Maria Psyllaki

Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndrome associated with chromosome 5q deletion

Background Lenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodysplastic syndrome and interstitial deletion of the long arm of chromosome 5 [del(5q)]. The aim of this study was to explore the effect of lenalidomide treatment on the reserves and functional characteristics of bone marrow hematopoietic progenitor/precursor cells, bone marrow stromal cells and peripheral blood lymphocytes in patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q). Design and Methods We evaluated the number and clonogenic potential of bone marrow erythroid/myeloid/megakaryocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesion molecule expression of CD34+ cells by flow cytometry, the hematopoiesis-supporting capacity of bone marrow stromal cells using long-term bone marrow cultures and the number and activation status of peripheral blood lymphocytes in ten patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q) receiving lenalidomide. Results Compared to baseline, lenalidomide treatment significantly decreased the proportion of bone marrow CD34+ cells, increased the proportion of CD36+/GlycoA+ and CD36−/GlycoA+ erythroid cells and the percentage of apoptotic cells within these cell compartments. Treatment significantly improved the clonogenic potential of bone marrow erythroid, myeloid, megakaryocytic colony-forming cells and increased the proportion of CD34+ cells expressing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48. The hematopoiesis-supporting capacity of bone marrow stroma improved significantly following treatment, as demonstrated by the number of colony-forming cells and the level of stromal-derived factor-1α and intercellular adhesion molecule-1 in long-term bone marrow culture supernatants. Lenalidomide treatment also increased the proportion of activated peripheral blood T lymphocytes. Conclusions The beneficial effect of lenalidomide in patients with lower risk myelodysplastic syndrome with del(5q) is associated with significant increases in the proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant alterations in the adhesion profile of bone marrow CD34+ cells.

Effect of cA2 anti -tumor necrosis factor-α antibody therapy on hematopoiesis of patients with myelodysplastic syndromes

Purpose: Tumor necrosis factor α (TNF-α) plays a prominent role in the pathophysiology of myelodysplastic syndromes (MDS). The aim of this study was to explore the biological and immunoregulatory effect of the treatment with the anti–tumor necrosis factor-α monoclonal antibody cA2 on bone marrow (BM) progenitor/precursor and stromal cells and lymphocyte subsets, as well as the clinical response in MDS patients. Experimental Design: Ten low-intermediate risk MDS patients received i.v. cA2 (3 mg/kg) at weeks 0, 2, 6, and 12. The number, survival, and clonogenic potential of BM progenitor/precursor cells, the hematopoiesis-supporting capacity of BM stromal cells, and the lymphocyte activation status were investigated in the patients at baseline and following treatment using flow cytometry, clonogenic assays, and long-term BM cultures (LTBMC). Clinical response was evaluated according to standardized criteria. Results: cA2 administration reduced the proportion of apoptotic and Fas+ cells in the CD34+ cell compartment (P = 0.0215 and P = 0.0344, respectively) and increased the clonogenic potential of BM mononuclear and CD34+ cells (P = 0.0399 and P = 0.0304, respectively) compared with baseline. The antibody reduced tumor necrosis factor-α levels in LTBMC supernatants (P = 0.0043) and significantly improved the hematopoiesis-supporting capacity of LTBMC adherent cells. The proportion of activated peripheral blood and BM T-lymphocytes decreased significantly after treatment, suggesting an immunomodulatory effect of cA2. Two patients displayed minor hematologic responses whereas the remaining patients displayed stable disease with no disease progression. Conclusions: The encouraging biological insights from cA2 administration may be useful in conducting further clinical trials using cA2 for selected MDS patients, particularly those with evidence of immune-mediated inhibition of hematopoiesis.

Alendronate reduces serum TNFα and IL-1β, increases neutrophil counts, and improves bone mineral density and bone metabolism indices in patients with chronic idiopathic neutropenia (CIN)-associated osteopenia/osteoporosis

The current study was undertaken to investigate the effect of alendronate on bone mineral density (BMD), bone metabolism markers, and serum bone-resorbing cytokines in patients with chronic idiopathic neutropenia (CIN)-associated osteopenia/osteoporosis. Sixteen randomly selected women, 7 with CIN-associated osteoporosis and 9 with CIN-associated osteopenia, and 14 age- and menopausal status-matched healthy volunteers, were enrolled in the study. Patients received 10 mg alendronate daily per os for 360 sdays and studies were done before treatment (day 0) and at varying time points during the study. We found that patients’ BMD measurements increased by 5.32% after treatment, and that the elevated serum osteocalcin (OC), a bone formation marker, decreased by day 30, normalized by day 90, and increased again by day 270 of treatment. Elevated values of patients’ urine deoxypyridinoline (Dpd) and N-telopeptide of type I of collagen (NTx), two bone resorption markers, returned to the control range by day 30 and decreased thereafter. Increased levels of patients’ serum tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), two bone resorbing cytokines, returned to the control range by day 30 and decreased thereafter. Peripheral blood neutrophil counts increased by day 30 and continued to rise thereafter, reaching a mean value higher than 2650 neutrophils per µl of blood on day 360. Interestingly, alendronate-induced changes in the levels of both cytokines correlated inversely with the respective changes in neutrophil counts and BMD measurements, and positively with the changes in the respective means of urine NTx and Dpd values. All these findings indicate that alendronate is effective in treating CIN-associated osteopenia/osteoporosis, and that the beneficial effect of the compound may lie, at least in part, in its property to inhibit the production of TNFα and IL-1β by cells of the monocyte/macrophage system, in which osteoclasts are included.

Impaired clearance of apoptotic cells leads to HMGB1 release in the bone marrow of patients with myelodysplastic syndromes and induces TLR4-mediated cytokine production

Excessive pro-inflammatory cytokine production in the bone marrow has been associated with the pathogenesis of myelodysplastic syndromes. We herein investigated the involvement of toll-like receptors and their endogenous ligands in the induction/maintenance of the inflammatory process in the marrow of patients with myelodysplastic syndromes. We evaluated the expression of toll-like receptors in marrow monocytes of patients (n=27) and healthy controls (n=25) by flow-cytometry and also assessed the activation of the respective signaling using a real-time polymerase chain reaction-based array. We measured the high mobility group box-1 protein, a toll-like receptor-4 ligand, in marrow plasma and long-term bone marrow culture supernatants by an enzyme-linked immunosorbent assay and we performed cross-over experiments using marrow plasma from patients and controls in the presence/absence of a toll-like receptor-4 inhibitor to evaluate the pro-inflammatory cytokine production by chemiluminescence. We assessed the apoptotic cell clearance capacity of patients’ macrophages using a fluorescence microscopy-based assay. We found over-expression of toll-like receptor-4 in patients’ marrow monocytes compared to that in controls; this over-expression was associated with up-modulation of 53 genes related to the respective signaling. Incubation of patients’ monocytes with autologous, but not with normal, marrow plasma resulted in over-production of pro-inflammatory cytokines, an effect that was abrogated by the toll-like receptor-4 inhibitor suggesting that the pro-inflammatory cytokine production in myelodysplastic syndromes is largely mediated through toll-like receptor-4. The levels of high mobility group box-1 protein were increased in patients’ marrow plasma and culture supernatants compared to the levels in controls. Patients’ macrophages displayed an impaired capacity to engulf apoptotic cells and this defect was associated with excessive release of high mobility group box-1 protein by dying cells. A primary apoptotic cell clearance defect of marrow macrophages in myelodysplastic syndromes may contribute to the induction/maintenance of the inflammatory process through aberrant release of molecules inducing toll-like receptor-4 such as high mobility group box-1 protein.

Impaired megakaryopoiesis in patients with chronic idiopathic neutropenia is associated with increased transforming growth factor β1 production in the bone marrow

Patients with chronic idiopathic neutropenia (CIN) display relatively low peripheral blood platelet counts and hypo‐lobulated megakaryocytes in the bone marrow (BM). The underlying pathogenetic mechanismswere probed by studying the reserves and clonogenic potential of BM megakaryocytic progenitor cells using flow‐cytometry and a collagen‐based clonogenic assay for the identification of megakaryocyte colony‐forming units (CFU‐Meg). Thrombopoietin (TPO) and transforming growth factor‐β1 (TGFβ1) levels were also evaluated in long‐term BM culture supernatants using an enzyme‐linked immunosorbent assay. CIN patients (n = 39) showed a low proportion of BM CD34+/CD61+ megakaryocytic progenitor cells and low frequency of early and mixed CFU‐Meg in the BM mononuclear, but not CD34+, cell fraction, compared with healthy controls (n = 20). TPO and TGFβ1 levels were significantly higher in patients compared with controls. TPO levels inversely correlated with platelet counts whereas TGFβ1 values correlated inversely with CD34+/CD61+ and CFU‐Meg megakaryocytic progenitor cell numbers and positively with TPO levels. The addition of an anti‐TGFβ1 neutralising antibody significantly increased the numbers of CFU‐Meg in CIN patients but not in controls, compared with baseline. These data suggest that increased local production of TGFβ1 probably affects the BM megakaryocytic progenitor cell growth in CIN whereas the compensatory production of TPO finally balances the TGFβ1‐induced inhibitory effect.

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with severe congenital neutropenia.

Abstract: Objective: To investigate further the cellular defect responsible for impaired granulopoiesis in severe congenital neutropenia (SCN), we have evaluated bone marrow (BM) stem cell reserve and function and BM stromal cell myelopoiesis supporting capacity in two patients with SCN. Methods: BM primitive stem cells and myeloid progenitor cells were assessed using flow cytometry, limiting dilution assay, clonogenic assays, and long‐term BM cultures (LTBMC). BM stroma function was assessed by evaluating the ability of irradiated stromal layers from the patients to induce granulocyte‐macrophage colony formation (CFU‐GM) by normal CD34+ cells. Results: Compared to the normal controls (n = 37), SCN patients displayed a low percentage of CD34+/CD38+ cells (P < 0.05), low CFU‐GM colony formation by highly purified CD34+ cells (P < 0.05), low CFU‐GM recovery in LTBMC (P < 0.05), and normal primitive stem cells as indicated by the frequency of CD34+/CD38– cells and the number of long‐term culture initiating cells. Patient BM stromal layers exhibited normal myelopoiesis supporting capacity as shown by the CFU‐GM content of irradiated LTBMC recharged with normal CD34+ cells. In addition, patient LTBMC supernatants displayed 20‐fold normal granulocyte colony stimulating factor and 2‐fold normal granulocyte‐macrophage colony stimulating factor levels. Conclusion: These data show that primitive BM stem cells and stromal cells are not affected in SCN patients, while they support further the concept of a primary defect at the myeloid progenitor cell level. To know the differentiation stage at which the underlying defect causes the malfunction will be relevant for further elucidation of its nature at the molecular level.

Anti-thrombopoietin antibodies suppress megakaryocytic colony formation in vitro in patients with systemic lupus erythaematosus

Autoantibodies against thrombopoietin (TPO) have been implicated in systemic lupus erythaematosus (SLE) thrombocytopoenia.1 2 To investigate their pathophysiological significance, we have evaluated the effect of SLE sera on megakaryocytic colony formation in vitro. A total of 35 randomly selected SLE sera were initially screened for the presence of antiplatelet antibodies (APAs), using a commercially available assay (PAK12, GTI Diagnostics, Wauksesha, Wisconsin, USA). In all, 16 sera tested positive and were excluded from the analysis. Only sera negative for APAs have been evaluated in the current study, since APAs have been reported to inhibit megakaryopoiesis in vitro.3 Of the remaining 19 samples, 7 tested positive for anti-TPO antibodies and 12 were negative. TPO concentration was then measured by a …

Increased Frequency and Specific Reactivity of Serum Antinuclear Antibodies in Patients with Nonimmune Chronic Idiopathic Neutropenia of Adults

This study describes the frequency of serum organ-specific and organ-nonspecific autoantibodies in 157 patients with nonimmune chronic idiopathic neutropenia of adults (NI-CINA). Forty-two age- and gender-matched volunteers were used as controls. We found that patients with NI-CINA had increased frequency of antinuclear antibodies (ANA) compared to controls (33.1 vs. 9.5%, p = 0.0025), and that ANA positivity inversely correlated with the number of circulating neutrophils (r = –0.2765, p < 0.0001). Speckled pattern of reactivity was seen in 84.6% of ANA-positive patients, and diffuse pattern in the remaining 15.4%. Patients had also increased levels of circulating immune complexes compared to controls (3.30 ± 2.41 vs. 1.70 ± 1.19 µg/ml, p = 0.0042), which inversely correlated with the number of circulating neutrophils (r = –0.2405, p = 0.0154) but not with the titer of ANA positivity. No significant differences were found between the patients and the normal controls in the frequency of positive tests for antibodies to dsDNA, Sm, nRNP, SSA, SSB and Scl-70 antigens, or for parietal cell antibodies, anti-neutrophil cytoplasmic antibodies (ANCA), anti-cardiolipin and anti-thyroid antibodies. Serum levels of rheumatoid factor, C-reactive protein (CRP) and complement factors C3 and C4 ranged within normal limits in the patients studied, but a highly significant correlation was noted between the levels of CRP and ANA positivity (r = 0.3936, p < 0.0001). These findings are suggestive of a chronic inflammation in NI-CINA patients which provides the antigenic stimulus for ANA production, and they further support our previously reported suggestion for the possible involvement of such a low-grade chronic inflammatory process in the pathogenesis of neutropenia in the affected subjects.