Impact of 5-Fu/oxaliplatin on mouse dendritic cells and synergetic effect with a colon cancer vaccine.

OBJECTIVE: The aim of the present study was to investigate the effects of 5-fluorouracil (5-Fu) and oxaliplatin on the function and activation pathways of mouse dendritic cells (DCs), and to clarify whether 5-Fu/oxaliplatin combined with the CD1d-MC38/α-galactosylceramide (α-GC) tumor vaccine exhibits synergistic effects on the treatment of colon cancer in mice. METHODS: The combination of the Toll like receptor (TLR) ligands and/or 5-Fu/oxaliplatin was added into myeloid-derived DCs in vitro culture. DC phenotypic changes were detected by flow cytometry, and the secretion of DC cytokines was detected by cytometric bead array (CBA). A MC38 mouse colon cancer model was constructed and the DCs were isolated from the spleen, tumor tissue and lymph nodes following intraperitoneal injection of 5-Fu/oxaliplatin. The cell phenotypes were detected by flow cytometry. The tumor infiltrating leukocytes, splenocytes and lymph node cells were co-cultured with the dead MC38 tumor cells, and the secretion levels of interferon-γ (IFN-γ) were detected. 5-Fu/oxaliplatin combined with our previously developed CD1d-MC38/α-GC tumor vaccine was used to inhibit the growth of MC38 colon cancer in mice, and the tumor growth rate and survival time were recorded. RESULTS: 5-Fu/oxaliplatin exerted no significant effect on the expression of the stimulating phenotypes of DCs in vitro, while it could reduce the expression of programmed death ligand 1/2 (PD-L1/L2) and promote interleukin-12 (IL-12) secretion by DCs. Furthermore 5-Fu/oxaliplatin was beneficial to the differentiation of T-helper 1 (Th1) cells. 5-Fu/oxaliplatin further enhanced the stimulating phenotypic expression of DCs in tumor bearing mice, decreased PD-L1/L2 expression, and specifically activated the lymphocytes. The CD1d-MC38/α-GC tumor vaccine combined with 5-Fu/oxaliplatin could exert a synergistic role that resulted in a significant delay of the tumor growth rate, and an increase in the survival time of tumor bearing mice. CONCLUSIONS: 5-Fu/oxaliplatin decreased the expression of the DC inhibitory phenotypes PD-L1/L2, promoted DC phenotypic maturation in tumor bearing mice, activated the lymphocytes of tumor bearing mice, and exerted synergistic effects with the CD1d-MC38/α-GC colon cancer tumor vaccine.

Early activated hepatic stellate cell-derived paracrine molecules modulate acute liver injury and regeneration.

The effects of paracrine action from early activated hepatic stellate cells (HSCs) on resident liver epithelium cells are not clear. Here, we investigated whether a systemic infusion of early activated HSC-derived paracrine factors (HSC-CM) would evoke an enhanced liver protective response in acetaminophen (APAP)-induced acute liver injury (ALI) in mice and explored the possible underlying mechanisms. The survival rate, liver injury, and liver regeneration were analyzed in mice with or without HSC-CM treatment in vivo. A systemic infusion of HSC-CM provided a significant survival benefit in APAP-induced ALI. HSC-CM therapy resulted in a reduction of hepatocellular death and increased numbers of both proliferating hepatocytes and adult hepatic progenitor cells (AHPCs) with up-regulation of liver regeneration relevant genes. The HSC-CM treatment reduced leukocyte infiltration and down-regulated systemic inflammation with decreases in IFN-γ, IL-1ra, IL-1ß, TNF-α, and increases in IL-10. The direct anti-death and pro-regeneration effects of HSC-CM on AHPCs were demonstrated using in vitro assays. Treatment with HSC-CM promoted AHPCs proliferation and resulted in increased pAkt expression in vitro, and this effect was abolished by the PI3K/Akt inhibitor LY294002. These data provide evidence that early activated HSC-CM therapy offered trophic support to the acutely injured liver by inhibiting liver cell death and stimulating regeneration, potentially creating a new method for the treatment of ALI.

Knockdown of calponin 2 suppressed cell growth in gastric cancer cells.

Calponin family members are actin filament-associated regulatory proteins with distinct expression patterns. Previous studies on CNN2 (calponin 2) have demonstrated that CNN2 is expressed in a broad range of tissues and cell types, exhibiting potential regulatory roles in a number of cellular activities, including cell proliferation, cell migration, and platelet adhesion. In this work, we found that both messenger RNA and protein expression levels of CNN2 were remarkably upregulated in 60%-70% of gastric cancer tissues by comparison with those of neighboring non-tumorous mucosa. By utilizing specific shCNN2 (small hairpin RNA targeting CNN2), the potential role of CNN2 in regulating AGS gastric cancer cell growth was then further investigated. AGS cells infected with shCNN2 exhibited significantly decreased cell growth ability by comparison with control cells in vitro. Moreover, while there was no obvious difference in cell cycle distribution between two groups, enhanced cell apoptosis was detected in cells with reduced CNN2 expression. Consistently, caspase 3/7 activity was also remarkably activated upon shCNN2 lentivirus infection. Taken together, our results demonstrated that knockdown of endogenous CNN2 in AGS cells could significantly activate cell apoptosis pathway and therefore suppress cell growth in vitro. The deletion of CNN2 might be a potential therapeutic approach to inhibit aggressive growth of gastric cancer.

Co-operation of α-galactosylceramide-loaded tumour cells and TLR9 agonists induce potent anti-tumour responses in a murine colon cancer model.

Dendritic cells (DCs) and invariant natural killer T (iNKT) cells play important roles in linking innate immunity and adaptive immunity. Mature DCs activated by Toll-like receptor (TLR) agonists directly activate iNKT cells and the iNKT ligand α-galactosylceramide (α-Galcer) can induce DC maturation, resulting in enhanced protective immune responses. In the present study, we aimed to boost anti-tumour immunity in a murine colon cancer model by synergizing DCs and iNKT cells using α-Galcer-loaded tumour cells (tumour-Gal) and the TLR9 agonist cytosine-phosphorothioate-guanine (CpG1826). The vaccine strategy was sufficient to inhibit growth of established tumours and prolonged survival of tumour-bearing mice. Importantly, the immunization induced an adaptive memory immune response as the survivors from primary tumour inoculations were resistant to a tumour re-challenge. Furthermore, injection of tumour-Gal with CpG1826 resulted in iNKT cell activation and DC maturation as defined by interferon (IFN)-γ secretion by iNKT, natural killer (NK) cells and interleukin (IL)-12 by DCs. Immunohistochemistry analysis revealed that cluster of differentiation (CD)4(+) T-cells and CD8(+) T-cells played important roles in anti-tumour immunity. Additionally, the vaccine redirected Th2 (T-helper cell type 2) responses toward Th1 (T-helper cell type 1) responses with increases in IL-2, IFN-γ expression and decreases in IL-4 and IL-5 expression after immunization with tumour-Gal with CpG1826. Taken together, our results demonstrated a novel vaccination by synergizing tumour-Gal and CpG1826 against murine colon cancer, which can be further developed as tumour-specific immunotherapy against human cancer.

Randomized Controlled Trial of Intraportal Chemotherapy Combined With Adjuvant Chemotherapy (mFOLFOX6) for Stage II and III Colon Cancer.

OBJECTIVES: The optimal time to initiate adjuvant chemotherapy after surgery in patients with colon cancer is not clear. We investigated the benefit of combined intraportal chemotherapy administered during radical surgery with adjuvant chemotherapy for treating stage II and III colon cancer. METHODS: Patients were randomly assigned to OCTREE arm (intraportal chemotherapy plus mFOLFOX6) or a standard adjuvant chemotherapy arm (mFOLFOX6). The primary study endpoint was disease-free survival. The secondary endpoints included metastasis-free survival, overall survival, and safety. RESULTS: The intent-to-treat population comprised 237 patients. With a median follow-up of 44 months, the hazard ratio (OCTREE vs mFOLFOX6) was 0.66 (95% confidence interval, 0.43-0.90), a 34% risk reduction in favor of OCTREE (P = 0.016). The 3-year disease-free survival rate was 85.2% for OCTREE and 75.6% for mFOLFOX6 alone (P = 0.030). The 3-year metastasis-free survival rates were 87.6% for OCTREE and 78.0% for mFOLFOX6 (P = 0.035). Patients had lower distant metastatic rate in the OCTREE arm (12.7% vs 22.7%; P = 0.044), when compared with the mFOLFOX6 arm. The 3-year overall survival was no significant difference between 2 arms (P = 0.178). Neutropenia occurred in 12.7% of the patients receiving OCTREE and in 2.5% of the patients receiving mFOLFOX6 (P = 0.003) within 2 weeks of surgery, and grade 3 or 4 toxicity event was no difference between 2 regimens. CONCLUSIONS: Combination of intraoperative intraportal chemotherapy with mFOLFOX6 reduced the occurrence of distant metastases and improved disease-free survival in patients with stage II and stage III colon cancer.

Isolation and culture of hepatic stellate cells from mouse liver.

Hepatic stellate cells (HSCs) are the primary extracellular matrix-producing cells within the liver and have numerous vital functions. A robust protocol for the isolation and culture of HSCs is important for further investigations of cell functions and related mechanisms in liver disease. The volume of the mouse liver is much smaller than that of the rat liver, which makes it much more difficult to isolate mouse HSCs (mHSCs) than rat HSCs. At present, isolating mHSCs is still a challenge because there is no efficient, robust method to isolate and culture these cells. In the present study, C57BL/6J mice were intravenously injected with liposome-encapsulated dichloromethylene diphosphate (CL2MDP) to selectively eliminate Kupffer cells from the liver. The mouse livers were then perfused in situ, and the mHSCs were isolated with an optimized density gradient centrifugation technique. In the phosphate buffer solution (PBS)-liposome group, the yield of mHSCs was (1.37 ± 0.23) × 10(6)/g liver, the cell purity was (90.18 ± 1.61)%, and the cell survival rate was (94.51 ± 1.61)%. While in the CL2MDP-liposome group, the yield of mHSCs was (1.62 ± 0.34) × 10(6)/g liver, the cell purity was (94.44 ± 1.89)%, and the cell survival rate was (94.41 ± 1.50)%. Based on the yield and purity of mHSCs, the CL2MDP-liposome treatment was superior to the PBS-liposome treatment (P < 0.05, P < 0.01). This study established successfully a robust and efficient protocol for the separation and purification of mHSCs, and both a high purity and an adequate yield of mHSCs were obtained.

Neddylation pathway regulates the proliferation and survival of macrophages.

Neddylation is a new type of protein post-translational modification which adds the ubiquitin-like molecule Nedd8 to target proteins. The well-identified targets of neddylation are cullins, which serve as essential components of Cullin-RING E3 ligases (CRL). It is reported that inhibition of neddylation repressed NF-κB-mediated proinflammatory cytokine production in macrophages. However, the role of neddylation in the proliferation and survival of macrophages has not been well defined. Here we report that partial inactivation of the neddylation pathway by a specific Nedd8-activating enzyme E1 (NAE) inhibitor MLN4924 reduced LPS-induced production of the proinflammatory cytokines TNF-α and IL-6 without obvious impairment of cell viability. However, persistent and severe inactivation of neddylation by MLN4924 significantly inhibited cell proliferation by inducing G2 phase cell-cycle arrest and further triggered cell death by inducing apoptosis in RAW264.7 macrophages. Mechanistic analysis revealed that inactivation of neddylation blocked cullin neddylation, inhibited CRL E3 ligase activity, and thus led to the accumulation of CRL substrates, resulting in cell-cycle arrest, DNA damage response and apoptosis. The findings revealed that neddylation serves as an important signaling pathway regulating the proliferation and survival of macrophages.

Synergistical toll-like receptors activated dendritic cells induce antitumor effects against carcinoembryonic antigen-expressing colon cancer.

PURPOSE: Dendritic cell (DC)-based cancer vaccine represents a promising immunotherapy against cancer. There has been recent evidence which have suggested that toll-like receptor (TLR) ligands may be critical for DC preparation; this was usually omitted in the past. Our study is designed to investigate if the vaccination of synergistical toll-like receptors activated DCs can induce more potent cytotoxic T Lymphocytes (CTL) responses and antitumor activity in carcinoembryonic antigen (CEA) transgenic mouse tumor models. METHODS: We involved combination of TLR3 and TLR7/8 ligands in culture protocol of DCs. The DCs' surface molecules expression, IL-12 secretion and proliferation capacity of lymphocytes were tested. We also investigate the CTL activity against MC38-CEA colon tumor cells and the prophylactic and therapeutic effects of DC vaccination in subcutaneous mouse colon tumor models. RESULTS: Compared with conventionally generated DCs, we showed synergistic TLR-activated DCs exhibited higher surface molecule expression, significantly higher secretion of IL-12 and more potent proliferating capacity of lymphocytes. Synergistic TLR-activated DCs were also able to induce lymphocytes possessing the specific cytotoxicity against MC38-CEA cells in vitro. Vaccination with CEA epitope pulsed TLR-activated DCs elicited antigen-specific preventive effect on MC38-CEA tumors, but failed to cure the tumor-bearing mice, that may be due to the suboptimal epitope selected and host immunosuppression. CONCLUSIONS: Our results have proved that combined activation of TLRs can lead to better maturation status of DCs and also induce more effective antitumor immune responses against colon cancer, suggesting this may be a potential strategy to develop more powerful DC cancer vaccines.

A Brain Network Analysis-Based Double Way Deep Neural Network for Emotion Recognition.

Constructing reliable and effective models to recognize human emotional states has become an important issue in recent years. In this article, we propose a double way deep residual neural network combined with brain network analysis, which enables the classification of multiple emotional states. To begin with, we transform the emotional EEG signals into five frequency bands by wavelet transform and construct brain networks by inter-channel correlation coefficients. These brain networks are then fed into a subsequent deep neural network block which contains several modules with residual connection and enhanced by channel attention mechanism and spatial attention mechanism. In the second way of the model, we feed the emotional EEG signals directly into another deep neural network block to extract temporal features. At the end of the two ways, the features are concatenated for classification. To verify the effectiveness of our proposed model, we carried out a series of experiments to collect emotional EEG from eight subjects. The average accuracy of the proposed model on our emotional dataset is 94.57%. In addition, the evaluation results on public databases SEED and SEED-IV are 94.55% and 78.91%, respectively, demonstrating the superiority of our model in emotion recognition tasks.

Enhanced Recovery After Surgery (ERAS) program attenuates stress and accelerates recovery in patients after radical resection for colorectal cancer: a prospective randomized controlled trial.

BACKGROUND: The aim of this trial was to compare the Enhanced Recovery After Surgery (ERAS) program with conventional perioperative management in patients who underwent radical resection for colorectal cancer. METHODS: A combination of evidence-based and consensus methodology was used to develop the ERAS protocol. Five hundred ninety-seven consecutive patients who underwent elective colorectal resection were randomized to either the ERAS (n = 299) or the control group (n = 298). Outcomes relating to nutrition and metabolism index, stress index, and recovery index were measured and recorded. RESULTS: Demographic and operative data were similar between the two groups. Patients in the ERAS group showed improved nutritional status when compared with those of the control group. On postoperative day (POD) 1, the HOMA-IR (insulin resistance index) of the ERAS group was lower than that of the control group (p < 0.001). The cortisol level of the control group was elevated on both POD 1 (p = 0.007) and POD 5 (p = 0.002) compared to the preoperative level. However, the cortisol level of the ERAS group was not increased until POD 5 (p = 0.001). Reduced levels of TNF-α, IL-1ß, IL-6, and IFN-γ in the ERAS group indicated less postoperative stress responses. In addition, ERAS was associated with accelerated recovery of gastrointestinal function. The postoperative length of stay (p < 0.001) and expense (p < 0.001) for the ERAS group were reduced in comparison to the controls. Twenty-eight cases in the control group and twenty-nine in the ERAS group suffered complications, which was not significantly different. CONCLUSION: The ERAS protocol attenuates the surgical stress response and accelerates postoperative recovery without compromising patient safety.