxinellone and sauchinone on anti-inflammatory actions in dsRNA-stimulated microglia. Both compounds inhibited dsRNA-induced inducible nitric oxide synthase (iNOS) expression, a major pro-inflammatory enzyme. To demonstrate the mechanism of inhibitory effect on iNOS expression, we further examined the signaling pathway induced by dsRNA in microglia. Our data show that dsRNA promotes the expression of signal transducers and activators of transcription (STAT)1/3 identified as major inflammatory transcription factors as well as activates c-Jun N-terminal kinase (JNK) in an early time. Moreover, both compounds suppressed activation of JNK-STAT1/3 signaling pathway. These results suggest that an anti-inflammatory effect by fraxinellone and sauchinone is mediated via blockade of the JNK-STAT1/3-iNOS signaling pathway in viral-infected microglia.

Polarity formation in preimplantation embryos is controversial. To investigate the embryonic-abembryonic axis in the pig, porcine parthenotes were used to prevent the topological change caused by polyspermy as well as to avoid the influences of sperm entry position. For lineage tracing, DiI, a fluorescence dye, was injected into only a blastomere of the 2-cell stage embryos. If the first blastomere to divide was labeled, the embryo was included in the leading group, and while all others were included in the lagging group. In 60.5% of the blastocysts in the lagging group, the progeny of the labeled blastomeres formed the inner cell mass (ICM) and adjacent trophectoderm (TE) hemisphere; 62.1% of the blastocysts in the leading group had progeny of the labeled blastomeres distributed only to the TE (opposite of ICM). The rest of the lagging and leading groups showed random distributions. Unlike murine parthenotes, biased mitochondrial distribution was also found in porcine parthenotes (38.1%). Our findings indicate that the ;leading' blastomere of the 2-cell porcine parthenote forms the distal TE (abembryonic) and that the 'lagging' blastomere forms the remaining portion of the blastocyst, including the ICM (embryonic). Biased distribution of mitochondria in each 2-cell blastomere may contribute partly to this event.

Recent studies reported that the direct transformation of one differentiated somatic cell type into another is possible. In the present study, we were able to modulate the cell fate of somatic cells to take on male germ cell function by introducing cell extracts derived from porcine testis tissue. Fibroblasts were treated with streptolysin O, which reversibly permeabilises the plasma membrane, and incubated with testis extracts. Our results showed that the testis extracts (TE) could activate expression of male germ cell-specific genes, implying that TE can provide regulatory components required for altering the cell fate of fibroblasts. Male germ cell function was sustained for more than 10 days after the introduction of TE. In addition, a single TE-treated cell was injected directly into the cytoplasm of in vitro-matured porcine oocytes. The rate of blastocyst formation was significantly higher in the TE-treated nuclear donor cell group than in the control cell group. The expression level of Nanog, Sox9 and Eomes was drastically increased when altered cells were used as donor nuclei. Our results suggest that TE can be used to alter the cell fate of fibroblasts to express male germ cell function and improve the developmental efficiency of the nuclear transfer porcine embryos.

Somatic cell cloning has promise for medical treatment using embryonic stem cells derived from cloned embryos. However, porcine cloning by somatic cell nuclear transfer has been inefficient and, even after birth, cloned pigs are found to carry a variety of abnormalities. Moreover, recent molecular analyses of cloned embryos have revealed abnormal epigenetic modifications. Therefore, the prevention of epigenetic errors is expected to lead to the improvement of the success rate in animal cloning. Reports of recent studies indicate that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. Therefore, in this study, we modulated the cell fate of somatic donor cells by introducing cell extracts derived from porcine testis. Several porcine somatic cells, including primary and stabilized porcine fibroblasts or epithelial kidney cells, were treated with streptolysin O (SLO; 230 ng mL-1), which reversibly permeablizes plasma membrane, and incubated for 30 min with testis cell-derived cell extracts (4 mg mL-1). To reseal plasma membranes, cells were placed in DMEM containing 30% FBS and 2 mM CaCl2 for 30 min. After resealing the cell membranes, we incubated the cells for 3 weeks and analyzed the expression of testis-specific genes such as protamine 1, protamine 2, SOX 9, mullerian inhibitory substance (MIS), preproacrosine (ACR), phosphoglycerate kinase 2 (PGK-2), protein C, and c-kit ligand. In the reprogrammed primary porcine fibroblasts or epithelial kidney cells, the porcine testis extracts were able to activate the expression of the porcine testis sertoli cell-specific genes. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Then, in vitro-matured oocytes were enucleated and a single cell (either reprogrammed or intact) was injected directly into cytoplasm of the oocytes. The reconstructed embryos were activated electrically and cultured in vitro for 7 days. The rate of blastocyst formation was significantly higher (P < 0.05; chi-square test) in the reprogrammed nuclear donor cells (27/119; 22.7 � 5.0%) than in the control (intact) cells (11/83; 13.3 � 3.2%). Taken together, our results suggest that testis-derived cell extracts can be successfully used to reprogram fibroblasts to express male germ cell function, thus improving the developmental efficiency of the nuclear transfer embryos.

Embryonic axis formation is important in normal animal development. Some vertebrate and invertebrate eggs display undeniable polarity along a plane known as the animal–vegetal axis. However, axis formation in mammals, which is studied only in mice, is still a point of issue (Hiiragi and Solter 2004 Nature 430, 360–364; Zernicka-Goetz et al. 2005 Nature 434, 391–395). In the present study, we investigated the embryonic–abembryonic axis formation in porcine species. We used porcine parthenogenetic embryos to prevent a topological change of the 2 apposing pronuclei in the egg center caused by polyspermy and to avoid the influence of the fertilization cone, which indicates the sperm entry position. For lineage tracing, DiI, a lipophilic fluorescence dye, was injected into one blastomere per embryo at the 2-cell stage. After this process, as embryos developed into the 3-cell stage, all the embryos were divided into 2 groups named Leading and Lagging, following the location of the DiI oil drop (Leading: the oil drop was positioned in the first-dividing blastomere; Lagging: the drop was positioned in the late-dividing blastomere). The embryos developed into blastocysts after 6 to 7 days of culture in vitro. Only the inner cell mass (ICM) was labeled in 50% (8/16) of the Lagging group. In some Lagging embryos (2/16), ICMs with a small portion of adjacent trophectodermal cells (TE) were labeled and 62.5% (10/16) of Lagging embryos formed the ICM part in total. On the other hand, 60% (6/10) of the Leading embryos formed only distal TE (opposite side of the ICM). Only one of the Leading embryos formed the ICM. The rest of Lagging embryos (37.5%, 6/16) or the Leading group (40.0%, 4/10) showed an even distribution of blastocysts, regardless of the ICM or TE. Oocytes, zygotes, and 2-cell-stage embryos in porcine species tended to show unequally distributed lipid contents and could be distinguished by their different colors and brightness, although not all of them showed significant differences. We injected DiI into a relatively brighter blastomere of the 2-cell-stage embryos and the results were very similar to those obtained from the Leading group (distal TE: 70.6%, 24/34). High lipid contents may be the cause of delayed development of the blastomere of an embryo. Our findings indicated that the Leading blastomere of 2-cell porcine parthenotes formed distal TE first, and that afterward, the Lagging blastomere not only filled the rest of the TE but also contributed to the ICM. This study was supported by the Research Project on the Production of Bio-organs (No. 200506030601) and Technology Developmental Program (High-Technology Development Project No. 204117-3) for Agriculture and Forestry, Ministry of Agriculture and Forestry, Republic of Korea