Effect of tofacitinib on the phenotype and activity of Caco­2 cells in a model of inflammatory bowel disease.

Reports have indicated that autoimmune bowel disorders affect an increasing number of people on every continent; therefore, it is important to better understand inflammatory bowel disease (IBD) and to explore new treatment options for patients suffering from it. Research has indicated the important role of enterocytes in IBD. Understanding the role of the intestinal epithelium in the pathogenesis of IBD may contribute to a better understanding of the inflammatory processes and aid in the identification of potential therapeutic treatments. The present study aimed to evaluate the effects of tofacitinib on Caco-2 cells cultured in an inflammatory environment induced using cytokines naturally found in patients with ulcerative colitis. Tofacitinib is an orally administered inhibitor of Janus kinases (JAKs) which, by modifying the JAK/STAT signaling pathway, reduces the effect of inflammatory cytokines in the gut. Caco-2 cells were used to model the intestinal epithelium and the culture conditions included the proinflammatory cytokine TNFα and tofacitinib. At the end of the culture period, enzymes involved in oxidative stress (superoxide dismutase 1, catalase, nicotinamide adenine dinucleotide phosphate), a marker of apoptosis (Bcl-2) and a key player in intracellular inflammatory signaling (nuclear factor κB) were assessed by quantitative PCR and western blotting. The in vitro phenotype of Caco-2 cells exposed to an inflammatory environment was observed to be similar to that observed in ulcerative colitis. Notably, tofacitinib was able to improve TNFα-induced changes in an in vitro model of ulcerative colitis, and a reduction in the activity of enzymes associated with oxidative stress was observed. In addition, tofacitinib-induced upregulation of Bcl-2 and claudin-1 may contribute to the beneficial effects of tofacitinib on the intestinal epithelium. Tofacitinib appears to have a protective effect on Caco-2 cells. Notably, in the present study, exposure to TNFα stimulated oxidative stress and apoptotic effects, and disrupted intercellular connectivity. The addition of tofacitinib decreased the activity of the examined parameters of oxidative and apoptotic stress, while increasing the activity of the parameter examined to evaluate the degree of intercellular connections. In conclusion, the inhibitory effects of tofacitinib on oxidative stress, as well as its anti-apoptotic and regenerative effects, provide important information regarding the positive effect of tofacitinib on Caco-2 cells, and therefore constitute potential information about the beneficial effect of the evaluated drug in UC.

Exenatide prevents statin-related LDL receptor increase and improves insulin secretion in pancreatic beta cells (1.1E7) in a protein kinase A-dependent manner.

Statins are primary drugs in the treatment of hyperlipidemias. This group of drugs is known for its beneficial pleiotropic effects (e.g., reduction of inflammatory state). However, a growing body of evidence suggests its diabetogenic properties. The culpable mechanism is not completely understood and might be related to the damage to pancreatic beta cells. Therefore, we conceived an in vitro study to explore the impact of atorvastatin on pancreatic islet beta cells line (1.1.E7). We evaluated the influence on viability, insulin, low-density lipoprotein (LDL) receptor, and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. A significant drop in mRNA for proinsulin and insulin expression was noted. Concurrently, a rise in LDL receptor at the protein level in cells exposed to atorvastatin was noted. Further experiments have shown that exenatide - belonging to glucagon-like peptide 1 (GLP-1) analogs that are used in a treatment of diabetes and known for its weight reducing properties - can alleviate the observed alterations. In this case, the mechanism of action of exenatide was dependent on a protein kinase A pathway. In conclusion, our results support the hypothesis that statin may have diabetogenic properties, which according to our study is related to reduced insulin expression. The concomitant use of GLP-1 receptor agonist seemed to successfully revert insulin expression.

Epicardial, paracardial, and perivascular fat quantity, gene expressions, and serum cytokines in patients with coronary artery disease and diabetes.

INTRODUCTION: Obesity and diabetes mellitus (DM) are common disorders that increase cardiovascular risk and lead to coronary artery disease (CAD). OBJECTIVES: The aim of our study was to assess the link between epicardial fat (EF) volume and paracardial fat (PF) volume, relative expressions of several genes in epicardial, paracardial, and perivascular fat and corresponding serum cytokines in patients with CAD in relation to DM. PATIENTS AND METHODS: A total of 66 consecutive patients (33 with DM) with multivessel CAD were included. We obtained cardiac magnetic resonance, serum cytokines levels, and their relative mRNA expressions in EF, PF, and perivascular fat samples of the following: adrenomedullin (ADM), fibroblast growth factor 21 (FGF21), transforming growth factor ß (TGFß), phospholipid transfer protein (PLTP), receptor for advanced glycation endproducts (RAGE), thrombospondin 1 (THSB1), and uncoupling protein 1 (UCP1). RESULTS: There were no differences in the anthropometric parameters or fat depots, except for higher epicardial fat volume in patients with DM (mean [SD], 105.6 [38.5] ml vs 84 [29.2] ml; P = 0.02). Patients with DM exhibited a significantly increased RAGE expression in EF (median [Q1-Q3], 0.17 [0.06-1.48] AU vs 0.08 [0.02-0.24] AU, P = 0.03). Diabetes was also associated with increased expression of ADM in EF and PF and decreased expression of FGF21 compared with patients without DM. CONCLUSIONS: Patients with multivessel CAD and DM revealed increased volume and more dysfunctional profile of gene expressions in EF and significantly decreased expression of cardioprotective FGF21.

The impact of exenatide (a GLP-1 agonist) on markers of inflammation and oxidative stress in normal human astrocytes subjected to various glycemic conditions.

GLP-1 agonists such as exenatide and liraglutide are novel drugs for the treatment of diabetes and obesity. While improvements in glycemic control can rely on an incretin effect, the mechanisms behind the loss of weight following therapy have yet to be completely elucidated, and seem to be associated with alterations in eating habits, resulting from changes in cytokines e.g. interleukin 1ß (IL-1ß) and oxidative signaling in the central nervous system (CNS). Increased levels of IL-1ß and reactive oxygen species have been demonstrated to exert anorexigenic properties, and astrocytes appear to actively participate in maintaining the integrity of the CNS, which includes the paracrine secretion of inflammatory cytokines and involvement in the redox status. Therefore, the present study decided to explore the influence of exenatide [a glucagon-like peptide 1 (GLP-1 agonist)] on inflammatory and oxidative stress markers in cultured human astrocytes as a potential target for weight reduction therapies. In an experimental setting, normal human astrocytes were subjected to various glycemic conditions, including 40 mg/dl-hypoglycemic, 100 mg/dl-normoglycemic and 400 mg/dl-hyperglycemic, and exenatide, which is a GLP-1 agonist. The involvement of intracellular signaling by a protein kinase A (PKA) in the action of exenatide was estimated using a specific PKA inhibitor-PKI (14-22). The expression levels of IL-1ß, nuclear factor kappa κB (NFκB), glial-fibrillary acidic protein (GFAP), p22 NADPH oxidase, glutathione peroxidase, catalase, superoxide dismutase 1, and reactive oxidative species were measured. The present study demonstrated that varying glucose concentrations in the culture media did not affect the protein expression or the level of reactive oxygen species. Conversely, exenatide led to an increase in IL-1ß in normoglycemic culture conditions, which was accompanied by the increased expression of p22, glutathione peroxidase and the reduced expression of GFAP. Changes in the expression of IL-1ß and p22 were dependent on the activation of PKA. The present study concluded that exenatide predominantly affected astrocytes in normoglycemic conditions, and hypothesize that this impact demonstrated one of novel mechanisms associated with astrocyte signaling that may contribute to weight loss.

Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA.

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.

A Novel, Highly Selective RT-QPCR Method for Quantification of MSRV Using PNA Clamping Syncytin-1 (ERVWE1).

HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1 (coding syncytin-1, which is a glycoprotein essential in the formation of the placenta) and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV env gene and a peptide nucleic acid (PNA) probe, fully complementary to the ERVWE1 sequence fragment that efficiently blocks the polymerase action on ERVWE1 templates. The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e(2) to 1 × 10 e(8) copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1% in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.

A peptide nucleic acid (PNA)-mediated polymerase chain reaction clamping allows the selective inhibition of the ERVWE1 gene amplification.

A new peptide nucleic acid (PNA) mediated QPCR technique for the detection and quantification of the Multiple Sclerosis-Associated Retrovirus (MSRV) belonging to the human endogenous retrovirus-W (HERV-W) family has been developed. The assay utilizes a PNA probe which is fully complementary to the ERVWE1 sequence, another member of the HERV-W family which is closely related to MSRV. Due to its excellent affinity to a completely matched template, PNA probe selectively blocks the amplification of ERVWE1 thus allowing the specific and exclusive detection and quantification of the MSRV as PNA does not interfere with the amplification of MSRV. Using this newly developed method we found that MSRV is predominantly expressed over ERWVE1 in astrocyte-derived U-87 MG cell line but not in human umbilical vein endothelial cells or human placental cells.