Identification of a rare copy number polymorphic gain at 3q12.2 with candidate genes for familial endometriosis.

Endometriosis is a complex disease that affects 10-15% of women of reproductive age. Familial studies show that relatives of affected patients have a higher risk of developing the disease, implicating a genetic role for this disorder. Little is known about the impact of germline genomic copy number variant (CNV) polymorphisms on the heredity of the disease. In this study, we describe a rare CNV identified in two sisters with familial endometriosis, which contain genes that may increase the susceptibility and progression of this disease. We investigated the presence of CNVs from the endometrium and blood of the sisters with endometriosis and normal endometrium of five women as controls without the disease using array-CGH through the Agilent 2x400K platform. We excluded common CNVs that were present in the database of genomic variation. We identified, in both sisters, a rare CNV gain affecting 113kb at band 3q12.2 involving two candidate genes: ADGRG7 and TFG. The CNV gain was validated by qPCR. ADGRG7 is located at 3q12.2 and encodes a G protein-coupled receptor influencing the NF-kappaß pathway. TFG participates in chromosomal translocations associated with hematologic tumor and soft tissue sarcomas, and is also involved in the NF-kappa B pathway. The CNV gain in this family provides a new candidate genetic marker for future familial endometriosis studies. Additional longitudinal studies of affected families must confirm any associations between this rare CNV gain and genes involved in the NF-kappaß pathway in predisposition to endometriosis.

Identification of a rare copy number polymorphic gain at 3q12.2 with candidate genes for familial endometriosis

Abstract Endometriosis is a complex disease that affects 10-15% of women of reproductive age. Familial studies show that relatives of affected patients have a higher risk of developing the disease, implicating a genetic role for this disorder. Little is known about the impact of germline genomic copy number variant (CNV) polymorphisms on the heredity of the disease. In this study, we describe a rare CNV identified in two sisters with familial endometriosis, which contain genes that may increase the susceptibility and progression of this disease. We investigated the presence of CNVs from the endometrium and blood of the sisters with endometriosis and normal endometrium of five women as controls without the disease using array-CGH through the Agilent 2x400K platform. We excluded common CNVs that were present in the database of genomic variation. We identified, in both sisters, a rare CNV gain affecting 113kb at band 3q12.2 involving two candidate genes: ADGRG7 and TFG. The CNV gain was validated by qPCR. ADGRG7 is located at 3q12.2 and encodes a G protein-coupled receptor influencing the NF-kappaβ pathway. TFG participates in chromosomal translocations associated with hematologic tumor and soft tissue sarcomas, and is also involved in the NF-kappa B pathway. The CNV gain in this family provides a new candidate genetic marker for future familial endometriosis studies. Additional longitudinal studies of affected families must confirm any associations between this rare CNV gain and genes involved in the NF-kappaβ pathway in predisposition to endometriosis.

MicroRNA expression profile predicts prognosis of pediatric adrenocortical tumors.

Pediatric adrenocortical tumors (ACT) are rare aggressive neoplasms with heterogeneous prognosis. Despite extensive efforts, identifying reliable prognostic factors for pediatric patients with ACT remains a challenge. MicroRNA (miRNA) signatures have been associated with cancer diagnosis, treatment response, and prognosis of several types of cancer. However, the role of miRNAs has been poorly explored in pediatric ACT. In this study, we performed miRNA microarray profiling on a cohort of 37 pediatric ACT and nine nonneoplastic adrenal (NNA) samples and evaluated the prognostic significance of abnormally expressed miRNAs using Kaplan-Meier plots, log-rank test, and Cox regression analysis. We identified a total of 98 abnormally expressed miRNAs; their expression profile discriminated ACT from NNAs. Among the 98 deregulated miRNAs, 17 presented significant associations with patients' survival. In addition, higher expression levels of hsa-miR-630, -139-3p, -125a-3p, -574-5p, -596, -564, -1321, and -423-5p and lower expression levels of hsa-miR-377-3p, -126-3p, -410, -136-3p, -29b-3p, -29a-3p, -337-5p, -143-3p, and 140-5p were significantly associated with poor prognosis, tumor relapse, and/or death. Importantly, the expression profile of these 17 miRNAs stratified patients into two groups of ACTs with different clinical outcomes. Although some individual miRNAs exhibit potential prognostic values in ACTs, only the 17 miRNA-based expression clustering was considered an independent prognostic factor for 5-year event-free survival (EFS) compared to other clinicopathological features. In conclusion, our study reports for the first time associations between miRNA profiles and childhood ACT prognosis, providing evidence that miRNAs could be useful biomarkers to discriminate patients with favorable and unfavorable clinical outcomes.

ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.

High-throughput microRNA profile in adult and pediatric primary glioblastomas: the role of miR-10b-5p and miR-630 in the tumor aggressiveness.

Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system and, despite the standard therapy; the patients' prognoses remain dismal. The miRNA expression profiles have been associated with patient prognosis, suggesting that they may be helpful for tumor diagnosis and classification as well as predictive of tumor response to treatment. We described the microRNA expression profile of 29 primary GBM samples (9 pediatric GBMs) and 11 non-neoplastic white matter samples as controls (WM) by microarray analysis and we performed functional in vitro assays on these 2 most differentially expressed miRNAs. Hierarchical clustering analysis showed 3 distinct miRNA profiles, two of them in the GBM samples and a group consisting only of cerebral white matter. When adult and pediatric GBMs were compared to WM, 37 human miRNAs were found to be differentially expressed, with miR-10b-5p being the most overexpressed and miR-630 the most underexpressed. The overexpression of miR-630 was associated with reduced cell proliferation and invasion in the U87 GBM cell line, whereas the inhibition of miR-10b-5p reduced cell proliferation and colony formation in the U251 GBM cell line, suggesting that these miRNAs may act as tumor-suppressive and oncogenic miRNAs, respectively. The present study highlights the distinct epigenetic profiling of adult and pediatric GBMs and underscores the biological importance of mir-10b-5p and miR-630 for the pathobiology of these lethal tumors.

GVHD-derived plasma as a priming strategy of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. METHODS: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1ß, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. RESULTS: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. CONCLUSIONS: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases.

Proteomics analysis reveals the role of ubiquitin specific protease (USP47) in Epithelial to Mesenchymal Transition (EMT) induced by TGFß2 in breast cells.

Epithelial to Mesenchymal Transition (EMT) is a normal cellular process that is also triggered during cancer progression and metastasis. EMT induces cellular and microenviromental changes, resulting in loss of epithelial features and acquisition of mesenchymal phenotypes. The growth factor TGFß and the transcription factor SNAIL1 (SNAIL) have been described as inducers of EMT. Here, we carried out an EMT model with non-tumorigenic cell line MCF-10A induced with the TGFß2 specific isoform of TGF protein family. The model was validated by molecular, morphological and functional experiments and showed correlation with the up-regulation of SNAIL. In order to identify additional regulators of EMT in this non-tumorigenic model, we explored quantitative proteomics, which revealed the Ubiquitin carboxyl-terminal hydrolase 47 (USP47) as one of the top up-regulated proteins. USP47 has a known role in cell growth and genome integrity, but not previously correlated to EMT. After validating USP47 alterations using MRM and antibody-based assays, we demonstrated that the chemical inhibition of USP47 with the inhibitor P5091 reduced expression of EMT markers and reverted morphological changes in MCF-10A cells undergoing EMT. These results support the involvement of USP47 in our EMT model as well as potential applications of deubiquitinases as therapeutic targets for cancer progression management. BIOLOGICAL SIGNIFICANCE: Metastasis is responsible for most cancer-associated mortality. Additionally, metastasis requires special attention, as the cellular transformations make treatment at this stage very difficult or occasionally impossible. Early steps in cancer metastasis involve the ability to detach from the solid tumor mass and invade the surrounding stromal tissues through cohesive migration, or a mesenchymal or amoeboid invasion. One of the first steps for metastatic cascade is denominated epithelial to mesenchymal transition (EMT), which can be triggered by different factors. Here, our efforts were directed to better understand this process and identify new pathways that contributes for acquisition of EMT, mainly focused on post translational modifications related to ubiquitin proteasome system. Our model of EMT induction by TGFß2 mimics early stage of metastatic cancer in epithelial breast cells and a proteomic study carried out for such model demonstrates that the deubiquitinase enzyme USP47 acts in SNAIL stabilization, one of the most important transcription factors for EMT phenotype acquisition and consequent metastasis. In addition, the inhibiton of USP47 with P5091, reverted the EMT phenotype. Together the knowledge of such processes of cancer progression and regulation can help in designing new strategies for combined therapies for control of cancer in early stages.

A High-Content Screening Approach to Identify MicroRNAs Against Head and Neck Cancer Cell Survival and EMT in an Inflammatory Microenvironment.

Head and neck squamous cell carcinoma (HNSCC) is among the most common cancer types. Metastasis, the main cause of death by cancer, can be promoted by an inflammatory microenvironment, which induces epithelial-mesenchymal transition (EMT) through a NF-κB-mediated stabilization of Snail. Here, we aimed to explore how microRNAs (miRs) can affect cell survival and EMT in HNSCC cells under an inflammatory microenvironment. By using a high-content screening (HCS) approach, we evaluated alterations in morphometric parameters, as well as expression/localization of Snail/Slug, in HNSCC cells primed with TNF-α. Based on those quantitation, we established the optimal experimental conditions of EMT induction driven by TNF-α. Those conditions were applied to cells transfected with distinct miRs (N = 31), followed by clusterization of miRs based on alterations related to cell survival and EMT. The signaling pathways enriched with molecular targets from each group of miRs were identified by in silico analyses. Finally, cells were transfected with siRNAs against signaling pathways targeted by miRs with anti-survival/EMT effect and evaluated for alterations in cell survival and EMT. Overall, we observed that TNF-α, at 20 ng/ml, induced EMT-related changes in cell morphology, Snail/Slug expression, and cell migration. Predicted targets of miRs with anti-survival/EMT effect were enriched with targets of NF-κB, PI3K/ATK, and Wnt/beta catenin pathways. Strikingly, individual gene silencing of elements from those pathways, namely RELA (NF-kB), AKT1 (PI3K/AKT), and CTNNB1 (Wnt/beta catenin) reduced cell survival and/or expression of Snail/Slug in cells stimulated with TNF-α. As a whole, our HCS approach allowed for the identification of miRs capable of inhibiting cell survival and EMT considering the presence of an inflammatory microenvironment, also indicating the common signaling pathways and molecular targets most likely to underlie those alterations. These findings may contribute to the development of targeted therapies against HNSCC.

Focused screening reveals functional effects of microRNAs differentially expressed in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs in carcinogenesis. Several microRNAs are described as aberrantly expressed in CRC tissues and in the serum of patients. However, functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs aberrantly expressed in CRC samples in the proliferation and cell death of a CRC cell line. METHODS: We transfected 31 microRNA mimics into HCT116 cells. Total number of live propidium iodide negative (PI-) and dead (PI+) cells were measured 4 days post-transfection by using a high content screening (HCS) approach. HCS was further used to evaluate apoptosis (via Annexin V and PI staining), and to discern between intrinsic and extrinsic apoptotic pathways, by detecting cleaved Caspase 9 and 8, respectively. To reveal mRNA targets and potentially involved mechanisms, we performed microarray gene expression and functional pathway enrichment analysis. Quantitative PCR and western blot were used to validate potential mRNA targets. RESULTS: Twenty microRNAs altered the proliferation of HCT116 cells in comparison to control. miR-22-3p, miR-24-3p, and miR-101-3p significantly repressed cell proliferation and induced cell death. Interestingly, all anti-proliferative microRNAs in our study had been previously described as poorly expressed in the CRC samples. Predicted miR-101-3p targets that were also downregulated by in our microarray were enriched for genes associated with Wnt and cancer pathways, including MCL-1, a member of the BCL-2 family, involved in apoptosis. Interestingly, miR-101-3p preferentially downregulated the long anti-apoptotic MCL-1 L isoform, and reduced cell survival specifically by activating the intrinsic apoptosis pathway. Moreover, miR-101-3p also downregulated IL6ST, STAT3A/B, and MYC mRNA levels, genes associated with stemness properties of CRC cells. CONCLUSIONS: microRNAs upregulated in CRC tend to induce proliferation in vitro, whereas microRNAs poorly expressed in CRC halt proliferation and induce cell death. We provide novel evidence linking preferential inhibition of the anti-apoptotic MCL-1 L isoform by miR-101-3p and consequent activation of the intrinsic apoptotic pathway as potential mechanisms for its antitumoral activity, likely due to the inhibition of the IL-6/JAK/STAT signaling pathway.

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation.

BACKGROUND: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. METHODS: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. RESULTS: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states. CONCLUSIONS: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation.

Arrayed functional genetic screenings in pluripotency reprogramming and differentiation.

Thoroughly understanding the molecular mechanisms responsible for the biological properties of pluripotent stem cells, as well as for the processes involved in reprograming, differentiation, and transition between Naïve and Primed pluripotent states, is of great interest in basic and applied research. Although pluripotent cells have been extensively characterized in terms of their transcriptome and miRNome, a comprehensive understanding of how these gene products specifically impact their biology, depends on gain- or loss-of-function experimental approaches capable to systematically interrogate their function. We review all studies carried up to date that used arrayed screening approaches to explore the function of these genetic elements on those biological contexts, using focused or genome-wide genetic libraries. We further discuss the limitations and advantages of approaches based on assays with population-level primary readouts, derived from single-parameter plate readers, or cell-level primary readouts, obtained using multiparametric flow cytometry or quantitative fluorescence microscopy (i.e., high-content screening). Finally, we discuss technical limitation and future perspectives, highlighting how the integration of screening data may lead to major advances in the field of stem cell research and therapy.

MicroRNA profile of pediatric pilocytic astrocytomas identifies two tumor-specific signatures when compared to non-neoplastic white matter.

PURPOSES: Pilocytic astrocytoma (PA) is a low-grade neoplasm frequently found in childhood. PA is characterized by slow growth and a relatively good prognosis. Genetic mechanisms such as activation of MAPK, BRAF gene deregulation and neurofibromatosis type 1 (NF1) syndrome have been associated with PA development. Epigenetic signature and miRNA expression profile are providing new insights about different types of tumor, including PAs. METHODS: In the present study we evaluated global miRNA expression in 16 microdissected pediatric PA specimens, three NF1-associated PAs and 11 cerebral white matter (WM) samples by the microarray method. An additional cohort of 20 PAs was used to validate by qRT-PCR the expression of six miRNAs differentially expressed in the microarray data. RESULTS: Unsupervised hierarchical clustering analysis distinguished one cluster with nine PAs, including all NF1 cases and a second group consisting of the WM samples and seven PAs. Among 88 differentially expressed miRNAs between PAs and WM samples, the most underexpressed ones regulate classical pathways of tumorigenesis, while the most overexpressed miRNAs are related to pathways such as focal adhesion, P53 signaling pathway and gliomagenesis. The PAs/NF1 presented a subset of underexpressed miRNAs, which was also associated with known deregulated pathways in cancer such as cell cycle and hippo pathway. CONCLUSIONS: In summary, our data demonstrate that PA harbors at least two distinct miRNA signatures, including a subgroup of patients with NF1/PA lesions.

TGF-beta/atRA-induced Tregs express a selected set of microRNAs involved in the repression of transcripts related to Th17 differentiation.

Regulatory T cells (Tregs) are essential regulators of immune tolerance. atRA and TGF-ß can inhibit the polarization of naïve T cells into inflammatory Th17 cells, favoring the generation of stable iTregs, however the regulatory mechanisms involved are not fully understood. In this context, the roles of individual microRNAs in Tregs are largely unexplored. Naïve T cells were immunomagnetically isolated from umbilical cord blood and activated with anti-human CD2/CD3/CD28 beads in the presence of IL-2 alone (CD4Med) or with the addition of TGF-ß and atRA (CD4TGF/atRA). As compared to CD4Med, the CD4TGF/atRA condition allowed the generation of highly suppressive CD4+CD25hiCD127-FOXP3hi iTregs. Microarray profiling allowed the identification of a set of microRNAs that are exclusively expressed upon TGF-ß/atRA treatment and that are predicted to target a set of transcripts concordantly downregulated. This set of predicted targets were enriched for central components of IL-6/JAK/STAT and AKT-mTOR signaling, whose inhibition is known to play important roles in the generation and function of regulatory lymphocytes. Finally, we show that mimics of exclusively expressed miRs (namely miR-1299 and miR-30a-5p) can reduce the levels of its target transcripts, IL6R and IL6ST (GP130), and increase the percentage of FoxP3+ cells among CD4+CD25+/hi cells.

Modulation of Immunoregulatory Properties of Mesenchymal Stromal Cells by Toll-Like Receptors: Potential Applications on GVHD.

In the last decade, the immunomodulatory properties of mesenchymal stromal cells (MSCs) have attracted a lot of attention, due to their potential applicability in the treatment of graft-versus-host disease (GVHD), a condition frequently associated with opportunistic infections. The present review addresses how Pathogen-Associated Molecular Patterns (PAMPS) modulate the immunosuppressive phenotype of human MSCs by signaling through Toll-like receptors (TLRs). Overall, we observed that regardless of the source tissue, human MSCs express TLR2, TLR3, TLR4, and TLR9. Stimulation of distinct TLRs on MSCs elicits distinct inflammatory signaling pathways, differentially influencing the expression of inflammatory factors and the ability of MSCs to suppress the proliferation of immune system cells. The capacity to enhance the immunosuppressive phenotype of MSCs through TLRs stimulation might be properly elucidated in order to improve the MSC-based immunotherapy against GVHD.

HOX genes: potential candidates for the progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype of the head and neck squamous cell carcinoma (HNSCC). Despite significant advances in the understanding and treatment of cancer, prognosis of patients with LSCC has not improved recently. In the present study, we sought to understand better the genetic mechanisms underlying LSCC development. Thirty-two tumor samples were collected from patients undergoing surgical resection of LSCC. The samples were submitted to whole-genome cDNA microarray analysis aiming to identify genetic targets in LSCC. We also employed bioinformatic approaches to expand our findings using the TCGA database and further performed functional assays, using human HNSCC cell lines, to evaluate viability, cell proliferation, and cell migration after silencing of selected genes. Eight members of the homeobox gene family (HOX) were identified to be overexpressed in LSCC samples when compared to normal larynx tissue. Quantitative RT-PCR analysis validated the overexpression of HOX gene family members in LSCC. Receiver operating characteristic (ROC) statistical method curve showed that the expression level of seven members of HOX gene family can distinguish tumor from nontumor tissue. Correlation analysis of clinical and gene expression data revealed that HOXC8 and HOXD11 genes were associated with the differentiation degree of tumors and regional lymph node metastases, respectively. Additionally, siRNA assays confirmed that HOXC8, HOXD10, and HOXD11 genes might be critical for cell colony proliferation and cell migration. According to our findings, several members of the HOX genes were overexpressed in LSCC samples and seem to be required in biological processes involved in tumor development. This suggests that HOX genes might play a critical role in the physiopathology of LSCC tumors.

TNF-alpha and Notch signaling regulates the expression of HOXB4 and GATA3 during early T lymphopoiesis.

During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34+ HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3ß inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.

The expression of Death Inducer-Obliterator (DIDO) variants in Myeloproliferative Neoplasms.

Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms.

DSP30 enhances the immunosuppressive properties of mesenchymal stromal cells and protects their suppressive potential from lipopolysaccharide effects: A potential role of adenosine.

Multipotent mesenchymal stromal cells (MSC) are imbued with an immunosuppressive phenotype that extends to several immune system cells. In this study, we evaluated how distinct Toll-like receptor (TLR) agonists impact immunosuppressive properties of bone marrow (BM)-MSC and explored the potential mechanisms involved. We show that TLR4 stimulation by lipopolysaccharide (LPS) restricted the ability of MSC to suppress the proliferation of T lymphocytes, increasing the gene expression of interleukin (IL)-1ß and IL-6. In contrast, stimulation of TLR9 by DSP30 induced proliferation and the suppressive potential of BM-MSC, coinciding with reducing tumor necrosis factor (TNF)-α expression, increased expression of transforming growth factor (TGF)-ß1, increased percentages of BM-MSC double positive for the ectonucleotidases CD39+CD73+ and adenosine levels. Importantly, following simultaneous stimulation with LPS and DSP30, BM-MSC's ability to suppress T lymphocyte proliferation was comparable with that of non-stimulated BM-MSC levels. Moreover, stimulation of BM-MSC with LPS reduced significantly the gene expression levels, on co-cultured T lymphocyte, of IL-10 and interferon (IFN)γ, a cytokine with potential to enhance the immunosuppression mediated by MSC and ameliorate the clinical outcome of patients with graft-versus-host disease (GVHD). Altogether, our findings reiterate the harmful effects of LPS on MSC immunosuppression, besides indicating that DSP30 could provide a protective effect against LPS circulating in the blood of GVHD patients who receive BM-MSC infusions, ensuring a more predictable immunosuppressive effect. The novel effects and potential mechanisms following the stimulation of BM-MSC by DSP30 might impact their clinical use, by allowing the derivation of optimal "licensing" protocols for obtaining therapeutically efficient MSC.

LL-37 boosts immunosuppressive function of placenta-derived mesenchymal stromal cells.

BACKGROUND: Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces important challenges. For instance, increasing MSC migratory capacity as well as potentializing immune response suppression are of interest. For GvHD management, preventing opportunistic infections is also a valuable strategy, since immunocompromised patients are easy targets for infections. LL-37 is a host defense peptide (HDP) that has been deeply investigated due to its immunomodulatory function. In this scenario, the combination of MSC and LL-37 may result in a robust combination to be clinically used. METHODS: In the present study, the effects of LL-37 upon the proliferation and migratory capacity of human placenta-derived MSCs (pMSCs) were assessed by MTT and wound scratch assays. The influence of LL-37 over the immunosuppressive function of pMSCs was then investigated using CFSE cell division kit. Flow cytometry and real-time PCR were used to investigate the molecular mechanisms involved in the effects observed. RESULTS: LL-37 had no detrimental effects over MSC proliferation and viability, as assessed by MTT assay. Moreover, the peptide promoted increased migratory behavior of pMSCs and enhanced their immunomodulatory function over activated human PBMCs. Strikingly, our data shows that LL-37 treatment leads to increased TLR3 levels, as shown by flow cytometry, and to an increased expression of factors classically related to immunosuppression, namely IDO, IL-10, TGF-ß, IL-6, and IL-1ß. CONCLUSIONS: Taken together, our observations may serve as groundwork for the development of new therapeutic strategies based on the combined use of LL-37 and MSCs, which may provide patients not only with an enhanced immunosuppression regime, but also with an agent to prevent opportunistic infections.

Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile.

Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3(+) lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols.