RESUMO
FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (Treg) cells. CD4+ T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous Treg-like cells, some very similar to normal Treg cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4+ T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal Treg cells exerted dominant suppression, quenching the disease signature and revealing in mutant Treg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes Treg cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering Treg cell dysfunction. Accordingly, interleukin-2 treatment improved the Treg-like compartment and survival.
Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/genética , Fatores de Transcrição Forkhead/deficiência , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Adolescente , Animais , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Conjuntos de Dados como Assunto , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diarreia/sangue , Diarreia/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Doenças do Sistema Imunitário/sangue , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Mutação , RNA-Seq , Análise de Célula Única , Linfócitos T Reguladores/metabolismo , Adulto JovemRESUMO
Acute promyelocytic leukemia (APL) with variant RARA translocation is linked to over 15 partner genes. Recent publications encompassing 6 cases have expanded the spectrum of RARA partners to torque teno mini virus (TTMV). This entity is likely underrecognized due to the lack of clinician and pathologist familiarity, inability to detect the fusion using routine testing modalities, and informatic challenges in its recognition within next-generation sequencing (NGS) data. We describe a clinicopathologic approach and provide the necessary tools to screen and diagnose APL with TTMV::RARA using existing clinical DNA- or RNA-based NGS assays, which led to the identification of 4 cases, all without other known cytogenetic/molecular drivers. One was identified prospectively and 3 retrospectively, including 2 from custom automated screening of multiple data sets (50,257 cases of hematopoietic malignancy, including 4809 acute myeloid leukemia/myeloid sarcoma/APL cases). Two cases presented as myeloid sarcoma, including 1 with multiple relapses after acute myeloid leukemia-type chemotherapy and hematopoietic stem cell transplant. Two cases presented as leukemia, had a poor response to induction chemotherapy, but achieved remission upon reinduction (including all-trans retinoic acid in 1 case) and subsequent hematopoietic stem cell transplant. Neoplastic cells demonstrated features of APL including frequent azurophilic granules and dim/absent CD34 and HLA-DR expression. RARA rearrangement was not detected by karyotype or fluorescent in situ hybridization. Custom analysis of NGS fusion panel data identified TTMV::RARA rearrangements and, in the prospectively identified case, facilitated monitoring in sequential bone marrow samples. APL with TTMV::RARA is a rare leukemia with a high rate of treatment failure in described cases. The diagnosis should be considered in leukemias with features of APL that lack detectable RARA fusions and other drivers, and may be confirmed by appropriate NGS tests with custom informatics. Incorporation of all-trans retinoic acid may have a role in treatment but requires accurate recognition of the fusion for appropriate classification as APL.
Assuntos
Leucemia Promielocítica Aguda , Proteínas de Fusão Oncogênica , Receptor alfa de Ácido Retinoico , Torque teno virus , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/diagnóstico , Receptor alfa de Ácido Retinoico/genética , Masculino , Torque teno virus/genética , Proteínas de Fusão Oncogênica/genética , Feminino , Adulto , Pessoa de Meia-Idade , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD.
Assuntos
Anemia Falciforme , Modelos Animais de Doenças , Traumatismo por Reperfusão , Animais , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Anemia Falciforme/complicações , Camundongos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Humanos , Masculino , Hipóxia/metabolismo , Hipóxia/tratamento farmacológicoRESUMO
Several of the complications observed in sickle cell disease (SCD) are influenced by variation in hematologic traits (HT), such as fetal hemoglobin (HbF) level and neutrophil count. Previous large-scale genome-wide association studies carried out in largely healthy individuals have identified thousands of variants associated with HT, which have then been used to develop multi-ancestry polygenic trait scores (PTS). Here, we tested whether these PTS associate with HT in SCD patients and if they can improve statistical models associated with SCD-related complications. In 2,056 SCD patients, we found that the PTS predicted less HT variance than in non-SCD individuals of African ancestry. This was particularly striking at the Duffy/DARC locus, where we observed an epistatic interaction between the SCD genotype and the Duffy null variant (rs2814778) that led to a two-fold weaker effect on neutrophil count. PTS for these HT which are measured as part of routine practice were not associated with complications in SCD. In contrast, we found that a simple PTS for HbF that includes only six variants explained a large fraction of the phenotypic variation (20.5-27.1%), associated with acute chest syndrome and stroke risk, and improved the statistical modeling of the vaso-occlusive crisis rate. Using Mendelian randomization, we found that increasing HbF by 4.8% reduces stroke risk by 39% (P=0.0006). Taken together, our results highlight the importance of validating PTS in large diseased populations before proposing their implementation in the context of precision medicine initiatives.
Assuntos
Anemia Falciforme , Acidente Vascular Cerebral , Humanos , Herança Multifatorial , Estudo de Associação Genômica Ampla , Anemia Falciforme/genética , Anemia Falciforme/complicações , Genótipo , Hemoglobina Fetal/genéticaRESUMO
Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.
Assuntos
Anemia Falciforme , Simportadores , Ácido Acético , Anemia Falciforme/tratamento farmacológico , Animais , Calcimicina , Cloretos/metabolismo , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Camundongos , Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Simportadores/metabolismoRESUMO
Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.
Assuntos
Anemia Falciforme , Reticulócitos , Trifosfato de Adenosina/metabolismo , Anemia Falciforme/metabolismo , Apirase/metabolismo , Cátions/metabolismo , Células Cultivadas , Eritrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hipóxia/metabolismo , Probenecid/metabolismo , Reticulócitos/metabolismo , Suramina/metabolismo , Suramina/farmacologiaRESUMO
The molecular identity of Psickle, the deoxygenation-activated cation conductance of the human sickle erythrocyte, remains unknown. We observed in human sickle red cells that inhibitors of TRPA1 and TRPV1 inhibited Psickle, whereas a TRPV1 agonist activated a Psickle-like cation current. These observations prompted us to test the roles of TRPV1 and TRPA1 in Psickle in red cells of the SAD mouse model of sickle cell disease. We generated SAD mice genetically deficient in either TRPV1 or TRPA1. SAD;Trpv1-/- and SAD;Trpa1-/- mice were indistinguishable in appearance, hematological indices, and osmotic fragility from SAD mice. We found that deoxygenation-activated cation currents remained robust in SAD;Trpa1-/- and SAD;Trpv1-/- mice. In addition, 45Ca2+ influx into SAD mouse red cells during prolonged deoxygenation was not reduced in red cells from SAD;Trpa1-/- and SAD;Trpv1-/- mice. We conclude that the nonspecific cation channels TRPA1 and TRPV1 are not required for deoxygenation to stimulate Psickle-like activity in red cells of the SAD mouse model of sickle cell disease. (159).
Assuntos
Anemia Falciforme/metabolismo , Eritrócitos/patologia , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Cátions/metabolismo , Modelos Animais de Doenças , Eritrócitos/metabolismo , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/genéticaRESUMO
In a recent clinical trial, the metabolite l-glutamine was shown to reduce painful crises in sickle cell disease (SCD) patients. To support this observation and identify other metabolites implicated in SCD clinical heterogeneity, we profiled 129 metabolites in the plasma of 705 SCD patients. We tested correlations between metabolite levels and six SCD-related complications (painful crises, cholecystectomy, retinopathy, leg ulcer, priapism, aseptic necrosis) or estimated glomerular filtration rate (eGFR), and used Mendelian randomization (MR) to assess causality. We found a potential causal relationship between l-glutamine levels and painful crises (N = 1278, odds ratio (OR) [95% confidence interval] = 0.68 [0.52-0.89], P = 0.0048). In two smaller SCD cohorts (N = 299 and 406), the protective effect of l-glutamine was observed (OR = 0.82 [0.50-1.34]), although the MR result was not significant (P = 0.44). We identified 66 significant correlations between the levels of other metabolites and SCD-related complications or eGFR. We tested these correlations for causality using MR analyses and found no significant causal relationship. The baseline levels of quinolinic acid were associated with prospectively ascertained survival in SCD patients, and this effect was dependent on eGFR. Metabolomics provide a promising approach to prioritize small molecules that may serve as biomarkers or drug targets in SCD.
Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/complicações , Glutamina/sangue , Dor/etiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Dor/sangue , Adulto JovemRESUMO
Resolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R -RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.
Assuntos
Anemia Falciforme/complicações , Anti-Inflamatórios não Esteroides/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Nefropatias/prevenção & controle , Pneumonia/prevenção & controle , Animais , Citocinas/metabolismo , Humanos , Nefropatias/etiologia , Nefropatias/patologia , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Pneumonia/etiologia , Pneumonia/patologiaRESUMO
Hematology analyzers capable of performing complete blood count (CBC) have lagged in their prevalence at the point-of-care. Sight OLO (Sight Diagnostics, Israel) is a novel hematological platform which provides a 19-parameter, five-part differential CBC, and is designed to address the limitations in current point-of-care hematology analyzers using recent advances in artificial intelligence (AI) and computer vision. Accuracy, repeatability, and flagging capabilities of OLO were compared with the Sysmex XN-Series System (Sysmex, Japan). Matrix studies compared performance using venous, capillary and direct-from-fingerprick blood samples. Regression analysis shows strong concordance between OLO and the Sysmex XN, demonstrating that OLO performs with high accuracy for all CBC parameters. High repeatability and reproducibility were demonstrated for most of the testing parameters. The analytical performance of the OLO hematology analyzer was validated in a multicenter clinical laboratory setting, demonstrating its accuracy and comparability to clinical laboratory-based hematology analyzers. Furthermore, the study demonstrated the validity of CBC analysis of samples collected directly from fingerpricks.
Assuntos
Inteligência Artificial , Contagem de Células Sanguíneas/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Contagem de Células Sanguíneas/métodos , Desenho de Equipamento , Humanos , Reprodutibilidade dos TestesRESUMO
ß-thalassemia (ß-Thal) is caused by defective ß-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress. We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of ß-thalassemia intermedia. In the absence of these transporters, the anemia of ß-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of ß-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved ß-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of ß-thalassemia.
Assuntos
Simportadores/genética , Talassemia beta/genética , Anemia/prevenção & controle , Animais , Desidratação , Modelos Animais de Doenças , Eritrócitos/química , Eritrócitos/patologia , Camundongos , Fragilidade Osmótica , Fenótipo , Esplenomegalia , Simportadores/metabolismo , Talassemia beta/patologia , Cotransportadores de K e Cl-RESUMO
BACKGROUND: Many clinical decisions depend on estimating patient risk of clinical outcomes by interpreting test results relative to reference intervals, but standard application of reference intervals suffers from two major limitations that reduce the accuracy of clinical decisions: (1) each test result is assessed separately relative to a univariate reference interval, ignoring the rich pathophysiologic information in multivariate relationships, and (2) reference intervals are intended to reflect a population's biological characteristics and are not calibrated for outcome prediction. METHODS: We developed a combined reference region (CRR), derived CRRs for some pairs of complete blood count (CBC) indices (RBC, MCH, RDW, WBC, PLT), and assessed whether the CRR could enhance the univariate reference interval's prediction of a general clinical outcome, 5-year mortality risk (MR). RESULTS: The CRR significantly improved MR estimation for 21/21 patient subsets defined by current univariate reference intervals. The CRR identified individuals with >2-fold increase in MR in many cases and uniformly improved the accuracy for all five pairs of tests considered. Overall, the 95% CRR identified individuals with a >7× increase in 5-year MR. CONCLUSIONS: The CRR enhances the accuracy of the prediction of 5-year MR relative to current univariate reference intervals. The CRR generalizes to higher numbers of tests or biomarkers, as well as to clinical outcomes more specific than MR, and may provide a general way to use existing data to enhance the accuracy and precision of clinical decisions.
Assuntos
Contagem de Células Sanguíneas/métodos , Contagem de Células Sanguíneas/normas , Adulto , Biomarcadores/sangue , Testes de Química Clínica/métodos , Testes de Química Clínica/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes , Estatísticas não ParamétricasRESUMO
Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.
Assuntos
Anemia Hemolítica Congênita/sangue , Eritrócitos/metabolismo , Hidropisia Fetal/sangue , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/sangue , Potássio/sangue , Anemia Hemolítica Congênita/diagnóstico , Anemia Hemolítica Congênita/genética , Sinalização do Cálcio , Estudos de Casos e Controles , Índices de Eritrócitos , Predisposição Genética para Doença , Humanos , Hidropisia Fetal/diagnóstico , Hidropisia Fetal/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais Iônicos/sangue , Canais Iônicos/genética , Potenciais da Membrana , Mutação de Sentido Incorreto , Fragilidade Osmótica , FenótipoRESUMO
Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.
Assuntos
Anemia Falciforme/sangue , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Animais , Tamanho Celular/efeitos dos fármacos , Desidratação/tratamento farmacológico , Modelos Animais de Doenças , Eritrócitos/patologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Camundongos , Simportadores/deficiência , Simportadores/genética , Cotransportadores de K e Cl-RESUMO
Sickle cell disease (SCD) is the most common monogenic disorder in the world. Notably, there is extensive clinical heterogeneity in SCD that cannot be fully accounted for by known factors, and in particular, the extent to which the phenotypic diversity of SCD can be explained by genetic variation has not been reliably quantified. Here, in a family-based cohort of 449 patients with SCD and 755 relatives, we first show that 5 known modifiers affect 11 adverse outcomes in SCD to varying degrees. We then utilize a restricted maximum likelihood procedure to estimate the heritability of 20 hematologic traits, including fetal hemoglobin (HbF) and white blood cell count (WBC), in the clinically relevant context of inheritance from healthy carriers to SCD patients. We report novel estimations of heritability for HbF at 31.6% (±5.4%) and WBC at 41.2% (±6.8%) in our cohort. Finally, we demonstrate shared genetic bases between HbF, WBC, and other hematologic traits, but surprisingly little overlap between HbF and WBC themselves. In total, our analyses show that HbF and WBC have significant heritable components among individuals with SCD and their relatives, demonstrating the value of using family-based studies to better understand modifiers of SCD.
Assuntos
Anemia Falciforme/genética , Família , Hemoglobina Fetal/genética , Característica Quantitativa Herdável , Adulto , Anemia Falciforme/sangue , Estudos de Coortes , Feminino , Hemoglobina Fetal/metabolismo , Humanos , Contagem de Leucócitos , MasculinoRESUMO
BACKGROUND: Regulation of body iron occurs at cellular, tissue, and systemic levels. In healthy individuals, iron absorption and losses are minimal, creating a virtually closed system. In the setting of chronic kidney disease and hemodialysis (HD), increased iron losses, reduced iron absorption, and limited iron availability lead to iron deficiency. Intravenous (IV) iron therapy is frequently prescribed to replace lost iron, but determining an individual's iron balance and stores can be challenging and imprecise, contributing to uncertainty about the long-term safety of IV iron therapy. SUMMARY: Patients on HD receiving judicious doses of IV iron are likely to be in a state of positive iron balance, yet this does not appear to confer an overt risk for clinically relevant iron toxicity. The concomitant use of iron with erythropoiesis-stimulating agents, the use of maintenance iron dosing regimens, and the reticuloendothelial distribution of hepatic iron deposition likely minimize the potential for iron toxicity in patients on HD. Key Messages: Because no single diagnostic test can, at present, accurately assess iron status and risk for toxicity, clinicians need to take an integrative approach to avoid iron doses that impose excessive exposure while ensuring sufficient replenishment of iron stores capable of overcoming hepcidin blockade and allowing for effective erythropoiesis.