Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 µM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Red blood cells microparticles are associated with hemolysis markers and may contribute to clinical events among sickle cell disease patients.

Microparticles are sub-micron vesicles possessing protein and other materials derived from the plasma membrane of their parent cells, and literature suggests that they may have a role in the pathophysiology and downstream manifestations of sickle cell disease (SCD). The contributions of red blood cells microparticles (RMP) to the pathogenic mechanisms and clinical phenotypes of SCD are largely unknown. There is a controversy as to whether the proportions of intravascular hemolysis (approximately ≤ 30% of total hemolysis) would be enough to explain some complications seen in patients with SCD. We investigated RMP among 138 SCD patients and 39 HbAA individuals. Plasma RMPs were quantified by flow cytometry, plasma hemoglobin and heme by colorimetric assays, and haptoglobin and hemopexin by ELISA. The patients had higher RMP, plasma hemoglobin, and heme compared to the controls. On the contrary, haptoglobin and hemopexin were depleted in the patients. The RMP correlated positively with heme, lactate dehydrogenase, plasma hemoglobin, serum bilirubin, reticulocyte counts, and tricuspid regurgitant jet velocity of the patients. Contrarily, it correlated negatively with HbF, hemopexin, red blood cells counts, hemoglobin concentration, and haptoglobin. Although patients treated with hydroxyurea had lower RMP, this did not attain statistical significance. Patients with sickle leg ulcer and elevated tricuspid regurgitant jet velocity had higher levels of RMP. In conclusion, these data suggest that RMPs are associated with hemolysis and may have important roles in the pathophysiology and downstream complications of SCD.

Rabbit antithymocyte globulin dose does not affect response or survival as first-line therapy for acquired aplastic anemia: a multicenter retrospective study.

In a prospective randomized study, treatment for aplastic anemia (AA) with rabbit antithymocyte globulin (r-ATG) and cyclosporine showed inferior hematological response and survival in comparison to horse antithymocyte globulin (h-ATG) and cyclosporine. However, h-ATG was discontinued in most Asian, South American, and European countries, where r-ATG became the only ATG formulation available. We retrospectively evaluated consecutive patients with acquired AA who received either rabbit (n = 170) or horse (n = 85) ATG and cyclosporine for first-line treatment from 1992 to 2014 in seven referral centers in Brazil and Argentina. Overall response at 3 months was 17% (95%CI, 11-23%) for r-ATG and 44% (95%CI, 33-55%) for h-ATG (p < 0.001). At 6 months, it was 31% (95%CI, 34-39%) for r-ATG and 59% (95%CI, 48-69%) for h-ATG (p < 0.001). Overall survival at 5 years was 57% (95%CI, 47-65%) for r-ATG and 80% (95%CI, 69-87%) for h-ATG (log-rank = 0.001). Relapse was significantly higher in patients receiving h-ATG (28%; 95%CI, 17-43%) as compared to r-ATG (9.4%; 95%CI, 4-21%; log-rank, p = 0.01). The type of ATG was the only factor associated with both response and survival. The r-ATG dose varied from 1 to 5 mg/kg/day, but it did not correlate with outcomes. In summary, this is the largest multicenter study comparing the two ATG formulations in AA. Our results indicate that the dose of r-ATG does not influence hematologic response or survival in first-line therapy for acquired AA. Considering the toxicity and costs of r-ATG, our findings challenge its aggregate benefit to cyclosporine therapy and further strengthen that h-ATG should remain standard therapy in AA.

Coinheritance of Hb Bristol-Alesha [ß67(E11)Val→Met; HBB: c.202G>A] and the α212 Patchwork Allele in a Brazilian Child with Severe Congenital Hemolytic Anemia.

Hb Bristol-Alesha [HBB: c.202G>A; ß 67 Val>Met] is a rare structural variant of hemoglobin (Hb) resulting from a GTG>ATG substitution at codon 67 of the ß-globin gene that leads to the replacement of valine by methionine in the corresponding position of the ß-globin chain. The methionine residue is subsequently modified to aspartic acid [ß67(E11)Val-Met→Asp], possibly by autoxidation mechanisms. This substitution prevents normal non-polar binding of Val67 to the heme group, resulting in molecular instability and severe hemolysis. We identified Hb Bristol-Alesha (in the heterozygous state), as the cause of severe congenital hemolytic anemia in an 11-month-old girl of mixed (native Indian and European) ethnic origin from the Midwestern region of Brazil, whose parents were clinically and hematologically normal. The mutation on the ß-globin gene was found to have been coinherited with the α212 patchwork allele.

Reduced rate of sickle-related complications in Brazilian patients carrying HbF-promoting alleles at the BCL11A and HMIP-2 loci.

The presence of high levels of fetal haemoglobin (HbF) provides well-validated clinical benefits to patients with sickle cell anaemia (SCA). Nevertheless it has been difficult to show clear direct effects of the known genetic HbF modifiers, such as the enhancer polymorphisms for haematopoietic transcription factors BCL11A and MYB, on SCA severity. Investigating SCA patients from Brazil, with a high degree of European genetic admixture, we have detected strong effects of these variants on HbF levels. Critically, we have shown, for the first time, that the presence of such HbF-promoting variants leads to a reduced rate of SCA complications, especially stroke.

An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis.

Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G --> C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.

ARHGAP21 protein, a new partner of α-tubulin involved in cell-cell adhesion formation and essential for epithelial-mesenchymal transition.

Cell-cell adhesions and the cytoskeletons play important and coordinated roles in cell biology, including cell differentiation, development, and migration. Adhesion and cytoskeletal dynamics are regulated by Rho-GTPases. ARHGAP21 is a negative regulator of Rho-GTPases, particularly Cdc42. Here we assess the function of ARHGAP21 in cell-cell adhesion, cell migration, and scattering. We find that ARHGAP21 is localized in the nucleus, cytoplasm, or perinuclear region but is transiently redistributed to cell-cell junctions 4 h after initiation of cell-cell adhesion. ARHGAP21 interacts with Cdc42, and decreased Cdc42 activity coincides with the appearance of ARHGAP21 at the cell-cell junctions. Cells lacking ARHGAP21 expression show weaker cell-cell adhesions, increased cell migration, and a diminished ability to undergo hepatocyte growth factor-induced epithelial-mesenchymal transition (EMT). In addition, ARHGAP21 interacts with α-tubulin, and it is essential for α-tubulin acetylation in EMT. Our findings indicate that ARHGAP21 is a Rho-GAP involved in cell-cell junction remodeling and that ARHGAP21 affects migration and EMT through α-tubulin interaction and acetylation.

The Cratylia mollis seed lectin induces membrane permeability transition in isolated rat liver mitochondria and a cyclosporine a-insensitive permeability transition in Trypanosoma cruzi mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 µM Ca(2+) was significantly decreased by 50 µg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 µM Ca(2+) this lectin, at 50 µg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Viability of umbilical cord blood mononuclear cell subsets until 96 hours after collection.

BACKGROUND: Umbilical cord blood (UCB) is a good source of hematopoietic stem cells for transplantation and cell therapy. In 2006, the Brazilian Public Network of Cord Blood Banks was founded; however, because our country is large, logistic problems could hamper the collection of numerous samples. Our aim was to evaluate the viability of several UCB cell subsets until 96 hours after collection, to examine whether this delay would be acceptable for processing and freezing the samples. STUDY DESIGN AND METHODS: Two experiments were performed: in the first one, volume reduction of the UCB units was carried out before analysis. In the second one, analysis was carried out with no previous manipulation. Samples were stored at room temperature and one aliquot was taken daily for analysis. We examined CD34+ cell, B-cell precursor, mature B and T lymphocyte, monocyte, granulocyte, and mesenchymal stem cell (MSCs) concentrations. RESULTS: Thirty-six UCB units were analyzed. CD34+ cells and mature T lymphocytes increased (viability 99%). Mature B lymphocytes and MSCs decreased, maintaining viability. Granulocytes decreased with loss of viability. Monocytes and immature B lymphocytes remained stable. Clonogenic assays showed a decrease in colony-forming unit (CFU) number in UCB units stored for 96 hours. CONCLUSION: UCB manipulation did not influence cell viability. All cell subsets remained viable until 96 hours after collection. CD34+ cells and T lymphocytes increased, probably due to the loss of other subsets. CFU growth during the period analyzed and confirmed stem cell functionality, despite the decrease at 96 hours. Results demonstrated that UCB units could probably be processed up to 96 hours after collection.

PML/RARa leukemia induced murine model for immunotherapy evaluation.

Even though leukemia murine models are valuable tools for new drug therapy studies, most of these models consist of immunocompromised mice, which do not exhibit immune responses. In order to obtain an adequate leukemia model, we established an acute promyelocytic leukemia transplantation-based model (PML/RARa) in immunocompetent BALB/c mice, thus making it possible to study drug-induced cellular immune responses in leukemia. The development of PML/RARa leukemia was confirmed by leukocytosis (76.27 ± 21.8 vs. 3.40 ± 1.06; P < 0.0001), anemia (7.46 ± 1.86 vs. 15.10 ± 0.96; P < 0.0001), and thrombocytopenia (131.85 ± 39.32 vs. 839.50 ± 171.20; P < 0.0001), and the presence of blasts in the peripheral blood of mice (approximately 50% blasts; P < 0.0001), 15 days after the transplants. These findings were corroborated through differential counts, flow cytometry, and in vivo imaging, which indicated increased number of immature cells in the bone marrow (15.75 ± 3.30 vs 6.69 ± 0.55; P < 0.001), peripheral blood (7.88 ± 2.67 vs 1.22 ± 0.89; P < 0.001), and spleen (35.21 ± 4.12 vs 1.35 ± 0.86; P < 0.0001), as well as promyelocytes in the bone marrow (41.23 ± 4.80 vs 5.73 ± 1.50; P < 0.0001), peripheral blood (46.08 ± 7.52 vs 1.10 ± 0.59; P < 0.0001) and spleen (35.31 ± 8.26 vs 2.49 ± 0.29; P < 0.0001) of PML/RARa mice. Compared to basal conditions of untransplanted mice, the PML/RARa mice exhibited frequencies of T lymphocytes CD4 helper = 14.85 ± 2.91 vs 20.77 ± 2.9 in the peripheral blood (P < 0.05); 12.75 ± 1.33 vs 45.90 ± 2.02 in the spleen (P < 0.0001); CD8 cytotoxic = 11.27 ± 3.44 vs 11.05 ± 1.22 in the peripheral blood (P > 0.05); 10.48 ± 1.16 vs 30.02 ± 1.80 in the spleen (P < 0.0001); natural killer (NK) cells = 3.68 ± 1.35 vs 6.84 ± 0.52 in the peripheral blood (P < 0.001); 4.43 ± 0.57 vs 6.40 ± 1.14 in the spleen (P < 0.05); B cells 2.50 ± 0.60 vs 15.20 ± 5.34 in the peripheral blood (P < 0.001); 17.77 ± 4.39 vs 46.90 ± 5.92 in the spleen (P < 0.0001); neutrophils = 5.97% ± 1.88 vs 31.57 ± 9.14 (P < 0.0001); and monocytes = 6.45 ± 2.97 vs 15.85 ± 2.57 (P < 0.001), selected as classical (3.33 ± 3.40 vs 57.80 ± 16.51, P < 0.0001), intermediate (57.42 ± 10.61 vs 21.75 ± 5.90, P < 0.0001), and non-classical monocytes (37.51 ± 10.85 vs 18.08 ± 7.13, P < 0.05) in the peripheral blood; and as classically activated (M1) within in the bone marrow (3.70 ± 0.94 vs 1.88 ± 0.39, P < 0.05) and spleen 15.19 ± 3.32 vs 9.47 ± 1.61, P < 0.05), in addition to alternatively activated (M2) macrophages within the bone marrow (23.06 ± 5.25 vs 1.76 ± 0.74, P < 0.0001) and spleen (46.51 ± 11.18 vs 30.58 ± 2.64, P < 0.05) compartments. All-trans retinoic acid (ATRA) treatment of PML/RARa mice reduced blast (immature cells) in the bone marrow (8.62 ± 1.81 vs 15.76 ± 1.25; P < 0.05) and spleen (8.75 ± 1.31 vs 35.21 ± 1.55; P < 0.0001) with no changes in the peripheral blood (10.13 ± 3.33 vs 7.88 ± 1.01; P > 0.05), as well as reduced promyelocytes in the bone marrow (19.79 ± 4.84 vs 41.23 ± 1.81; P < 0.05), peripheral blood (31.65 ± 3.92 vs 46.09 ± 2.84; P < 0.05) and spleen (24.84 ± 2.03 vs 41.46 ± 2.39; P < 0.001), and increased neutrophils of the peripheral blood (35.48 ± 7.24 vs 7.83 ± 1.40; P < 0.05) which was corroborated by reducing of immature cells and increase of neutrophil in the stained smears from PML/RARa mice, thus confirming that this model can be used in drug development studies. Our results show the effective induction of PML/RARa leukemia in BALB/c mice, thus producing a low-priced and reliable tool for investigating cellular immune responses in leukemia.

Cytoprotective effect of Fridericia chica (Bonpl.) L.G. Lohmann extract associated with geranylgeraniol enriched-fraction from Bixa orellana L. on epithelial cells treated with bisphosphonate.

Bisphosphonates are drugs used to treat bone disorders. The chronic use of bisphosphonates is associated with the occurrence of medication-related osteonecrosis of the jaw (MRONJ). Previous data reported the positive effects of Geranylgeraniol on different cell types treated with Bisphosphonates. Foregoing work done by our research group demonstrated the wound healing capacity of Fridericia chica (Bonpl.) L.G.Lohmann standardized ethanol extract. Herein in vitro cytoprotective synergistic effect of the association of F. chica extract associated with an enriched geranylgeraniol fraction on keratinocytes exposed to zoledronic acid is reported. An association of F. chica at 1 and 5 µg/mL with geranylgeraniol at 15 µg/mL, increased cell viability by 73.5% and 71.1%, respectively. This treatment did not increase tumor cells viability; whereas the clonogenic potential assessment showed that, the association with F. chica (5 µg/mL) reversed the effects of zoledronic acid on the cells. This study provides data for a potential treatment for MRONJ.

Elevated plasma levels and platelet-associated expression of the pro-thrombotic and pro-inflammatory protein, TNFSF14 (LIGHT), in sickle cell disease.

Chronic vascular inflammation and endothelial activation may initiate vaso-occlusion in sickle cell disease (SCD). TNFSF14 (CD258; LIGHT), a recently-identified pro-thrombotic and pro-inflammatory tumour necrosis factor (TNF)-superfamily cytokine, has a potent activating effect on endothelial cells. We evaluated whether TNFSF14 production is altered in SCD and whether platelets contribute to this production. TNFSF14 was measured in platelet-free plasma from healthy-control individuals (CON), steady-state sickle cell anaemia (SCA), SCA on hydroxycarbamide therapy (SCAHC) and haemoglobin SC (HbSC) patients. Mean plasma TNFSF14 was significantly increased in SCA, SCAHC and HbSC, compared to CON individuals. In SCA/SCAHC patients, plasma TNFSF14, showed no correlation with haematological variables, but was significantly correlated with serum lactate dehydrogenase and inflammatory markers (CD40LG , IL8 and ICAM1). Platelet-membrane TNFSF14 expression was significantly augmented on SCA platelets, and correlated with platelet activation; furthermore, measurement of platelet TNFSF14 release indicated that platelets may be a major source of circulating TNFSF14 in SCA. Interestingly, high plasma TNFSF14 was significantly associated with elevated tricuspid regurgitant velocity (≥2·5 m/s) in a population of SCA/SCAHC patients. The pro-inflammatory and atherogenic cytokine, TNFSF14, could contribute to endothelial activation and inflammation in SCA; future investigations may confirm whether this protein contributes to major clinical complications of the disease, such as pulmonary hypertension, and represents a potential therapeutic target.

Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin.

BACKGROUND: Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis. DESIGN AND METHODS: Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays. RESULTS: Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils. CONCLUSIONS: The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.

Inhibition of phosphodiesterase 9A reduces cytokine-stimulated in vitro adhesion of neutrophils from sickle cell anemia individuals.

OBJECTIVE: Leukocyte adhesion to vessel walls may initiate vaso-occlusion in sickle cell anemia (SCA); however, the extent to which inflammation participates in this mechanism is not understood. This in vitro study investigated whether inflammatory molecules, commonly augmented in SCA, can affect neutrophil adhesive properties and whether cyclic guanosine monophosphate (cGMP)-elevating agents can inhibit such adhesion. SUBJECTS AND METHODS: Effects of Interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines, BAY 73-6691 [phosphodiesterase (PDE)-9A-inhibitor], and BAY 41-2271 (guanylate-cylase stimulator) on the adhesive properties of neutrophils from healthy control (CON) and steady-state SCA individuals were determined using static-adhesion assays. RESULTS: SCA neutrophils demonstrated increased adhesive properties, compared to CON neutrophils; IL-8, TNF-α and GM-CSF increased CON neutrophil adhesion and further increased SCA neutrophil adhesion to fibronectin (FN). The PDE9A inhibitor, BAY-73-6691, significantly reduced basal CON neutrophil and SCA neutrophil adhesion; this was accompanied by decreased SCA neutrophil surface expressions of the L-selectin and CD11b adhesion molecules. BAY-73-6691 also significantly reduced cytokine-stimulated CON neutrophil and SCA neutrophil adhesion to FN; however, this was not accompanied by alterations in adhesion-molecule presentation. CONCLUSIONS: The chronic inflammatory nature of SCA may contribute to leukocyte adhesive functions in SCA. Furthermore, elevation of leukocyte cGMP may be an interesting approach for inhibition of leukocyte adhesion to the vessel wall, even in the presence of inflammatory stimuli.

3D Scaffolds to Model the Hematopoietic Stem Cell Niche: Applications and Perspectives.

Hematopoietic stem cells (HSC) are responsible for the production of blood and immune cells during life. HSC fate decisions are dependent on signals from specialized microenvironments in the bone marrow, termed niches. The HSC niche is a tridimensional environment that comprises cellular, chemical, and physical elements. Introductorily, we will revise the current knowledge of some relevant elements of the niche. Despite the importance of the niche in HSC function, most experimental approaches to study human HSCs use bidimensional models. Probably, this contributes to the failure in translating many in vitro findings into a clinical setting. Recreating the complexity of the bone marrow microenvironment in vitro would provide a powerful tool to achieve in vitro production of HSCs for transplantation, develop more effective therapies for hematologic malignancies and provide deeper insight into the HSC niche. We previously demonstrated that an optimized decellularization method can preserve with striking detail the ECM architecture of the bone marrow niche and support HSC culture. We will discuss the potential of this decellularized scaffold as HSC niche model. Besides decellularized scaffolds, several other methods have been reported to mimic some characteristics of the HSC niche. In this review, we will examine these models and their applications, advantages, and limitations.

Increased adhesive properties of platelets in sickle cell disease: roles for alphaIIb beta3-mediated ligand binding, diminished cAMP signalling and increased phosphodiesterase 3A activity.

Whilst high pro-coagulant activity is reported in sickle cell disease (SCD), the precise role of platelets (PLTs) in SCD inflammatory and vaso-occlusive processes is unclear. Adhesion of PLTs from healthy controls (CON), SCD individuals (SCD) and SCD patients on hydroxycarbamide (SCDHC) to fibrinogen (FB) was compared using static adhesion assays. PLT adhesion molecules and intraplatelet cyclic adenosine monophosphate (icAMP) were observed by flow cytometry and enzyme-linked immunosorbent assay. SCD-PLTs demonstrated significantly greater adhesion than CON-PLTs to FB. Participation of the alpha(IIb)beta(3)-integrin in SCD-PLT adhesion was implicated by increased alpha(IIb)beta(3) activation and data showing that an alpha(IIb)beta(3)-function-inhibiting antibody significantly diminished SCD-PLT adhesion to FB. Platelet activation was potentiated by reductions in icAMP; cAMP levels were decreased in SCD-PLTs, being comparable to those of thrombin-stimulated CON-PLTs. Furthermore, SCD-PLT adhesion to FB was significantly reduced by cilostazol, an inhibitor of cAMP-hydrolyzing phosphodiesterase 3A (PDE3A). Both alpha(IIb)beta(3)-integrin activation and icAMP correlated significantly with fetal haemoglobin in SCD. Accordingly, hydroxycarbamide therapy was associated with lower PLT adhesion and higher icAMP. SCD-PLTs may be capable of adhering to proteins encountered on the inflamed vascular wall and, potentially, participate in vaso-occlusive processes. Hydroxycarbamide and, speculatively, nitric oxide donor or cyclic-nucleotide-targeted therapies may aid in the reversal of PLT adhesive properties in SCD.

CXCR4hi effector neutrophils in sickle cell anemia: potential role for elevated circulating serotonin (5-HT) in CXCR4hi neutrophil polarization.

Leukocyte recruitment and heterocellular aggregate formation drive the inflammatory vaso-occlusive processes associated with sickle cell anemia (SCA). We characterized neutrophils in a population of patients with SCA and investigated whether platelet-derived molecules can induce phenotypic alterations in this cell type. Imaging flow cytometry analysis demonstrated that the frequency of circulating CXCR4hi neutrophils was significantly higher in steady-state SCA individuals than in healthy control individuals and that these cells presented increased CD11b activation and toll-like receptor-4 expression. SCA neutrophils display increased neutrophil-platelet aggregation, and CXCR4hi neutrophils demonstrated augmented neutrophil-platelet aggregate frequency with a higher mean number of platelets adhered per neutrophil. Importantly, incubation of neutrophils with platelets significantly elevated their CXCR4 expression, while SCA plasma was found to induce CXCR4hi neutrophil polarization significantly more than control plasma. SCA individuals had significantly increased plasma levels of serotonin (5-HT), and serotonin molecule and SCA plasma induced neutrophil CXCR4 expression in a serotonin-receptor-dependent manner. Thus, the augmented CXCR4hi neutrophil population may contribute to mechanisms that promote vaso-occlusion in SCA; furthermore, circulating serotonin, derived from platelet activation, may play a role in the polarization of neutrophils, suggesting that serotonin-receptor antagonists or serotonin reuptake inhibitors could represent therapeutic approaches to reduce neutrophil activation in SCA.

PIP4KIIA and beta-globin: transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease.

We are reporting here the results of differential gene expression experiments comparing two siblings, a 21-yr-old male and a 19-yr-old female, with the same alpha-thalassemia genotype (-alpha(3.7)/(--SEA)) and quite different levels of Hb H in the peripheral blood (18.7 and 5%, respectively). By using mRNA differential-display reverse-transcription-PCR and suppression subtractive hybridization, two main transcripts were selected in both procedures and validated by qRT-PCR, one corresponding to the phosphatidylinositol phosphate 4-kinase type II-alpha (PIP4KIIA) gene and the other to the beta-globin gene, both over expressed in the patient with the higher percentage of Hb H. Type II PIP kinases produce phosphatidylinositol 4,5 biphosphate, a critical and pleiotropic regulatory molecule involved in diverse cellular activities, including gene expression. Our results suggest that PIP4KIIA may be one of the factors related to the regulation of the beta-globin gene expression and the different levels of Hb H in alpha-thalassemic patients.

High levels of human gamma-globin are expressed in adult mice carrying a transgene of the Brazilian type of hereditary persistence of fetal hemoglobin ((A)gamma -195).

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by increased levels of Hb F during adult life. Nondeletional forms of HPFH are characterized by single base mutations in the (A)gamma and (G)gamma promoters, resulting in an increase of Hb F ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the (A)gamma -195 (C>G) mutation that causes the Brazilian type of HPFH (HPFH-B). To better understand this mechanism, we have developed HPFH-B transgenic mice. mRNA levels of human gamma-globin of -195 transgenic mice were clearly higher when compared with control transgenic mice bearing a wild type sequence of the gamma promoter. Thus, our data indicate that the -195 mutation is the unique cause of elevation of Hb F in Brazilian HPFH. These results could provide us with an opportunity to study the modifying effects of the Hb F in the phenotype of sickle cell disease and beta-thalassemia (beta-thal).