IL-36 cytokines imprint a colitogenic phenotype on CD4+ T helper cells.

IL-36 cytokines are emerging as potent orchestrators of intestinal inflammation and are being implicated in the pathogenesis of inflammatory bowel diseases (IBD). However, the mechanisms through which these cytokines mediate these effects remain to be fully uncovered. Here, we report specifically elevated expression of IL-36α, and not IL-36ß or IL-36γ in the serum of newly diagnosed, treatment naïve, paediatric IBD patients and identify T cells as primary cellular mediators of IL-36 responses in the inflamed gut. IL-36R expression on CD4+ T cells was found to promote intestinal pathology in a murine model of colitis. Consistent with these effects, IL-36R can act as a potent instructor of CD4+ T cell differentiation in vivo, enhancing Th1 responses, while inhibiting the generation of Tregs. In addition, loss of IL-36 responsiveness significantly reduced the migration of pathogenic CD4+ T cells towards intestinal tissues and IL-36 was found to act, uniquely among IL-1 family members, to induce the expression of gut homing receptors in proinflammatory murine and human CD4+ T cells. These data reveal an important role for IL-36 cytokines in driving the colitogenic potential of CD4+ T cells and identify a new mechanism through which they may contribute to disease pathogenesis.

2-Deoxy-D-Glucose inhibits aggressive triple-negative breast cancer cells by targeting glycolysis and the cancer stem cell phenotype.

Due to limited availability of pharmacological therapies, triple-negative breast cancer (TNBC) is the subtype with worst outcome. We hypothesised that 2-Deoxy-D-Glucose (2-DG), a glucose analogue, may hold potential as a therapy for particularly aggressive TNBC. We investigated 2-DG's effects on TNBC cell line variants, Hs578T parental cells and their isogenic more aggressive Hs578Ts(i)8 variant, using migration, invasion and anoikis assays. We assessed their bioenergetics by Seahorse. We evaluated metabolic alterations using a Seahorse XF Analyzer, citrate synthase assay, immunoblotting and flow cytometry. We assessed the cancer stem cell (CSC) phenotype of the variants and 2-DG's effects on CSCs. 2-DG significantly inhibited migration and invasion of Hs578Ts(i)8 versus Hs578T and significantly decreased their ability to resist anoikis. Investigating 2-DG's preferential inhibitory effect on the more aggressive cells, we found Hs578Ts(i)8 also had significantly decreased oxidative phosphorylation and increased glycolysis compared to Hs578T. This is likely due to mitochondrial dysfunction in Hs578Ts(i)8, shown by their significantly decreased mitochondrial membrane potential. Furthermore, Hs578Ts(i)8 had a significantly increased proportion of cells with CSC phenotype, which was significantly decreased by 2-DG. 2-DG may have benefit as a therapy for TNBC with a particularly aggressive phenotype, by targeting increased glycolysis. Studies of more cell lines and patients' specimens are warranted.

miR-758-3p: a blood-based biomarker that's influence on the expression of CERP/ABCA1 may contribute to the progression of obesity to metabolic syndrome.

Due to increasing prevalence of obesity, a simple method or methods for the diagnosis of metabolic syndrome are urgently required to reduce the risk of associated cardiovascular disease, diabetes and cancer. This study aimed to identify a miRNA biomarker that may distinguish metabolic syndrome from obesity and to investigate if such a miRNA may have functional relevance for metabolic syndrome. 52 adults with clinical obesity (n=26) or metabolic syndrome (n=26) were recruited. Plasma specimens were procured from all and were randomly designated to discovery and validation cohorts. miRNA discovery profiling was performed, using array technology, on plasma RNA. Validation was performed by quantitative polymerase chain reaction. The functional effect of miR-758-3p on its predicted target, cholesterol efflux regulatory protein/ATP-binding cassette transporter, was investigated using HepG2 liver cells. Custom miRNA profiling of 25 miRNAs in the discovery cohort found miR-758-3p to be detected in the obese cohort but undetected in the metabolic syndrome cohort. miR-758-3p was subsequently validated as a potential biomarker for metabolic syndrome by quantitative polymerase chain reaction. Bioinformatics analysis identified cholesterol efflux regulatory protein/ATP-binding cassette transporter as miR-758-3p's predicted target. Specifically, mimicking miR-758-3p in HepG2 cells suppressed cholesterol efflux regulatory protein/ATP-binding cassette transporter protein expression; conversely, inhibiting miR-758-3p increased cholesterol efflux regulatory protein/ATP-binding cassette transporter protein expression. miR-758-3p holds potential as a blood-based biomarker for distinguishing progression from obesity to metabolic syndrome and as a driver in controlling cholesterol efflux regulatory protein/ATP-binding cassette transporter expression, indicating it potential role in cholesterol control in metabolic syndrome.

Profiling Circulating miRNAs from the Plasma of Individuals with Metabolic Syndrome.

The technique of RT-qPCR (real time-quantitative polymerase chain reaction) is invaluable in miRNA research both at the profiling and individual RT-qPCR stages. At the profiling stage, numerous miRNAs are looked at in the plasma of numerous individuals from two or more cohorts (i.e., control vs. case). The miRNAs of interest would be either upregulated or downregulated by more than twofold in the case cohort compared to the control cohort. Profiling human specimens for miRNA biomarkers has exploded over the last decade, with researchers profiling plasma, serum, urine, and also the miRNA content of extracellular vesicles, which are also isolated from human specimens. RT-qPCR is a relatively easy technique; however, sample preparation from plasma to RNA to RNA input in RT reaction requires accuracy and precision.

Resistance to HER2-targeted anti-cancer drugs is associated with immune evasion in cancer cells and their derived extracellular vesicles.

Neuromedin U (NmU) -a neuropeptide belonging to the neuromedin family- plays a substantial role in HER2-positive breast cancer, correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients. However, the mechanism through which it exerts these effects remains unclear. To elucidate this, initially we used HER2-positive breast cancer cells stably over-expressing NmU. These cells and their released extracellular vesicles (EVs) had increased amounts of the immunosuppressive cytokine TGFß1 and the lymphocyte activation inhibitor PD-L1. Furthermore, these cells also showed enhanced resistance to antibody-dependent cell cytotoxicity (ADCC) mediated by trastuzumab, indicating a role of NmU in enhancing immune evasion. All these features were also found in HER2-targeted drug-resistant cells which we previously found to express higher levels of NmU than their drug-sensitive counterparts. Interestingly, EVs from drug-resistant cells were able to increase levels of TGFß1 in drug-sensitive cells. In our neo-adjuvant clinical trial, TGFß1 levels were significantly higher in EVs isolated from the serum of patients with HER2-overexpressing breast cancers who went on to not respond to HER2-targeted drug treatment, compared with those who experienced complete or partial response. Taken together, our results report a new mechanism-of-action for NmU in HER2-overexpressing breast cancer that enhances resistance to the anti-tumor immune response. Furthermore, EV levels of TGFß1 correlating with patients' response versus resistance to HER2-targeted drugs suggests a potential use of EV-TGFß1 as a minimally-invasive companion diagnostic for such treatment in breast cancer.

Blood-Based Biomarkers for Metabolic Syndrome.

Metabolic syndrome (MetS) is a constellation of factors increasing the risk of type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), and cancer. MetS diagnosis is cumbersome and the precise diagnosis differs throughout the world. Efforts are underway to find MetS biomarkers that could all be analysed in a single blood sample. Here we review recent advances, including progress on circulating exosomes and microvesicles and their molecular contents, as well as DNA, RNAs, and proteins taken directly from blood samples. While additional research is now warranted to advance upon these findings, there is reason for optimising that such blood-based entities will be beneficial for MetS diagnosis and will help reduce risk of T2DM, CVD, and cancers, contributing both societal and economic benefit.

Dairy proteins, dairy lipids, and postprandial lipemia in persons with abdominal obesity (DairyHealth): a 12-wk, randomized, parallel-controlled, double-blinded, diet intervention study.

BACKGROUND: Abdominal obesity and exaggerated postprandial lipemia are independent risk factors for cardiovascular disease (CVD) and mortality, and both are affected by dietary behavior. OBJECTIVE: We investigated whether dietary supplementation with whey protein and medium-chain saturated fatty acids (MC-SFAs) improved postprandial lipid metabolism in humans with abdominal obesity. DESIGN: We conducted a 12-wk, randomized, double-blinded, diet intervention study. Sixty-three adults were randomly allocated to one of 4 diets in a 2 × 2 factorial design. Participants consumed 60 g milk protein (whey or casein) and 63 g milk fat (with high or low MC-SFA content) daily. Before and after the intervention, a high-fat meal test was performed. We measured changes from baseline in fasting and postprandial triacylglycerol, apolipoprotein B-48 (apoB-48; reflecting chylomicrons of intestinal origin), free fatty acids (FFAs), insulin, glucose, glucagon, glucagon-like peptide 1 (GLP-1), and gastric inhibitory polypeptide (GIP). Furthermore, changes in the expression of adipose tissue genes involved in lipid metabolism were investigated. Two-factor ANOVA was used to examine the difference between protein types and fatty acid compositions, as well as any interaction between the two. RESULTS: Fifty-two participants completed the study. We found that the postprandial apoB-48 response decreased significantly after whey compared with casein (P = 0.025) independently of fatty acid composition. Furthermore, supplementation with casein resulted in a significant increase in the postprandial GLP-1 response compared with whey (P = 0.003). We found no difference in postprandial triacylglycerol, FFA, insulin, glucose, glucagon, or GIP related to protein type or MC-SFA content. We observed no interaction between milk protein and milk fat on postprandial lipemia. CONCLUSION: We found that a whey protein supplement decreased the postprandial chylomicron response compared with casein in persons with abdominal obesity, thereby indicating a beneficial impact on CVD risk. This trial was registered at clinicaltrials.gov as NCT01472666.