The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Gamma Irradiation-Inactivated Respiratory Syncytial Virus Vaccine Provides Protection but Exacerbates Pulmonary Inflammation by Switching from Prefusion to Postfusion F Protein.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.

Low-dose radiation therapy suppresses viral pneumonia by enhancing broad-spectrum anti-inflammatory responses via transforming growth factor-ß production.

Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-ß (TGF-ß) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-ß reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-ß production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-ß may be an alternative therapy for viral pneumonia.

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells.

There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.

18:0 Lyso PC Derived by Bioactivity-Based Molecular Networking from Lentil Mutant Lines and Its Effects on High-Fat Diet-Induced Obese Mice.

Lentil (Lens culinaris; Fabaceae), one of the major pulse crops in the world, is an important source of proteins, prebiotics, lipids, and essential minerals as well as functional components such as flavonoids, polyphenols, and phenolic acids. To improve crop nutritional and medicinal traits, hybridization and mutation are widely used in plant breeding research. In this study, mutant lentil populations were generated by γ-irradiation for the development of new cultivars by inducing genetic diversity. Molecular networking via Global Natural Product Social Molecular Networking web platform and dipeptidyl peptide-IV inhibitor screening assay were utilized as tools for structure-based discovery of active components in active mutant lines selected among the lentil population. The bioactivity-based molecular networking analysis resulted in the annotation of the molecular class of phosphatidylcholine (PC) from the most active mutant line. Among PCs, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0 Lyso PC) was selected for further in vivo study of anti-obesity effect in a high-fat diet (HFD)-induced obese mouse model. The administration of 18:0 Lyso PC not only prevented body weight gain and decreased relative gonadal adipose tissue weight, but also attenuated the levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, and leptin in the sera of HFD-induced obese mice. Additionally, 18:0 Lyso PC treatment inhibited the increase of adipocyte area and crown-like structures in adipose tissue. Therefore, these results suggest that 18:0 Lyso PC is a potential compound to have protective effects against obesity, improving obese phenotype induced by HFD.

Depletion of NK Cells Resistant to Ionizing Radiation Increases Mutations in Mice After Whole-body Irradiation.

BACKGROUND: Ionizing radiation is a very powerful genetic mutagenic agent. Although immune cells are very sensitive to radiation, their sensitivity varies between different types of immune cell. We hypothesized that radiation-resistant immune cells survive after irradiation and then play a role in removing mutant cells. MATERIALS AND METHODS: Splenic lymphocytes and mice were irradiated with γ-rays. Cell populations were analyzed using flow cytometry after dyeing with antibodies and expression of B-cell lymphoma 2 (BCL2) was measured by western blot analysis. To deplete natural killer (NK) cells, anti-asialo GM1 antiserum was used. Micronuclei in polychromatic erythrocytes were measured by May-Grunwald/Giemsa staining. H-2Kb loss variant in T-cells induced by irradiation of B6C3F1 mice were detected by flow cytometry. RESULTS: When splenic lymphocytes were irradiated in vitro, B cells notably died, while NK cells did not. In vivo, on the third day after whole-body irradiation, the total number of lymphocytes in the spleen decreased rapidly, but the proportion of NK cells was approximately three times higher than that of the normal control group. In addition, it was confirmed that high expression of BCL2 in NK cells was maintained after irradiation, whereas that of B-cells was not. Removal of NK cells by injection with anti-asialo GM1 antiserum immediately after irradiation increased the micronuclei of polychromatic erythrocytes in the bone marrow and the variant fraction with H-2kb loss in the spleen. CONCLUSION: These results provide important evidence that radioresistant NK cells apparently survive by escaping apoptosis in the early stages after irradiation, and work to eliminate mutant cells resulting from γ-ray irradiation. Future studies are needed to reveal why NK cells are resistant to radiation and the in-depth mechanisms involved in the elimination of radiation-induced mutant cells.

Repeated Irradiation with γ-Ray Induces Cancer Stemness through TGF-ß-DLX2 Signaling in the A549 Human Lung Cancer Cell Line.

Cancer stem cells (CSCs) play an important role in cancer recurrence and metastasis. It is suggested that the CSC properties in heterogeneous cancer cells can be induced by ionizing radiation (IR). This study investigated the role of DLX2 in the radioresistance and CSC properties induced by IR in NSCLC cancer cells. Here, A549 cells were exposed to fractionated irradiation at a cumulative dose of 52 Gy (4 Gy × 13 times) for a generation of radioresistant cells. After fractionated irradiation, surviving A549 cells exhibited resistance to IR and enhanced expression of various cancer stem cell markers. They also showed upregulation of mesenchymal molecular markers and downregulation of epithelial molecular markers, correlating with an increase in the migration and invasion. Fractionated irradiation triggered the secretion of TGF-ß1 and DLX2 expression. Interestingly, the increased DLX2 following fractionated irradiation seemed to induce the expression of the gene for the EGFR-ligand betacellulin via Smad2/3 signaling. To contrast, DLX2 knockdown dramatically decreased the expression of CSC markers, migration, and proliferation. Moreover, A549 cells expressing DLX2 shRNA formed tumors with a significantly smaller volume compared to those expressing control shDNA in a mouse xenograft assay. These results suggest that DLX2 overexpression in surviving NSCLC cancer cells after fractionated IR exposure is involved in the cancer stemness, radioresistance, EMT, tumor survival, and tumorigenic capability.

Synergistic Anti-cancer Activity of MH-30 in a Murine Melanoma Model Treated With Cisplatin and its Alleviated Effects Against Cisplatin-induced Toxicity in Mice.

BACKGROUND/AIM: Although cisplatin is an effective anticancer drug, its toxic effects on normal tissues limit its use. We developed a herbal formula, MH-30, with increased fat-soluble polyphenols by improving the manufacturing method of HemoHIM. In this study, we examined whether the combination of MH-30 with cisplatin exerts synergistic antitumor effect while it reduces cisplatin-induced toxicities. MATERIALS AND METHODS: MH-30 was produced by adding the ethanol-insoluble fraction to its extract after decocting herbs in 30% ethanol and water. We used a melanoma-bearing mice model to investigate synergistic anticancer effects. The NK cell activity and cytokine levels were measured by Cr51-release assay and ELISA. The AST, ALT, BUN, and creatinine levels were estimated in the serum. RESULTS: MH-30 effectively inhibited melanoma growth in vitro. Furthermore, MH-30 had a synergistic effect in combination with cisplatin on melanoma growth inhibition in vitro and in vivo. In melanoma-bearing mice, cisplatin alone decreased the activity of NK cells and the levels of IL-2 and IFN-γ, which were effectively restored by the combination of MH-30 with cisplatin. Combined treatment with MH-30 and cisplatin significantly inhibited the cisplatin-induced increase in the levels of AST, ALT, BUN, and creatinine. CONCLUSION: Combination of MH-30 with cisplatin may be a beneficial anticancer treatment with reduced adverse effects.

Ionizing Radiation Promotes Epithelial-to-Mesenchymal Transition in Lung Epithelial Cells by TGF-ß-producing M2 Macrophages.

BACKGROUND/AIM: Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-ß-secreting M2 macrophages in association with ionizing radiation-induced EMT. MATERIALS AND METHODS: The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. RESULTS: In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1ß, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-ß and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-ß. Moreover, the elimination of TGF-ß from M2 macrophages attenuated mesenchymal transition of MLE12. CONCLUSION: TGF-ß-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.

Protective effects of a standardized extract (HemoHIM) using indomethacin- and ethanol/HCl-induced gastric mucosal injury models.

Context: HemoHIM is a medicinal herbal preparation of Angelica gigas Nakai (Apiaceae), Cnidium officinale Makino (Umbelliferae), and Paeonia japonica Miyabe (Paeoniaceae) developed for immune regulation. HemoHIM has been investigated for its ability to enhance tissue self-renewal and stimulate immune systems. To date, studies on the protective effects of HemoHIM against gastritis and gastric ulcers have not been conducted. Objective: The protective effects of HemoHIM using models of indomethacin and ethanol/hydrochloric acid (EtOH/HCl)-induced gastric mucosal injury were investigated. Materials and methods: Rats were divided into five groups (n = 10): control, indomethacin, or EtOH/HCl groups, HemoHIM 250, 500 mg kg-1, and cimetidine 100 mg kg-1, respectively. Indomethacin (80 mg kg-1) and 60% EtOH/150 mM HCl were administered orally 1 h after the administration of samples and rats were anesthetized 3 h after induction. The lesion area (%), inhibition ratio (%), and total acidity were investigated, and tissues were histopathologically analyzed using hematoxylin and-eosin (H&E) staining. Results: HemoHIM significantly reduced gastric injury in indomethacin-induced model (250 and 500 mg kg-1; 64.30% and 67.75%, p < 0.001) compared to indomethacin group. In the EtOH/HCl-induced model, HemoHIM reduced gastric lesion (250 and 500 mg kg-1; 61.05% and 73.37%, p < 0.001) and gastric acidity (250 and 500 mg kg-1; 37.80 and 45.20 meq L-1, p < 0.001) compared to EtOH/HCl group. H&E staining of the gastric mucosa showed decreased erosion and hemorrhage in HemoHIM group compared to EtOH/HCl group. Discussion and conclusions: Based on the results, HemoHIM is potential candidate for the treatment of gastritis and gastric ulcers.

Diospyros kaki calyx inhibits immediate-type hypersensitivity via the reduction of mast cell activation.

CONTEXT: Diospyros kaki L. (Ebenaceae) fruit is widely distributed in Asia and is known to exert anti-inflammatory and antithrombotic effects. OBJECTIVE: We evaluated the inhibitory effect of aqueous extract of D. kaki calyx (AEDKC) on mast cell-mediated immediate-type hypersensitivity and underlying mechanism of action. MATERIALS AND METHODS: For in vivo, ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) and immunoglobulin (Ig) E-mediated passive cutaneous anaphylaxis (PCA) models were used. In the ASA, AEDKC (1-100 mg/kg) was orally administered 3 times during 14 days. In the PCA, AEDKC was orally treated 1 h before the antigen challenge. The control drug dexamethasone was used to compare the effectiveness of AEDKC. For in vitro, IgE-stimulated RBL-2H3 cells and primary cultured peritoneal mast cells were used to determine the role of AEDKC (0.01-1 mg/mL). RESULTS: Oral administration of AEDKC dose dependently suppressed rectal temperature decrease and increases in serum histamine, total IgE, OVA-specific IgE, and interleukin (IL)-4 in the ASA. In the PCA, AEDKC reduced Evans blue pigmentation. Compared to dexamethasone (10 mg/kg), AEDKC (100 mg/kg) showed similar inhibitory effects in vivo. AEDKC concentration dependently suppressed the release of histamine and ß-hexosaminidase through the reduction of intracellular calcium in mast cells. In addition, AEDKC decreased the expression and secretion of tumour necrosis factor-α and IL-4 by the reduction of nuclear factor-κB. The inhibitory potential of AEDKC (1 mg/mL) was similar with dexamethasone (10 µM) in vitro. CONCLUSIONS: We suggest that AEDKC may be a potential candidate for the treatment of mast cell-mediated allergic diseases.

2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway.

Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 µmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

Ionizing Radiation Induces Altered Neuronal Differentiation by mGluR1 through PI3K-STAT3 Signaling in C17.2 Mouse Neural Stem-Like Cells.

Most studies of IR effects on neural cells and tissues in the brain are still focused on loss of neural stem cells. On the other hand, the effects of IR on neuronal differentiation and its implication in IR-induced brain damage are not well defined. To investigate the effects of IR on C17.2 mouse neural stem-like cells and mouse primary neural stem cells, neurite outgrowth and expression of neuronal markers and neuronal function-related genes were examined. To understand this process, the signaling pathways including PI3K, STAT3, metabotrophic glutamate receptor 1 (mGluR1) and p53 were investigated. In C17.2 cells, irradiation significantly increased the neurite outgrowth, a morphological hallmark of neuronal differentiation, in a dose-dependent manner. Also, the expression levels of neuronal marker proteins, ß-III tubulin were increased by IR. To investigate whether IR-induced differentiation is normal, the expression of neuronal function-related genes including synaptophysin, a synaptic vesicle forming proteins, synaptotagmin1, a calcium ion sensor, γ-aminobutyric acid (GABA) receptors, inhibitory neurotransmitter receptors and glutamate receptors, excitatory neurotransmitter receptors was examined and compared to that of neurotrophin-stimulated differentiation. IR increased the expression of synaptophysin, synaptotagmin1 and GABA receptors mRNA similarly to normal differentiation by stimulation of neurotrophin. Interestingly, the overall expression of glutamate receptors was significantly higher in irradiated group than normal differentiation group, suggesting that the IR-induced neuronal differentiation may cause altered neuronal function in C17.2 cells. Next, the molecular mechanism of the altered neuronal differentiation induced by IR was studied by investigating signaling pathways including p53, mGluR1, STAT3 and PI3K. Increases of neurite outgrowth, neuronal marker and neuronal function-related gene expressions by IR were abolished by inhibition of p53, mGluR-1, STAT3 or PI3K. The inhibition of PI3K blocked both p53 signaling and STAT3-mGluR1 signaling but inhibition of p53 did not affect STAT3-mGluR1 signaling in irradiated C17.2 cells. Finally, these results of the IR-induced altered differentiation in C17.2 cells were verified in ex vivo experiments using mouse primary neural stem cells. In conclusion, the results of this study demonstrated that IR is able to trigger the altered neuronal differentiation in undifferentiated neural stem-like cells through PI3K-STAT3-mGluR1 and PI3K-p53 signaling. It is suggested that the IR-induced altered neuronal differentiation may play a role in the brain dysfunction caused by IR.

Herbal preparation (HemoHIM) enhanced functional maturation of bone marrow-derived dendritic cells mediated toll-like receptor 4.

BACKGROUND: HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells. RESULTS: In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1ß, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4(+) and CD8(+) T cell responses. CONCLUSIONS: Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4(+) and CD8(+) T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses.

Smad2/3-Regulated Expression of DLX2 Is Associated with Radiation-Induced Epithelial-Mesenchymal Transition and Radioresistance of A549 and MDA-MB-231 Human Cancer Cell Lines.

The control of radioresistance and metastatic potential of surviving cancer cells is important for improving cancer eradication by radiotheraphy. The distal-less homeobox2 (DLX2) gene encodes for a homeobox transcription factor involved in morphogenesis and its deregulation was found in human solid tumors and hematologic malignancies. Here we investigated the role of DLX2 in association with radiation-induced epithelial to mesenchymal transition (EMT) and stem cell-like properties and its regulation by Smad2/3 signaling in irradiated A549 and MDA-MB-231 human cancer cell lines. In irradiated A549 and MDA-MB-231 cells, EMT was induced as demonstrated by EMT marker expression, phosphorylation of Smad2/3, and migratory and invasive ability. Also, irradiated A549 and MDA-MB-231 cells showed increased cancer stem cells (CSCs) marker. Interestingly, DLX2 was overexpressed upon irradiation. Therefore, we examined the role of DLX2 in radiation-induced EMT and radioresistance. The overexpression of DLX2 alone induced EMT, migration and invasion, and CSC marker expression. The reduced colony-forming ability in irradiated cells was partially restored by DLX2 overexpression. On the other hand, the depletion of DLX2 using si-RNA abolished radiation-induced EMT, CSC marker expression, and phosphorylation of Smad2/3 in irradiated A549 and MDA-MB-231 cells. Also, depletion of DLX2 increased the radiation sensitivity in both cell lines. Moreover, knockdown of Smad2/3, a key activator of TGF-ß1 pathway, abrogated the radiation-induced DLX2 expression, indicating that radiation-induced DLX2 expression is dependent on Smad2/3 signaling. These results demonstrated that DLX2 plays a crucial role in radioresistance, radiation-induced EMT and CSC marker expression, and the expression of DLX2 is regulated by Smad2/3 signaling in A549 and MDA-MB-231 cell lines.

Anti-allergic and anti-inflammatory effects of aqueous extract of Pogostemon cablin.

Allergic disease is caused by exposure to normally innocuous substances that activate mast cells. Mast cell-mediated allergic inflammation is closely related to a number of allergic disorders, such as anaphylaxis, allergic rhinitis, asthma and atopic dermatitis. The discovery of drugs for treating allergic disease is an interesting subject and important to human health. The aim of the present study was to investigate the anti­allergic and anti-inflammatory effects of the aqueous extract of Pogostemon cablin (Blanco) Benth (AEPC) (a member of the Labiatae family) using mast cells, and also to determine its possible mechanisms of action. An intraperitoneal injection of compound 48/80 or a serial injection of immunoglobulin E and antigen was used to induce anaphylaxis in mice. We found that AEPC inhibited compound 48/80­induced systemic and immunoglobulin E-mediated cutaneous anaphylaxis in a dose-dependent manner. The release of histamine from mast cells was reduced by AEPC, and this suppressive effect was associated with the regulation of calcium influx. In addition, AEPC attenuated the phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated expression of pro-inflammatory cytokines in mast cells. The inhibitory effects of AEPC on pro-inflammatory cytokines were dependent on the activation of nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). AEPC blocked the PMACI-induced translocation of NF-κB into the nucleus by hindering the degradation of IκBα and the phosphorylation of p38 MAPK. Our results thus indicate that AEPC inhibits mast cell­mediated allergic inflammation by suppressing mast cell degranulation and the expression of pro-inflammatory cytokines caused by reduced intracellular calcium levels and the activation of NF-κB and p38 MAPK.

Epicardial fat thickness is associated to type 2 diabetes mellitus in Korean men: a cross-sectional study.

BACKGROUND: Visceral fat, including epicardial fat (EF) is recognized as a responsible factor of cardiovascular disease. The aim of this study was to investigate the relationship between EF and diabetes in Korean men. METHODS: EF thickness was measured in the left main coronary artery fat tissue (LMCA-fat) by low-dose chest CT scans in 1,048 Korean men (age above 20 years). LMCA-fat values were divided into quartiles and the prevalence of diabetes was analyzed based on the quartiles of LMCA-fat values using logistic regression. RESULTS: There were significant correlations between LMCA-fat and body mass index (r = 0.169, p = 0.004), waist circumference (r = 0.172, p < 0.001), fasting glucose (r = 0.106, p = 0.037) and HbA1c (r = 0.176, p < 0.001). The patients in the higher LMCA-fat quartiles were associated with higher prevalence of diabetes (p for trend <0.001). Even after adjustment for multiple covariates, this association still remained statistically significant (p for trend = 0.022). The highest LMCA-fat quartile group was significantly associated with diabetes compared to the lowest quartile group. (OR = 3.26, 95% CI = 1.17-9.12). CONCLUSION: These findings indicate that increased EF thickness is independently associated with the prevalence of diabetes in Korean men.

Ionizing radiation induces neuronal differentiation of Neuro-2a cells via PI3-kinase and p53-dependent pathways.

PURPOSE: The influence of ionizing radiation (IR) on neuronal differentiation is not well defined. In this study, we investigated the effects of IR on the differentiation of Neuro-2a mouse neuroblastoma cells and the involvement of tumor protein 53 (p53) and mitogen-activated protein kinases (MAPK) during this process. MATERIALS AND METHODS: The mouse neuroblastoma Neuro-2a cells were exposed to (137)Cs γ-rays at 4, 8 or 16 Gy. After incubation for 72 h with or without inhibitors of p53, phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) and other kinases, the neuronal differentiation of irradiated Neuro-2a cells was examined through analyzing neurite outgrowth and neuronal maker expression and the activation of related signaling proteins by western blotting and immunocytochemistry. Mouse primary neural stem cells (NSC) were exposed to IR at 1 Gy. The change of neuronal marker was examined using immunocytochemistry. RESULTS: The irradiation of Neuro-2a cells significantly increased the neurite outgrowth and the expression of neuronal markers (neuronal nuclei [NeuN], microtubule-associated protein 2 [Map2], growth associated protein-43 [GAP-43], and Ras-related protein 13 [Rab13]). Immunocytochemistry revealed that neuronal class III beta-tubulin (Tuj-1) positive cells were increased and nestin positive cells were decreased by IR in Neuro-2a cells, which supported the IR-induced neuronal differentiation. However, the IR-induced neuronal differentiation was significantly attenuated when p53 was inhibited by pifithrin-α (PFT-α) or p53-small interfering RNA (siRNA). The PI3K inhibitor, LY294002, also suppressed the IR-induced neurite outgrowth, the activation of p53, the expression of GAP-43 and Rab13, and the increase of Tuj-1 positive cells. The increase of neurite outgrowth and Tuj-1 positive cells by IR and its suppression by LY294002 were also observed in mouse primary NSC. CONCLUSION: These results suggest that IR is able to trigger the neuronal differentiation of Neuro-2a cells and the activation of p53 via PI3K is an important step for the IR-induced differentiation of Neuro-2a cells.

A design for evaluation of the trauma apportionment in cerebral infarction after trauma.

OBJECTIVE: Posttraumatic cerebral infarction (CI) is a well-known complication of traumatic brain injury (TBI). However, the causation and apportionment of trauma in patients with CI after TBI is not easy. There is a scoring method, so-called trauma apportionment score (TAS) for CI, consisted with the age, the interval, and the severity of the TBI. We evaluated the reliability of this score. METHODS: We selected two typical cases of traumatic CI. We also selected consecutive 50 patients due to spontaneous CI. We calculated TAS in both patients with traumatic and spontaneous CI. To enhance the reliability, we revised TAS (rTAS) adding three more items, such as systemic illness, bad health habits, and doctor's opinion. We also calculated rTAS in the same patients. RESULTS: Even in 50 patients with spontaneous CI, the TAS was 4 in 44 patients, and 5 in 6 patients. TAS could not assess the apportionment of trauma efficiently. We recalculated the rTAS in the same patients. The rTAS was not more than 11 in more than 70% of the spontaneous CI. Compared to TAS, rTAS definitely enhanced the discriminating ability. However, there were still significant overlapping areas. CONCLUSION: TAS alone is insufficient to differentiate the cause or apportionment of trauma in some obscure cases of CI. Although the rTAS may enhance the reliability, it also should be used with cautions.

Classification of ginseng berry (Panax ginseng C.A. MEYER) extract using 1H NMR spectroscopy and its inhibition of lipid accumulation in 3 T3-L1 cells.

BACKGROUND: Panax ginseng is a famous traditional medicine in Korea for its beneficial effect on obesity, cardiac and liver associated diseases. The aim of this study was to investigate the metabolite in Panax ginseng (P. ginseng, Aralicaceae) berries depending on the ripen stages and evaluate its potential inhibition on adipocyte differentiation in 3 T3-L1 cells. METHODS: Different ripening stage samples of P. ginseng berry were analyzed through global metabolite profiling by NMR spectroscopy. Lipid accumulation in the cells was analyzed by Oil Red O staining. RESULTS: The PLS-DA clearly distinguished P. ginseng berry extract (PGBE) according to the partial ripe (PR), ripe(R) and fully ripe (FR) stage. Lipid accumulation of PGBE was examined by measuring triglyceride content and Oil-Red O staining. These results suggested that the FR stage of PGBE decrease in lipid accumulation during adipocyte differentiation and the amount of threonine, asparagine, fumarate, tyraine, tyrosine, and phenylalanine increased with longer ripening of ginseng berries. CONCLUSION: Metabolite profiling of P. ginseng was identified by 1H NMR spectra. P. ginseng extract efficiently inhibits adipogenesis in 3 T3-L1 adipocytes concluded that the P. ginseng has the antiobesity properties.