To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication

Objective To study the effect of Itk down regulation on PBMC cell proliferation and inflammatory cytokines production in a Coxsackievirus-induced myocarditis model.Methods BALB/c mice were injected via caudal vein with plasmid Itk-shRNA then infected with CVB3.The change of Itk protein expression,cell proliferation,cytokines production and mice survival rate of mice were observed in the fourth days after infected.Results Itk mRNA in groups of mice transfected with Itk-shRNA was reduced about 40％ in spleen cells,compared with that in control groups or shRNAnon groups (P＜0.05).PBMC proliferation and serum cytokines were significantly inhibited by transfected with Itk-shRNA.Conclusion Knocking down Itk expression can inhibit mice inflammatory reaction.

Objective To evaluate the possibility of short interfering RNA (siRNA) inhibiting Coxsackievirns B3 (CVB3) infection in vitro, and discover the mechanism initially. Methods We obtained proper effective dosage of siRNA by observing cytopathic effect (CPE). Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR. Results Results showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77 % and its effect can last for 48 h stably in cells. 0.6 μmol/L siRNA-3753 got a high inhibiting effect of virus and didn't show any toxicity to cells. So we consider this concentration as the experimental concentration, siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R (PKR). Conclusion The data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.

OBJECTIVETo evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA).

METHODSAntiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR.

RESULTSEight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection.

CONCLUSIONSiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

Coxsackievirus Infections ; therapy ; virology ; Cytopathogenic Effect, Viral ; drug effects ; Enterovirus ; genetics ; physiology ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; Virus Replication ; drug effects

Adolescent ; Adult ; Antiviral Agents ; administration & dosage ; therapeutic use ; Child ; DNA, Viral ; blood ; Drug Therapy, Combination ; Enzyme-Linked Immunosorbent Assay ; Female ; Follow-Up Studies ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Interferon-alpha ; administration & dosage ; therapeutic use ; Lamivudine ; administration & dosage ; therapeutic use ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome ; Young Adult

To study the inhibitory effect and function characteristics of small interfering RNA (siRNA) on cosxackievirus B3(CVB3) infection by RNA interference technique, siRNA-2B against the viral 2B region was synthesized and transfected into HeLa cell, which was then infected with CVB3. The efficiency of siRNA transfection was examined by FCM, the cell toxicity of siRNA-2B by MTT, and the antiviral ability of siRNA-2B by cytopathic effect (CPE), plaque reduction assay and RT-PCR. The results showed that siRNA-2B could be transfected efficiently into HeLa cell and lasted at least 48h. High concentration of siRNA-2B didn't show any sign of toxicity to cells. siRNA-2B exhibited a significant protective effect on cell viability by specific inhibition of viral replication. It showed a close relationship between the concentrations of siRNA-2B and the antiviral effects. siRNA-2B led to dramatical reduction of viral titers in supernatant of cell culture and weakened the reinfection ability of the virus. These data proposed that siRNA-2B, targeting 2B protein, can effectively inhibit CVB3 infection in HeLa cell and exhibits its transfection efficiency, viral inhibition specificity and adose-dependant manner, suggesting its potential role in prevention and treatement of CVB3 infection.
Enterovirus ; genetics ; growth & development ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Microscopy, Fluorescence ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Viral Nonstructural Proteins ; genetics ; Virus Replication ; genetics ; physiology

Objective To study the effects of early intervention on neonatal jaundice and body weight, thereby preventing the occurrence of hyperbilirubinemia in newborns. Methods 320 healthy newborns were randomly divided into the two groups of the intervention group and the control group, with 160 cases in each group. All neonates of the intervention group were given the early intervention, including early breast-feeding, formula milk before the adequate breast-feeding, swimming and massaging. There was no nursing intervention for the control group, only the general nursing. The first defecation time, the first yellow defecation time, body weight in everyday, bilirubin value per cutem were measured and recorded. Results The bilirnbin value of the intervention group was much lower than that of the control group. The difference had statistical meaning (P < 0. 01). The body weight of newborns in the intervention group was much higher than that of the control group 5 days after born. There was statistical difference between the two groups (P < 0. 01). Conclusions Early nursing intervention can mitigate the bilirubin value effectively, reduce the incidence rate of hyperbilirnbinemia in newborns, reduce the decrease of body weight in newborns, and raise the body weight on discharge.

Objective By using the RNAi method to inhibit Itk protein expression specificity,to observe lymphocytes proliferation and cytokines production, verify its function as a drug target. Methods Designed siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA. Results Itk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-T. They all have statistical differenc (P < O. 05). Conclusion Itk has animportant immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines. This can supply an experimental basis to regard Irk as drug target for inflammation therapy.

Objective To study the effect of Itk down regulation on Jurkat cell proliferation and inflammatory cytokines production, and provide useful data for Itk as an attractive target for potential drugs.Methods Three shRNAs against different region of Itk were constructed and cotransfected with pEGFP-C1-hItk. The shRNA, which can knock down Itk, was selected and packed into lentivirus. After Jurkat cells were transfected with shRNA lentivirus, the change of Itk protein expression, cell proliferation and cytokines production was observed. Results Itk mRNA was reduced about 55% in Jurkat cells transfected with ItkshRNA1, compared with that in control cells shRNAnon (P ＜ 0. 05 ). Knocking down Itk expression had a profound inhibitory effect on Jurkat cell proliferation. In addition, there was a substantial decrease in level of cytokines, such as IL-2, IL-5, IL-10 and IFN-γ, produced by cell transfected with Itk-shRNA1.Conclusion Knocking down Itk expression can inhibit Jurkat cell proliferation and inflammatory cytokines production.

OBJECTIVETo evaluate the effect and safety of BCG vaccine on initially treated pulmonary tuberculosis and its controlling effect on multidrug-resistant tuberculosis.

METHODSAll 360 volunteers with initially treated pulmonary tuberculosis of positive smear and culture were divided into immunotherapy group (180 cases, also BCG group) and control group (180 cases) at random pair. The patients in BCG group were treated with chemotherapy of a regimen of 2HRZ/2HR and immunotherapy with BCG for 4 months,and the first BCG vaccine was given a month after chemotherapy. Meanwhile, the patients in the control group were treated with chemotherapy of 2HRZ/4HR only.

RESULTS(1) The negative conversion rate of sputum smear in BCG group was 98.3% (177/180), and it was 97.2% (175/180) in control group. There was no significant difference between the two groups both at the ends of 4 and 6 months after treatment (chi2 = 0.1278, P > 0.05). (2) The positive conversion rate of sputum smear in BCG group was 2.3% (4/177), and it was 6.9% (12/175) in control group followed up for 5 years. The successful rate was 96.1% (173/180) in BCG group, and it was significantly higher than that of 90.6% (163/180) in control group (chi2 = 4.4643, P < 0.05). (3) In the 5-year follow up, bacteriologic result was similar to that of X-ray. (4) The occurrence rate of multidrug-resistant tuberculosis was 2.3% (4/177) in BCG group,significantly lower than that of 7.3% (13/178) in the control group (chi2 = 4.9513, P < 0.05).

CONCLUSIONAs an adjunct chemotherapy,immunotherapy with BCG vaccine should be helpful for patients with initially treated pulmonary tuberculosis. It would further strengthen the effects of chemotherapy and reduce the occurrence rate of multidrug-resistant tuberculosis.

Adjuvants, Immunologic ; therapeutic use ; Adolescent ; Adult ; Aged ; Antitubercular Agents ; therapeutic use ; BCG Vaccine ; therapeutic use ; Child ; Female ; Follow-Up Studies ; Humans ; Immunotherapy, Active ; Male ; Middle Aged ; Tuberculosis, Multidrug-Resistant ; prevention & control ; Tuberculosis, Pulmonary ; therapy