The aim of this paper was to analyze the microarray data between ulcerative colitis(UC) patients and healthy people by bioinformatics technology, screen the differentially expressed genes of UC, and predict the potential Chinese medicines for UC. The GSE36807 gene expression profile was downloaded from the gene expression database(GEO) and the differentially expressed(both up-regulated and down-regulated) genes(DEGs) were analyzed by using R language software. The core genes in the DEGs were obtained by using String database, Cytoscape software and its plug-in analysis, and the gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) were used to analyze the core genes. Moreover, the core genes and the medical ontology information retrieval platform(Coremine Medical) were mapped to each other to screen the traditional Chinese medicines and its active ingredients for treating UC. A total of 648 DEGs were screened, including 397 up-regulated genes and 251 down-regulated genes. Up-regulation of DEGs yielded 15 core genes including CXCL8, IL1 B, MMP9, CXCL1, CXCL10, CXCL9, CXCL2, CXCL5, TIMP1, CXCL11, STAT1,LCN2, IL1 RN, MMP1 and IDO1. Their biological processes and pathways were mainly enriched in interleukins, chemokine ligands and cytokines, chemokine-mediated signaling pathways, and were closely related to inflammatory responses, defense responses, cell chemotaxis, secretory granules, IL17 signaling pathways, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and TNF signaling pathway. Potential Chinese medicines for the treatment of UC include Curcumae Longae Rhizoma, Coptidis Rhizoma, Scutellariae Radix, Dendrobii Caulis, Sanguisorbae Radix, Phellodendri Chinensis Cortex, Bletillae Rhizoma and Atractylodis Rhizoma. The analysis of DEGs and core genes could promote our understanding on pathogenesis of UC. This study provides potential gene targets and research ideas for the development of new drugs of Chinese medicine intervention for UC.

Keywords: Chinese medicine prediction; Kyoto encyclopedia of genes and genomes(KEGG); differentially expressed genes; gene ontology(GO); hub genes; protein-protein interaction; ulcerative colitis.

In tick-borne encephalitis (TBE) the cerebrospinal fluid (CSF) cytosis is dominated by T CD3+CD4+ and T CD3+CD8+ lymphocytes, but their pathogenetic roles and mechanisms of migration into central nervous system (CNS) are unclear. Currently, we have studied CSF lymphocyte subsets and chemotactic axes in TBE patients stratified according to the clinical presentation. Blood and CSF were obtained from 51 patients with TBE (presenting as meningitis in 30, meningoencephalitis in 18 and meningoencephalomyelitis in 3), 20 with non-TBE meningitis and 11 healthy controls. We have studied: (1) abundances of the main lymphocyte subsets and (2) CXCR3 and CCR5 expression on CD3+CD4+ and CD3+CD8+ lymphocytes cytometrically with fluorochrome-stained monoclonal antibodies; (3) concentrations of chemotactic cytokines: CCL5 (CCR5 ligand), CXCL10 (CXCR3 ligand), IL-16, CCL2, CCL20 and CXCL5 with ELISA. Cytokine concentrations were additionally studied in 8 pediatric TBE patients. Data were analyzed with non-parametric tests, p < 0.05 considered significant. The higher CSF lymphocyte counts were associated with symptoms of CNS involvement, especially with altered consciousness (B, Th and Tc cells) and focal neurologic deficits (B cells). The minor fraction of double-positive T CD4+CD8+ cells was unique in associating negatively with encephalitis and altered consciousness. CSF CD3+CD4+ and CD3+CD8+ lymphocyte population was enriched in CCR5-positive cells and CCL5 concentration in CSF was increased and associated with a milder presentation. Although CXCL10 was vividly up-regulated intrathecally and correlated with CSF T lymphocyte counts, the CXCR3 expression in CSF T lymphocytes was low. Serum and CSF concentrations of CCL2, CXCL5 and IL-16 were increased in adult TBE patients, CCL2 created a chemotactic gradient towards CSF and both CCL2 and IL-16 concentrations correlated positively with CSF lymphocyte counts. The particular lymphoid cell populations in CSF associate differently with the clinical presentation of TBE, suggesting their distinct roles in pathogenesis. CCR5/CCL5 axis probably contributes to T lymphocyte migration into CNS. CXCL10 mediates the intrathecal immune response, but is probably not directly responsible for T cell migration. Additional chemotactic factors must be involved, probably including CCL2 and IL-16.

Keywords: Chemotaxis; Lymphocytes; Pleocytosis; tick–borne encephalitis.

Objective: To study subclinical inflammation in intercritical gout patients and its relation to the estimated size of monosodium urate crystal deposition and cardiovascular risk factors.

Methods: We performed a secretome analysis and the quantification of cytokine and adipokine plasma levels (IL-1β, IL-18, IL-6, sIL-6R, TNFα, CXCL5, RANTES, leptin, resistin and adiponectin) to analize subclinical inflammation in intercritical gout patients. Since it is currently not feasible to determinate the whole body deposit of monosodium urate crystals, we created an indirect clinical classification to estimate it. Then, we compared cytokine levels in controls and gout patients, and in patients with different crystal deposition size. We also studied the association between cytokine-levels and the number of cardiovascular risk factors.

Results: Ninety consecutive patients attending a Crystal Arthritis Unit were studied. IL-18, sIL-6R, RANTES, leptin and adiponectin were higher in intercritical gout patients than in controls. An association was observed between IL-18, sIL6-R and RANTES levels and the size of crystal deposition. IL-18, sIL6-R, RANTES and leptin were higher in patients with no cardiovascular risk factors when compared with controls with no risk factors.

Conclusion: Our results showed that the levels of some proinflammatory cytokines and metabolic proteins are elevated in intercritical gout patients. The levels of certain cytokines was related to the estimated size of the monosodium urate crystal deposition and to the number of cardiovascular risk factors. These cytokine changes may help to explain the increase of the cardiovascular events in gout patients.

Keywords: Gout; cardiovascular diseases; cytokine; inflammation.

Backgrounds: Tumor microenvironment (TME) plays a crucial role in the initiation and progression of Hepatocellular Carcinoma (HCC), especially immune infiltrates. However, there is still a challenge in understanding the modulation of the immune and stromal components in TME, especially TME related genes.

Methods: The proportion of tumor-infiltrating immune cells (TICs) and the immune and stromal scores in 374 HCC patients from The Cancer Genome Atlas (TCGA) database were determined using CIBERSORT and ESTIMATE computational methods. The final screened genes were confirmed by the PPI network and univariate Cox regression of the differentially expressed genes based on different immune or stromal scores. The correlation between the expression levels of the final gene interactions and the clinical characteristics was based on TCGA database and local hospital data. Gene set enrichment analysis (GSEA) and the effect of CXCL5 expression on TICs were conducted.

Results: There were correlations between the expression of CXCL5 and survival of HCC patients and TMN classification both in TCGA database and local hospital data. The immune-related activities were enriched in the high-expression group; however, the metabolic pathways were enriched in the low-expression group. The result of CIBERSORT analyzing had indicated that CXCL5 expression were correlated with the proportion of NK cells activated, macrophages M0, Mast cells resting, Neutrophils.

Conclusions: CXCL5 was a potential prognostic marker for HCC and provides clues regarding immune infiltrates, which offers extra insight for therapeutics of HCC, however, more independent cohorts and functional experiments of CXCL5 are warranted.

Keywords: CXCL5; hepatocellular carcinoma; immune infiltration; prognosis; tumor microenvironment.

OBJECTIVE

To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs).

METHODS

CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs.

RESULTS

CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels.

CONCLUSIONS

CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Purpose: Gut microbiota has been reported to contribute to either prevent or promote colorectal cancer (CRC), and treatment with probiotics might be a promising intervention method. The present study aimed to evaluate the potential anti-CRC effects of Lactobacillus coryniformis MXJ32 on a colitis-associated (CA)-CRC mouse model.

Methods: The CA-CRC mouse model was induced by a single intraperitoneal injection of 10 mg/kg azoxymethane and followed by three 7-day cycles of 2% dextran sulfate sodium in drinking water with a 14-day recovery period. Mice were supplemented with L. coryniformis MXJ32 by oral gavage (1 × 10⁹ CFU/day/mouse). The CA-CRC attenuating effects of this probiotic were assessed via intestinal barrier integrity, inflammation, and gut microenvironment.

Results: Treatment with L. coryniformis MXJ32 could significantly inhibit the total number of tumors and the average tumor diameter. This probiotic administration prevented the damage of intestinal barrier function by enhancing the expression of tight junction proteins (Occludin, Claudin-1, and ZO-1) and recovering the loss of goblet cells. Moreover, L. coryniformis MXJ32 alleviated intestinal inflammation via down-regulating the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-γ, and IL-17a) and chemokines (Cxcl1, Cxcl2, Cxcl3, Cxcl5, and Ccl7). In addition, L. coryniformis MXJ32 supplementation increased the abundance of some beneficial bacteria (such as SCFAs-producing bacteria, Lactobacillus, Bifidobacterium, Akkermansia, and Faecalibaculum) and decreased the abundance of some harmful bacteria (such as pro-inflammatory bacteria, Desulfovibrio and Helicobacter), which in turn attenuated the overexpression of inflammation.

Conclusion: Lactobacillus coryniformis MXJ32 could effectively ameliorate CA-CRC via regulating intestinal microenvironment, alleviating inflammation, and intestinal barrier damage, which further suggested that L. coryniformis MXJ32 could be considered as a functional food ingredient for the alleviation of CA-CRC.

Keywords: Colorectal cancer; Gut microbiota; Inflammation; Intestinal permeability; Lactobacillus coryniformis.

Purpose: The study aimed to predict and explore the possible clinical value and mechanism of genetic markers in adrenal cortical carcinoma using a bioinformatics analysis method. Methods: The RNA-seqs and miRNAs data were downloaded from TCGA database to identify the differentially expressed genes and differentially expressed miRNAs. The hub-genes were screened by building protein-protein interaction sub-networks with 12 topological analysis methods. We conducted the receiver operating characteristic curve to elevate the diagnostic value of hub-genes in distinguishing the death and alive groups. The survival analysis of hub-genes and key miRNAs were conducted using Kaplan-Meier curves. Furthermore, most significant small molecules were identified as therapeutic candidates for adrenal cortical carcinoma by the CMap analysis. Results: Compared to survival group, we found 475 up-regulated genes and 354 genes and the key pathways leading to the death of different ACC individual patients. Then we used 12 topological analysis methods to found the most possible 22 hub-genes. Among these hub-genes, nine hub-genes (C3, CXCL5, CX3CR1, GRM8, HCAR2, HTR1B, SUCNR1, PTGER3 and SSTR1) could be used to distinguish the death and survival groups for patients. We also revealed that mRNA expressions of 12 genes (C3, CXCL8, CX3CR1, GNAT3, GNGT1, GRM8, HCAR2, HTR1B, HTR1D, PTGER3, SSTR1 and SUCNR1) and four key miRNAs (hsa-mir-330, hsa-mir-489, hsa-mir-508 and hsa-mir-513b) were related to survival. Three most small molecules were identified (H-9, AZ-628 and phensuximide) as potential therapeutic drugs for adrenal cortical carcinoma. Conclusion: The hub-genes expression was significant useful in adrenal cortical carcinoma, provide new diagnostic, prognosis and therapeutic approaches for adrenal cortical carcinoma. Furthermore, we also explore the possible miRNAs involved in regulation of hub-genes.

Keywords: adrenal cortical carcinoma; biomarkers; prognosis; treatment.

Background: Hepatocellular carcinoma (HCC) is a common malignant cancer worldwide. CXCL5 has a role in inhibiting cell viability and metastasis in many tumors. In the present study, we investigated the role of CXCL5 in HCC and explored the underlying mechanism. Material and Methods. RT-qPCR and western blot were performed to evaluate the mRNA and protein levels of CXCL5. CCK-8 and transwell assay were applied to measure the proliferative and invasive abilities. Meanwhile, the Kaplan-Meier method was used to assess the survival of HCC patients.

Results: CXCL5 was upregulated in HCC tissues, which predicted a shorter overall survival in HCC. CXCL5 was a target gene of miR-577, and its expression was mediated by miR-577 in HCC. Knockdown of CXCL5 suppressed HuH-7 cell proliferation, invasion, and EMT and inhibited the NF-κB signaling pathway in cells. Moreover, knockdown of CXCL5 inhibited the xenograft growth of HuH-7 cells.

Conclusion: Overexpression of CXCL5 predicts poor prognosis in HCC patients. Knockdown of CXCL5 inhibits cell proliferation and invasion through the NF-κB signaling pathway in HCC. The newly identified role of the CXCL5/miR-577/NF-κB axis provides novel insights into the targeted therapy of HCC.

Introduction: As one of the most prevalent and malignant brain cancers, glioblastoma multiforme (GBM) presents a poor prognosis and the molecular mechanisms remain poorly understood. Consequently, molecular research, including various biomarkers, is essential to exploit the occurrence and development of glioma.

Methods: Weighted gene co-expression network analysis (WGCNA) was used to construct gene co-expression modules and networks based on the Chinese Glioma Genome Atlas (CGGA) glioblastoma specimens. Then, protein-protein interaction (PPI) and gene ontology (GO) analyses were performed to mine hub genes. RT-PCR and immunohistochemistry were employed to examine the expression level of GRPR, CXCL5, and CXCL11 in glioma patients.

Results: We confirmed two gene modules by protein-protein interaction networks. Functional enrichment analysis was performed to identify the significance of gene modules. Prognostic biomarkers GRPR, CXCL5, and CXCL11 related to the survival time of GBM samples were mined in The Cancer Genome Atlas (TCGA) dataset. qRT-PCR revealed that GRPR, CXCL5, and CXCL11 led to a significant increase in GBM sample compared to control.

Conclusion: In this study, we developed and confirmed three mRNA signatures (GRPR, CXCL5, and CXCL11) for evaluating overall survival in GBM patients. Our research assists in existing understanding of GBM diagnosis and prognosis.

Keywords: CGGA; GBM; TCGA; WGCNA; biomarkers.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/Mϕ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/Mϕ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/Mϕ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.

Acute, strenuous physical exertion requiring high levels of energy production induces the production of reactive oxygen species and metabolic disturbances that can damage the mitochondria. Thus, selective autophagic elimination of defective mitochondria may improve resistance to oxidative stress and potentially to inflammation. The main goal of this study was to evaluate the impacts of intense effort on changes in the expression of select genes related to post-effort inflammation and autophagy. Thirty-five men aged 16-21 years were recruited to the study. The impacts of both aerobic (endurance) and anaerobic (speed) efforts on selected genes encoding chemokines (<i><em>CXCL5</em></i>, <i>8-12</i>) were analyzed. Significant increases in the expression of all studied genes excluding <i>CXCL12</i> were observed. Moreover, both types of effort induced an increase in the expression of genes encoding IL-2, -4, -6, -10, IFN-γ and TNF-α, excluding IL-17A. Generally, these efforts caused a significant increase in the relative expression of apoptosis- (<i>BCL2</i> and <i>BAX</i>) and autophagy- (<i>BNIP3</i>, <i>BECN1</i>, <i>MAP1LC3B</i>, <i>ATG5</i>, <i>ATG7</i>, <i>ATG12</i>, <i>ATG16L1</i> and <i>SQSTM1</i>) related genes. It seems that the duration of physical activity and its bioenergetic cost has an important impact on the degree of increase in expression of this panel of autophagy-related genes. Anaerobic effort is more strenuous than aerobic effort and requires a higher bioenergetic investment. This may explain the stronger impact of anaerobic effort on the expression of the studied genes. This observation seems to support the protective role of autophagy proposed in prior studies.

Keywords: cell death; chemokines; cytokines; effort; gene expression; inflammation; leukocytes.

Selective estrogen receptor modulators (SERMs) have been developed to achieve beneficial effects of estrogens while minimizing their side effects. In this context, we decided to evaluate the protective effect of genistein, a natural SERM, on skin flap viability in rats and in a series of in vitro experiments on endothelial cells (migration, proliferation, antioxidant properties, and gene expression profiling following genistein treatment). Our results showed that administration of genistein increased skin flap viability, but importantly, the difference is only significant when treatment is started 3 days prior the flap surgery. Based on our in vitro experiments, it may be hypothesized that the underlying mechanism may rather by mediated by increasing SOD activity and Bcl-2 expression. The gene expression profiling further revealed 9 up-regulated genes (angiogenesis/inflammation promoting: CTGF, CXCL5, IL-6, ITGB3, MMP-14, and VEGF-A; angiogenesis inhibiting: COL18A1, TIMP-2, and TIMP-3). In conclusion, we observed a protective effect of genistein on skin flap viability which could be potentially applied in plastic surgery to women undergoing a reconstructive and/or plastic intervention. Nevertheless, further research is needed to explain the exact underlying mechanism and to find the optimal treatment protocol.

Importin α1/KPNA1 is a member of the Importin α family widely present in the mammalian brain and has been characterized as a regulator of neuronal differentiation, synaptic functionality, and anxiety-like behavior. In humans, a de novo mutation of the KPNA1 (human Importin α5) gene has been linked with schizophrenia; however, the precise roles of KPNA1 in disorder-related behaviors are still unknown. Moreover, as recent studies have highlighted the importance of gene-environment interactions in the development of psychiatric disorders, we investigated the effects of Kpna1 deletion and social isolation stress, a paradigm that models social stress factors found in human patients, on psychiatric disorder-related behaviors in mice. Through assessment in a behavioral battery, we found that Kpna1 knockout resulted in the following behavioral phenotype: (1) decreased anxiety-like behavior in an elevated plus maze test, (2) short term memory deficits in novel object recognition test (3) impaired sensorimotor gating in a prepulse inhibition test. Importantly, exposure to social isolation stress resulted in additional behavioral abnormalities where isolated Kpna1 knockout mice exhibited: (1) impaired aversive learning and/or memory in the inhibitory avoidance test, as well as (2) increased depression-like behavior in the forced swim test. Furthermore, we investigated whether mice showed alterations in plasma levels of stress-associated signal molecules (corticosterone, cytokines, hormones, receptors), and found that Kpna1 knockout significantly altered levels of corticosterone and LIX (CXCL5). Moreover, significant decreases in the level of prolactin were found in all groups except for group-housed wild type mice. Our findings demonstrate that Kpna1 deletion can trigger widespread behavioral abnormalities associated with psychiatric disorders, some of which were further exacerbated by exposure to adolescent social isolation. The use of Kpna1 knockout mice as a model for psychiatric disorders may show promise for further investigation of gene-environment interactions involved in the pathogenesis of psychiatric disorders.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease due to axonal damage of the corticospinal secondary to an inflammatory response against infected T-cells. In the present work, we aimed to evaluate biomarkers of neurodegeneration and neuroinflammation in the definition of HAM/TSP prognosis. Neurofilament light (NfL) and phosphorylated heavy (pNfH) chains, total Tau protein, cellular prion protein (PrPc), inflammatory chemokines, and neopterin were quantified in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n=21), HTLV-1 asymptomatic carriers (AC) (n=13), and HTLV-1 seronegative individuals with non-inflammatory non-degenerative neurological disease (normal-pressure hydrocephalus) (n=9) as a control group. HTLV-1 proviral load in peripheral blood mononuclear cells and the expression of chemokine receptors CCR4, CCR5, and CXCR3 in infected CD4⁺ T-cells (HTLV-1 Tax⁺ cells) were also assessed. CSF levels of Tau, NfL, and pNfH were similar between groups, but PrPc and neopterin were elevated in HAM/TSP patients. Most individuals in the control group and all HTLV-1 AC had CSF/serum neopterin ratio < 1.0, and two-thirds of HAM/TSP patients had ratio values > 1.0, which positively correlated with the speed of disease progression and pNfH levels, indicating active neuroinflammation. HAM/TSP patients showed high serum levels of CXCR3-binding chemokines (CXCL9, CXCL10, and CXCL11) and elevated CSF levels of CCL2, CCL3, CCL4, CCL17, CXCL5, CXCL10, and CXCL11. Indeed, CXCL10 concentration in CSF of HAM/TSP patients was 5.8-fold and 8.7-fold higher in than in HTLV-1 AC and controls, respectively, and correlated with CSF cell counts. HAM/TSP patients with typical/rapid disease progression had CSF/serum CXCL10 ratio > 1.0 and a higher frequency of CXCR3⁺Tax⁺CD4⁺ T-cells in blood, which indicated a positive gradient for the migration of infected cells and infiltration into the central nervous system. In conclusion, the slow progression of HAM/TSP abrogates the usefulness of biomarkers of neuronal injury for the disease prognosis. Thus, markers of inflammation provide stronger evidence for HAM/TSP progression, particularly the CSF/serum neopterin ratio, which may contribute to overcome differences between laboratory assays.

Keywords: HTLV-1; Tau protein; biomarkers; cellular prion protein; chemokines; neopterin; neurofilament; neuroinflammation.

Several distinct classes of drugs, such as anticonvulsants, immunosuppressants, and calcium channel blockers, caused gingival overgrowth. One of the main drugs associated with the gingival overgrowth is the anti-epileptic such as phenytoin, which affects gingival tissues by altering extracellular matrix metabolism. In our study, we evaluate the effect of phenytoin, a drug whose active substance is phenytoin, on gingival fibroblasts of healthy volunteers. Gene expression of 29 genes was investigated in gingival fibroblasts' cell culture treated with phenytoin compared with untreated cells. Among the studied genes, only 13 genes (CXCL5, CXCL10, CCR1, CCR3, CCR5, CCR6, IL-1A, IL-1B, IL-5, IL-7, IL-6R, BMP-2, and TNFSF-10) were statistically significant. All but one gene resulted downregulated after 24 h of treatment with phenytoin. BPM2 was the only, although weakly, up-expressed gene. Probably, we have not highlighted overexpression of the other inflammatory molecules because the study was performed on healthy people. Many studies show that phenytoin induces the overexpression of these cytokines but, probably, in our study, the drug does not have the same effect because we used gingival fibroblasts of healthy people.

Purpose: To investigate potential target genes associated with the diabetic condition in mouse cavernous endothelial cells (MCECs) for the treatment of diabetes-induced erectile dysfunction (ED).

Materials and methods: Mouse cavernous tissue was embedded into Matrigel, and sprouted cells were subcultivated for other studies. To mimic diabetic conditions, MCECs were exposed to normal-glucose (NG, 5 mmoL) or high-glucose (HG, 30 mmoL) conditions for 72 hours. An RNA-sequencing assay was performed to evaluate gene expression profiling, and RT-PCR was used to validate the sequencing data.

Results: We isolated MCECs exposed to the two glucose conditions. MCECs showed well-organized tubes and dynamic migration in the NG condition, whereas tube formation and migration were significantly decreased in the HG condition. RNA-sequencing analysis showed that MCECs had different gene profiles in the NG and HG conditions. Among the significantly changed genes, which we classified into 14 major gene categories, we identified that aging-related (9.22%) and angiogenesis-related (9.06%) genes were changed the most. Thirteen genes from the two gene categories showed consistent changes on the RNA-sequencing assay, and these findings were validated by RT-PCR.

Conclusions: Our gene expression profiling studies showed that Cyp1a1, Gclm, Igfbp5, Nqo1, Il6, Cxcl5, Olr1, Ctgf, Hbegf, Serpine1, Cyr61, Angptl4, and Loxl2 may play a critical role in diabetes-induced ED through aging and angiogenesis signaling. Additional research is necessary to help us understand the potential mechanisms by which these genes influence diabetes-induced ED.

Keywords: Diabetes mellitus; Erectile dysfunction; Gene expression; Penis; RNA sequencing.

The inflammatory human chemokine CXCL5 interacts with the G protein-coupled receptor CXCR2 to induce chemotaxis and activation of neutrophils. CXCL5 also has weak agonist activity toward CXCR1. The N-terminus of CXCL5 can be modified by proteolytic cleavage or deimination of Arg⁹ to citrulline (Cit), and these modifications can occur separately or together. Here, we chemically synthesized native CXCL5(1-78), truncated CXCL5 [CXCL5(9-78)], and the citrullinated (Cit⁹) versions and characterized their functions in vitro and in vivo. Compared with full-length CXCL5, N-terminal truncation resulted in enhanced potency to induce G protein signaling and β-arrestin recruitment through CXCR2, increased CXCL5-initiated internalization of CXCR2, and greater Ca²⁺ signaling downstream of not only CXCR2 but also CXCR1. Citrullination did not affect the capacity of CXCL5 to activate classical or alternative signaling pathways. Administering the various CXCL5 forms to mice revealed that in addition to neutrophils, CXCL5 exerted chemotactic activity toward monocytes and that this activity was increased by N-terminal truncation. These findings were confirmed by in vitro chemotaxis and Ca²⁺ signaling assays with primary human CD14⁺ monocytes and human THP-1 monocytes. In vitro and in vivo analyses suggested that CXCL5 targeted monocytes through CXCR1 and CXCR2. Thus, truncation of the N-terminus makes CXCL5 a more potent chemoattractant for both neutrophils and monocytes that acts through CXCR1 and CXCR2.

Mogamulizumab shows cytotoxicity against CCR4+ lymphoma cells by antibody-dependent cell-mediated cytotoxicity (ADCC) in advanced cutaneous T cell lymphoma (CTCL) patients. Although mogamulizumab is used as one of the anchor drugsfor the treatment of advanced CTCL, its efficacy is unsatisfactory, especially in mycosis fungoides (MF). Therefore, additional drugs to enhance the antitumor effects of mogamulizumab is needed to further optimize its use for the treatment of MF. In this report,two cases of mogamulizumab-resistant MF successfully treated with additional administration of etoposide are presented. Moreover, the possible mechanisms of mogamulizumab-etoposide combined therapy for the treatment of MF were investigated based on the modulation of chemokine profiles in vivo using an EL-4 mouse T cell lymphoma model.Intraperitoneal administration of etoposide significantly increased the mRNA expressions of CCL17, CXCL5, and CXCL10, suggesting that CCR4+ CTCL cells gather around the TAMs. Furthermore, the immunomodulatory effects of etoposide on the mRNA expressions of these chemokines were validated using monocyte-derived M2 macrophages in vitro. Since mogamulizumab showscytotoxicity against CCR4+ lymphoma cells by ADCC that depends on the contact between the lymphoma cellsand the effector cells, these chemokines could enhance the therapeutic effect of mogamulizumab. This article is protected by copyright. All rights reserved.

Metastasis is a major risk for lung adenocarcinoma-related mortality. Accumulating evidence raises the possibility that anticancer therapies might be more sensitive by targeting premetastatic niches in addition to the cancer cells themselves. Here, we identified a subpopulation of metastatic lung adenocarcinoma, which was characterized by EMT-related markers such as E-cadherin, Twist, SMAD, and β-catenin. EMT⁺ cases exhibited poorer prognosis than EMT^- patients, reflecting the pro-metastatic features of EMT. Immunohistochemical staining decorated CD15⁺ PMN-MDSCs surrounding EMT⁺ cancer cells in lymph nodes. Metastatic tissues secreted high levels of chemokines, including CXCL1, CXCL5, and CCL2, into the circulation to recruit histidine decarboxylase (Hdc)-positive PMN-MDSCs into metastatic colonies through upregulated CXCR2. The percentage of Hdc⁺ PMN-MDSCs increased in the setting of metastasis. Hdc⁺ PMN-MDSCs obtained from EMT⁺ metastatic masses expressed a higher level of TGF-β1, rather than TGF-β2 and TGF-β3, compared to EMT^- counterparts. The depletion of Hdc⁺ PMN-MDSCs or downregulation of TGF-β1 significantly decreased EMT⁺ percentage and, thus, hampered the metastasis process in murine models. Together, our findings suggest that metastatic tumor secretes high levels of chemokines to recruit Hdc⁺ PMN-MDSCs, which, in turn, express TGF-β1 to induce cancer cells to undergo EMT at metastatic sites.

Keywords: MDSCs; TGF-β; epithelial-mesenchymal transition; lung adenocarcinoma; metastasis.

Although chemokines have been believed to exert a pivotal role in pathogenesis of primary biliary cholangitis (PBC), comprehensive analysis of circulating chemokine profile in PBC has been little performed. The aim of this study is to determine serum chemokine profile and to explore its association with the development and progression of PBC. Sixty PBC patients and 30 healthy controls (HC) were enrolled. The sera were detected for 14 chemokines using multiplex immunoassay. The expression of CD3 and CD68 in the portal area of liver tissues was determined by immunohistochemistry in 6 PBC patients. The characteristics of chemokine profile in PBC were analyzed. Serum concentrations of most chemokines were higher, but TARC/CCL17, MDC/CCL22 and ENA-78/CXCL5 were lower in PBC patients than those in HC (all P < 0.05). Most of increased chemokines remained significant in both early and advanced PBC patients. PBC stage was correlated inversely with MCP-4/CCL13(r = -0.373), TARC/CCL17(r = -0.365), ENA-78/CXCL5 (r = -0.418) and I-TAC/CXCL11(r = -0.262), but positively with fractalkine/CX3CL1(r = 0.325). There were significant correlations between serum levels of IP-10/CXCL10 (r = 0.971, P = 0.001) and I-TAC/CXCL11 (r = 0.883, P = 0.020) and CD3 expression within the portal area, and between MIP-3α/CCL20 and CD68 expression within the portal area (r = 0.886, P = 0.019). In PBC patients, there were significant correlations among each other of MCP-4/CCL13, TARC/CCL17, MDC/CCL22, CTACK/CCL27, ENA-78/CXCL5, IP-10/CXCL10, I-TAC/CXCL11, excepting correlations of CTACK/CCL27 with IP-10/CXCL10 and I-TAC/CXCL11. Also, there were strong correlations among each other of CCL3/MIP-1α, CCL4/MIP-1β and IL-8/CXCL8. The only negative correlation was found in the serum between fractalkine/CX3CL1 and TARC/CCL17 (r = -0.311, P = 0.016). Serum levels of most chemokines are increasing throughout the development and progression of PBC, with the exception of chemokines, mainly attractive for neutrophil and eosinophil (e.g. ENA-78/CXCL5, MCP-4/CCL13), decreasing in advanced PBC, and of chemokines, dominantly responsible for Th2 chemotaxis (e.g. TARC/CCL17), decreasing in early PBC and associating negatively with PBC progression.

Keywords: Chemokine; Immune cell; Primary biliary cholangitis.

The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity. Upon MDC activation, MPO is secreted into phagosomes where it catalyzes the production of hypochlorous acid (HOCl), a potent chlorinating oxidant. Here, we demonstrated that the myeloid lineage-restricted MPO-HOCl system had antitumor effects in early melanoma growth in aged mice. Orthotopic melanomas grew more slowly in immunocompetent MPO^+/+ host mice compared to age-matched syngeneic MPO^-/- mice. Real-time intravital tumor imaging in vivo and in cell cocultures revealed a cell-cell proximity-dependent association between MDC-derived MPO enzyme activity and blockade of ligand-induced IκBα degradation in tumor cells. HOCl directly trans-inhibited IκB kinase (IKK) activity in tumor cells, thereby decreasing nuclear factor κB (NF-κB) transcriptional activation and inducing changes in the expression of genes involved in metabolic pathways, cell cycle progression, and DNA replication. By contrast, HOCl induced transcriptional changes in CD8⁺ T cells related to ion transport and the MAPK and PI3K-AKT signaling pathways that are associated with T cell activation. MPO increased the circulating concentrations of the myeloid cell-attracting cytokines CXCL1 and CXCL5, enhanced local infiltration by CD8⁺ cytotoxic T cells, and decreased tumor growth. Overall, these data reveal a role for MDC-derived HOCl as a small-molecule paracrine signaling factor that trans-inhibits IKK in melanoma tumor cells, mediating antitumor responses during early tumor progression.

Human respiratory syncytial virus (RSV) is one of the major causes of childhood acute lower respiratory tract infection worldwide. Autophagy is an intracellular pathway involved in nutrient recycling. Recently, autophagy has been reported to play a role in regulating host cytokine response to several viruses, including vesicular stomatitis virus and human immunodeficiency virus. Previous in vivo studies using mouse model has shown that inhibition of autophagy reduces RSV-induced cytokine production. However, the role of autophagy in modulating RSV-induced cytokine response in human cells has not been reported. We investigated the role of autophagy in regulating the production of the cytokines C-X-C motif ligand 8 (CXCL8) and C-C motif ligand 5 (CCL5), in RSV-infected human bronchial epithelium BEAS-2B cells. Fluorescent microscopic analysis showed that RSV infection induced autophagosome formation in BEAS-2B cells. This autophagy inducing ability of RSV was further confirmed by flow cytometry. The effects of pharmacological inhibition of autophagy by SAR405 or chloroquine on cell death and cytokine release were quantified using lactate dehydrogenase assay and enzyme-linked immunosorbent assay (ELISA), respectively. We found that SAR405 or chloroquine did not cause cell death. Importantly, ELISA analysis showed that pharmacological inhibition of autophagy by SAR405 or chloroquine did not affect the productions of both CXCL5 and CXCL8. In contrast to the previous studies using mouse model, our data suggest that pharmacological inhibition of autophagy may not be a suitable strategy in controlling RSV-induced airway inflammation.

Keywords: airway inflammation; autophagy; cytokines; human lung epithelial cells; respiratory syncytial virus.

Purpose: Mechanical loading of bone defects through rehabilitation is a promising approach to stimulate repair and reduce nonunion risk; however, little is known about how therapeutic mechanical stimuli modulate early-stage repair before mineralized bone formation. The objective of this study was to investigate the early effects of osteogenic loading on cytokine expression and angiogenesis during the first 3 weeks of BMP-2 mediated segmental bone defect repair.Materials and Methods: A rat model of BMP-2 mediated bone defect repair was subjected to an osteogenic mechanical loading protocol using ambulatory rehabilitation and a compliant, load-sharing fixator with an integrated implantable strain sensor. The effect of fixator load-sharing on local tissue strain, angiogenesis, and cytokine expression was evaluated.Results: Using sensor readings for local measurements of boundary conditions, finite element simulations showed strain became amplified in remaining soft tissue regions between 1 and 3 weeks (Week 3: load-sharing: -1.89 ± 0.35% and load-shielded: -1.38 ± 0.35% vs. Week 1: load-sharing: -1.54 ± 0.17%; load-shielded: -0.76 ± 0.06%). Multivariate analysis of cytokine arrays revealed that load-sharing significantly altered expression profiles in the defect tissue at 2 weeks compared to load-shielded defects. Specifically, loading reduced VEGF (p = 0.052) and increased CXCL5 (LIX) levels. Subsequently, vascular volume in loaded defects was reduced relative to load-shielded defects but similar to intact bone at 3 weeks. Endochondral bone repair was also observed histologically in loaded defects at 3 weeks.Conclusions: Together, these results demonstrate that moderate ambulatory strains previously shown to stimulate bone regeneration significantly alter early angiogenic and cytokine signaling and may promote endochondral ossification.

Keywords: Bone repair; angiogenesis word; immune response; mechanobiology; rehabilitation.