Woon Won Jung

Formaldehyde exposure induces airway inflammation by increasing eosinophil infiltrations through the regulation of reactive oxygen species production

Formaldehyde (FA) is a well-known cytotoxic irritant to the airways, but the mechanism of airway inflammation due to FA has not been clarified. In the present study, C57BL/6 mice were exposed to two concentrations (5 and 10ppm) of FA for 6h/day, 5days/week, for 2 weeks. The FA-exposed mice had much higher number of CCR3(+) eosinophils than control mice, and showed upregulated gene expression of CC-chemokine receptor-3 (CCR3), eotaxin and intercellular adhesion molecules-1 (ICAM-1) as well as an increased expression of proinflammatory and Th2 cytokines, such as interleukin (IL)-1β, IL-4 and IL-5. In addition, FA exposure revealed a considerable increase in the serum levels of IgG1, IgG3, IgA and IgE compared to controls. Histopathological analysis of the lung tissues demonstrated eosinophils and mononuclear cell infiltration of the alveolar cell walls and alveolar spaces. Gene expression of thioredoxin (TRX), redox-regulating antioxidant proteins, was markedly suppressed in FA-exposed mice, and thereby intracellular ROS levels were increased along with increased FA concentration. These results were consistent with an increase in the number of CCR3-expressing eosinophils, and indicate that FA-induced ROS was generated from eosinophils recruited to the inflammatory sites of the airways.

Complete Freund's adjuvant-induced intervertebral discitis as an animal model for discogenic low back pain.

BACKGROUND: Although numerous animal models for low back pain associated with intervertebral disk (IVD) degeneration have been proposed, insufficient data have been provided to make any conclusions regarding pain. Our aim in this study was to determine the reliability of complete Freund’s adjuvant (CFA) injection into the rat spine as an animal model representing human discogenic pain. METHODS: We studied IVD degenerative changes with pain development after a 10-&mgr;L CFA injection into the L5-6 IVD of adult rats using behavioral, histologic, and biochemical studies. Serial histologic changes were analyzed to detect degenerative changes. Expression of calcitonin gene-related peptide (CGRP), prostaglandin E (PGE), and inducible nitric oxide synthase (iNOS) were determined using immunohistochemistry or real-time polymerase chain reaction as support data for pain development. In addition, CGRP immunoreactivity (ir) at the IVD was considered indirect evidence of neural ingrowth into the IVD. RESULTS: There was a significant increase of the hindpaw withdrawal response in the CFA group until 7 wk postoperatively (P < 0.05). Histologic analyses revealed progressive degenerative changes of the disks without any damage in adjacent structures, including nerve roots. In the CGRP-ir staining study, the bilateral dorsal horns and IVD had positive ir after intradiscal CFA injection. CGRP mRNA expression was increased in the dorsal root ganglion (DRG) at 2 and 4 wk, whereas PGE and iNOS mRNAs were markedly increased at 2 wk. The increment of CGRP expression was higher in allodynic rats compared with nonallodynic rats. CONCLUSION: Intradiscal CFA injection led to chronic disk degeneration with allodynia, which was suggested by pain behavior and expression of pain-related mediators. The increment of CGRP, PGE, and iNOS also suggest pain-related signal processing between the IVD and the neural pathway in this animal model. This animal model may be useful for future research related to the pathophysiology and development of novel treatment for spine-related pain.

An evaluation of the neonatal immune system using a listeria infection model.

Background: T helper 1 (Th1)/T helper 2 (Th2)-biased cytokine regulation may be another reason that neonates are much more susceptible to infectious disease than are adults. Objectives: We attempted to determine the ability of neonatal mice to direct the Th1 phenotype against Listeria monocytogenes (LM), because LM, an intracellular Gram-positive bacterium, induces profound cellular immunity by Th1 cells in vivo. Methods: In order to determine whether neonatal mice evidence strong Th1 activity during LM infection, neonatal mice were compared with adult mice with regard to susceptibility to LM, cytotoxic T lymphocyte activity, and cytokine profiles. Neonatal gene profiles relevant to Th1 and Th2 differentiation during LM infection were also compared between neonatal and adult mice, via real-time PCR and RT-PCR. Results: Neonatal mice were found to be far more susceptible to LM infection than adult mice, due to a lack in the induction of cytotoxic T cell activity, coupled with poor IFN-γ secretion. Further, LM-infected neonatal mice evidenced much lower levels of expression of Th1-type immune components, including IL-12, IFN-γ, Delta-4 and T-bet, as compared to those features in adult mice. These results may be due to the comparably lower expressions of mannose-bind lectins and some of toll-like receptors (TLRs) such as TLR-5, -6 and -9, necessary mediators to develop Th1 immune responses. Conclusions: Neonatal mice may not mount an adequate Th1 type immune response due to a significantly lower expression of Th1-type immune components as compared to adult mice, even when forced into a Th1-prone environment.

Intervertebral disc degeneration-induced expression of pain-related molecules: glial cell-derived neurotropic factor as a key factor.

Background Discogenic low back pain has been shown to develop into chronic intractable pain due to an unknown pathogenesis. To study the mechanism of discogenic pain, we analyzed the serial expression of pain-related molecules in the dorsal root ganglia (DRG) and thalamus using a newly developed rat model of disc degeneration. Methods Ten microliters of complete Freunds adjuvant was injected into the L5-6 disc of male Sprague-Dawley rats for 10 minutes using a 26-gauge needle. Using a behavioral test, rats with significant pain were selected and subsequently serial gene expression of pain-related molecules in the DRG and the thalamus was analyzed by reverse transcriptase polymerase chain reaction. Results The expression of tumor necrosis factor-&agr; and interleukin-1&bgr; significantly increased at 4 and 8 weeks in the DRG of rats with pain. Furthermore, interleukin-6 was significantly increased at 4 weeks in the DRG; however, these cytokines did not show a significant change in the thalamus. Calcitonin gene-related peptide and substance P were significantly increased in DRG at 4 and 8 weeks and in the thalamus at 2 and 4 weeks. The level of nerve growth factor-&bgr; did not significantly increase in the DRG or thalamus, whereas glial cell line-derived neurotropic factor (GDNF) was significantly increased at 2 weeks and was sustained through 8 weeks in both the DRG and thalamus. Conclusions The disc degeneration rat model described herein led to significant pain of a chronic nature. The gradual and persistent increase of GDNF in both the thalamus and DRG suggests that GDNF might be a key factor in the development of intractable, chronic discogenic pain.

Evaluation of plasma carcinogenic markers in rat hepatic tumors models induced by rat hepatoma N1-S1 cells and benzo[a]pyrene

Benzo[a]pyrene (BaP) is a known carcinogen. Grilled or smoked meat is the major source of BaP intake for human beings. Previously, we established hepatic tumor animal models by injecting rat hepatoma N1-S1 cells or concomitant injection of N1-S1 cells and BaP into healthy Sprague-Dawley rats. In this study, we performed proteomic analyses of rat plasma collected from a hepatic tumor model and compared them to controls using a 3–10 pI range and large two dimensional gel electrophoreses. Proteomic analyses of rat plasma with hepatic tumors induced by the injection of N1-S1 cells resolved 1295 protein spots. Among them, 10 proteins were identified by ESI-MS-MS; four proteins were up-regulated and six proteins were down-regulated as compared to the controls. In addition, 1295 protein spots were also resolved from rats with hepatic tumors by the injection of N1-S1 cells plus BaP; five proteins were upregulated, and seven proteins were down-regulated. Of these 12 proteins, 10 proteins were identified by ESI-MS-MS. Out of 20 identified proteins, alpha-1-inhibitor 3 and zero beta-1 globin were down-regulated in both rats with hepatic tumors by N1-S1 cell-only and rats with hepatic tumors by the injection of N1-S1 cell plus BaP as compared to the controls. In addition, the identities of four proteins, including dermcidin, serum amyloid P-component (SAP), proteasome subunit alpha type-4 and glutathione peroxidase 3 (GPX-3) were confirmed by western blot analysis. Therefore, the importance of those proteins as candidate biomarkers for the development of hepatic tumors should be further elucidated.

Overexpression of metastatic tumor antigen in osteosarcoma: comparison between conventional high-grade and central low-grade osteosarcoma.

PURPOSE The metastatic tumor antigen (MTA) gene is a recently identified metastasis-associated gene which has implications in the signal transduction or regulation of gene expression. However, the expression of MTA in osteosarcoma and its potential relationship with metastasis have not been examined, forming the basis of this study. MATERIALS AND METHODS We compared the expression levels of the MTA1 protein between 32 cases of high-grade osteosarcomas and 21 cases of low-grade osteosarcomas by immunohistochemistry. In addition, the mRNA expression levels of MTA1, 2, 3 in these osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative PCR. RESULTS MTA1 immunoreactivity was present in 81.25% of high-grade osteosarcoma specimens. Its expression was predominantly localized to the nucleus or cytoplasm of osteosarcoma cells. Thirteen (86.6%) of 15 patients who died of osteosarcomas displayed strong MTA1 expression. Both primary bone and pulmonary metastatic lesions exhibited MTA1 expression. All low-grade osteosarcomas were negative for MTA1 except for focal weak reactivity in two cases. The tested high-grade osteosarcoma cell lines showed marked amplification of MTA1 and MTA2 mRNA compared to control cells. CONCLUSION These results suggest that MTA might be involved in the progression of high-grade osteosarcoma, particularly in hematogenous metastasis of osteosarcoma.

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two-dimensional electrophoresis.

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH4HCO3 + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.

Expression of vascular endothelial growth factor-C and its receptor in osteosarcomas.

Vascular endothelial growth factor-C (VEGF-C) and its receptor, vascular endothelial growth factor receptor-3 (VEGFR-3), have been implicated as important factors in the formation of lymphatic vessels, but its role in osteosarcomas has not yet been fully investigated. This study aims to define the expression of VEGF-C and VEGFR-3 in primary and metastatic osteosarcomas and their relationship to various clinicopathologic parameters. Thirty-three primary osteosarcomas and two pulmonary metastatic samples were immunostained for VEGF-C and VEGFR-3. In addition, VEGF-C and vascular endothelial growth factor-D (VEGF-D) mRNA expression levels in three different human osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative polymerase chain reaction (PCR). Both VEGF-C and VEGFR-3 were expressed mainly in the cytoplasm of the tumor cells. Of the 35 patients with osteosarcoma, 16 patients (45.7%) showed strong positive reaction with VEGF-C. Four cases (11.4%) were negative, and 15 cases (42.9%) showed weak immunoreactivity. For VEGFR-3, 12 patients (34.3%) showed strong positive reaction. Fifteen cases (42.9%) were negative, and eight cases (22.8%) showed weak immunoreactivity. A significant, positive correlation (Rs=0.42, p=0.01) was found between the expression of VEGF-C and VEGFR-3 in osteosarcomas. The expression of VEGF-C was significantly associated with the osteoblastic subtype and high histologic grade in osteosarcomas. However, the expression of VEGF-C showed no significant correlation with the presence of metastasis. Expression of VEGFR-3 was not related to any clinicopathologic features analyzed. Two of the three osteosarcoma cell lines tested showed amplification of VEGF-C mRNA compared with control cells. No amplification of VEGF-D was noted in these cell lines. Our data suggest that VEGF-C and its receptor are expressed in osteosarcomas. Although the level of VEGF-C was high, it does not seem to have a direct influence on tumor metastasis in osteosarcomas.

Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression

Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (Ab) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G1 to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27KIP1. Saponins also increased stability of p27KIP1 in Th cells after antigenic stimulation.

E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.