Amélia G. Araújo

Mesenchymal stromal cells up-regulate CD39 and increase adenosine production to suppress activated T-lymphocytes.

Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A(2a) (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases.

Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling.

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa‐B (NF‐κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3+ T cells were activated and cultured in the presence or absence of MSCs. CD4+ cell mRNA expression was then characterized by microarray analysis. The drug BAY11‐7082 (BAY) and a siRNA against v‐rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non‐canonical NF‐κB signalling, respectively. Flow cytometry and real‐time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non‐canonical NF‐κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co‐cultured with MSCs. The frequency of CD69+ cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69+ cells increased significantly following activation in lymphocytes co‐cultured with MSCs. Inhibition of canonical NF‐κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF‐κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF‐κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non‐canonical pathway and is inhibited by canonical NF‐κB signalling.

Halofuginone has anti-proliferative effects in acute promyelocytic leukemia by modulating the transforming growth factor beta signaling pathway

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (P = 0.002) after a 24-hour incubation. Addition of TGFβ revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFβ target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFβ protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFβ values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFβ blockade in APL. Since loss of the TGFβ response in leukemic cells may be an important second oncogenic hit, modulation of TGFβ signaling may be of therapeutic interest.

Potential roles of microRNA-29a in the molecular pathophysiology of T-cell acute lymphoblastic leukemia

Recent evidence has shown that deregulated expression of members of the microRNA‐29 (miR‐29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR‐29 in the molecular pathophysiology of T‐cell acute lymphoblastic leukemia (T‐ALL) has not been investigated. Here, we show that lower levels of miR‐29a were significantly associated with higher blast counts in the bone marrow and with increased disease‐free survival in T‐ALL patients. Furthermore, miR‐29a levels are extremely reduced in T‐ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR‐29a mimics into Jurkat cells revealed the downregulation of several predicted targets (CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR‐29a levels in Jurkat and Molt‐4 T‐ALL cells led to the demethylation of many genes commonly methylated in T‐ALL. Overall, our results suggest that reduced miR‐29a levels may contribute to the altered epigenetic status of T‐ALL, highlighting its relevance in the physiopathology of this disease.

The expression of ΔNTP73, TATP73 and TP53 genes in acute myeloid leukaemia is associated with recurrent cytogenetic abnormalities and in vitro susceptibility to cytarabine cytotoxicity

TP73 encodes for two proteins: full‐length TAp73 and ΔNp73, which have little transcriptional activity and exert dominant‐negative function towards TP53 and TAp73. We compared TATP73 and ΔNTP73 expression in acute myeloid leukaemia (AML) samples and normal CD34+ progenitors. Both forms were more highly expressed in leukaemic cells. Amongst AML blasts, TATP73 was more expressed in AML harbouring the recurrent genetic abnormalities (RGA): PML‐RARA, RUNX1‐RUNX1T1 and CBFB‐MYH11, whereas higher ΔNTP73 expression was detected in non‐RGA cases. TP53 expression did not vary according to ΔNTP73/TATP73 expression ratio. Leukaemic cells with higher ΔNTP73/TATP73 ratios were significantly more resistant to cytarabine‐induced apoptosis.

Increased Levels of NOTCH1, NF-κB, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

As previously shown, higher levels of NOTCH1 and increased NF-kappaB signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow (BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells (CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency (than expected by chance) of NF-kappaB-binding sites (BS), including potentially novel NF-kappaB targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappaB, and other important TFs on more primitive HSC sets.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology.

Heterogeneous ethnic distribution of the factor v leiden mutation

Resistencia a proteina C ativada associada a mutacao do fator V Leiden (FVL) e a mais prevalente causa genetica de trombose venosa conhecida, sendo encontrada em 20 a 60% dos pacientes com trombofilia. Adicionalmente, uma associacao entre a mutacao do FVL e predisposicao aumentada para doenca cardiovascular prematura em mulheres foi recentemente descrita. No presente estudo nos determinamos a prevalencia da mutacao do FVL em 440 individuos (880 cromossomos) de 4 grupos etnicos diferentes: caucasoides, negros africanos, asiaticos e amerindios. Amplificacao por PCR seguida de digestao com a enzima de restricao MnlI foi utilizada para definicao do genotipo. A mutacao do FVL foi encontrada em heterozigose em 4 de 152 caucasoides (2,6%), 1 de 151 amerindios (0,6%) e esteve ausente em 97 negros africanos e 40 asiaticos. Nossos resultados confirmam que o FVL apresenta distribuicao heterogenea em diferentes populacoes humanas, fato que pode contribuir para diferencas etnicas e geograficas na prevalencia de doencas tromboticas. Adicionalmente, estes dados podem ser uteis para a definicao de estrategias de rastreamento desta mutacao em individuos sob risco para desenvolvimento de trombose, na populacao brasileira.

Hydroxycarbamide modulates components involved in the regulation of adenosine levels in blood cells from sickle-cell anemia patients

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

Effects of Light-Emitting Diode Therapy on Muscle Hypertrophy, Gene Expression, Performance, Damage, and Delayed-Onset Muscle Soreness: Case-control Study with a Pair of Identical Twins.

ObjectiveThe aim of this study was to verify how a pair of monozygotic twins would respond to light-emitting diode therapy (LEDT) or placebo combined with a strength-training program during 12 weeks. DesignThis case-control study enrolled a pair of male monozygotic twins, allocated randomly to LEDT or placebo therapies. Light-emitting diode therapy or placebo was applied from a flexible light-emitting diode array (&lgr; = 850 nm, total energy = 75 J, t = 15 seconds) to both quadriceps femoris muscles of each twin immediately after each strength training session (3 times/wk for 12 weeks) consisting of leg press and leg extension exercises with load of 80% and 50% of the 1-repetition maximum test, respectively. Muscle biopsies, magnetic resonance imaging, maximal load, and fatigue resistance tests were conducted before and after the training program to assess gene expression, muscle hypertrophy and performance, respectively. Creatine kinase levels in blood and visual analog scale assessed muscle damage and delayed-onset muscle soreness, respectively, during the training program. ResultsCompared with placebo, LEDT increased the maximal load in exercise and reduced fatigue, creatine kinase, and visual analog scale. Gene expression analyses showed decreases in markers of inflammation (interleukin 1&bgr;) and muscle atrophy (myostatin) with LEDT. Protein synthesis (mammalian target of rapamycin) and oxidative stress defense (SOD2 [mitochondrial superoxide dismutase]) were up-regulated with LEDT, together with increases in thigh muscle hypertrophy. ConclusionsLight-emitting diode therapy can be useful to reduce muscle damage, pain, and atrophy, as well as to increase muscle mass, recovery, and athletic performance in rehabilitation programs and sports medicine.