Neonatal influenza virus infection affects myelination in influenza-recovered mouse brain

Influenza virus infection is a zoonosis that has great socioeconomic effects worldwide. Influenza infection induces respiratory symptoms, while the influenza virus can infect brain and leave central nervous system sequelae. As children are more vulnerable to infection, they are at risk of long-term neurological effects once their brains are infected. We previously demonstrated that functional changes in hippocampal neurons were observed in mice recovered from neonatal influenza infection. In this study, we investigated changes in myelination properties that could affect neural dysfunction. Mice were infected with the influenza virus on postnatal day 5. Tissues were harvested from recovered mice 21-days post-infection. The expression levels for myelin basic protein (MBP) were determined, and immunohistochemical staining and transmission electron microscopy were performed. Real-time polymerase chain reaction and Western blot analyses showed that mRNA and protein expressions increased in the hippocampus and cerebellum of recovered mice. Increased MBP-staining signal was observed in the recovered mouse brain. By calculating the relative thickness of myelin sheath in relation to nerve fiber diameter (G-ratio) from electron photomicrographs, an increased G-ratio was observed in both the hippocampus and cerebellum of recovered mice. Influenza infection in oligodendrocyte-enriched primary brain cell cultures showed that proinflammatory cytokines may induce MBP upregulation. These results suggested that increased MBP expression could be a compensatory change related to hypomyelination, which may underlie neural dysfunction in recovered mice. In summary, the present results demonstrate that influenza infection during the neonatal period affects myelination and further induces functional changes in influenza-recovered mouse brain.

An immunohistochemical study on the initial development of 155 kd neurofascin in rat sciatic nerve / 대한수의학회지

Neurofascin, one of the members of L1CAM, has been known to have some important roles during the development of nerve fibers. In order to investigate the role of neurofascin associated with the development of nerve fibers in the rat sciatic nerve, the initial development of NF155 in the paranode was studied with immuno-fluorescence and immuno-electron microscopy. The result of the present study showed NF155 was not detected in the fetal sciatic nerve and began to reveal at the postnatal day 0 (P0) and dramatically increased by time lapse until postnatal day 7 (P7). NF155 was prominently localized in the axolemma of paranode and not detected in the central region of node of Ranvier. According to the present study, NF155 is likely to have some relationships with the formation of paranode and myelin sheath.

Effect of 5-FU and MTX on the Expression of Drug-resistance Related Cancer Stem Cell Markers in Non-small Cell Lung Cancer Cells

Cancer stem cells (CSCs) are often characterized by the elevated expression of drug-resistance related stem-cell surface markers, such as CD133 and ABCG2. Recently, we reported that CSCs have a high level of expression of the IL-6 receptor (IL-6R). The purpose of this study was to investigate the effect of anticancer drugs on the expression of the drug resistance-related cancer stem cell markers, ABCG2, IL-6R, and CD133 in non-small cell lung cancer (NSCLC) cell lines. A549, H460, and H23 NSCLC cell lines were treated with the anticancer drugs 5-fluorouracil (5-FU; 25 microg/ml) and methotrexate (MTX; 50 microg/ml), and the expression of putative CSC markers was analyzed by fluorescent activated cell sorter (FACS) and the gene expression level of abcg2, il-6r and cd133 by reverse transcriptasepolymerase chain reaction (RT-PCR). We found that the fraction of ABCG2-positive(+) cells was significantly increased by treatment with both 5-FU and MTX in NSCLC cells, and the elevation of abcg2, il-6r and cd133 expressions in response to these drugs was also confirmed using RT-PCR. Also, the number of IL-6R(+) cells was increased by MTX in the 3 cell lines mentioned and increased by 5-FU in the H460 cell line. The number of CD133(+) cells was also significantly increased by both 5-FU and MTX treatment in all of the cell lines tested. These results indicate that 5-FU and MTX considerably enhance the expression of drug-resistance related CSC markers in NSCLC cell lines. Thus, we suggest that antimetabolite cancer drugs, such as 5-FU and MTX, can lead to the propagation of CSCs through altering the expression of CSC markers.

Expression patterns of influenza virus receptors in the respiratory tracts of four species of poultry

The primary determinant of influenza virus infectivity is the type of linkage between sialic acid and oligosaccharides on the host cells. Hemagglutinin of avian influenza viruses preferentially binds to sialic acids linked to galactose by an alpha-2,3 linkage whereas hemagglutinin of human influenza viruses binds to sialic acids with an alpha-2,6 linkage. The distribution patterns of influenza receptors in the avian respiratory tracts are of particular interest because these are important for initial viral attachment, replication, and transmission to other species. In this study, we examined the distribution patterns of influenza receptors in the respiratory tract of chickens, ducks, pheasants, and quails because these species have been known to act as intermediate hosts in interspecies transmission. Lectin histochemistry was performed to detect receptor-bearing cells. Cell-specific distribution of the receptors was determined and expression densities were compared. We observed species-, site-, and cell-specific variations in receptor expression. In general, receptor expression was the highest in quails and lowest in ducks. Pheasants and quails had abundant expression of both types of receptors throughout the respiratory tract. These results indicate that pheasants and quails may play important roles as intermediate hosts for the generation of influenza viruses with pandemic potential.

Characterization of canine oral papillomavirus by histopathological and genetic analysis in Korea

In August 2008, forty dogs out of 400 developed oral warts in a breeding farm in Korea. Canine oral papilloma infection is a common disease in dogs. However, there has been no report of an outbreak of canine oral papillomavirus (COPV) in a group of dogs or in dog breeding farms in Korea, and the genetic analysis of COPV in Korea has yet to be performed. This study diagnosed canine oral papilloma from the oral samples of these dogs based on histopathological examination and immunohistochemistry. Polymerase chain reaction was applied to amplify the corresponding products using pre-existing primer sets for COPV and a universal human papillomavirus targeting L1 gene. Further genetic analysis of the major viral capsid gene L1 confirms the sequences of Korean COPV, which shows a close relationship to previously reported COPV. This study describes the histopathological and immunohistochemical characteristics of canine oral papilloma in a group of breeding dogs in Korea and discloses the complete L1 gene sequences of Korean COPV.

The Effects of air-borne particulate matters on the Alveolar Macrophages for the iNOS Expression and Nitric Oxide with Nitrotyrosilated-proteins Formation / 결핵및호흡기질환

BACKGROUND: Particulate matters (PM) when inhaled is known to induce pulmonary diseases including asthma and chronic bronchitis when inhaled. Despite the epidemiological proofevidence, the pathogenesis of PM-related pulmonary diseases is unclearremain poorly understood. METHODS: Primary alveolar macrophages were harvested from the SPF and inflammatory rats by bronchioalveolar lavage (BAL). The cultured primary alveolar macrophages were treated with the medium only, PM only (5~40 microgram/cm2), LPS (5ng/ml) only, and PM with LPS for 24 and 48 hours. The level of secreted nitric oxide (NO) was assayed from the cultured medium by using the Griess reaction. The cultured cells were utilized for the western blotting against the inducible nitric oxide synthase (iNOS) proteins. Immunocyto- chemical staining against the iNOS and NT-proteins were performed in cells that cultured in the Lab-Tek(R) chamber slide after treatments. RESULTS: The PM that utilizein this experiments induced NO formation with iNOS expression in the cultured SPF and inflammatory rats alveolar macrophages, by itself. When the cells were co-treated with PM and LPS, there was a statistically significant synergistic effect on NO formation and iNOS expression over the LPS effect. The cells from the sham control showed minimal immunoreactivity for the NT-proteins. Significantly higher quantities of NT-proteins were detected in the PM and PM with LPS co-treated cells than from the sham control. CONCLUSION: Increased iNOS expression and NO formation with increased NT-proteins formation might be involved in the pathogenesis of PM-induced lung injury.

The Effects of Air-borne Particulate Matters on the Alveolar Macrophages for the TNF-alpha and IL-1beta Secretion / 결핵및호흡기질환

BACKGROUND: PM is known to induce various pulmonary diseases, including asthma, cancer, fibrosis and chronic bronchitis. Despite the epidemiological evidence the pathogenesis of PM-related pulmonary diseases is unclear. METHODS: This study examined the effects of PM exposure on the secretion of TNF-alpha and IL-1beta in the cultured alveolar macrophages. The cultured primary alveolar macrophages were treated with the medium, PM (5~20 microgram/cm2), LPS (5ng/ml), and PM with LPS for 24h and 48h respectively. ELISA was used to assay the secreted TNF-alpha and IL-beta in the culture medium. Western blotting was used to identify and determine the level of proteins isolated from the culture cells. The cells cultured in the Lab-Tek(R) chamber slides were stained with immunocytochemical stains. RESULTS: PM induced TNF-alpha and IL-1beta secretion in the culturing alveolar macrophages, collected from the SPF and inflammatory rats. However, the effects were only dose-dependent in the inflammatory macrophages. When the cells were co-treated with PM and LPS, there was a significant synergistic effect compared with the LPS in the both cell types. CONCLUSION: PM might be play an important role in the induction and/or potentiation of various lung diseases by oversecretion of TNF-alpha and IL-1beta.

Effect of Etomidate on the Expressions of c-JUN and Heat Shock Protein 70 Induced by Kainic Acid in Rats / 대한마취과학회지

BACKGROUND: The present investigation was undertaken to evaluate the neuroprotective effect of etomidate against kainic acid (KA) induced neurotoxicity in rats by Western immunoblotting for heat shock protein 70 (HSP-70), c-JUN, and by acid-fuchsin histochemistry. METHODS: Etomidate (20 mg/kg, i.p.) was administered in sequence, first being just one hour after KA (10 mg/kg, i.p.) injection, then three times one hour intervals. Neuronal damage in hippocampus was evaluated by using acid-fuchsin stain to detect cell death and HSP-70 and c-JUN induction as an index of cell injury at 3, 6, 24 and 48 h after the administration of KA. RESULTS: Acid fuchsin positive neurons were increased in the CA1 and CA3 regions of the hippocampus after KA injection, but were significantly decreased by etomidate injection (P <0.01). Etomidate administration also significantly suppressed the KA-induced induction of c-JUN and HSP-70 in both regions of the rat hippocampus. CONCLUSIONS: These results suggest that etomidate has a protective effect on hippocampal neurons against KA-induced neurotoxicity.

Expression of PSD95 in the Rat Sciatic Nerve

This study was designed to elucidate the existence of PSD95 in the rat sciatic nerve. Immunohistochemical stains of cryosection and teased fiber of sciatic nerves were performed with goat polyclonal antibody against PSD95. Western blot analysis was also accomplished with the same antibody. We got an interesting result that the rat sciatic nerve obviously showed PSD95 immunoreactivity especially in the nodal and paranodal regions, and we also identified a distinct band of PSD95 by western blot. These results suggest PSD95 exists in the sciatic nerve as well as it does in the central nervous system. We suppose PSD95 may have some important roles in ion channel clustering, junctional plasticity and signal transduction in the peripheral nerves as well.

Translocational changes of localization of synapsin in axonal sprouts of regenerating rat sciatic nerves after ligation crush injury

Time-dependent translocational changes of Synapsin I (SyI), a synaptic vesicle-associated phosphoprotein and its involvement in the axonal transport were investigated in the regenerating axonal sprouts. A weak SyI immunoreactivity (IR) was found in the axoplasm of normal axons. Rat sciatic nerves were crush-injured by ligating with 1-0 silk thread at the mid-thigh level and released from the ligation 24 h later. At various times after release, immunocytochemistry was performed. SyI was translocated from the proximal to the distal site of ligation and also involved in the sprouting of regenerating axons. The distribution patterns of SyI IR were changed in the crush-injured nerves. SyI immunoreactive thin processes were strongly appeared in the proximal region from 1 h after release. After 3 h, a very strong IR was expressed. The intense SyI immunoreactive thin processes were elongated distally and were changed the distribution pattern by time-lapse. After 12 h, strong immunoreactive processes were extended to the ligation crush site. At 1 day, a very intense IR was expressed. At 2 days, immunoreactive thin processes extended into the distal region over the ligation crush site and strong IR was observed after 3 days. SyI was accumulated in the proximal region at the early phases after release. These results suggest that SyI may be related to the translocation of vesicles to the elongated membranes by a fast axonal transport in the regenerating sprouts.

A study on the immunocytochemical localization of neurofascin in rat sciatic nerve

We examined the localization of neurofascin (NF) in the sciatic nerve of rat. In the myelinated fibers, neurofascin localizes strongly in the nodal axolemma except the small central cleft and also expresses in the paranodes, and weakly in the Schmidt-Lanterman incisures. In the paranodes, NF localizes around the axolemma and it expresses in the apposing membrane of paranodal loops. Axoplasm, compact myelin and cytoplasm of Schwann cell do not express NF at all. In the Schmidt-Lanterman incisures, NF is expressed weakly along the Schwann cell membrane. We propose that neurofascin may be a plasmalemmal integral protein of Schwann cell in the paranode and plays some important roles for the maintenance of axo-glial junctions at the paranode. It may also have some roles for maintaining the structure of Schmidt-Lanterman incisure and have some relations with proteins localizing in the node.