BackgroundThrough negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies.Methodology/Principal FindingsIn a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept.Conclusions/SignificanceDespite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient qu...

• Peptidic C3 inhibitors of the compstatin family (Cp40) efficiently prevent hemolysis and opsonization of PNH erythrocytes in vitro.• Pharmacokinetic studies show that sustained therapeutic concentrations can be achieved with both Cp40 and its PEGylated derivative, PEG-Cp40.Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis due to the lack of CD55 and CD59 on affected erythrocytes. The anti-C5 antibody eculizumab has proven clinically effective, but uncontrolled C3 activation due to CD55 absence may result in opsonization of erythrocytes, possibly leading to clinically meaningful extravascular hemolysis. We investigated the effect of the peptidic C3 inhibitor, compstatin Cp40, and its long-acting form (polyethylene glycol [PEG]-Cp40) on hemolysis and opsonization of PNH erythrocytes in an established in vitro system. Both compounds demonstrated dose-dependent inhibition of hemolysis with IC 50 ∼4 mM and full inhibition at 6 mM. Protective levels of either Cp40 or PEG-Cp40 also efficiently prevented deposition of C3 fragments on PNH erythrocytes. We further explored the potential of both inhibitors for systemic administration and performed pharmacokinetic evaluation in nonhuman primates. A single intravenous injection of PEG-Cp40 resulted in a prolonged elimination half-life of >5 days but may potentially affect the plasma levels of C3. Despite faster elimination kinetics, saturating inhibitor concentration could be reached with unmodified Cp40 through repetitive subcutaneous administration. In conclusion, peptide inhibitors of C3 activation effectively prevent hemolysis and C3 opsonization of PNH erythrocytes, and are excellent, and potentially cost-effective, candidates for further clinical investigation. (Blood. 2014;123(13):2094-2101 IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a complex hematologic disorder characterized by the expansion of hematopoietic cells deficient in glycophosphatidylinositol-anchored surface proteins, including the complement regulators CD55 and CD59. 1 Affected erythrocytes suffer from uncontrolled complement activation on their surface, and subsequent membrane attack complex (MAC)-mediated intravascular hemolysis.2 The therapeutic anti-C5 antibody eculizumab (Soliris, Alexion) has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in a majority of PNH patients.3,4 Yet, persistent C3 activation occurring during eculizumab treatment may lead to progressive deposition of C3 fragments on affected erythrocytes and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematologic benefit of anti-C5 treatment. 5,6 Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current complement-targeted treatment. 7,8 Indeed, it has been recently documented that protein inhibitors of the alternative pathway (AP) of complement activation, such as the CD21/factor H (FH) fusion protein TT30 (Alexion) or the engin...

Endoglin (CD105) is a cell membrane glycoprotein over-expressed on highly proliferating endothelial cells in culture, and on endothelial cells of angiogenetic blood vessels within benign and malignant tissues. CD105 binds several factors of the Transforming Growth Factor (TGF)-b superfamily, and its over-expression modulates cellular responses to TGF-b1. The complex of experimental ®ndings accumulated in the last few years strongly indicate that CD105 is a powerful marker of angiogenesis, and that it might play a critical role in the pathogenesis of vascular diseases and in tumor progression. In this paper, we will review the structural, biological and functional features of CD105, as well as its distribution within normal and neoplastic tissues, emphasizing its foreseeable role as a molecular target for new diagnostic and bioimmunotherapeutic approaches in human malignancies.

Secreted phospholipases A2 (sPLA2) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA2s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA2s induced TNF-α and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA2s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA2 (bromophenacyl bromide-inactivated human sPLA2 and the H48Q mutant of the porcine sPLA2) were as effective as the catalytically active sPLA2s in inducing cytokine production. HLM expressed the M-type receptor for sPLA2s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA2 activity as well as binding to the M-type receptor, suppressed sPLA2-induced cytokine production. Incubation of HLM with the sPLA2s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA2s. In conclusion, two distinct sPLA2s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.

Many cell types release extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, which play a role in physiology and diseases. Presence and phenotype of circulating EVs in hematological malignancies (HMs) remain largely unexplored.The aim of this study was to characterize EVs in peripheral blood of HM patients compared to healthy subjects (controls). We isolated serum EVs from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia (WM), Hodgkin's lymphoma (HL), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and controls. EVs were isolated from serum of peripheral blood by ultracentrifuge steps and analyzed by flow cytometry to define count, size, and immunophenotype. MV levels were significantly elevated in WM, HL, MM, AML, and some MPNs and, though at a lesser degree, in CLL and NHL as compared to healthy controls. HL, MM, and MPNs generated a population of MVs characterized by lower size (below 0.3 μm) when compared to controls. MVs from patients specifically expressed tumor-related antigens, such as CD19 in B cell neoplasms, CD38 in MM, CD13 in myeloid tumors, and CD30 in HL. Both total and antigen-specific count of MVs significantly correlated with different HM clinical features such as Rai stage in CLL, International Prognostic Scoring System in WM, International Staging System in MM, and clinical stage in HL. MVs may represent a novel biomarker in HMs.

Summary. Acute promyelocytic leukaemia (APL), characterized by a specific PML-RARa fusion gene resulting from translocation t(15;17) and by a high response rate to differentiation therapy with all-trans retinoic acid, presents clinical (varying WBC counts, age and treatment outcome), morphological (hypergranular M3 and hypogranular M3V) and molecular (three isoforms of PML breakpoint) heterogeneity.We correlated leukaemic immunophenotype with these aspects in 196 molecularly confirmed APLs (63 children and 133 adults) in Italy. The bcr3 isoform (P ¼ 0·05) and FAB M3V (P ¼ 0·05) were more frequent in children. We confirmed in APL an immunophenotype characterized by frequent expression of CD13, CD33 and CD9 and rare expression of HLA-DR, CD10, CD7 and CD11b. However, we recognized CD2 in 28%, CD34 in 23% and CD19 in 11% of cases and demonstrated by double labelling that CD34 and CD2 may be co-expressed. CD2, CD34 and CD19 were significantly intercorrelated, and variably associated to other features: CD2 and CD34 with PML bcr3 (P < 0·001 and P < 0·001, respectively) and with M3V (P < 0·001 and P ¼ 0·002), whereas only CD19 was directly correlated with WBC counts and only CD2 positively influenced CR rate (logistic model) and event-free survival (Cox model). We conclude that immunophenotype plays a role in the determination of the biological and clinical heterogeneity of childhood and adult APL.

The expression of CD56 is significantly associated with inferior CR duration and survival in patients with APL who were treated with modern frontline treatment that included ATRA and simultaneous chemotherapy. Combined with other well-established prognostic factors such as WBC count, CD56 expression at diagnosis might be used to build prognostic scores for risk-adapted therapy in APL.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complementmediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membranetargeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membranetargeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients. (Blood. 2012;119(26):6307-6316) IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a blood disorder clinically characterized by intravascular hemolysis, thrombophilia, and bone marrow failure. 1-3 A unique feature of PNH is the presence of clonal populations of blood cells that are defective in glycosylphosphatidylinositol (GPI)-anchor biosynthesis, 4 because they derive from stem cells harboring an acquired somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene. 5,6 GPIlinked surface proteins include CD55 (also known as decayaccelerating factor [DAF]) 7,8 and CD59 (or membrane inhibitor of reactive lysis [MIRL]), 9,10 2 major complement regulators on the cell surface. Because of the lack of these 2 regulators, PNH erythrocytes (red blood cells [RBCs]) are exquisitely vulnerable to complement activation. Indeed, the main mechanism of hemolysis in PNH is the intravascular destruction of CD59 deficient RBCs by the membrane attack complex (MAC; supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). 11 MAC formation in patients can be abrupt and massive when complement is triggered by specific conditions, as with an infection, explaining the paroxysms of hemoglobinuria that have given PNH its name. However, at a lower rate, hemolysis in PNH is continuous, and it is accounted for by the so-called tickover of the complement alternative pathway (CAP). Tickover occurs through spontaneous hydrolysis of C3, binding of factor B to this form of C3, and subsequent formation of a C3 cleavage and activating multiprotein complex designated a C3 convertase (supplemental Figure 1). 12,1...