Fibrinogen-like protein 2 promotes the accumulation of myeloid-derived suppressor cells in the hepatocellular carcinoma tumor microenvironment.

The tumor microenvironment in hepatocellular carcinoma can be classified into cellular and non-cellular components. Myeloid-derived suppressor cells (MDSCs) are cellular components of this microenvironment that serve an important role in the progression of hepatocellular carcinoma. Fibrinogen-like protein 2 (FGL2) has been demonstrated to promote tumor progression by regulating cellular components of the tumor microenvironment in various types of malignant tumor. The present study aimed to determine the expression of FGL2 in hepatocellular carcinoma and its effect on the tumor microenvironment in order to determine novel targets for liver cancer treatment. Immunohistochemistry and reverse transcription quantitative PCR were performed to determine the expression level of FGL2 and the correlation with surface markers of human MDSCs in hepatocellular carcinoma. Furthermore, a mouse hepatocellular carcinoma cell line overexpressing FGL2 was established by stable transfection of a lentivirus expressing FGL2. In addition, fresh bone marrow cells extracted from mouse femurs were in vitro cultured using conditioned medium derived from the cell line overexpressing FGL2. An orthotopic hepatocellular carcinoma mouse model was also established. The results demonstrated that FGL2 expression level in hepatocellular carcinoma tissues was closely associated with tumor size. FGL2 level was positively correlated with the expression level of the MDSC surface markers CD11b and CD33 in hepatocellular carcinoma. The in vitro results demonstrated that FGL2 could maintain the undifferentiated state of bone marrow cells, therefore promoting MDSC accumulation. Furthermore, in the orthotopic hepatocellular carcinoma mouse model, we observed that overexpression of FGL2 could promote tumor growth and significantly increase the number of MDSCs in the tumors and spleen. Taken together, these findings suggested that FGL2 may promote hepatocellular carcinoma tumor growth by promoting the accumulation of MDSCs in the tumor microenvironment.

Toll-Like Receptor 3 Activator Preconditioning Enhances Modulatory Function of Adipose­Derived Mesenchymal Stem Cells in a Fully MHC-Mismatched Murine Model of Heterotopic Heart Transplantation.

BACKGROUND Donor-specific tolerance is the ultimate goal in organ transplantation. Diverse approaches, including the use of mesenchymal stem cells (MSCs), have been investigated to induce graft tolerance. Non-stimulated MSCs showed limited regulatory functions through interaction with multiple immune-regulatory cells, such as regulatory T cells (Tregs). To augment their functions, MSCs have been preconditioned with toll-like receptor (TLR3/4) agonist in autoimmune disease models, but results were conflicting. MATERIAL AND METHODS We evaluated the immunomodulatory effects of mouse adipose-derived mesenchymal stem cells (ADSCs) preconditioned with various combinations of TLR3/4 agonist and antagonists, including polyinosinic-polycytidylic acid poly(I:C)-TLR3 agonist, lipopolysaccharide (LPS) -TLR4 agonist, and TAK242-TLR4 antagonist. In vitro and in vivo experiments including mixed lymphocyte reaction, cytokines measurement, Tregs analysis, and a fully mismatched MHC heterotopic heart transplantation in mice (BALB/c to C57BL/6) were conducted. RESULTS ADSCs preconditioned with poly(I:C) showed the highest efficiency in inhibiting lymphocyte proliferation, which was correlated with the upregulation of fibrinogen-like protein 2 (FGL2), an effector molecule of Tregs. The mean survival of cardiac allografts was extended from 8 to 12 days by intravenous injection of a single dose of ADSCs preconditioned with TLR3 agonist. The proportion of Tregs in the recipient's spleen was significantly increased by injecting the poly(I:C)-stimulated ADSCs. CONCLUSIONS These results show that short-term TLR3 agonist preconditioning enhances the immunomodulatory efficacy of ADSCs, which can induce the generation of Tregs and upregulate the expression of FGL2, thereby improving the outcome of patients receiving organ transplantation.

A Self-Assembling Lipidic Peptide and Selective Partial V2 Receptor Agonist Inhibits Urine Production.

Lipidised analgesic peptide prodrugs self-assemble into peptide nanofibers; with the nanofiber morphology protecting the peptide from plasma degradation and improving therapeutic efficacy. Extending this learning, we hypothesised that a self-assembling lipidized peptide arginine vasopressin (AVP) receptor agonist, that had not been designed as a prodrug, could prove pharmacologically active and control urine production. The only approved AVP receptor agonist, desmopressin is indicated for the treatment of central diabetes insipidus (DI), bedwetting, haemophilia A and von Willebrand disease. Desmopressin is well tolerated by most patients, however adverse effects, such as hyponatraemia and water intoxication necessitate a strict fluid intake, thus motivating the search for alternative DI treatments. Selective V2 receptor agonism is required for anti-DI activity and we hypothesised that our new lipidized peptide (METx) would lead to selective AVP receptor agonism. METx was synthesised and characterised and then tested for activity against the V2, V1a and OT uterine receptors and not tested against the V1b receptor as METx was not expected to cross the blood brain barrier. METx was also tested in vivo in a healthy rat model. METx forms nanofibers and is a partial V2 receptor agonist (determined by measuring MDCK cell line cAMP accumulation), producing 57% of AVP's maximal activity (EC50 = 2.7 nM) and is not a V1a agonist up to a concentration of 1 µM (determined by measuring A7r5 cell line D-myo-inositol-1-phosphate accumulation). METx is a weak OT receptor antagonist, reducing the frequency of OT induced contractions (EC50 = 350 nM) and increasing the OT EC50 from 0.081 nM to 21 nM at a concentration of 600 nM. METx (41 nM) had no effect on spontaneous uterine contractions and METx (100 nM) had no effect on OT induced uterine contractions. Simulated binding studies show that binding avidity to the receptors follows the trend: V2 > OT > V1a. On intravenous injection, a nanoparticle formulation of METx reduced urine production in a healthy rat model in a dose responsive manner, with 40 mg kg-1 METx resulting in no urine production over 4 hours. The lipidized self-assembling peptide - METx - is a selective competitive V2 receptor agonist and an anti-diuretic.

Low mir-372 expression correlates with poor prognosis and tumor metastasis in hepatocellular carcinoma.

BACKGROUND: Recent studies have shown that miR-372 plays important roles in hepatocellular carcinoma (HCC) progression. However, results have been conflicting regarding its expression levels and role in HCC. METHODS: RT-PCR and in situ hybridization was used to evaluate miR-372 expression in HCC tissues and cell lines. The methylation status of neighboring CpG islands upstream of the miR-372 promoter was analyzed by methylation-specific PCR (MSP). Transfection of miR-372 mimic into HCC cell lines was used to evaluate cellular proliferation and invasion. Prognostic significance was analyzed by the Kaplan-Meier survival method and Cox regression. RESULTS: miR-372 was expressed at lower levels in HCC tissues compared with controls and was related to tumor metastasis and poor prognosis. Hypermethylation of miR-372 was detected in HCC cell lines and tissues, and miR-372 expression was restored upon 5-aza-dCyd treatment. Upregulated expression by mir-372 mimic transfection inhibited proliferation and invasion capacity in HCC cells. CONCLUSIONS: miR-372 may play an important role in hepatic carcinogenesis and may serve as a new target or method to detect and treat HCC in the future.

Acute toxicity and bioconcentration of pyrimorph in zebrafish, Brachydanio rerio.

BACKGROUND: Pyrimorph is a novel fungicide being developed in China that shows high antifungal activity against diseases caused by Phytophthora infestans, Phytophthora capsici, Rhizoctonia solani, Peronophythora litchi and Pseudoperonospora cubensis. Until now, no information on the toxicity of pyrimorph to untargeted organisms has been reported. To assess the potential environmental impacts of pyrimorph in fish, the acute toxicity and bioconcentration of pyrimorph in zebrafish were studied in this paper. RESULTS: When tested by the semi-static method, the 48, 72 and 96 h median lethal concentration (LC50 ) values of pyrimorph to zebrafish were 24.33, 22.61 and 19.79 mg L(-1) respectively. To study the bioconcentration of pyrimorph in zebrafish, the fish were exposed to sublethal concentrations of pyrimorph (2.00 and 0.25 mg L(-1) ) for 192 h, a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method was applied to extract pyrimorph from samples and the residues of pyrimorph in the water and fish were determined by high-performance liquid chromatography (HPLC). The highest bioconcentration factor (BCF) of pyrimorph in the fish was 1.07 × 10(2) (144 h) and 23.1 (96 h) after exposure to 2.00 and 0.25 mg L(-1) of pyrimorph respectively. CONCLUSIONS: The novel fungicide pyrimorph has low toxicity to zebrafish; however, it showed medium BCF to zebrafish. Therefore, more attention should be paid to the residue of pyrimorph in fish, as well as to its long-term ecological effects.