Effects of replacing inorganic salts of trace minerals with organic trace minerals in the diet of prepartum cows on quality of colostrum and immunity of newborn calves.

Our objectives were to evaluate the impact of supplementary trace mineral (TM) form-inorganic salts (STM; Co, Cu, Mn, Zn sulfates, and Na selenite) or organic (OTM; Co, Cu, Mn, Zn proteinates, and selenized yeast)-in the prepartum diet on quantity and quality of colostrum, passive immunity, antioxidant biomarkers, cytokine responses to lipopolysaccharide (LPS), health, and growth of newborn calves. Pregnant heifers (n = 100) and cows (n = 173) were enrolled at 45 d before calving, blocked by parity and body condition score, and allocated randomly to STM (50 heifers; 86 cows) or OTM (50 heifers; 87 cows) supplementation. Cows in both treatments were fed the same diet, except for the source of supplementary TM. Within 2 h of calving, dams and calves were separated, colostrum was harvested, the yield was measured, and a sample was saved for posterior analyses of colostrum quality. A subgroup of calves (n = 68) had a blood sample collected before colostrum feeding. After colostrum feeding, all samples and data collection were limited to 163 calves (STM = 82; OTM = 81) fed 3 L of good quality (Brix% >22) maternal colostrum via nipple bottle minutes after harvesting. Concentration of IgG in colostrum and serum was determined 24 h after colostrum feeding using radial immunodiffusion. Concentration of TM in colostrum and serum were performed by inductively coupled plasma mass spectrometry. Activity of glutathione peroxidase, ferric reducing ability of plasma, and concentration of superoxide dismutase were evaluated in plasma by colorimetric assays. Ex vivo whole blood stimulation with LPS was performed on d 7 of life to evaluate cytokine responses in a subgroup of 66 calves. Health events were recorded from birth to weaning, and body weight was recorded at birth (all calves) and on d 30 and 60 (heifers only). Continuous variables were analyzed by ANOVA and binary responses were analyzed by logistic regression. Complete replacement of STM by OTM in prepartum diet resulted in greater concentration of Se (461 vs. 543 ± 7 µg/g; ± SEM) but did not alter the concentration or total mass of other TM and IgG in colostrum. Female calves of the OTM group had greater concentration of Se in serum at birth (0.23 vs. 0.37 ± 0.05 µg/mL), were lighter in weight at birth (40.9 vs. 38.8 ± 0.6 kg) and weaning (93.2 vs. 89.7 ± 1.6 kg) than those of the STM group. Maternal treatments did not affect passive immunity or antioxidant biomarkers. On d 7, basal concentrations (log10 of concentration in pg/mL) of IFNγ (0.70 vs. 0.95 ± 0.083) and LPS-stimulated concentrations of CC chemokine ligand 2 (CCL2; 2.45 vs. 2.54 ± 0.026), CC chemokine ligand 3 (CCL3; 2.63 vs. 2.76 ± 0.038), IL-1α (2.32 vs. 2.49 ± 0.054), and IL-1ß (3.62 vs. 3.86 ± 0.067) were greater in OTM than in STM. Supplementation with OTM in pregnant heifers, but not in pregnant cows, reduced the incidence of preweaning health problems in their calves (36.4 vs. 11.5%). Complete replacement of STM by OTM in the prepartum diet did not cause major changes in colostrum quality, passive immunity, and antioxidant capacity, but increased cytokine and chemokine responses to LPS on d 7 of life and benefited preweaning health of calves born to primiparous cows.

The survival of epidemic and sporadic MRSA on human skin mimics is determined by both host and bacterial factors.

Bacterial survival on, and interactions with, human skin may explain the epidemiological success of MRSA strains. We evaluated the bacterial counts for 27 epidemic and 31 sporadic MRSA strains on 3D epidermal models based on N/TERT cells (NEMs) after 1, 2 and 8 days. In addition, the expression of antimicrobial peptides (hBD-2, RNase 7), inflammatory cytokines (IL-1ß, IL-6) and chemokine IL-8 by NEMs was assessed using immunoassays and the expression of 43 S. aureus virulence factors was determined by a multiplex competitive Luminex assay. To explore donor variation, bacterial counts for five epidemic and seven sporadic MRSA strains were determined on 3D primary keratinocyte models (LEMs) from three human donors. Bacterial survival was comparable on NEMs between the two groups, but on LEMs, sporadic strains showed significantly lower survival numbers compared to epidemic strains. Both groups triggered the expression of immune factors. Upon interaction with NEMs, only the epidemic MRSA strains expressed pore-forming toxins, including alpha-hemolysin (Hla), gamma-hemolysin (HlgB), Panton-Valentine leucocidin (LukS) and LukED. Together, these data indicate that the outcome of the interaction between MRSA and human skin mimics, depends on the unique combination of bacterial strain and host factors.

Performance of a Standardized Clinical Assay for Urinary C-C Motif Chemokine Ligand 14 (CCL14) for Persistent Severe Acute Kidney Injury.

Background: Clinical use of biomarkers requires the development of standardized assays and establishment of cutoffs. Urinary C-C motif chemokine ligand 14 (CCL14) has been validated to predict persistent severe AKI in critically ill patients with established AKI. We now report on the performance of standardized cutoffs using a clinical assay. Methods: A second aim of the multicenter RUBY Study was to establish two cutoffs for the prediction of persistent severe AKI (defined as KDIGO stage 3 AKI for at least 72 consecutive hours). Patients who received renal replacement therapy (RRT) or died before achieving 72 hours in stage 3 AKI were also considered to have reached the end point. Results: A cutoff value for urinary CCL14 of 1.3 ng/ml was determined to achieve high sensitivity (91%; 95% CI, 84% to 96%), and 13 ng/ml achieved high specificity (93%; 95% CI, 89% to 96%). The cutoff of 1.3 ng/ml identifies the majority (91%) of patients who developed persistent severe AKI with a negative predictive value of 92%. The cutoff at 13 ng/ml had a positive predictive value of 72% (with a negative predictive value of 75%). In multivariable adjusted analyses, a CCL14 concentration between 1.3 and 13 ng/ml had an adjusted odds ratio (aOR) of 3.82 (95% CI, 1.73 to 9.12; P=0.001) for the development of persistent severe AKI compared with those with a CCL14 ≤1.3 ng/ml, whereas a CCL14 >13 ng/ml had an aOR of 10.4 (95% CI, 3.89 to 29.9; P<0.001). Conclusions: Using a clinical assay, these standardized cutoffs (1.3 and 13 ng/ml) allow for the identification of patients at high risk for the development of persistent severe AKI. These results have immediate utility in helping to guide AKI patient care and may facilitate future clinical trials.Clinical Trial registry name and registration number: Identification and Validation of Biomarkers of Acute Kidney Injury Recovery, NCT01868724.

Identification of early diagnostic biomarkers via WGCNA in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the world's second-leading cause of cancer-related mortality, continuing to make it a serious healthcare concern. Even though the prevalence of GC reduces, the prognosis for GC patients remains poor in terms of a lack of reliable biomarkers to diagnose early GC and predict chemosensitivity and recurrence. METHODS AND MATERIAL: We integrated the gene expression patterns of gastric cancers from four RNAseq datasets (GSE113255, GSE142000, GSE118897, and GSE130823) from Gene Expression Omnibus (GEO) database to recognize differentially expressed genes (DEGs) between normal and GC samples. A gene co-expression network was built using weighted co-expression network analysis (WGCNA). Furthermore, RT-qPCR was performed to validate the in silico results. RESULTS: The red modules in GSE113255, Turquoise in GSE142000, Brown in GSE118897, and the green-yellow module in GSE130823 datasets were found to be highly correlated with the anatomical site of GC. ITGAX, CCL14, ADHFE1, and HOXB13) as the hub gene are differentially expressed in tumor and non-tumor gastric tissues in this study. RT-qPCR demonstrated a high level of the expression of this gene. CONCLUSION: The expression levels of ITGAX, CCL14, ADHFE1, and HOXB13 in GC tumor tissues are considerably greater than in adjacent normal tissues. Systems biology approaches identified that these genes could be possible GC marker genes, providing ideas for other experimental studies in the future.

Expressions of CXCL12, CXCL10 and CCL18 in Warthin tumors characterized pathologically by having a lymphoid stroma with germinal centers.

The Warthin tumor is a benign neoplasm of the salivary glands, histologically, the tumor has an oncocytic epithelial component forming uniform rows of cells surrounded by cystic spaces associated with a lymphoid stroma often showing the presence of germinal centers. The lymphoid stroma is a representative microscopic finding. If this lymphocytic accumulation is active, some sort of transmitter should exist between the Warthin tumor cells and lymphocytes. C-X-C motif chemokine ligand (CXCL12) 12, CXCL10 and C-C motif chemokine ligand 18 (CCL18) are a chemoattractant for lymphocytes in vivo. There is no report on the relationship between these chemokines and Warthin tumors. In this study, we investigated these chemokines expressions in 20 Warthin tumors using immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). For comparison, we also enrolled samples of pleomorphic adenoma, which is another benign salivary gland tumor type without prominent lymphocytic infiltration. All Warthin tumors were immunopositive for CXCL12 and CXCL10, and these reactivities were diffuse. Meanwhile, the majority of pleomorphic adenomas were immunonegative for CXCL12 (95%), CXCL10 (80%) and CCL18 (85%). Warthin tumor and pleomorphic adenoma cases were significantly different in these immunostaining expressions (CXCL12, p<0.001; CXCL10, p<0.001; CCL18, p=0.024). We examined CXCL12, CXCL10 and CCL18 mRNA expressions of 3 representative Warthin tumor samples, each having these chemokines immunopositive areas detected by RT-PCR. Finding CXCL12 and CXCL10 expressions indicate that these chemokines may play a part in the formation of a lymphoid stroma within Warthin tumors. In regards to this phenomenon, the participation of CCL18 might be restrictive compared to CXCL12 and CXCL10.

External validation of urinary C-C motif chemokine ligand 14 (CCL14) for prediction of persistent acute kidney injury.

BACKGROUND: Persistent acute kidney injury (AKI) portends worse clinical outcomes and remains a therapeutic challenge for clinicians. A recent study found that urinary C-C motif chemokine ligand 14 (CCL14) can predict the development of persistent AKI. We aimed to externally validate urinary CCL14 for the prediction of persistent AKI in critically ill patients. METHODS: This was a secondary analysis of the prospective multi-center SAPPHIRE study. We evaluated critically ill patients with cardiac and/or respiratory dysfunction who developed Kidney Disease: Improving Global Outcomes (KDIGO) stage 2-3 AKI within one week of enrollment. The main exposure was the urinary concentration of CCL14 measured at the onset of AKI stage 2-3. The primary endpoint was the development of persistent severe AKI, defined as ≥ 72 h of KDIGO stage 3 AKI or death or renal-replacement therapy (RRT) prior to 72 h. The secondary endpoint was a composite of RRT and/or death by 90 days. We used receiver operating characteristic (ROC) curve analysis to assess discriminative ability of urinary CCL14 for the development of persistent severe AKI and multivariate analysis to compare tertiles of urinary CCL14 and outcomes. RESULTS: We included 195 patients who developed KDIGO stage 2-3 AKI. Of these, 28 (14%) developed persistent severe AKI, of whom 15 had AKI ≥ 72 h, 12 received RRT and 1 died prior to ≥ 72 h of KDIGO stage 3 AKI. Persistent severe AKI was associated with chronic kidney disease, diabetes mellitus, higher non-renal APACHE III score, greater fluid balance, vasopressor use, and greater change in baseline serum creatinine. The AUC for urinary CCL14 to predict persistent severe AKI was 0.81 (95% CI, 0.72-0.89). The risk of persistent severe AKI increased with higher values of urinary CCL14. RRT and/or death at 90 days increased within tertiles of urinary CCL14 concentration. CONCLUSIONS: This secondary analysis externally validates urinary CCL14 to predict persistent severe AKI in critically ill patients.

Cytokines and chemokines associated with Treg/Th17 response in chronic inflammatory periapical disease.

Cytokines and chemokines have a fundamental role in the maintenance of inflammation and bone response, which culminate in the development of chronic periapical lesions. Regulatory (Treg) and Th17 cytokines play a key role in regulating the immune response involved in this process. The aim of this study was to investigate the role of Treg and Th17 cells in chronic inflammatory periapical disease, by comparing the expression of the immunoregulatory mediators TGF-ß, IL-10, CCL4, and the proinflammatory IL-17 and CCL20 in the periapical tissue of teeth with pulp necrosis, with and without associated chronic lesions. Eighty-six periapical tissue samples were obtained from human teeth. The samples were divided into three groups: pulp necrosis with a periapical lesion (n=26); pulp necrosis without a periapical lesion (n=30), and control (n=30). All samples were submitted to histopathological analysis and cytokine and chemokine measurement through ELISA. Statistical analyses were done with Kruskal-Wallis and Mann-Whitney tests and Spearman correlation. The group with pulp necrosis and a periapical lesion showed a higher expression of CCL4 and TGF-ß in comparison with pulp necrosis without a lesion. CCL20 was higher in the group with a periapical lesion when compared to the control. In all groups there was a weak positive correlation between IL-17/CCL20, IL-10/CCL4, and IL-17/TGF-ß. Both types of cytokines, pro-inflammatory and immunoregulatory, occur simultaneously in periapical tissue. However, a rise in immunosuppressive cytokines and chemokines (CCL4 and TGF-ß) in periapical lesions suggests a role of these cytokines in stable periapical disease.

Comprehensive analysis of vitreous humor chemokines in type 2 diabetic patients with and without diabetic retinopathy.

AIMS: To compare the vitreous levels of chemokines in diabetic patients with and without retinopathy. To find the relationship between stages of diabetic retinopathy (DR) and levels of vitreous chemokines. METHODS: The study involved 20 non-diabetic and 20 diabetic patients without clinical signs of DR (NDR) and 40 diabetic patients with proliferative diabetic retinopathy (PDR). The vitreous humor was collected and the levels of 40 chemokines were measured using magnetic color-bead-based multiplex assay. RESULTS: The control group, NDR group, PDR with vitreous hemorrhage (VH) group, and PDR with tractional retinal detachment group comprised 20, 20, 21, and 19 eyes, respectively. Only the concentration of CCL3 was significantly higher in the NDR group compared with the controls (p = 0.038). Twenty-five types of chemokines were statistically higher in the PDR with VH group in comparison to NDR group (all p < 0.05). All chemokines were statistically higher in the PDR with TRD group in comparison to NDR group (all p < 0.05) apart from 3 chemokines: GM-CSF, MIF, and CCL3(p = 0.086, p = 0.109, p = 0.094, respectively). The concentration of CCL21, CCL15 in PDR with TRD group was significantly higher compared with PDR with VH group, while other 36 chemokines were not significantly different between PDR with VH group and PDR with TRD group. CONCLUSIONS: The inflammation gradually worsen with the progression of DR. CCL3 may be associated with the onset of early diabetic retinal damage, and CCL15 and CCL21 may be closely related to the formation of fibrovascular membrane and the progression of the end stage of DR.

Cytokines and chemokines associated with Treg/Th17 response in chronic inflammatory periapical disease

Abstract Cytokines and chemokines have a fundamental role in the maintenance of inflammation and bone response, which culminate in the development of chronic periapical lesions. Regulatory (Treg) and Th17 cytokines play a key role in regulating the immune response involved in this process. The aim of this study was to investigate the role of Treg and Th17 cells in chronic inflammatory periapical disease, by comparing the expression of the immunoregulatory mediators TGF-β, IL-10, CCL4, and the proinflammatory IL-17 and CCL20 in the periapical tissue of teeth with pulp necrosis, with and without associated chronic lesions. Eighty-six periapical tissue samples were obtained from human teeth. The samples were divided into three groups: pulp necrosis with a periapical lesion (n=26); pulp necrosis without a periapical lesion (n=30), and control (n=30). All samples were submitted to histopathological analysis and cytokine and chemokine measurement through ELISA. Statistical analyses were done with Kruskal-Wallis and Mann-Whitney tests and Spearman correlation. The group with pulp necrosis and a periapical lesion showed a higher expression of CCL4 and TGF-β in comparison with pulp necrosis without a lesion. CCL20 was higher in the group with a periapical lesion when compared to the control. In all groups there was a weak positive correlation between IL-17/CCL20, IL-10/CCL4, and IL-17/TGF-β. Both types of cytokines, pro-inflammatory and immunoregulatory, occur simultaneously in periapical tissue. However, a rise in immunosuppressive cytokines and chemokines (CCL4 and TGF-β) in periapical lesions suggests a role of these cytokines in stable periapical disease.

Tumor Necrosis Factor-α and C-C Motif Chemokine Ligand 18 Associate with Atherosclerotic Lipid Accumulation In situ and In vitro.

Atherosclerosis is regarded as a chronic inflammatory disease associated with changes in the innate immune system functioning and cytokine disturbances. Local inflammation in the arterial wall is an important component in the development and growth of atherosclerotic plaques. Inside the lesions, both pro- and antiinflammatory cytokines were detected, highlighting the complexity of the atherosclerotic process. However, little is known about the expression of these signaling molecules in early human atherosclerotic lesions. In this study, we explored localization of a pro-inflammatory cytokine, tumor necrosis factor-α (TNFα), and anti-inflammatory chemokine, C-C motif chemokine ligand 18 (CCL18), in the arterial wall of human aorta. We noticed differences in the intensity of staining for TNFα and CCL18 in atherosclerotic lesions and grossly normal areas, as well as differences in their localization. While CCL18 prevailed in the areas close to the aortic lumen, TNFα was localized in deeper layers of the intima. We next studied the expression of TNFα and CCL18 mRNA in lesions corresponding to different stages of atherosclerosis progression and found that it was maximal in lipofibrous plaques that are most enriched in lipids. To test the hypothesis that cytokine expression can be associated with lipid accumulation, we studied the TNFα and CCL18 expression profiles in primary human monocyte-derived macrophages after inducing lipid accumulation by incubating cultured cells with atherogenic LDL. We found that intracellular cholesterol accumulation was associated with upregulation of both TNFα and CCL18, confirming our hypothesis. These results encourage further investigation of cytokine expression in human atherosclerotic lesions and its role in the atherosclerosis progression.

Significance of molecular biomarkers in idiopathic pulmonary fibrosis: A mini review.

Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible condition with poor prognosis that is characterized by a variable clinical course in each patient, which renders it a complex disease with unknown causes. Despite the proven efficacy of novel antifibrotic therapies, including pirfenidone and nintedanib, the diagnosis and follow-up of IPF remain challenging. Hence, the identification of molecular biomarkers for early detection of IPF and to predict biologically determined individual clinical courses, has recently piqued the interest of researchers. Previous studies have demonstrated the diagnostic and prognostic efficacy of blood proteins such as KL-6, Surfactant protein (SP)-A, and SP-D, in patients with IPF. Due to their use in clinical practice in Japan, for approximately twenty years, a significant amount of data about these biomarkers has been accumulated. This paper reviews the recent literature on molecular biomarkers for IPF that have been developed in Japan as well as other potential molecular biomarkers.

Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice.

Severe acute pancreatitis is a lethal inflammatory disease frequently accompanied by pancreatic necrosis. We aimed to identify a key regulator in the development of pancreatic necrosis. A cytokine/chemokine array using sera from patients with acute pancreatitis (AP) revealed that serum CXCL16 levels were elevated according to the severity of pancreatitis. In a mouse model of AP, Cxcl16 expression was induced in pancreatic acini in the late phase with the development of pancreatic necrosis. Cxcl16-/- mice revealed similar sensitivity as wild-type (WT) mice to the onset of pancreatitis, but better resisted development of acinar cell necrosis with attenuated neutrophil infiltration. A cytokine array and immunohistochemistry revealed lower expression of Ccl9, a neutrophil chemoattractant, in the pancreatic acini of Cxcl16-/- mice than WT mice. Ccl9 mRNA expression was induced by stimulation with Cxcl16 protein in pancreatic acinar cells in vitro, suggesting a Cxcl16/Ccl9 cascade. Neutralizing antibody against Cxcl16 ameliorated pancreatic injury in the mouse AP model with decreased Ccl9 expression and less neutrophil accumulation. In conclusion, Cxcl16 expressed in pancreatic acini contributes to the development of acinar cell necrosis through the induction of Ccl9 and subsequent neutrophil infiltration. CXCL16 could be a new therapeutic target in AP.

An initial investigation into endothelial CC chemokine expression in the human rheumatoid synovium.

Rheumatoid arthritis (RA) is a destructive and chronic autoimmune inflammatory disease. Synovial inflammation is a major feature of RA and is associated with leukocyte recruitment. Leukocytes cross the endothelial cells (ECs) into the synovial tissue and fluid and this migration is mediated via a range of chemokines and adhesion molecules on the ECs. As important mediators of leukocyte extravasation, a number of chemokines from each of the chemokine families have been established as expressed in the RA joint. However, as little information is available on which chemokines are expressed/presented by the ECs themselves, the purpose of the study was to ascertain which of the CC chemokines were localised in RA ECs. Immunofluoresence was used to assess the presence of the CC-family chemokines in RA synovial ECs using von-Willebrand factor (VWF) as a pan-endothelial marker and a range of human chemokine antibodies. The percentage of VWF positive vessels which were positive for the chemokines was determined. The presence of the four most highly expressed novel chemokines were further investigated in non-RA synovial ECs and the sera and synovial fluid (SF) from patients with RA and osteoarthritis (OA). Statistical analysis of immunofluorescence data was carried out by Student's t-test. For analysis of ELISA data, Kruskal-Wallis ANOVA followed by Dunn's multiple comparison test was utilised to analyse differences in sera and SF levels for each chemokine between RA and OA. Spearman rank correlations of sera and SF chemokine levels with a range of clinical variables were also performed. Chemokine detection varied, the least abundant being CCL27 which was present in 8.3% of RA blood vessels and the most abundant being CCL19 which was present in 80%. Of the 26 chemokines studied, 19 have not been previously observed in RA ECs. Four of these novel chemokines, namely CCL7, CCL14, CCL16 and CCL22 were present on ≥60% of vessels. CCL14 and CCL22 were shown to be increased in RA ECs compared to non-RA ECs, p=0.0041 and p=0.014 respectively. EC chemokines CCL7, CCL14, CCL16 and CCL22 also occurred in RA synovial fluid and sera as established by ELISA. CCL7 was shown to be significantly increased in sera and SF from RA patients compared to that from osteoarthritis (OA) patients (p<0.01), and to have a highly significant correlation with the level of anti-CCP (R=0.93, p=0.001). Less abundant chemokines shown to be present in RA ECs were CCL1-3, CCL5, CCL10-13, CCL15, CCL17, CCL18, CCL20, CCL21 and CCL23-28. In conclusion, this initial study is the first to show the presence of a number of CC chemokines in RA ECs. It provides evidence that further validation and investigation into the presence and functionality of these novel chemokines expressed at RA synovial ECs may be warranted.

Immunohistochemical differentiation of eosinophilic esophageal myositis from eosinophilic esophagitis.

BACKGROUND AND AIM: Eosinophilic esophagitis (EoE) is a Th2-mediated allergic disease of the esophageal epithelium, associated with antigen. We previously reported a case series for eosinophilic esophageal myositis (EoEM)-a novel eosinophilic gastrointestinal disorder defined as eosinophilic infiltration localized in the esophageal muscle layer-and diagnosed it by peroral endoscopic muscle biopsy. Here, we investigated the immunopathology of EoEM to differentiate it from EoE. METHODS: Histological analysis was performed for three cases of EoEM and EoE, respectively. The results were compared with those of two control samples (non-eosinophilic gastrointestinal disorder full-layer esophagus). Using immunofluorescence, we analyzed the expression of the chemokine receptor CCR3 and its ligands eotaxin-1 and eotaxin-3 to investigate the eosinophilic reaction. Additionally, we determined the expression patterns of desmoglein-1 in the esophageal epithelium, which shows dysregulated expression in EoE. RESULTS: Eosinophil infiltration was observed in the muscle layer (maximum number, 30, 36, 73/high-power field) and the epithelium (50, 44, 40/high-power field) for EoEM and EoE, respectively. In EoE esophageal epithelium, the number of eotaxin-3-positive epithelial cells was significantly increased together with CCR3-positive infiltrating cells. However, in EoEM, a number of eotaxin-1-positive and eotaxin-3-positive myocytes and vascular endothelial cells were increased in the esophageal muscle layer. A significant loss of desmoglein-1 expression was only observed in EoE, not in EoEM. CONCLUSIONS: Eotaxin-1 and eotaxin-3 expression on the smooth muscle and vessels plays a role in the pathogenesis of EoEM, while EoE shows an epithelial eotaxin-3-dominant immunoreaction. Thus, the EoEM immunological pattern displays clear differences from that of EoE.

[Establishment and verification of detecting multiple biomarkers for ovarian cancer by suspension array technology].

Objective: Establish and validation of combined detecting of CCL18, CXCL1, C1D, TM4SF1, FXR1, TIZ suspension array technology. Methods: (1)CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ protein were coupled with polyethylene microspheres. Biotinylated CCL18, CXCL1 polyclonal antibody and sheep anti-human IgG polyclonal antibody were prepared simultaneously. The best packaged concentrations of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigens were optimized. The best packaged concentrations of CCL18, CXCL1 polyclonal antibodys and C1D, TM4SF1, FXR1, TIZ sheep anti-human IgG polyclonal antibody were optimized to establish a stable detected suspension array.(2)Sixty patients confirmed by pathological examination with ovarian cancer(ovarian cancer group)which treated in Affiliated Tumor Hospital of Guangxi Medical University, 30 patients with ovarian benign tumor(benign group)and 30 cases of healthy women(control group)were chosen between September 2003 and December 2003. Suspension array technology and ELISA method were used to detect expression of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1 and TIZ IgG autoantibody contented in 3 groups of serum, then to compare the diagnostic efficiency and diagnostic accuracy of two methods(coefficient of variation between batch and batch). Results: (1)This research successfully established stable detecting system of CCL18, CXCL1, C1D, TM4SF1, FXR1 and TIZ IgG autoantibody. The best concentration of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigen package were 8, 8, 12, 8, 4 and 8 µg/ml; the best detection of CCL18, CXCL1 biotin polyclonal antibody and C1D, TM4SF1, FXR1, TIZ sheep anti-huamne IgG polyclonal antibody were respectively 4, 2, 2, 4, 4 and 2 µg/ml.(2)Suspension array technology and ELISA method were used to detect CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody of three groups in serum were similar(P>0.05).(3)The comparison of two methods in the diagnosis of efficiency: the diagnostic accuracy of two methods were 99.2%(119/120)and 94.2%(113/120), the difference was statistically significant(P=0.031). The sensitivity of the diagnosis of ovarian cancer of two methods were 100.0%(60/60)and 93.3%(56/60), specific degrees were 100.0%(59/59)and 93.4%(57/61), positive predictive value was 100.0%(60/60)and 93.3%(56/60), negative predictive value was 98.3%(59/60)and 95.0%(57/60), the difference was statistically significant(P<0.05).(4)The detected results of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody shown that the diagnostic accuracy of suspension array technology was superior to those of ELISA method(all P<0.05). Conclusion: The study has established the stable detection of suspension array technology, and the diagnostic efficiency and diagnostic accuracy was much better than that by ELISA.

CC chemokine ligand 18(CCL18) promotes migration and invasion of lung cancer cells by binding to Nir1 through Nir1-ELMO1/DOC180 signaling pathway.

Non-small cell lung cancer (NSCLC) comprises nearly 80% of lung cancers and the poor prognosis is due to its high invasiveness and metastasis. CC chemokine ligand 18 (CCL18) is predominantly secreted by M2-tumor associated macrophages (TAMs) and promotes malignant behaviors of various human cancer types. In this study, we report that the high expression of CCL18 in TAMs of NSCLC tissues and increased expression of CCL18 in TAMs is correlated with the lymph node metastasis, distant metastasis, and poor prognosis NSCLC patients. CCL18 can increase the invasive ability of NSCLC cells by binding to its receptor Nir1. In addition, CCL18 is capable of modulating cell migration and invasion by regulating the activation of RAC1 which resulted in cytoskeleton reorganization in an ELMO1 dependent manner. Furthermore, we found that CCL18 could enhance adhesion of NSCLC cells via activating ELMO1-integrin ß1 signaling. Thus, CCL18 and its downstream molecules may be used as targets to develop novel NSCLC therapy. © 2016 Wiley Periodicals, Inc.

Inflammatory potential in relation to the microbial content of settled dust samples collected from moisture-damaged and reference schools: results of HITEA study.

Aiming to identify factors causing the adverse health effects associated with moisture-damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture-damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n = 25) collected from moisture-damaged and reference schools in Spain, the Netherlands, and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor-α (TNF-)α, interleukin (IL)-6, and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis, and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers such as ergosterol, 3-hydroxy fatty acids, muramic acid, endotoxins, and glucans were measured as markers of exposure. Dust from moisture-damaged schools in Spain and the Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL-6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram-positive bacteria.

CCL15 overexpression predicts poor prognosis for hepatocellular carcinoma.

OBJECTIVES: The purpose of this study was to study the expression of CCL15 in hepatocellular carcinoma (HCC) and explore its clinicopathological significance, and study relationships between expressions of CCL15 and malignant behaviors of HCC. METHODS: The SP immunohistochemical method was used to detect expression of CCL15 in routinely paraffin-embedded sections from 80 cases of HCC, 80 of adjacent cancerous specimens and 50 of normal liver tissue. In these patients with HCC, Kaplan-Meier was used to assess survival outcomes. RESULTS: The positive rates and scores of CCL15 were significantly higher in HCC than adjacent cancerous specimens and normal liver tissue (p < 0.05), but not significantly higher between adjacent cancerous specimens and normal liver tissue (p > 0.05). The expression of CCL15 was significantly correlated to tumor size, tumor thrombi in portal vein of HCC, capsule and TNM stage (p < 0.05), but not to sex, age, liver cirrhosis and the level of AFP so on (p > 0.05). Survival time of the patients with positive CCL15 expression was significantly decreased, and multivariate analysis indicated CCL15 expression was one of the independent predictors of survival (p = 0.042). CONCLUSION: The expression of CCL15 was significantly correlated with malignant behaviors of HCC, and CCL15 might be important biological markers for reflecting the carcinogenesis, progression, biological behaviors and prognosis of HCC.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes.

UNLABELLED: In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. OBJECTIVE: This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. MATERIAL AND METHODS: Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. RESULTS: Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. CONCLUSION: A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.

Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production.Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the differentA. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation.Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the differentA. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels.Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b.Conclusion A T-lymphocyte response biased towards a Th1- and Th17-pattern of CCL and CCR expression was detected under stimulation with the serotype b ofA. actinomycetemcomitans.