Feline infectious peritonitis virus ORF7a is a virulence factor involved in inflammatory pathology in cats.

A hyperinflammatory response is a prominent feature of feline infectious peritonitis (FIP), but the mechanisms behind the feline infectious peritonitis virus (FIPV)-induced cytokine storm in the host have not been clarified. Studies have shown that coronaviruses encode accessory proteins that are involved in viral replication and associated with viral virulence, the inflammatory response and immune regulation. Here, we found that FIPV ORF7a gene plays a key role in viral infection and host proinflammatory responses. The recombinant FIPV strains lacking ORF7a (rQS-79Δ7a) exhibit low replication rates in macrophages and do not induce dramatic upregulation of inflammatory factors. Furthermore, through animal experiments, we found that the rQS-79Δ7a strain is nonpathogenic and do not cause symptoms of FIP in cats. Unexpectedly, after three vaccinations with rQS-79Δ7a strain, humoral and cellular immunity was increased and provided protection against virulent strains in cats, and the protection rate reaches 40%. Importantly, our results demonstrated that ORF7a is a key virulence factor that exacerbates FIPV infection and inflammatory responses. Besides, our findings will provide novel implications for future development of live attenuated FIPV vaccines.

The inhibitory effect of KIAA1456 on the proliferation and metastasis of epithelial ovarian cancer through SSX1 and AKT signaling pathway.

Background: KIAA1456 is effective in the inhibition of tumorigenesis. We previously confirmed that KIAA1456 inhibits cell proliferation and metastasis in epithelial ovarian cancer (EOC). In the current study, the specific molecular mechanisms and clinical significance of KIAA1456 underlying the repression of EOC were investigated. Methods: Immunohistochemistry was used to evaluate the protein expression of KIAA1456 and SSX1 in EOC and normal ovarian tissues. The relationship of KIAA1456 and SSX1 with overall survival of patients with EOC was analysed with Kaplan-Meier survival curve and log-rank tests. KIAA1456 was overexpressed and silenced in HO8910PM cells with lentivirus. Anticancer activities of KIAA1456 was tested by CCK8, plate clone formation assay, flow cytometry, wound healing assay and Transwell invasion assay. Xenograft tumour models were used to investigate the effects of KIAA1456 on tumour growth in vivo. Bioinformatics analyses of microarray profiling indicated that SSX1 and the PI3K/AKT were differentially expressed in KIAA1456-overexpressing and control cells. The downstream factors of PI3K/AKT that are related to cell growth and apoptosis. Results: KIAA1456 expression was lower in EOC than in normal ovarian tissues. Its expression negatively correlated with pathological grade. Pearson's correlation analysis showed that KIAA1456 negatively correlated with SSX1 expression. The overexpression of KIAA1456 in HO8910PM cells inhibited proliferation, migration and invasion and promoted apoptosis. The silencing of KIAA1456 resulted in the opposite behaviour. A xenograft tumour experiment showed that KIAA1456 overexpression inhibited tumour growth in vivo. The overexpression of KIAA1456 inhibited SSX1 and AKT phosphorylation in HO8910PM cells, causing the inactivation of the AKT pathway and eventually reducing the expression of PCNA, CyclinD1, MMP9 and Bcl2. The silencing of KIAA1456 resulted in the opposite behaviour. SSX1 overexpression could partially reverse the KIAA1456-induced biological effect. Conclusion: KIAA1456 may serve as a tumour suppressor via the inactivation of SSX1 and the AKT pathway, providing a promising therapeutic target for EOC.

Human amniotic mesenchymal stem cells promote endometrium regeneration in a rat model of intrauterine adhesion.

Human amniotic transplantation has been proposed to improve the therapeutic efficacy of intrauterine adhesions (IUAs). Human amniotic mesenchymal stem stromal cells (hAMSCs) can differentiate into multiple tissue types. This study aimed to investigate the mechanism by which hAMSCs transplantation promotes endometrial regeneration. The rat models with IUA were established through mechanical and infective methods, and PKH26-labeled hAMSCs were transplanted through the tail vein (combined with/without estrogen). Under three different conditions, hAMSCs differentiated into endometrium-like cells. HE and Mason staining assays, and immunohistochemistry were used to compare the changes in rat models treated with hAMSCs and/or estrogen transplantation. To define the induction of hAMSCs to endometrium-like cells in vitro, an induction medium (cytokines, estrogen) was used to investigate the differentiation of hAMSCs into endometrium-like cells. qRT-polymerase chain reaction (PCR) and western blotting were performed to detect the differentiation of hAMSCs into endometrium-like cells. A greater number of glands, fewer endometrial fibrotic areas, and stronger expression of vascular endothelial growth factor and cytokeratin in the combined group (hAMSCs transplantation combined with estrogen) than in the other treatment groups were observed. hAMSCs could be induced into endometrium-like cells by cytokine treatment (TGF-ß1, EGF, and PDGF-BB). Transplantation of hAMSCs is an effective alternative for endometrial regeneration after injury in rats. The differentiation protocol for hAMSCs will be useful for further studies on human endometrial regeneration.

Human amniotic mesenchymal stem cells combined with PPCNg facilitate injured endometrial regeneration.

BACKGROUND: Caused by the injury to the endometrial basal layer, intrauterine adhesions (IUA) are characterized by uterine cavity obliteration, leading to impaired fertility. Human amniotic mesenchymal stem cells (hAMSCs) have the potential to promote endometrial regeneration mainly through paracrine ability. PPCNg is a thermoresponsive biomaterial consisted of Poly (polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin, which has been reported as a scaffold for stem cell transplantation. This study aims to investigate the therapeutic effect of hAMSCs combined with PPCNg transplantation in promoting the regeneration of injured endometrium. METHODS: hAMSCs were cultured in different concentrates of PPCNg in vitro, and their proliferation, apoptosis and cell cycle were examined by CCK-8 assay and flow cytometry. Immunofluorescence was used to determine the MSCs specific surface markers. The expression of pluripotent genes was analyzed by qRT-PCR. The multiple-lineage differentiation potential was further evaluated by detecting the differentiation-related genes using qRT-PCR and specific staining. The Sprague-Dawley (SD) rat IUA model was established with 95% ethanol. hAMSCs combined with PPCNg were transplanted through intrauterine injection. The retention of DiR-labeled hAMSCs was observed by vivo fluorescence imaging. The endometrium morphology was assessed using hematoxylin and eosin (H&E) and Masson staining. Immunohistochemistry staining was performed to detect biomarkers related to endometrial proliferation, re-epithelialization, angiogenesis and endometrial receptivity. The function of regenerated endometrium was evaluated by pregnancy tests. RESULTS: hAMSCs maintained normal cell proliferation, apoptosis and cell cycle in PPCNg. Immunofluorescence and qRT-PCR showed that hAMSCs cultured in PPCNg and hAMSCs cultured alone expressed the same surface markers and pluripotent genes. hAMSCs exhibited normal multilineage differentiation potential in PPCNg. Vivo fluorescence imaging results revealed that the fluorescence intensity of hAMSCs combined with PPCNg intrauterine transplantation was stronger than that of direct hAMSCs intrauterine transplantation. Histological assays showed the increase in the thickness of endometrial and the number of endometrial glands, and the remarkably decrease in the fibrosis area in the PPCNg/hAMSCs group. The expressions of Ki-67, CK7, CK19, VEGF, ER and PR were significantly increased in the PPCNg/hAMSCs group. Moreover, the number of implanted embryos and pregnancy rate were significantly higher in the PPCNg/hAMSCs group than in the hAMSCs group. CONCLUSIONS: PPCNg is suitable for growth, phenotype maintenance and multilineage differentiation of hAMSCs. hAMSCs combined with PPCNg intrauterine transplantation can facilitate the regeneration of injured endometrium by improving utilization rates of hAMSCs, and eventually restore reproductive capacity.

Management of intrauterine adhesions using human amniotic mesenchymal stromal cells to promote endometrial regeneration and repair through Notch signalling.

Intrauterine adhesions (IUAs) severely hamper women's reproductive functions. Human amniotic mesenchymal stromal cell (hAMSC) transplantation is effective in treating IUAs. Here, we examined the function of Notch signalling in IUA treatment with hAMSC transplantation. Forty-five Sprague-Dawley female rats were randomly divided into the sham operation, IUA, IUA + E2, IUA + hAMSCs and IUA + hAMSCs + E2 groups. After IUA induction in the rats, hAMSCs promoted endometrial regeneration and repair via differentiation into endometrial epithelial cells. In all groups, the expression of key proteins in Notch signalling was detected in the uterus by immunohistochemistry. The results indicated Notch signalling activation in the hAMSCs and hAMSCs + E2 groups. We could also induce hAMSC differentiation to generate endometrial epithelial cells in vitro. Furthermore, the inhibition of Notch signalling using the AdR-dnNotch1 vector suppressed hAMSC differentiation (assessed by epithelial and mesenchymal marker levels), whereas its activation using the AdR-Jagged1 vector increased differentiation. The above findings indicate Notch signalling mediates the differentiation of hAMSCs into endometrial epithelial cells, thus promoting endometrial regeneration and repair; Notch signalling could have an important function in IUA treatment.

The study on the safety and efficacy of amnion graft for preventing the recurrence of moderate to severe intrauterine adhesions.

Transcervical resection of adhesion (TCRA) is the standard treatment for the intrauterine adhesions, but the recurrence of adhesions is a tough problem for the gynecologist. In addition, the therapeutic strategy after TCRA about prevention of recurrence remains controversial especially for the patients with moderate to severe intrauterine adhesions (IUAs). Hence, we designed this study to explore the safety and efficacy of fresh amnion grafts for preventing the recurrence after TCRA for patients with moderate to severe IUAs. One hundred patients with moderate to severe IUAs who presented with a history of hypomenorrhea, amenorrhea and infertility were included in the study from January 2015 to December 2017. Patients were divided into amnion group (52 patients) and chitosan group (48 patients). Fresh amnion grafts or intrauterine injections of chitosan were administered after TCRA. Transvaginal ultrasonography (TVUS) and hysteroscopy were performed at the first and third month after the operation. The surgical procedures for all patients were completed successfully without relevant complications. In amnion group, 8 patients exhibited relapse in the first month and 2 patients in three months after surgery; in chitosan group, 23 women exhibited relapse in the first month and 18 patients in three months after surgery. Statistical analysis revealed that the recurrence rate of adhesion in amnion group was significantly lower than those of chitosan group in the first and three months after surgery (P 1  = 0.000, P 2  = 0.000). After TCRA, fresh amnion graft plays a significant role in preventing further adhesions than injections of chitosan.