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The aim of this study was to perform an immunohistochemical analysis from 100 megakaryocytes per sample, analyzing positivity and intensity levels of anti-LAP human TGF-β1 (or Latent TGF-β1) and anti-TGF-β1 (or Active TGF-β1) antibodies from 18 essential thrombocythemia (ET) and 38 primary myelofibrosis (PMF) patients (being 19 pre-fibrotic and 19 fibrotic). Six bone marrow donor biopsies were used as controls. Fibrosis in bone marrow biopsies (BMB) was evaluated according to the European Consensus. The average fibrosis grade differed between each group (P=0.001 or P=0.003). Latent TGF-β1 values differed significantly between pre-fibrotic (P=0.018) and fibrotic (P=0.031) groups when compared with the control group. The high immunoexpression level of Latent TGF-β1 in the megakaryocytes from patients with myelofibrosis, which was not observed in patients with essential thrombocythemia, may be associated with the development of bone marrow fibrosis.

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Experiments were undertaken to assess the role of amifostine in the activation of latent TGF␤1 and in the smad proteins cascade (smad 2/3, smad4, smad7), focusing on megakaryocytes, in the bone marrow irradiated in vivo. Non-irradiated megakaryocytes were negative for active TGF␤1. Immunopositivity to active TGF␤1 was detected in megakaryocytes 10 days after irradiation in amifostine-treated and untreated marrows. Smad 2/3 and smad 4 were strongly positive in the nucleus of megakaryocytes 10 days after irradiation. At the same time, a predominant hypocellular bone marrow with foci of hematopoiesis was observed with few megakaryocytes. An increase in the number of reticulin fibers was also seen. In amifostine-treated marrows, smad 2/3 and smad4 were not detected in the nucleus but were positive in the cytoplasm of megakaryocytes 10 days after irradiation. Coincidentally, bone marrows were cellular with megakaryocytes. Smad7 immunoexpression was detected in the cytoplasm of megakaryocytes in the non-irradiated, amifostine-treated and in the irradiated, amifostine-treated marrows. Data indicate that amifostine does not prevent latent TGF␤1 activation in irradiated megakaryocytes. While TGF␤1 signal transduction occurs in megakaryocytes in untreated bone marrows, it is inhibited in megakaryocytes in amifostine-treated marrows due to the induction of smad 7 activation. This is the first report showing smad 7 activation by amifostine. Our results also suggest a role for TGF␤1 as an inhibitor of megakaryocytes in vivo. Am. J. Hematol. 71:143-151, 2002.

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Background: The mobilization the Peripheral Blood Stem Cells (PBSC) from bone marrow (BM) to peripheral blood (PB) is a complex process yet. This process is related to mobilization growth factor and/or chemotherapy protocol and disease status. CXCR4, receptor of SDF-1 and VLA-4 searched in primitive CD34+ and CD34+Thy+ cells have shown some functions in migration of CD34+ cells. Marrow disease infiltration and grade of fibrosis seem to be important features related to CD34+ cells trafficking from BM to PB. The objective of this study was to compare the cellularities, degrees of disease infiltration, and grades of fibrosis of hematopoietic stem cells (HSCs) in BM biopsies before and after mobilization and to correlate the results with the success of the mobilization in patients with hematologic malignancies and in donors. Patients and Methods: Fifty-nine patients and 21 PB stem cell (PBSC) donors participated in this prospective study between January/2003 and May/2006. The patients included 21 with non-Hodgkin lymphoma (NHL), 21 with multiple myeloma (MM), 10 with Hodgkin lymphoma (HL), and 7 with leukemia (5 AML, 1 ALL, and 1 CLL). The mobilization regimen consisted of 5 g/m2 of cyclophosphamide in 29 patients (21 MM, 4 NHL, 3 HL, and 1 CLL), DHAP in 8 (7 NHL and 1 NHL), ICE in 14 (9 NHL and 5 HL), high-dose Ara-C in 5 (all with AML), and G-CSF in 24 (21 donors, 1 NHL, 1 HL, and 1 ALL). The mean numbesr for previous chemotherapy cycles were 11, 10, 6, and 5 for NHL, HL, MM, and leukemia, respectively. We collected PBSC using a Spectra-Cobe device (Cobe, Lakewood, CO, USA). Large-volume leukoapheresis (LVL) was used in all cases. Mobilization success was defined as > 8 × 106 CD34+ cells/L in PB before collection, and adequate LVL yield was defined as 2 × 106 CD34+ cells/kg in LVL products. Overall BM and WBC precursor cellularities were visually assessed as normal (20–50%), decreased (<20%), and increased (> 50%). Fibrosis was graded as absent, slight (grades 1/2), or severe (grades 3/4). The clonal patterns of infiltration were confirmed by immunohistochemicaly analyses using monoclonal antibodies. Results: Eighty proposals (59 patients and 21 donors) were presented for mobilization. There were 49 men and 31 women; the median age and mean duration of disease at time of mobilization were 40.3 years (range 25–68 years) and 15.9 months (range 10.3–20.1 months). Eighteen patients were complete responders, 37 were partial responders, and 4 had refractory disease. The median duration of G-CSF usage was 5 days. Mobilization failed in 17 patients (2 MM, 8 NHL, 3 HL, and 4 leukemia) and 1 donor. The mean numbers of CD+34 cells/μL in the PB at time of LVL were 110 in MM (1.1–647), 64.5 in NHL (0–211), 29.3 in HL (2–80), and 16.2 in leukemia (1.3–65). The mean yield of PBSC × 106/Kg in LVL were 15.3 in MM (1.1–85.5), 9.4 in NHL (2.4–26.6), 6.3 in HL (1.3–14.1), 6.1 in donors (1.9–11.9), and 5.0 in leukemia (2.9–7.3). There were correlations between successful mobilization and high overall cellularity pre- and post-mobilization (p=0.015 and p=0.002, respectfully) and successful mobilization and high WBC precursors post-mobilization (p=0.009). Conclusion: BM biopsy is a method important to re-statement at pre transplant, should be continue searched by overall and WBS precursor’s celularities, fibrosis and clonal infiltration before mobilization.