Effect and Mechanism of Fuzheng Touxie Prescription on Immune Homeostasis of Drug-resistant Pseudomonas aeruginosa Lung Infection Rats / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of Fuzheng Touxie prescription （FZTX） on the immune homeostasis of drug-resistant <italic>Pseudomonas aeruginosa</italic> lung infection in rats at different time points. Method:A total of 168 rats were divided into a blank group （<italic>n</italic>=8），a model group （<italic>n</italic>=40），a Touxie （TX） group （<italic>n</italic>=40），an early Fuzheng （FZ） group （<italic>n</italic>=40）， and a delayed FZ group （<italic>n</italic>=40）. The blank group was given distilled water by gavage， the model group was given distilled water by gavage after infection，the TX group was given clear heat and penetrate evil drug free decoction granules（3.5 g·kg^-1） by gavage after infection， the early FZ group was given Fuzheng Touxie whole formula free decoction granules（10.75 g·kg^-1） by gavage after infection， the delayed FZ group was given clear heat and penetrate evil drug free decoction granules by gavage after infection， on the third day plus Fuzheng drug free decoction granules［（3.5+10.75） g·kg^-1］ by gavage， the three treatment groups were gavaged twice a day， 2 mL each time .Each drug treatment group was divided into five groups according to five time points （3 h，1 d，3 d，5 d， and 7 d）， with eight rats in each group. The levels of tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>），high mobility group protein 1（HMGB1），interleukin-10（IL-10）， and tumor necrosis factor -<italic>α</italic>-induced protein-8-like2 （TIPE2） were measured by enzyme-linked immunosorbent assay （ELISA）， and HMGB1 protein expression level by Western blot. Result:At 3 h，the TNF-<italic>α</italic> content in the drug treatment groups was higher than that in the blank group and the model group （<italic>P</italic><0.05）. At 3 d，the TNF-<italic>α</italic> content in the early FZ group and the delayed FZ group was lower than that in the model group （<italic>P</italic><0.05） and the TX group （<italic>P</italic><0.05）. At 1 d，the HMGB1 content in the TX group and the delayed FZ group was higher than that in the model group （<italic>P</italic><0.05）. At 5 d，the HMGB1 content was lower in the delayed FZ group than in the model group （<italic>P</italic><0.05）. At 7 d，HMGB1 protein expression in the model group was higher than that in the blank group （<italic>P</italic><0.05） and the early FZ group （<italic>P</italic><0.05）. At 3 d，the IL-10 content was significantly higher in both the early FZ group and the delayed FZ group than that in the model group （<italic>P</italic><0.05）. At 5 d，the IL-10 content was higher in the early FZ group than that in the TX group （<italic>P</italic><0.05）. At 7 d，the IL-10 content in the early FZ group and the delayed FZ group was lower than that in the TX group （<italic>P</italic><0.05）. At 5 d，the TIPE2 content in the early FZ group was lower than that in the model group （<italic>P</italic><0.05）. At 7 d，the TIPE2 content in the TX group and the delayed FZ group was lower than that in the model group （<italic>P</italic><0.05）. Conclusion:FZTX or modified prescription can promote the inflammatory response to eliminate pathogenic bacteria in the early stage and suppress the inflammatory response in the late stage to avoid the inflammatory cascade effect and lung tissue damage，indicating that Fuzheng drugs have an important role in maintaining the immune homeostasis of the body after infection.

Study of coxsackievirus A6 infection in Mongolian gerbils / 中华实验和临床病毒学杂志

Objective@#To study infection of coxsackievirus A6 (CV-A6) in Mongolian gerbils.@*Methods@#To screen the optimal ages of Mongolian gerbils, five groups with different ages were infected with 1×105 TCID50 dose of CV-A6 XS45 strain by intraperitoneal, and symptom scores of Mongolian gerbils were collected. Then to estimate the dose-effect, three doses of virus were injected to the Mongolian gerbils. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry(IHC) were used to determine virus load and tissues infection in muscle, brain and intestinal tract.@*Results@#Mongolian gerbils infected with 1×105 TCID50 dose CV-A6 consistently exhibited clinical signs, and the morbidity (death) rates of five age groups were up to 100%. There was a positive correlation between the trend of symptom scores changes and ages. The morbidity (death) rates of three doses (1×103 TCID50, 1×104 TCID50, 1×105 TCID50) also were up to 100% in 28 days Mongolian gerbils. The correlation between the trend of symptom scores changes and doses were negative. Virus loads were detected in muscle, brain and intestinal tract of pathogenesis animal. The virus loads of muscle were higher than others. IHC results showed virus infection and cytopathic effects in three tissues.@*Conclusions@#Mongolian gerbils had high susceptibility to CV-A6, and were best for animal model of CV-A6 infection.

Study on the Effects of Maggot Oil on the Healing of Rat with Acute Skin Trauma and Infection and Its Mechanism / 中国药房

OBJECTIVE:To study the effects of maggot oil on the healing of rat with acute skin trauma and infection and its mechanism,in order and to provide reference for further development of maggot oil. METHODS:SD rats were randomly divided into normal group,model group,maggot oil group and Jingwanhong treatment group (positive control),with 70 mice in each group. Except for normal group,acute skin trauma and infection model was induced in other groups by smearing Staphylococus aureus suspension at the wound. After modeling,normal group and model group were given normal saline,and maggot oil group and Jingwanhong treatment group were given relevant medicine 0.3 mL/100 g,at 9:00 and 17:00,for consecutive 15 d. Wound and wound healing time of rats were observed in each group. The content of hydroxyproline in wound was determined in 10 rats of each group after 1,2,4,6,8 d of administration. The content of water in wound was determined after 2,4,6,8 d after administration. 2 h after last administration,the content of lysozyme,the levels of inflammatory factors (TNF-α,IL-6),the expression of NF-κB p65(in cytoplasm and nucleus)and p-IκB-α(in cytoplasm)protein were determined in 10 rats of each group. RESULTS:Compared with normal group,wound edema of model group was obvious,and wound healing time was(14.3±2.1)d. After 4,6,8 d of medication,the content of hydroxyproline in wound of rats was decreased significantly in model group (P<0.05 or P<0.01). After 2,4,6,8 d of medication,the content of water in wound was increased significantly in model group(P<0.01). After 15 d of medication,the serum contents of lysozyme,TNF-α and IL-6 in rats were increased significantly in model group (P<0.01). The expression of NF-κB p65 (in cytoplasm) in wound was decreased significantly (P<0.01),while the expressions of NF-κB p65 (in nucleus) and p-IκB-α(in nucleus) protein were increased significantly (P<0.01). Compared with model group,above indicators of administration groups were improved significantly (P<0.05 or P<0.01). CONCLUSIONS:Maggot oil could protect tissue injury induced by acute skin wound infection,promote wound healing. The possible mechanism might play anti-inflammatory effect through promoting collagen production,increasing lysozyme content,regulating NF-κB signal pathway.

Breeding and application of SPF Bama miniature pig / 中国比较医学杂志

In this study, a Specific Pathogen-Free (SPF) Bama miniature pig herd was established.The standards for breeding experimental pigs, genetic test and microbiological quality control had been drafted.The local standards of Heilongjiang province SPF technical specifications for microbiological monitoring of pigs had been formulated.The genetic and microbiological quality criterion had been used to control of the SPF pig herd.Classical swine fever virus (CSFV) and highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) infection models of Bama miniature pigs were established, especially the infection miniature pig model of PRRSV was used to evaluated commercially vaccine.The comparison of the cytokine homology between Bama miniature pigs and domestic pigs showed that the Bama miniature pigs can be used to instead of domestic pigs as an ideal experiental animal.At present the SPF Bama miniature pig colonies have been widely used to study the mechanism of prevention and pathogenic in the classical swine fever virus, porcine reproductive and respiratory syndrome virus and porcine transmissible gastroenteritis virus and porcine circovirus and so on.The completion of the project solved the bottleneck problem of the experimental pig usage which provided a new ideal experimental animal for animal disease and life science research.

Establishment of a EV71 virus infection model of tree shrew primary renal cells / 中国实验动物学报

Objective To establish an enterovirus 71(EV71) infection model of tree shrew primary renal cells.Methods Tree shrew primary renal cells were obtained by trypsin digestion.After subculture and purification,EV71 virus was used to infect these primary cells.The culture supernatant of these EV71-infected cells was collected for virus titer detection at 1,2,4,6 and 8 days post-infection.The cells were collected for detection of EV71 VP1 protein by Western blot assay.Furthermore,the expression and location of VP1 protein in the infected cells were detected by indirect immunofluorescence assay.Vero cells were taken as positive control to evaluate the infectivity of EV71 virus to tree shrew primary renal cells.Results Morphologically,the cultured cells were proved to be majorly consisted of the primary renal cells after subculture and purification.The obtained primary cells were infected by EV71 virus.The virus titer was up to 1.3×106 TCID 50/mL during 48-96 h post-infection,proving that EV71 virus infected and proliferated in the tree shrew primary renal cells.Western blot showed that the viral VP1 protein was detected from infected primary cells at 2 to 8 d post infection.VP1 protein was also observed in the cytoplasm at 2 to 6 d post infection by indirect immunofluorescence.Compared with Vero cells,the infectivity of EV71 virus to tree shrew primary renal cells and its proliferation were confirmed.Conclusions Based on the successful establishment of cell culture of tree shrew primary renal cells,the infectivity to the obtained cells and proliferation of EV71 virus in the cells are confirmed.The model of EV71 virus-infected tree shrew primary renal cells is initially established.

Establishment of human papillomavirus pseudovirion infection model in mouse for potency evaluation of HPV16 VLP Vaccine / 中国生化药物杂志

Objective To establish a mouse model of genital human papillomavirus (HPV) pseudovirion (PsV) transmission and evaluate the protective potency of HPV16 VLP vaccine.Methods HPV16 PsV with the encapsidated luciferase expressing plasmid Luc were generated from 293FT cells and purified by size-exclusion chromatography.The endpoint titers of HPV16 PsV-Luc were determined on 293FT cells, denoted as TRLU/mL.For in vivo genital challenge, mice were synchronized in a diestrus-like status by a subcutaneous injection with 0.1 μg β-estradiol and then with 3mg DepoProvera after 24 hours.Six hours prior to HPV16 PsV-Luc challenge, deeply anesthetized mice were intravaginally pretreated with 50 μL of 4%nonoxynol-9 ( N-9 ).HPV16 PsV-Luc was thoroughly mixed with 20 μL solution containing 4%carboxymethylcellulose ( CMC ) and intravaginally instilled using a positive-displacement pipette.Forty-eight hours after PsV-Luc challenge, mice were anesthetized and D luciferin was intravaginally instilled.After 3 minites, bioluminescence was measured with a cryogenically cooled Xenogen IVIS camera system.Then,the murine genital challenge model was used to determine the potency that HPV16 VLP vaccine is efficient at preventing HPV infection.Results HPV16 PsV-Luc was generated and purified from 293FT cells.HPV16 PsV-Luc was verified to containe L1 and L2 protein by Western blot.HPV 16 PsV-Luc successfully infected vaginal epithelial cells of mouse and the murine genital challenge model was established.To achieve consistent bioluminescence, the minimal dose of HPV16 PsV-Luc was 1.7 ×104 TRLU.The protective potency of HPV16 VLP vaccine was shown using this murine model.Our data showed that immune serum with over neutralizing antibody titer of 256-fold was sufficient to confer protection against HPV PsV genital infection .Conclusion The murine genital challenge model of HPV16 was successfully established, and the model is used to evaluate the potency of HPV16 VLP vaccine against in vivo genital HPV16 PsV challenge.Our model will benefit for the investigation of HPV neutralization and the potency evaluation of the HPV vaccine .

Establishment and evaluation of hamster model of Clostridium difficile-associat-ed diarrhea / 中国感染与化疗杂志

Objective To explore a method for establishing the hamster model of Clostridium difficile-associated diarrhea (CDAD)and the indicators for its evaluation.Methods Clindamycin was administered to hamsters subcutaneously (day 1),and 24 h later infected with C.difficile clinical isolates KH1 (ribotype 027,106-108 CFU/mL)or SH9 (ribotype 001 ,108-1010 CFU/mL)by gavage.Animals were observed for CDAD symptoms such as diarrhea,weight loss and death.At the end of ob-servation period (day 7 or death),the cecum was collected from each animal for histological evaluation of inflammation.Results Following a single dose of 100 mg/kg clindamycin subcutaneously,all the animals challenged with KH1 (108 CFU/mL)devel-oped diarrhea and then died within 5 days.All the hamsters challenged with SH9 (1010 CFU/mL)developed diarrhea as well but only 66.7% died at the end of observation period.Among other groups,only one or none developed diarrhea and then died. The symptoms of hamsters with diarrhea included loose stool,wet tail and weight loss.On histological examination,conges-tion,hemorrhage and neutrophil infiltration of the mucosa were observed in the hamsters died of CDAD.Conclusions We have successfully established a hamster CDAD model that allows for future investigations.

Study of the relationship between antibody level and protection induced by human papillomavirus(HPV) L2-peptide / 中华微生物学和免疫学杂志

Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection.

The establishment of the mouse model based on the pseudovirious of humnn papillomavirus types 16,45 and 58 / 中华微生物学和免疫学杂志

Objective To establish the mouse model for human papillomavirus types 16, 45 and 58 by the corresponding pseudovirions. Methods The 293FT cells were co-transfected with eodon-modified HPV eapsids genes together with a reporter plasmid containing the luciferase gene. The cells were collected and lysed, then the pseudovirus was collected and the titration was performed. The mouse was subcutaneous-ly injected with Depo-Provera. After 4 d and intravaginally injected with nonoxynol-9, and 6 h later pseud-oviruses were inoculated in intravaginal. After 7 d, the mouse was instilled luciferin substrate intravaginally, and the expression level of the lueiferase gene was detected by the in vivo Imaging System (IVIS). Results Three types (HPV16, HPV45 and HPV58) of pseudoviruses had been produced and the titer was 3.7×108 TU/ml, 1.5×108 TU/ml and 1.2×108 TU/ml, respectively. The luminescent regions could be detected in the mice which were infected with the pseudovirions, the luminescent signal intensity for types 16,45 and 58 was 1.779×106p/s, 5.738×105×p/s and 1.829×106p/s, respectively. Conclusion The mouse models for HPV16, 45 and 58 have been successfully established based on pseudovirions, which will be very useful for the research of HPV infection intervention, the evaluation of HPV vaccines and the screening of the prophylactic agents.

Possibility of Zebrafish as New infection model for Leprosy / 나학회지

The zebrafish emerged as a new model of developmental research and infection. Recently zebrafish were used for various mycobacterial infection studies. In this study, we investigated a possibility of the zebrafish as an infection model of Mycobacterium leprae infection. We injected Mycobacterium leprae (10(5)/30microliter) to the peritoneum of zebrafish. After 4 weeks, we found two fish evident to the subsistence and multiplication of Mycobacterium leprae, among total 25 fish. This study thus demonstrates that zebrafish may become an animal model for M. leprae infection.

In Vitro Cultivation Model of Cryptosporidium parvum in MDCK Cells and its Development / 中国寄生虫学与寄生虫病杂志

Objective To develop an in vitro culture system for Cryptosporidium parvum in Madin-Darby canine kidney(MDCK) cell and observe its life cycle(from desquamate to oocyst).Methods Oocysts of C.parvum were co-cultured with MDCK cells in vitro.Culture condition was optimized and the life cycle of C.parvum investigated.Results The optimal culture conditions for C.parvum in MDCK cells were 2.0?105 cells cultured for 12 h, and infected by 1.0?105 oocysts in the Dulbecco′s Modified Eagle Medium with 5% FBS.Following 72 h co-culture, desquamate, sporozoites, trophozoites, meronts, microgametocytes, macrogametocytes, zygote, thin-wall oocyst, and thick-wall oocyst appeared orderly.Between the 60th and 72th hour, many oocysts emerged.Inoculated by the C.parvum-infected cell culture supernatant at the 48th hour, the immunosuppressed mice became infected.Conclusion The culture system provides a model for propagation of the parasites and demonstrates a complete in vitro life cycle of C.parvum.