Maddalena Raia

Peptide inhibitors of C3 activation as a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal hemoglobinuria

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 IC50 ∼4 µM and full inhibition at 6 µM. 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.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks.

Fe65 is a binding partner of the Alzheimers β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites.

CD200/OX2, a cell surface molecule with immuno-regulatory function, is consistently expressed on hairy cell leukaemia neoplastic cells

CD200 (formerly called OX2) is a transmembrane glycoprotein with immunosuppressive functions. It is expressed on normal B-lymphocytes, T-lymphocytes, dendritic cells and several solid tissues (Kawasaki & Farrar, 2008). CD200 receptor expression is limited to myeloid leucocytes and a subset of Tlymphocytes (Kawasaki & Farrar, 2008). In mouse systems, the binding of CD200 to its receptor (i) decreases the production of T-helper cell type 1 (Th1)-like cytokines, such as interleukin (IL)-2 and interferon c, (ii) increases the release of Th2-like cytokines, such as IL-10 and IL-4 (Gorczynski, 2001) and (iii) promotes the in vitro differentiation of T cells toward CD4CD25Foxp3 Treg lymphocytes (Gorczynski et al, 2008). CD200 is constantly overexpressed on chronic lymphocytic leukaemia (CLL) cells (McWhirter et al, 2006). The addition of CLL cells to mixed lymphocyte reactions causes an immunological shift from a Th1-like response to a Th2-like response, confirming that CD200 plays an important role in controlling T-cytotoxic immune response (McWhirter et al, 2006). Starting from these data, we extended the investigation of CD200 expression to another B-chronic lymphoproliferative disorder i.e. hairy cell leukaemia (HCL). Hairy cell leukaemia is a distinct disease entity in the World Health Organization (WHO) classification, displaying unique clinico-pathological and biological features (Tiacci et al, 2006). As hairy cells display a specific immunophenotype, multicolour flow cytometry is currently the best tool for HCL diagnosis. A total of 10 specimens (six peripheral blood samples and four bone marrow aspirates), collected from 10 patients with newly diagnosed HCL, were studied. As normal controls we analysed 10 peripheral blood specimens and two bone marrow aspirates from 12 healthy donors. An aliquot (50 ll) of each sample was incubated at 4 C for 30 min in the presence of appropriate amounts of monoclonal antibodies. The mixtures were then diluted 1:20 in ammonium chloride lysing solution, incubated at room temperature for 10 min and finally washed prior to flow cytometric analysis with the FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA). The following antigens were analysed: CD200, SmIg-kappa, SmIg-lambda, CD45, CD19, CD5, CD23, CD20, CD22, CD103, CD11c, CD25, CD43, CD10, CD3, CD56 and CD81. Hairy cells were gated as CD45CD19 ‘monocytoid cells’ (i.e. cells with light scatter features typical of monocytes). In addition, in the majority of cases, we also were able to perform a full immunological gate on CD45 CD19CD103CD11c cells. With regard to the normal controls included in our study, B-lymphocytes were simply gated as CD45CD19 cells. In all specimens cell doublets and debris were excluded from our analysis by forward-scatter versus side-scatter dotplot examination. To set the cut-off point to distinguish between CD200 negative and positive cells, we used the ‘Fluorescence Minus One’ technique as described by Perfetto et al (2004). A single case was arbitrarily judged CD200 positive when the percentage of positive cells (PPC) was higher than 30%. All HCL samples were CD200 positive with PPC and median fluorescence intensity (MFI) median values of 99 (25th–75th percentile 92–99) and 3016 (25th–75th percentile 1382–5430), respectively. Although CD200 was positive in 12 out of 12 normal controls, the PPC and MFI median values were of 71 (25th–75th percentile 64–83) and 582 (25th–75th percentile 406–725), respectively. Differences in PPCs and MFIs between HCLs and normal controls were statistically significant (Mann–Whitney U, two-tailed testing, P < 0Æ0001). Data regarding MFI analysis are shown in Fig 1. Whereas normal controls showed weak CD200 fluorescence intensity with a bimodal distribution, HCL samples showed bright CD200 expression in a homogeneous pattern (Fig 2). This is the first documented direct evidence of CD200 overexpression in HCL. As described above, CD200 promotes Th2-like cytokines synthesis. IL-4 and IL-10 are reported to reduce anti-tumour cytotoxic T cell response (McWhirter

Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphomas.

Among histological aggressive non-Hodgkin lymphomas (NHL), the overall risk of central nervous system (CNS) relapse is approximately 5%, a figure which is too low to offer prophylaxis to all patients. The aim of this work is to demonstrate the utility of flow cytometry (FCM) in detecting occult leptomeningeal disease in this subtype of NHL. We studied cerebrospinal fluid (CSF) involvement in 42 newly diagnosed aggressive NHL patients at risk for CNS involvement. We used multicolour FCM to detect CSF infiltrating neoplastic cells. Among the 42 patients studied, 11 had CSF involvement as detected by FCM. Of these, only four were also positive for conventional morphology (p=0.046). These results designate that FCM as the first choice technique in NHL CSF clinical cell analysis.

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies.

BackgroundAldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients.ResultsIn normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA).ConclusionOur study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia.

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

Miniaturized flow cytometry-based BCR-ABL immunoassay in detecting leptomeningeal disease

Despite central nervous system (CNS) prophylactic programs limit leptomeningeal involvement in acute lymphoblastic leukemia (ALL), it can still occur in a restricted percentage of cases. The exact risk rate remains still unknown, and several factors are associated with an increased probability to develop CNS involvement. Among them, Philadelphia (Ph)-positive genotype seems to play a relevant role. Recently, a flow cytometric assay to detect BCR-ABL protein has been developed, but little is known about its possible employment in leptomeningeal disease. Here, we show the miniaturized application of the original assay for BCR-ABL oncoprotein detection in cerebrospinal fluid (CSF) samples.

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations

The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.

High Aminopeptidase N/CD13 Levels Characterize Human Amniotic Mesenchymal Stem Cells and Drive Their Increased Adipogenic Potential in Obese Women

Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.