Xenopus: An in vivo model for studying skin response to ultraviolet B irradiation.

Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.

Circulating microRNA profile in response to remdesivir treatment in coronavirus disease 2019 (COVID-19) patients.

Coronavirus disease 2019 (COVID-19), a serious infectious disease caused by the recently discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a major global health crisis. Although no specific antiviral drugs have been proven to be fully effective against COVID-19, remdesivir (GS-5734), a nucleoside analogue prodrug, has shown beneficial effects when used to treat severe hospitalized COVID-19 cases. The molecular mechanism underlying this beneficial therapeutic effect is still vaguely understood. In this study, we assessed the effect of remdesivir treatment on the pattern of circulating miRNAs in the plasma of COVID-19 patients, which was analyzed using MiRCURY LNA miRNA miRNome qPCR Panels and confirmed by quantitative real-time RT-PCR (qRT-PCR). The results revealed that remdesivir treatment can restore the levels of miRNAs that are upregulated in COVID-19 patients to the range observed in healthy subjects. Bioinformatics analysis revealed that these miRNAs are involved in diverse biological processes, including the transforming growth factor beta (TGF-ß), hippo, P53, mucin-type O-glycan biosynthesis, and glycosaminoglycan biosynthesis signaling pathways. On the other hand, three miRNAs (hsa-miR-7-5p, hsa-miR-10b-5p, and hsa-miR-130b-3p) were found to be upregulated in patients receiving remdesivir treatment and in patients who experienced natural remission. These upregulated miRNAs could serve as biomarkers of COVID-19 remission. This study highlights that the therapeutic potential of remdesivir involves alteration of certain miRNA-regulated biological processes. Targeting of these miRNAs should therefore be considered for future COVID-19 treatment strategies.

High-throughput synthetic rescue for exhaustive characterization of suppressor mutations in human genes.

Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment. Here, we first review all known suppressor mutations' mechanisms characterized by genetic screens on model species like yeast or flies. However, human suppressor mutations are scarce, despite some being discovered based on orthologue genes. Because of recent advances in high-throughput screening, developing an inventory of human suppressor mutations for pathological processes seems achievable. In addition, we review several screening methods for suppressor mutations in cultured human cells through knock-out, knock-down or random mutagenesis screens on large scale. We provide examples of studies published over the past years that opened new therapeutic avenues, particularly in oncology.

Mapping and characterization of G-quadruplexes in the genome of the social amoeba Dictyostelium discoideum.

G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. 'Dicty' is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249-1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a 'G4-poor' model for studies on G-quadruplexes.

Isoconazole and Clemizole Hydrochloride Partially Reverse the Xeroderma Pigmentosum C Phenotype.

Xeroderma Pigmentosum protein C (XPC) is involved in recognition and repair of bulky DNA damage such as lesions induced by Ultra Violet (UV) radiation. XPC-mutated cells are, therefore, photosensitive and accumulate UVB-induced pyrimidine dimers leading to increased cancer incidence. Here, we performed a high-throughput screen to identify chemicals capable of normalizing the XP-C phenotype (hyper-photosensitivity and accumulation of photoproducts). Fibroblasts from XP-C patients were treated with a library of approved chemical drugs. Out of 1280 tested chemicals, 16 showed ≥25% photo-resistance with RZscore above 2.6 and two drugs were able to favor repair of 6-4 pyrimidine pyrimidone photoproducts (6-4PP). Among these two compounds, Isoconazole could partially inhibit apoptosis of the irradiated cells especially when cells were post-treated directly after UV irradiation while Clemizole Hydrochloride-mediated increase in viability was dependent on both pre and post treatment. No synergistic effect was recorded following combined drug treatment and the compounds exerted no effect on the proliferative capacity of the cells post UV exposure. Amelioration of XP-C phenotype is a pave way towards understanding the accelerated skin cancer initiation in XP-C patients. Further examination is required to decipher the molecular mechanisms targeted by these two chemicals.

Targeting Lipid Metabolism in Liver Cancer.

Cancer cells are highly dependent on different metabolic pathways for sustaining their survival, growth, and proliferation. Lipid metabolism not only provides the energetic needs of the cells but also provides the raw material for cellular growth and the signaling molecules for many oncogenic pathways. Mainly processed in the liver, lipids play an essential role in the physiology of this organ and in the pathological progression of many diseases such as metabolic syndrome and hepatocellular carcinoma (HCC). The progression of HCC is associated with inflammation and complex metabolic reprogramming, and its prognosis remains poor because of the lack of effective therapies despite many years of dedicated research. Defects in hepatic lipid metabolism induce abnormal gene expression and rewire many cellular pathways involved in oncogenesis and metastasis, implying that interfering with lipid metabolism within the tumor and the surrounding microenvironment may be a novel therapeutic approach for treating liver cancer patients. Therefore, this review focuses on the latest advances in drugs targeting lipid metabolism and leading to promising outcomes in preclinical studies and some ongoing clinical trials.

Triple negative breast cancer: microRNA expression profile and novel discriminators according to BRCA1 status.

Triple-negative breast cancer (TNBC) represents 15% of breast carcinomas. More than 80% of women with a breast cancer associated with a breast cancer type 1 (BRCA1) mutation develop a TNBC. microRNAs (miRNAs) play critical roles in diverse biological processes and are aberrantly expressed in several human neoplasms including breast cancer, where they function as actors of tumor onset, behavior, and progression. However, an extensive microRNA profile has not yet been determined for TNBC. Taqman low-density arrays (TLDAs) were used to screen the expression level of 667 miRNAs in TNBC versus normal breast tissues. Our TLDA results revealed 20 differentially expressed miRNAs among which 14 (10 upregulated and four downregulated) were confirmed by an individual quantitative real-time polymerase chain reaction. Interestingly, a novel link between BRCA1 status and miRNA expression level was identified through miR-96 and miR-10b that were very important discriminators between TNBC with mutated BRCA1 and TNBC with wild type BRCA1. This study promises discoveries of new pathological pathways at work in this dreadful disease and clearly warrants validation in large prospective studies with the aim of identifying novel biomarkers for diagnosis and targets for clinical interventions.

The effects of different bisphenol derivatives on oxidative stress, DNA damage and DNA repair in RWPE-1 cells: A comparative study.

Bisphenol A (BPA) is a well-known endocrine disruptor and it is widely used mainly in the plastics industry. Due to recent reports on its possible impact on health (particularly on the male reproductive system), bisphenol F (BPF) and bisphenol S (BPS) are now being used as alternatives. In this study, RWPE-1 cells were used as a model to compare cytotoxicity, oxidative stress-causing potential and genotoxicity of these chemicals. In addition, the effects of the bisphenol derivatives were assessed on DNA repair proteins. RWPE-1 cells were incubated with BPA, BPF, and BPS at concentrations of 0-600 µM for 24 h. The inhibitory concentration 20 (IC20 , concentration that causes 20% of cell viability loss) values for BPA, BPF, and BPS were 45, 65, and 108 µM, respectively. These results indicated that cytotoxicity potentials were ranked as BPA > BPF > BPS. We also found alterations in superoxide dismutase, glutathione peroxidase and glutathione reductase activities, and glutathione and total antioxidant capacity in all bisphenol-exposed groups. In the standard and modified Comet assay, BPS produced significantly higher levels of DNA damage vs the control. DNA repair proteins (OGG1, Ape-1, and MyH) involved in the base excision repair pathway, as well as p53 protein levels were down-regulated in all of the bisphenol-exposed groups. We found that the BPA alternatives were also cytotoxic and genotoxic, and changed the expressions of DNA repair enzymes. Therefore, further studies are needed to assess whether they can be used safely as alternatives to BPA or not.

Identification of Acute Myeloid Leukemia Bone Marrow Circulating MicroRNAs.

BACKGROUND: In addition to their roles in different biological processes, microRNAs in the tumor microenvironment appear to be potential diagnostic and prognostic biomarkers for various malignant diseases, including acute myeloid leukemia (AML). To date, no screening of circulating miRNAs has been carried out in the bone marrow compartment of AML. Accordingly, we investigated the circulating miRNA profile in AML bone marrow at diagnosis (AMLD) and first complete remission post treatment (AMLPT) in comparison to healthy donors (HD). METHODS: Circulating miRNAs were isolated from AML bone marrow aspirations, and a low-density TaqMan miRNA array was performed to identify deregulated miRNAs followed by quantitative RT-PCR to validate the results. Bioinformatic analysis was conducted to evaluate the diagnostic and prognostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s). RESULTS: We found several deregulated miRNAs between the AMLD vs. HD vs. AMLPT groups, which were involved in tumor progression and immune suppression pathways. We also identified significant diagnostic and prognostic signatures with the ability to predict AML patient treatment response. CONCLUSIONS: This study provides a possible role of enriched circulating bone marrow miRNAs in the initiation and progression of AML and highlights new markers for prognosis and treatment monitoring.

Th17 immune response to adipose tissue-derived mesenchymal stromal cells.

Adipose tissue-derived mesenchymal stromal cells (ASCs) hold the promise of achieving successful immunotherapeutic results due to their ability to regulate different T-cell fate. ASCs also show significant adaptability to environmental stresses by modulating their immunologic profile. Cell-based therapy for inflammatory diseases requires a detailed understanding of the molecular relation between ASCs and Th17 lymphocytes taking into account the influence of inflammation and cell ratio on such interaction. Accordingly, a dose-dependent increase in Th17 generation was only observed in high MSC:T-cell ratio with no significant impact of inflammatory priming. IL-23 receptor (IL-23R) expression by T cells was not modulated by ASCs when compared to levels in activated T cells, while ROR-γt expression was significantly increased reaching a maximum in high (1:5) unprimed ASC:T-cell ratio. Finally, multiplex immunoassay showed substantial changes in the secretory profile of 15 cytokines involved in the Th17 immune response (IL-1ß, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-22, IL-21, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40, and TNF-α), which was modulated by both cell ratio and inflammatory priming. These findings suggest that Th17 lymphocyte pathway is significantly modulated by ASCs that may lead to immunological changes. Therefore, future ASC-based immunotherapy should take into account the complex and detailed molecular interactions that depend on several factors including inflammatory priming and cell ratio.

Human CD8+ CD25 + CD127 low regulatory T cells: microRNA signature and impact on TGF-ß and IL-10 expression.

Regulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune-related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8+ CD25 + CD127 low Tregs has not been described yet. We thus identified, using TaqMan low-density array (TLDA) technique followed by individual quantitative real-time polymerase chain reaction (qRT-PCR) confirmation, 14 miRNAs, among which 12 were downregulated whereas two were upregulated in CD8 + CD25 + CD127 low Tregs in comparison to CD8 + CD25 - T cells. In the next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3'-untranslated region (3'-UTR) of key Treg cell-immunomodulatory genes with a special focus on interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Having identified potential miR target sites in the 3'-UTR of IL-10 (miR-27b-3p and miR-340-5p) and TGF-ß (miR-330-3p), we showed through transfection and transduction assays that the overexpression of two underexpressed miRNAs, miR-27b-3p and miR-340-5p, downregulated IL-10 expression upon targeting its 3'-UTR. Similarly, overexpression of miR-330-3p negatively regulated TGF-ß expression. These results highlighted an important impact of the CD8 + Treg mirnome on the expression of genes with significant implication on immunosuppression. These observations could help in better understanding the mechanism(s) orchestrating Treg immunosuppressive function toward unraveling new targets for treating autoimmune pathologies and cancer.

MiR302c, Sp1, and NFATc2 regulate interleukin-21 expression in human CD4+CD45RO+ T lymphocytes.

Interleukin-21 (IL-21) is a cytokine with potent regulatory effects on different immune cells. Recently, IL-21 has been contemplated for use in the treatment of cancers. However, the molecular mechanisms regulating human IL-21 gene expression has not yet been described. In this study, we initially studied the promoter region and identified the transcription start site. We thereafter described the essential region upstream of the transcription start site and showed the in vivo binding of NFATc2 and SP1 transcription factors to this region, in addition to their positive role in IL-21 expression. We also studied the role of microRNAs (miRNAs) in regulating IL-21 expression. We, thus, established the miRNA profile of CD4+CD45RO+ versus CD4+CD45RA+ isolated from healthy volunteers and identified a signature composed of 12 differentially expressed miRNAs. We showed that miR-302c is able to negatively regulate IL-21 expression by binding directly to its target site in the 3'-untranslated region. Moreover, after using fresh human CD4-positive T cells, we observed the high acetylation level of histone H4, an observation well in line with the already described high expression of IL-21 in CD4+CD45RO+ versus CD4+CD45RA+ T cells. Altogether, our data identified different molecular mechanisms regulating IL-21 expression.

Immunological modulation following bone marrow-derived mesenchymal stromal cells and Th17 lymphocyte co-cultures.

OBJECTIVE AND DESIGN: The objective of the study is to uncover the influence of human bone marrow-derived mesenchymal stem cells (BM-MSCs) on the generation of Th17 lymphocytes in co-cultures of both BM-MSCs and T cells. MATERIALS AND METHODS: BM-MSCs, characterized according to the international society for cellular therapy (ISCT) criteria, were co-cultured with T cells isolated from peripheral blood. The expression levels of IL-17 receptor, RORγt and IL-23 receptor were evaluated using flow cytometry. The levels of cytokines involved in Th17 immunomodulation were measured using multiplex assay. TREATMENT: Inflammatory primed and non-primed BM-MSCs were co-cultured with either activated or non-activated T cells either at (1/80) and (1/5) ratio respectively. RESULTS: MSC/T-cell ratio and inflammation significantly influenced the effect of BM-MSCs on the generation of Th17 lymphocytes. Cocultures of either primed or non-primed BM-MSCs with activated T cells significantly induced IL-17A-expressing lymphocytes. Interestingly, the expression of the transcription factor RORγt was significantly increased when compared to levels in activated T cells. Finally, both cell ratio and priming of BM-MSCs with cytokines substantially influenced the cytokine profile of BM-MSCs and T cells. CONCLUSION: Our findings suggest that BM-MSCs significantly modulate the Th17 lymphocyte pathway in a complex manner.

Immunomodulatory effects of foreskin mesenchymal stromal cells on natural killer cells.

Foreskin-mesenchymal stromal cells (FSK-MSCs) are immune-privileged thus making them valuable immunotherapeutic cell product. Characterization of the relationship between FSK-MSCs and natural killer (NK) cells is essential to improve cell-based therapy. In the present study, we studied for the first time FSK-MSCs-NK interaction and showed that the result of such cross talk was robustly dependent on the type of cytokines (IL-2, IL-12, IL-15, and IL-21) employed to activate NK cells. Distinctly activated-NK cells showed uneven cytotoxicity against FSK-MSCs, triggering their death in fine. The expression of different cell-surface ligands (CD112, CD155, ULPB-3) and receptors (LAIR, KIRs) ensuring such interaction was altered following co-culture of both populations. Despite their partial negative effect on NK cell proliferation, FSK-MSCs boosted the capacity of activated NK-cells to secrete IFN-γ and TNF-α. Moreover, FSK-MSCs enhanced degranulation of NK cells, reinforced secretion of perforin and granzymes, while only modestly increased ROS production. On the other hand, FSK-MSCs-mediated expression of C1 and B9 serpins was significantly lowered in the presence of activated NK cells. Altogether, our results highlight major immunological changes following FSK-MSCs-NK interaction. Understanding these outcomes will therefore enhance the value of the therapeutic strategy.

Characterization and assessment of potential microRNAs involved in phosphate-induced aortic calcification.

Medial artery calcification, a hallmark of type 2 diabetes mellitus and chronic kidney disease (CKD), is known as an independent risk factor for cardiovascular mortality and morbidity. Hyperphosphatemia associated with CKD is a strong stimulator of vascular calcification but the molecular mechanisms regulating this process remain not fully understood. We showed that calcification was induced after exposing Sprague-Dawley rat aortic explants to high inorganic phosphate level (Pi , 6 mM) as examined by Alizarin red and Von Kossa staining. This calcification was associated with high Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity, vascular smooth muscle cells de-differentiation, manifested by downregulation of smooth muscle 22 alpha (SM22α) protein expression which was assessed by immunoblot analysis, immunofluorescence, and trans-differentiation into osteo-chondrocyte-like cells revealed by upregulation of Runt related transcription factor 2 (Runx2), TNAP, osteocalcin, and osteopontin mRNA levels which were determined by quantitative real-time PCR. To unravel the possible mechanism(s) involved in this process, microRNA (miR) expression profile, which was assessed using TLDA technique and thereafter confirmed by individual qRT-PCR, revealed differential expression 10 miRs, five at day 3 and 5 at day 6 post Pi treatment versus control untreated aortas. At day 3, miR-200c, -155, 322 were upregulated and miR-708 and 331 were downregulated. After 6 days of treatment, miR-328, -546, -301a were upregulated while miR-409 and miR-542 were downregulated. Our results indicate that high Pi levels trigger aortic calcification and modulation of certain miRs. These observations suggest that mechanisms regulating aortic calcification might involve miRs, which warrant further investigations in future studies.

First-line treatment of Helicobacter pylori in Lebanon: Comparison of bismuth-containing quadruple therapy versus 14-days sequential therapy.

Helicobacter Pylori (H. Pylori) is the most common cause of peptic ulcer disease (PUD) and represents a strong risk factor for gastric cancer. Treatment of H. Pylori is, therefore, a persistent need to avoid serious medical complications. Resistance to antibiotics remains to be the major challenge for H. Pylori eradication. In this study, we determined the prevalence of H. pylori infection and evaluated H. pylori eradication efficacy of bismuth-containing quadruple therapy (Pylera) versus 14-days sequential therapy in treatment naïve-Lebanese patients. 1030 patients, showing symptoms of peptic ulcer (PU) and gastritis, underwent 14C-Urea Breath Test and esophagogastroduodenoscopy to examine H. Pylori infection and gastrointestinal disorders. Among the H. Pylori-positive patients 60 individuals were randomly selected, separated into two groups (each consisting of 30 patients) and treated with either bismuth-containing quadruple therapy or 14-days sequential therapy. We show that of the 1050 patients tested: 46.2% were H. pylori-positive, 55% had gastritis, 46.2% had both gastritis and H. pylori infection, 8.8% had gastritis but no H. pylori infection, 44.9% had neither gastritis nor H. pylori infection. Following the 14-days sequential therapy, the eradication rate was significantly higher than that obtained upon using bismuth-containing quadruple therapy [80% (24/30) versus 50% (15/30), χ2 = 5.93, P = 0.015]. In conclusion, we determined H. pylori and gastritis prevalence among Lebanese PU-patients and showed that 14-days sequential therapy is more efficient than bismuth-containing quadruple therapy in terms of H. Pylori-eradication.

Immunological impact of Wharton's Jelly mesenchymal stromal cells and natural killer cell co-culture.

Due to their easier isolation, multilineage potential, and immunomodulatory capacity, Wharton's Jelly-derived mesenchymal stromal cells (WJ-MSCs) exhibit promising efficacy in the field of regenerative medicine and immunotherapy. Characterization of WJ-MSCs-natural killer (NK) cells crosstalk is required for ameliorating the medicinal value of WJ-MSCs. Here, we revealed that the outcome of WJ-MSCs-NK cells crosstalk varied according to the type of cytokines (IL-2, IL-12, IL-15 and IL-21) utilized to activate NK cells. Differently activated NK cells exerted distinct cytotoxicities against WJ-MSCs causing their probable death. Cell surface ligands (CD112, CD155, ULPB-3) and receptors (LAIR, CD226, CD314, CD335, CD336 and CD337) governing the interaction between NK cells and their targets, exhibited altered expression profiles following the co-culture with WJ-MSCs. Although partly inhibited NK cell proliferation, WJ-MSCs enhanced activated NK-cell-mediated secretion of IFN-γ and TNF-α. Moreover, WJ-MSCs reinforced NK cells' degranulation as well as secretion of perforin and granzymes. On the other hand, WJ-MSCs displayed only slight increase in ROS generation but significant decrease in A1 and C1 serpins expression following co-culture with activated NK cells. Altogether, our results highlight that WJ-MSCs-NK cells interaction may affect both cell type features and, therefore, their therapeutic properties.

fMLP-dependent activation of Akt and ERK1/2 through ROS/Rho A pathways is mediated through restricted activation of the FPRL1 (FPR2) receptor.

OBJECTIVE AND DESIGN: The objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte. MATERIALS OR SUBJECTS: Freshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP. TREATMENT: Time-course and dose-response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases. METHODS: Freshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media. RESULTS: fMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H2O2 release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H2O2-induced activation of Akt and RhoA-GTPase. CONCLUSION: Collectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.

The micronome of mesenchymal stromal cells is partially responsive to inflammation.

Mesenchymal stromal cells (MSCs) display a special immunological profile that allows their potential use as immunotherapeutic cells. Nowadays, foreskin (FSK) represents a valuable reservoir of MSCs with International Society for Cellular Therapy (ISCT) compliant criteria and relevant functional properties. However, their mode of action is poorly understood and needs to be more elucidated to optimize their therapeutic use. Because microRNAs (miRNAs) act as key regulators in a wide variety of biological processes, we decided to establish the micronome of FSK-MSCs, the influence of inflammation and the predicted target pathways. Here, we provide the full list of unchanged and additional four differentially expressed miRNAs, miR-199b, -296-3p and -589-5p being downregulated whilst miR-146-3p being upregulated, in MSCs following their exposure to a cocktail of proinflammatory cytokines. MicroRNA target prediction in addition to Pathway enrichment analysis performed using miRNet, showed that miR-296-3p is linked to antigen processing and presentation pathway. Collectively, our data indicate that the micronome of FSK-MSCs is partially responsive to inflammation. Differentially expressed miRNAs are subsequently modulated by inflammation and seem to be involved in regulating the immunological fate of FSK-MSCs. These miRNAs deserve more attention in order to optimize MSC-based therapy and achieve the appropriate therapeutic effect.

Study of the microRNA expression profile of foreskin derived mesenchymal stromal cells following inflammation priming.

BACKGROUND: Due to their self-renewal capacity, multi-lineage potential, and immunomodulatory properties, mesenchymal stromal cells (MSCs) are an attractive tool for different therapeutic strategies. Foreskin (FSK), considered as a biological waste material, has already been shown to be a valuable source of MSCs. Besides their typical fibroblast like morphology and International Society for cellular Therapy compliant phenotype, foreskin-MSCs (FSK-MSCs) are clonogenic, and highly proliferative cells with multi-lineage and strong immunomodulatory capacities. Of importance, FSK-MSCs properly adjust their fate following exposure to inflammatory signals. Being potent regulators of gene expression, miRNAs are involved in modulating nearly all cellular processes and in orchestrating the roles of different immune cells. In this study, we characterized the miRNome of FSK-MSCs by determining the expression profile of 380 different miRNAs in inflammation primed vs. control non-primed cells. METHODS: TaqMan low density array (TLDA) was performed to identify dysregulated miRNAs after exposing FSK-MSCs to inflammatory signals. Quantitative real-time RT-PCR was carried out to validate the observations. DIANA-miRPath analysis web server was used to identify potential pathways that could be targeted by the dysregulated miRNAs. RESULTS: Sixteen miRNAs were differentially expressed in inflammation-primed vs. non-primed FSK-MSCs. The expression level of miR-27a, -145, -149, -194, -199a, -221, -328, -345, -423-5p, -485-3p, -485-5p, -615-5p and -758 was downregulated whilst that of miR-155, -363 and -886-3p was upregulated. Target pathway prediction of those differentially expressed miRNAs identified different inflammation linked pathways. CONCLUSIONS: After determining their miRNome, we identified a striking effect of inflammatory signals on the miRNAs' expression levels in FSK-MSCs. Our results highlight a potential role of miRNAs in modulating the transcription programs of FSK-MSCs in response to inflammatory signals. Further, we propose that specific miRNAs could serve as interesting targets to manipulate some functions of FSK-MSCs, thus ameliorating their therapeutic potential.