OBJECTIVE: To evaluate the sub-acute oral effect of titanium dioxide (TiO₂) nanoparticles on the oxidation/antioxidation biomarkers and inflammatory cytokines in blood, liver, intestine, and colon in rats. METHODS: Twenty four 4-week-old clean-grade Sprague Dawley (SD) rats were randomly devided into 4 groups by body weight (n=6, control, low, middle, and high), in which the rats were orally exposed to TiO₂ nanoparticles at doses of 0, 2, 10 and 50 mg/kg body weight/day for 28 consecutive days separately. Food intake, body weight and abnormal behaviors during the experiment were recorded. The rats were euthanized on the 29th day. The blood was collected via abdominal aortic method and centrifuged to collect the serum. Tissues from liver, intestine and colon were collected and homogenated. Then enzyme-linked immunosorbent assay (ELISA) and microwell plate methods were used to detect oxidation/antioxidation biomarkers including superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), total mercapto (T-SH), glutathione disulfide (GSSG), malomdialdehvde (MDA) and inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the serum, liver, intestine and colon in the rats. RESULTS: Compared with the control group, no significant differences in body weight, food intake and organ coefficients were observed in all the three groups after TiO₂ gavage. No significant changes in GSH, GSH-Px, T-SH, and IL-6 were observed. Compared with the control group, significant increase of SOD activity in serum in high dose group, signi-ficant increase of GSSG concentration in intestine in middle and high dose group and significant increase of MDA concentration in liver in low and high dose group were observed. Compared with the control group, a significant increase of TNF-α in liver in middle and high dose group was observed. CONCLUSION TiO₂ nanoparticle can increase antioxidant enzymes activities in blood, increase oxidative biomarkers in liver and intestine, increase inflammatory cytokines in liver in rats after a 28-day sub-acute orally administration. Among blood, liver, intestine, and colon, liver is most sensitive to the toxicity induced by TiO₂ nanoparticles, followed by intestine, blood, and colon in sequence.
Animals ; Antioxidants ; Biomarkers ; Cytokines ; Nanoparticles ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Titanium

OBJECTIVETo construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system.

METHODSSelf-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells (5 mug/100 mm culture dish) and injecting by high-pressure tail vein injection into Kunming mice (10 - 30 mug/mouse). Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems.

RESULTSHCV RNA (1 - 3 * 10(6) copies/ml) was detected in the supernatant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supernatant were able to infect nave Huh7.5 cells. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing.

CONCLUSIONThe constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However, the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.

Animals ; Base Sequence ; Cell Line ; Genetic Vectors ; Genome, Viral ; Hepacivirus ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred Strains ; RNA, Catalytic ; genetics ; Recombination, Genetic ; Replicon ; Virus Replication ; genetics

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.
Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Mutagenesis, Site-Directed ; methods ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

BACKGROUNDOsteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis.

METHODSAdenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated.

RESULTSIntra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction.

CONCLUSIONThe enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae ; genetics ; Animals ; Arthritis, Experimental ; genetics ; therapy ; Cartilage ; metabolism ; pathology ; Cartilage, Articular ; metabolism ; pathology ; Cell Line ; Cells, Cultured ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Osteoarthritis ; genetics ; therapy ; Rabbits ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; physiology ; Sialoglycoproteins ; genetics ; physiology ; Synovial Fluid ; metabolism ; Synovial Membrane ; cytology ; metabolism ; Transfection ; methods

BACKGROUNDRemodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-beta1 (TGFbeta1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFbeta1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.

METHODSAdenoviral vector containing TGFbeta1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFbeta1, the expression of VEGF165 and TGFbeta1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFbeta1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.

RESULTSThe results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFbeta1 and VEGF165 genes. Co-expression of TGFbeta1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.

CONCLUSIONCo-expression of TGFbeta1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

Adenoviridae ; genetics ; Animals ; Anterior Cruciate Ligament ; cytology ; metabolism ; Cell Movement ; Cells, Cultured ; Collagen ; genetics ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; physiology ; Fibronectins ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Rabbits ; Transforming Growth Factor beta1 ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; Wound Healing

To study the effect of simian vacuolating virus 40 (SV40) on development and differentiation of dendritic cells (DC) from rhesus macaque, the peripheral blood-derived dendritic cells from rhesus monkey were pulsed with inactivated SV40 and infective SV40, respectively at the 5th day post DC cultivation. Expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface at the 7th, 9th day post DC cultivation were analyzed by flow cytometry (FCM). The results showed that expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface in the inactivated SV40-pulsed experimental group were higher than those in the infective SV40-pulsed experimental group (P < 0.05). These cell surface molecules represented characteristic development and differentiation phase of DC. Down-regulation of expressions of these cell surface molecules indicated that infective SV40 might hamper differentiation and maturation of dendritic cells from rhesus monkey.
Animals ; Antigens, CD ; metabolism ; Antigens, CD1 ; metabolism ; B7-2 Antigen ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; virology ; Flow Cytometry ; HLA-DR Antigens ; metabolism ; Immunoglobulins ; metabolism ; Macaca mulatta ; Membrane Glycoproteins ; metabolism ; Polyomavirus Infections ; physiopathology ; Simian virus 40 ; physiology

OBJECTIVE: To establish a database of high-fall death cases for future forensic study and practice, based on the scene investigation, injury characteristics and other informations. METHODS: Five hundred and four cases of high-fall death from 5 provinces and cities were included in the study. Data including personal information of the deceased, scene investigation, autopsy findings, history of mental illness and the results of toxicology were collected and analyzed. RESULTS: The male accidental death rate was significantly higher than that of female. No case of suicide was found in the 0-10 age group, while the suicide rate was apparently higher in the 60 years or over age group than that of accident. Most of the accident cases occurred at workplace, with head landing first and foot or lower-extremity landing first observed from height below 10 m and between 10-25 m, respectively. CONCLUSION The majority of cases have obvious conclusions. A substantial set of the cases, however, is still difficult to determine the mechanism of injury and the manner of death. So further study should be performed.
Accidental Falls ; Adolescent ; Adult ; Age Distribution ; Aged ; Aged, 80 and over ; Cause of Death ; Child ; Child, Preschool ; Female ; Forensic Pathology ; Homicide/statistics & numerical data* ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Sex Distribution ; Suicide/statistics & numerical data* ; Wounds and Injuries/pathology* ; Young Adult

To evaluate the safety and efficacy of andrographolide drop-pill in treatment of acute upper respiratory tract infection with external wind-heat syndrome.

Humans ; Infant, Low Birth Weight ; physiology ; Infant, Newborn ; Urination

Objective To understand the health status and potential impact resulted in the second stage of Three Gorges Reservoir Areas sluicing. Methods Data were collected on deaths, prevalence rates of infectious and endemic diseases, as well as on vector surveillance through the project entitled 'Three Gorges Population Health Survey System'. Results The main causes of death in the population living in the Three Gorges Reservoir Areas would include: circulatory system diseases, tumors, respiratory system diseases, injuries/poison and digestive system diseases. The number of deaths caused by the above said five kind of diseases accounted for 90.94% of the total number of deaths. The prevalence rates on Water-born diseases related to the sluicing of reservoir and zoonosis-bome diseases related to the changes of vectors were still low. The indoor and outdoor densities of rodents were 3.11% and 3.16%, both were higher than that in 2006 but lower than the average numbers in the five years prior to the sluicing. The constituent ratio of Apodemus agrarius had constantly risen since 2006. The density of mosquitoes found in livestock barns and human households was higher than that in 2006 but lower than the average number of the five-year studies prior to the sluicing. Conclusion Environment change after the sluicing of the Three Gorges Reservoir Areas did not seem to have obvious impact on the health status of the people living in the areas. However, to strengthen the surveillance on the biological features of the vectors which might have related to the transmission of diseases would be highly recommended.