Platelet-derived Growth Factor Receptor-α Induces Contraction Knots and Inflammatory Pain-like Behavior in a Rat Model of Myofascial Trigger Points.

BACKGROUND: Myofascial trigger points (MTrPs) are the primary etiological characteristics of chronic myofascial pain syndrome. Receptor tyrosine kinases (RTKs) are associated with signal transduction in the central mechanisms of chronic pain, but the role of RTKs in the peripheral mechanisms of MTrPs remains unclear. The current study aimed to identify RTKs expression in MTrPs and elucidate the molecular mechanisms through which platelet-derived growth factor receptor-α (PDGFR-α) induces contraction knots and inflammatory pain-like behavior in a rat model of myofascial trigger points. METHODS: MTrPs tissue samples were obtained from the trapezius muscles of patients with myofascial pain syndrome through needle biopsy, and PDGFR-α activation was analyzed by microarray, enzyme-linked immunosorbent assay, and histological staining. Sprague-Dawley rats (male and female) were used to investigate PDGFR-α signaling, assessing pain-like behaviors with Randall-Selitto and nest-building tests. Muscle fiber and sarcomere morphologies were observed using histology and electron microscopy. The PDGFR-α binding protein was identified by coimmunoprecipitation, liquid chromatograph mass spectrometer, and molecular docking. PDGFR-α-related protein or gene levels, muscle contraction, and inflammatory markers were determined by Western blot and reverse-transcription quantitative polymerase chain reaction. RESULTS: PDGFR-α phosphorylation levels were elevated in the MTrPs tissues of individuals with trapezius muscle pain and were positively correlated with pain intensity. In rats, PDGFR-α activation caused pain-like behaviors and muscle contraction via the Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathway. JAK2/STAT3 inhibitors reversed the pain-like behaviors and muscle contraction induced by PDGFR-α activation. Collagen type I α 1 (COL1A1) binds to PDGFR-α and promotes its phosphorylation, which contributed to pain-like behaviors and muscle contraction. CONCLUSIONS: COL1A1-induced phosphorylation of PDGFR-α and the subsequent activation of the JAK2/STAT3 pathway may induce dysfunctional muscle contraction and increased nociception at MTrPs.

APOC1 as a novel diagnostic biomarker for DN based on machine learning algorithms and experiment.

Introduction: Diabetic nephropathy is the leading cause of end-stage renal disease, which imposes a huge economic burden on individuals and society, but effective and reliable diagnostic markers are still not available. Methods: Differentially expressed genes (DEGs) were characterized and functional enrichment analysis was performed in DN patients. Meanwhile, a weighted gene co-expression network (WGCNA) was also constructed. For further, algorithms Lasso and SVM-RFE were applied to screening the DN core secreted genes. Lastly, WB, IHC, IF, and Elias experiments were applied to demonstrate the hub gene expression in DN, and the research results were confirmed in mouse models and clinical specimens. Results: 17 hub secretion genes were identified in this research by analyzing the DEGs, the important module genes in WGCNA, and the secretion genes. 6 hub secretory genes (APOC1, CCL21, INHBA, RNASE6, TGFBI, VEGFC) were obtained by Lasso and SVM-RFE algorithms. APOC1 was discovered to exhibit elevated expression in renal tissue of a DN mouse model, and APOC1 is probably a core secretory gene in DN. Clinical data demonstrate that APOC1 expression is associated significantly with proteinuria and GFR in DN patients. APOC1 expression in the serum of DN patients was 1.358±0.1292µg/ml, compared to 0.3683±0.08119µg/ml in the healthy population. APOC1 was significantly elevated in the sera of DN patients and the difference was statistical significant (P > 0.001). The ROC curve of APOC1 in DN gave an AUC = 92.5%, sensitivity = 95%, and specificity = 97% (P < 0.001). Conclusions: Our research indicates that APOC1 might be a novel diagnostic biomarker for diabetic nephropathy for the first time and suggest that APOC1 may be available as a candidate intervention target for DN.

Peri-tumoral infiltrate associates with postoperative prognosis of patients with OSCC: Stronger association in HPV negative patients.

PURPOSE: The current data on the relationship between local inflammatory infiltration and prognosis in oral squamous cell carcinoma (OSCC) are limited and controversial, especially in different HPV status. In this study, we analyzed the relationship between peri-tumoral inflammatory infiltrate (PTI) and HPV status and prognosis of patients with OSCC after surgery. METHODS: A retrospective cohort of 99 primary OSCC patients who underwent surgery was constructed. P16 immunohistochemistry was used to determine HPV status. PTI was determined by hematoxylin-eosin staining and quantified into four levels: none (Score 0), weak (Score 1), moderate (Score 2) and strong (Score 3). The associations of PTI with clinico-pathological characteristics, HPV status and survival were examined. RESULTS: Most OSCC patients had weak to moderate PTI. PTI was significantly associated with lymph node metastasis (P = 0.041), and patients with moderate PTI had significantly better OS (P = 0.009) than those with no PTI. In HPV negative OSCC, patients with moderate PTI also had significantly better OS (P = 0.019) than those with no PTI. However, PTI was not significantly associated with survival in HPV positive OSCC. CONCLUSIONS: In HPV negative OSCC, moderate PTI may suggest a better postoperative prognosis than no PTI.

Identification and validation of a prognostic signature of autophagy, apoptosis and pyroptosis-related genes for head and neck squamous cell carcinoma: to imply therapeutic choices of HPV negative patients.

Introduction: An effective tool is needed to predict the prognosis of head and neck squamous cell carcinoma (HNSCC). Human papillomavirus (HPV) positive HNSCC patients generally have a favorable survival and a promising responsiveness to radiotherapy, chemoradiotherapy and checkpoint blockades. However, HPV negative patients, the majority of HNSCC patients, have been largely overlooked. Cell death has been involved in the therapeutic resistance of cancers. To this end, we aimed to identify the association of autophagy, apoptosis and pyroptosis-related genes with the prognosis of HNSCC, and construct a prognostic signature to predict the prognosis for HNSCC, especially for HPV negative HNSCC. Methods: Autophagy and apoptosis-related genes were obtained from Gene Set Enrichment Analysis (GSEA) website, and pyroptosis-related genes were obtained from GSEA and Gene Ontology (GO) database. We established the cell death index (CDI) based on RNA sequencing (RNA-seq) data and clinicopathological information from The Cancer Genome Atlas (TCGA) dataset. The prognostic value of CDI was verified by Kaplan-Meier, receiver operating characteristic (ROC) and univariate and multivariate Cox regression analyses in TCGA dataset, and validated with the datasets from Gene Expression Omnibus (GEO) and Qilu Hospital of Shandong University. We further assessed the immune microenvironment of patients with high and low CDI scores. Moreover, the expression of the signature genes in HNSCC cell lines were explored. Results: We found that CDI was an independent prognostic indicator for overall survival (hazard ratio 3.80, 95% confidential interval: 2.70-5.40, P < 0.001). Furthermore, HNSCC patients with high CDI scores obtained increased overall survival post radiation indicating benefits from radiotherapy of this subgroup. On the other hand, HPV negative HNSCC patients with low CDI exhibited increased checkpoint gene expressions, an inflamed tumor microenvironment and an enriched immune response-related functions, suggesting the potential benefits from checkpoint immunotherapies of this subgroup. Moreover, we validated the baseline and induced expressions of above 16 genes in two HPV negative HNSCC cell lines, CAL27 and SCC-15. Discussion: We established a prognostic signature and emphasized its implements in the therapeutic choices of HPV negative HNSCC patients, the majority and the poor outcome population of HNSCC.

Dysbiotic tumor microbiota associates with head and neck squamous cell carcinoma outcomes.

BACKGROUND: The need for an effective tool to predict prognosis of head and neck squamous cell carcinoma (HNSCC) patients is critical and unmet. Microbiota has recently been found involved in tumor progression and response to immunotherapy. However, the association of microbiota with the prognosis of HNSCC patients remains obscure. This study aims to investigate the association between tumor microbiota and outcomes of HNSCC patients. METHODS: A retrospective study including 129 primary tumors of HNSCC was conducted. Using 16S rRNA sequencing, the profile and the composition of tumor microbiota were measured and their associations with overall survival (OS) and disease free survival (DFS) were examined. RESULTS: We observed a reduced richness and enriched abundances of genera Schlegelella and Methyloversatilis in tumor microbiota of HNSCC patients with poor prognosis. However, a richer tumor microbiota with greater abundances of genera Bacillus, and Lactobacillus and Sphingomonas was characterized in the patients with favorable prognosis.The ratio of these differentially abundant taxa, microbial dysbiosis index (MDI), was significantly associated with OS (hazard ratio [HR], 4.67, 95% confidence interval [CI], 2.51 to 8.69,P < 0.001) and DFS (HR, 2.89; 95% CI, 1.74 to 4.80, P < 0.001) independently of age, tumor size, lymph node metastasis, differentiation and p16 status. The risk score of multivariate Cox regression exhibited an excellent performance for estimating three-year OS (AUC of 0.826). We also found a richer tumor microbiota was correlated with moderate peritumoral inflammatory infiltration. CONCLUSION: These results indicate that tumor microbiota associates with outcomes of HNSCC patients.

Whole transcriptome analysis of trophoblasts under hypoxia.

INTRODUCTION: A physiological hypoxia environment exists at maternal-fetal interface during early pregnancy. In addition, there is a pathological hypoxic microenvironment in patients with preeclampsia. Therefore, investigating the hypoxic adaptation and the effects of hypoxia on trophoblasts transcriptome is helpful to better understand the function and regulatory mechanism of trophoblasts at the maternal-fetal interface. METHODS: Trophoblast cell line HTR-8/SVneo was cultured under normoxia and hypoxia for 24 h, the full transcriptome was analyzed via RNA-Seq. GO and KEGG enrichment were performed on differentially expressed mRNA, adjacent genes of differentially expressed lncRNA, host genes of differentially expressed circRNA and target genes of differential expressed miRNA. RESULTS: The results showed that hypoxia differentially regulated 373 mRNAs, 334 lncRNAs, 71 circRNAs and 33 miRNAs. GO and KEGG enrichment showed that hypoxia negatively regulated TLR3 and PI3K-Akt signaling pathways. Consistently, we found hypoxia significantly inhibited TLR3 agonist-induced cytokines expression and the phosphorylation of Akt and mTOR. DISCUSSION: Our study obtained the full transcriptome data and potential regulatory network of trophoblasts under hypoxia, providing supportive data for revealing the function of trophoblasts.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Vitamin E succinate exerts anti-tumour effects on human cervical cancer cells via the CD47-SIRPɑ pathway both in vivo and in vitro.

Vitamin E succinate (RRR-a-tocopheryl succinate, VES) acts as a potent agent for cancer therapy and has no toxic and side effects on normal tissue cells. However, the mechanism by which VES mediates the effects are not yet fully understood. Here, we hypothesised that VES mediates antitumour activity on human cervical cancer cells via the CD47-SIRPɑ pathway in vivo and in vitro. Results indicated that the human cervical cancer HeLa cells treated with VES were more efficiently engulfed by THP-1-derived macrophages. In response to VES, the protein expression of CD47 on cell membranes and the mRNA level of CD47 in different human cervical cancer cells significantly decreased. And the level of calreticulin (CRT) mRNA in the VES-treated cells increased. By contrast, CRT protein expression was not altered. miRNA-155, miRNA-133 and miRNA-326 were up-regulated in the VES-treated HeLa cells. Knocking down miRNA-155 and miRNA-133 by RNA interference increased CD47 protein expression in the VES-treated cells. In vivo efficacy was determined in BALB/C nude mice with HeLa xenografts. Results showed that VES reduced tumour growth, increased overall survival and inhibited CD47 in the tumour transcriptionally and translationally. Furthermore, inflammatory factors (TNF-α, IL-12, IFN-γ, IL-2 and IL-10) in the spleen were altered because of VES treatment. Our results suggest that VES-induced antitumour activity is coupled to the CD47-SIRPɑ pathway in human cervical HeLa cancer cells.

Systemic Inflammatory Response Markers Associated with Infertility and Endometrioma or Uterine Leiomyoma in Endometriosis.

PURPOSE: The aim of this study was to find the most useful marker of endometriosis-related infertility and evaluate predictive and diagnostic values of systemic inflammatory response markers (preoperative white blood-cell subtypes, neutrophil:lymphocyte ratio [NLR], platelet:lymphocyte ratio [PLR], and monocyte:lymphocyte ratio [MLR]) and CA125 levels in endometriosis patients. METHODS: This study comprised 662 women who had undergone laparoscopic surgery and been pathologically confirmed as having endometriosis and 83 patients pathologically confirmed with benign ovarian tumors. Related inflammatory factors in endometriosis complicated by infertility were analyzed via logistic regression analysis. Diagnostic values of the inflammatory response markers were obtained by receiver operating-characteristic analysis. RESULTS: We firstly identified that lower NLR level was an independent risk factor of infertility. Serum lymphocytes were significantly higher in endometriosis patients, while serum CA125, NLR, MLR, and PLR were elevated. For differentiating endometriosis from other benign ovarian tumors, the combination of NLR and CA125 achieved greater sensitivity than CA125 alone. In addition, both CA125 and NLR were positively correlated with stage, oviduct adhesion, and diameter of ovarian ectopic cysts. CONCLUSION: NLR may be used as a simple and easily obtained predictive marker for endometriosis with infertility. Moreover, NLR can be a neoadjuvant biomarker for serum CA125 to diagnose endometriosis.

Neutrophils infiltration and its correlation with human papillomavirus status in the oral squamous cell carcinoma.

Purpose: One of the cardinal etiological factors for oral squamous cell carcinoma (OSCC) is Human papillomavirus (HPV). Neutrophils were potential targets of immune therapy for patients with OSCC. The objective of this study was to determine if neutrophils density and HPV status can be used to define a high-risk category of patients in OSCC and to investigate the possible relationship between them. Patients and methods: We performed immunohistochemistry to probe neutrophils infiltration and HPV (P16) expression in 81 patients with OSCC. Prognostic factors for cancer-related survival were evaluated by univariate and multivariate analyses. We used the detection of cytokines to investigate the possible molecular mechanisms between neutrophils infiltration and HPV status. Results: There were significantly higher numbers of CD15+ neutrophils infiltration in OSCC tissues. Higher numbers of CD15+ neutrophils infiltration was related to stage Ⅲ，Ⅳ (p<0.001), poor grade (p<0.001), lymph node metastasis (p=0.014), and the higher preoperative neutrophil-lymphocyte ratio (NLR) (p<0.001). HPV-negative status was also associated with stage Ⅲ，Ⅳ (p=0.001), poor grade (p=0.002), lymph node metastasis (p=0.005), radiotherapy (p=0.038), and the higher NLR (p=0.002). The high density of neutrophils was associated with worse cancer-related survival time (p<0.001) and was an independent prognostic factor for OSCC, while the HPV-positive group was associated with better cancer-related survival time. Moreover, high density of neutrophils was correlated with HPV-negative status in OSCC (p<0.001). Detection of cytokines and chemokines revealed that one of the chemotactic factors of neutrophils, IL-8, was exhibited relatively low expression by HPV-positive OSCC cells, whereas HPV-negative OSCC cells were found to drive an IL-8 secretion profile. Conclusion: Neutrophils infiltration and HPV status appear to be prognostic parameters for OSCC. Overexpression of HPV18 E7 on OSCC cells may participate in depressing neutrophils infiltration to some extent through downregulating expression of IL-8.

Macrophage ERα promoted invasion of endometrial cancer cell by mTOR/KIF5B-mediated epithelial to mesenchymal transition.

Tumor-associated macrophages (TAMs) exert tumor-promoting effects. There have been reports that estrogen receptors (ERs) are expressed on the infiltrating macrophages of endometriosis, ovarian cancer and lung cancer. However, the role of ERs in macrophages is not well characterized. In this study, we identified that ER alpha (ERα) expression on the macrophages of human endometrial cancer was positively correlated with cancer progression. Conditioned medium from selective ERα agonist-treated M2 macrophages induced the epithelial to mesenchymal transition (EMT) in endometrial cancer cells. However, this effect can be inhibited by ERα antagonist. Here, we showed that macrophages ERα-engaged abundantly produced chemokine (C-C motif) ligand 18 (CCL18), and its expression promoted the invasion of endometrial cancer cells by activating the extracellular signal-regulated kinase 1/2 pathway, whereas suppressing CCL18 abrogated these effects. Furthermore, we identified that CCL18 derived from TAMs upregulated KIF5B expression to promote EMT via activating the PI3K/AKT/mTOR signaling pathway in endometrial cancer. Overall, our findings show how ERα-engaged infiltrating macrophages initiate chronic inflammation and promote the aggressive progression of endometrial cancer cells. ERα-positive TAMs act as drivers of endometrial cancer, which may become a potential therapeutic target.

Preoperative neutrophil lymphocyte ratio but not platelet lymphocyte ratio predicts survival and early relapse in patients with oral, pharyngeal, and lip cancer.

BACKGROUND: To evaluate the prognostic value of preoperative neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) in oral, pharyngeal, and lip cancer for survival and relapse. METHODS: Clinic-pathologic and hematological records were retrospectively retrieved. Patients completed follow-up period were included for survival and relapse analysis. RESULTS: The preoperative NLR value was a prognostic factor for both overall survival and relapse-free survival. The high NLR group demonstrated higher total relapse rate, higher local relapse rate, and higher relapse rate within 12 months. However, the preoperative PLR did not associate with survival or relapse. CONCLUSIONS: The preoperative NLR, not PLR, is an independent prognostic indicator of survival. It also exhibits predictive value for relapse, particularly early relapse within 12 months. The preoperative NLR value might be recommended as a useful tool for predicting the outcomes and stratifying patients for different management strategies.

Preoperative Tim­3 expression on peripheral NK cells is correlated with pathologic TNM staging in colorectal cancer.

Previous research has indicated that T cell immunoglobulin and mucin domain 3 (Tim-3) serves an important regulatory role in lymphocytes and in several cancers. However, the association between Tim­3 expression on various lymphocyte subsets and human colorectal cancer (CRC) has not been elucidated. The present study aimed to characterize Tim­3 expression on peripheral lymphocytes, including cluster of differentiation CD3+CD56­ T cells, CD3­CD56+ natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells, in patients with CRC. The frequency of T cells, NK cells and NKT cells expressing Tim­3 was assessed by multicolor flow cytometry of peripheral blood collected from 36 preoperative CRC patients and 38 healthy donors. The expression of Tim­3 on lymphocyte subsets from 53 postoperative blood samples of CRC patients was also analyzed. There were fewer circulating NK cells in patients with CRC compared with healthy controls (P=0.0027); NK cell expression of Tim­3 was also significantly decreased (P=0.0239). The frequency of circulating NK cells and Tim­3+ NK cells was negatively correlated with clinical cancer stage, compared with healthy controls, but not with other clinicopathological parameters or serum concentrations of CRC biomarkers. Furthermore, the expression of Tim­3 in NK cells was higher in CRC patients without metastasis. Notably, NK cell Tim­3 expression in CRC patients was significantly restored following surgical resection of the primary tumor. In conclusion, the present study indicates the presence of an altered frequency and expression of Tim­3 in peripheral NK cells in CRC patients. Preoperative Tim­3 expression on peripheral NK cells is correlated with differential staging in colorectal cancer, and may be useful as a serum biomarker.

Kif4 regulates the expression of VEGFR1 through the PI3K/Akt signaling pathway in RAW264.7 monocytes/macrophages.

Kinesin superfamily protein 4 (Kif4), a microtubule-based motor protein, has been shown to participate in a number of critical cellular processes, such as cell division, the intracellular transport of membranous vesicles and signal transduction. However, whether KIF4 regulates vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) expression remains unknown. Thus, in this study, in order to examine the effects of Kif4 on the expression of VEGFR1 in RAW264.7 monocytes/macrophages, Kif4 was silenced using siRNA. RT-qPCR, western blot analysis and ELISA were used to assess the expression of Kif4 and VEGFR1 up- and downstream signaling molecules, including VEGF-A, VEGFR1, soluble form of VEGFR1 (sVEGFR1), phosphorylated (p-)Akt and Akt. The silencing Kif4 inhibited the mRNA expression of VEGF (P<0.01) and p-Akt (P<0.05); however, the level of VEGF-A was increased (P<0.05) compared with the negative control siRNA-transfected group. The silencing of Kif4 decreased the VEGFR1 mRNA (P<0.05), VEGFR1 protein and sVEGFR1 levels in the cell supernatant (P<0.01). Following the application of insulin-like growth factor-1 (100 ng/ml), the specific agonist of PI3K/Akt in the Kif4 siRNA-transfected group, the VEGFR1 mRNA levels (P<0.001), the VEGFR1 protein levels and the sVEGFR1 (P<0.01) levels significantly increased; however, the levels of VEGF in the cell supernatant were decreased (P<0.05). Taken together, these findings suggest that Kif4 regulates the expression of VEGFR1 in RAW264.7 cells and that the PI3K/Akt pathway is involved in this process.

M2-like tumor-associated macrophages drive vasculogenic mimicry through amplification of IL-6 expression in glioma cells.

Vasculogenic mimicry (VM) has offered a new horizon for understanding tumor angiogenesis, but the mechanisms of VM in glioma progression have not been studied explicitly until now. As a significant component of immune infiltration in tumor microenvironment, macrophages have been demonstrated to play an important role in tumor growth and angiogenesis. However, whether macrophages could play a potential key role in glioma VM is still poorly understood. Herein we reported that both VM and CD163+ cells were associated with WHO grade and reduced patient survival, and VM channel counting was correlated to the number of infiltrated CD163+ cells in glioma specimens. In vitro studies of glioma cell lines implicated that M2-like macrophages (M2) promoted glioma VM. We found that conditional medium derived from M2 amplified IL-6 expression in glioma cells. Furthermore, our data indicated that IL-6 could promote glioma VM, as blocking IL-6 with neutralizing antibodies abrogated M2-mediated VM enhancement. In addition, the potent PKC inhibitor bisindolylmaleimide I could prevent M2-induced IL-6 upregulation and further inhibited glioma VM facilitation. Taken together, our results suggested that M2-like macrophages drove glioma VM through amplifying IL-6 secretion in glioma cells via PKC pathway.

Salvianolic acid B reduced the formation of epidural fibrosis in an experimental rat model.

BACKGROUND: Salvianolic acid B (Sal B) was newly reported to be able to attenuate fibrosis in the animal model. The aim of the present study was to investigate the effect of the intragastric application of Sal B on the prevention of epidural fibrosis (EF). METHODS: Forty healthy adult male Wistar rats were divided into four treatment groups (n = 10 per group): (1) 10 mg/kg Sal B, (2) 30 mg/kg Sal B, (3) 50 mg/kg Sal B and (4) Saline (vehicle treatment, control group). All animals underwent a laminectomy at the lumbar 1-2 (L 1-2) level. After intragastric treatment, all rats were sacrificed at post-operative week 8. The extent of the epidural scar, the regeneration of the vasculature and the expression levels of vascular endothelial growth factor (VEGF) were analysed. RESULTS: The animals' recovery was uneventful during the experimental period. The extent of the epidural scar, the regeneration of the vasculature and the expression levels of VEGF suggested better outcomes in the Sal B-treated groups. Sal B exerted the ability to prevent the formation of an epidural scar and vascularization at the laminectomy sites. The effects of Sal B were dose-dependent, with the 50 mg/kg Sal B group showing the best outcomes compared with the other groups. CONCLUSIONS: Post-operative intragastric application of Sal B can prevent the formation of epidural scarring. Sal B exerted these effects in a dose-dependent manner, and 50 mg/kg dose was shown to be the best effect in the present study. The results of this study reveal that Sal B could be a potential therapy for EF and valuable for further research.

Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer.

In tumor microenvironment, macrophages as a polarized M2 population promote tumor progression via releasing multiple cytokines and chemokines. A brown seaweed fucose-rich polysaccharide, fucoidan has antitumor activity and immune modulation through affecting tumor cells and lymphocytes. Here, we focused on the effect of fucoidan on macrophages especially M2 subtype. Our results demonstrated that fucoidan down-regulated partial cytokines and chemokines, especially a M2-type chemokine CCL22. Furthermore, fucoidan inhibited tumor cells migration and CD4+ T lymphocytes, especially Treg cells, recruitment induced by M2 macrophages conditioned medium through suppression of CCL22. Mechanismly, fucoidan inhibited CCL22 via suppressing p65-NF-κB phosphorylation and nuclear translocation. In addition, p38-MAPK and PI3K-AKT also affected the expression of CCL22 through differential modulation of NF-κB transcriptional activity. Taken together, we reveal an interesting result that fucoidan can inhibit tumor cell migration and lymphocytes recruitment by suppressing CCL22 in M2 macrophages via NF-κB-dependent transcription, which may be a novel and promising mechanism for tumor immunotherapy.

Interactions between colon cancer cells and tumor-infiltrated macrophages depending on cancer cell-derived colony stimulating factor 1.

Tumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Colony stimulating factor 1 (CSF1) is a primary chemoattractant and functional regulator for macrophages, and therefore would be a feasible intervention for the macrophage-targeting therapeutics. However, the expression of CSF1 in colon cancer microenvironment and its roles in cancer development is largely unknown. In the present study, we found that CSF1 was over-expressed exclusively in colon cancer cells and was correlated with macrophages infiltration. The high CSF1 expression and macrophages infiltration were related to the tumor-node-metastasis (TNM) stage of colon cancer, and suggested to be positively associated with survival of colon cancer patients. In the in vitro studies based on an indirect Transwell system, we found that co-culture with macrophage promoted CSF1 production in colon cancer cells. Further investigation on regulatory mechanisms suggested that CSF1 production in colon cancer cells was dependent on PKC pathway, which was activated by IL-8, mainly produced by macrophages. Moreover, colon cancer cell-derived CSF1 drove the recruitment of macrophages and re-educated their secretion profile, including the augment of IL-8 production. The mice tumor xenografts study also found that over-expression of CSF1 in colon cancer cells promoted intratumoral infiltration of macrophages, and partially suppressed tumor growth. In all, our results demonstrated that CSF1 was an important factor in the colon cancer microenvironment, involving in the interactions between colon cancer cells and tumor-infiltrated macrophages.

Reduced Expression of Galectin-9 Contributes to a Poor Outcome in Colon Cancer by Inhibiting NK Cell Chemotaxis Partially through the Rho/ROCK1 Signaling Pathway.

Galectin-9 is a widely expressed protein that is involved in immune regulation and tumorpathogenesis and serves as a marker of a poor prognosis in various types of cancers. However, the clinical impact and the precise mechanism by which this protein contributes to colon tumor progression are unclear. In the present study, we detected the expression of galectin-9 and CD56 cells using immunohistochemistry. Spearman's rank correlation was used to clarify the association between galectin-9 expression and natural killer (NK) cell infiltration. The influence of galectin-9 on NK-92 cell migration was evaluated in vitro using transwell chemotaxis assays. The role of rh-galectin-9 in F-actin polarization in NK-92 cells was investigated using laser scanning confocal microscopy. We showed that galectin-9 was expressed in 101 (78.91%) colon tumor tissues and that was expressed at lower levels in these tissues than in para-tumor tissues. Low levels of galectin-9 expression were positively correlated with a poor histological grade and lymph node metastasis (P<0.05). A Kaplan-Meier method and Cox proportional hazards regression analysis showed that overall survival was longer in patients with high galectin-9 expression in an 8-year follow-up (P<0.05). Spearman's rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56+ NK cell infiltration (R(2) = 0.658; P<0.0001). Galectin-9 stimulated migration in human NK-92 cells by affecting F-actin polarization through the Rho/ROCK1 signaling pathway. These results suggest that galectin-9 expression potentially represents a novel mechanism for tumors to escape immune surveillance in colon tumors.

Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway.

NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3(+) dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-ß1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.