Interleukin-10 Contributes to Therapeutic Effect of Mesenchymal Stem Cells for Acute Liver Failure via Signal Transducer and Activator of Transcription 3 Signaling Pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation has been proven to have therapeutic potential for acute liver failure (ALF). However, the mechanism remains controversial. Recently, modulation of inflammation by MSCs has been regarded as a crucial mechanism. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects in ALF. METHODS: MSCs isolated from Sprague-Dawley rats were identified by fluorescence-activated cell sorting analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. MSCs and MSCs-CM were transplanted into rats with D-galactosamine-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, anti-rat IL-10 antibody, and AG490 (signal transducer and activator of transcription 3 [STAT3] signaling pathway inhibitor) were administered to explore the therapeutic mechanism of MSCs-CM. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by the independent-sample t-test. RESULTS: There are statistical differences of the survival curve between ALF+MSCs group and ALF+Dulbecco's modified Eagle's medium (DMEM) group, as well as ALF+MSCs-CM group and ALF+DMEM group (all P < 0.05). Serum alanine aminotransferase (ALT) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (865.53±52.80 vs. 1709.75±372.12 U/L and 964.72±414.59 vs. 1709.75±372.12 U/L, respectively, all P < 0.05); meanwhile, serum aspartate aminotransferase (AST) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (2440.83±511.94 vs. 4234.35±807.30 U/L and 2739.83±587.33 vs. 4234.35±807.30 U/L, respectively, all P < 0.05). Furthermore, MSCs or MSCs-CM treatment significantly reduced serum interferon-γ (IFN-γ), IL-1ß, IL-6 levels and increased serum IL-10 level compared with DMEM (all P < 0.05). Proteome profile analysis of MSCs-CM indicated the presence of anti-inflammatory factors and IL-10 was the most distinct. Blocking of IL-10 confirmed the therapeutic significance of this cytokine. Phosphorylated STAT3 was upregulated after IL-10 infusion and inhibition of STAT3 by AG490 reversed the therapeutic effect of IL-10. CONCLUSIONS: The factors released by MSCs, especially IL-10, have the potential for therapeutic recovery of ALF, and the STAT3 signaling pathway may mediate the anti-inflammatory effect of IL-10.

Small antibody fusion proteins with complementarity-determining regions and lidamycin for tumor targeting therapy.

Gelatinases are overexpressed in several types of maligancies and tumor stromal cells. Lidamycin is an enediyne antitumor antibiotic, which is composed of an apoprotein (LDP) and an active chromophore (AE). It is known that the heavy-chain complementarity-determining region-3 (CDR3) domain of scFv is important in antibody affinity. The aim of this study was to prepare the enediyne-energized fusion proteins with a heavy-chain CDR3 domain of anti-gelatinases scFv and lidamycin, and to evaluate their antitumor efficiency. Fusion proteins comprising the CDR3 domain and the lidamycin apoprotein were generated, and ELISA, immunofluorescence and FACS were used to analyze the binding of the fusion protein with antigen gelatinases. The purified fusion proteins were assembled with the lidamycin chromophore, and the antitumor effects were evaluated in vitro and in vivo. It was found that the CDR3-LDP and CDR3-LDP-CDR3 fusion proteins demonstrated high affinity towards antigen gelatinases. Following stimulation of CDR3-LDP with enediyne, the results of MTT showed potent cytotoxicity towards tumor cells; the IC50 values of CDR3-LDP-AE to HepG2 and Bel-7402 tumor cells were 1.05×10-11 and 6.6×10-14 M, respectively. In addition, CDR3-LDP-AE displayed a potent antitumor effect in H22 cell xenografts in mice; the combination of CDR3-LDP (10 mg/kg) and CDR3-LDP-AE (0.25 and 0.5 mg/kg) revealed that the tumor inhibitory rates were 85.2 and 92.7%, respectively (P<0.05 compared with CDR3-LDP-AE). In conclusion, these results suggest that the CDR3-LDP fusion protein and its analog CDR3-LDP-AE may both be promising candidates for tumor targeting therapy.

Antitumor activities of dFv-LDP-AE: An enediyne-energized fusion protein targeting tumor-associated antigen gelatinases.

Gelatinases play an important role in tumor growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumors. Lidamycin is an enediyne antitumor antibiotic with potent cytotoxicity. We previously reported that a tandem scFv format (dFv-LDP-AE) showed enhanced binding ability with gelatinases compared with the scFv-lidamycin conjugate (Fv-LDP-AE). In this study, the antitumor activities of dFv-LDP-AE on hepatocellular carcinoma (HCC) were evaluated in vitro and in vivo. By SDS-PAGE analysis, it was found that partial fusion protein dFv-LDP existed as dimer; the results of ELISA and immunofluorescence demonstrated that the fusion protein dFv-LDP could efficiently bind to hepatoma cells in vitro. The apparent arrest of cell cycle at G2/M phase and induction of apoptosis at nanomole levels indicated that the dFv-LDP-AE was very potent against HCC. In in vivo experiments, dFv-LDP-AE shown enhanced cytotoxic effects compared to those of LDM. Administration at mouse tolerable dosage level, the inhibition rate of tumor growth was 89.5% of dFv-LDP-AE vs. 73.6% of LDM on transplantable H22 in mice (P<0.05) and, 87.3% of dFv-LDP-AE vs. 63.4% of LDM on hepatoma Bel-7402 in athymic mice (P<0.01). Small animal optical imaging showed that the FITC-labeled dFv-LDP preferentially localized in the tumor site in less than 30 min, which demonstrated remarkable tumor-targeting properties. Taken together with the above findings, the enediyne-energized fusion protein dFv-LDP-AE showed potential application as a new agent for therapeutic appications in HCC.

[Effects of continuous cropping of vegetables on ammonia oxidizers community structure].

Investigations were conducted on the effects of intensive application of chemical fertilizers in crop production on soil nitrifier communities and the relationship between nitrifier communities and soil nitrification ability. Two series of vegetable soils were selected from Huangxing, Changsha, reflecting continuous vegetable cropping with about 20 years and new vegetable field with only about 2 years vegetable growing history. In each series five independent topsoils (0-20 cm) were sampled and each soil was a mixture of 10 cores randomly taken in the same field. Terminal restriction fragment length polymorphism (T-RFLP) and quantity PCR (Q-PCR) were used to determine the composition and abundance of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) communities. Results indicated that long-term and continuous vegetable cropping obviously changed the compositions of both AOB and AOA amoA gene, soil pH and Olsen-P content were the dominant factors affecting the composition of AOB amoA. In the vegetable soils, although the copy number of AOA amoA gene was about 5 times higher than AOB amoA gene, no significant correlation was detected between AOA amoA gene abundance and soil nitrification rate. It was not sure whether long-term and continuous vegetable cropping could shift the abundance of AOB and AOA, but it resulted in the enrichment of some dominant AOB species and increase of soil nitrification potential (PNF).

[Effect of long-term application of nitrogen fertilizer on the diversity of nitrifying genes (amoA and hao) in paddy soil].

The aim of this study was to determine the effect of long-term (16 years) application of nitrogen fertilizer on the diversity of nitrifying genes (amoA and hao) in paddy soil on the basis of long-term paddy field experimental station (started in 1990) located in Taoyuan, with the molecular approaches of PCR, constructing libraries and sequencing. The fertilizer was urea and no fertilizer was as control. The Shannon index showed that long-term application of nitrogen fertilizer made the diversity of amoA gene descend while no effect on the diversity of hao gene. The LIBSHUFF statistical analyses demonstrated that both amoA and hao libraries of CK and N treatments were significantly different from each other and the rarefaction curves of libraries failed to meet the plateaus indicating that there were lots kinds of genes haven't been detected. The results of blasting with GenBank and the phylogenetic tree showed that the amoA genes detected in our study had a similarity with the uncultured gene of amoA, which showed some similar to Nitrosospira. Otherwise, the hao genes cloned showed a relationship to the genes of cultured bacteria such as Silicibacteria, Nitrosospira and Methylococcus, and the hao genes found in the N treatment dominated in alpha-Proteobacteria. These results suggest that long-term fertilization of nitrogen had significant impacts on the diversity or community of amoA and hao genes.

[Effects of rice straw on the diversity of nitrifying genes (amoA and hao) in paddy soil].

The effects of long-term (16 years) fertilization on the diversity and community structure of soil ammonia-oxidizing gene (amoA) and hydroxylamine-oxidizing gene (hao) in paddy soil were evaluated using the methods of polymerase chain reaction, cloning and sequencing. The soil samples were collected from the treatments of NPK (CK) and NPK plus rice straw (SR) of the long-term field fertilization experiment in Taoyuan Agro-ecological Experimental Station. The Shannon Indices showed that the diversity of amoA and hao in SR treatment was lower than that in CK, and LUBSHUFF statistical analyses demonstrated that the sequence compositions of both amoA and huo libraries were significantly different between CK and SR. The phylogenetic trees indicated that some clusters appeared in SR treatment but were not detected in CK treatment. As to amoA, only Nitrosospira besides the uncultured amoA sequences were cloned from the two treatments, while no Nitrosomonas species were detected. As to hao, the strains from Silicibacter and Methylococcus were dominant in CK, while in SR the strains from Nitrosospira and Nitrosomonas were dominant. Sum up, the long-term rice straw application has caused a remarkable impact on the diversity and community structure on Nitrosobacteria.

[Effect of long-term fertilization on the diversity of nitrite reductase genes (nirK and nirS) in paddy soil].

In order to investigate the effects of long-term application of nitrogen fertilizer on soil denitrifying communities, the diversities of nir genes (nirK and nirS) were studied using molecular approaches in the long-term paddy field experiment (started in 1990) located in Taoyuan. Analysis of clone sequences indicated that the nirK fragments from paddy soil showed close similarity (90.7%) to the nirK sequences registered in GenBank database, but were not related to any known strain. Whereas, most of the airS clones showed low similarity (74.7%) to the nirS gene fragments registered in GenBank. The Chao1 estimates showed that the diversity of nirK gene 13) OTUs] than in N treatment [(49 +/- 9) OTUs], but the difference was not significant. However, application of nitrogen fertilizer resulted in significant difference of nirS-community compared to CK. Nitrogen fertilizer had obvious effect on tbe community structure of nirK-denitrifiers (p < 0.022), but the nirS-containing community was not affected. Based on phylogenetic analysis, nirK clones grouped into three clusters with aggregations of some OTUs cloned from N treatment. Although nirS clones grouped into four clusters, the majority of the clones were attributed in one cluster. The results suggested that application of nitrogen fertilizer had a greater influence on the diversity of nirS-containing bacterial community than that of the nirK. However, the community structure of nirK-containing denitrifiers was more sensitive to nitrogen fertilization than that of the mrS.

[Relationship between soil fungistasis and bacterial community structure].

As a natural attribute of clean and healthy soil, fungistasis is an important indicator of soil quality, and has positive ecological significance to the inhibition of plant disease eruption caused by soil-borne fungal pathogens. In this study, soil samples (0-15 cm) were collected from the abandoned land at Shenyang Experimental Station of Ecology, Chinese Academy of Sciences, to which pesticides and fertilizers have never been applied for nearly 10 years. A series of soil samples with gradient fungistasis was obtained by heating (CK, 100 degrees C, 110 degrees C, and 121 degrees C for 4 min, respectively), and bacterial community structure was analyzed by Polymerase Chain Reaction coupled with Denaturing Gradient Gel Electrophoresis method (PCR-GGGE). The results showed that there was a significant correlation between soil fungistasis and bacterial community composition. Treatment CK showed the strongest capacity to control the growth of target soil-borne pathogenic fungi. The further the bacterial community structure of treated soil deviated from that of CK, the lower the soil fungistasis was. Sequencing and the following phylogenetic analysis of special bands in DGGE indicated that Sphingomonas asaccharolytica, Nitrospira sp., Hyphomicrobiaceae sp., Bacillus megaterium, and Micrococcus sp. could be involved in soil fungistasis.