OBJECTIVE: Conventional methods for organotypic hippocampal tissue slice culture (OHSC) have shown several disadvantages or limitations regarding age of animals used, duration of culture and difficulty using neurodegenerative models. Therefore, we tried to establish OHSC from old 3xTg-Alzheimer’s disease (AD) mice for longer period (over 4 weeks) and to validate utility of this system as a valid platform for translational neuroscience of AD. METHODS: OHSC was performed with old 3xTg-AD mice (12–14 months), old wild type mice (12–14 months) and young 3xTg-AD mice (2–4 months) using serum-free medium for 4 weeks. Hippocampal structure was evaluated by 4’, 6-diamidino-2-phenylindole (DAPI) intensity and neuronal metabolism was measured by Alamarblue assay. Pathologic characteristics of AD were also investigated; β-amyloid levels by ELISA, amyloid plaque deposition by Thioflavin-S staining, and glial activation by immunohistochemistry. RESULTS: Following 4-week culture in serum-free media, hippocampal cells and layers were well preserved in cultured slices from old AD mice as was in those from young AD and old wild type mice. On the contrary, excessive regression of total visible cells was observed in conventional serum-containing medium regardless of genotype of mice. In parallel with this well preserved structure, major pathologic characteristics of AD were also well manifested in hippocampal slices from old AD mice. CONCLUSION: Our findings suggest that long-term OHSC from old 3xTg-AD mouse can serve as a promising ex vivo system for studies on pathophysiology of AD, especially with the minimum number of sacrifice of experimental animals.
Alzheimer Disease ; Animals ; Culture Media, Serum-Free ; Enzyme-Linked Immunosorbent Assay ; Genotype ; Hippocampus ; Immunohistochemistry ; Metabolism ; Mice ; Neurons ; Neurosciences ; Plaque, Amyloid

OBJECTIVES: Deposition of the beta-amyloid(Abeta) peptide in the senile plaque has been thought as a major etiologic factor for the development of Alzheimer's disease. Among the hypotheses suggested to explain the mechanism by which Abeta causes Alzheimer's disease, the immune processes have been considered as crucial events in the pathophysiology of the Alzheimer's disease. This study examined the effects of Abeta on the proliferation and the production of IL-1beta(interleukin-1beta) and TNF-alpha(tumor necrosis factor-alpha) in peripheral blood mononuclear cells isolated from the patients with Alzheimer's disease, vascular dementia, and normal elderly control subjects. METHODS: Nineteen patients with Alzheimer's disease, 22 patients with vascular dementia, and 19 controls were participated in this study. Peripheral blood mononuclear cells were obtained from each donors, and subjected to the proliferation assays in response to the stimulation of phytohemagglutinin-P(PHA-P) and Abeta. The levels of IL-1beta and TNF-alpha from the culture supernatants of the cells before and after the stimulation of Abeta were also determined by enzyme linked immunosorbent assay. RESULTS: The results were as follows: 1) The proliferation of mononuclear cells in response to PHA-P were not different among three groups. 2) When compared to PHA-P, the proliferation responses of mononuclear cells to Abeta were insignificant in all experimental groups. However Alzheimer's disease group showed greater stimulation index than vascular dementia and controls. 3) IL-1beta production was higher in the vascular dementia group than Alzheimer's disease and control groups both before and after the stimulation of Abeta. However the stimulation ratio of before and after Abeta stimulation was highest in Alzheimer's disease group. 4) TNF-alpha production was higher in Alzheimer's disease group than controls both before and after the stimulation of Abeta. CONCLUSION: These findings suggest that the immune responses to the stimulation of Abeta may be enhanced in patients with Alzheimer's disease compared to vascular dementia and control groups, supporting the immune hypothesis for the pathophysiology of Alzheimer's disease.
Aged ; Alzheimer Disease ; Amyloid ; Dementia* ; Dementia, Vascular ; Enzyme-Linked Immunosorbent Assay ; Humans ; Necrosis ; Plaque, Amyloid ; Self-Help Groups ; Tissue Donors ; Tumor Necrosis Factor-alpha

OBJECTIVETo compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method.

METHODSFour recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed.

RESULTSNo significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml).

CONCLUSIONGFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.

Adenoviridae ; isolation & purification ; metabolism ; physiology ; Capsid Proteins ; metabolism ; DNA, Viral ; isolation & purification ; Green Fluorescent Proteins ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Viral Plaque Assay ; methods ; Virus Replication

BACKGROUND: LIGHT (TNFSF14) is a member of tumor necrosis factor superfamily and is the ligand for TR2 (TNFRSF14/HVEM). LIGHT is known to have pro- inflammatory roles in atherosclerosis. METHODS: To find out the expression pattern of LIGHT in atherosclerotic plaques, immunohistochemical analysis was performed on human carotid atherosclerotic plaque specimens. LIGHT induced atherogenic events using human monocytic cell line THP-1 were also investigated. RESULTS: Immunohistochemical analysis revealed expression of LIGHT and TR2 in foam cell rich regions in the atherosclerotic plaques. Double immunohistochemical analysis further confirmed the expression of LIGHT in foam cells. Stimulation of THP-1 cells, which express TR2, with either recombinant LIGHT or immobilized anti-TR2 monoclonal antibody induced interleukin-8 and matrix metalloproteinase(MMP)-9. Electrophoretic mobility shift assay demonstrated that LIGHT induces nuclear localization of transcription factor, nuclear factor (NF)-kappaB. LIGHT induced activation of MMP-9 is mediated by NF-kappaB, since treatment of THP-1 cells with the NF-kappaB inhibitor PDTC (pyrrolidine dithiocarbamate) completely blocked the activation of MMP-9. CONCLUSION: These data indicate that LIGHT is expressed in foam cells in atherosclerotic plaques and is involved in atherogenesis through activation of pro-atherogenic cytokine IL-8 and destabilization of plaque by inducing matrix degrading enzyme.
Atherosclerosis ; Cell Line ; Electrophoretic Mobility Shift Assay ; Foam Cells* ; Humans ; Inflammation ; Interleukin-8* ; Matrix Metalloproteinase 9* ; NF-kappa B ; Plaque, Atherosclerotic* ; Transcription Factors ; Tumor Necrosis Factor-alpha

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.
Animals ; Cell Line ; China ; Culicidae ; virology ; Encephalitis Virus, Japanese ; genetics ; isolation & purification ; pathogenicity ; Encephalitis, Japanese ; virology ; Genotype ; Humans ; Mice ; Phenotype ; Viral Plaque Assay ; Virulence

Acquired von Willebrand syndrome (AvWS) is a relatively rare acquired bleeding disorder similar to inherited von Willebrand disease in terms of laboratory findings, and occurs without a personal or family history of bleeding. A 23-year-old man with no previous disease history and no family history of hemorrhagic diathesis was referred to our hospital because of recurrent epistaxis and intramuscular hematoma. He was diagnosed as having AvWS because of an almost complete absence of ristocetin cofactor activity (vWF : RCo) despite normal vWF antigen level. Furthermore, anti-vWF antibody was detected in his serum using home-brewed ELISA. Finally, the amyloid deposit was found in muscle biopsy. He was diagnosed with AvWS which is associated with amyloidosis. AvWS should be considered in patients with current bleeding diatheses and no past history of bleeding.
Amyloidosis ; Autoantibodies ; Biopsy ; Disease Susceptibility ; Enzyme-Linked Immunosorbent Assay ; Epistaxis ; Hematoma ; Hemorrhage ; Hemorrhagic Disorders ; Humans ; Muscles ; Plaque, Amyloid ; von Willebrand Diseases ; von Willebrand Factor ; Young Adult

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Geese ; Host-Pathogen Interactions ; Newcastle Disease ; virology ; Newcastle disease virus ; growth & development ; isolation & purification ; physiology ; ultrastructure ; Poultry Diseases ; virology ; Vero Cells ; Viral Plaque Assay

BACKGROUND: It was recently shown that human atherosclerotic plaque contains large numbers of T lymphocytes : this indicates that immune and inflammatory mechanism may be important factors in the pathogenesis of atherosclerosis. By measuring the soluble interleukin 2 receptor(sIL-2R) level we can evaluate the activation of T lymphocyte. The purpose of this study is to evaluate relationship between T cell activation and ischemic heart disease by measuring the soluble interleukin 2 receptor (sIL-2R) level in patient with ischemic heart disease. METHOD: Seventy-two patients(40 males and 32 female, mean age : 56.5+/-9.9 years) who were taken coronary angiography were included in this study. Among them, 49 patients showed abnormal coronary angiographic findings and 23 patients showed normal coronary angiographic findings. Ten mililiters of arterial blood was drawn at the time of coronary angiography. The blood was allowed to coagulate and then the serum was removed and tested in duplicate for soluble interleukin 2 receptor (sIL-2R) level by ELISA. RESULTS: 1) The soluble interleukin 2 receptor (sIL-2R) level was significantly different between abnormal coronary angiographic findings and normal coronary angiographic findings (P < 0.001). 2) According to clinical severity of ischemic heart disease (i.e. stable angina, unstable angina, acute myocardial infarction.), soluble interleukin 2 receptor (sIL-2R) level was not significantly different between single vessel disease group and multivessels disease groups (p > 0.05), but showed increasing tendency with clinical severity. 3) According to numbers of involved coronary vessels, soluble interleukin 2 receptor (sIL-2R) level was not significantly different between single vessel disease group and multivessels disease groups (p > 0.05). CONCLUSION: T lymphocyte activation, as reflected in elevated soluble interlekin 2 receptor (sIL-2R) level, is frequent in patient with ischemic heart disease. In the further we will investigate relationship between clinical diagnosis of ischemic heart disease of the numbers of involved coronary vessels and T cell activation.
Angina, Stable ; Angina, Unstable ; Atherosclerosis ; Coronary Angiography ; Coronary Vessels ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-2 ; Lymphocyte Activation ; Lymphocytes ; Male ; Myocardial Ischemia* ; Plaque, Atherosclerotic ; Receptors, Interleukin-2 ; T-Lymphocytes

BACKGROUNDVulnerable plaques play an important role in the onset of sudden cardiac events and strokes. How to stabilize vulnerable plaques is still a challenge to medical science. Alprostadil is a biologically active substance with strong activity on vessel. Our study assessed the stabilizing effects of an alprostadil liposome microsphere preparation (ALMP) on vulnerable plaques in the brachiocephalic artery of apolipoprotein E (Apo E) knockout mice.

METHODSSeventy-two male Apo E-knockout mice were fed a high-fat diet beginning at eight weeks of age. At week 17, they were divided randomly into groups for treatment with a high dose (3.6 µg×kg(-1)×d(-1)) or low dose (1.8 µg×kg(-1)×d(-1)) of an ALMP, or 0.2 ml/d normal saline (control group). The drug was administered using a micro-capsule pump. Twenty weeks after drug administration, pathological changes in the vulnerable plaques within the brachiocephalic artery were assessed, and levels of anti-mouse monocyte/macrophage monoclonal antibody (MOMA-2) and superoxide anions in the plaques were detected using immunofluorescence. The soluble intercellular adhesion molecule-1 (ICAM-1) expression was measured by ELISA, and the expression of matrix metalloproteinase-9 (MMP-9) and CD40 mRNA was measured using RT-PCR. Thrombospindin-1 (TSP-1) expression was detected using Western blotting.

RESULTSCompared with the control group, ALMP treatment significantly reduced the plaque area in the brachiocephalic artery (P < 0.01), significantly lowered the contents of the lipid core (P < 0.01), significantly reduced the number of ruptured fibrous caps (P < 0.05), and increased the thickness of the fibrous cap and significantly reduced the incidence of intra-plaque hemorrhage (P < 0.05). ALMP treatment significantly reduced the expression of MOMA-2, superoxide anion, MMP-9, ICAM-1 and CD40 in the plaques (P < 0.01), decreased plasma ICAM-1 expression (P < 0.01), and increased the expression of TSP-1.

CONCLUSIONSTreatment with ALMP can stabilize vulnerable plaques by inhibiting inflammation.

Alprostadil ; chemistry ; therapeutic use ; Animals ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; drug therapy ; metabolism ; pathology ; Intercellular Adhesion Molecule-1 ; metabolism ; Liposomes ; chemistry ; Male ; Mice ; Mice, Knockout ; Microscopy, Fluorescence ; Microspheres ; Plaque, Atherosclerotic ; drug therapy ; metabolism ; pathology ; Polymerase Chain Reaction

BACKGROUNDTo determine if the attenuated Japanese encephalitis (JE) virus SA14-14-2 vaccine strain interacts efficiently with Culex tritaeniorhynchus and Culex pipiens quinquefasciatus, and further to acquire a new knowledge of its characteristics and safety for human beings.

METHODSLaboratory colonies of the two species of mosquitoes were set up and were inoculated intrathoracically with the attenuated vaccine virus and wild JE virus (Nak), both of which were used with different dilution from 10(-1) to 10(-9). Subsequently, the virus titers in the mosquitoes were detected by the plaque assay.

RESULTSInoculated with the vaccine strain, two species of mosquitoes were infected with the titers ranged from 10(0)-10(-3), and the maximum titers in Culex tritaeniorhynchus and Culex pipiens quinquefasciatus were 4.48 logPFU/ml and 5.63 logPFU/ml, respectively. Inoculated with wild JE virus, Culex pipiens quinquefasciatus was infected with titers ranged from 10(0)-10(-5), and the maximum titer in the mosquitoes was 6.59; Culex tritaeniorhynchus was infected with titers ranged from 10(0)-10(-4) and the maximum titer was 5.74 logPFU/ml.

CONCLUSIONBy intrathoracic infection, the attenuated JE virus SA14-14-2 vaccine strain can replicate in both species of mosquitoes.

Animals ; Culex ; classification ; virology ; Encephalitis Virus, Japanese ; genetics ; growth & development ; immunology ; Encephalitis, Japanese ; virology ; Humans ; Insect Vectors ; virology ; Japanese Encephalitis Vaccines ; immunology ; Species Specificity ; Vaccines, Attenuated ; immunology ; Viral Plaque Assay