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In recent years fetal adnexa and fluids have been recognized as important sources of mesenchymal stem cells (MSCs). The aim of this study was to characterize cell populations of bovine amniotic fluid studying phenotypic characterization, RNA expression and differentiation potential of samples after in vitro culture for different length of time following trypsinization and expansion (passage). Amniotic fluid samples were recovered at slaughterhouse from 25 pregnant cows and harvested cells were cultured in DMEM-TCM199 (1:1) plus 10% FBS in 5%CO2 at 38.5 °C. At passages P3 and P7, a sample for each of the 4 population found was characterized. Immunophenotypic characterization was performed for MSC (CD90, CD105, CD44) and hematopoietic (CD14, CD34) markers by flow cytometry (FACS). Immunocytochemistry (ICC) was performed for Oct4, SSEA4 and α-SMA and ratio between positive cells and total nuclei was evaluated. Gene expression profile was analyzed by RT-PCR for pluripotency markers (Oct4, Nanog, Sox2). At the same passages chondrogenic, osteogenic and adipogenic differentiation were induced and evaluated morphologically and cytologically using respectively: Alcian blue to identify glycosaminoglycans of cartilage matrix, Von Kossa for extracellular calcium deposition and Oil Red O for intracellular lipid droplets. Cell population appeared heterogeneous and we could identify 2 main cell types: round (R) and spindle-shaped (S) cells. Each isolated sample was classified into one of the following 4 types depending on percentages of R or S cells: prevalence of S-cells (S), prevalence of R-cells (R) and samples showing both morphologies with about 10% of S-cells (S10) or 40% S-cells (S40). S-cell percentage decreased with passages in S10 and S40. After FACS all lines were positive for CD90, CD105, CD44 and CD34 and negative for CD14 both at P3 and at P7. After ICC, Oct4 was negative in all samples analysed, few S-type cells stained for SSEA4 (8%) at P3 but increased at P7 to 22%; R, S10 and S40 did not express SSEA4 both at P3 and at P7. α-SMA was expressed in all samples at P3 (9.4% S; 0.9% R; 2.5% S10; 27% S40) but not at P7 (27.5% S; 0% R; 0% S10; 0% S40). After RT-PCR analyses Oct4 was negative in all samples, at P3 Nanog was clearly positive in S cells, weak in S40 and negative in R and S10, but all samples turned negative at P7. Sox2 was weakly expressed (S) or not expressed (S10, S40, R) at P3 and it was negative in all cells at P7. Only S-cells showed high differentiation potential into all 3 lineages at both P3 and P7, R-cells had the lowest differentiation potential while S10 and S40 were intermediate at both end points. In conclusion, bovine amniotic fluid showed heterogeneous cell populations and S-type had the characteristics of MSCs. S10 and S40 showed more MSC markers at P3, when S population was still present, and this aspect suggests that S population is the presumptive MSC one. Although prevalent, R-type showed only some MSC characteristics. Further studies are under way to improve S-type isolation, purification, culture and to determine the lifespan of these cell types. Work supported by grant PRIN2009.

Introduction - Amniotic fluid (AF) is a reservoir of multipotent mesenchymal stem cells (MSCs), very promising cells for tissue engineering in clinical application. The aim of this work was to isolate and characterize bovine MSCs from AF (bAF-MSCs) as alternative sources of primitive multipotent stem cells in a species that could be an attractive large-animal model for biomedical and biotechnology researches. Materials and Methods - Samples were recovered, at slaughterhouse, from 18 pregnant cows at different gestational ages established on crown-rump length (1: 0-105 d; 2: 106-160 d; 3: 161-200 d; 4: 201-240 d; 5: 241 d-term of pregnancy). Cells were isolated by centrifugation and cultured in DMEM-TCM 199 (1:1) plus 10% FBS. At passage (P) 4, chondrogenic, osteogenic and adipogenic differentiation were induced (n=16) and evaluated morphologically and cytologically (Alcian Blue, Von Kossa and Oil Red O stainings respectively). Phenotypic characterization for stem cell markers was performed at P3 or P4 by flow cytometry for CD105, CD90, CD45 and CD14 (n=16), by immunocytochemistry (ICC) for Oct4, SSEA1, SSEA4 and α-SMA (n=4) and by RT-PCR analysis for Oct4, Nanog and Sox2 (n=7). In both ICC and RT-PCR, bovine fibroblasts were used as negative control. Results – Mean collected AF volume was 35 ml and 200-3636 cells/ml were recovered. Cells were isolated from 16/18 samples. At P0, cell populations appeared heterogeneous and we could identify 4 different cell types: round, spindle-shaped, fibroblastoid and large-flat cells. These different populations were observed at every gestational age analyzed. Through passages, only round cells (n=15/16) predominated. In only one sample, spindle-shaped cells prevailed. After 30 d of culture mean cell doublings were 14,1±3,2 and mean doubling time was 57,7±27,9 hrs without differences related to gestational age or sample (p>0.05). Cells were able to differentiate into osteocytes and adipocytes; chondrogenesis was clearly obtained only from samples recovered within 140 d of pregnancy (n=9). Percentage of cells positive for CD90 was 73,0±17,2, for CD105 45,8±35,7, for CD14 5,6±11,2 and for CD45 13,4±11,3; no differences were observed among different gestational ages (p>0,05). ICC demonstrated low presence of SSEA4 positive cells (0,05%) in 1 sample, the presence of α-SMA in all 4 samples (mean 11,8±14.1%, range 2.5-32.4%) and the lack of Oct4 and SSEA1. RT-PCR analysis, performed at P4, showed expression of Nanog in 1/7 sample and weak expression in 4/7 samples. Low expression of SOX2 was detected in 2/7 samples. Conclusions - Based on these preliminary results, in the bovine AF there is an heterogeneous cell population containing also multipotent MSCs, identified by their differentiation ability and phenotypic characteristics typical of MSCs. Unlike human AF-MSCs, only a fraction of bAF-MSCs are positive for some pluripotency markers. Current work is ongoing to further characterize other cell phenotypes selected after long term culture.

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton’s Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0G0.6 days) than those isolated from CB (2.6G1.3 days) and AF (2.3G1.0 days) (P!0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.

The purpose of this study was to evaluate the possible involvement of the phosphoinositide 3-kinase (PI3K)/Akt survival pathway in determining resistance to arsenic trioxide (As2O3)-induced apoptosis. We employed a HL60 cell clone (HL60AR) with a constitutively active PI3K/Akt survival pathway, as well as U937 and K562 cells. In addition, we used parental (PT) HL60 cells overexpressing a constitutively active Akt. Selective pharmacological inhibitors of the PI3K/Akt axis (LY294002, wortmannin) were employed to influence the sensitivity to As2O3. While HL60PT cells were sensitive to 2.5 microM As2O3 and died of apoptosis, HL60AR cells were resistant up to 5 microM As2O3. Treatment with either LY294002 or wortmannin lowered resistance of HL60AR cells to As2O3. Also in U937 and K562 cells, inhibitors of the PI3K/Akt axis caused a decrease in As2O3 resistance. Overexpression of constitutively active Akt in HL60PT cells caused the induction of resistance to 2.5 microM As2O3. Conversely, forced expression of a dominant negative Akt in HL60AR cells resulted in a decrease in As2O3 resistance. Moreover, HL60 cell resistance to 2.5 microM As2O3 could be significantly reduced by incubation with SN50, a peptide inhibitor selective for the NF-kappaB transcription factor. Taken together our findings suggest that a constitutive activation of the PI3K/Akt pathway, which is increasingly detected in some types of acute myeloid leukemia, may contribute to As2O3 resistance, most likely through NF-kappaB activation. Selective pharmacological inhibitors of this survival pathway, as well as of NF-kappaB, might be usefully employed in the future to reverse resistance to this treatment.

Myelofibrosis (MF) is characterized by chronic inflammation and hyper-activation of the JAK-STAT pathway. Infections are one of the main causes of morbidity/mortality. Therapy with Ruxolitinib (RUX), a JAK1/2 inhibitor, may further increase the infectious risk. Monocytes are critical players in inflammation/immunity through cytokine production and release of bioactive extracellular vesicles. However, the functional behavior of MF monocytes, particularly during RUX therapy, is still unclear. In this study, we found that monocytes from JAK2V617F-mutated MF patients show an altered expression of chemokine (CCR2, CXCR3, CCR5) and cytokine (TNF-α-R, IL10-R, IL1β-R, IL6-R) receptors. Furthermore, their ability to produce and secrete free and extracellular vesicles-linked cytokines (IL1β, TNF-α, IL6, IL10) under lipopolysaccharides (LPS) stimulation is severely impaired. Interestingly, monocytes from RUX-treated patients show normal level of chemokine, IL10, IL1β, and IL6 receptors together with a restored ability to produce intracellular and to secrete extracellular vesicles-linked cytokines after LPS stimulation. Conversely, RUX therapy does not normalize TNF-R1/2 receptors expression and the LPS-driven secretion of free pro/anti-inflammatory cytokines. Accordingly, upon LPS stimulation, in vitro RUX treatment of monocytes from MF patients increases their secretion of extracellular vesicles-linked cytokines but inhibits the secretion of free pro/anti-inflammatory cytokines. In conclusion, we demonstrated that in MF the infection-driven response of circulating monocytes is defective. Importantly, RUX promotes their infection-driven cytokine production suggesting that infections following RUX therapy may not be due to monocyte failure. These findings contribute to better interpreting the immune vulnerability of MF and to envisaging strategies to improve the infection-driven immune response.

Domestic cats are preferred models for normal physiology and several human diseases. In the present study feline fetal fluids and membranes were evaluated as possible sources of MSCs. Samples were recovered from 4 pregnant queens after ovarian-hysterectomy. Gestational sacs were separated from uterine wall; after allantoic and amniotic fluids aspiration and chorion-allantois and amniotic membranes separation, all cell lineages were cultured into 25 cm2 flasks, in DMEM/TCM199, in a 5% CO2 incubator at 38.5° C. At passage 3, chondrogenic, osteogenic and adipogenic differentiation ability were evaluated by culturing cell monolayers in differentiating media for 21 days. Cellular characterization with CD90, CD44, CD105, CD73, CD34, CD14, CD45, was performed by flow cytometry. In all samples, adherent fibroblastoid spindle-shaped cells were observed. Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian blue staining of matrix glycosaminoglycans illustrated chondrogenesis, and positive Oil Red O lipid droplets within cell cytoplasm suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105 and negative for CD34, CD14 and CD45; as unexpected and different from human cells, feline cells resulted negative for CD73. Based on this preliminary results, fetal fluids and membranes could represent an alternative sources for mesenchymal stem cells in feline species.

Constitutively activated AKT kinase is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). Here, we report that the novel AKT inhibitor (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan2-amine (A443654) leads to rapid cell death of T-ALL lines and patient samples. Treatment of CEM, Jurkat, and MOLT-4 cells with nanomolar doses of the inhibitor led to AKT phosphorylation accompanied by dephosphorylation and activation of the downstream target, glycogen synthase kinase-3beta. Effects were time- and dose-dependent, resulting in apoptotic cell death. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -6, -8, and -9. Apoptotic cell death was mostly dependent on caspase-2 activation, as demonstrated by preincubation with a selective pharmacological inhibitor. It is remarkable that A443654 was highly effective against the drug-resistant cell line CEM-VBL100, which expresses 170-kDa P-glycoprotein. Moreover, A443654 synergized with the DNA-damaging agent etoposide in both drug-sensitive and drug-resistant cell lines when coadministered [combination index (CI) = 0.39] or when pretreated with etoposide followed by A443654 (CI = 0.689). The efficacy of A443654 was confirmed using blasts from six patients with T-ALL, all of whom displayed low levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and constitutive phosphorylation of Akt on Ser473. At 1 microM, the inhibitor was able to induce apoptotic cell death of T-ALL blast cells, as indicated by flow cytometric analysis of samples immunostained for active (cleaved) caspase-3. Because activated AKT is seen in a large percentage of patients with T-ALL, A443654, either alone or in combination with existing drugs, may be a useful therapy for primary and drug-resistant T-ALL.

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamycin (mTOR) signaling pathway plays a critical role in many cellular functions which are elicited by extracellular stimuli. However, constitutively active PI3K/Akt/mTOR signaling has also been firmly established as a major determinant for cell growth, proliferation, and survival in an wide array of human cancers. Thus, blocking the PI3K/AKT/mTOR signal transduction network could be an effective new strategy for targeted anticancer therapy. Pharmacological inhibitors of this signaling cascade are powerful antineoplastic agents in vitro and in xenografted models of tumors, and some of them are now being tested in clinical trials. Recent studies showed that PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug-resistance of these cells. Both the disease-free survival and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade may represent a target for innovative therapeutic treatments of AML patients. In this review, we discuss the possible mechanisms of activation of this pathway in AML cells and the downstream molecular targets of the PI3K/Akt/mTOR signaling network which are important for blocking apoptosis, enhancing proliferation, and promoting drug-resistance of leukemic cells. We also highlight several pharmacological inhibitors which have been used to block this pathway for targeted therapy of AML. These small molecules induce apoptosis or sensitize AML cells to existing drugs, and might be used in the future for improving the outcome of this hematological disorder.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34(+) cells cultured in the presence of IGF-I. Chemoresistant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me 2 SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 °C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49 ± 16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.

INTRODUCTION: This prospective study defined the immunological and clinical results after fresh and cryopreserved arterial homograft replacement due to graft infection. MATERIALS AND METHODS: Thirty patients who underwent ABO-compatible homograft transplantation were studied for anti-human leukocyte antigen (HLA): antibody production and CD3- and CD4- versus CD8-positive lymphocyte subsets. Nine patients (30%) received immunosuppressive treatment with cyclosporine (1 to 3 mg/kg/d). Immunological studies were performed preoperatively, and early (1, 3, 7 days) and late (1, 3, 6, 12, 24, 36, 48 months) during follow-up. Abdominal computed tomography scans were performed postoperatively at 1, 6, 12, 24, 36, and 48 months of follow-up. RESULTS: Preoperatively, antibodies were not detected. Postoperatively, a progressive increase in percent panel reactive antibodies was observed in all patients 1 month after the transplant. There were no difference between fresh and cryopreserved homografts. The antibody response among patients treated with cyclosporine was less pronounced and delayed. Recipient antibodies were directed against donor-specific antigens. During the immediate postoperative period (1, 3, 7 days) there was a slight increase in CD3- and CD4-positive T lymphocytes and a concomitant decrease in the CD8 subset. Later, CD3 and CD4 progressively decreased and the CD8 set increased. Clinically, no patients had signs of recurrent infection upon late follow-up. Four patients died (13%), but only one death was homograft-related (rupture of the graft). At 2-year follow-up, two patients showed stenotic lesions due to chronic rejection. Clinically, no differences were noted between fresh and cryopreserved homografts, or between patients treated with or without cyclosporine. CONCLUSIONS: Fresh and cryopreserved arterial homografts are immunogenic; they induce a strong anti-HLA antibody response, similar to chronic rejection.

Gene expression profiles of bone marrow (BM) CD34-derived megakaryocytic cells (MKs) were compared in patients with essential thrombocythemia (ET) and healthy subjects using oligonucleotide microarray analysis to identify differentially expressed genes and disease-specific transcripts. We found that proapoptotic genes such as BAX, BNIP3, and BNIP3L were down-regulated in ET MKs together with genes that are components of the mitochondrial permeability transition pore complex, a system with a pivotal role in apoptosis. Conversely, antiapoptotic genes such as IGF1-R and CFLAR were up-regulated in the malignant cells, as was the SDF1 gene, which favors cell survival. On the basis of the array results, we characterized apoptosis of normal and ET MKs by time-course evaluation of annexin-V and sub-G1 peak DNA stainings of immature and mature MKs after culture in serum-free medium with an optimal thrombopoietin concentration, and annexin-V–positive MKs only, with decreasing thrombopoietin concentrations. ET MKs were more resistant to apoptosis than their normal counterparts. We conclude that imbalance between proliferation and apoptosis seems to be an important step in malignant ET megakaryocytopoiesis.

SUMMARYT-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting.

Peripheral T-cell lymphomas not otherwise specified (PTCL/NOS) are very aggressive tumors characterized by consistent aberrant expression of platelet-derived growth factor receptor alpha (PDGFRA). In this study, we aimed to identify the determinants of PDGFRA activity in PTCL/NOS and to elucidate the biological consequences of its activation. We observed overexpression of the PDGFRA gene by gene expression profiling in most of the tested PTCLs and confirmed the expression of PDGFRA and phospho-PDGFRA using immunohistochemistry. The integrity of the PDFGRA locus was demonstrated using several different approaches, including massive parallel sequencing and Sanger sequencing. PDGF-AA was found to be expressed and secreted by PTCL/NOS cells and to be necessary and sufficient for PDGFRA phosphorylation ex vivo by sustaining an autocrine stimulation. We documented consistently high PDGF-A expression in primary biopsies and patients' plasma and tracked PDGFRA signaling in primary tumors, achieving evidence of its activation. Indeed, we found that STAT1 and STAT5 are implicated in PDGFRA signaling transduction. Finally, we demonstrated that PDGFRA activation supported tumor cell proliferation and provided the first evidence of the anti-lymphoma activity of PDGRA inhibition in a PTCL/NOS patient. Altogether, our data demonstrated that PDGFRA activity fosters PTCL/NOS proliferation via an autocrine loop. © 2014 Macmillan Publishers Limited. All rights reserved.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3 alpha/b and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200–1,000 ng/ml), whereas lower Tat concentrations (1–100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat-related modulation of adipogenesis was tackled by treatment with antagonists of Tat-specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF-induced endothelial markers such as Flt-1, KDR, and vWF. The treatment of MSCs with Tat-derived peptides corresponding to the cysteine-rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat-related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV-infected patients. J. Cell. Biochem. 113: 1132–1141, 2012. © 2011 Wiley Periodicals, Inc.

Background HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs). Results MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. Conclusions The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

Abstract 2596 Although intensive combination chemotherapy is effective in inducing complete remission in most patients with AML, the majority of responders subsequently relapse and ultimately die of the disease. This is particularly true of elderly (>60 years old) patients. The development of new therapies is therefore vital and, in this regard, targeting signaling pathways that are upregulated in AML represents a rapidly expanding research field. Constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway is a common finding in AML and preclinical studies have highlighted a critical role for PI3K and its downstream effectors, Akt and mammalian target of rapamycin (mTOR), for leukemic cell survival. Especially mTOR complex 1 (mTORC1) represents a highly attractive target for cancer therapy, as it controls cap-dependent mRNA translation, a process frequently deregulated in tumor cells and that strongly contributes to oncogenesis. Preclinical data suggest that allosteric mTORC1 inhibition with rapamycin impairs leukemia initiating cell (LIC) function in AML. While PI3K/Akt inhibitors are just beginning to be tested in the clinic, mTORC1 inhibitors have already been investigated as antileukemic agents. Recently, a phase II clinical trial combining temsirolimus (an mTORC1 inhibitor) and clofarabine has been performed in elderly patients with relapsed or refractory AML (NCT007755903). Clofarabine is a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. Here, we report the results of our pre-clinical evaluation of the efficacy of such a drug combination on AML cells. The combination of clofarabine and temsirolimus (CLO-TOR; drug concentration range: 6.125 nM–100 nm; ratio 1:1) was cytotoxic to a panel of AML cell lines (U937, OCI-AML3, HL60, THP1), as documented by MTT assays. The combination was highly synergistic, with combination indexes (CIs) ranging from 0.05 at the lowest drug concentration to 0.79 at the highest concentration. Treatment with CLO-TOR induced a G1 phase cell cycle arrest and apoptotic cell death of leukemic cells, as demonstrated by Annexin V/propidium iodide staining and cytofluorimetric analysis. Western blot analysis documented a dose-dependent cleavage of caspase-3, -8, -9. The combined treatment inhibited phosphorylation of Ser 473 p-Akt and of mTORC1 downstream targets (S6RP and 4E-BP1) more than either drug alone. Moreover, CLO-TOR affected the MEK/ERK pathway, as documented by dephosphorylation of p-ERK 1/2 and 90-kDa ribosomal S6 kinase. The effectiveness of the CLO-TOR treatment was not influenced by overexpression of 170-kDa P-glycoprotein (P-gp, one of the main determinants of drug-resistance), as documented by MTT assays performed on drug-sensitive and drug-resistant (i.e. overexpressing P-gp) CEM leukemic cells. The CLO-TOR combination, when employed at the same concentrations as for AML cell lines, was also cytotoxic to primary cells from AML patients (CIs: 0.11–0.25). CLO-TOR was highly effective in inducing apoptosis in an AML patient cell subpopulation (CD34+/CD38−/CD123+) which is enriched in putative LIC, as documented by quadruple staining and flow cytometric analysis of Annexin V-positive cells. The percentage of apoptotic cells in the CD34+/CD38−/CD123+ cell subset treated with CLO-TOR ranged between 90% and 97%, and was much higher than in samples treated with TOR alone (about 45%) or CLO alone (about 70%). The combined treatment also resulted in Akt, S6RP, and 4E-BP1 dephosphorylation in this cell subpopulation. In summary, the CLO-TOR combination could represent a valuable innovative treatment for AML patients, also in light of its efficacy against putative LIC. Disclosures: No relevant conflicts of interest to declare.

Background. Persistent mixed chimerism represents a state wherein recipient and donor cells stably co-exist after haematopoietic stem cell transplantation. However, since in mostly of the studies reported in literature the engraftment state was observed in the nucleated cells, in this paper we determined the donor origin in the mature erythrocytes of patients with persistent mixed chimerism after transplantation for haemoglobinopathies. Results were compared with the engraftment state observed in singularly picked-up burst-forming unit-erythroid colonies and in the nucleated cells collected from the peripheral blood and from the marrow. Design and Methods. The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocytes suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Short tandem repeats analysis was used to determine the donor origin of nucleated cells and burst-forming unit-erythroid colonies singularly picked up after 14 days incubation. Results. A proportion of donor-derived nucleated cells of 71%, 46%, 15% and 25% was observed at day 1364, 1385, 1314 and 932 respectively, in four transplanted patients affected by haemoglobinopathies. Similar results were also obtained in the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit-erythroid colonies, while on the contrary, at the same days of observation, a proportion of 100%, 100%, 73% and 90% donor-derived erythrocytes was observed in the four patients with persistent mixed chimerism. Conclusions. Our results showed that mostly of the erythrocytes present in four long-term transplanted patients affected by haemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient to clinical control the disease in patients affected by haemoglobinopathies is relevant, although the biological mechanisms underlying these observations need to be further investigated.