BACKGROUND

Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells (MEC) using Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA) bacterial cell wall components are well - characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC (pgMEC). We performed quantitative real-time RT-PCR (qPCR) to measure mRNA levels of 9 genes involved in transcriptional regulation or antibacterial activity: Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), prostaglandin-endoperoxide synthase 2 (PTGS2), interferon induced protein with tetratricopeptide repeats 3 (IFIT3), interferon regulatory factor 3 (IRF3), myeloid differentiation primary response 88 (MYD88), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1), Toll interacting protein (TOLLIP), and lactoferrin (LTF). Furthermore, we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 6 (CXCL6), interleukin 8 (CXCL8), interleukin 1 beta (IL1B), interleukin 6 (IL6), and tumor necrosis factor alpha (TNF).

RESULTS

Stimulation of pgMEC with LPS for 3 h led to an increase in expression of CCL2, CXCL6, IL6, CXCL8, PTGS2, IFIT3, MYD88, NFKB1, and TLR4 (P < 0.05). Except for IL6, and PTGS2, the same genes had greater expression than controls at 6 h post-LPS (P < 0.05). Expression of CCL5, PTGS2, IFIT3, NFKB1, TLR4, and TOLLIP was greater than controls after 3 h of incubation with LTA (P < 0.05). Compared to controls, stimulation with LTA for 6 h led to greater expression of PTGS2, IFIT3, NFKB1, and TOLLIP (P < 0.05) whereas the expression of CXCL6, CXCL8, and TLR4 was lower (P < 0.05). At 3 h incubation with both toxins compared to controls a greater expression (P < 0.05) of CCL2, CCL5, CXCL6, CXCL8, IL6, PTGS2, IFIT3, IRF3, MYD88, and NFKB1 was detected. After 6 h of incubation with both toxins, the expression of CCL2, CXCL6, IFIT3, MYD88, NFKB1, and TLR4 was higher than the controls (P < 0.05).

CONCLUSIONS

Data indicate that in the goat MEC, LTA induces a weaker inflammatory response than LPS. This may be related to the observation that gram-positive bacteria cause chronic mastitis more often than gram-negative infections.

Pattern recognition receptors (PRRs) are important components of the innate immune system whose ligands are specific pathogen associated molecular patterns (PAMPs). Considering the scarcity of studies on transcription of PRRs in the pregnant uterus of cows, and its response to PAMPs and microorganisms that cause abortion in cattle, this study aimed to characterize the transcription of TLR1-10, NOD1, NOD2 and MD2 in bovine uterus throughout gestation and to investigate the sensitivity of different uterine tissues at third trimester of pregnancy to purified TLR ligands or heat-killed Brucella abortus, Salmonella enterica serotype Dublin (S. Dublin), Listeria monocytogenes, and Aspergillus fumigatus, by assessing chemokine transcription. RNA extracted from endometrium, placentome and intercotiledonary region of cows at the first (n=6), second (n=6), and third (n=6) trimesters of pregnancy were subjected to real time RT-PCR. After stimulation of endometrium and intercotiledonary regions with purified TLR ligands or heat-killed microorganisms, gene transcription was assessed by real time RT-PCR. In the placentome, there was no significant variation in TLRs transcription throughout the three trimesters of pregnancy. In the endometrium, there was significant variation in TLR4 and TLR5 transcription during the three stages of gestation; i.e. TLR4 transcription was higher during the third trimester, whereas TLR5 transcription was higher during the last two trimesters. In the intercotiledonary region, there was significant variation in transcription of TLR1/6, TLR7, and TLR8, which were more strongly expressed during the first trimester of pregnancy. At the third trimester of gestation, significant transcription of CXCL6 and CXCL8 was detected mostly in endometrial tissues in response to purified TLR4 and TLR2 ligands. Transcription of these chemokines was induced in the endometrium and intercotiledonary region at the third trimester of pregnancy when stimulated with heat-killed B. abortus or S. Dublin. Therefore, this study demonstrates that some PRRs are expressed in the uterus during pregnancy, which coincides with its ability to respond to stimulation with TLRs ligands as well as heat-killed organisms known to cause abortion in cattle.

Lipopolysaccharide (endotoxin, LPS) is a strong inducer of the innate immune response. It is widespread in our environment, e.g. in house dust and contributes to asthma. Compared to humans, horses are even more sensitive to LPS. However, data on LPS effects on the equine transcriptome are very limited. Using RNA-seq we analysed LPS-induced differences in the gene expression in equine peripheral blood mononuclear cells at the gene and gene-network level in two half-sib families and one group of unrelated horses.

24 h-LPS challenge of equine immune cells resulted in substantial changes in the transcriptomic profile (1,265 differentially expressed genes) showing partial overlap with human data. One of the half-sib families showed a specific response different from the other two groups of horses. We also identified co-expressed gene modules that clearly differentiated 24 h-LPS- from non-stimulated samples. These modules consisted of 934 highly interconnected genes and included genes involved in the immune response (e.g. IL6, CCL22, CXCL6, CXCL2), however, none of the top ten hub genes of the modules have been annotated as responsive to LPS in gene ontology.

Using weighted gene co-expression network analysis we identified ten co-expressed gene modules significantly regulated by in vitro stimulation with LPS. Apart from 47 genes (5%) all other genes highly interconnected within the most up- and down-regulated modules were also significantly differentially expressed (FDR < 0.05). The LPS-regulated module hub genes have not yet been described as having a role in the immune response to LPS (e.g. VAT1 and TTC25).

Prevalence of swine respiratory disease causes poor growth performance in and serious economic losses to the swine industry. In this study, a categorical trait of enzootic pneumonia-like (EPL) score representing the infection gradient of a respiratory disease, more likely enzootic pneumonia, was recorded in a herd of 332 Chinese Erhualian pigs. According to their EPL scores and the disease effect on weight gains, these pigs were grouped into controls (EPL score ≤ 1) and cases (EPL score>> 1). The weight gain of the case group reduced significantly at days 180, 210, 240 and 300 as compared to the control group. The heritability of EPL score was estimated to be 0.24 based on the pedigree information using a linear mixed model. All 332 Erhualian pigs and their nine sire parents were genotyped with Illumina Porcine 60K SNP chips. Two genome-wide association studies were performed under a generalized linear mixed model and a case-control model respectively. In total, five loci surpassed the suggestive significance level (P = 2.98 × 10-5 ) on chromosomes 2, 8, 12 and 14. CXCL6, CXCL8, KIT and CTBP2 were highlighted as candidate genes that might play important roles in determining resistance/susceptibility to swine EP-like respiratory disease. The findings advance understanding of the genetic basis of resistance/susceptibility to respiratory disease in pigs.

Neutrophils operate as part of the innate defence in the skin and may eliminate the Borrelia spirochaete via phagocytosis, oxidative bursts, and hydrolytic enzymes. However, their importance in Lyme neuroborreliosis (LNB) is unclear. Neutrophil extracellular trap (NET) formation, which is associated with the production of reactive oxygen species, involves the extrusion of the neutrophil DNA to form traps that incapacitate bacteria and immobilise viruses. Meanwhile, NET formation has recently been studied in pneumococcal meningitis, the role of NETs in other central nervous system (CNS) infections has previously not been studied. Here, cerebrospinal fluid (CSF) samples from clinically well-characterised children (N = 111) and adults (N = 64) with LNB and other CNS infections were analysed for NETs (DNA/myeloperoxidase complexes) and elastase activity. NETs were detected more frequently in the children than the adults (p = 0.01). NET presence was associated with higher CSF levels of CXCL1 (p < 0.001), CXCL6 (p = 0.007), CXCL8 (p = 0.003), CXCL10 (p < 0.001), MMP-9 (p = 0.002), TNF (p = 0.02), IL-6 (p < 0.001), and IL-17A (p = 0.03). NETs were associated with fever (p = 0.002) and correlated with polynuclear pleocytosis (r_s = 0.53, p < 0.0001). We show that neutrophil activation and active NET formation occur in the CSF samples of children and adults with CNS infections, mainly caused by Borrelia and neurotropic viruses. The role of NETs in the early phase of viral/bacterial CNS infections warrants further investigation.

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex-vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells.hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.

Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis, and S. aureus generally causes subclinical mastitis which is more persistent and resistant to treatment. Peptidoglycan (PGN) and lipoteichoic acid (LTA) are cell wall components of S. aureus. Although the roles of PGN and LTA in causing inflammation are well studied, the epigenetic mechanisms of the effects of PGN and LTA on the inflammation and lactation remain poorly understood. This study characterized the gene expression profiling by RNA sequencing and investigated DNA methylation and histone acetylation in relation to inflammation and lactation in the immortalized bovine mammary epithelial cell line (MAC-T). The cells were cultured for 24 h with neither PGN nor LTA (CON), PGN (30 μg/mL), LTA (30 μg/mL), and PGN (30 μg/mL) + LTA (30 μg/mL), respectively. The number of differentially expressed genes (DEGs) and the expression of proinflammatory factors including interleukin (IL)-1β, IL-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6 of the treatments increased in the following order: CON < PGN < LTA < PGN + LTA, and the DEGs mainly enriched on the cytokine-cytokine receptor interaction and chemokine signaling pathway. LTA and PGN + LTA induced hypomethylation of global DNA by suppressing DNA methyltransferase (DNMT) activity. PGN and LTA, alone or combined, decreased the mRNA expression of casein genes (CSN1S1, CSN2, and CSN3) and the expression of two caseins (CSN2 and CSN3), and reduced histone H3 acetylation by suppressing histone acetyltransferase (HAT) activity and promoting histone deacetylase (HDAC) activity. Collectively, this study revealed that PGN and LTA induced inflammation probably due to decreasing DNA methylation through regulating DNMT activity, and decreased lactation possibly through reducing histone H3 acetylation by regulating HAT and HDAC activity in bovine mammary epithelial cells.

BACKGROUND

Meniscus tears are classified as traumatic or degenerative based on the tear pattern. There is little evidence demonstrating biological differences between the 2 tear types.

OBJECTIVE

Gene expression signatures in the injured meniscus are different between traumatic (vertical) and degenerative (complex, horizontal, or flap) tears.

METHODS

Controlled laboratory study.

METHODS

Samples of the torn meniscus from the white-white zone were removed at the time of clinically indicated partial meniscectomy from 48 patients (37 with degenerative tears and 11 with traumatic tears). mRNA expression in the injured menisci was measured by quantitative real-time polymerase chain reaction for selected molecular markers of osteoarthritis, inflammation, and cartilage homeostasis (eg, cytokines/chemokines, aggrecanases/metalloproteinases, transcription factors, cartilage matrix genes, and adipokines). The tear pattern (traumatic or degenerative) and location (medial or lateral) were recorded for each patient. Gene expression differences between degenerative and traumatic tears were computed after adjusting for patients' age, sex, and body mass index and for location of the resected meniscus (medial/lateral).

RESULTS

Gene expression in meniscus tears varied by pattern. Chemokines ( IL8 [ P < .001] and CXCL6 [ P < .001]) and matrix metalloproteinases ( MMP1 [ P = .011] and MMP3 [ P = .016]) were expressed at a significantly higher level in traumatic tears compared with degenerative tears. In contrast, COL1A1 was expressed at a lower level in traumatic tears compared with degenerative tears ( P = .058). None of the genes tested demonstrated significant differences between medial and lateral meniscus tears.

CONCLUSIONS

Traumatic meniscus tears overall exhibited a higher inflammatory/catabolic response as evidenced by higher levels of chemokine and matrix metalloproteinase expression than degenerative tears. These findings suggest that there is a (molecular) biological distinction between traumatic and degenerative tears.

CONCLUSIONS

The catabolic/inflammatory differences between traumatic and degenerative tears may be relevant to treatment decisions regarding the meniscus as well as advance our understanding of how meniscus tears relate to the development of knee osteoarthritis.

Background: Ulcerative colitis (UC) is an inflammatory bowel disease that is difficult to diagnose and treat. To date, the degree of inflammation in patients with UC has mainly been determined by measuring the levels of nonspecific indicators, such as C-reactive protein and the erythrocyte sedimentation rate, but these indicators have an unsatisfactory specificity. In this study, we performed bioinformatics analysis using data from the National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) databases and verified the selected core genes in a mouse model of dextran sulfate sodium (DSS)-induced colitis.

Aim: To identify UC-related differentially expressed genes (DEGs) using a bioinformatics analysis and verify them in vivo and to identify novel biomarkers and the underlying mechanisms of UC.

Methods: Two microarray datasets from the NCBI-GEO database were used, and DEGs between patients with UC and healthy controls were analyzed using GEO2R and Venn diagrams. We annotated these genes based on their functions and signaling pathways, and then protein-protein interactions (PPIs) were identified using the Search Tool for the Retrieval of Interacting Genes. The data were further analyzed with Cytoscape software and the Molecular Complex Detection (MCODE) app. The core genes were selected and a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed. Finally, colitis model mice were established by administering DSS, and the top three core genes were verified in colitis mice using real-time polymerase chain reaction (PCR).

Results: One hundred and seventy-seven DEGs, 118 upregulated and 59 downregulated, were initially identified from the GEO2R analysis and predominantly participated in inflammation-related pathways. Seven clusters with close interactions in UC formed: Seventeen core genes were upregulated [C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine receptor 2 (CXCR2), CXCL9, CXCL5, C-C motif chemokine ligand 18, interleukin 1 beta, matrix metallopeptidase 9, CXCL3, formyl peptide receptor 1, complement component 3, CXCL8, CXCL1, CXCL10, CXCL2, CXCL6, CXCL11 and hydroxycarboxylic acid receptor 3] and one was downregulated [neuropeptide Y receptor Y1 (NYP1R)] in the top cluster according to the PPI and MCODE analyses. These genes were substantially enriched in the cytokine-cytokine receptor interaction and chemokine signaling pathways. The top three core genes (CXCL13, NYP1R, and CXCR2) were selected and verified in a mouse model of colitis using real-time PCR Increased expression was observed compared with the control mice, but only CXCR2 expression was significantly different.

Conclusion: Core DEGs identified in UC are related to inflammation and immunity inflammation, indicating that these reactions are core features of the pathogenesis of UC. CXCR2 may reflect the degree of inflammation in patients with UC.

Keywords: Bioinformatics analysis; C-X-C motif chemokine ligand 13; C-X-C motif chemokine receptor 2; Colitis model mice; Neuropeptide Y receptor Y1; Ulcerative colitis.

Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".

BACKGROUND

To recruit leucocytes to an inflammatory site, chemokine binding to glycosaminoglycans (GAGs) is critical. Therefore, strategies to interfere with this interaction, aiming at the production of anti-inflammatory agents, were developed. These include production of modified chemokines without affinity for G protein-coupled receptors but with enhanced affinity for GAGs. Such modified chemokines compete with functional chemokines for GAG binding, prevent chemokine immobilization and presentation, and inhibit leucocyte migration. In addition to modified chemokines, a GAG-binding peptide consisting of the 30 COOH-terminal residues of CXCL9, that is CXCL9(74-103), inhibited CXCL8- and monosodium urate crystal-induced neutrophil migration.

OBJECTIVE

We wanted to explore whether interference with chemokine-GAG interactions by CXCL9(74-103) reduces inflammation in neutrophil-dependent dinitrofluorobenzene-induced contact hypersensitivity.

METHODS

For this study, we evaluated several inflammatory parameters, including ear swelling and the levels of chemokines, cytokines, proteases and neutrophils in the ears of dinitrofluorobenzene-induced mice treated with CXCL9(74-103) or buffer.

RESULTS

One intravenous injection of CXCL9(74-103), just before painting with dinitrofluorobenzene on the ear, did not affect protein levels of the major murine neutrophil attractant, that is CXCL6, in this contact hypersensitivity model. However, IL-6, CXCL1, CCL2 and matrix metalloproteinase-9 (MMP-9) protein concentrations and peroxidase activity in challenged ears were reduced. In addition, intravenous injection of the CXCL9-derived peptide led to a reduced ear swelling response, indicating that the locally produced chemokines were hindered to attract leucocytes. The inhibiting potential of CXCL9(74-103) was explained by its competition for GAG binding with CXCL1, CXCL6 and CCL3 and inhibition of transendothelial migration of neutrophils to CXCL6.

CONCLUSIONS

The CXCL9(74-103) peptide inhibited dinitrofluorobenzene-induced infiltration of neutrophils and neutrophil-dependent inflammation in ears. Therefore, CXCL9(74-103) may be a lead molecule for the development of therapeutic peptides or peptide derivatives that compete with functional chemokines for GAG binding.

This study investigates if treatment with a peptide corresponding to the 30 C-terminal amino acids of CXCL9, CXCL9(74-103), ameliorates joint inflammation in a murine model of antigen-induced arthritis (AIA). AIA was induced in male C57BL/6J mice. Intravenous injection of CXCL9(74-103), simultaneously performed with a tibiofemoral challenge with methylated BSA (mBSA) as antigen in mice immunized with mBSA, diminished the accumulation of leukocytes, in particular neutrophils, in the synovial cavity. The levels of the chemokines CXCL1, CXCL2, and CXCL6 and of the cytokine IL-6 were decreased in inflamed periarticular tissue of mice treated with the CXCL9-derived peptide compared to non-treated AIA mice. In addition, CXCL9(74-103) treatment substantially reduced joint and cartilage damage. CXCL9(74-103) competes with CXCL6 and CCL3 for binding to the glycosaminoglycans heparan sulfate and chondroitin sulfate in vitro. In vivo, CXCL9(74-103) quickly binds to blood vessels in joints as observed by confocal microscopy. Next, we evaluated if later treatment with CXCL9(74-103) had a beneficial impact on joint inflammation. CXCL9(74-103) injection 6 h after mBSA challenge still reduced neutrophil accumulation in the joint, although it did not reduce chemokine and IL-6 concentrations. However, a delay of treatment until 12 h after challenge had no effect on cell recruitment and chemokine and IL-6 levels. Taken together, we demonstrated that treatment with a peptide, which interferes with the interaction between chemokines and glycosaminoglycans, from the beginning of the disease controlled the massive accumulation of neutrophils in the joint of AIA mice, greatly impacting on joint inflammation and tissue damage.

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

The molecular changes in gene expression following ablative laser treatment of skin lesions, such as atrophic scars and UV-damaged skin, are not completely understood. A standardized in vitro model of human skin, to study the effects of laser treatment on human skin, has been recently developed. Therefore, the aim of the investigation was to examine morphological and molecular changes caused by fractional ablative erbium:YAG laser treatment on an in vitro full-thickness 3D standardized organotypic model of human skin. A fractional ablative erbium:YAG laser was used to irradiate organotypic human 3D models. Laser treatments were performed at four different settings using a variety of stacked pulses with similar cumulative total energy fluence (60 J/cm2). Specimens were harvested at specified time points and real-time PCR (qRT-PCR) and microarray studies were performed. Frozen sections were examined histologically. Three days after erbium:YAG laser treatment, a significantly increased mRNA expression of matrix metalloproteinases and their inhibitors (MMP1, MMP2, MMP3, TIMP1, and TIMP2), chemokines (CXCL1, CXCL2, CXCL5, and CXCL6), and cytokines such as IL6, IL8, and IL24 could be detected. qRT-PCR studies confirmed the enhanced mRNA expression of IL6, IL8, IL24, CXCLs, and MMPs. In contrast, the mRNA expression of epidermal differentiation markers, such as keratin-associated protein 4, filaggrin, filaggrin 2, and loricrin, and antimicrobial peptides (S100A7A, S100A9, and S100A12) as well as CASP14, DSG2, IL18, and IL36β was reduced. Four different settings with similar cumulative doses have been tested (N10%, C10%, E10%, and W25%). These laser treatments resulted in different morphological changes and effects on gene regulations. Longer pulse durations (1000 μs) especially had the strongest impact on gene expression and resulted in an upregulation of genes, such as collagen-1A2, collagen-5A2, and collagen-6A2, as well as FGF2. Histologically, all treatment settings resulted in a complete regeneration of the epidermis 3 days after irradiation. Fractional ablative erbium:YAG laser treatment with a pulse stacking technique resulted in histological alterations and shifts in the expression of various genes related to epidermal differentiation, inflammation, and dermal remodeling depending on the treatment setting applied. A standardized in vitro 3D model of human skin proved to be a useful tool for exploring the effects of various laser settings both on skin morphology and gene expression during wound healing. It provides novel data on the gene expression and microscopic architecture of the exposed skin. This may enhance our understanding of laser treatment at a molecular level.

Clinical factors and liver biopsy cannot accurately predict the risk of developing cirrhosis in chronic hepatitis B (CHB).This study was to develop a predictive gene signature for cirrhosis in CHB patients. A total of 183 untreated CHB patients were enrolled. GeneChip, significant analysis of microarray (SAM) and prediction analysis of microarray (PAM) were used to select predictor genes (PGs) in liver tissues. The Cirrhosis Risk Score (CRS) was calculated based on 6 PG variables and the predictive value of CRS was evaluated. Firstly differentially expressed genes were filtered from a genome scan and SAM, and 87 significant genes were selected for the signature building. Secondly a signature consisting of 6 PGs (CD24, CXCL6, EHF, ITGBL1, LUM and SOX9) most predictive for cirrhosis risk in CHB patients was developed in the selection set (n=40) by use of PAM and PCR approach. Finally the CRS was calculated to estimate the risk of developing cirrhosis and then tested in validation cohort (n=143). The area under the ROC curves (AUROC) of the CRS was 0.944 and exceeded to 6 PGs and clinical factors. A low CRS cutoff of 6.43 to identify low-risk patients would misclassify only 8.16% of high-risk patients, while a high cutoff of 8.32 to identify high-risk patients would misclassify 0% of low-risk patients. So CRS is a better predictor than clinical factors in differentiating high-risk versus low-risk for cirrhosis and application of CRS in clinical practice could help to reduce the rate of liver biopsy in patients with CHB.

Schistosomiasis is a significant cause of human morbidity and mortality. We performed a genome-wide transcriptional survey of liver biopsies obtained from Chinese patients with chronic schistosomiasis only, or chronic schistosomiasis with a current or past history of viral hepatitis B. Both disease groups were compared with patients with no prior history or indicators of any liver disease. Analysis showed in the main, downregulation in gene expression, particularly those involved in signal transduction via EIF2 signalling and mTOR signalling, as were genes associated with cellular remodelling. Focusing on immune associated pathways, genes were generally downregulated. However, a set of three genes associated with granulocytes, MMP7, CLDN7, CXCL6 were upregulated. Differential gene profiles unique to schistosomiasis included the gene Granulin which was decreased despite being generally considered a marker for liver disease, and IGBP2 which is associated with increased liver size, and was the most upregulated gene in schistosomiasis only patients, all of which presented with hepatomegaly. The unique features of gene expression, in conjunction with previous reports in the murine model of the cellular composition of granulomas, granuloma formation and recovery, provide an increased understanding of the molecular immunopathology and general physiological processes underlying hepatic schistosomiasis.

BACKGROUND

The pathophysiological events inhibited by prophylactic digital hypothermia that result in reduction of the severity of acute laminitis are unknown.

OBJECTIVE

To determine if digital hypothermia inhibits lamellar inflammatory signalling during development of oligofructose (OF) induced laminitis.

METHODS

Fourteen Standardbred horses were given 10 g/kg bwt OF by nasogastric tube with one forelimb (CRYO) continuously cooled by immersion in ice and water and one forelimb (NON-RX) at ambient temperature. Lamellae were harvested prior to the onset of lameness (24 h post OF administration, DEV group, n = 7) or at the onset of lameness (OG1 group, n = 7). Lamellar mRNA was purified and cDNA produced for real time-quantitative PCR analysis of mRNA concentrations of cytokines (IL-6, IL-1β, IL-10), chemokines (CXCL1, CXCL6, CXCL8/IL-8, MCP-1, MCP-2), cell adhesion molecules (ICAM-1, E-selectin), COX-2 and 3 housekeeping genes. Data were analysed (NON-RX vs. CRYO, NON-RX vs. archived control [CON, n = 7] lamellar tissue) using nonparametric tests.

RESULTS

Compared with CON, the OG1 NON-RX had increased (P<0.05) lamellar mRNA concentrations of all measured mediators except IL-10, IL-1β and MCP-1/2, whereas only CXCL8 was increased (P<0.05) in DEV NON-RX. Within the OG1 group, CRYO limbs (compared with NON-RX) had decreased (P<0.05) mRNA concentrations of the majority of measured inflammatory mediators (no change in MCP-1 and IL-10). Within the DEV group, mRNA concentrations of CXCL-1, ICAM-1, IL-1β, CXCL8 and MCP-2 were decreased (P<0.05) and the anti-inflammatory cytokine IL-10 was increased (compared with NON-RX limbs; P<0.05).

CONCLUSIONS

Digital hypothermia effectively blocked early lamellar inflammatory events likely to play an important role in lamellar injury including the expression of chemokines, proinflammatory cytokines, COX-2 and endothelial adhesion molecules.

CONCLUSIONS

This study demonstrates a potential mechanism by which hypothermia reduces the severity of acute laminitis, and may help identify molecular targets for future laminitis intervention.

The use of C(60) fullerenes is expected to increase in various industrial fields. Little is known about the potential toxicological mechanism of action of water-soluble C(60) fullerenes. In our previous research, gene expression profiling of the rat lung was performed after whole-body inhalation exposure to C(60) fullerenes to gain insights into the molecular events. These DNA microarray-based data closely matched the pathological findings that C(60) fullerenes caused no serious adverse pulmonary effects under the inhalation exposure condition. Taking advantage of this, we attempted to characterize time-dependent changes in the gene expression profiles after intratracheal instillation with C(60) fullerenes at different dosages and to identify the candidate expressed genes as potential biomarkers. The hierarchical cluster analysis revealed that the up- or downregulation of genes after intratracheal instillation with 1.0 mg C(60) fullerene particles in rat lung tissue was significantly over-represented in the "response to stimulus" and "response to chemical stimulus" categories of biological processes and in the "extracellular space" category of the cellular component. These results were remarkable for 1 week after the instillation with C(60) fullerenes. In the lung tissues instilled with 1.0 mg C(60) fullerene particles, many representative genes involved in "inflammatory response," such as the Cxcl2, Cxcl6, Orm1, and Spp1 genes, and in "matrix metalloproteinase activity," such as the Mmp7 and Mmp12 genes, were upregulated for over 6 months. The expression levels of 89 and 21 genes were positively correlated with the C(60) fullerene dose at 1 week and 6 months after the instillation, respectively. Most of them were involved in "inflammatory response", and the Ccl17, Ctsk, Cxcl2, Cxcl6, Lcn6, Orm1, Rnase9, Slc26a4, Spp1, Mmp7, and Mmp12 genes were overlapped. Meanwhile, the expression levels of 16 and 4 genes were negatively correlated with the C(60) fullerene dose at 1 week and 6 months after the instillation, respectively. Microarray-based gene expression profiling suggested that the expression of some genes is correlated with the dose of intratracheally instilled C(60) fullerenes. We propose that these genes are useful for identifying potential biomarkers in acute-phase or persistent responses to C(60) fullerenes in the lung tissue.

Hepatocellular carcinoma (HCC) is a common liver malignancy worldwide accompanying with the high rate of recurrence. Accumulating reports have documented the significance of circular RNAs (circRNAs) in carcinogenesis and development of HCC. This study aimed to establish the mechanism underlying circ-HOMER1 involvement in HCC. To this end, we identified a binding site for miR-1322 via bioinformatics, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and dual-luciferase reporter assays providing evidence of a direct link between circ-HOMER1 and miR-1322. Similarly, the target gene of miR-1322 was investigated. Moreover, we determined the specific function of circ-HOMER1 in HCC with the aid of qRT-PCR based on patient clinical records, Cell Counting Kit-8, acridine orange/ethidium bromide double fluorescence staining, flow cytometry, and wound-healing and transwell assays. Notably, circ-HOMER1 was upregulated in both HCC cells and tissues. This aberrant expression pattern was closely correlated with larger tumor size, higher tumor-node-metastasis stage, and poorer prognosis for the patients with HCC. Moreover, silenced circ-HOMER1 inhibited cell proliferation, migration, and invasion concomitant with the promotion of apoptosis in HCC cells, and vice versa. Mechanistically, circ-HOMER1 enhanced the inhibition of miR-1322 on CXCL6 in HCC. Furthermore, we found that circ-HOMER1 promoted HCC cell growth and aggressiveness by miR-1322/CXCL6 axis. This study may provide a potential prognostic indicator and therapeutic target for patients with HCC.

BACKGROUND

Chronic inflammation disrupts dental pulp regeneration by disintegrating the recruitment process of progenitors for repair. Bone marrow-derived mesenchymal stem cells (BM-MSCs) share the common features with dental pulp stem cells (DPSCs). The aim of the study was to investigate the migration of BM-MSCs toward DPSCs in response to inflammatory chemoattractants. Additionally, our studies also delineated the signaling mechanisms from BM-MSCs in mediating the proliferation and differentiation of DPSCs in vitro.

METHODS

Human DPSCs and BM-MSCs between passages 2 and 4 were used and were grown in odontogenic differentiation medium. Mineralization was determined by alizarin red staining analysis. Migration was assessed using crystal violet staining in cells grown in Boyden chamber Transwell inserts (Corning Inc Foundation, Tewksbury, MA). The mineralization potential of DPSCs was evaluated using alkaline phosphatase activity assay. Real-time polymerase chain reaction analysis was performed to assess the gene expression profile of chemokine (C-X-C motif) ligand (Cxcl) 3, 5, 6, 10, 11, 12, 14, and 16; stromal cell-derived factor (SDF) α; vascular endothelial growth factor; and fibroblast growth factor.

RESULTS

Interferon gamma (FN-γ) treatment significantly abrogated the differentiation potential of DPSCs as shown by using alizarin red and alkaline phosphatase activity analysis. An increase in the migration of BM-MSCs was documented when cocultured with IFN-γ-treated DPSCs. RNA expression studies showed an increase in the levels of Cxcl6 and Cxcl12 in BM-MSCs when cocultured with IFN-γ-treated DPSCs. Additionally, an up-regulation of proangiogenic factors vascular endothelial growth factor and fibroblast growth factor were observed in DPSCs exposed to IFN-γ.

CONCLUSIONS

Our findings indicate that inflamed IFN-γ-treated DPSCs release factors (presumably Cxcl6 and 12) that contribute to the homing of MSCs. This model might provide a potential research tool for studying MSC-DPSC cross talk and for future studies involving the recruitment and sustainability of progenitor stem cells sustaining the inflammatory cascade to treat pulp inflammation.

The roles of the pro-adipogenic ligands of the transcription factor Peroxisome Proliferator Activated Receptor gamma (PPARG) in regulating innate immune responses in bovine mammary epithelial cells (bMEC) were investigated using quantitative real-time PCR. The analyses revealed that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) enhanced the expression of Interleukin 8 (IL-8) and Chemokine (C-X-C motif) ligand 6 (CXCL6) in these cells in a dose-dependent manner. 15d-PGJ2 also induced the expression of transcripts encoding proteins involved in oxidative stress, including Ferritin heavy chain and Superoxide dismutase 1, as well as substantial microfilament reorganization. In contrast, synthetic PPARG agonists displayed a different and much smaller range of effects on the cells, causing down-regulation of Interleukin 1-beta, Interleukin 6 and IL-8 and increased expression of Chemokine (C-C motif) ligand 2 (CCL2) and Tumour necrosis factor alpha (TNFalpha). In an independent analysis, the cells were pre-incubated with PPARG agonists followed by lipopolysaccharide stimulation. This study revealed that troglitazone increased the responsiveness of the cells to lipopolysaccharide resulting in up-regulation of Interleukin 1-beta, TNFalpha, IL-8, CCL2 and CXCL6 while 15d-PGJ2 caused down-regulation of TNFalpha, CCL2 and CXCL6. These findings are relevant to understanding the anti-inflammatory potential of the PPARG ligands and underline different mechanisms of action of 15d-PGJ2 and troglitazone in bMEC. Furthermore, the present results demonstrate that the generation of pro-inflammatory mediators can be modulated by currently available therapeutic agents and may therefore be of value in the treatment of mastitis in ruminants.