Mohammad Fayyad-Kazan

Circulating miR-150 and miR-342 in plasma are novel potential biomarkers for acute myeloid leukemia

BackgroundMicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet.MethodsTaqMan miRNA microarray was performed to identify deregulated miRNAs in the plasma of AML patients. Quantitative real-time RT-PCR was used to validate the results. Receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the highly and significantly identified deregulated miRNA(s) as potential candidate biomarker(s).ResultsThe plasma expression level of let-7d, miR-150, miR-339, and miR-342 was down-regulated whilst that of let-7b, and miR-523 was up-regulated in the AML group at diagnosis compared to healthy controls. ROC curve analyses revealed an AUC (the areas under the ROC curve) of 0.835 (95% CI: 0.7119– 0.9581; P<0.0001) and 0.8125 (95% CI: 0.6796–0.9454; P=0.0005) for miR-150, and miR-342 respectively. Combined ROC analyses using these 2 miRNAs revealed an elevated AUC of 0.86 (95% CI: 0.7819–0.94; P<0.0001) indicating the additive effect in the diagnostic value of these 2 miRNAs. QRT-PCR results showed that the expression level of these two miRs in complete remission AML patients resembled that of healthy controls.ConclusionsOur findings indicated that plasma miR-150 and miR-342 are novel important promising biomarkers in the diagnosis of AML. These novel and promising markers warrant validation in larger prospective studies.

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function

Background: Regulatory T cells are a subset of T cells with immunosuppressive properties, crucial for immune tolerance, which are also associated with cancer development. Results: The human circulating CD4+ Treg microRNA signature was identified. Conclusion: Differentially expressed microRNAs from the Treg miR signature directly and indirectly regulate crucial Treg genes (FOXP3 and CTLA-4). Significance: Identifying novel regulatory mechanisms of crucial Treg genes expression provides better insight into their biology and offers potential new targets for immunomodulatory therapies. Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4+ Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3′-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3′-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4+ adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3′-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4+ Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

Yeast nitrogen catabolite repression is sustained by signals distinct from glutamine and glutamate reservoirs.

Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling bakers yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1‐mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP‐glutamate dehydrogenase activity of Gdh1 negatively regulates NCR‐sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation.

Components of Golgi-to-vacuole trafficking are required for nitrogen- and TORC1-responsive regulation of the yeast GATA factors.

Nitrogen catabolite repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae responds to the available nitrogen status and selectively utilizes rich nitrogen sources in preference to poor ones. Expression of NCR‐sensitive genes is mediated by two transcription activators, Gln3 and Gat1, in response to provision of a poorly used nitrogen source or following treatment with the TORC1 inhibitor, rapamycin. During nitrogen excess, the transcription activators are sequestered in the cytoplasm in a Ure2‐dependent fashion. Here, we show that Vps components are required for Gln3 localization and function in response to rapamycin treatment when cells are grown in defined yeast nitrogen base but not in complex yeast peptone dextrose medium. On the other hand, Gat1 function was altered in vps mutants in all conditions tested. A significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. Further, our results are consistent with the possibility that Ure2 might function downstream of the Vps components during the control of GATA factor‐mediated gene expression. These observations demonstrate distinct media‐dependent requirements of vesicular trafficking components for wild‐type responses of GATA factor localization and function. As a result, the current model describing participation of Vps system components in events associated with translocation of Gln3 into the nucleus following rapamycin treatment or growth in nitrogen‐poor medium requires modification.

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

BackgroundDue 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.MethodsTaqMan 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.ResultsSixteen 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.ConclusionsAfter 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.

The potential of mesenchymal stromal cells in immunotherapy

Over the past years, extensive studies have been conducted to develop safe and effective therapeutic strategies to treat human diseases. Immunomodulation refers to the manipulation of the immune system either by enhancing (immunopotentiation strategy), or supressing (immunosuppression strategy) the immune responses. Immunomodulation can be achieved by different agents, referred to as immunomodulators, including monoclonal antibodies, cytokines, glucocorticoids, adjuvants, ultraviolet light, plasmapheresis and many others [1]. Mesenchymal stromal cells (MSCs) are immunomodulators of special interest and have been suggested as promising therapeutics for several autoimmune diseases and transplantation complications [2]. According to the International Society for Cellular Therapy, MSCs are defined as plastic-adherent cells with a specific surface antigen expression profile (CD73, CD90, CD105, CD34, CD45, CD11b, CD14, CD19, CD79a and HLA DR) and have the potential to differentiate, under standard in vitro differentiation conditions, into adipocytes, chondroblasts and osteoblasts [3]. Being a major component of the hematopoietic stem cell (HSC) niche, MSCs were first isolated from the bone marrow and identified for their capacities to support hematopoiesis [4], thus being of special importance for treating hematological disorders. Later, MSC-like cells were found and isolated from a variety of adult and fetal tissues including adipose tissue, umbilical cord and placenta [5]. This wide distribution in different parts of the organism highlighted a potential role for MSCs in tissue repair, and regeneration throughout individual’s life. The discovery of the superior immuneregulatory capacities of MSCs over different immune cells, raised their medical importance as they are used, nowadays, in preclinical and clinical studies as a potential therapy for treating autoimmune diseases, and modulating inflammatory responses [6]. The rationale of MSC-based therapy, is derived from their special immunologic profile and potent immunomodulatory capacity. Although MSCs isolated from different tissues exhibit differences in their expression profiles of some endothelialand stromal-associated markers, all MSCs show low expression levels of different surface antigens, including MHC class I and the costimulatory molecules (CD40, CD80 and CD86) as well as no MHC class II expression [7]. Thanks to this special immunologic profile, MSCs can evade being rejected by host immune responses, thus contributing for their hypoimmunogenicity. Besides not being recognized as alloantigens, MSCs can actively sense the surrounding inflammatory microenvironment and modulate, accordingly, the function of different immune cells within the host [8]. In fact, different parameters including the concentration The potential of mesenchymal stromal cells in immunotherapy

Identification and Evaluation of New Immunoregulatory Genes in Mesenchymal Stromal Cells of Different Origins: Comparison of Normal and Inflammatory Conditions.

Background Mesenchymal stromal cells (MSCs) possess potent immunomodulatory properties that increase their value as a cell-based therapeutic tool for managing various immune-based disorders. Over the past years, accumulated results from trials using MSCs-based therapy have shown substantial contradictions. Although the reasons underlying these discrepancies are still not completely understood, it is well known that the immunomodulatory activities mediated by distinct MSCs differ in a manner dependent on their tissue origin and adequate response to inflammation priming. Thus, characterization of new molecular pathway(s) through which distinct MSC populations can exert their immunomodulatory effects, particularly during inflammation, will undoubtedly enhance their therapeutic potential. Material/Methods After confirming their compliance with ISCT criteria, quantitative real time-PCR (qRT-PCR) was used to screen new immunoregulatory genes in MSCs, derived from adipose tissue, foreskin, Wharton’s jelly or the bone-marrow, after being cultivated under normal and inflammatory conditions. Results FGL2, GAL, SEMA4D, SEMA7A, and IDO1 genes appeared to be differentially transcribed in the different MSC populations. Moreover, these genes were not similarly modulated following MSCs-exposure to inflammatory signals. Conclusions Our observations suggest that these identified immunoregulatory genes may be considered as potential candidates to be targeted in order to enhance the immunomodulatory properties of MSCs towards more efficient clinical use.

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.

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.

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.