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.

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.

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.

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.

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.

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.

S100A8 acts as an autocrine priming signal for heme-induced human Mϕ pro-inflammatory responses in hemolytic inflammation.

Intravascular hemolysis, in addition to reducing red cell counts, incurs extensive vascular inflammation and oxidative stress. One product of hemolysis, heme, is a potent danger associated molecular pattern (DAMP), activating leukocytes and inducing cytokine expression and processing, among other pro-inflammatory effects. We explored pathways by which heme-induced inflammation may be amplified under sterile conditions. Incubation of human Mϕs, differentiated from CD14+ cells, with heme induced time- and concentration-dependent gene and protein expression of S100A8, a myeloid cell-derived alarmin. Human Mϕ stimulation with recombinant S100A8, in turn, induced robust pro-IL-1ß expression that was dependent upon NF-κB activation, gene transcription, and partially dependent upon TLR4-mediated signaling. Moreover, heme itself stimulated significant Mϕ pro-IL-1ß gene and protein expression via an S100A8-mediated mechanism and greatly amplified S100A8-driven NLRP3 inflammasome-mediated IL-1ß secretion. In vivo, induction of acute intravascular hemolysis in mice induced a rapid elevation of plasma S100A8 that could be abolished by hemopexin, a heme scavenger. Finally, plasma S100A8 levels were found to be significantly elevated in patients with the inherited hemolytic anemia, sickle cell anemia, when compared with levels in healthy individuals. In conclusion, we demonstrate that hemolytic processes are associated with S100A8 generation and that some of the inflammatory effects of heme may be amplified by autocrine S100A8 production. Findings suggest a mechanism by which hemolytic inflammation could be propagated via leukocyte priming by endogenous proteins, even in sterile inflammatory environments such as those that occur in the hemolytic diseases. S100A8 may represent a therapeutic target for reducing inflammation in hemolytic disorders.

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.

Lithium, a classic drug in psychiatry, improves nilotinib-mediated antileukemic effects.

Although Tyrosine kinase inhibitors (TKIs) that target Bcr-Abl play a key role in Chronic Myeloid Leukemia (CML) therapy, they do not eradicate CML-initiating cells, which lead to the emergence of drug resistance. Here we used the lithium, a GSK-3 inhibitor, to attempt to potentiate the effects of nilotinib against leukemia cells. For this purpose, a K562 leukemia cell line and bone marrow cells from untreated Chronic Myeloid Leukemia (CML) patients, prior to any exposure to TKIs, were used as a model. Our results demonstrated that the combination of lithium + nilotinib (L + N) induced K562-cell death and cleaved caspase-3 when compared to lithium or nilotinib alone, accompanied by GSK-3ß phosphorylation and Bcr-Abl oncoprotein levels reduction. Interestingly, these events were related to autophagy induction, expressed by increased LC3II protein levels in the group treated with L + N. Furthermore, the clonogenic capacity of progenitor cells from CML patients was drastically reduced by L + N, as well as lithium and nilotinib when used separately. The number of cell aggregates (clusters), were increased by all treatments (L + N, lithium, and nilotinib). This pioneering research has demonstrated that lithium might be of therapeutic value when targeting Bcr-Abl cells with nilotinib because it triggers cell death in addition to exerting classical antiproliferative effects, opening new perspectives for novel target and therapeutic approaches to eradicate CML.

Hematopoietic defects in response to reduced Arhgap21.

Arhgap21 is a member of the Rho GTPase activating protein (RhoGAP) family, which function as negative regulators of Rho GTPases. Arhgap21 has been implicated in adhesion and migration of cancer cells. However, the role of Arhgap21 has never been investigated in hematopoietic cells. Herein, we evaluated functional aspects of hematopoietic stem and progenitor cells (HSPC) using a haploinsufficient (Arhgap21+/-) mouse. Our results show that Arhgap21+/- mice have an increased frequency of phenotypic HSC, impaired ability to form progenitor colonies in vitro and decreased hematopoietic engraftment in vivo, along with a decrease in LSK cell frequency during serial bone marrow transplantation. Arhgap21+/- hematopoietic progenitor cells have impaired adhesion and enhanced mobilization of immature LSK and myeloid progenitors. Arhgap21+/- mice also exhibit reduced erythroid commitment and differentiation, which was recapitulated in human primary cells, in which knockdown of ARHGAP21 in CMP and MEP resulted in decreased erythroid commitment. Finally, we observed enhanced RhoC activity in the bone marrow cells of Arhgap21+/- mice, indicating that Arhgap21 functions in hematopoiesis may be at least partially mediated by RhoC inactivation.

Probiotics modulate gut microbiota and improve insulin sensitivity in DIO mice.

Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.

Deferasirox associated with liver failure and death in a sickle cell anemia patient homozygous for the -1774delG polymorphism in the Abcc2 gene.

This manuscript describes the case of a patient with sickle cell anemia who died of fulminant hepatitis after therapy with the iron chelator Deferasirox. The patient was homozygous for the -1774delG polymorphism in the Abcc2 gene, which raises the concern about the use of hepatotoxic drugs in this specific context.

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.

Overexpression and Characterization of The C-Terminal Domain of Human Siva1, A Proapoptotic Factor and Cytoskeleton Binding Protein.

Siva1 protein interacts with tumor protein p53 and with the member of the tumor necrosis factor receptor superfamily, stathmin, among others. These proteins are related to several pathways involved in cancer and are therefore strong candidate targets for drug design. This study aimed to characterize the biophysical properties of Siva 1 C- terminal domain to contribute to the discovery of new target directed drugs. Siva1 protein interacts with tumor protein p53 and with the member of the tumor necrosis factor receptor superfamily, stathmin, among others. These proteins are related to several pathways involved in cancer and are therefore strong candidate targets for drug design. This study aimed to characterize the biophysical properties of Siva 1 C- terminal domain to contribute to the discovery of new target directed drugs. The C-terminus Siva1 domain (residues 84-175) was fused to glutathione Stransferase (GST) and expressed in an E coli system and the recombinant GST-Siva C-terminus was purified by GSTTagged Protein affinity and gel filtration chromatography. We tested the biological activity of the purified Siva Cterminus domain in a Jurkat extract cell line and found that the protein interacted with natural binders. Biophysical and biochemical assays have demonstrated monodispersion of the protein in solution with a predominant unfolded and elongated shape. However, at high concentrations, the protein showed a tendency to form soluble aggregates. These results are expected to lead to further progress in the understanding of Siva1 properties and target-directed drug design.

Molecular matching for Rh and K reduces red blood cell alloimmunisation in patients with myelodysplastic syndrome.

BACKGROUND: Matching for Rh and K antigens has been used in an attempt to reduce antibody formation in patients receiving chronic transfusions but an extended phenotype matching including Fy(a) and Jk(a) antigens has also been recommended. The aim of this study was to identify an efficient transfusion protocol of genotype matching for patients with myelodysplastic syndrome (MDS) or chronic myelomonocytic leukaemia. We also examined a possible association of HLA class II alleles with red blood cell (RBC) alloimmunisation. MATERIALS AND METHODS: We evaluated 43 patients with MDS undergoing transfusion therapy with and without antibody formation. We investigated antigen-matched RBC units for ABO, D, C, c, E, e, K, Fy(a), Fy(b), Jk(a), Jk(b), S, s, Do(a), Do(b) and Di(a) on the patients' samples and on the donor units serologically matched for them based on their ABO, Rh and K phenotypes and presence of antibodies. We also determined the frequencies of HLA-DRB1 alleles in the alloimmunised and non-alloimmunised patients. RESULTS: Seventeen of the 43 patients had discrepancies or mismatches for multiple antigens between their genotype-predicted profile and the antigen profile of the units of blood serologically matched for them. We verified that 36.8% of patients had more than one RBC alloantibody and 10.5% of patients had autoantibodies. Although we were able to find a better match for the patients in our extended genotyped/phenotyped units, we verified that matching for Rh and K would be sufficient for most of the patients. We also observed an over-representation of the HLA-DRB1*13 allele in the non-alloimmunised group of patients with MDS. DISCUSSION: In our population molecular matching for C, c, E, e, K was able to reduce RBC alloimmunisation in MDS patients. An association of HLA-DRB1*13 and protection from RBC alloimmunisation should be confirmed.

In vitro microfluidic model for the study of vaso-occlusive processes.

Vaso-occlusion, responsible for much of the morbidity of sickle-cell disease, is a complex multicellular process, apparently triggered by leukocyte adhesion to the vessel wall. The microcirculation represents a major site of leukocyte-endothelial interactions and vaso-occlusive processes. We have developed a biochip with subdividing interconnecting microchannels that decrease in size (40 µm to 10 µm in width), for use in conjunction with a precise microfluidic device, to mimic cell flow and adhesion through channels of sizes that approach those of the microcirculation. The biochips were utilized to observe the dynamics of the passage of neutrophils and red blood cells, isolated from healthy and sickle-cell anemia (SCA) individuals, through laminin or endothelial adhesion molecule-coated microchannels at physiologically relevant rates of flow and shear stress. Obstruction of E-selectin/intercellular adhesion molecule 1-coated biochip microchannels by SCA neutrophils was significantly greater than that observed for healthy neutrophils, particularly in the microchannels of 40-15 µm in width. Whereas SCA red blood cells alone did not significantly adhere to, or obstruct, microchannels, mixed suspensions of SCA neutrophils and red blood cells significantly adhered to and obstructed laminin-coated channels. Results from this in vitro microfluidic model support a primary role for leukocytes in the initiation of SCA occlusive processes in the microcirculation. This assay represents an easy-to-use and reproducible in vitro technique for understanding molecular mechanisms and cellular interactions occurring in subdividing microchannels of widths approaching those observed in the microvasculature. The assay could hold potential for testing drugs developed to inhibit occlusive mechanisms such as those observed in SCA and thrombotic diseases.

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.

Immunophenotyping in myelodysplastic syndromes can add prognostic information to well-established and new clinical scores.

BACKGROUND: myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic clonal disorders. So, prognostic variables are important to separate patients with a similar biology and clinical outcome. We compared the importance of risk stratification in primary MDS of IPSS and WPSS with the just described revision of IPSS (IPSS-R), and examined if variables obtained by bone marrow immunophenotyping could add prognostic information to any of the scores. METHODS: In this prospective study of 101 cases of primary MDS we compared the relation of patients' overall survival with WHO types, IPSS, IPSS-R, WPSS and phenotypic abnormalities of hematopoietic precursors. We examined aberrancies in myelomonocytic precursors and CD34(+) cells. Patients were censored when receiving chemotherapy or BM transplantation. Survival analysis was made by Cox regressions and stability of the models was examined by bootstrap resampling. RESULTS: MEDIAN AGE: 64 years (15-93). WHO types: 2 cases of 5q- syndrome, 7 of RA, 64 of RCDM and 28 of RAEB. In the univariate Cox analysis, increasing risk category of all scores, degree of anemia, higher percentage of BM blasts, higher number of CD34(+) cells and their myeloid fractions besides increasing number of phenotypic abnormalities detected were significantly associated with a shorter survival. In the multivariate analysis comparing the three scores, IPSS-R was the only independent risk factor. Comparing WPSS with phenotypic variables (CD34(+)/CD13(+) cells, CD34(+)/CD13(-) cells and "total alterations") the score and "CD34(+)/CD13(+) cells" remained in the model. When IPSS was tested together with these phenotypic variables, only "CD34(+)/CD13(+) cells", and "total alterations" remained in the model. Testing IPSS-R with the phenotypic variables studied, only the score and "CD34(+)/CD13(+) cells" entered the model. CONCLUSIONS: Immunophenotypic analysis of myelomonocytic progenitors provides additional prognostic information to all clinical scores studied. IPSS-R improved risk stratification in MDS compared to the former scores.