Diana Campioni

Darbepoetin alpha for the treatment of anaemia in low-intermediate risk myelodysplastic syndromes

Thirty‐seven anaemic subjects with low‐to‐intermediate risk myelodysplastic syndrome (MDS) received the highly glycosylated, long‐acting erythropoiesis‐stimulating molecule darbepoetin‐alpha (DPO) at the single, weekly dose of 150 μg s.c. for at least 12 weeks. Fifteen patients (40·5%) achieved an erythroid response (13 major and two minor improvements, respectively, according to International Working Group criteria). Such results are currently maintained after 7–22 months in 13 of the responders, one of whom required iron substitutive therapy during the treatment. One patient relapsed after 4 months. Another responder died after 5 months because of causes unrelated to the treatment. No relevant side‐effects were recorded. At multivariate analysis, significant predictive factors of response were baseline serum levels of endogenous erythropoietin <100 IU/l, absent or limited transfusional needs, no excess of blasts and hypoplastic bone marrow. This study suggests that DPO, at the dose and schedule used, can be safely given in low‐intermediate risk MDS and may be effective in a significant proportion of these patients.

Evidence for a Role of TNF-Related Apoptosis- Inducing Ligand (TRAIL) in the Anemia of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are characterized by impaired erythropoiesis, possibly caused by proapoptotic cytokines. We focused our study on the cytokine TRAIL (TNF-related apoptosis-inducing ligand), which has been shown to exhibit an anti-differentiation activity on erythroid maturation. Immunocytochemical analysis of bone marrow mononuclear cells (BMMC) showed an increased expression of TRAIL in MDS patients with respect to acute myeloid leukemia (AML) patients and normal BM donors. TRAIL expression was increased predominantly in myeloid precursors of granulocytic lineage and in a subset of monocytes and pro-erythroblasts. Significant levels of soluble TRAIL were released in 21 of 68 BMMC culture supernatants from MDS patients. On the other hand, TRAIL was detected less frequently in the culture supernatants of AML (4 of 33) and normal BMMC (0 of 22). Analysis of peripheral blood parameters revealed significantly lower levels of peripheral red blood cells and hemoglobin in the subset of patients whose BMMC released TRAIL in culture supernatants compared to the subgroup of patients who did not release TRAIL. Moreover, TRAIL-positive BMMC culture supernatants inhibited the differentiation of normal glycophorin A+ erythroblasts generated in serum-free liquid phase. Thus, increased expression and release of TRAIL at the bone marrow level is likely to impair erythropoiesis and to contribute to the degree of anemia, the major clinical feature of MDS.

A decreased positivity for CD90 on human mesenchymal stromal cells (MSCs) is associated with a loss of immunosuppressive activity by MSCs

Biologic and clinical interest in human mesenchymal stromal cells (hMSC) has risen over the last years, mainly due to their immunosuppressive properties. In this study, we investigated the basis of immunomodulant possible variability using hMSC from different sources (amniotic membrane, chorion, and bone marrow from either healthy subjects or patients with hematological malignancies, HM) and having discordant positivity for several immunological markers. The CD90+ hMSC reduced lymphoproliferative response in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) via sHLA‐G and IL‐10 up‐modulation. On the contrary, hMSC showing a significantly lower expression for CD90 antigen, elicited a lymphoproliferative allogeneic response in PHA/PBMCs without any increase in soluble HLA‐G and IL‐10 levels. These data seems to suggest that CD90 molecule may be considered a novel predictive marker for hMSC inhibitory ability, and might cooperate with HLA‐G molecule in regulating suppressive versus stimulatory properties of hMSC. These results may have clinical implication in either transplantation or in regenerative medicine fields.

A functional role for soluble HLA-G antigens in immune modulation mediated by mesenchymal stromal cells.

BACKGROUND It has been suggested that soluble factors produced by bone marrow (BM) mesenchymal stromal cells (MSC) play a fundamental role in mediating immune modulation. HLA-G antigens (Ag) are major histocompatibility complex (MHC) class Ib molecules characterized by a limited polymorphism and a splicing mechanism that regulates the production of membrane-bound and soluble isoforms. Interleukin-10 (IL-10) cytokine is one of the main up-modulators of soluble HLA-G Ag (sHLA-G) production by CD14+ peripheral blood monocyte cells and increased IL-10 levels are reported to be associated with MSC immune modulation. METHODS We investigated, by specific enzyme-linked immunosorbent assay (ELISA), the possible role of sHLA-G molecules in the inhibition of the peripheral blood mononuclear cell (PBMC) response to phytohemagglutinin (PHA) mediated by MSC from different sources. RESULTS There was a significant correlation between the presence of increased levels of sHLA-G and IL-10 in the MSC/PBMC/PHA culture supernatants and lymphoproliferative inhibition. Neutralizing experiments performed with monoclonal Ab directed against HLA-G and IL-10 molecules confirmed the inhibitory ability of sHLA-G Ag. Furthermore, exogenous IL-10 induced sHLA-G molecule secretion by MSC alone in a polymorphic way, while a longitudinal analysis confirmed the loss of MSC inhibitory functions in relation to in vitro MSC aging. DISCUSSION Overall the results obtained suggest a functional role for sHLA-G molecules in inhibiting the PBMC response mediated by MSC. Moreover, the ability of IL-10 to induce sHLA-G Ag production by MSC alone could be proposed as a marker of MSC functional ability.

Promotion of tumour metastases and induction of angiogenesis by native HIV-1 Tat protein from BK virus/tat transgenic mice

ObjectiveTo characterize the T53 cell line and its clones derived from an adenocarcinoma of BK virus (BKV)/tat transgenic mice and to establish the role of native Tat in tumorigenicity, induction of metastases and angiogenesis. Design and methodsTat was quantified by flow cytometry and chloramphenicol acetyltransferase (CAT) assays. Tumorigenicity and metastatic ability of cell lines were assayed in nude mice. Production of proteases was evaluated by a plasmin chromogenic assay and gelatinase zymography. The angiogenic effect was studied in vivo with conditioned medium from tumour cell lines. ResultsTat protein was detected in tumour cell lines in amounts from 600–7000 molecules/cell. Conditioned medium from tumour cell lines was able to transactivate an LTR-CAT in HL3T1 cells, indicating release of extracellular Tat. Tumour cell lines, inoculated into nude mice, induced angiogenic tumours with remarkable recruitment of host endothelial cells. Metastases were detected in lymph nodes, lungs, kidneys, and heart. Cell lines produced relevant amounts of proteases. Conditioned medium implanted in mice with matrigel induced an angiogenic response, enhanced by addition of heparin. Preincubation with an anti-Tat antibody abolished the angiogenic effect. ConclusionsTat from cells from BKV/tat transgenic mice promotes tumorigenesis and formation of metastases and induces angiogenic activity. Angiogenesis occurs at physiological concentrations of Tat lower than 20 ng/ml. The effects of Tat on induction of metastases and angiogenesis appear to be mediated by activation of proteases.

Loss of Thy-1 (CD90) antigen expression on mesenchymal stromal cells from hematologic malignancies is induced by in vitro angiogenic stimuli and is associated with peculiar functional and phenotypic characteristics

BACKGROUND Little is known about human mesenchymal stromal cell (hMSC) phenotypic and functional subsets in response to environmental stimuli. The strategy used in this study focused on defining hMSC functional subpopulations based in particular on their Thy-1 (CD90) antigen (Ag) surface expression. METHODS The effect of different in vitro microenvironmental conditions on the isolation and expansion of bone marrow-derived (BM) hMSC from hematologic malignancies (HM) and normal samples (NS) was assayed. hMSC clonogenic and differentiation potential, phenotypic profile and long-term capacity to sustain in vitro hemopoiesis were considered in relation to the different expansion protocols. RESULTS The results showed that angiogenic supplements used in combination with low serum content gave rise to the appearance of Thy-1(-) HM-MSC with high proliferative potential, capable of restoring the typical HM stromal impairment. The expression of the CD271 was partially maintained. We further report an enhancement towards the osteogenic and adipogenic differentiation capacity by the Thy-1(-) HM-MSC subset. Despite the angiogenic treatment, the Thy-1(-) MSC stopped short of full endothelial differentiation. DISCUSSION In this paper we provide evidence that in vitro angiogenic stimuli generate HM-MSC lacking CD90 Ag expression. The Thy-1(-) MSC subset is characterized by peculiar functional and phenotypic characteristics, thus supporting the role played by the microenvironment in selecting particular hMSC subsets maintaining normal tissue homeostasis or inducing pathologic processes.

Angiogenesis in multiple myeloma: correlation between in vitro endothelial colonies growth (CFU-En) and clinical–biological features

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/106 BM mononucleated cells in untreated MM samples (2.07 s.d. ± 1.3) was significantly higher than in normal BM (0.28 ± 0.48), while no difference was seen between normal BM and MGUS (0.28 ± 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 ± 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 ± 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.

CD34+ cell subsets and long-term culture colony-forming cells evaluated on both autologous and normal bone marrow stroma predict long-term hematopoietic engraftment in patients undergoing autologous peripheral blood stem cell transplantation

OBJECTIVE The aim of this study was to evaluate which CD34(+) cell subset contained in leukapheresis products could be regarded as the most predictive of long-term hematopoietic recovery after autologous peripheral blood stem cell transplantation (auto-PBSCT). MATERIALS AND METHODS Based on data from 34 patients with hematologic malignancies, doses of CD34(+) cells and CD34(+) cell subsets, defined by the expression of HLA-DR, CD38, CD117 (c-kit/R), CD123 (alpha subunit of IL-3/R), CD133 (AC133), and CD90 (Thy-1) antigens, were correlated with the number of short-term (i.e., colony-forming cells [CFC]) and long-term culture CFC (LTC-CFC) (generated at week 5 of culture) and with the kinetics of hematopoietic engraftment following auto-PBSCT. The capacity of autologous stroma (AS), normal human bone marrow stroma, and M2-10B4 murine cell line to sustain CD34(+) cell growth was comparatively evaluated in the LTC assay. RESULTS Our data demonstrated that some of the most primitive progenitor subsets (CD34(+)CD117(-)HLA-DR(-), and CD34(+)CD38(+)HLA-DR(-)) showed the strongest correlation with LTC-CFC numbers generated within the AS, whereas no significant correlation was noted using normal bone marrow stroma. Multivariate analysis showed that the only CD34 cell subset independently associated with long-term (3 to 6 months) platelet engraftment after auto-bone marrow transplantation was the CD34(+)CD117(-)HLA-DR(-) phenotype; long-term erythrocyte engraftment was correlated with CD34(+)CD38(+)HLA-DR(-) cell content. The latter further influenced platelet engraftment in the first 3 months after auto-PBSCT. The most predictive parameters for neutrophil engraftment were CD34(+)CD38(+)HLA-DR(-) cell subtype and the total LTC-CFC quantity infused. CONCLUSIONS These data further support the hypothesis that the type of stromal feeders influences the frequency of LTC-CFC, possibly because they differ in their ability to interact with distinct subsets of hematopoietic stem cells. Furthermore, as the use of AS in LTC assay can mimic in vitro the human bone marrow microenvironment, it can be speculated that this culture system could be a useful means to study the kinetics of recovery of bone marrow stroma following chemotherapy and PBSCT. From these results, it can be concluded that some CD34(+) cell subsets appear to be more reliable predictors of long-term hematopoietic recovery rates than total CD34(+) cell quantity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF-α promote the NF-κB-dependent maturation of normal and leukemic myeloid cells

Tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) and TNF‐α induced monocytic maturation of primary normal CD34‐derived myeloid precursors and of the M2/M3‐type acute myeloid leukemia HL‐60 cell line, associated to increased nuclear factor (NF)‐κB activity and nuclear translocation of p75, p65, and p50 NF‐κB family members. Consistently, both cytokines also induced the degradation of the NF‐κB inhibitors, IκBα and IκBɛ, and up‐regulated the surface expression of TRAIL‐R3, a known NF‐κB target. However, NF‐κB activation and IκB degradation occurred with different time‐courses, since TNF‐α was more potent, rapid, and transient than TRAIL. Of the two TRAIL receptors constitutively expressed by HL‐60 (TRAIL‐R1 and TRAIL‐R2), only the former was involved in IκB degradation, as demonstrated by using agonistic anti‐TRAIL receptor antibodies. Moreover, NF‐κB nuclear translocation induced by TRAIL but not by TNF‐α was abrogated by z‐IETD‐fmk, a caspase‐8‐specific inhibitor. The key role of NF‐κB in mediating the biological effects of TNF‐α and TRAIL was demonstrated by the ability of unrelated pharmacological inhibitors of the NF‐κB pathway (parthenolide and MG‐132) to abrogate TNF‐α‐ and TRAIL‐induced monocytic maturation. These findings demonstrate that NF‐κB is essential for monocytic maturation and is activated via distinct pathways, involving or not involving caspases, by the related cytokines TRAIL and TNF‐α.

Functional and immunophenotypic characteristics of isolated CD105(+) and fibroblast(+) stromal cells from AML: implications for their plasticity along endothelial lineage.

BACKGROUND In vitro cultures of BM cells from newly diagnosed patients with AML displayed a defective BM stromal compartment, with a reduced number of fibroblast-colony-forming unit (CFU-F: 1 +/- 1.25 SD) and a decreased proliferative ability. The purposes of our study were: 1). to select BM mesenchymal stem cells (MSC) and BM-derived stromal cells (BMDSCs) from AML patients at diagnosis and from healthy subjects, using an immunomagnetic system and either anti-CD105 or anti-fibroblast MAbs; 2). to study the immunophenotypic and functional properties of freshly isolated and cultured mesenchymal cells; 3). to test the in vitro plasticity of the selected cells to differentiate towards an endothelial phenotype. METHODS Fresh mononuclear cells obtained from BM of 20 patients newly diagnosed with AML and from eight healthy subjects were selected by using anti-fibroblast and anti-CD105 MAbs. Freshly isolated cells were analyzed, characterized by flow cytometry using a wide panel of MAbs and seeded in long-term culture medium to assess CFU-F formation. The level of confluence after 30 days and functional capacity in a long-term colony-forming cell culture (LTC-CFC) were tested. Furthermore, the cultured selected cell populations were assayed for their ability to differentiate into an endothelial-like cell phenotype with the addition of vascular endothelial growth factor (VEFG) and endothelial cell growth supplement (ECGS). RESULTS In normal subjects the selection produced an increase of the CFU-F number of 2.6-fold with anti-fibroblast MAb and 2.7-fold with the anti-CD105 MAb. Anti-fibroblast and anti-CD105 MAb selection from AML BM cells resulted in a statistically significant greater count of CFU-F that was respectively 10.6-fold (P = 0.04) and 14.4-fold (P = 0.00001) higher in comparison with the unselected AML samples. Interestingly, in 80% of AML samples immunoselection was also able to restore the capacity of the CFU-F to proliferate and form confluent stromal layers. The isolation of those layers sustained the proliferation and differentiation of hematopoietic stem cells in the LTC-CFC. The phenotypic profile of cultured BMDSCs was different from that of the freshly isolated cells, and changed in relation to the culture conditions: CD105+ selected cells cultured with VEGF and ECGS expressed endothelial markers, a finding that suggests that this cell subpopulation may have the potential to differentiate toward an endothelial-like phenotype. DISCUSSION We report that immunomagnetic selection represents a valid tool for the selection of BM mesenchymal cells in samples obtained from both healthy subjects and patients with AML. This technique was able to rescue two functional and immunophenotypic compartments related to two different selected populations. In particular, the CD105+ cells isolated in AML displayed, after stimulation with VEGF and ECGS, the ability to change towards an endothelial-like cell phenotype, thus revealing an unexpected plasticity. Both CD105+ and fibroblast+ cells once successfully isolated might represent sources of mesenchymal cells populations useful for in vitro investigations and, above all, as therapeutic devices.