Search

Tumor-associated macrophages (TAMs) constitute a large population of glioblastoma and facilitate tumor growth and invasion of tumor cells, but the underlying mechanism remains undefined. In this study, we demonstrate that chemokine (C-C motif) ligand 8 (CCL8) is highly expressed by TAMs and contributes to pseudopodia formation by GBM cells. The presence of CCL8 in the glioma microenvironment promotes progression of tumor cells. Moreover, CCL8 induces invasion and stem-like traits of GBM cells, and CCR1 and CCR5 are the main receptors that mediate CCL8-induced biological behavior. Finally, CCL8 dramatically activates ERK1/2 phosphorylation in GBM cells, and blocking TAM-secreted CCL8 by neutralized antibody significantly decreases invasion of glioma cells. Taken together, our data reveal that CCL8 is a TAM-associated factor to mediate invasion and stemness of GBM, and targeting CCL8 may provide an insight strategy for GBM treatment.

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

Acute lung injury (ALI) is a common and devastating clinical disorder featured by excessive inflammatory responses. Stimulator of interferon genes (STING) is an indispensable molecule for regulating inflammation and immune response in multiple diseases, but the role of STING in the ALI pathogenesis is not well elucidated. In this study, we explored the molecular mechanisms of STING in regulating lipopolysaccharide (LPS)-induced lung injury. Mice were pretreated with a STING inhibitor C-176 (15, 30 mg/kg, i.p.) before LPS inhalation to induce ALI. We showed that LPS inhalation significantly increased STING expression in the lung tissues, whereas C-176 pretreatment dose-dependently suppressed the expression of STING, decreased the production of inflammatory cytokines including TNF-α, IL-6, IL-12, and IL-1β, and restrained the expression of chemokines and adhesion molecule vascular cell adhesion protein-1 (VCAM-1) in the lung tissues. Consistently, in vitro experiments conducted in TNF-α-stimulated HMEC-1cells (common and classic vascular endothelial cells) revealed that human STING inhibitor H-151 or STING siRNA downregulated the expression levels of adhesion molecule and chemokines in HMEC-1cells, accompanied by decreased adhesive ability and chemotaxis of immunocytes upon TNF-α stimulation. We further revealed that STING inhibitor H-151 or STING knockdown significantly decreased the phosphorylation of transcription factor STAT1, which subsequently influenced its binding to chemokine CCL2 and adhesive molecule VCAM-1 gene promoter. Collectively, STING inhibitor can alleviate LPS-induced ALI in mice by preventing vascular endothelial cells-mediated immune cell chemotaxis and adhesion, suggesting that STING may be a promising therapeutic target for the treatment of ALI.

By establishing C57-BABL/c mice allogeneic skin transplantation model, to analyze the immune system function with the administration of thymosin al (Tal) in mouse after skin transplantation; and to explore the mechanism of specific immune tolerance induced by Tal in vivo.80 C57 mice and 80 BABL/c mice were used as donor and acceptor respectively to establish C57-BABL/c mice allogeneic skin transplantation model and divided into four groups: Group A, control group (without any treatment, n=20); Group B, CsA treatment group (CsA 10 mg/kg, n=20); Group C, Tal treatment group (Tal 400 microg/kg, n=20) and Group D, combination therapy group (CsA 10 mg/kgTalphal 400 microg/kg, n=20). In the three experimental group, the drug of each group were respectively administrated by intraperitoneal injection daily, for 21 d. The survival of skin graft were observed and recorded, the Luminexx MAP for cytokine detection were performed in 1, 7, 14, 21 d after treatment, skin grafts were taking for HE staining, and flow cytometry were performed for lymphocyte phenotype.After transplantation, in 1, 7, 14, 21 d, the cytokine of Group B, C and ID compared to Group A, as well as Group D to BC respectively, shows a decreaing of IL-1, IL-2, IL-6, IL-17 value and increasing of IL-10 significantly (P0.05) at the same time point; While no statistical significance shows between Group B and C. Compared with other groups, Group D have a high ratio of CD4/CD8, and a high percentage of CD4+ CD25+ T cells (P0.05).Administrated Tal after transplantation, can decrease the graft damage from T cells, but could not prevent rejection.

Background The objective of this paper was to study antimicrobial activity and safety of Enterococcus faecium KQ 2.6 (E. faecium KQ 2.6) isolated from peacock feces. Methods Agar well diffusion method was adopted in antimicrobial activity assay. Disk diffusion test was used to determine the antibiotic resistance. The identification and virulence potential of E. faecium KQ 2.6 were investigated using PCR amplification. Results The results indicated that cell free supernatant (CFS) of the strain had the good antimicrobial activity against selected gram-positive and gram-negative bacteria. The biochemical characteristics of antimicrobial substances were investigated. The results indicated that the antimicrobial substances were still active after treatment with catalase and proteinase, respectively. Moreover, the stability of antimicrobial substances did not change after heat treatment at 40, 50, 60, 70 and 80°C for 30 min, respectively. The activity of antimicrobial substances remained stable at 4 and −20°C after long time storage. The antimicrobial activity of CFS was compared with that of the buffer with similar strength and pH. The inhibitory zone of the buffer was apparently smaller than that of CFS, which meant that the acid in CFS was not the only factor that was contributed to antibacterial activity of CFS. The antibiotic resistance and virulence potential were evaluated using disk diffusion test and PCR amplification. The results showed that E. faecium KQ 2.6 did not harbor any tested virulence genes such as gelE, esp, asa1, cylA, efaA and hyl. It was susceptible to most of tested antibiotics except for vancomycin and polymyxin B. Conclusion E. faecium KQ 2.6 may be used as bio-preservative cultures for the production of fermented foods.

Pregnant adult C57BL/6J mice were randomly assigned to four groups: one sham and three irradiated groups that were exposed to β-irradiation from tritiated water (HTO) by a single intraperitoneal injection on Day 12.5 of gestation. The offspring received cumulative doses of 0.036, 0.071, and 0.213 Gy, respectively. The litters were observed for postnatal growth (body weight, brain weight), the development of four physiologic milestones (pinna detachment, eye opening, testes decent, vaginal opening), the acquisition age of several reflexes (cliff avoidance, air righting) and sensory functions (auditory startle, thermal reflex), movement and coordination functions and activity (pivoting, foot splay, continuous corridor activity), learning and memory performance (shock avoidance, conditioning reflex), and the density of CA1–CA4 hippocampal pyramidal neurons. Modest but significant dose-dependent neuronal death and functional impairment were seen in both 0.071 and 0.213 Gy groups. In conclusion, even prenatal low-dose β-radiation may impair murine central nervous system (CNS) development suggesting the potential importance of minimizing environmental exposure during human pregnancy.

To analysis the effects of Talpha1 on the immune effector molecules in mouse immune system.Sixty five BABL/c mice were divided into four groups: CsA group (n=20), Talpha1 group (n= 20), CsA+Talpha1 group (n=20) and control group (n=5). In the 3 experimental groups, 10 mg/kg CsA, 400 microg/ kg Talpha1, 10 mg/kg CsA+400 microg/kg Talpha1 were respectively administrated by intraperitoneal injection daily. Luminex was performed for cytokine detection at 1 d, 7 d, 14 d, 21 d day after the above treatments. Lymphocyte culture was prepared with the mouse spleen suspension, and then treated with 0. 25 mg/mL CsA, 10 microg/mL Talpha1 or 0.25 mg/mL CsA+10 microg/mL Talpha1 in vitro, respectively. Three days later, OD values of each treated lymphocyte culture and several cytokines in the culture were measured.Compared with other groups, CsA+Talpha1 group had significant lower IL-1alpha, IL-2, IL-6, IL-17, and significant higher IL-10 at 1 d, 7 d, 14 d, 21 d after the treatments (P0.05). Three days after the culture, OD value in the control group was significantly higher than that in Talppha1 group, CsA group, and CsA+ Talpha1 group (P0.05). IL-1alpa and IL-6 in the control group were significantly higher than those in the experiment groups (P0.05), while IL-10 in the control group was significantly lower than that in the experiment groups (P0.05). IL-2 and IL-17 were similar.Talpha1 show regulatory effect on the immune effector molecules which could promote Th1 cells transforming to Th2 cells.

To explore the molecular mechanism of interleukin cytokines IL-6, IL-17, IL-23, IL-23R in the onset of acute pancreatitis in rats pancreatitis models.Thirty six SD rats were randomized into 3 groups: control group, model group, and intervention group (n = 12). The rats in model group and intervention group were induced by intraperitoneal injection of 1-arginine, and those in intervention group were treated by tail intravenous administration of drugs at the same time. Then the rats were sacrified at 3rd, 6th, and 12th h. after the modeling. The levels of IL-6 ,IL-17, IL-23, IL-23R in blood, pancreas, lung and kidney were checked by ELISA.The IL-6 levels of serum, pancreas, lung and kidney in AP Model group were obviously higher than those in the control group and intervention group. This trend increased with time. Similarly, the levels of IL-17 and IL-23 in AP group were obviously higher than those in the control group and intervention group. Compared with the control and intervention group, AP group showed higher IL-23R levels in serum, pancreas, but lower IL-23R levels in lung and kidney.IL-23 is a mediator involved in the formation of IL-17 and IL-6, they all can promote acute pancreatitis.

To investigate the effects of NKG2D mAb on the survival of allogeneic transplanted islets nonobese diabetic (NOD) mice, and to find if CD154 mAb has synergistic effects.Spontaneous diabetic NOD mice transplanted with allogeneic islets of BALB/c mice were divided into 4 groups. Group A was control group, Group B were treated with anti-NKG2D monoclonal antibody (mAb), Group C were treated with CD154 mAb (MR1), Group D were treated with NKG2D mAb and MR1. Glucose levels were monitored at regular intervals through caudal vein, and islet function was evaluated by glycemia. Histological study was performed at graft rejection or at day 120. Spleen cell suspension was prepared for mixed lymphocyte cultivation. The kidneys hosting the islet graft were prepared with HE staining and immuno-histochemistry staining of CD3, CD4 and CD8 was performed.MR1 therapy alone significantly prolonged the survival of islet grafts when compared to NKG2D mAb group and the control group: median graft survival was 41 days versus 8 days (P0.05) and 8 days (P0.05), respectively. Combination therapy with NKG2D mAb and MR1 prolonged islet grafts survival when compared to MR1 therapy alone: median graft survival was 51 days versus 41 days (P0.05).NKG2D mAb alone did not result in the prolongation of islet graft survival, whereas CD154 mAb increased graft survival. When both antibodies were administered, a synergistic effect was obtained, but did not provide permanent protection from diabetes recurrence.

To study the role of Th17 cells in the Immune rejection of islet transplantation, explore the feasibility of immune tolerance of islet transplantation induced by the combination applying of IL-23R antibody and Anti-CD154mAb.The in vitro experiments were divided into 5 groups: Blank control group, SD rat islet cells were cultured alone; A group, co-culture of rat pancreatic islet cells and lymphocytes, without IL-23R antibodies; B, C, D groups, co-culture of rat pancreatic islet cells and lymphocytes, respectively with IL-23R antibodies 0.1 microg/mL, 0.5 microg/mL, 1.0 microg/mL. Cells were harvested for Acridine orange (AO)/propidium iodide (PD) fluorescence staining, insulin and glucagon staining and glucose-stimulated insulin secretion test. The in vivo experiments (the purified islet to be transplanted under the left kidney capsule of the mice) were divided into four groups: Control group, BABL/c mice were transplanted with islets of SD rats with no treatments, IL-23R antibody (200 microg) treatment alone, anti-CD154mAb (200 microg) treatment alone and a combination of both. The blood glucose of the transplanted mice were monitored. The kidney of islet grafts were sliced for HE staining and insulin and glucagon immunohistochemical detection.Three days after mixed cultivation, the glucose stimulation index was 3.66 +/- 0.10 in blank control group, which was higher than that of other groups. Stimulation index of D group was 1.95 +/- 0.75, which was significantly higher than that of other groups. The functional graft survival of all experimental groups were significantly better than that of control group as demonstrated by immunohistochemical staining of insulin and glucagon, as well as in vitro and in vivo experiments. After three days of islet transplantation, the blood glucose of control group was higher than that of experimental groups, but no significant difference was observed among experimental groups.Th17 cells were involved in the islet transplant rejection. The expression of IL-17 could be considerably reduced through the block of the IL-23R, the effect of the block had a positive correlation in a dose-dependent manner. The combination of Anti-CD154 mAb and IL-23R antibody could prevent the acute rejection to some extent. However, there's no significant difference compared with the Anti-CD154mAb alone.

To assess the value of perforin mRNA and granzyme B mRNA in lymphocytes as an early diagnostic indicator for acute rejection of pancreatic transplantation.Twenty-four hybrid landraces were divided into three groups (control group, transplantation group, transplantation plus immunosuppressant group) randomly. The landraces in the transplantation group were treated with pancreatoduodenal allotranplantation. The landraces in the transplantation plus immunosuppressant group underwent the same surgery and were intervened simultaneously with CsA, MMF, and Metrisone. Peripheral venous bloods were sampled via subclavic veins one day before the operations and on the 1st, 3rd, 5th and 7th day after the operations. The RNA was extracted from the blood samples. The mRNA of perforin and granzyme B expressed in the lymphocytes were detected by RT-PCR. The serum glucose, insulin and glucagons were also measured with routine methods. The pancreatic graft tissues were collected for pathologic examinations at the 1st, 3rd, 5th and 7th day after the operations.No differences in serum glucose, insulin and glucagons had been detected among the three groups (P0.05). The landraces in the transplantation plus immunosuppressant group had significantly lower expression of the mRNA of perforin and granzyme B in the lymphocytes and the local transplanted pancreas than those in the transplantation group (P0.05). The change of mRNA expression of perforin and granzyme B in the lymphocytes occurred 2-4 days earlier than the pathological changes when the acute rejection started.Monitoring the change of mRNA expression of perforin and granzyme B in the lymphocytes from the peripheral blood can help the early diagnosis of acute rejection of pancreatic transplantation.

To study the immune tolerance induced by human Ad-CTLA-41g after pancreaticoduodenal transplantation of rats.Wistar rats with diabetes were induced by caudal intravenous administration of streptozotocin (STZ) at a single dose of 60 mg/kg. Pancreaticoduodenal transplantations were performed with the SD rats as donors and the diabete Wistar rats as recipients. The SD pancreaticoduodenal grafts with human Ad-CTLA-41g infection via arterial perfusion in vitro were transplanted into Wistar rats with diabetes. The blood glucose of the rats was measured after transplantation. The graft functional survival time (GFST) was recorded. The concentration of IL-2 was measured by ELISA on day 3 and day 10 after operation. The human CTLA-41g was detected by Western blot after operation.The GFST of control group without gene transfer was (11+/- 1) d, while that of gene transfer group was (33 +/-4) d (P0. 01). The expression of serum human CTLA-41g was detected in rats of gene transfer group on the 10th day after transplantation, while no serum human CTLA-4Ig was detected in rats of control group without gene transfer. On the 3rd day after transplantation, the level of serum IL-2 was (33. 710+/-7. 803) pg/mL in rats of transfer group, and was (44. 772+/-9. 102) pg/mL in rats of control group without gene transfer. On the 10th day, the level of serum IL-2 was (47. 846+/-14. 050) pg/mL in rats of transfer group, and was (80. 806+/-18. 629) pg/mL in rats of control group without gene transfer. The levels of serum IL-2 in rats of transfer group was much lower than those of the control group without gene transfer (PO. 01).In vitro perfusion with human Ad-CTLA-41g is an effective method to transfer the target gene into pancreaticoduodenal graft. Ad-CTLA-41g infection can induce immune tolerance in rat with pancreaticoduodenal transplantation.

To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2-P30 antigen by gene engineering.The gene fragment encoding P30 was amplified by PCR from T. gondii RH strain and subcloned into the recombinant plasmid pUC119/ROP2 already constructed. The recombinant plasmid pUC119/ROP2-P30 was digested by Sac I/HindIII and inserted into the same site of expression vector pET28b. The recombinant plasmid of pET28b/ROP2-P30 was transformed to E. coli and expressed under the induction of IPTG.The gene fragment 700 bp encoding P30 was obtained from the total DNA of T. gondii by PCR. The recombinant plasmid pET28b/ROP2-P30 was successfully constructed, which was highly expressed in E. coli, a fusion protein with molecular weight of 69000.The fusion gene encoding the rhoptry protein ROP2 and the major surface protein P30 of T. gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2-P30 with molecular weight 69000.

To develop a method for detecting the genotype of Plasmodium vivax merozoite surface protein 1 (PvMSP-1) alleles.According to the sequence characteristic of PvMSP-1, nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to amplify the polymorphic region of ICB5-ICB6 which contains Q repeats and PvuII restriction site (Sal-1 type). The PCR product was digested by PvuII restriction endonuclease and the digested fragments were observed by 2% agarose gel electrophoresis. The allelic type was determined according to the banding pattern.Bands in size of 400 bp (Belem type) and/or 470 bp (Sal-1 type) appeared in all 98 P. vivax isolates, no band was found in negative control. After PvuII digestion, two Sal-1 type fragments (120 bp and 350 bp) were obtained from 45 samples of 470 bp. Single-band of 400 bp appeared in 3 of 40 samples with 400 bp as Belem type, two bands of 120 bp and 280 bp appeared from other 35 samples as recombination type III, and another 2 bands with 120 bp and 240 bp as Korean isolate.The result showed that the nested PCR-RFLP may be applied in the detection and identification of the three PvMSP-1 allelic types in China.