Preclinical and dose-ranging assessment of hESC-derived dopaminergic progenitors for a clinical trial on Parkinson's disease.

Human embryonic stem cell (hESC)-derived midbrain dopaminergic (mDA) cell transplantation is a promising therapeutic strategy for Parkinson's disease (PD). Here, we present the derivation of high-purity mDA progenitors from clinical-grade hESCs on a large scale under rigorous good manufacturing practice (GMP) conditions. We also assessed the toxicity, biodistribution, and tumorigenicity of these cells in immunodeficient rats in good laboratory practice (GLP)-compliant facilities. Various doses of mDA progenitors were transplanted into hemi-parkinsonian rats, and a significant dose-dependent behavioral improvement was observed with a minimal effective dose range of 5,000-10,000 mDA progenitor cells. These results provided insights into determining a low cell dosage (3.15 million cells) for human clinical trials. Based on these results, approval for a phase 1/2a clinical trial for PD cell therapy was obtained from the Ministry of Food and Drug Safety in Korea, and a clinical trial for treating patients with PD has commenced.

An Anti-Cancer Drug Candidate CYC116 Suppresses Type I Hypersensitive Immune Responses through the Inhibition of Fyn Kinase in Mast Cells.

Mast cells are the most prominent effector cells of Type 1 hypersensitivity immune responses. CYC116 [4-(2-amino-4-methyl-1,3-thiazol-5-yl)-N-[4-(morpholin-4-yl)phenyl] pyrimidin-2-amine] is under development to be used as an anti-cancer drug, but the inhibitory effects of CYC116 on the activation of mast cells and related allergy diseases have not reported as of yet. In this study, we demonstrated, for the first time, that CYC116 inhibited the degranulation of mast cells by antigen stimulation (IC50, ~1.42 µM). CYC116 also inhibited the secretion of pro-inflammatory cytokines including TNF-α (IC50, ~1.10 µM), and IL-6 (IC50, ~1.24 µM). CYC116 inhibited the mast cell-mediated allergic responses, passive cutaneous anaphylaxis (ED50, ~22.5 mg/kg), and passive systemic anaphylaxis in a dose-dependent manner in laboratory experiments performed on mice. Specifically, CYC116 inhibited the activity of Fyn in mast cells and inhibited the activation of Syk and Syk-dependent signaling proteins including LAT, PLCγ, Akt, and MAP kinases. Our results suggest that CYC116 could be used as an alternative therapeutic medication for mast cell-mediated allergic disorders, such as atopic dermatitis and allergic rhinitis.

Polysaccharide isolated from Aloe vera gel suppresses ovalbumin-induced food allergy through inhibition of Th2 immunity in mice.

An allergic reaction occurs when the immune system overreacts to harmless substance called allergen that gains access to the body. Food allergy is a hypersensitive immune reaction to food proteins and the number of patients with food allergy has recently increased. Aloe Vera is used for wellness and medicinal purposes. In particular, Aloe vera has been reported to enhance immunity. However, the effect of Aloe vera on food allergy is not yet known. In this study, we investigated the effects of processed Aloe vera gel (PAG) containing low molecular weight Aloe polysaccharide (AP) on ovalbumin (OVA)-induced food allergy in mice. Allergic symptoms, rectal temperature, and diarrhea were measured in OVA-induced food allergy mice. Other allergic parameters were also analyzed by RT-PCR, ELISA, flow cytometry, and other biochemical methods. As the results, PAG suppressed the decrease of body temperature, diarrhea, and allergic symptoms in OVA-induced food allergy mice. PAG also reduced serum concentrations of type 2 helper T cell (Th2) cytokines (Interleukin-(IL)-4, IL-5, and IL-13) as well as histamine, mast cell protease-1 (MCP-1), and immunoglobulin (Ig)E. PAG blocked the degranulation of mast cells and infiltration of eosinophils in intestine. Furthermore, PAG suppressed the population of Th2 cells in spleen and mesenteric lymph nodes. PAG also increased the production of IL-10 and population of type 1 regulatory T (Tr1) cells in mice with food allergy. Taken together, our findings suggest that PAG suppressed Th2 immune responses through, at least partially, stimulating the secretion of IL-10 in food allergy mice.

Furaltadone suppresses IgE-mediated allergic response through the inhibition of Lyn/Syk pathway in mast cells.

Mast cells are critical cells that prompt various allergic response-inducing factors, contributing to allergic diseases. While used as an antibiotic for livestock, there is no study on the effect of furaltadone on allergic response. This study investigated the effect of furaltadone on mast cells and passive cutaneous anaphylaxis (PCA). Furaltadone inhibited the degranulation of mast cells stimulated by antigen (IC50, ~ 3.9 µM), and also suppressed the production of tumor necrosis factor (TNF)-α and interleukin (IL)-4 in a concentration dependent manner. In addition, furaltadone inhibited allergic responses in an acute allergy animal model, PCA. Further investigation on the mechanism for these inhibitory effects of furaltadone found that the activities of Lyn/Syk and Syk-dependent downstream proteins such as mitogen-activated protein (MAP) kinases were inhibited by furaltadone in mast cells. Taken together, this study demonstrates that furaltadone inhibits the activation of mast cells by antigen via the suppression of the Lyn/Syk pathway and ameliorates allergic responses in vivo.

JQ1, a BET inhibitor, controls TLR4-induced IL-10 production in regulatory B cells by BRD4-NF-κB axis.

Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomaincontaining protein 4 (BRD4) was associated with NF-κB on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with NF-κB on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer. [BMB Reports 2017; 50(12): 640-646].

DJ-1 controls bone homeostasis through the regulation of osteoclast differentiation.

Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis.

Suppression of IgE-mediated mast cell activation and mouse anaphylaxis via inhibition of Syk activation by 8-formyl-7-hydroxy-4-methylcoumarin, 4µ8C.

Mast cells trigger IgE-mediated allergic reactions by releasing various allergic mediators. 8-Formyl-7-hydroxy-4-methylcoumarin, also called 4µ8C, was originally known as an inositol-requiring enzyme 1 (IRE1) suppressant, but no study has examined its relationship with mast cells and allergic diseases. Therefore, the purpose of this study was to determine whether 4µ8C is effective in suppressing allergic reactions in mast cells and in IgE-mediated allergic animal model. 4µ8C suppressed the degranulation of IgE-mediated mast cells (IC50=3.2µM) and the production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in a dose-dependent manner. 4µ8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50=25.1mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4µ8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4µ8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4µ8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE-mediated signaling pathway. In summary, for the first time, this study shows that 4µ8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4µ8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis.

The Extract of Chrysanthemum zawadskii var. latilobum Ameliorates Collagen-Induced Arthritis in Mice.

Chrysanthemum zawadskii var. latilobum (CZ) has been used for beverage or tea and also as folk medicine for the remedy of diverse inflammatory diseases. Nevertheless, the therapeutic effect of CZ on arthritis remains to be unknown. In this paper we aim to investigate the CZ's antiarthritic effect and mechanism of action both in vitro and in vivo. To assess CZ's antiarthritic effect, mouse models of type II collagen-induced arthritis (CIA) were used. Mice were used to gauge clinical arthritis index and histopathological changes. Reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, electrophoretic mobility shift assay (EMSA), and other biological methods were adopted to measure CZ's effect on arthritis and to understand the veiled mechanism of action. CZ greatly suppressed CIA, histopathological score, bone erosion, and osteoclast differentiation. Mechanistically, CZ inhibited the production of various inflammatory and arthritic mediators like inflammatory cytokines, matrix metalloproteinases (MMPs), and chemokines. Of note, CZ significantly suppressed the activation of the NF-κB pathway in vivo. CZ exerted an antiarthritic effect in CIA mice by curbing the production of crucial inflammatory and arthritis mediators. This study warrants further investigation of CZ for the use in human rheumatoid arthritis (RA).

NQO1-Knockout Mice Are Highly Sensitive to Clostridium Difficile Toxin A-Induced Enteritis.

Clostridium difficile toxin A causes acute gut inflammation in animals and humans. It is known to downregulate the tight junctions between colonic epithelial cells, allowing luminal contents to access body tissues and trigger acute immune responses. However, it is not yet known whether this loss of the barrier function is a critical factor in the progression of toxin A-induced pseudomembranous colitis. We previously showed that NADH:quinone oxidoreductase 1 (NQO1) KO (knockout) mice spontaneously display weak gut inflammation and a marked loss of colonic epithelial tight junctions. Moreover, NQO1 KO mice exhibited highly increased inflammatory responses compared with NQO1 WT (wild-type) control mice when subjected to DSS-induced experimental colitis. Here, we tested whether toxin A could also trigger more severe inflammatory responses in NQO1 KO mice compared with NQO1 WT mice. Indeed, our results show that C. difficile toxin A-mediated enteritis is significantly enhanced in NQO1 KO mice compared with NQO1 WT mice. The levels of fluid secretion, villus disruption, and epithelial cell apoptosis were also higher in toxin A-treated NQO1 KO mice compared with WT mice. The previous and present results collectively show that NQO1 is involved in the formation of tight junctions in the small intestine, and that defects in NQO1 enhance C. difficile toxin A-induced acute inflammatory responses, presumably via the loss of epithelial cell tight junctions.

Mesenteric IL-10-producing CD5+ regulatory B cells suppress cow's milk casein-induced allergic responses in mice.

Food allergy is a hypersensitive immune reaction to food proteins. We have previously demonstrated the presence of IL-10-producing CD5(+) B cells and suggested their potential role in regulating cow's milk casein allergy in humans and IgE-mediated anaphylaxis in mice. In this study, we determined whether IL-10-producing CD5(+) regulatory B cells control casein-induced food allergic responses in mice and, if so, the underlying mechanisms. The induction of oral tolerance (OT) by casein suppressed casein-induced allergic responses including the decrease of body temperature, symptom score, diarrhea, recruitment of mast cells and eosinophils into jejunum, and other biological parameters in mice. Notably, the population of IL-10-producing CD5(+) B cells was increased in mesenteric lymph node (MLN), but not in spleen or peritoneal cavity (PeC) in OT mice. The adoptive transfer of CD5(+) B cells from MLN, but not those from spleen and PeC, suppressed the casein-induced allergic responses in an allergen-specific and IL-10-dependent manner. The inhibitory effect of IL-10-producing CD5(+) B cells on casein-induced allergic response was dependent on Foxp3(+) regulatory T cells. Taken together, mesenteric IL-10-producing regulatory B cells control food allergy via Foxp3(+) regulatory T cells and could potentially act as a therapeutic regulator for food allergy.

The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut.

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21(Cip1/Waf1), and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21(Cip1/Waf1). Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.

Antimicrobial Peptide, Lumbricusin, Ameliorates Motor Dysfunction and Dopaminergic Neurodegeneration in a Mouse Model of Parkinson's Disease.

We recently reported that the antimicrobial peptide Lumbricusin (NH2-RNRRWCIDQQA), isolated from the earthworm, increases cell proliferation in neuroblastoma SH-SY5Y cells. Here, we investigated whether Lumbricusin has neurotropic activity in mouse neural stem cells (MNSCs) and a protective effect in a mouse model of Parkinson's disease (PD). In MNSCs isolated from mouse brains, Lumbricusin treatment significantly increased cell proliferation (up to 12%) and reduced the protein expression of p27(Kip1) through proteasomal protein degradation but not transcriptional regulation. Lumbricusin inhibited the 6-OHDA-induced apoptosis of MNSCs, and also showed neuroprotective effects in a mouse PD model, ameliorating the motor impairments seen in the pole, elevated body swing, and rotation tests. These results suggest that the Lumbricusin-induced promotion of neural cell proliferation via p27(Kip1) degradation has a protective effect in an experimental PD model. Thus, the antimicrobial peptide Lumbricusin could possibly be developed as a potential therapeutic agent for the treatment of PD.

An indoxyl compound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice.

Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC50, ~3.8µM) and human mast cells (IC50, ~3.0µM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED50 27.9mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells.

Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40(hi)CD5(+) regulatory B cells in vitro and in vivo.

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40(hi)CD5(+) B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40(hi) is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10(-/-)CD5(+)CD19(+) B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40(hi)CD5(+) Breg cells in mice. However, the population of CD40(hi)CD5(+) B cells was minimal in IL-10(-/-) mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40(hi)CD5(+) B cells and the autocrine effect of IL-10 is critical to the formation of CD40(hi)CD5(+) Breg cells.

Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin.

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH2-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson's disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27(Kip1) protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27(Kip1) significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27(Kip1) degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.

Effect of antisera from Clostridium difficile-infected mice on toxin-A-induced colonic epithelial cell death signaling.

Clostridium difficile causes mucosal damage and diarrhea by releasing two exotoxins: toxin A and toxin B. C. difficile colitis is associated with alterations in bowel flora and the failure to mount an effective antibody response. The aim of the current study was to investigate whether antitoxin sera prevent toxin-A-induced apoptosis, cytoskeletal disaggregation, cell detachment, and tight junction loss in cultured colonic epithelial cells. Serum samples were isolated from mice that survived a C. difficile infection following antibiotic treatment, and the antitoxin effects of these samples were investigated in toxin-A-exposed HT29 colonic epithelial cells and a toxin-A-induced animal model of gut inflammation. Unchallenged mice did not produce IgG against toxin A, whereas serum (antiserum) from C. difficile-challenged mice showed significant IgG responses against toxin A. Treatment with the antiserum markedly inhibited mucosal damage and inflammation in the toxin-A-treated mouse model. In contrast to control mouse serum, the antiserum also markedly inhibited toxin-A-induced DNA fragmentation, dephosphorylation of paxillin and Epo receptor (EpoR), deacetylation of tubulin, and upregulation of p21(WAF1/CIP1) and p53. Taken together, these results reveal that the generated antitoxin serum has biotherapeutic effects in preventing various C. difficile toxin-A-induced cellular toxicities.

Role of NADH: quinone oxidoreductase-1 in the tight junctions of colonic epithelial cells.

NADH:quinone oxidoreductase 1 (NQO1) is known to be involved in the regulation of energy synthesis and metabolism, and the functional studies of NQO1 have largely focused on metabolic disorders. Here, we show for the first time that compared to NQO1-WT mice, NQO1-KO mice exhibited a marked increase of permeability and spontaneous inflammation in the gut. In the DSS-induced colitis model, NQO1-KO mice showed more severe inflammatory responses than NQO1-WT mice. Interestingly, the transcript levels of claudin and occludin, the major tight junction molecules of gut epithelial cells, were significantly decreased in NQO1-KO mice. The colons of NQO1-KO mice also showed high levels of reactive oxygen species (ROS) and histone deacetylase (HDAC) activity, which are known to affect transcriptional regulation. Taken together, these novel findings indicate that NQO1 contributes to the barrier function of gut epithelial cells by regulating the transcription of tight junction molecules.

Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis.

We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer's disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

Clostridium difficile toxin A inhibits erythropoietin receptor-mediated colonocyte focal adhesion through inactivation of Janus Kinase-2.

Previously, we demonstrated that the erythropoietin receptor (EpoR) is present on fibroblasts, where it regulates focal contact. Here, we assessed whether this action of EpoR is involved in the reduced cell adhesion observed in colonocytes exposed to Clostridium difficile toxin A. EpoR was present and functionally active in cells of the human colonic epithelial cell line HT29 and epithelial cells of human colon tissues. Toxin A significantly decreased activating phosphorylations of EpoR and its downstream signaling molecules JAK-2 (Janus kinase 2) and STAT5 (signal transducer and activator of transcription 5). In vitro kinase assays confirmed that toxin A inhibited JAK 2 kinase activity. Pharmacological inhibition of JAK2 (with AG490) abrogated activating phosphorylations of EpoR and also decreased focal contacts in association with inactivation of paxillin, an essential focal adhesion molecule. In addition, AG490 treatment significantly decreased expression of occludin (a tight junction molecule) and tight junction levels. Taken together, these data suggest that inhibition of JAK2 by toxin A in colonocytes causes inactivation of EpoR, thereby enhancing the inhibition of focal contact formation and loss of tight junctions known to be associated with the enzymatic activity of toxin A.