Studying dynamic stress effects on the behaviour of THP-1 cells by microfluidic channels

Atherosclerosis is a long-term disease process of the vascular system that is characterized by the formation of atherosclerotic plaques, which are inflammatory regions on medium and large-sized arteries. There are many factors contributing to plaque formation, such as changes in shear stress levels, rupture of endothelial cells, accumulation of lipids, and recruitment of leukocytes. Shear stress is one of the main factors that regulates the homeostasis of the circulatory system; therefore, sudden and chronic changes in shear stress may cause severe pathological conditions. In this study, microfluidic channels with cavitations were designed to mimic the shape of the atherosclerotic blood vessel, where the shear stress and pressure difference depend on design of the microchannels. Changes in the inflammatory-related molecules ICAM-1 and IL-8 were investigated in THP-1 cells in response to applied shear stresses in an continuous cycling system through microfluidic channels with periodic cavitations. ICAM-1 mRNA expression and IL-8 release were analyzed by qRT-PCR and ELISA, respectively. Additionally, the adhesion behavior of sheared THP-1 cells to endothelial cells was examined by fluorescence microscopy. The results showed that 15 Pa shear stress significantly increases expression of ICAM-1 gene and IL-8 release in THP-1 cells, whereas it decreases the adhesion between THP-1 cells and endothelial cells

Multiplex-PCR-based screening and computational modeling of virulence factors and t-cell mediated immunity in helicobacter pylori infections for accurate clinical diagnosis

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer—based models

Doxorubicin inhibits miR-140 expression and upregulates PD-L1 expression in HCT116 cells, opposite to its effects on MDA-MB-231 cells

One of the most challenging problems in colorectal cancer (CRC) is resistance to chemotherapy drugs such as doxorubicin (DOX). The programmed death ligand-1 (PD-L1) is related to chemoresistance and is overexpressed in several human cancer cell types. Here, we investigated the changes in the expression of PD-L1 in DOX-treated CRC and breast cancer (BRC) cells. Also, to address PD-L1 regulation, we assessed expression levels of miR-140 and miR-34a, two microRNAs that can target the 3’ UTR region of the gene encoding PD-L1. HCT116 CRC and MDA-MB-231 BRC cells were treated with various doses of DOX in culture and PD-L1 expression was quantified using qRT-PCR, flow cytometry, and western blot analysis. We also evaluated PD-L1 localization in HCT116 cells by immunofluorescence. Next, we assessed expression of miR-140 and miR-34a in DOX-treated HCT116 and MDA-MB-231 cells. Finally, we investigated whether miR-140 targets the 3’ UTR of the gene encoding PD-L1 in HCT116 cells using the p2FP-RNAi RNAi reporter vector system. PD-L1 expression in HCT116 cells, while low at baseline, can be induced by treatment with 0.5 µM DOX. MDA-MB-231 baseline PD-L1 expression exceeded HCT116 cell maximal expression and decreased following DOX treatment. We further demonstrated that PD-L1 localizes to the cell surface in DOX-treated HCT116 cells. While miR-140 expression decreased in DOX-treated HCT116 cells, it increased in DOX-treated MDA-MB-231 cells. MiR-34a expression increased in both DOX-treated cell types. Finally, we present evidence for the regulation of PD-L1 by miR-140 in HCT116 cells. PD-L1 expression can increase following treatment with DOX in HCT116 cells but decrease in MDA-MB-231 cells, suggesting a distinct response to DOX in these two different cancer types. Also, a negative correlation between PD-L1 and miR-140 was observed in DOX-treated HCT116 cells, but not in MDA-MB-231 cells

The cytokine response in THP-1 (monocyte) and HL-60 (neutrophil-differentiated) cells infected with different genotypes of Helicobacter pylori strains

Background/Aims: Helicobacter pylori (H. pylori) is a microaerophilic bacterium related with peptic ulcer and gastric cancer. Its virulence factors include cytotoxin-associated gene A (CagA) and vacuolating cytotoxin gene A (VacA) proteins. Cytokine release inducted by H. pylori colonization has an important role in pathogenesis of H. pylori. The severity of gastric pathologies depends on the H. pylori genotypes found in different geographical regions. We aimed to determine the relationship between different H. pylori genotypes and their effects on the cytokine release levels

Multiplex-PCR primers designed for the amplification of <i>H</i>. <i>pylori</i> virulence genes.

<p>Multiplex-PCR primers designed for the amplification of <i>H</i>. <i>pylori</i> virulence genes.</p

Expert-derived models for diagnostic prediction of gastric diseases using <i>H</i>. <i>pylori</i> virulence factors and host immune responses.

<p><b>(a)</b> shows that a possible relationship prediction between the <i>vacAm1/m2</i>, <i>cagA</i> and <i>ureA</i> and IL-17, FOXP3 and patient’s clinical outcomes and <b>(b)</b> shows that the possible relationship prediction between <i>vacAm1/m2</i>, <i>cagA</i> and <i>ureA</i> and IL-17, FOXP3 and IFN-γ and patient’s clinical outcomes.</p

Quantitative RT-PCR primers designed for the detection of Th1, Th17 and Treg cell response.

<p>Quantitative RT-PCR primers designed for the detection of Th1, Th17 and Treg cell response.</p