The incidence of endometrial cancer (EC) has increased over the past years and mainly affects women above the age of 45 years. Metabolic diseases such as obesity and type II diabetes mellitus as well as associated conditions like polycystic ovary syndrome (PCOS), insulin resistance and hyperinsulinemia lead to elevated levels of circulating estrogens. Increased estrogen concentrations, in turn, further trigger the proliferation of endometrial cells and thus promote EC development and progression, especially in the absence of progesterone as seen in postmenopausal women. Elevated blood glucose levels in diabetic patients further contribute to the risk of EC development. Metformin is an insulin-sensitizing biguanide drug, commonly used in the treatment of type II diabetes mellitus, especially in obese patients. Besides its effects on glucose metabolism, metformin displayed anti-cancer effects in various cancer types, including EC. Direct anti-cancer effects of metformin target signaling pathways that are involved in cellular growth and proliferation, e.g. the AKT/PKB/mTOR pathway. Further proteins and pathways have been suggested as potential targets, but the underlying mechanism of action of metformin's anti-cancer activity is still not completely understood. In the present study, the effects of metformin on protein expression were investigated in the human EC cell line HEC-1A using an affinity proteomic approach. Cells were treated with 0.5 mmol/L metformin over a period of 7 days and changes in the expression pattern of 1,300 different proteins were compared to the expression in untreated control cells as well as insulin-treated cells. Insulin treatment (100 ng/mL) was incorporated into the study in order to implement a model for insulin resistance and associated hyperinsulinemia, conditions that are often observed in obese and diabetic patients. Furthermore, the culture medium was supplemented with 10 nmol/L ß-estradiol (E2) during treatments to mimic increased estrogen levels, a common risk factor for EC development. Based on the most prominent and significant changes in expression, a set of 80 proteins was selected and subjected to a more detailed analysis. The data revealed that metformin and insulin targeted similar pathways in the present study and mostly acted on proteins related to proliferation, migration and tumor immune response. These pathways may be affected in a tumor-promoting as well as a tumor-suppressing way by either metformin treatment or insulin supplementation. The consequences for the cells resulting from the detected expression changes were discussed in detail for several proteins. The presented data helps identify potential targets affected by metformin treatment in EC and allows for a better understanding of the mechanism of action of the biguanide drug's anti-cancer activity. However, further investigations are necessary to confirm the observations and conclusions drawn from the presented data after metformin administration, especially for proteins that were regulated in a favorable way, i.e. AKT3, CCND2, CD63, CD81, GFAP, IL5, IL17A, IRF4, PI3, and VTCN1. Further proteins might be of interest, where metformin counteracted unfavorable effects that have been induced by hyperinsulinemia.

Breast cancer (BrC) affects millions of women yearly. Mast cells (MCs) are common components of breast tumors with documented agonistic and antagonistic roles in tumor progression. Understanding the participation of MCs in BrC may lead to new therapies to control tumor growth. In this study, we looked into mechanistic models of MC responses triggered by BrC cells (BrCC), assessing both early degranulation and late transcriptional activities. We used aggressive and non-aggressive BrCC to model the progressive staging of the disease over HMC1 and LAD-2 human MC lines. We found that both MC lines were chemoattracted by all BrCC, but their activation was preferentially induced by aggressive lines, finding differences in their active transcriptional programs, both at basal level and after stimulation. Among those genes with altered expression were down-regulated SPP1, PDCD1, IL17A and TGFB1 and up-regulated KITLG and IFNG. A low expression of SPP1 and a high expression of KITLG and IFNG were associated with increased overall survival of BrC patients from public databases. The set of altered genes is more often associated with tumor stromas enriched with anti-tumoral signals, suggesting that MCs may participate in tumor control.

Keywords: IFNγ; anti-tumor functions; breast cancer; mast cells; tumor stroma.

BACKGROUND

The genes encoding IL9, IL9R, IL17A, and IL17F have recently been implicated in the genetic basis of rhinitis and allergy.

OBJECTIVE

The purpose of this study was to assess the association of the single nucleotide polymorphisms (SNPs) of IL9, IL9R, IL17A, and IL17F and potential interaction of these genes with the determination of IgE levels in women with allergic rhinitis (AR) in Shahrekord, Iran.

METHODS

In a case-control study, SNPs from the IL9, IL9R, IL17A, and IL17F were genotyped in 394 random samples including 195 AR patients and 199 normal controls. Enzyme-linked immunosorbent assay was performed for the determination of serum total IgE levels. The Student's t-test was used to compare the differences. The Chi-square test was performed to compare proportions of cases with different clinical features among cases with different genotypes. The genotype and allele frequencies were obtained by direct counting. Hardy-Weinberg equilibrium was tested between cases and controls separately. The relative risk associated with rare alleles was estimated as an odds ratio with 95% confidence interval. P ≤ 0.05 was considered statistically significant.

RESULTS

The rs731476 SNP in the IL9R was significantly associated with the AR phenotype in women. No association was found between any of the other SNPs in IL9, IL17A, and IL17F genes and AR. In the gene-gene interaction analysis, we found that IL9R/IL9 genotype rs731476 T-/rs2069885 G conferred a higher risk for AR phenotype development. We also did not find a significant association in terms of IgE levels between cases and controls.

CONCLUSIONS

Our result suggests that the rs731476 SNP located in the IL9R is associated with an increased susceptibility to AR in females. In a subsequent gene-gene interaction analysis, the rs731476 T-/rs2069885 G-genotype combination (IL9R/IL9) has significantly been associated with the development of the AR phenotype.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is the most spread clinical form of leishmaniasis in Brazil. However, only a few part of the people infected develop clinically perceptive disease, suggesting the influence of human genetic components in the CL pathogeny. The rs2275913 SNP is the nucleotide variant of the IL17A gene. The A allele is associated with a vast number of infectious and non-infectious diseases. Here, we investigated the association of the rs2275913 SNP (G/A) from IL-17A and two forms of susceptibility to CL in Brazil by case-control study. Furthermore, we evaluated the functional relevance of this SNP during the immune response of the host and analyzed its impact in the parasite elimination. Weak associations of A allele with susceptibility to L. braziliensis infection or to symptomatic CL were observed, and a tendency of A allele carriers to be more susceptible to infection and cutaneous disease. Functional analysis of the Th17 cell phenotypes revealed lower frequencies of CD4+ IL-17+ cells in samples of infected people with AA/AG genotypes. Furthermore, people carrying the A allele maintain higher parasite loads, reinforcing the genetic susceptibility findings. This study adds knowledge about the influence of a significant genetic variation on IL-17 promoter on CL pathogenesis, and may contribute to enhance the knowledge about the role of IL-17 in the L. braziliensis infections.

Schistosomiasis remains the fourth most prevalent parasitic disease affecting over 200 million people worldwide. Control efforts have focussed on the disruption of the life cycle targeting the parasite, vector and human host. Parasite burdens are highly skewed, and the majority of eggs are shed into the environment by a minority of the infected population. Most morbidity results from hepatic fibrosis leading to portal hypertension and is not well-correlated with worm burden. Genetics as well as environmental factors may play a role in these skewed distributions and understanding the genetic risk factors for intensity of infection and morbidity may help improve control measures. In this review, we focus on how genetic factors may influence parasite load, hepatic fibrosis and portal hypertension. We found 28 studies on the genetics of human infection and 20 studies on the genetics of pathology in humans. <i>S. mansoni</i> and <i>S. haematobium</i> infection intensity have been showed to be controlled by a major quantitative trait locus <i>SM1</i>, on chromosome 5q31-q33 containing several genes involved in the T_h2 immune response, and three other loci of smaller effect on chromosomes 1, 6, and 7. The most common pathology associated with schistosomiasis is hepatic and portal vein fibroses and the <i>SM2</i> quantitative trait locus on chromosome six has been linked to intensity of fibrosis. Although there has been an emphasis on T_h2 cytokines in candidate gene studies, we found that four of the five QTL regions contain T_h17 pathway genes that have been included in schistosomiasis studies: <i>IL17B</i> and <i>IL12B</i> in <i>SM1, <em>IL17A</em></i> and <i>IL17F</i> in 6p21-q2, <i>IL6R</i> in 1p21-q23 and <i>IL22RA2</i> in <i>SM2</i>. The T_h17 pathway is known to be involved in response to schistosome infection and hepatic fibrosis but variants in this pathway have not been tested for any effect on the regulation of these phenotypes. These should be priorities for future studies.

Keywords: QTL; Th17; fibrosis; intensity of infection; linkage; schistosomiasis.

We recently developed a partial peptide of receptor activator of nuclear factor-кB ligand (RANKL) known as microglial healing peptide 1 (MHP1-AcN), that inhibits Toll-like receptor (TLR)-related inflammation through RANKL/RANK signaling in microglia and macrophages without promoting osteoclast activation. The abnormal activation of TLRs contributes to the initiation and maintenance of psoriasis, which is a chronic inflammatory skin disease that involves the aberrant expression of proinflammatory cytokines and the subsequent dermal γδ T cell and T helper 17 (Th17) cell responses. The inhibition of TLR-mediated inflammation provides an important strategy to treat psoriasis. Here, we examined the preventative effects of MHP1-AcN in a mouse model of imiquimod (a TLR 7/8 agonist)-induced psoriasis. Topical imiquimod application induced psoriasis-like skin lesions on the ear and dorsal skin. Systemic administration of MHP1-AcN by daily subcutaneous injection significantly prevented the development of skin lesions, including erythema, scaling and thickening. Mice treated with MHP1-AcN showed reduced levels of skin Il6 mRNA at 32 h and reduced levels of Il23 and Il17a mRNA at d9. Serum levels of IL-6 and IL-23 were reduced at 32 h, and IL-17A was reduced at d9. These results indicated that MHP1-AcN could decrease imiquimod-induced IL-6, IL-23 and IL-17A production. MHP1-AcN is potentially an alternative treatment for psoriasis.

Bovine tuberculosis (bTB), a zoonosis mainly caused by Mycobacterium bovis has severe socio-economic consequences and impact on animal health. Host-pathogen interactions during M. bovis infection are poorly understood, especially early events which are difficult to follow in vivo. This study describes the utilisation of an in vitro co-culture model, comprising immortalised bovine alveolar type II (BATII) epithelial cells and bovine pulmonary arterial endothelial cells (BPAECs). When cultured at air-liquid interface, it was possible to follow the migration of live M. bovis Bacille Calmette-Guérin (BCG) and to observe interactions with each cell type, alongside cytokine release. Infection with BCG was shown to exert a detrimental effect primarily upon epithelial cells, with corresponding increases in IL8, TNFα, IL22 and IL17a cytokine release, quantified by ELISA. BCG infection increased expression of CD54, MHC Class I and II molecules in endothelial but not epithelial cells, which exhibited constitutive expression. The effect of peripheral blood mononuclear cell conditioned medium from vaccinated cattle upon apical-basolateral migration of BCG was examined by quantifying recovered BCG from the apical, membrane and basolateral fractions over time. The numbers of recovered BCG in each fraction were unaffected by the presence of PBMC conditioned medium, with no observable differences between vaccinated and naïve animals.

The present paradigm of psoriasis pathogenesis revolves around the IL-23/IL-17A axis. Dual-secreting Th17 T cells presumably are the predominant sources of the psoriasis phenotype-driving cytokines, IL-17A and IL-22. We thus conducted a meta-analysis of independently acquired RNA-seq psoriasis datasets to explore the relationship between the expression of IL17A and IL22. This analysis failed to support the existence of dual secreting IL-17A/IL-22 Th17 cells as a major source of these cytokines. However, variable relationships amongst the expression of psoriasis susceptibility genes and of IL17A, IL22, and IL23A were identified. Additionally, to shed light on gene expression relationships in psoriasis, we applied a machine learning nonlinear dimensionality reduction strategy (t-SNE) to display the entire psoriasis transcriptome as a 2-dimensonal image. This analysis revealed a variety of gene clusters, relevant to psoriasis pathophysiology but failed to support a relationship between IL17A and IL22. These results support existing theories on alternative sources of IL-17A and IL-22 in psoriasis such as a Th22 cells and non-T cell populations.

Staphylococcus aureus- induced mastitis is one of the most intractable problems for the dairy industry, which causes loss of milk yield and early slaughter of cows worldwide. Few studies have used a comprehensive approach based on the integrative analysis of miRNA and mRNA expression profiles to explore molecular mechanism in bovine mastitis caused by S. aureus. In this study, S. aureus (A1, B1 and C1) and sterile phosphate buffered saline (PBS) (A2, B2 and C2) were introduced to different udder quarters of three individual cows, and transcriptome sequencing and microarrays were utilized to detected miRNA and gene expression in mammary glands from the challenged and control groups. A total of 77 differentially expressed microRNAs (DE miRNAs) and 1625 differentially expressed genes (DEGs) were identified. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that multiple DEGs were enriched in significant terms and pathways associated with immunity and inflammation. Integrative analysis between DE miRNAs and DEGs proved that miR-664b, miR-23b-3p, miR-331-5p, miR-19b and miR-2431-3p were potential factors regulating the expression levels of CD14 Molecule (CD14), G protein subunit gamma 2 (GNG2), interleukin 17A (IL17A), collagen type IV alpha 1 chain (COL4A1), microtubule associated protein RP/EB family member 2 (MAPRE2), member of RAS oncogene family (RAP1B), LDOC1 regulator of NFKB signaling (LDOC1), low-density lipoprotein receptor (LDLR) and S100 calcium binding protein A9 (S100A9) in bovine mastitis caused by S. aureus. These findings could enhance the understanding of the underlying immune response in bovine mammary glands against S. aureus infection and provide a useful foundation for future application of the miRNA-mRNA-based genetic regulatory network in the breeding cows resistant to S. aureus.

Keywords: Staphylococcus aureus; bovine mastitis; differential expression microRNAs; differentially expressed genes; integrative analysis.

OBJECTIVE

Evaluate the participation of IL-17 pathway in T1D pathogenesis. T helper 17 cells are potent, highly inflammatory cells that produce interleukin 17A (IL-17A), considered a mediator of various immune disorders. However, their role in Type 1 diabetes (T1D) pathogenesis in humans is not totally elucidated.

METHODS

The expression of IL-17 Receptor A (IL-17RA) in peripheral T lymphocytes and IL-17A serum levels in recent-onset patients with T1D were compared with healthy controls. IL-17A gene variants were evaluated in a greater cohort.

RESULTS

Patients with recent-onset T1D (less than 6 months of diagnosis) exhibited lower expression of IL-17RA in CD3+ T (% of cells = 31.3% × 43.6%; p = .041) and CD4+ T cells (11.1% × 25.2%; p = .0019) and lower number of IL-17RA in CD4+ T cells (MFI = 1.16 × 4.56; p = .03) than controls. IL-17RA expression in CD8+ T cells and IL-17A serum levels were similar in both groups. The coding regions and boundary intron sequences of IL17A were sequenced. Seventeen allelic variants, including three novel variants in exon 3 (3'UTR n) were identified, but no one was associated with T1D susceptibility, as well as the resulting haplotypes and diplotypes. The expression of IL-17RA was not correlated with metabolic variables (glucose and HbA1c levels) or pancreatic autoantibodies titers.

CONCLUSIONS

The lower expression of IL-17RA in CD3+ and CD4+ T cells suggests a reduced effect of IL-17A in immune response of recent-onset T1D patients, at least at peripheral tissues. IL-17A allelic variants were not related with T1D susceptibility.

Background: Cachexia is defined as an involuntary decrease in body weight, which can increase the risk of death in cancer patients and reduce the quality of life. Cachexia-inducing factors (CIFs) have been reported in colorectal cancer and pancreatic adenocarcinoma, but their value in diffuse large B-cell lymphoma (DLBCL) requires further genetic research.

Methods: We used gene expression data from Gene Expression Omnibus to evaluate the expression landscape of 25 known CIFs in DLBCL patients and compared them with normal lymphoma tissues from two cohorts [GSE56315 (n = 88) and GSE12195 (n = 136)]. The mutational status of CIFs were also evaluated in The Cancer Genome Atlas database. Based on the expression profiles of 25 CIFs, a single exploratory dataset which was merged by the datasets of GSE10846 (n = 420) and GSE31312 (n = 498) were divided into two molecular subtypes by using the method of consensus clustering. Immune microenvironment between different subtypes were assessed via single-sample gene set enrichment analysis and the CIBERSORT algorithm. The treatment response of commonly used chemotherapeutic drugs was predicted and gene set variation analysis was utilized to reveal the divergence in activated pathways for distinct subtypes. A risk signature was derived by univariate Cox regression and LASSO regression in the merged dataset (n = 882), and two independent cohorts [GSE87371 (n = 221) and GSE32918 (n = 244)] were used for validation, respectively.

Results: Clustering analysis with CIFs further divided the cases into two molecular subtypes (cluster A and cluster B) associated with distinct prognosis, immunological landscape, chemosensitivity, and biological process. A risk-prognostic signature based on CCL2, CSF2, IL15, IL17A, IL4, TGFA, and TNFSF10 for DLBCL was developed, and significant differences in overall survival analysis were found between the low- and high-risk groups in the training dataset and another two independent validation datasets. Multivariate regression showed that the risk signature was an independently prognostic factor in contrast to other clinical characteristics.

Conclusion: This study demonstrated that CIFs further contribute to the observed heterogeneity of DLBCL, and molecular classification and a risk signature based on CIFs are both promising tools for prognostic stratification, which may provide important clues for precision medicine and tumor-targeted therapy.

Keywords: cachexia-inducing factors; diffuse large B-cell lymphoma; molecular subtype; prognosis-related; signature.

Anti-nuclear antibodies (ANA) are frequently detected in patients with psoriasis (Ps) and psoriatic arthritis (PsA), but their target autoantigens remain unknown. We assessed antibody (ab) reactivity against 23 known nuclear antigens in patients with Ps and PsA and assess the effects of secukinumab (anti-IL17A) treatment on ANA levels. A total of 201 patients, 101 with Ps and 100 with PsA, and 50 ANA-negative healthy controls (HCs) were tested for ANAs by a line immunoassay testing reactivity to 23 nuclear antigens. Ab reactivity to at least 1 antigen was found in 20.4% psoriatic disease patients (25.7% Ps and 15% PsA) compared to 8% HCs (p=ns), the most frequent being against dense fine speckled 70 (DFS70) (6.5%). In Ps and PsA patients with secukinumab-induced remission, anti-DFS70 and other antigen-specific autoantibodies were diminished over time. No decline was noted for IgG abs against antigens from pathogens such as cytomegalovirus, Epstein-Barr virus and Helicobacter pylori. Autoantibody decrease was associated with significant reduction of plasmablasts, follicular B and follicular T cells. In conclusion, one third of antigen-specific ANA patients with psoriatic disease recognize DFS70. Secukinumab decreases nuclear antigen autoreactivity, plasmablasts, follicular B and follicular T cells, highlighting a new mechanism of its action.

Keywords: anti-IL-17; antinuclear antibody; autoantibody; biologics; psoriasis; psoriatic arthritis.

Objectives: Inflammatory bowel disease (IBD) is more complex in children and they will have to live with the disease for much longer. For this reason, it is necessary to optimize treatment. The polymorphisms associated with the response to anti-tumor necrosis factor (TNF) drugs in adults with IBD have not been analyzed in children. The aim of the study was to identify genetic variants associated with the long-term response to anti-TNF drugs in children with IBD.

Methods: An observational, longitudinal, ambispective cohort's study was conducted. We recruited 209 anti-TNF-treated children diagnosed with IBD and genotyped 21 polymorphisms previously studied in adults with Crohn disease (CD) using real-time PCR. The association between single-nucleotide polymorphisms (SNPs) and time-to-failure was analyzed using the log-rank test.

Results: After multivariate analysis, 3 SNPs in IL10, IL17A and IL6 were significantly associated with response to anti-TNF treatment among patients diagnosed with CD (rs1800872-HR, 4.749 (95% confidence interval [CI] 1.156-19.517), P value < 0.05; rs2275913-HR, 0.320 [95% CI 0.111-0.920], P value < 0.05; and rs10499563-HR, 0.210 [95% CI 0.047-0.947], P value 0.05, respectively). None of these SNPs were associated with response to infliximab in adults diagnosed with CD. Among patients diagnosed with ulcerative colitis (UC), 1 SNP in LY96 was significantly associated with response to anti-TNF treatment (rs-11465996-HR, 10.220 [95% CI 1.849-56.504] P value < 0.05).

Conclusions: Genotyping of these DNA variants before starting treatment may help to identify children who are long-term responders to anti-TNF drugs, and thus tailor treatment of pediatric IBD.

Lignosus rhinocerotis Cooke. (L. rhinocerotis) is a medicinal mushroom traditionally used in the treatment of asthma and several other diseases by the indigenous communities in Malaysia. In this study, the effects of L. rhinocerotis on allergic airway inflammation and hyperresponsiveness were investigated. L. rhinocerotis extract (LRE) was prepared by hot water extraction using soxhlet. Airway hyperresponsiveness (AHR) study was performed in house dust mite (HDM)-induced asthma in Balb/c mice while airway inflammation study was performed in ovalbumin (OVA)-induced asthma in Sprague-Dawley rats. Treatment with different doses of LRE (125, 250 and 500 mg/kg) significantly inhibited AHR in HDM-induced mice. Treatment with LRE also significantly decreased the elevated IgE in serum, Th2 cytokines in bronchoalveolar lavage fluid and ameliorated OVA-induced histological changes in rats by attenuating leukocyte infiltration, mucus hypersecretion and goblet cell hyperplasia in the lungs. LRE also significantly reduced the number of eosinophils and neutrophils in BALF. Interestingly, a significant reduction of the FOXP3+ regulatory T lymphocytes was observed following OVA induction, but the cells were significantly elevated with LRE treatment. Subsequent analyses on gene expression revealed regulation of several important genes i.e. IL17A, ADAM33, CCL5, IL4, CCR3, CCR8, PMCH, CCL22, IFNG, CCL17, CCR4, PRG2, FCER1A, CLCA1, CHIA and Cma1 which were up-regulated following OVA induction but down-regulated following treatment with LRE. In conclusion, LRE alleviates allergy airway inflammation and hyperresponsiveness, thus suggesting its therapeutic potential as a new armamentarium against allergic asthma.

Bacterial flagellar hook and recombinant flagellar hook protein E (FlgE) were reportedly immunostimulatory in mammalian cells or tissues. Current study focused on the mechanisms underlying FlgE stimulation. In an acute lung injury model induced by intranasal FlgE challenge, neutrophils were the predominant infiltrates in lungs, and depletion of neutrophils with anti-Ly6G antibody attenuated FlgE-induced lung damage. However, the FlgE-induced neutrophils recruitment, neutrophils reactive oxygen species (ROS) generation, and neutrophil extracellular traps (NETs) formation were significantly impaired in Il17a-/- mice compared with those in wild-type (WT) mice. In FlgE-treated lung organoids and isolated neutrophils, the phosphorylation levels of signal transfer and activator of transcription protein 3 (STAT3), which was involved in neutrophils functions, were upregulated, but this upregulation was partly impaired upon IL17A deficiency or by IL6 neutralisation. When neutrophils isolated from WT mice were treated with FlgE, the expression of IL17A/IL17RC was increased, but the activation was blocked by STAT3 inhibitor. The NETs formation in FlgE-treated neutrophils was not affected by the ROS inhibitor or recombinant IL17A alone but partly impaired in the presence of STAT3 pathway inhibition. In conclusion, we propose that the pro-inflammatory activities of FlgE are mediated by activating STAT3 phosphorylation and IL17A/IL17R expression and by promoting a ROS-independent NETs formation.

Mounting evidence supports that the malignant phenotypes of cancers are defined not only by the intrinsic activity of tumor cells but also by immune cells that are recruited and activated in tumor-related microenvironment. Here, we developed a diagnostic and prognostic model for colon cancer, based on expression profiles of immune-related genes and immune cell component. As a result, we found that B cell infiltration ratio, CD4⁺ T cells, as well as immune-related genes of TRIB3, CHGA, CASP7, LGALS4, LEP, NOX4, IL17A, and HSPD1 may be highly relevant with clinical outcome of colon cancer.

Keywords: bioinformatics; colon cancer; immune cell infiltration; immune-related gene; prognostic model.

Subclinical endometritis (SE) in cattle is defined as clinically unapparent inflammation of the endometrium. It is reported to impair fertility in affected cows and causes economic loss within the dairy industry. A gold standard for diagnosis of SE has not been set. Uterine cytology and histopathology are both applied, but low agreement between these methods has been described. The objective of the present study was to assess the capability of uterine secretions (US) as a new medium for diagnosis of SE. A novel sampling tool was applied to retrieve US as well as cytological, histological and bacteriological samples of the endometrium after a singular passage through the cervix in 108 dairy cows (43-62 days post-partum [dpp]). To assess the quality of the US samples, a proteome analysis of samples from five healthy donors was performed, demonstrating that in vivo sampling of US was feasible and generated samples suitable for diagnostic purposes. Diagnosis of SE was realized by the combination of clinical, cytological, and histopathological findings. Quantitative analysis of pro- and anti-inflammatory cytokines (interleukin (IL)1B, IL6, IL8, IL17A, IL10) in US was conducted using AlphaLISA-technology. RNAlater-fixed endometrial biopsies were used for gene expression analysis of the cytokines IL1B, IL6, IL8, IL10 and tumor necrosis factor alpha (TNFα) as well as the prostaglandin-endoperoxide synthase 2 (PTGS2) and the antimicrobial peptide S100A9 by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Cows were assigned to groups according to their uterine health status. A large group of animals (n = 83) displayed no signs of endometritis (E.NEG). Cytological and histopathological examination revealed low agreement; hence, animals with SE were differentiated into SE(cyto) and SE(histo) groups (n = 7 and n = 13, respectively). One animal in group SE(cyto + histo) as well as four animals with signs of clinical endometritis (CE) were excluded from further analysis. SE(cyto) showed significantly higher median concentrations of IL1B, IL8 and IL17A in US as well as a significantly higher median expression of IL1B, IL8 and IL10 in endometrial biopsies compared to E.NEG. No significant differences were found for IL6 and IL10 in US and IL6, TNFα, PTGS2 and S100A9 in endometrial tissue between these groups. SE(histo) presented no differences concerning the analyzed parameters compared to E.NEG. In conclusion, a method to sample US was successfully established in dairy cows. The cytokines IL1B, IL8 and IL17A are promising candidates in diagnosing cytological endometritis by US. Further assessment of US might contribute to a better understanding of the pathological mechanisms leading to chronic endometrial inflammation and to impaired fertility in affected cows.

Background: Chronic obstructive pulmonary disease (COPD) is commonly associated with both a pro-inflammatory and a T-helper 1 (Th1) immune response. It was hypothesized that cannabis oil extract can alleviate COPD symptoms by eliciting an anti-inflammatory Th2 immune response. Accordingly, the effects of cannabis oil extract on the expression of 84 Th2 and related immune response genes in human small airways epithelial cells (HSAEpC) were investigated.

Methods: HSAEpC from a single donor were treated with three dilutions of a standardized cannabis oil extract (1:400, 1:800 and 1:1600) along with a solvent control (0.25% [2.5 ul/ml] ethanol) for 24 h. There were four replicates per treatment dilution, and six for the control. RNA isolated from cells were employed in pathway-focused quantitative polymerase chain reaction (qPCR) microarray assays.

Results: The extract induced significant (P < 0.05) changes in expression of 37 tested genes. Six genes (CSF2, IL1RL1, IL4, IL13RA2, IL17A and PPARG) were up-regulated at all three dilutions. Another two (CCL22 and TSLP) were up-regulated while six (CLCA1, CMA1, EPX, LTB4R, MAF and PMCH) were down-regulated at the 1:400 and 1:800 dilutions. The relationship of differentially-expressed genes of interest to biologic pathways was explored using the Database for Annotation, Visualization and Integrated Discovery (DAVID).

Conclusions: This exploratory investigation indicates that cannabis oil extract may affect expression of specific airway epithelial cell genes that could modulate pro-inflammatory or Th1 processes in COPD. These results provide a basis for further investigations and have prompted in vivo studies of the effects of cannabis oil extract on pulmonary function.

Trial registration: NONE (all in vitro experiments).

Keywords: Anti-inflammatory; Cannabis; Chronic obstructive pulmonary disease (COPD); Gene expression profiling; HSAEpC (human small airways epithelial cells); KEGG pathway analysis; Th1 and Th2 immune response.

The interplay between tumors and their immune microenvironment is critical for cancer development and progression. The discovery of tumor heterogeneity has provided a window into a complex interplay between tumors, their secreted products, and host immune responses at the cellular and molecular levels. Tumor heterogeneity can also act as a driving force in promoting treatment resistance and correlates with distinct tumor-mediated acquired immune responses. A prevailing question is how genetic aberrations in solid tumors can shape the immune landscape, resulting in pro-tumor or anti-tumor activities. Here we review evidence for clinical and pathophysiological mechanisms that underlie different types of non-small cell lung cancer (NSCLC) and provide new insights for future immunomodulatory-based therapies. Some of the more common driver mutations in NSCLC heterogeneity includes the opposing immune responses in oncogenic mutations in K-ras vs. non-K-ras models and their pro-inflammatory cytokines such as interleukin (IL)17A. We will discuss possible molecular and metabolic mechanisms that may govern the opposing immune responses observed in distinct genetic models of NSCLCs. A deeper understanding of how tumor heterogeneity modulates immune response can improve current therapeutic strategies and provide precise treatment to individual lung cancer patients.

Staphylococcus aureus (S. aureus) is an important pathogen causing various limited or systemic infections. Methicillin resistant S. aureus (MRSA) in particular presents a major clinical and public health problem. Toxic shock syndrome toxin-1 (TSST-1) encoded by the gene tst is an important virulence factor of tst positive S. aureus, leading to multi-organ malfunction. However, the mechanism of TSST-1 in pathogenesis is only partly clear. In this study, we investigated the prevalence of the tst gene in clinical isolates of S. aureus. Then, animal experiments were performed to further evaluate the influence of the presence of the tst gene associated Staphylococcus aureus Pathogenicity Island (SaPI) on body weight, serum cytokine concentrations and the bacterial load in different organs. In addition, macrophages were used to analyze the secretion of cytokines in vitro and bacterial survival in the cytoplasm. Finally, pathological analysis was carried out to evaluate organ tissue impairment. The results demonstrated that the prevalence of tst gene was approximately 17.8% of the bacterial strains examined. BALB/c mice infected with tst gene associated SaPI positive isolates exhibited a severe loss of body weight and a high bacterial load in the liver, heart, kidney and spleen. Pathological analysis demonstrated that tissue impairment was more severe after infection with tst gene associated SaPI positive isolates. Moreover, the secretion of IL-6, IL-2 and IL17A by macrophages infected with tst gene associated SaPI positive isolates clearly increased. Notably, IL-6 secretion in BALB/c mice infected with tst gene associated SaPI positive isolates was higher than that in BALB/c mice infected with negative ones. Together, these results indicated that the tst gene associated SaPI may play a critical role in the pathological process of infection via a direct and persistent toxic function, and by promoting the secretion of inflammatory cytokines that indirectly induce immune suppression.

Restriction in antimicrobial use in broiler chicken production is driving the exploration of alternative feed additives that will support growth through the promotion of gastrointestinal health and development. The objective of this study was to determine the effects of dietary inclusion of laminarin on growth performance, the expression of nutrient transporters, markers of inflammation and intestinal integrity in the small intestine and composition of the caecal microbiota in broiler chickens. Two-hundred-and-forty day-old male Ross 308 broiler chicks (40.64 (3.43 SD) g) were randomly assigned to: (T1) basal diet (control); (T2) basal diet + 150 ppm laminarin; (T3) basal diet + 300 ppm laminarin (5 bird/pen; 16 pens/treatment). The basal diet was supplemented with a laminarin-rich Laminaria spp. extract (65% laminarin) to achieve the two laminarin inclusion levels (150 and 300 ppm). Chick weights and feed intake was recorded weekly. After 35 days of supplementation, one bird per pen from the control and best performing (300 ppm) laminarin groups were euthanized. Duodenal, jejunal and ileal tissues were collected for gene expression analysis. Caecal digesta was collected for microbiota analysis (high-throughput sequencing and QPCR). Dietary supplementation with 300 ppm laminarin increased both final body weight (2033 vs. 1906 ± 30.4, P < 0.05) and average daily gain (62.3 vs. 58.2 ± 0.95, P < 0.05) compared to the control group and average daily feed intake (114.1 vs. 106.0 and 104.5 ± 1.77, P < 0.05) compared to all other groups. Laminarin supplementation at 300 ppm increased the relative and absolute abundance of Bifidobacterium (P < 0.05) in the caecum. Laminarin supplementation increased the expression of interleukin 17A (IL17A) in the duodenum, claudin 1 (CLDN1) and toll-like receptor 2 (TLR2) in the jejunum and IL17A, CLDN1 and SLC15A1/peptide transporter 1 (SLC15A1/PepT1) in the ileum (P < 0.05). In conclusion, supplementation with laminarin is a promising dietary strategy to enhance growth performance and 300 ppm was the optimal inclusion level with which to promote a beneficial profile of the gastrointestinal microbiota in broiler chickens.

Keywords: broiler; gastrointestinal health; laminarin; microbiota; performance.