A disintegrin and metalloprotease 15 (ADAM15) is involved in several malignancies. In this study, we investigated the role of ADAM15 in rheumatoid arthritis (RA) angiogenesis. Soluble ADAM15 (s-ADAM15) in serum from RA and normal (NL) subjects was measured using ELISA. To determine membrane-anchored ADAM15 (ADAM15) expression in RA synovial tissues, immunohistochemistry was performed. To examine the role of ADAM15 in angiogenesis, we performed in vitro Matrigel assays and monocyte adhesion assays using human umbilical vein endothelial cells (HUVECs) transfected with ADAM15 siRNA. Finally, to investigate whether angiogenic mediators were affected by ADAM15, cytokines in ADAM15 siRNA-transfected HUVEC-conditioned medium were measured. ADAM15 was significantly higher in RA serum than in NL serum. ADAM15 was also expressed on RAST endothelial cells. ADAM15 siRNA-treated HUVECs had decreased EC tube formation in response to RA synovial fluids compared with non-treated HUVECs. The adhesion index of ADAM15 siRNA-transfected HUVECs was significantly lower than the adhesion index of control siRNA-transfected HUVECs. ENA-78/CXCL5 and ICAM-1 were decreased in tumor necrosis factor (TNF)-α-stimulated ADAM15 siRNA-transfected HUVEC-conditioned medium compared with TNF-α-stimulated control siRNA-transfected HUVEC-conditioned medium. These data show that ADAM15 plays a role in RA angiogenesis, suggesting that ADAM15 might be a potential target in inflammatory diseases such as RA.

Viral replication and interstitial inflammation play important roles in the pathogenesis of HIV-associated nephropathy. Cell-cell interactions between renal tubule epithelial cells (RTECs) and HIV-infected T cells can trigger efficient virus internalization and viral gene expression by RTEC. To understand how HIV replication initiates HIV-associated nephropathy, we studied the cellular response of RTECs to HIV, examining the transcriptional profiles of primary RTECs exposed to cell-free HIV or HIV-infected T cells.

HIV-induced gene expression in hRTECs was examined in vitro by Illumina RNA deep sequencing and revealed an innate response to HIV, which was subclassified by gene ontology biological process terms. Chemokine responses were examined by CD4 T-cell chemotaxis assays.

As compared with cell-free virus infection, exposure to HIV-infected T cells elicited a stronger upregulation of inflammatory and immune response genes. A major category of upregulated genes are chemokine/cytokine families involved in inflammation and immune response, including inflammatory cytokines CCL20, IL6 and IL8-related chemokines: IL8, CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6. Supernatants from virus-exposed RTECs contained strong chemoattractant activity on primary CD4 T cells, which was potently blocked by a CXCR2 antagonist that antagonizes IL8-related chemokines. We observed a preferential migration of CXCR2-expressing, central memory CD4 T cells in response to HIV infection of RTECs.

Interactions between primary RTECs and HIV-infected T cells result in potent induction of inflammatory response genes and release of cytokines/chemokines from RTECs that can attract additional T cells. Activation of these genes reflects an innate response to HIV by nonimmune cells.

Tofacitinib (CP-690550), an oral Janus kinase inhibitor, has shown significant efficacy in the treatment of rheumatoid arthritis through blocking the signaling pathways of pro-inflammatory cytokines. However, recent evidence suggests that long-term tofacitinib treatment is associated with increased risk of infection (e.g. tuberculosis) in patients. In the present study, we illustrate that tofacitinib administration significantly reduced the survival rate of mice given lethal or sub-lethal dose challenge with Candida albicans. This was related to the ability of tofacitinib to reverse TNFα- and IFNγ-enhanced candidacidal activity of murine polymorph nuclear cells (PMNs) and also to suppress chemokine CXCL5 expression and PMN infiltration in the infected tissues of mice. More importantly, tofacitinib significantly antagonized the ability of TNFα, IFNγ and GM-CSF to boost human PMNs in phagocytosis and direct killing of C. albicans in vitro. It also down-regulated reactive oxygen production and neutrophil extracellular trap formation by human PMNs stimulated with yeast-derived β-glucans in the presence of TNFα, IFNγ or GM-CSF. Our data emphasizes a significantly increased risk for opportunistic fungal infection associated long-term tofacitinib treatment in humans, likely through antagonizing the PMN-boosting effect of pro-inflammatory cytokines.

Colorectal cancer is a heterogeneous disease. Although many risk factors are used to predict colorectal cancer patients' prognosis after surgical resection, new prognostic factors are still needed to be defined to promote predictive efficacy of prognosis and further guide therapies. Herein, we identified the prognostic significance of CXCR2 in colorectal cancer patients. We retrospectively analysed 134 patients with colorectal cancer who underwent minimally invasive surgery between 2010 and 2011. The overall cohort was divided into a training set (n = 78) and a validation set (n = 56). We detected CXCR2 expression using immunohistochemical staining and defined the cut-off value using X-tile program. Next, we analysed the association between CXCR2 expression and clinicopathologic features in training and validation sets. High expression of CXCR2 was associated with Dukes stage (P = 0.018), tumor invasion (P = 0.018) and liver metastasis (P = 0.047). Multivariate COX regression analyses confirmed that high CXCR2 level was an independent prognostic risk factor for both overall survival and disease free survival. Kaplan-Meier survival analysis demonstrated that patients with high expression of CXCR2 had a poor overall survival and disease free survival even in low-risk group (I + II). This indicated that CXCR2 can help to refine individual risk stratification. In addition, we established Nomograms of all significant factors to predict 3- or 5-years overall survival and disease free survival. Moreover, we found the combination of CXCR2 and its ligand CXCL5 had more significant value in predicting the prognosis than single CXCR2 factor.

OBJECTIVE

Xerostomia is a common radiation sequela, which has a negative impact on the quality of life of patients with head and neck cancer. Current treatment strategies offer only partial relief. Botulinum toxins (BTX) have been successfully used in treating a variety of radiation sequelae such as cystitis, proctitis, fibrosis, and facial pain. The purpose of this study was to evaluate the effect of BTX on radiation-induced salivary gland damage.

METHODS

We used a previously established model for murine salivary gland irradiation (IR). The submandibular glands (SMGs) of C5BL/6 mice (n=6/group) were injected with saline or BTX 72 hours before receiving 15 Gy of focal irradiation. Saliva flow was measured 3, 7, and 28 days after treatment. The SMGs were collected for immunohistochemistry, confocal microscopy, and Western blotting. A cytokine array consisting of 40 different mouse cytokines was used to evaluate cytokine profiles after radiation treatment.

RESULTS

Irradiated mice showed a 50% reduction in saliva flow after 3 days, whereas mice preinjected with BTX had 25% reduction in saliva flow (P<.05). Cell death detected by TUNEL staining was similar in SMG sections of both groups. However, neutrophil infiltrate, detected by myeloperoxidase staining, was 3-fold lower for the BTX treated mice. A cytokine array showed a 2-fold upregulation of LPS-induced chemokine (LIX/CXCL5) 3 days after IR. BTX pretreatment reduced LIX levels by 40%. At 4 weeks after IR, the saline (control) group showed a 40% reduction in basal SMG weight, compared with 20% in the BTX group. Histologically, BTX-pretreated glands showed relative preservation of acinar structures after radiation.

CONCLUSIONS

These data suggest that BTX pretreatment ameliorates radiation-induced saliva dysfunction. Moreover, we demonstrate a novel role for CXCL5 in the acute phase of salivary gland damage after radiation. These results carry important clinical implications for the treatment of xerostomia in patients with head and neck cancer.

Endoplasmic reticulum (ER) stress has been reported to be associated with metastasis in many malignant tumors. PKR-like ER kinase-phosphorylated eukaryotic translation initiation factor 2α (PERK-p-eIF2α) pathway is one of the three main signal pathways in ER stress, however, its mechanism in regulating breast cancer (BC) relapse or metastasis was still not completely understood. Besides, drug resistance was an important factor influencing the effect of tumor treatment and whether PERK-p-eIF2α pathway was involved in the drug resistance to BC treatment also needs to be explored. The authors conducted survival analysis of ER stress-related genes in the The Cancer Genome Atlas (TCGA) database to find the candidate molecule and found that eIF2α was significantly correlated with relapse-free survival in BC patients, especially in the triple-negative BC (TNBC) patients. Furthermore, BC cell lines were used to study the downstream target of PERK-p-eIF2α. In this study, p-eIF2α could negatively regulate the expression of programmed death ligand 1 (PDL1) and C-X-C motif chemokine ligand 5 (CXCL5), which were important ligands of the immune cells such as T cells and myeloid-derived suppressor cells in the tumor microenvironment. Besides, p-eIF2α expression in highly metastatic human TNBC cells after treatment of carboplatin was significantly decreased. The data indicated the possible novel immune-related mechanism of PERK-p-eIF2α in regulating TNBC metastasis and drug resistance of carboplatin in highly metastatic TNBC.

OBJECTIVE

Cardiovascular diseases are significantly correlated with chronic periodontitis. The aim of this study was to evaluate bone-loss level, neutrophil migration, CXCL2/CINC-2α, CXCL5/LIX, CCL20/MIP-3α and tumor necrosis factor-α (TNF-α) production, inducible nitric oxide synthase (iNOS) expression and C-reactive protein (CRP) release in spontaneously hypertensive rats (SHRs) and normotensive (WTK) rats after experimental induction of periodontal disease.

METHODS

Periodontitis was induced by placement of silk yarn ligatures around the first molar counterparts. The levels of CRP, CCL20/MIP-3α and CXCL5/LIX were evaluated in the peripheral blood, and bone-loss level, neutrophil recruitment, the production of myeloperoxidase, CXCL2, CXCL5, CCL20 and TNF-α, and the expression of iNOS were evaluated in the gingival tissue. Histological sections were taken to evaluate and measure bone resorption and neutrophil recruitment in the furcation region.

RESULTS

Rats with periodontitis had alveolar bone resorption. SHRs with periodontitis showed marked bone loss and increased neutrophil infiltration in comparison with WTK rats. SHRs with periodontitis showed increased levels of TNF-α and CXCL2, and a slight tendency for increased levels of CXCL5, in the gingival tissue but no increase in the level of CCL20. In SHRs, even without periodontitis, the levels of TNF-α, CXCL2, CXCL5 and CCL20 showed a slight tendency to increase. In the WTK rats, TNF-α, CXCL2 and CXCL5 levels were increased with periodontitis, but the level of CCL20 was not. iNOS was expressed in the gingival tissue of WTK rats and SHRs with periodontitis; however, SHRs appeared to express a higher level of iNOS than did WKT rats. The CRP level was elevated in both types of rats with periodontitis; however, the CRP level was higher in SHRs with periodontitis than in WTK rats with periodontitis.

CONCLUSIONS

In SHRs, the hypertensive condition per se seems to favor the inflammatory processes that become potentiated with periodontitis, when compared with WKT rats.

Homozygous mutations in Triggering Receptor Expressed on Myeloid cells 2 gene (TREM2) are one of the major causes of Nasu Hakola Disease (NHD). We analysed Peripheral Blood Mononuclear Cells (PBMC) profile of 164 inflammatory factors in patients with NHD carrying the TREM2 Q33X mutation as compared with heterozygous and wild type individuals. Several molecules related to bone formation and angiogenesis were altered in NHD compared to non-carriers: Bone Morphogenetic Protein (BMP)-1 mRNA levels were significantly increased in PBMC (2.32 fold-increase; P = 0.01), as were Transforming Growth Factor Beta (TGFB)3 levels (1.51 fold-increase; P = 0.02). Conversely, CXCL5 and Pro Platelet Basic Protein (PPBP) were strongly downregulated (-28.26, -9.85 fold-decrease over non-carriers, respectively, P = 0.01), as well as Platelet Factor 4 Variant 1 (PF4V1; -41.44, P = 0.03). Among other inflammatory factors evaluated, Interleukin (IL)-15 and Tumor Necrosis Factor Superfamily Member (TNFSF)4 mRNA levels were decreased in NHD as compared with non-carriers (-2.25 and -3.87 fold-decrease, P = 0.01 and 0.001, respectively). In heterozygous individuals, no significant differences were observed, apart from IL-15 mRNA levels, that were decreased at the same extent as NHD (-2.05 fold-decrease over non-carriers, P = 0.002). We identified a signature in PBMC from patients with NHD consisting of strongly decreased mRNA levels of CXCL5, PPBP, PF4V1, mildly decreased IL-15 and TNFSF4 and mildly increased BMP-1 and TGFB3.

Ionising radiation is a carcinogen capable of inducing tumours, including colorectal cancer, in both humans and animals. By backcrossing a recombinant line of Apc(Min/+) mice to the inbred BALB/c mouse strain, which is unusually sensitive to radiation-induced tumour development, we obtained panels of 2Gy-irradiated and sham-irradiated N2 Apc(Min/+) mice for genotyping with a genome-wide panel of microsatellites at approximately 15 cM density and phenotyping by counting adenomas in the small intestine. Interval and composite interval mapping along with permutation testing identified five significant susceptibility quantitative trait loci (QTLs) responsible for radiation induced tumour multiplicity in the small intestine. These were defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (log of odds, LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p<0.00001, 6.2, p<0.00001) and two regions within chromosome 16 respectively (LOD 4.1, p = 4x10(-5), 4.8, p<0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p<0.005). Genes containing BALB/c specific non-synonymous polymorphisms were identified within Mrip regions and prediction programming used to locate potentially functional polymorphisms. Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in Apc(Min/+) mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Casc5, and Bub1), DNA repair (Recc1 and Prkdc) or inflammation (Duox2, Itgb2l and Cxcl5). Our study demonstrates use of in silico analysis in candidate gene identification as a way of reducing large-scale backcross breeding programmes.

BACKGROUND

Periodontal disease is a major complication of type 1 diabetes mellitus (T1DM). The aim of the present study is to investigate the relationship between glycated hemoglobin and circulating levels of interleukin (IL)-6, IL-8, and C-X-C motif chemokine ligand 5 (CXCL5) in non-smoking patients suffering from T1DM, with and without periodontitis. In addition, to determine the effect of advanced glycation end products (AGE) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) on IL-6, IL-8, and CXCL5 expression by THP-1 monocytes and OKF6/TERT-2 cells.

METHODS

There were 104 participants in the study: 19 healthy volunteers, 23 patients with periodontitis, 28 patients with T1DM, and 34 patients with T1DM and periodontitis. Levels of blood glucose/glycated hemoglobin (International Federation of Clinical Chemistry [IFCC]) were determined by high-performance liquid chromatography. Levels of IL-6, IL-8, and CXCL5 in plasma were determined by enzyme-linked immunosorbent assay (ELISA). In vitro stimulation of OKF6/TERT-2 cells and THP-1 monocytes was performed with combinations of AGE and P. gingivalis LPS. Changes in expression of IL-6, IL-8, and CXCL5 were monitored by ELISA and real-time polymerase chain reaction.

RESULTS

Patients with diabetes and periodontitis had higher plasma levels of IL-8 than patients with periodontitis alone. Plasma levels of IL-8 correlated significantly with IFCC units, clinical probing depth, and attachment loss. AGE and LPS, alone or in combination, stimulated IL-6, IL-8, and CXCL5 expression in both OKF6/TERT-2 cells and THP-1 monocytes.

CONCLUSIONS

Elevated plasma levels of IL-8 potentially contribute to the cross-susceptibility between periodontitis and T1DM. P. gingivalis LPS and AGE in combination caused significantly greater expression of IL-6, IL-8, and CXCL5 from THP-1 monocytes and OKF6/TERT-2 cells than LPS alone.

Context/Objective: Clinical disease activity in multiple sclerosis (MS) may manifest as predominant involvement of optic nerves and spinal cord, as exemplified by opticospinal multiple sclerosis (OSMS) often encountered in Asian countries. Our aim was to compare the clinical features, neuropsychological profile and cytokine/chemokine levels of patients with conventional MS (CMS) and MS presenting predominantly with spinal cord and optic nerve attacks (MS-SCON). Design: Cross-sectional study. Setting: MS Outpatient Clinic. Participants: Fourteen MS-SCON patients, 20 CMS patients without myelitis and optic neuritis attacks and 21 healthy individuals. Outcome measures: IL-8, IL-10, IFN-γ, IL-17 and TNF-α levels were measured by multiplex assay and CXCL2 and CXCL5 levels were measured by ELISA. A panel of neuropsychological tests, Beck depression inventory, 9-hole peg and timed 25-foot walk tests were employed. Results: CMS and MS-SCON patients showed similar clinical features. Both CMS and MS-SCON patients displayed reduced IL-8 and CXCL2 and increased TNF-α levels, while IL-10 and CXCL5 levels were identical among all groups. Conclusion: Neuropsychological and motor function test performances of CMS and MS-SCON patients were highly comparable. CMS and MS-SCON present with similar clinical, neuropsychological and immunological features. Therefore, optic nerve and spinal cord-dominant form of MS does not necessarily establish a distinct entity in our region. Cognitive networks of the central nervous system may be damaged during the disease course of MS, despite the absence of cerebral or cerebellar clinical attacks.

Despite the introduction of biologics, topical dithranol (anthralin) has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e. IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), antimicrobial peptides (e.g. ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), chemotactic factors for neutrophils (e.g. CXCL5, CXCL8) and neutrophilic infiltration was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs.

Trial registration: ClinicalTrials.gov NCT02752672.

Keywords: AMPs; IL-36; anthralin; dithranol; human; immunology; inflammation; keratinocytes; mouse; psoriasis.

We hypothesized that WNT5A could contribute to the enhanced migration and invasiveness of rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), which is one of the incompletely understood aspects of the RA FLS aggressive phenotype. This hypothesis is based on the previous evidence of a WNT5A role in both, RA and cell migration. Migration and invasion of RA FLS were assessed after incubation with recombinant Wnt5a (rWnt5a) or silencing of the endogenous WNT5A expression. The expression of WNT5A, WNT receptors, cytokines, chemokines, and metalloproteinases was quantified with RT-PCR. The WNT pathway was explored with gene silencing, antibody and pharmacological inhibition followed by migration assays and phosphoprotein western blots. Here, we reported that rWnt5a promoted migration and invasion of RA FLS, whereas knockdown of the endogenous WNT5A reduced them. These effects were specific to the RA FLS since they were not observed in FLS from osteoarthritis (OA) patients. Also, rWnt5a induced the expression of IL6, IL8, CCL2, CXCL5, MMP1, MMP3, MMP9, and MMP13 from baseline or potentiating the TNF induction, WNT5A signaling required the RYK receptor and was mediated through the WNT/Ca²⁺ and the ROCK pathway. These pathways involved the RYK and ROCK dependent activation of the p38, ERK, AKT, and GSK3β kinases, but not the activation of JNK. Together these findings indicate that WNT5A contributes to the enhanced migration and invasiveness of RA FLS through RYK and the specific activation of ROCK and downstream kinases.

Keywords: MAPK; RYK; WNT5A; fibroblast-like synoviocytes; inflammatory response; invasion; migration; rheumatoid arthritis.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening clinical conditions predominantly arising from uncontrolled inflammatory reactions. It has been found that the administration of astaxanthin (AST) can exert protective effects against lipopolysaccharide (LPS)-induced ALI; however, the robust genetic signatures underlying LPS induction and AST treatment remain obscure. Here we performed a statistical meta-analysis of five publicly available gene expression datasets from LPS-induced ALI mouse models, conducted RNA-sequencing (RNA-seq) to screen differentially expressed genes (DEGs) in response to LPS administration and AST treatment, and integrative analysis to determine robust genetic signatures associated with LPS-induced ALI onset and AST administration. Both the meta-analyses and our experimental data identified a total of 198 DEGs in response to LPS administration, and 11 core DEGs (Timp1, Ly6i, Cxcl13, Irf7, Cxcl5, Ccl7, Isg15, Saa3, Saa1, Tgtp1, and Gbp11) were identified to be associated with AST therapeutic effects. Further, the 11 core DEGs were verified by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), and functional enrichment analysis revealed that these genes are primarily associated with neutrophils and chemokines. Collectively, these findings unearthed the robust genetic signatures underlying LPS administration and the molecular targets of AST for ameliorating ALI/ARDS which provide directions for further research.

Keywords: RNA sequencing; acute lung injury; astaxanthin; genetic signatures; integrative analysis.

Tyrosine sulfation, a post-translational modification found on many chemokine receptors, typically increases receptor affinity for the chemokine ligand. A previous bioinformatics analysis suggested that a sulfotyrosine (sY)-binding site on the surface of the chemokine CXCL12 may be conserved throughout the chemokine family. However, the extent to which receptor tyrosine sulfation contributes to chemokine binding has been examined in only a few instances. Computational solvent mapping correctly identified the conserved sulfotyrosine-binding sites on CXCL12 and CCL21 detected by nuclear magnetic resonance (NMR) spectroscopy, demonstrating its utility for hot spot analysis in the chemokine family. In this study, we analyzed five chemokines that bind to CXCR2, a subset of which also bind to CXCR1, to identify hot spots that could participate in receptor binding. A cleft containing the predicted sulfotyrosine-binding pocket was identified as a principal hot spot for ligand binding on the structures of CXCL1, CXCL2, CXCL7, and CXCL8, but not CXCL5. Sulfotyrosine titrations monitored via NMR spectroscopy showed specific binding to CXCL8, but not to CXCL5, which is consistent with the predictions from the computational solvent mapping. The lack of CXCL5-sulfotyrosine interaction and the presence of CXCL8-sulfotyrosine binding suggests a role for receptor post-translational modifications regulating ligand selectivity.

Driving nanomaterials to specific cell populations is still a major challenge for different biomedical applications. Several strategies to improve cell binding and uptake have been tried thus far by intrinsic material modifications or decoration with active molecules onto their surface. In the present work, we covalently bound the chemokine CXCL5 on fluorescently labeled amino-functionalized SiO₂ nanoparticles to precisely targeting CXCR2⁺ immune cells. We synthesized and precisely characterized the physicochemical features of the modified particles. The presence of CXCL5 on the surface was detected by z-potential variation and CXCL5-specific electron microscopy immunogold labeling. CXCL5-amino SiO₂ nanoparticle cell binding and internalization performances were analyzed in CXCR2⁺ THP-1 cells by flow cytometry and confocal microscopy. We showed improved internalization of the chemokine modified particles in the absence or the presence of serum. This internalization was reduced by cell pre-treatment with free CXCL5. Furthermore, we demonstrated CXCR2⁺ cell preferential targeting by comparing particle uptake in THP-1 vs. low-CXCR2 expressing HeLa cells. Our results provide the proof of principle that chemokine decorated nanomaterials enhance uptake and allow precise cell subset localization. The possibility to aim at selective chemokine receptor-expressing cells can be beneficial for the diverse pathological conditions involving immune reactions.

Assessment of incised wound age in skeletal muscles is important because fatal injuries are often complicated with muscle involvement. Transcriptome of injured skeletal muscle along with histopathological and immunohistochemistry staining, were analyzed to explore the biological effect of incised injuries using a mouse incised injury model. An incisional wound was made at the biceps femoris muscle of anesthetized mice, and the muscles were sampled at 6, 12, 24, 36 and 48h post-injury. DNA microarray analysis using RNA extracted from the muscle samples of 12h post-injury identified 3,655 upregulated and 3,583 downregulated genes. Referring to the results of the gene ontology and gene expression pathway analysis, time course expression of five cytokines, namely chemokine (C-C motif) ligand 4 (CCL4), chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin-1 beta (IL-1β), interleukin- 6 (IL-6) and interleukin-7 (IL-7), were analyzed by quantative reverse transcription PCR (qRT-PCR). CXCL5 was the most upregulated gene throughout the post-injury period with higher expression from 6 through 36h post injury. Upregulation of CCL4 and IL-1β was also persisted until 36h post injury. IL-6 mRNA was highly and rapidly expressed at 6h post-injury followed by significant decrease at 12h. Unlike other four cytokines, IL-7 showed slow and steady increasing over time until 48h post-injury. Immunohistochemical staining of post-injury samples showed gradual mild increase of staining intensity proportional to increasing time points especially around the wound edges. The present study highlights the unique dynamics of each cytokine and reflects their roles in the process of muscle wound healing, and suggests the potential of them as a tool for forensic wound age estimation.

Influenza A viruses (IAV) mutate rapidly and cause seasonal epidemics and occasional pandemics, which result in substantial number of patient visits to the doctors and even hospitalizations. We aimed here to identify inflammatory proteins, which levels correlated to clinical severity of the disease. For this we analysed 102 cytokines and growth factors in human nasopharyngeal aspirate (NPA) samples of 27 hospitalized and 27 outpatients diagnosed with influenza A(H1N1)pdm09 virus infection. We found that the relative levels of monocyte differentiation antigen CD14, lipocalin-2 (LCN2), C-C-motif chemokine 20 (CCL20), CD147, urokinase plasminogen activator surface receptor (uPAR), pro-epidermal growth factor (EGF), trefoil factor 3 (TFF3), and macrophage migration inhibitory factor (MIF) were significantly lower (p<0.008), whereas levels of retinol-binding protein 4 (RBP4), C-X-C motif chemokine 5 (CXCL5), interleukin-8 (IL-8), complement factor D (CFD), adiponectin, and chitinase-3-like 1 (CHI3L1) were significantly higher (p<0.008) in NPA samples of hospitalized than non-hospitalized patients. While changes in CD14, LCN2, CCL20, uPAR, EGF, MIF, CXCL5, IL-8, adiponectin and CHI3L1 levels have already been correlated with severity of IAV infection in mice and humans, our study is the first to describe association of CD147, RBP4, TFF3, and CFD with hospitalization of IAV-infected patients. Thus, we identified local innate immune profiles, which were associated with the clinical severity of influenza infections.

GNA13, the alpha subunit of a heterotrimeric G protein, mediates signaling through G protein-coupled-receptors (GPCRs). GNA13 is up-regulated in many solid tumors, including prostate cancers, where it contributes to tumor initiation, drug resistance and metastasis. To better understand how GNA13 contributes to tumorigenesis and tumor progression, we compared the entire transcriptome of PC3 prostate cancer cells with those cells in which GNA13 expression had been silenced. This analysis revealed that GNA13 levels affected multiple CXC-family chemokines. Further investigation in three different prostate cancer cell lines singled out pro-tumorigenic C-X-C motif chemokine ligand 5 (CXCL5) as a target of GNA13 signaling. Elevation of GNA13 levels consistently induced CXCL5 RNA and protein expression in all three cell lines. Analysis of the CXCL5 promoter revealed that the -505/+62 region was both highly active and influenced by GNA13, and a single NF-κB site within this region of the promoter was critical for GNA13-dependent promoter activity. Chromatin immunoprecipitation experiments revealed that, upon induction of GNA13 expression, occupancy at the CXCL5 promoter was significantly enriched for the p65 component of NF-ĸB. GNA13 knockdown suppressed both p65 phosphorylation and the activity of a specific NF-κB reporter, and p65 silencing impaired the GNA13-enhanced expression of CXCL5. Finally, blockade of Rho GTPase activity eliminated the impact of GNA13 on NF-κB transcriptional activity and CXCL5 expression. Together, these findings suggest that GNA13 drives CXCL5 expression by transactivating NF-ĸB in a Rho-dependent manner in prostate cancer cells.

Infertility remains the most prevalent reason for cattle being removed from production environments. We utilized metabolomic profiling to identify metabolites in the blood plasma that may be useful in identifying infertile heifers at the time of artificial insemination (AI). Prior to AI, phenotypic parameters including body condition, weight, and reproductive organ measurements were collected. These were determined not effective at differentiating between fertile and infertile heifers. Analysis of the resulting metabolomic profiles revealed 15 metabolites at significantly different levels (T-test P ≤ 0.05), with seven metabolites having a greater than 2-fold difference (T-test P ≤ 0.05, fold change ≥2, ROC-AUC ≥ 0.80) between infertile and fertile heifers. We further characterized the utility of using the levels of these metabolites in the blood plasma to discriminate between fertile and infertile heifers. Finally, we investigated the potential role inflammation may play by comparing the expression of inflammatory cytokines in the white blood cells of infertile heifers to that of fertile heifers. We found significantly higher expression in infertile heifers of the proinflammatory markers tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), and the C-X-C motif chemokine 5 (CXCL5). Our work offers potentially valuable information regarding the diagnosis of fertility problems in heifers undergoing AI.

Background: Circular RNAs (circRNA) have been shown to be involved in the pathogenesis of colorectal cancer (CRC). CircCTNNA1 was found to be one of the upregulated circRNAs in CRC. However, there are few studies on circCTNNA1, so it is necessary to carry out further studies.

Methods: The expression of circCTNNA1, microRNA (miR)-363-3p, and chemokine C-X-C motif ligand 5 (CXCL5) was detected by quantitative real-time PCR (qRT-PCR). The protein levels of CXCL5 and metastasis markers were measured using western blot (WB) analysis. Cell proliferation, apoptosis, cell cycle, migration, and invasion were determined by cell counting kit 8 (CCK8) assay, colony formation assay, flow cytometry, and transwell assay. The relationship between miR-363-3p and circCTNNA1 or CXCL5 was evaluated via dual-luciferase reporter assay and RNA immunoprecipitation assay. Animal study was performed to explore the function of circCTNNA1 on CRC tumorigenesis.

Results: CircCTNNA1 and CXCL5 were highly expressed in CRC. Knockdown of circCTNNA1 could inhibit the proliferation, cell cycle, metastasis, and promote the apoptosis of CRC cells. MiR-363-3p could be sponged by circCTNNA1, and the inhibition effect of circCTNNA1 silencing on CRC progression could be reversed by miR-363-3p inhibitor. Moreover, miR-363-3p could interact with CXCL5, and CXCL5 overexpression also could reverse the suppressive effect of miR-363-3p on CRC progression. Downregulation of circCTNNA1 also could hinder the tumor growth of CRC in vivo.

Conclusion: CircCTNNA1 enhanced CRC progression via regulating the miR-363-3p/CXCL5 axis.

Keywords: CXCL5; Colorectal cancer; circCTNNA1; miR-363-3p.