Copper/zinc chelation by clioquinol reduces spinal cord white matter damage and behavioral deficits in a murine MOG-induced multiple sclerosis model.

The present study aimed to evaluate the therapeutic potential of clioquinol (CQ), a metal chelator, on multiple sclerosis pathogenesis. Experimental autoimmune encephalomyelitis was induced by immunization with myelin oligodendrocyte glycoprotein (MOG(35-55)) in female mice. Three weeks after the initial immunization, demyelination and immune cell infiltration in the spinal cord were analyzed. CQ (30mg/kg) was given by gavage once per day for the entire experimental course. CQ profoundly reduced the daily clinical score and incidence rate of EAE mice. The CQ-mediated inhibition of the clinical course of EAE was accompanied by suppression of demyelination and reduced infiltration by encephalitogenic immune cells including CD4, CD8, CD20 and F4/80 positive cells. CQ also remarkably inhibited EAE-associated BBB disruption and MMP-9 activation. Autophagy contributes to clearance of aggregated proteins in astrocytes and neurons. The present study found that EAE increased the induction of autophagy and CQ further increased this expression. Furthermore, the present study found that post-treatment with CQ also reduced the clinical score of EAE and spinal cord demyelination. These results demonstrate that CQ inhibits the clinical features and neuropathological changes associated with EAE. The present study suggests that transition metals may be involved in several steps of multiple sclerosis pathogenesis.

Comparison of methods for the repair of acute tympanic membrane perforations: Silk patch vs. paper patch.

We investigated the effects of repairing large tympanic membrane (TM) perforations in rats with a thin silk patch compared with the commonly used paper patch. We performed bilateral myringotomies of 1.8 mm in diameter on 50 adult Sprague-Dawley rats with intact TMs. The perforations in the right ears of 40 rats were treated with a silk patch, and the perforations in the left ears of the same rats were treated with a paper patch. Ten rats acted as controls. The mean healing times of the TM perforations on the silk-patch-treated ears and the paper-patch-treated ears were 7.2+/-1.48 and 9.1+/-1.11 days, respectively (control 10.38+/-1.70 days). The difference between silk-patch- and paper-patch-treated ears was statistically significant, with a mean difference of 1.9 days (0.6-4.5 days). The mean perforation closure times were significantly shorter in silk-patch- and paper-patch-treated ears than in the control animals. The endoscopic and histological findings of this study provide evidence that silk-patch treatment accelerates wound healing and shortens TM perforation closure time. We suggest that the silk patch may prove to be an effective material for repairing TM perforations in human patients in an outpatient clinical setting.

2,4-Dinitrofluorobenzene modifies cellular proteins and induces macrophage inflammatory protein-2 gene expression via reactive oxygen species production in RAW 264.7 cells.

The skin sensitizer 2,4-dinitrofluorobenzene (DNFB) provokes delayed hypersensitivity responses as a result of topical application to the skin. Here, we demonstrate that DNFB modifies proteins in RAW 264.7 cells and skin tissues in NC/Nga mice; we also show the functional involvement of DNFB-induced modification of cellular proteins in the DNFB-induced macrophage inflammatory protein (MIP)-2 gene expression in RAW 264.7 cells. In addition, we demonstrate that DNFB strongly induces reactive oxygen species (ROS) production. Our RT-PCR analysis and reporter gene assays reveal that the DNFB-induced intracellular ROS production is necessary for MIP-2 gene expression by DNFB. We observed that the vitamin C and chemical oxidant scavenger N-acetyl-cysteine have an inhibitory effect on the generation of ROS, the activation of MAP kinase pathways, and the MIP-2 gene expression in DNFB-treated RAW 264.7 cells. These results provide insight into the mechanisms involved in DNFB-induced contact hypersensitivity.

Effects of 60 Hz 14 microT magnetic field on the apoptosis of testicular germ cell in mice.

We recently reported that continuous exposure, for 8 weeks, of extremely low frequency (ELF) magnetic field (MF) of 0.1 or 0.5 mT might induce testicular germ cell apoptosis in BALB/c mice. In that report, the ELF MF exposure did not significantly affect the body weight or testicular weight, but significantly increased the incidence of testicular germ cell death. In the present study, we aimed to further characterize the effect of a 16-week continuous exposure to ELF MF of 14 or 200 microT on testicular germ cell apoptosis in mice. There were no significant effects of MF on body weight and testosterone levels in mice. In TUNEL staining (In situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling), germ cells showed a significantly higher apoptotic rate in exposed mice than in sham controls (P < 0.001). TUNEL-positive cells were mainly spermatogonia. In an electron microscopic study, degenerating spermatogonia showed condensation of nuclear chromatin similar to apoptosis. These results indicate that apoptosis may be induced in spermatogenic cells in mice by continuous exposure to 60 Hz MF of 14 microT.

Delayed allograft rejection by the suppression of class II transactivator.

(CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenicity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.

Involvement of lymphocytes in dextran sulfate sodium-induced experimental colitis.

AIM: To investigate the roles of lymphocytes in the development of dextran sulfate sodium-induced colitis. METHODS: Using various doses of dextran sulfate sodium (DSS), we induced colitis in wild-type B6 control and Rag-1 knockout (H-2b haplotype) mice, and evaluated the colitis in terms of symptomatic and histologic parameters, such as weight loss, survival, severity of diarrhea, shortage of colon length and histological changes. Symptomatic parameters were checked daily and histological changes were scored. RESULTS: Although development of colitis in Rag-1 knockout mice treated with high dose (5%) of DSS was comparable to that in B6 control mice, colitis progression was much more tolerable in Rag-1 knockout mice compared to than in B6 mice treated with low dose (1.5%) DSS. Symptomatic parameters as well as histopathologic changes were improved in Rag-1 knockout mice. CONCLUSION: These results indicate that the presence of lymphocytes contributes to colitis progression at low dose of DSS stimulation. Lymphocytes may play roles as an aggravating factor in DSS-induced colitis.

Prevention and therapy of hepatocellular carcinoma by vaccination with TM4SF5 epitope-CpG-DNA-liposome complex without carriers.

Although peptide vaccines have been actively studied in various animal models, their efficacy in treatment is limited. To improve the efficacy of peptide vaccines, we previously formulated an efficacious peptide vaccine without carriers using the natural phosphodiester bond CpG-DNA and a special liposome complex (Lipoplex(O)). Here, we show that immunization of mice with a complex consisting of peptide and Lipoplex(O) without carriers significantly induces peptide-specific IgG2a production in a CD4(+) cells- and Th1 differentiation-dependent manner. The transmembrane 4 superfamily member 5 protein (TM4SF5) has gained attention as a target for hepatocellular carcinoma (HCC) therapy because it induces uncontrolled growth of human HCC cells via the loss of contact inhibition. Monoclonal antibodies specific to an epitope of human TM4SF5 (hTM4SF5R2-3) can recognize native mouse TM4SF5 and induce functional effects on mouse cancer cells. Pre-immunization with a complex of the hTM4SF5R2-3 epitope and Lipoplex(O) had prophylactic effects against tumor formation by HCC cells implanted in an mouse tumor model. Furthermore, therapeutic effects were revealed regarding the growth of HCC when the vaccine was injected into mice after tumor formation. These results suggest that our improved peptide vaccine technology provides a novel prophylaxis measure as well as therapy for HCC patients with TM4SF5-positive tumors.

Immunization with a hemagglutinin-derived synthetic peptide formulated with a CpG-DNA-liposome complex induced protection against lethal influenza virus infection in mice.

Whole-virus vaccines, including inactivated or live-attenuated influenza vaccines, have been conventionally developed and supported as a prophylaxis. These currently available virus-based influenza vaccines are widely used in the clinic, but the vaccine production takes a long time and a huge number of embryonated chicken eggs. To overcome the imperfection of egg-based influenza vaccines, epitope-based peptide vaccines have been studied as an alternative approach. Here, we formulated an efficacious peptide vaccine without carriers using phosphodiester CpG-DNA and a special liposome complex. Potential epitope peptides predicted from the hemagglutinin (HA) protein of the H5N1 A/Viet Nam/1203/2004 strain (NCBI database, AAW80717) were used to immunize mice along with phosphodiester CpG-DNA co-encapsulated in a phosphatidyl-ß-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex (Lipoplex(O)) without carriers. We identified a B cell epitope peptide (hH5N1 HA233 epitope, 14 amino acids) that can potently induce epitope-specific antibodies. Furthermore, immunization with a complex of the B cell epitope and Lipoplex(O) completely protects mice challenged with a lethal dose of recombinant H5N1 virus. These results suggest that our improved peptide vaccine technology can be promptly applied to vaccine development against pandemic influenza. Furthermore our results suggest that potent epitopes, which cannot be easily found using proteins or a virus as an antigen, can be screened when we use a complex of peptide epitopes and Lipoplex(O).

Effects of lipopolysaccharide and CpG-DNA on burn-induced skin injury.

Destruction of the skin barrier by thermal injury induces microbial invasion, which can lead to the development of systemic infection and septic shock. Microbial pathogens possess pathogen-associated molecular patterns (PAMPs), which are recognized by conserved receptors. To understand the role of PAMPs in thermal injury-induced mice, LPS or CpG-DNA were topically applied to dorsal skin after thermal injury. We observed an increase in the number of inflammatory cell infiltrates as well as thickening in the dermis upon treatment with LPS or CpG-DNA. We also found that expression of IL-1ß, MIP-2, and RANTES induced by thermal injury was enhanced by LPS or CpG-DNA. In addition, the proportions of CD4(+) and CD8(+) T cells in the spleen and lymph nodes were altered by LPS or CpG-DNA. These results provide important information concerning PAMPs-induced inflammation upon thermal injury and provide a basis for studying the role of PAMPs in thermal injury-induced complications.

Protection of burn-induced skin injuries by the flavonoid kaempferol.

Thermal burn injury induces inflammatory cell infiltrates in the dermis and thickening of the epidermis. Following a burn injury, various mediators, including reactive oxygen species (ROS), are produced in macrophages and neutrophils, exposing all tissues to oxidative injury. The anti-oxidant activities of flavonoids have been widely exploited to scavenge ROS. In this study, we observed that several flavonoids-kaempferol, quercetin, fisetin, and chrysin-inhibit LPS-induced IL-8 promoter activation in RAW 264.7 cells. In contrast with quercetin and fisetin, pretreatment of kaempferol and chrysin did not decrease cell viability. Inflammatory cell infiltrates in the dermis and thickening of the epidermis induced by burn injuries in mice was relieved by kaempferol treatment. However, the injury was worsened by fisetin, quercetin, and chrysin. Expression of TNF-a induced by burn injuries was decreased by kaempferol. These findings suggest the potential use of kaempferol as a therapeutic in thermal burn-induced skin injuries. [BMB reports 2010; 43(1): 46-51].

Expression pattern of immunoproteasome subunits in human thymus.

The expression pattern of immunoproteasomes in human thymus has not been analyzed but may have important consequences during thymic selection. Here we examined the expression patterns of immunoproteasome subunits in fetal and adult thymic tissues by immunohistochemistry and found that all three subunits are expressed in both cortical and medullary stromal cells. These data suggest that thymic selection in human can be affected by peptide repertoires generated by immunoproteasomes.

Diethylnitrosamine (DEN) induces irreversible hepatocellular carcinogenesis through overexpression of G1/S-phase regulatory proteins in rat.

Hepatocellular carcinoma (HCC) is the fifth most frequent cause of cancer deaths in males and was the third most frequent cause of cancer deaths in 2007 throughout the world. The incidence rate is 2-3 times higher in developing countries than in developed countries. Animal models have enabled study of the mechanism of HCC and the development of possible strategies for treatment. Diethylnitrosamine (DEN) is a representative chemical carcinogen with the potential to cause tumors in various organs, including the liver, skin, gastrointestinal tract, and respiratory system. Specifically in HCC, DEN is a complete carcinogen. Many lines of evidence have demonstrated a relationship between carcinogenesis and cell cycle regulation. In this study we found that cell cycle regulatory proteins were critically involved in cancer initiation and promotion by DEN. Cyclin D1, cyclin E, cdk4, and p21(CIP1/WAF1) are factors whose expression levels may be useful as criteria for the classification of hepatic disease. In particular, cdk4 had a pivotal role in the transition to the neoplastic stage. In conclusion, we suggest that changes in the level of cdk4 may be useful as a biomarker for detection of HCC.

Regulation of macrophage inflammatory protein-2 gene expression in response to 2,4-dinitrofluorobenzene in RAW 264.7 cells.

Several skin sensitizers, like 2,4-dinitrofluorobenzene (DNFB), are known to provoke contact hypersensitivity responses after topical application. Here, we show that DNFB can upregulate macrophage inflammatory protein-2 (MIP-2) expression in RAW 264.7 cells via a mechanism that is largely dependent on mitogen-activated protein kinase (MAPK) signaling pathways. ELISA-based transcription factor activation assays and chromatin immunoprecipitation assays revealed that functional interaction between AP-1 and MIP-2 promoter element is necessary for MIP-2 gene expression by DNFB. Interestingly, topical application of DNFB to NC/Nga mice increased MIP-2 expression in dermis, suggesting that MIP-2 contributes to the leukocyte infiltration associated with atopic dermatitis. These results provide additional insight of the mechanism of contact hypersensitivity induced by contact sensitizers.