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1.
Transfusion ; 62(4): 770-782, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35274303

RESUMO

BACKGROUND: In hematologic and transfusion medicine research, measurement of red blood cell (RBC) in vivo kinetics must be safe and accurate. Recent reports indicate use of biotin-labeled RBC (BioRBC) to determine red cell survival (RCS) offers substantial advantages over 51 Cr and other labeling methods. Occasional induction of BioRBC antibodies has been reported. STUDY DESIGN AND METHODS: To investigate the causes and consequences of BioRBC immunization, we reexposed three previously immunized adults to BioRBC and evaluated the safety, antibody emergence, and RCS of BioRBC. RESULTS: BioRBC re-exposure caused an anamnestic increase of plasma BioRBC antibodies at 5-7 days; all were subclass IgG1 and neutralized by biotinylated albumin, thus indicating structural specificity for the biotin epitope. Concurrently, specific antibody binding to BioRBC was observed in each subject. As biotin label density increased, the proportion of BioRBC that bound increased antibody also increased; the latter was associated with proportional accelerated removal of BioRBC labeled at density 6 µg/mL. In contrast, only one of three subjects exhibited accelerated removal of BioRBC density 2 µg/mL. No adverse clinical or laboratory events were observed. Among three control subjects who did not develop BioRBC antibodies following initial BioRBC exposure, re-exposure induced neither antibody emergence nor accelerated BioRBC removal. DISCUSSION: We conclude re-exposure of immunized subjects to BioRBC can induce anamnestic antibody response that can cause an underestimation of RCS. To minimize chances of antibody induction and underestimation of RCS, we recommend an initial BioRBC exposure volume of ≤10 mL and label densities of ≤18 µg/mL.


Assuntos
Biotina , Eritrócitos , Adulto , Anticorpos/metabolismo , Biotina/química , Sobrevivência Celular , Contagem de Eritrócitos , Eritrócitos/metabolismo , Humanos
2.
Mol Ther ; 28(8): 1876-1886, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32516572

RESUMO

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS.


Assuntos
Antineoplásicos/administração & dosagem , Desoxicitidina/análogos & derivados , Nanopartículas , Fosfatidilserinas/administração & dosagem , Animais , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/administração & dosagem , Modelos Animais de Doenças , Citometria de Fluxo , Expressão Gênica , Humanos , Camundongos , Nanopartículas/química , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
3.
Transfusion ; 58(8): 2068-2081, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29770455

RESUMO

The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-51 (51 Cr) RBC labeling for determining RBC volume, 24-hour posttransfusion RBC recovery, and long-term RBC survival. Here we provide evidence supporting adoption of a method for kinetics that uses the biotin-labeled RBCs (BioRBCs) as a superior, versatile method for both regulatory and investigational purposes. RBC kinetic analysis using BioRBCs has important methodologic, analytical, and safety advantages over 51 Cr-labeled RBCs. We critically review recent advances in labeling human RBCs at multiple and progressively lower biotin label densities for concurrent, accurate, and sensitive determination of both autologous and allogeneic RBC population kinetics. BioRBC methods valid for RBC kinetic studies, including successful variations used by the authors, are presented along with pharmacokinetic modeling approaches for the accurate determination of RBC pharmacokinetic variables in health and disease. The advantages and limitations of the BioRBC method-including its capability of determining multiple BioRBC densities simultaneously in the same individual throughout the entire RBC life span-are presented and compared with the 51 Cr method. Finally, potential applications and limitations of kinetic BioRBC determinations are discussed.


Assuntos
Biotinilação/métodos , Eritrócitos/metabolismo , Cinética , Prática Clínica Baseada em Evidências , Humanos
4.
Transfusion ; 57(6): 1488-1496, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28261808

RESUMO

BACKGROUND: Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dosing occasionally induces antibodies, a sensitive and specific anti-BioRBC detection assay is needed. STUDY DESIGN AND METHODS: Aims were to 1) develop a gel card assay to evaluate existing, naturally occurring and BioRBC-induced plasma antibodies, 2) compare gel card and tube agglutination detection results, and 3) test for a relationship of antibody induction and BioRBC dose. Reagent BioRBCs were prepared using sulfo-NHS biotin ranging from densities 18 (BioRBC-18) to 1458 (BioRBC-1458) µg/mL RBCs. RESULTS: Among BioRBC-exposed subjects, gel card and tube agglutination results were concordant in 21 of 22 adults and all 19 infant plasma samples. Gel card antibody detection sensitivity was more than 10-fold greater than tube agglutination. Twelve to 16 weeks after BioRBC exposure, induced anti-antibodies were detected by gel card in three of 26 adults (12%) at reagent densities BioRBC-256 or less, but in none of 41 infants. Importantly, induced anti-BioRBC antibodies were associated with higher BioRBC dose (p = 0.008); no antibodies were detected in 18 subjects who received BioRBC doses less than or equal to BioRBC-18. For noninduced BioRBC antibodies, six of 1125 naïve adults (0.3%) and none of 46 naïve infants demonstrated existing anti-BioRBC antibodies using reagent BioRBC-140 or -162. Existing anti-BioRBCs were all neutralized by biotin compounds, while induced antibodies were not. CONCLUSIONS: The gel card assay is more sensitive than the tube agglutination assay. We recommend reagent BioRBC-256 for identifying anti-BioRBCs. Use of a low total RBC biotin label dose (≤ BioRBC-18) may minimize antibody induction.


Assuntos
Anticorpos/imunologia , Biotina/química , Eritrócitos/imunologia , Adulto , Testes de Aglutinação , Bioensaio/métodos , Biotinilação , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Succinimidas/química
5.
Circulation ; 132(20): 1898-908, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26467254

RESUMO

BACKGROUND: High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). METHODS AND RESULTS: A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC(-/-) mice. In RBCs from HFD-fed wild-type and DARC(-/-) mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. CONCLUSIONS: RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.


Assuntos
Aterosclerose/metabolismo , Dieta Hiperlipídica/efeitos adversos , Eritrócitos/metabolismo , Obesidade/metabolismo , Animais , Aterosclerose/etiologia , Aterosclerose/patologia , Eritrócitos/patologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/patologia , Fagocitose/fisiologia
6.
Am J Hematol ; 91(12): 1195-1201, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27648808

RESUMO

Hemolysis is a key feature of sickle cell anemia (HbSS). Direct quantitation of hemolysis could be used as an objective outcome in clinical trials of new therapeutics for HbSS and would also enable better human studies of the pathogenesis of complications of HbSS that are ostensibly hemolysis-related, such as pulmonary hypertension. However, contemporary human studies in HbSS have used only surrogate markers of hemolysis rather than direct measurements of RBC survival. We directly quantified hemolysis in HbSS by measuring survival of an age cohort of RBCs labeled with a stable isotope, administered orally as 15 N-glycine, a metabolic precursor of heme. The atomic excess of 15 N in heme extracted from blood was monitored by mass spectrometry over time. We performed 13 labeling experiments in 11 individuals with HbSS. Mean RBC survival was 31.9 days (range 14.1-53.6). Both HbF level, a known determinant of hemolysis, and absolute reticulocyte count (ARC), an index of the marrow's response to hemolysis, correlated with directly measured RBC survival (r = 0.61, P < 0.002; r = -0.84, P < 0.001). However, commonly used biochemical surrogates of hemolysis (LDH, AST, bilirubin, and plasma free hemoglobin) did not correlate with directly measured RBC survival. These biochemical surrogates should be interpreted cautiously, at best, in clinical trials and human physiologic studies in HbSS. ARC was the best correlate of total hemolysis, but only 70% of the variation in RBC survival was reflected in this marker. If greater accuracy is required in human studies, 15 N-glycine RBC labeling can directly and accurately quantify hemolysis. Am. J. Hematol. 91:1195-1201, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Anemia Falciforme/patologia , Biomarcadores/sangue , Sobrevivência Celular , Eritrócitos/patologia , Hemólise , Isótopos de Nitrogênio/administração & dosagem , Adolescente , Adulto , Feminino , Hemoglobina Fetal , Glicina/administração & dosagem , Humanos , Marcação por Isótopo , Masculino , Espectrometria de Massas , Contagem de Reticulócitos , Adulto Jovem
7.
Mol Cancer ; 14: 78, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25889084

RESUMO

BACKGROUND: High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro. METHODS: In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells. RESULTS: SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release. CONCLUSIONS: Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy.


Assuntos
Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Neuroblastoma/tratamento farmacológico , Fosfatidilserinas/farmacologia , Saposinas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Ciclosporina/metabolismo , Citocromos c/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Nanopartículas/administração & dosagem , Neuroblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo
8.
Eur J Haematol ; 95(5): 405-10, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25600460

RESUMO

Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.


Assuntos
Eritrócitos/enzimologia , Fosfatidilserinas/farmacologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino
9.
Am J Hematol ; 90(1): 50-55, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25293624

RESUMO

HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC ) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral (15) N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15-20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R(2) = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c.


Assuntos
Senescência Celular , Diabetes Mellitus/sangue , Eritrócitos/citologia , Hemoglobinas Glicadas/análise , Glicina/administração & dosagem , Glicemia/análise , Sobrevivência Celular , Eritrócitos/metabolismo , Feminino , Glicina/química , Heme/química , Humanos , Masculino , Isótopos de Nitrogênio
10.
FASEB J ; 27(2): 750-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23085996

RESUMO

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.


Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Calpaína/antagonistas & inibidores , Eritrócitos Anormais/efeitos dos fármacos , Eritrócitos Anormais/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/sangue , Anemia Falciforme/genética , Animais , Proteínas de Ligação ao Cálcio/sangue , Calpaína/sangue , Inibidores de Cisteína Proteinase/farmacologia , Desidratação/sangue , Desidratação/prevenção & controle , Modelos Animais de Doenças , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Sulfonamidas/farmacologia
11.
Prim Care Diabetes ; 18(2): 151-156, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38172007

RESUMO

AIMS: Although diabetes management decisions in primary care are typically based largely on HbA1c, mismatches between HbA1c and other measures of glycemia that are increasingly more available present challenges to optimal management. This study aimed to assess a systematic approach to identify the frequency of mismatches of potential clinical significance amongst various measures of glycemia in a primary care setting. METHODS: Following screening to exclude conditions known to affect HbA1c interpretation, HbA1c, and fructosamine were obtained and repeated after ∼90 days on 53 adults with prediabetes or type 2 diabetes. A subset of 13 participants with repeat labs wore continuous glucose monitoring (CGM) for 10 days. RESULTS: As expected, HbA1c and fructosamine only modestly correlated (initial R2 = 0.768/repeat R2 = 0.655). The HbA1c/fructosamine mismatch frequency of ± 0.5% (using the following regression HbA1c = 0.015 *fructosamine + 2.994 calculated from the initial sample) was 27.0%. Of the 13 participants with CGM data, HbA1c and CGM-based Glucose Management Indicator correlated at R2 = 0.786 with a mismatch frequency of ± 0.5% at 46.2% compared to a HbA1c/fructosamine mismatch frequency of ± 0.5% at 30.8%. CONCLUSIONS: HbA1c is frequently mismatched with fructosamine and CGM data. As each of the measures has strengths and weaknesses, the utilization of multiple different measures of glycemia may be informative for diabetes assessment in the clinical setting.


Assuntos
Diabetes Mellitus Tipo 2 , Adulto , Humanos , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/terapia , Hemoglobinas Glicadas , Glicemia , Automonitorização da Glicemia , Frutosamina , Atenção Primária à Saúde
12.
Am J Hematol ; 88(1): 44-51, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23115087

RESUMO

The changes in red blood cells (RBC) as they age and the mechanisms for their eventual removal have been of interest for many years. Proposed age-related changes include dehydration with increased density and decreased size, increased membrane IgG, loss of membrane phospholipid asymmetry, and decreased activity of KCl cotransport. The biotin RBC label allows unambiguous identification of older cells and exploration of their properties as they age. Autologous normal human RBC were labeled ex vivo and, after reinfusion, compared with unlabeled RBC throughout their lifespan. RBC density increased with age, with most of the change in the first weeks. Near the end of their lifespan, RBC had increased surface IgG. However, there was no evidence for elevated external phosphatidylserine (PS) even though older RBC had significantly lower activity of aminophospholipid translocase (APLT). KCl cotransport activity persisted well past the reticulocyte stage, but eventually decreased as the RBC became older. These studies place limitations on the use of density fractionation for the study of older human RBC, and do not support loss of phospholipid asymmetry as a mechanism for human RBC senescence. However, increased levels of IgG were associated with older RBC, and may contribute to their removal from the circulation.


Assuntos
Senescência Celular/fisiologia , Membrana Eritrocítica/metabolismo , Fosfatidilserinas/metabolismo , Transfusão de Sangue Autóloga , Transfusão de Eritrócitos , Feminino , Humanos , Imunoglobulina G/metabolismo , Masculino , Proteínas de Transferência de Fosfolipídeos/metabolismo
13.
J Biol Chem ; 286(35): 30492-30503, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21733850

RESUMO

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.


Assuntos
Eritrócitos/metabolismo , Simportadores/biossíntese , Anemia Falciforme/metabolismo , Animais , Ânions , Diferenciação Celular , Linhagem Celular Tumoral , Cloretos/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Reticulócitos/citologia , Simportadores/química , Simportadores/metabolismo , Cotransportadores de K e Cl-
14.
Transfus Med Hemother ; 39(5): 302-7, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23801920

RESUMO

SUMMARY: The measurement of red blood cell (RBC) survival has a long history, and a wide variety of methods have been utilized for this purpose. Current methods are of 2 types. First, those that label a representative sample of RBCs of all ages from the blood and then measure their rate of disappearance upon reinfusion. This category includes the (51)Cr and biotin labels. Second, those that use a metabolic precursor or product to determine the turnover of hemoglobin. Examples of these are carbon monoxide production and incorporation of labeled glycine. Recent studies with the covalent, nonradioactive biotin label show its unique suitability for both the accurate measurement of red cell survival and the determination of changes in red cell properties as they age in vivo.

17.
Blood ; 112(10): 4284-91, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18694998

RESUMO

Although red blood cell (RBC) life span is a known determinant of percentage hemoglobin A1c (HbA1c), its variation has been considered insufficient to affect clinical decisions in hematologically normal persons. However, an unexplained discordance between HbA1c and other measures of glycemic control can be observed that could be, in part, the result of differences in RBC life span. To explore the hypothesis that variation in RBC life span could alter measured HbA1c sufficiently to explain some of this discordance, we determined RBC life span using a biotin label in 6 people with diabetes and 6 nondiabetic controls. Mean RBC age was calculated from the RBC survival curve for all circulating RBCs and for labeled RBCs at multiple time points as they aged. In addition, HbA1c in magnetically isolated labeled RBCs and in isolated transferrin receptor-positivereticulocytes was used to determine the in vivo synthetic rate of HbA1c. The mean age of circulating RBCs ranged from 39 to 56 days in diabetic subjects and 38 to 60 days in nondiabetic controls. HbA1c synthesis was linear and correlated with mean whole blood HbA1c (R(2) = 0.91). The observed variation in RBC survival was large enough to cause clinically important differences in HbA1c for a given mean blood glucose.


Assuntos
Senescência Celular , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Eritrócitos/metabolismo , Hemoglobinas Glicadas/metabolismo , Adulto , Glicemia/metabolismo , Sobrevivência Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
18.
Br J Haematol ; 146(4): 447-55, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19549270

RESUMO

Phosphatidylserine (PS) externalization may contribute to Sickle Cell Disease (SCD) characteristics including thrombogenesis, endothelial adhesion and shortened red blood cell (RBC) lifespan. Aminophospholipid translocase (APLT) returns externalized PS to the inner membrane, and phospholipid scramblase (PLSCR) equilibrates phospholipids (PL) across the membrane. APLT inhibition and PLSCR activation appear to be important for PS externalization. We examined relationships between APLT, PLSCR and external PS in mature sickle RBC and reticulocytes. Normally-hydrated sickle RBC without external PS had active APLT and inactive PLSCR. PS-exposing sickle RBC had inhibited APLT and active PLSCR. Sickle reticulocytes had active APLT and active PLSCR independent of external PS. Sickle RBC dehydrated in vivo had the highest proportion of PS-exposing RBC and markedly inhibited APLT. Normal and sickle RBC dehydrated in vitro had moderately decreased APLT. Rehydration resulted in significant recovery of APLT in RBC previously dehydrated in vitro, but not in sickle RBC dehydrated in vivo. These findings indicate that (i) PS externalization in mature sickle RBC depends on the balance between APLT and PLSCR activities, (ii) PS externalization in sickle reticulocytes depends primarily on PLSCR activation and (iii) APLT inhibition in sickle RBC dehydrated in vivo is due to dehydration itself and other factors.


Assuntos
Anemia Falciforme/sangue , Eritrócitos/enzimologia , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Estudos de Casos e Controles , Centrifugação com Gradiente de Concentração , Desidratação , Membrana Eritrocítica/metabolismo , Citometria de Fluxo/métodos , Humanos , Proteínas de Transferência de Fosfolipídeos/análise , Reticulócitos/enzimologia
19.
Am J Hematol ; 84(2): 109-14, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19025796

RESUMO

The measurement of red blood cell survival in the circulation has progressed from the original differential agglutination technique of Ashby to current isotopic and flow cytometric methods. While occasionally useful in the clinic, these methods find widespread use in a number of important research areas, including the evaluation of new red cell storage media in transfusion medicine and studies of the pathophysiology of sickle cell disease and diabetes. In this review, measurement techniques are placed in historical perspective and examined for relative merits and suitable application.


Assuntos
Envelhecimento Eritrocítico , Anemia/sangue , Animais , Biotinilação , Testes Respiratórios , Monóxido de Carbono/análise , Isótopos do Cromo/sangue , Radioisótopos de Cromo/sangue , Diabetes Mellitus/sangue , Eritropoese , Citometria de Fluxo , Hemoglobinas Glicadas/análise , Testes de Hemaglutinação , Doenças Hematológicas/sangue , Humanos , Recém-Nascido , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Coloração e Rotulagem/métodos
20.
Oncotarget ; 10(8): 856-868, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30783515

RESUMO

Normal living cells exhibit phosphatidylserine (PS) primarily within the intracellular leaflet of the plasma membrane. In contrast, viable cancer cells have high levels of PS on the external surface, and exhibit a broad range of surface PS, even within specific types of cancer. Agents that target surface PS have recently been developed to treat tumors and are expected to be more effective with higher surface PS levels. In this context, we examined whether surface PS is increased with irradiation. In vitro irradiation of cancer cell lines selected surviving cells that had higher surface PS in a dose- and time-dependent manner. This was more pronounced if surface PS was initially in the lower range for cancer cells. Radiation also increased the surface PS of tumor cells in subcutaneous xenografts in nude mice. We found an inverse relationship between steady state surface PS level of cancer cell lines and their sensitivity to radiation-induced cell death. In addition, serial irradiation, which selected surviving cells with higher surface PS, also increased resistance to radiation and to some chemotherapeutic drugs, suggesting a PS-dependent mechanism for development of resistance to therapy. On the other hand, fractionated radiation enhanced the effect of a novel anti-cancer, PS-targeting drug, SapC-DOPS, in some cancer cell lines. Our data suggest that we can group cancer cells into cells with low surface PS, which are sensitive to radiation, and high surface PS, which are sensitive to SapC-DOPS. Combination of these interventions may provide a potential new combination therapy.

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