miRNAs induced by white spot syndrome virus involve in immunity pathways in shrimp Litopenaeus vannamei.

White shrimp Litopenaeus vannamei are widely cultured in the world and white spot syndrome virus (WSSV) led to huge economic losses in the shrimp industry every year. In the present study, miRNAs involved in the response of shrimp L. vannamei to WSSV infection were obtained through the Illumina HiSeq 2500 high-throughput next-generation sequencing technique. A total number of 7 known miRNAs and 54 putative novel miRNAs were obtained. Among them, 14 DEMs were identified in the shrimp infected with WSSV. The putative target genes of these DEMs were related to host immune response or signaling pathways, indicating the importance of miRNAs in shrimp against WSSV infection. The results will provide information for further research on shrimp response to virus infection and contribute to the development of new strategies for effective protection against WSSV infections.

[Response surface methodology for optimizing the technology of induction and isolation of dendritic cells by multiple cytokines].

Objective To promote the induction and separation efficiency of bone marrow-derived dendritic cells (BMDCs) in vitro through optimizing the inducing and isolating process by multiple cytokines. Methods The factors to be optimized in single factor tests included recombinant mouse granulocyte macrophage-colony stimulating factor (rmGM-CSF), recombinant mouse interleukine 4 (rmIL-4), lipopolysaccharide (LPS), recombinant mouse tumor necrosis factor α (rmTNF-α) and inducing time. The numbers of immature dendritic cells (imDCs) and mature dendritic cells (mDCs) were investigated as the indicators. Box-Behnken experimental design-response surface methodology was used to analyze and verify the data. Morphological changes were observed using the inverted microscopy and the transmission electron microscopy. Surface molecules including CD11c and CD86 were detected using the flow cytometry. Results The optimum inducing conditions for imDCs were obtained as follows: rmGM-CSF was 46 ng/mL, rmIL-4 was 24 ng/mL, inducing time was 6 days, and the number of imDCs was (4.58±0.28)×106 cells, and the relative deviation was 4.00%. The optimum inducing conditions for mDCs were as follows: LPS was 1.4 µg/mL, rmTNF-α was 30 ng/mL, inducing time was 1 day, and the number of mDCs was (4.21±0.15)×106 cells, and the relative deviation was 3.80%. Sufficient typical imDCs and mDCs were obtained within 5-7 days of induction in vitro. Also, flow cytometry showed that the amplified imDCs had a high expression of CD11c (68.62%±2.3%) and a low expression of CD86 (37.95%±1.8%), and the mDCs had high expressions of both CD86 (90.34%±1.4%) and CD11c (82.05%±1.6%). Conclusion The combination of single factor tests and Box-Behnken design -response surface methodology could optimize the inducing and isolating method for DCs in vitro by multiple cytokines rapidly and efficiently, which provided basic experiment materials for further studies.

[Anti-inflammatory effect of recombinant human kallistatin in ulcerative colitis of mice].

This study was designed to evaluate the anti-inflammatory effect of recombinant human kallistatin (Kal) on ulcerative colitis (UC) in the mouse model. Acute colitis was induced by administration of 4% dextran sodium suffate (DSS) to KM mice for 7 days. The mice were then randomized into 5 groups: model control, Kal 0.2 mg·kg(-1)·d(-1), 1.0 mg·kg(-1)·d(-1) and 2.0 mg·kg-1·d(-1) group, salazosulfapyridine (SASP) group. Ten age-matched normal KM mouse were administered with saline in the normal control. The weight, colon length, inflammation factor (MPO/SOD/MDA) and TNF-α/IL-10 levels among the five groups of mice were determined. The results showed that histological index score and MPO/MDA/TNF-α levels of high-dose Kal treatment group and SASP group were significantly lower compared with the model group (P < 0.01), but the weight, colon length, IL-10 level and SOD activity were significant higher than the model group (P < 0.01), approaching the normal group. These parameters showed that Kal can significantly relieve the UC state in a dose-dependent manner. This study demonstrates that Kal significantly remits UC in mice, and participates in the regulation of inflammatory cytokines TNF-α/IL-10 levels and has some antioxidant activity.

[Adeno-associated vector mediated intracellular biological activity of human Kallistatin].

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.

Promising coagulation factor VIII bypassing strategies for patients with haemophilia A.

Haemophilia A is an X-linked recessive monogenic hereditary bleeding disorder caused by a deficiency or functional defect in coagulation factor VIII (FVIII). Typically, only 30% haemophilia A patients are treated with FVIII-specific products successfully. Therefore, other promising clotting factors and FVIII-bypassing factors exhibiting sufficient FVIII-independent activity, low immunogenicity and prolonged half-life are needed to conquer this malady. Here, we will systematically review the current status of the diverse FVIII-bypassing factors for the treatment of FVIII-insensitive haemophilia A patients.