A New Species of the Genus Takydromus (Squamata: Lacertidae) from Northeastern Guangxi, China.

During our collecting trip to Guangxi in 2016, we collected ten specimens of the genus Takydromus from the suburb of Guilin, northeastern Guangxi, South China, and found that they did not belong to any currently known species. Here, we described this new species, Takydromus guilinensis sp. nov., based on morphological and mitochondrial DNA (CO1 and cyt b) data. This new species is a sister taxon to T. intermedius with a p-distance of 0.070 in CO1 and 0.080 in cyt b. These two p-distances exceed not only the minimum value (0.067) between T. septentrionalis and T. stejnegeri but also the minimum value (0.079) between T. intermedius and T. yunkaiensis. Morphologically, this new species differs from other currently recognized Takydromus species from the same clade, more evidently in the longitudinal rows of dorsal scales, transverse rows of scales at the mid-body and mensural variables. The description of Takydromus guilinensis sp. nov. increases the total number of Takydromus species to 25, of which 16 can be found in China. Takydromus guilinensis sp. nov. is currently known only from Guilin, Guangxi, South China, where it is sympatric with the other four Takydromus species (T. septentrionalis, T. kuehnei, T. sexlineatus and T. intermedius).

Recombinant human IL-37 attenuates acute cardiac allograft rejection in mice.

BACKGROUND: Allograft rejection remains a major obstacle to long-term graft survival. Although previous studies have demonstrated that IL-37 exhibited significant immunomodulatory effects in various diseases, research on its role in solid organ transplantation has not been fully elucidated. In this study, the therapeutic effect of recombinant human IL-37 (rhIL-37) was evaluated in a mouse cardiac allotransplantation model. METHODS: The C57BL/6 recipients mouse receiving BALB/c donor hearts were treated with rhIL-37. Graft pathological and immunohistology changes, immune cell populations, and cytokine profiles were analyzed on postoperative day (POD) 7. The proliferative capacities of Th1, Th17, and Treg subpopulations were assessed in vitro. Furthermore, the role of the p-mTOR pathway in rhIL-37-induced CD4+ cell inhibition was also elucidated. RESULTS: Compared to untreated groups, treatment of rhIL-37 achieved long-term cardiac allograft survival and effectively alleviated allograft rejection indicated by markedly reduced infiltration of CD4+ and CD11c+ cells and ameliorated graft pathological changes. rhIL-37 displayed significantly less splenic populations of Th1 and Th17 cells, as well as matured dendritic cells. The percentages of Tregs in splenocytes were significantly increased in the therapy group. Furthermore, rhIL-37 markedly decreased the levels of TNF-α and IFN-γ, but increased the level of IL-10 in the recipients. In addition, rhIL-37 inhibited the expression of p-mTOR in CD4+ cells of splenocytes. In vitro, similar to the in vivo experiments, rhIL-37 caused a decrease in the proportion of Th1 and Th17, as well as an increase in the proportion of Treg and a reduction in p-mTOR expression in CD4+ cells. CONCLUSIONS: We demonstrated that rhIL-37 effectively suppress acute rejection and induce long-term allograft acceptance. The results highlight that IL-37 could be novel and promising candidate for prevention of allograft rejection.

A chemiluminescent magnetic microparticle immunoassay for the detection of antibody against African swine fever virus.

The p30 protein is abundantly expressed in the early stage of African swine fever virus (ASFV) infection. Thus, it is an ideal antigen candidate for serodiagnosis with the use of an immunoassay. In this study, a chemiluminescent magnetic microparticle immunoassay (CMIA) was developed for the detection of antibodies (Abs) against ASFV p30 protein in porcine serum. Purified p30 protein was coupled to magnetic beads, and the experimental conditions including concentration, temperature, incubation time, dilution ratio, buffers, and other relevant variables were evaluated and optimized. To evaluate the performance of the assay, a total of 178 pig serum samples (117 negative and 61 positive samples) were tested. According to receiver operator characteristic curve analysis, the cut-off value of the CMIA was 104,315 (area under the curve, 0.998; Youden's index, 0.974; 95% confidence interval: 99.45 to 100%). Sensitivity results showed that the dilution ratio of p30 Abs in ASFV-positive sera detected by the CMIA is much higher when compared to commercial blocking ELISA kit. Specificity testing showed that no cross-reactivity was observed with sera positive for other porcine disease viruses. The intraassay coefficient of variation (CV) was < 5%, and the interassay CV was < 10%. The p30-magnetic beads could be stored at 4 °C for more than 15 months without loss of activity. The kappa coefficient between CMIA and INGENASA blocking ELISA kit was 0.946, showing strong agreement. In conclusion, our method showed superiority with high sensitivity, specificity, reproducibility, and stability and potentialized its application in the development of a diagnostic kit for the detection of ASF in clinical samples. KEY POINTS: • ASFV tag-free p30 was successfully purified. • High sensitivity, specificity, relatively simple, and time-saving to detect antibody against ASFV were developed. • The development of CMIA will help the clinical diagnosis of ASFV and will be useful for large-scale serological test.

Study on the Hepatoprotective Effect Mechanism of Polysaccharides from Charred Angelica sinensis on the Layer Chickens Based on the Detection of the Intestinal Floras and Short-Chain Fatty Acids of Cecal Contents and Association Analysis.

To analyze the intervention mechanism of polysaccharides from charred Angelica sinensis (CASP) on the liver injury caused by Ceftiofur sodium (CS) and lipopolysaccharide (LPS) from the perspective of the intestine. Ninety-four one-day-old laying chickens underwent free feeding and drinking water for three days. Then, fourteen laying chickens were randomly selected as the control group, and sixteen laying chickens were selected as the model group. Sixteen laying chickens in the rest were randomly selected as the intervention group of CASP. Chickens in the intervention group were given CASP by the oral administration (0.25 g/kg/d) for 10 days, the control and model groups were given the same amount of physiological saline. During the 8th and 10th days, laying chickens in the model and CASP intervention group were subcutaneously injected with CS at the neck. In contrast, those in the control group were subcutaneously injected with the same amount of normal saline simultaneously. Except for the control group, the layer chickens in the model and CASP intervention groups were injected with LPS after CS injection on the 10th day of the experiment. In contrast, those in the control group were injected with the same amount of normal saline at the same time. 48 h after the experiment, the liver samples of each group were collected, and the liver injury was analyzed by hematoxylin-eosin (HE) staining and transmission electron microscopy. And the cecum contents of six-layer chickens in each group were collected, and the intervention mechanism of CASP on the liver injury from the perspective of the intestine was analyzed by the 16S rDNA amplicon sequencing technology and the short-chain fatty acids (SCFAs) detection of cecal contents based on Gas Chromatography-Mass Spectrometry (GC-MS), and their association analysis was carried out. The results showed that the structure of chicken liver in the normal control group was normal, while that in the model group was damaged. The structure of chicken liver in the CASP intervention group was similar to the normal control group. The intestinal floras in the model group were maladjusted compared to the normal control group. After the intervention of CASP, the diversity, and richness of chicken intestinal floras changed significantly. It was speculated that the intervention mechanism of CASP on the chicken liver injury might be related to the abundance and proportion of Bacteroidetes and Firmicutes. Compared with the model group, the indexes of ace, chao1, observed species, and PD whole tree of chicken cecum floras in the intervention group of CASP were significantly increased (p < 0.05). The contents of acetic acid, butyric acid, and total SCFAs in the intervention group of CASP were significantly lower than those in the model group (p < 0.05), and the contents of propionic acid and valeric acid in the intervention group of CASP were significantly lower than those in the model group (p < 0.05) and normal control group (p < 0.05). The correlation analysis showed that the changes in the intestinal floras were correlated with the changes in SCFAs in the cecum. It is confirmed that the liver-protecting effect of CASP is indeed related to the changes in the intestinal floras and SCFAs content in the cecum, which provides a basis for screening liver-protecting alternative antibiotics products for poultry.

Transcriptome Analysis of LLC-PK Cells Single or Coinfected with Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are the two most prevalent swine enteric coronaviruses worldwide. They commonly cause natural coinfections, which worsen as the disease progresses and cause increased mortality in piglets. To better understand the transcriptomic changes after PEDV and PDCoV coinfection, we compared LLC porcine kidney (LLC-PK) cells infected with PEDV and/or PDCoV and evaluated the differential expression of genes by transcriptomic analysis and real-time qPCR. The antiviral efficacy of interferon-stimulated gene 20 (ISG20) against PDCoV and PEDV infections was also assessed. Differentially expressed genes (DEGs) were detected in PEDV-, PDCoV-, and PEDV + PDCoV-infected cells at 6, 12, and 24 h post-infection (hpi), and at 24 hpi, the number of DEGs was the highest. Furthermore, changes in the expression of interferons, which are mainly related to apoptosis and activation of the host innate immune pathway, were found in the PEDV and PDCoV infection and coinfection groups. Additionally, 43 ISGs, including GBP2, IRF1, ISG20, and IFIT2, were upregulated during PEDV or PDCoV infection. Furthermore, we found that ISG20 significantly inhibited PEDV and PDCoV infection in LLC-PK cells. The transcriptomic profiles of cells coinfected with PEDV and PDCoV were reported, providing reference data for understanding the host response to PEDV and PDCoV coinfection.

Development and Efficacy Evaluation of a Novel Nano-Emulsion Adjuvant for a Foot-and-Mouth Disease Virus-like Particles Vaccine Based on Squalane.

The successful development of foot-and-mouth disease virus-like particles (FMD-VLPs) has opened a new direction for researching a novel subunit vaccine for foot-and-mouth disease (FMD). Therefore, it is urgent to develop an adjuvant that is highly effective and safe to facilitate a better immune response to be pair with the FMD-VLP vaccine. In this research, we prepared a new nano-emulsion adjuvant based on squalane (SNA) containing CpG using the pseudo-ternary phase diagram method and the phase transformation method. The SNA consisted of Span85, Tween60, squalane, polyethene glycol-400 (PEG400) and CpG aqueous solution. The average particle diameter of the SNA was about 95 nm, and it exhibited good resistance to centrifugation, thermal stability, and biocompatibility. Then, SNA was emulsified as an adjuvant to prepare foot-and-mouth disease virus-like particles vaccine, BALB/c mice and guinea pigs were immunized, and we evaluated the immunization effect. The immunization results in mice showed that the SNA-VLPs vaccine significantly increased specific antibody levels in mice within 4 weeks, including higher levels of IgG1 and IgG2a. In addition, it increased the levels of IFN-γ and IL-1ß in the immune serum of mice. Meanwhile, guinea pig-specific and neutralizing antibodies were considerably increased within 4 weeks when SNA was used as an adjuvant, thereby facilitating the proliferation of splenic lymphocytes. More importantly, in guinea pigs immunized with one dose of SNA-VLPs, challenged with FMDV 28 days after immunization, the protection rate can reach 83.3%, which is as high as in the ISA-206 control group. In conclusion, the novel squalane nano-emulsion adjuvant is an effective adjuvant for the FMD-VLPs vaccine, indicating a promising adjuvant for the future development of a novel FMD-VLPs vaccine.

[Mechanism of Tianhe Zhuifeng Ointment in treating rheumatoid arthritis with syndrome of internal obstruction and cold-dampness and compatibility principles based on "disease-syndrome-formula" association network].

This study aims to explore the mechanism of Tianhe Zhuifeng Ointment in treating rheumatoid arthritis(RA) with syndrome of internal obstruction and cold-dampness and the compatibility characteristics based on the "disease-syndrome-formula" association network. A gene set associated with the clinical symptoms of RA was collected from Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine v2.0(TCMIP v2.0). The different expression gene set of RA with syndrome of internal obstruction and cold-dampness was screened out by transcriptomic expression profile detection and bioinformatics data mining of the comparison of RA patients with syndrome of internal obstruction and cold-dampness and healthy volunteers. The chemical composition information of 35 Chinese medicines from Tianhe Zhuifeng Ointment was collected from TCMIP v2.0 and Traditional Chinese Medicine Bank(TCMBank). The candidate targets were predicted based on the similarity principle of compounds structure. The interactive network of "related gene of RA with syndrome of internal obstruction and cold-dampness-candidate target of Tianhe Zhuifeng Ointment" was constructed. The core network targets were screened out by topological characteristics of calculating network, and the functional exploration was carried out based on Kyoto Encyclopedia of Genes and Genomes(KEGG) and Reactome Pathway Database. The compatibility mechanisms of various efficacy groups of Tianhe Zhuifeng Ointment were further explored. The results showed that the candidate targets of Tianhe Zhuifeng Ointment were mainly involved into the modules of "immune-inflammation" regulation, nervous system function, cell function, and substance and energy metabolism, etc. The mechanisms of various efficacy groups emphasized on different aspects. The group of dispelling wind and removing dampness-dredging channels and activating collaterals, the group of extinguishing wind and stopping convulsions, and the group of pungent analgesia regulated "immune-inflammation" system by warming meridians and dissipating cold. The group of activating blood and resolving stasis and the group of strengthening sinews and bones regulated "immune-inflammation" system by activating blood and dredging channels. The group of dispelling wind and removing dampness-dredging channels and activating collaterals, the group of extinguishing wind and stopping convulsions, the group of activating blood and resolving stasis, the group of strengthening sinews and bones, and the group of clearing heat and draining water affected the nervous system by invigorating Qi-blood and benefiting spirit. The group of dispelling wind and removing dampness-dredging channels and activating collaterals and the group of extinguishing wind and stopping convulsions regulated cell function and substance and energy metabolism by dispelling wind and eliminating cold-dampness. The group of activating blood and resolving stasis and the group of strengthening sinews and bones regulated the cell function and substance and energy metabolism by activating blood and strengthening sinews and bones. The results showed that Tianhe Zhuifeng Ointment exerted the comprehensive efficacy of dispelling wind, removing dampness, activating blood, removing stasis, warming meridians, dredging channels, and strengthening sinews and bones through adjusting the imbalance of "immune-inflammation", regulating nervous system, cell function, and interfering with substance and energy metabolism, thus improving the syndrome of internal obstruction and cold-dampness. The findings of this study laid foundations for clarifying the therapeutic characteristics and clinical orientation of Tianhe Zhuifeng Ointment.

Tandem mass tag labeled quantitative proteomic analysis of differential protein expression on total alkaloid of Aconitum flavum Hand.-Mazz. against melophagus ovinus.

Melophagus ovinus disease is a common ectoparasitosis, which can lead to a decrease in animal production performance, product quality, and even death. Aconitum flavum Hand.-Mazz. has many pharmacological activities including insecticidal, heat-clearing, analgesic, and dehumidifying. However, there are few researches focused on the effects and related mechanism of Aconitum flavum Hand.-Mazz. in killing Melophagus ovinus. In this study, 11 alkaloids of Aconitum flavum Hand.-Mazz. were detected, and its total alkaloid activity was determined. The results showed when the total alkaloid concentration was 64 mg/ml and the treatment time was 16 h, the killing rate of Melophagus ovinus reached 100%. Through the observation of the differences in the surface of Melophagus ovinus in each experimental group, it was found that the morphology of the posterior end of the female Melophagus ovinus in the alkaloid treatment group was significantly different from that of the blank and positive control groups, and most of the epidermal tissue was obsessive and missing. Moreover, the enzyme activity determination results of 64 mg/ml group were significantly different when compared with the normal control group, while there was no significant difference in other groups. Then, the Melophagus ovinus gene library was established by the unreferenced genome transcriptome sequencing, the proteomic comparison was performed using tandem mass tag labeled protein detection technology, and finally, the samples were quantitatively analyzed by liquid chromatography-mass spectrometry tandem and bioinformatics methods. Based on the above experimental results, it was speculated that Aconitum flavum Hand.-Mazz. total alkaloids may cause the imbalance of protein disulfide isomerase expressions by affecting the regulation of Hsp40 cellular protein homeostasis and the oxidation of protein disulfide isomerase and related proteins. This would affect the selective recognition of signal sequence, the targeted transport of Sec 61, and the correct folding of the three-dimensional structure of amino acid chain, weakening the clearance of amino acid chains that cannot be correctly folded and eventually resulting in the killing of Melophagus ovinus. This study preliminarily revealed the mechanism of Aconitum flavum Hand.-Mazz. total alkaloids against Melophagus ovinus and provided a theoretical basis for the screening of Melophagus ovinus action targets and the development of new veterinary drugs.

Genomic characterization and pathogenicity analysis of a porcine deltacoronavirus strain isolated in western China.

Porcine deltacoronavirus (PDCoV) is an enteric virus that was first identified in 2012. Although PDCoV has been detected worldwide, there is little information about its circulation in western China. In this study, fecal samples were collected from piglets with watery diarrhea in western China between 2015 and 2018 for the detection of PDCoV. The positive rate was 29.9%. A PDCoV strain (CHN/CQ/BN23/2016, BN23) was isolated and selected for further investigation. Phylogenetic analysis showed that this strain formed an individual cluster between the early Chinese lineage and the Chinese lineage. RDP4 and SimPlot analysis demonstrated that strain BN23 is a recombinant of Thailand/S5015L/2015 and CHN-AH-2004. The pathogenicity of BN23 was evaluated in 3-day-old piglets. Challenged piglets developed serious clinical signs and died at 3 days post-inoculation. Our data show that PDCoV is prevalent in western China and that strain BN23 is highly pathogenic to newborn piglets. Therefore, more attention should be paid to emerging PDCoV strains in western China.

FoxJ1 inhibits African swine fever virus replication and viral S273R protein decreases the expression of FoxJ1 to impair its antiviral effect.

African swine fever (ASF) is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar, which is caused by the African swine fever virus (ASFV). ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health. To date, there is no safe and effective commercial vaccine against ASF. Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV. In the present study, RNA-sequencing, RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages (PAMs) infected by ASFV. RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes (ISGs) induced by poly(dA:dT). FoxJ1 revealed a function to positively regulate innate immune response, therefore, suppressing the replication of ASFV. In addition, Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway. Meanwhile, RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1. Altogether, we determined that FoxJ1 plays an antiviral role against ASFV replication, and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1. Our findings provide new insights into the antiviral function of FoxJ1, which might help design antiviral drugs or vaccines against ASFV infection.

Metabonomics study on the hepatoprotective effect mechanism of polysaccharides from different processed products of Angelica sinensis on layer chickens based on UPLC-Q/TOF-MS/MS, multivariate statistical analysis and conjoint analysis.

Chicken colibacillosis is one of the most severe diseases in the poultry industry. Ceftiofur sodium (CS) is often used to treat it in clinical practice and lipopolysaccharide (LPS) accumulates in the chicken's body. Previous experimental studies found that CS combined with LPS could induce liver injury in layer chickens, and polysaccharides from charred Angelica sinensis(CASP) had a better hepatoprotective effect than polysaccharides from unprocessed Angelica sinensis(UASP). However, the intervention mechanism was unclear. Thus, UPLC-Q/TOF-MS/MS-based metabonomics and transcriptomics were used in this study to clarify the hepatoprotective effect mechanism of CASP and UASP in layer chickens. Transcriptomics and enzyme-linked immunosorbent assay were used for biological verification of some critical mutual metabolic pathways screened with metabonomics. The comprehensive analysis results showed that in a layer chicken liver injury model built with LPS and CS, 12 critical metabolic pathways were disturbed, involving 10 important differential metabolites. The hepatoprotective effect mechanism of CASP is related to the arachidonic acid metabolism and mTOR signaling pathways, involving nine important differential metabolites. In contrast, the hepatoprotective effect mechanism of UASP is related to the arachidonic acid metabolism pathway, involving six important differential metabolites.

Galectin-9 Mediates the Therapeutic Effect of Mesenchymal Stem Cells on Experimental Endotoxemia.

Endotoxemia remains a major cause of mortality in the intensive care unit, but the therapeutic strategy is still lacking. Mesenchymal stem cell (MSC) was reported with a tissue-oriented differentiation ability and an excellent immunoregulatory capacity. However, the immunity signaling pathways that govern MSC modulation effect are not completely understood. In our current study, MSCs (2.5 × 105 /ml) were obtained and stimulated with IFN-γ (20 ng/ml) for 72 h. Gal-9 expression on MSCs was measured by ELISA, RT-PCR, flow cytometry, and immunofluorescence, respectively. Experimental endotoxemia was induced by LPS injection (10 mg/kg, i. p.) followed by the treatment with Gal-9 high-expressing MSCs, unmodified MSCs, and Gal-9 blocking MSCs. Therapeutic effects of MSCs were assessed by monitoring murine sepsis score, survival rate, splenocyte proportion rate, inflammatory mediator levels, and pathological manifestations. The results showed that Gal-9 expressed in MSCs, and this expression was increased in a dose-dependent manner after pre-stimulating with IFN-γ. Adoptive transfer of Gal-9 high-expressing MSCs into modeling mice significantly alleviated endotoxemia symptoms and multi-organ pathological damages. Splenocyte analysis indicated that Gal-9 high-expressing MSCs could promote macrophage polarization to M2-subtype and boost Treg generation. Moreover, there were also attenuated pro-inflammatory mediator expressions (TNF-α, IL-1ß, IFN-γ, and iNOS), and increased anti-inflammatory mediator expressions (T-SOD and IL-35) in the sera and damaged organ homogenates. Additionally, we found a higher expression of Gal-9 in liver, lung, and kidney homogenate. Taken together, this study reveals that the optimized immunoregulatory effect of MSCs is strongly correlated with Gal-9 high expression, which provides a novel idea for the investigation of MSC immunomodulatory mechanisms and offers a potential strategy for the treatment of endotoxemia in clinical settings.

Evaluation of Four Commercial Vaccines for the Protection of Piglets against the Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus (hp-PRRSV) QH-08 Strain.

Vaccination is the best way to prevent economic losses from highly pathogenic porcine reproductive and respiratory syndrome virus (hp-PRRSV) disease. However, the commercially available vaccines need to periodically evaluate their efficacy against infections caused by new hp-PRRSV variants. Therefore, the objective of this study was to evaluate the efficacy of four (two modified live vaccines (MLV) and two inactivated) PRRSV commercial vaccines in piglets challenged with QH-08 and to estimate the genetic distance of the vaccine strains from recently isolated (QH-08) filed strain. Randomly, piglets (n = 5) allocated in groups 1-4 were immunized with Ingelvac PRRS MLV, CH-1a, JXA1, and JXA1-RMLV vaccines, whereas the infected and non-infected control piglets in groups 5 and 6 (n = 3), respectively, were subjected to PBS. Results indicated that JXA1 and JXA1-R MLV vaccines showed complete protection, but Ingelvac PRRS MLV and CH-1α vaccines revealed partial protection against the QH-08 PRRSV challenge. Similarly, vaccinated and challenged pigs showed lower macroscopic and microscopic lesions than the pigs in group 5. Our findings demonstrated a new insight that the variation in ORF1a and 1b coding sequence could significantly affect PRRSV vaccines efficacy. In conclusion, QH-08 is a good candidate for the design and development of an innovative PRRSV vaccine that ultimately helps in the control and prevention strategies.

Oxymatrine protects cardiac allografts by regulating immunotolerant cells.

Organ transplantation is an effective treatment strategy for patients with irreversible organ failure or congenital organ dysfunction. Oxymatrine (OMT) is a quinolizidine alkaloid with protective and anti-inflammatory effects on tissues and organs. The objective of this study was to investigate whether OMT could exert protective effects in cardiac allografts by regulating immune cells. In vitro cell proliferation and co-culture experiments were used to measure the effects of OMT on splenocyte proliferation and differentiation. In the in vivo study, C57BL/6 mice transplanted with BALB/c cardiac grafts were randomly divided into untreated, low-dose OMT treated, middle-dose OMT treated, high-dose OMT treated, and rapamycin-treated groups. Haematoxylin and eosin and immunohistochemical staining were used to assess pathological changes in the grafts, and fluorescence-activated cell sorting analysis was performed to measure the percentages of immune cells. The results showed that, in the in vitro study, OMT inhibited splenocyte proliferation, decreased the percentage of mature dendritic cells (DCs), and increased the percentage of regulatory T cells (Tregs) and regulatory B cells (Bregs). In the in vivo study, OMT exerted allograft protective effects by prolonging survival time, alleviating pathological damages to the cardiac allograft, decreasing intragraft CD3+ cell and increasing intragraft Foxp3+ cell infiltration, decreasing the percentages of mature DCs, increasing the percentages of Tregs and Bregs, and inhibiting the function of DCs. In conclusion, our study demonstrates that OMT exerted a protective effect on cardiac allografts by regulating immunotolerant cells. More in-depth studies of OMT may provide additional insight into the use of immunosuppressive drugs as a post-transplantation treatment strategy.

IL-1ß pre-stimulation enhances the therapeutic effects of endometrial regenerative cells on experimental colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic, relapsing, and non-specific inflammatory bowel disease, and the current treatment strategies were mainly used to relieve symptoms or for maintenance. Endometrial regenerative cells (ERCs) are mesenchymal-like stromal cells and have been demonstrated to alleviate multiple immune-dysregulation diseases. Pro-inflammatory stimuli were reported to enhance the immunosuppressive functions of ERCs, but the mechanism underlined is not fully understood. Here, we have designed this study to investigate the therapeutic effects of IL-1ß-primed ERCs in the attenuation of experimental colitis. METHODS: BALB/c mice were given 3% dextran sodium sulfate (DSS) for 7 consecutive days and free tap water for 3 days sequentially to induce experimental colitis. PBS (200 µL), ERCs, and IL-1ß-primed ERCs (10ng/mL, 48 h) were injected (1 million/mouse/day, i.v.) on day 2, 5, and 8, respectively. Colonic and splenic samples were harvested on day 10 after DSS induction. RESULTS: It was found that IL-1ß-primed ERC treatment markedly attenuated colonic damage, body weight loss, and colon length shortening in colitis mice. Compared with other treatments, cell populations of CD4+IL-4+Th2 cells, CD4+CD25+FOXP3+ regulatory T cells (Tregs), and CD68+CD206+ macrophages in spleens were also significantly upregulated in the IL-1ß-primed ERC-treated group (p < 0.05). In addition, lower expression of pro-inflammatory (IFN-γ, IL-17, TNF-α, and IL-6), but higher levels of anti-inflammatory cytokines (IL-4 and IL-10) were detected in colons in the IL-1ß-primed ERC-treated group (p < 0.05 vs. other groups). Importantly, we also found that different generations of ERCs had an overall lower secretion of Dickkopf-1 (DKK1) by IL-1ß pre-stimulation (p < 0.05) and a higher expression of ß-catenin in colonic and splenic tissues after the administration of IL-1ß-primed ERCs. CONCLUSIONS: This study has demonstrated that IL-1ß pre-stimulation effectively downregulated DKK1 expression in ERCs, which in turn promoted the wnt/ß-catenin pathway activation in colonic and splenic tissues. Consequently, IL-1ß-primed ERCs exhibited an enhanced therapeutic effect in the attenuation of DSS-induced colitis.

IL-37 overexpression enhances the therapeutic effect of endometrial regenerative cells in concanavalin A-induced hepatitis.

BACKGROUND AIMS: Mesenchymal stromal cells and immunosuppressive factor IL-37 can both suppress concanavalin A (Con A)-induced hepatitis in mice. Endometrial regenerative cells (ERCs), novel types of mesenchymal-like stromal cells, possess powerful immunomodulatory effects and are effective in treating various diseases. The aim of this study was to explore the effects of ERCs in suppressing Con A-induced hepatitis and determine whether IL-37 overexpression could enhance the therapeutic effect of ERCs in this process. METHODS: ERCs were extracted from the menstrual blood of healthy female volunteer donors. The IL-37 gene was transferred into ERCs, and the expression of IL-37 in cells was detected by western blot and enzyme-linked immunosorbent assay. Hepatitis was induced by Con A in C57BL/6 mice that were randomly divided into groups treated with phosphate-buffered saline, ERCs, IL-37 or ERCs transfected with the IL-37 gene (IL-37-ERCs). Cell tracking, liver function, histopathological and immunohistological changes, immune cell proportions and levels of cytokines were measured 24 h after Con A administration. RESULTS: Compared with ERC or IL-37 treatment, IL-37-ERCs further reduced levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase) and improved histopathological changes in the liver. In addition, IL-37-ERC treatment further reduced the proportions of M1 macrophages and CD4+ T cells and increased the proportion of regulatory T cells. Moreover, IL-37-ERC treatment resulted in lower levels of IL-12 and interferon gamma, and higher level of transforming growth factor beta. CONCLUSIONS: The results of this study suggest that ERCs can effectively alleviate Con A-induced hepatitis. Furthermore, IL-37 overexpression can significantly enhance the therapeutic efficacy of ERCs by augmenting the immunomodulatory and anti-inflammatory properties of ERCs. This study may provide a promising strategy for treatment of T-cell-dependent hepatitis.

CD73 expression is critical to therapeutic effects of human endometrial regenerative cells in inhibition of cardiac allograft rejection in mice.

The newly found mesenchymal-like endometrial regenerative cells (ERCs) have been proved to induce immune tolerance in cardiac allograft transplantation. However, the therapeutic mechanism is not clear. The present study was undertaken to investigate whether ecto-5'-nucleotidase (CD73) expression on ERCs is critical to cardiac allograft protection. C57BL/6 mouse recipients receiving BALB/c mouse cardiac allografts were treated with unmodified ERCs or anti-CD73 monoclonal antibodies (mAb) pretreated ERCs, respectively. It has been found that CD73 expression was critical to ERC-induced attenuation of graft pathology. The blockage of CD73 expression on ERCs was related to the percentage decline of tolerogenic dendritic cells (Tol-DCs), macrophages type 2 (M2), and regulatory T cells (Tregs). As compared with anti-CD73 mAb pretreated ERCs group, CD73 expressing ERCs significantly increased the level of anti-inflammatory cytokine IL-10 but decreased levels of pro-inflammatory cytokines including IFN-γ and TNF-α. In addition, CD73 expressing ERCs showed tissue protective function via the regulation of adenosine receptor expression which was related to the infiltration of CD4+ and CD8+ cells in the allografts. Furthermore, significant increase of A2B receptors in the cardiac allograft was also associated with CD73 expressing ERC-induced prolongation of cardiac allograft survival.

Galectin-9 is required for endometrial regenerative cells to induce long-term cardiac allograft survival in mice.

BACKGROUND: Endometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cells, were identified as an attractive candidate for immunoregulation and induction of cardiac allograft tolerance. However, the underlying mechanisms of ERCs in immune regulation still remain largely unclear. The present study is designed to determine whether the expression of Galectin-9 (Gal-9), a soluble tandem-repeat member of the galectin family, is crucial for ERC-based immunomodulation. METHODS: In this study, we measured Gal-9 expression on ERCs and then co-cultured Gal-9-ERCs, ERCs, and ERCs+lactose (Gal-9 blocker) with activated C57BL/6-derived splenocytes. Furthermore, we performed mouse heart transplantation between BALB/c (H-2d) donor and C57BL/6 (H-2b) recipient. ERCs were administrated 24 h after the surgery, either alone or in combination with rapamycin. RESULTS: Our data demonstrate that ERCs express Gal-9, and this expression is increased by IFN-γ stimulation in a dose-dependent manner. Moreover, both in vitro and in vivo results show that Gal-9-ERC-mediated therapy significantly suppressed Th1 and Th17 cell response, inhibited CD8+ T cell proliferation, abrogated B cell activation, decreased donor-specific antibody production, and enhanced the Treg population. The therapeutic effect of ERCs was further verified by their roles in prolonging cardiac allograft survival and alleviating graft pathological changes. CONCLUSIONS: Taken together, these data indicate that Gal-9 is required for ERC-mediated immunomodulation and prevention of allograft rejection.

Establishment of a low-tumorigenic MDCK cell line and study of differential molecular networks.

Influenza is an acute respiratory infection caused by the influenza virus, and vaccination against influenza is considered the best way to prevent the onset and spread. MDCK (Madin-Darby canine kidney) cells are typically used to isolate the influenza virus, however, their high tumorigenicity is the main controversy in the production of influenza vaccines. Here, MDCK-C09 and MDCK-C35 monoclonal cell lines were established, which were proven to be low in tumorigenicity. RNA-seq of MDCK-C09, MDCK-C35, and MDCK-W73 cells was performed to investigate the putative tumorigenicity mechanisms. Tumor-related molecular interaction analysis of the differentially expressed genes indicates that hub genes, such as CUL3 and EGFR, may play essential roles in tumorigenicity differences between MDCK-C (MDCK-C09 and MDCK-C35) and MDCK-W (MDCK-W73) cells. Moreover, the analysis of cell proliferation regulation-associated molecular interaction shows that downregulated JUN and MYC, for instance, mediate increased proliferation of these cells. The present study provides a new low-tumorigenic MDCK cell line and describes the potential molecular mechanism for the low tumorigenicity and high proliferation rate.