Objective: There are few studies regarding vitamin B 12 deficiency in developing countries. In Brazil, a late diagnosis of vitamin B 12 deficiency progressing to severe neurological damage is common. Thus, the aim of the present study was to verify the frequency of vitamin B 12 deficiency in two Brazilian populations (elderly and adult participants) and to compare different methods of vitamin B 12 deficiency detection. Design: Five hundred participants were recruited from health centres from south-east Brazil and were separated into two groups: 60 years old or more and 30-59 years old. Vitamin B 12 and folate concentrations were measured using electrochemiluminescence immunoassay (ECI) and RIA. Methylmalonic acid (MMA) was measured by LC coupled to tandem MS. Full blood counts were acquired using standard methods. Results: All participants had normal blood count results and mean cell volume less than 99 fl; none of them presented folate deficiency according to the results, which were all greater than 3 ng/ml. Cobalamin levels less than 200 pmol/l were identified by one of the two or by both methods in 7?2 % of the participants aged 60 years or more and 6?4 % of the participants aged 30-59 years. MMA levels were higher in older subjects (P 5 0?007) compared with younger subjects. A greater correlation of MMA v. RIA was observed than of MMA v. ECI (P 5 0?0017 v. P 5 0?014). MMA quantification estimated that cobalamin deficiency was present in more than 11 % of the subjects for both studied groups. Conclusions: The study shows that vitamin B 12 deficiency is frequent in Brazilian adults and suggests that RIA is more sensitive than ECl for measuring cobalamin levels.
The sonic hedgehog (Shh) pathway is a regulatory network involved in the development of cancer. The Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) bind to the receptors Patched1 and Patched2, resulting in the inhibition of their repression on Smoothened (Smo). Smo is a seven-transmembrane protein, which inhibits Suppressor of Fused (SUFU), resulting in the stabilization and activation of the Gli family of transcription factors, followed by a transcriptional response to the “canonical Hh signaling”. SUFU is a negative regulator of the Hedgehog signaling pathway, through the restriction of Gli activity through cytoplasmic sequestration. Germline mutations in SUFU are involved in medulloblastoma. Dysregulation of Shh signaling has been reported to be involved in several malignancies, including chronic myeloid leukemia. To our knowledge, however, only one study has been carried out in myelodysplastic syndrome (MDS) and acute leukemia so far, and the role of this pathway in leukemogenesis is still unknown. Recently we showed an increased protein expression of Sonic and Dessert Hedgehog in addition to increased mRNA levels of PTCH and SMO in MDS bone marrow. Here, we investigated whether this increase was capable of activating Hedgehog pathway in total and in progenitor bone marrow cells. Bone marrow (BM) samples were collected from 39 MDS (25 low-risk, 14 high-risk, according WHO 2008) patients and 26 healthy donors. CD34+ cells were isolated from peripheral blood of 9 healthy donors and 20 (12 low-risk and 8 high risk) MDS patients. Relative expressions of PTCH, SMO, SUFU and GLI1 were obtained by Real time-PCR. Increased expression observed in ligands and receptors were not sufficient to upregulate the activity of Hedgehog pathway in MDS total bone marrow. The downstream effector GLI1 was under expressed in the disease as median [min-max] as follows: healthy donors 1.04[0.02-4.01]; low risk MDS 0.21[0.03-2.26]; high risk MDS 0.07[0.03-1.48] healthy donors vs low risk p=0.007; healthy donors vs high risk p=0.006. Furthermore, SUFU expression was similar in healthy donors 3.3[0.35-7.84] and in myelodysplastic (low risk 3.76[0.75-13, 2] and high risk 1.8[0.58-16.4]) bone marrows. Different from total bone marrow, the receptors Patched and SMO showed decreased expression in MDS CD34+ cells: Patched healthy donors 2.24[0.74-4.36]; low risk 0.6[0.1-3.34]; high risk 0.98[0.29-1.22] and SMO healthy donors 1.18[0.62-2.16]; low risk 0.79[0,11-1.87]; high risk 0.86[0.37-1.02]; healthy donors vs high risk MDS p=0.049. SUFU was overexpressed (healthy donors 1.44[0.75-2.39]; low risk 2.77[0.93-4.11]; high risk 1.68[1.07-3.43]; healthy donors vs low risk MDS p=.005). However, myelodysplastic progenitors had GLI1 overexpression (healthy donors 1.09[0.6-2.91]; low risk 2.93[0.75-14.52]; high risk 1.32[0.71-2.58]; healthy donors vs low risk p=0.03), indicating an activation of the Shh pathway in MDS progenitors. Recently, Kobune et al (Blood Cancer J. 2012 Sep 7) showed activation of Hedgehog signaling in primary CD34+ blasts from acute myeloid leukemia and MDS, as those cells expressed IHh, PTCH, SMO, GLI1 and GLI. Taken together, these results and ours led us to conclude that the Hedgehog pathway is activated in MDS. Since Shh pathway is being explored as a new opportunity for targeted therapies against tumors, our study suggests that the inhibition of this pathway could be strategic in the control of leukemic stem cell and MDS treatment. Disclosures: No relevant conflicts of interest to declare.
The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.
3817 Background: The Hedgehog pathway has an important role in self-renew of normal and leukemic stem cells and is upregulated in myeloid leukemias, however there are no studies regarding the Hedgehog pathway in myelodysplastic syndromes (MDS). Hedgehog ligands (Sonic hedgehog [SHh], Indian hedgehog [IHh], and Desert hedgehog [DHh]) are produced by stromal cells and bind to the receptor Patched (PTCH). This binding causes activation of Smoothened (SMO) receptor, resulting in downstream transcription of target genes. Aim: To evaluate Hedgehod pathway components in MDS. Methods: Bone marrow (BM) samples were collected from 39 MDS (25 low risk, 14 high risk-WHO 2008) patients and 26 healthy donors (HD). Relative expressions of PTCH and SMO were obtained by Real Time PCR. For immunohistochemistry, BM biopsies were collected from 21 MDS (17 low risk and 4 high risk) patients and 7 megaloblastic anemia (MA) were used as control. The BM sections were stained with antibodies for DHh, SHh and c-Kit and the percentage of stained nucleated cells was based on an average of 4 high-powered fields. Results: Hedeghog receptors PTCH and SMO were overexpressed in MDS BM cells compared to normal BM as follows; PTCH: [median (min-max)] healthy donors= 2.23 (0.42–9.22); low risk= 6.09 (0.41–25.28); high risk= 3.97 (0.42– 21.01); HD vs low risk and HD vs high risk p= 0.02; SMO: healthy donors= 2.33 (0.00–16.11); low risk= 14.79 (1.89– 41.93); high risk= 34.44 (8.04– 164.28)]; HD vs low risk and HD vs high risk p<0.001. Immunohistochemistry assays showed a higher expression of Hedgehog ligands in MDS bone marrow compared to control. For DHh, the number of stained cells in low risk MDS were higher than in MA [MA = 102(84 –183), low risk= 196(69 – 317) high risk 159(131 – 236)]; MA vs low risk p=0.018 and MA vs high risk p= 0.11. Furthermore, SHh staining was 2 and 2.5 fold higher in low and high risk MDS, respectively, compared to MA [MA= 47(18 – 74), low risk 98.25(53 – 129), high risk 116.25 (93 – 125)] MA vs low risk p= 0.001, MA vs high risk p=0.01. Similar to SHh, c-Kit staining was higher in MDS cases as follows: MA= 95.3(61 – 114), low risk= 106(57–136), high risk= 95.3(61 – 114). Interestingly, a high correlation was found between c-kit and DHh (p=0.002, r=0.63) or c-kit and SHh (p=0.001, r=0.7). Conclusion: To our knowledge, this is the first study of the Hedgehog pathway in MDS. We observed overexpression of Hedgehog receptors and ligands. Furthermore, there is a correlation between these ligands and a marker for hematopoietic stem/progenitors cells. According to these data, we propose that deregulation of Hedgehog in MDS may occur in abnormal progenitors found in MDS bone marrow. Disclosures: No relevant conflicts of interest to declare.
The main signaling pathway involved in migration, adhesion and homing of hematopoietic progenitor cells (HPC) is the CXCL-12, chemokine produced by bone marrow (BM) stromal cells that bind to its main receptor CXCR-4, expressed by HPC. ARHGAP21 is a RhoGAP member, negative regulators of RhoGTPAses. It was described that ARHGAP21 is involved in cellular migration and adhesion but its role in hematopoiesis is unknown. Researching Arhgap21 role in hematopoiesis, Arhgap21+/- mice were generated using Embryonic Stem cell obtained from GeneTrap consortium. Hematologic parameters were investigated by blood cell count, colony formation assay in methylcellulose medium and immunophenotipic characterization of hematopoietic populations. Transwell migration and adhesion of Lin- cells assay in the presence of CXCL12 were also performed. In vivo analysis of Arhgap21+/- HPC was achieved with homing and CFU-S assays in sublethally irradiated mice and hematopoiesis stress was generated by 5-fluoracil treatment. Arhgap21+/- mice presented more than 50% reduction in Arhgap21 expression (0.47 ± 0.13) in BM when compared to WT (1 ± 0.03) and live in normal conditions. On the other hand, Arhgap21 mice showed a reduction in BFU-E colonies and erythroid (Terr119+) committed cells in BM together with decreased peripheral blood (PB) Terr119+ cells and red blood cell number and increased medium corpuscular volume. In myeloid compartment, Arhgap21+/- mice presented less GM colonies and myeloid (Gr1/Mac1+) cells in BM followed by an increase of these cells in PB suggesting myeloid mobilization. Corroborating to this, whenArhgap21+/- BM was challenged for hematopoiesis stress with 5FU, the animals showed increased neutrophil number in peripheral blood 14 days (WT: 1887/mm3 ±721.9; Arhgap21+/-:3325/mm3 ±1640, p=0.02) and 21days (WT: 1264/mm3 ±635; Arhgap21+/-: 2182/mm3 ±854, p=0.01) after treatment, together with increased number of LSK cells in the BM (WT: 1.3% ±0.4; Arhgap21+/-: 1.9% ±0.3, p=0.006) 28 days after 5FU infusion. Erythroid and myeloid compartments reductions were observed together with an increase of Arhgap21+/- BM short term LSK( Arhgap21+/- : 0.34% ±0.13; WT: 0.18% ±0.07, p=0.03) and long term LSK (Arhgap21+/-: 0.003% ±0.001; WT: 0.002 ±0.0007, p=0.02) suggesting an attempt to restore normal hematopoietic levels. Arhgap21+/- HPC showed reduced CXCL-12-induced migration compared to WT (WT: 100%, Arhgap21+/-: 54.73% ± 13.57, p=0.01) in addition to decreased adhesion to fibronectin (Arhgap21+/-: 15% ± 3.8; WT: 27% ± 3.5, p=0.003) and a4b1 integrin expression (WT: 83.76 ± 4.35; Arhgap21+/-: 71.06 ± 7.00, p=0.008). In homing assay, the percentage of donor Lin- HPC from Arhgap21+/- mice that homed to BM (6.32 ± 2.41) or spleen (3.48 ± 1.57), were lower than those from WT mice (BM=10.09 ± 1.81;p=0.004; spleen=6.9 ± 1.48; p=0.0007) together with higher frequency of these cells in peripheral blood (WT: 6.39 ± 3.38; Arhgap21+/-: 13.95 ± 5.33, p=0.003), suggesting a retention of Arhgap21+/- HPC in the bloodstream, which inefficiently home to BM and spleen. Arhgap21+/- CFU-S capacity decline was also observed (Arhgap21+/-: 20.86 ± 2; WT: 29.29 ± 5.4 p=0.002). This is the first study showing that ARHGAP21 is involved in hematopoiesis and also shows that ARHGAP21 is an important protein for chemotaxis, adhesion, homing and short term reconstitution of HPC. Table. Hematological Parameters WT Arhgap21+/- Hemoglobin (g/dL) * 16.19 ± 0.35 15.62±0.32 RBC (10^6/uL) * 10.91 ± 0.3 10.46±0.25 Hematocrit (%) 54.78 ± 1.6 53.75± 1.3 MCV (fL) * 50 ±0.4 50.08±0.74 WBC (10^3/Ul) * 10.08 ±2 12.9±1.8 Platelets(10^3/uL) 1793 ±114.3 1770±96.5 Myeloid, Gr1+Mac1+ in BM (%) * 47.27 ±4.7 40.88 ±6.23 Erythroid, Terr119+ in BM (%) * 9.98 ±4.1 6.4 ±1.5 Short Term HSC, LSK in BM (%) * 0.18 ±0.07 0.34 ±0.13 Long Term HSC, LSK CD150+CD48- in BM (%) * 0.002 ±0.0007 0.003 ±0.001 Myeloid, Gr1+Mac1+ in PB (%) * 7.7 ±1.5 13.1 ±2.6 Erythroid, Terr119+ in PB (%) * 38.7 ±4.4 28.4 ±8.7 Myeloid Colonies, CFU-GM (number) 66.7 ±14.7 48.07 ±12.5 Erythroid Colonies, BFU-E (number) 5.5 ±2.2 3.2 ±1.5 Blood samples were collected from 9 WT and 8 Arhgap21+/- mice. RBC: red blood cell count; MCV: mean cell volume; WBC: White blood cell count. Hematopoietic populations were analyzed in flow cytometry and colony formation in methylcellulose assay. Data are expressed as mean ± standard deviation, analyzed by Student t test and considered statistically significant (*) if p ≤ 0.05. Disclosures No relevant conflicts of interest to declare.
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