Characterizing the Inflammatory Profile of Neutrophil-Rich Triple-Negative Breast Cancer.

Breast cancer (BC) is one of the most common types of cancer in women in the United Arab Emirates. Immunogenic tumours, such as triple-negative breast cancer (TNBC), show increased neutrophil infiltration, which is associated with poor prognosis and limited efficacy of immunotherapy. This study aims to investigate in vitro the bidirectional effect of neutrophils on metastatic TNBC (MDA-MB-231) compared to less-metastatic luminal breast cancer (MCF-7) cell lines. We found that BC cells or their conditioned medium (CM) reduced the viability of neutrophil-like cells (HL60). This was supported by increased cellular stress and NETosis in differentiated HL60 cells (dHL60) upon exposure to MDA-MB-231 compared to MCF-7-CM using nucleic acid staining essays. Flow cytometry showed comparable expression of inflammatory markers by polymorphonuclear cells (PMN) when treated with MDA-MB-231-CM and standard polarizing cocktails. Furthermore, MDA-MB-231-CM triggered an inflammatory pattern with evidence of stronger adhesion (CD62L) and degranulation (CD11b and CD66b) phenotypes. The proinflammatory polarization of dHL60 by MDA-MB-231-CM was additionally confirmed by the elevated CD54 expression, myeloperoxidase, and CD11b protein levels, which matched an increased transwell migratory capacity. In conclusion, BC might use neutrophils to their benefit through NETosis and complement system activation, which makes this crosstalk a potential mechanism for understanding tumour progression.

DNA methylation-mediated epigenetic regulation of oncogenic RPS2 as a novel therapeutic target and biomarker in hepatocellular carcinoma.

Ribosomal Protein S2 (RPS2) has emerged as a potential prognostic biomarker due to its involvement in key cellular processes and its altered expression pattern in certain types of cancer. However, its role in hepatocellular carcinoma (HCC) has yet to be investigated. Herein, we analyzed RPS2 mRNA expression and promoter methylation in HCC patient samples and HepG2 cells. Subsequently, loss-of-function experiments were conducted to determine the function of RPS2 in HCC cells in vitro. Our results revealed that RPS2 mRNA expression is significantly elevated, and its promoter is hypomethylated in HCC patient samples compared to controls. In addition, 5-Azacytidine treatment in HepG2 cells decreased RPS2 promoter methylation level and increased its mRNA expression. RPS2 knockdown in HepG2 cells suppressed cell proliferation and promoted apoptosis. Functional pathway analysis of genes positively and negatively associated with RPS2 expression in HCC showed enrichment in ribosomal biogenesis, translation machinery, cell cycle regulation, and DNA processing. Furthermore, utilizing drug-protein 3D docking, we found that doxorubicin, sorafenib, and 5-Fluorouracil, showed high affinity to the active sites of RPS2, and in vitro treatment with these drugs reduced RPS2 expression. For the first time, we report on DNA methylation-mediated epigenetic regulation of RPS2 and its oncogenic role in HCC. Our findings suggest that RPS2 plays a significant role in the development and progression of HCC, hence its potential prognostic and therapeutic utility. Moreover, as epigenetic changes happen early in cancer development, RPS2 may serve as a potential biomarker for tumor progression.

Comprehensive In silico analysis of chaperones identifies CRYAB and P4HA2 as potential therapeutic targets and their small-molecule inhibitors for the treatment of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a subtype of liver cancer with increasing incidence, poor prognosis, and limited treatment modalities. It is, therefore, imperative to identify novel therapeutic targets for better management of the disease. Chaperones are known to be significant regulators of carcinogenesis, however, their role in CCA remains unclear. This study aims to screen chaperones involved in CCA pathogenesis and identify drugs targeting key chaperones to improve the therapeutic response to the disease. To achieve this, first we mined the literature to create an atlas of human chaperone proteins. Next, their expression in CCA was determined by publicly available datasets of patients at mRNA and protein levels. In addition, our analysis involving protein-protein interaction and pathway analysis of eight key dysregulated chaperones revealed that they control crucial cancer-related pathways. Furthermore, topology analysis of the CCA network identified crystallin alpha-B protein (CRYAB) and prolyl-4-hydroxylase subunit 2 (P4HA2) as novel therapeutic targets for the disease. Finally, drug repurposing of 286 clinically approved anti-cancer drugs against these two chaperones performed by molecular docking and molecular dynamics simulations showed that tucatinib and regorafenib had a modulatory effect on them and could be potential inhibitors of CRYAB and P4HA2, respectively. Overall, our study, for the first time, provides insights into the pan-chaperone expression in CCA and explains the pathways that might drive CCA pathogenesis. Further, our identification of potential therapeutic targets and their inhibitors could provide new and complementary approaches to CCA treatment.

Molecular pathogenicity of 1-nonadecene and L-lactic acid, unique metabolites in radicular cysts and periapical granulomas.

Recently, 1-nonadecene and L-lactic acid were identified as unique metabolites in radicular cysts and periapical granuloma, respectively. However, the biological roles of these metabolites were unknown. Therefore, we aimed to investigate the inflammatory and mesenchymal-epithelial transition (MET) effects of 1-nonadecene, and the inflammatory and collagen precipitation effects of L-lactic acid on both periodontal ligament fibroblasts (PdLFs) and peripheral blood mononuclear cells (PBMCs). PdLFs and PBMCs were treated with 1-nonadecene and L-lactic acid. Cytokines' expression was measured using quantitative real-time polymerase chain reaction (qRT-PCR). E-cadherin, N-cadherin, and macrophage polarization markers were measured using flow cytometry. The collagen, matrix metalloproteinase (MMP)-1, and released cytokines were measured using collagen assay, western blot, and Luminex assay, respectively. In PdLFs, 1-nonadecene enhances inflammation through the upregulation of some inflammatory cytokines including IL-1ß, IL-6, IL-12A, monocyte chemoattractant protein (MCP)-1, and platelet-derived growth factor (PDGF) α. 1-Nonadecene also induced MET through the upregulation of E-cadherin and the downregulation of N-cadherin in PdLFs. 1-Nonadecene polarized macrophages to a pro-inflammatory phenotype and suppressed their cytokines' release. L-lactic acid exerted a differential impact on the inflammation and proliferation markers. Intriguingly, L-lactic acid induced fibrosis-like effects by enhancing collagen synthesis, while inhibiting MMP-1 release in PdLFs. These results provide a deeper understanding of 1-nonadecene and L-lactic acid's roles in modulating the microenvironment of the periapical area. Consequently, further clinical investigation can be employed for target therapy.

Titanium Particles Modulate Lymphocyte and Macrophage Polarization in Peri-Implant Gingival Tissues.

Titanium dental implants are one of the modalities to replace missing teeth. The release of titanium particles from the implant's surface may modulate the immune cells, resulting in implant failure. However, little is known about the immune microenvironment that plays a role in peri-implant inflammation as a consequence of titanium particles. In this study, the peri-implant gingival tissues were collected from patients with failed implants, successful implants and no implants, and then a whole transcriptome analysis was performed. The gene set enrichment analysis confirmed that macrophage M1/M2 polarization and lymphocyte proliferation were differentially expressed between the study groups. The functional clustering and pathway analysis of the differentially expressed genes between the failed implants and successful implants versus no implants revealed that the immune response pathways were the most common in both comparisons, implying the critical role of infiltrating immune cells in the peri-implant tissues. The H&E and IHC staining confirmed the presence of titanium particles and immune cells in the tissue samples, with an increase in the infiltration of lymphocytes and macrophages in the failed implant samples. The in vitro validation showed a significant increase in the level of IL-1ß, IL-8 and IL-18 expression by macrophages. Our findings showed evidence that titanium particles modulate lymphocyte and macrophage polarization in peri-implant gingival tissues, which can help in the understanding of the imbalance in osteoblast-osteoclast activity and failure of dental implant osseointegration.

Antimicrobial Activity of Biogenic Metal Oxide Nanoparticles and Their Synergistic Effect on Clinical Pathogens.

The rising prevalence of antibiotic-resistance is currently a grave issue; hence, novel antimicrobial agents are being explored and developed to address infections resulting from multiple drug-resistant pathogens. Biogenic CuO, ZnO, and WO3 nanoparticles can be considered as such agents. Clinical isolates of E. coli, S. aureus, methicillin-resistant S. aureus (MRSA), and Candida albicans from oral and vaginal samples were treated with single and combination metal nanoparticles incubated under dark and light conditions to understand the synergistic effect of the nanoparticles and their photocatalytic antimicrobial activity. Biogenic CuO and ZnO nanoparticles exhibited significant antimicrobial effects under dark incubation which did not alter on photoactivation. However, photoactivated WO3 nanoparticles significantly reduced the number of viable cells by 75% for all the test organisms, thus proving to be a promising antimicrobial agent. Combinations of CuO, ZnO, and WO3 nanoparticles demonstrated synergistic action as a significant increase in their antimicrobial property (>90%) was observed compared to the action of single elemental nanoparticles. The mechanism of the antimicrobial action of metal nanoparticles both in combination and in isolation was assessed with respect to lipid peroxidation due to ROS (reactive oxygen species) generation by measuring malondialdehyde (MDA) production, and the damage to cell integrity using live/dead staining and quantitating with the use of flow cytometry and fluorescence microscopy.

The Essential Role of 17-Octadecynoic Acid in the Pathogenesis of Periapical Abscesses.

INTRODUCTION: Periapical abscesses are 1 of the most frequent pathologic lesions in the alveolar bone. Recently, we have identified 17-octadecynoic acid (17-ODYA) as the highest unique metabolite in periapical abscesses. Therefore, the aim of this study was to investigate the immunologic and pathophysiological roles of this metabolite in the initiation and development of periapical abscesses. METHODS: Periodontal ligament fibroblasts and peripheral blood mononuclear cells were treated with 17-ODYA. Gene expression analysis and interleukin (IL)-8 release were determined using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophage polarization and cytokine release were also determined using flow cytometry and Luminex bioassay (R&D Systems, Minneapolis, MN), respectively. RESULTS: In periodontal ligament fibroblasts, 17-ODYA caused significant (P < .0001) up-regulation of IL-1α, IL-1ß, IL-6, matrix metalloproteinase-1, and monocyte chemoattractant protein-1 at 10 µmol/L after 6 days of treatment and up-regulation of platelet-derived growth factor alpha and vascular endothelial growth factor alpha at all tested concentrations after 2 days of treatment. In peripheral blood mononuclear cells, 17-ODYA significantly increased the expression of IL-1α, IL-1ß, IL-6, matrix metalloproteinase-1, and monocyte chemoattractant protein-1 at 10 µmol/L (P < .0001) and vascular endothelial growth factor alpha and platelet-derived growth factor alpha at 1 µmol/L 17-ODYA (P < .0001). 17-ODYA polarized macrophages toward a proinflammatory phenotype (M1) and suppressed the release of pro- and anti-inflammatory cytokines. 17-ODYA significantly enhanced the release of IL-8. CONCLUSIONS: This study was the first to identify the pathologic role of 17-ODYA in the development of periapical abscesses. The results of this study are important in shedding light on the pathogenesis of periapical abscesses in relation to microbial metabolites.

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen.

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9-1.5 kPa and 14-20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.

Cardiomyocyte-GSK-3ß deficiency induces cardiac progenitor cell proliferation in the ischemic heart through paracrine mechanisms.

Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here, we sought to identify the potential roles of glycogen synthase kinase-3ß (GSK-3ß), a critical regulator of cell proliferation and differentiation, in CPC proliferation post-myocardial infarction (MI). Cardiomyocyte-specific conditional GSK-3ß knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension and contractile functions were comparable at 2 weeks post-MI, cKO mice displayed significantly preserved LV chamber and contractile function versus control mice at 4 weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit-positive cells (KPCs) were observed in the cKO hearts at 4 and 6 weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage, as the majority of identified KPCs were negative for the hematopoietic lineage marker, CD45. Mechanistically, cardiomyocyte-GSK-3ß profoundly suppresses the expression and secretion of growth factors, including basic-fibroblast growth factor, angiopoietin-2, erythropoietin, stem cell factor, platelet-derived growth factor-BB, granulocyte colony-stimulating factor, and vascular endothelial growth factor, post-hypoxia. In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3ß promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocyte and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte-GSK-3ß could be helpful for the promotion of in situ cardiac regeneration post-ischemic injury.

Caspofungin-resistance in Candida auris is cell wall-dependent phenotype and potential prevention by zinc oxide nanoparticles.

Candida auris is an emergent nosocomial multi-drug-resistant yeast that represents a global health threat. Recently, C. auris clinical isolates with caspofungin resistance were identified. Mutation in FKS1 gene was determined as a mechanism of resistance. However, the ability of C. auris to develop acquired and cross-resistance has never been investigated. Herein, this resistance ability due to caspofungin and associate mechanisms were investigated. C. auris clinical isolate was successively cultured for ten generations in the presence of caspofungin compared to fluconazole-treatment and untreated controls. This was followed by the analysis of target gene expression and phenotypic changes. The obtained results showed that caspofungin-treated C. auris exhibited elevated MIC50(caspofungin), slower growth, elevated chitin content, overexpression of caspofungin target genes, and cross-resistance to fluconazole. Interestingly, caspofungin exposure induced cell-cell adhesion and biofilm formation. C. auris gradually lost caspofungin resistance after removal of antifungal pressure, while keeping the overexpression of fungal cell wall-related genes including ALS5. We propose that C. auris ageing in the presence of caspofungin caused the development of persistent phenotypic changes in the fungal cell wall, leading to acquired and physical cross-resistance mechanisms. Surprisingly, formulation of caspofungin in zinc oxide nanoparticles prevented the aforementioned behavioral changes regardless of the pathogen generations. LAY SUMMARY: Candida auris developed resistance against caspofungin. Our data indicated that this resistance mechanism is unique because of changes in the genes related to cell wall adhesions. Formulation of caspofungin in ZnO nanoparticles was able to overcome these phenotypic changes.

Estrogen-induced hypomethylation and overexpression of YAP1 facilitate breast cancer cell growth and survival.

Increased expression of Yes-associated protein-1 (YAP1) was shown to correlate with reduced survival in breast cancer (BC) patients. However, the exact mechanism of YAP1 regulation in BC cells remains ambiguous. Genomic sequence search showed that the promoter region of the YAP1 gene contains CpG Islands, hence the likelihood of epigenetic regulation by DNA methylation. To address this possibility, the effect of estrogen (17ß estradiol; E2) on YAP1 gene expression and YAP1 promoter methylation status was evaluated in BC cells. The functional consequences of E2 treatment in control and YAP1-silenced BC cells were also investigated. Our data showed that E2 modulates YAP1 expression by hypomethylation of its promoter region via downregulation of DNA methyltransferase 3B (DNMT3B); an effect that seems to facilitate tumor progression in BC cells. Although the effect of E2 on YAP1 expression was estrogen receptor (ER) dependent, E2 treatment also upregulated YAP1 expression in MDA-MB231 and SKBR3 cells, which are known ER-negative BC cell lines but expresses ERα. Functionally, E2 treatment resulted in increased cell proliferation, decreased apoptosis, cell cycle arrest, and autophagic flux in MCF7 cells. The knockdown of the YAP1 gene reversed these carcinogenic effects of E2 and inhibited E2-induced autophagy. Lastly, we showed that YAP1 is highly expressed and hypomethylated in human BC tissues and that increased YAP1 expression correlates negatively with DNMT3B expression but strongly associated with ER expression. Our data provide the basis for considering screening of YAP1 expression and its promoter methylation status in the diagnosis and prognosis of BC.

Azithromycin Differentially Alters TCR-Activated Helper T Cell Subset Phenotype and Effector Function.

In addition to their antibiotic activities, azithromycin (AZM) exhibits anti-inflammatory effects in various respiratory diseases. One of the potent anti-inflammatory mechanisms is through inhibition of CD4+ helper T (Th) cell effector function. However, their impact on specific Th subset is obscure. Herein, we demonstrate the cellular basis of phenotypic and functional alterations associated with Th subsets following AZM treatment in vitro. Using well-characterized Th subset specific chemokine receptors, we report significant suppression of T cell receptor (TCR)-stimulated hyperactivated CCR4+CXCR3+ (Th0) expansion compared to CCR4-CXCR3+ (Th1-like) and CCR4+CXCR3- (Th2-like) cells. Interestingly, this effect was associated with diminished cell proliferation. Furthermore, AZM significantly inhibited the inflammatory cytokines IFN-γ and IL-4 production, CCR4 and CXCR3 receptor expression, and viability of Th0, Th1-like, and Th2-like subsets. Our findings suggest that AZM differentially affects TCR-activated Th subsets phenotype and function, and CCR4 and CXCR3 downregulation and suppressed Th0 subset expansion could potentially influence their trafficking and differentiation into cytokine-producing effector cells.

Induction of CD4+CD25+ Regulatory T Cells from In Vitro Grown Human Mononuclear Cells by Sparteine Sulfate and Harpagoside.

Regulatory T cells (Tregs) are key players in the regulation of inflammatory responses. In this study, two natural molecules, namely, sparteine sulfate (SS) and harpagoside (Harp), were investigated for their ability to induce Tregs in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy volunteers and grown in the presence or absence of ConA, with TGF-beta, SS or Harp. Expression of the mRNA of FoxP3, TGF-beta, IL-10 and GAPDH was assessed via q-PCR. The expression of Treg markers including CD4, CD25, CD127 and FoxP3 was measured via flow cytometry. The secretion of IL-10 and TGF-beta by cultured cells was assessed by ELISA. Furthermore, the suppressive role of SS and Harp on PBMCs in vitro was tested via allogeneic mixed lymphocyte reaction (MLR). Data obtained show that both compounds increased the expression of FoxP3, TGF-beta and IL-10 mRNA in resting PBMCs but to a lesser extent in activated cells. Moreover, they significantly increased the percent of CD4+CD25+FoxP3+CD127- Tregs in activated and naïve PBMCs. Functionally, both compounds caused a significant reduction in the stimulation index in allogeneic MLR. Together, our data demonstrate for the first time that SS and Harp can induce human Tregs in vitro and therefore have great potential as anti-inflammatory agents.

Osteogenic and odontogenic differentiation potential of dental pulp stem cells isolated from inflamed dental pulp tissues (I-DPSCs) by two different methods.

Objective: The objective of the present study is to isolate stem cells from inflamed dental pulp tissues (I-DPSCs) and study the characteristic such as surface markers, osteo/odontogenic differentiation potential between the outgrowth (OG) and enzymatic digestion (COL) methods.Materials and methods: I-DPSCs harvested by both methods were analysed for Mesenchymal Stem Cell marker expression by flow cytometry. The metabolic activity of the isolated cells was assessed by MTT assay. The Alkaline Phosphatase (ALP) and Alizarin red staining was done to analyse the osteogenic potential of isolated cells. The osteo/odontogenic differentiation was done by checking the expression of Dentine Matrix Protein 1 (DMP1), Dentine Sialophosphoprotein (DSPP), ALP and Bone Gamma-Carboxyglutamate Protein (BGLAP) by Real time PCR.Results: The isolated cells were positive for MSC markers such as CD-90, CD-105 and CD-73 and negative for CD-14, CD-45 and STRO-1. MTT assay indicated that the I-DPSCs from OG method showed higher metabolic activity than cells from COL. However, the osteo/odontogenic differentiation was in favour of cells isolated by COL method.Conclusion: Although the cell metabolic rate was more in OG, the osteo/odontogenic differentiation was higher in COL, suggesting that the isolation method and culture conditions do affect the differentiation capacity of isolated cells.

Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1ß Release From NK92 Cells.

INTRODUCTION: Although natural killer (NK) are major cells used to treat cancer patients, recent clinical trials showed that NK92 cells can be also used for the same purpose due to their high anti-tumor activity. Here, we examined whether these cells might be inflammatory due to the release of interleukin-1ß (IL-1ß), and whether the anti-inflammatory molecules dimethyl fumarate (DMF), or monomethyl fumarate (MMF) impair this activity. METHODS: NK92 cells were examined for the synthesis and release of IL-1ß utilizing RT-PCR and ELISA assay, respectively. The expression of hydroxy-carboxylic acid receptors (HCA)1, HCA2 and HCA3 was detected by immunoblotting, flow cytometry, immunofluorescence and RT-PCR assays. The activation of caspase-1 and Gasdermin D (GSDMD) was evaluated by immunoblot assay. Pyroptosis was demonstrated by immunofluorescence imaging. Expression of DNA methyltransferases (DNMTs) mRNA was determined by whole transcriptome and immunoblot analyses. RESULTS: LPS-induced the release of IL-1ß from NK92 cells, whereas DMF or MMF inhibited this induction. The effect of these drugs was due to inhibiting the conversion of procaspase-1 into active caspase-1. NK92 cells highly expressed GSDMD, a pyroptotic-mediated molecule. However, LPS induced the distribution of GSDMD into the cell membranes, corroborated with the presence of pyroptotic bodies, an activity that was inhibited by DMF or MMF. These molecule also inhibited the generation of GSDMD through DNMT-mediated hypermethylation of the promoter region of GSDMD gene. These results were supported by increased expression of DNMTs mRNA as determined by whole transcriptome analysis. DISCUSSION: Our results are the first to show that NK92 cells utilize GSDMD pathway to release IL-1ß. Further, DMF and MMF which were previously shown to enhance NK cell cytotoxicity, also inhibit the inflammatory effects of these cells, making them most suitable for treating cancer patients. .