As a lymphoid organ, the spleen hosts a wide range of immune cell populations, which not only remove blood-borne antigens, but also generate and regulate antigen-specific immune responses. In ...particular, the splenic microenvironment has been demonstrated to play a prominent role in adaptive immune responses to enveloped viral infections and alloantigens. During both types of immunizations, antigen-specific immunoglobulins G (IgGs) have been characterized by the reduced amount of fucose present on N-linked glycans of the fragment crystallizable (Fc) region. These glycans are essential for mediating the induction of immune effector functions. Therefore, we hypothesized that a spleen may modulate humoral responses and serve as a preferential site for afucosylated IgG responses, which potentially play a role in immune thrombocytopenia (ITP) pathogenesis. To determine the role of the spleen in IgG-Fc glycosylation, we performed IgG subclass-specific liquid chromatography-mass spectrometry (LC-MS) analysis of Fc glycosylation in a large cohort of individuals splenectomized due to trauma, due to ITP, or spherocytosis. IgG-Fc fucosylation was consistently increased after splenectomy, while no effects for IgG-Fc galactosylation and sialylation were observed. An increase in IgG1- and IgG2/3-Fc fucosylation level upon splenectomy has been reported here for the first time, suggesting that immune responses occurring in the spleen may be particularly prone to generate afucosylated IgG responses. Surprisingly, the level of total IgG-Fc fucosylation was decreased in ITP patients compared to healthy controls. Overall, our results suggest a yet unrecognized role of the spleen in either the induction or maintenance of afucosylated IgG responses by B cells.
Hereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by ...membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, α-spectrin or β-spectrin using red cell indices, eosin-5-maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and ektacytometry to assess cell membrane stability, cellular density and deformability. Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellular heterogeneity. These cells show a prominent decrease in membrane stability and deformability and, as a consequence, are quickly removed from the circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular heterogeneity and density are strong markers of clinical severity in spherocytosis.
Blood rheology biomarkers in sickle cell disease Lu, Madeleine; Rab, Minke AE; Shevkoplyas, Sergey S ...
Experimental Biology and Medicine,
01/2020, Volume:
245, Issue:
2
Book Review, Journal Article
Peer reviewed
Open access
Sickle cell disease (SCD) is the most common inherited blood disorder, affecting approximately 100,000 patients in the U.S. and millions more worldwide. Patients with SCD experience a wide range of ...clinical complications, including frequent pain crises, stroke, and early mortality, all originating from a single-point mutation in the β-globin subunit. The RBC changes resulting from the sickle mutation lead to a host of rheological abnormalities that diminish microvascular blood flow, and produce severe anemia due to RBC hemolysis, and ischemia from vaso-occlusion initiated by sticky, rigid sickle RBCs. While the pathophysiology and mechanisms of SCD have been investigated for many years, therapies to treat the disease are limited. In addition to RBC transfusion, there are only two US Food and Drug Administration (FDA)-approved drugs to ameliorate SCD complications: hydroxyurea (HU) and L-glutamine (Endari™). The only curative therapy currently available is allogeneic hematopoietic stem cell transplantation (HSCT), which is generally reserved for individuals with a matched related donor, comprising only 10–15% of the total SCD population. Potentially curative advanced gene therapy approaches for SCD are under investigation in ongoing clinical trials. The ultimate goal of any curative treatment should be to repair the hemorheological abnormalities caused by SCD, and thus normalize blood flow and prevent clinical complications. Our mini-review highlights a set of key hemorheological biomarkers (and the current and emerging technologies used to measure them) that may be used to guide the development of novel curative and palliative therapies for SCD, and functionally assess outcomes.
Impact statement
Severe impairment of blood rheology is the hallmark of SCD pathophysiology, and one of the key factors predisposing SCD patients to pain crises, organ damage, and early mortality. As novel therapies emerge to treat or cure SCD, it is crucial that these treatments are functionally evaluated for their effect on blood rheology. This review describes a comprehensive panel of rheological biomarkers, their clinical uses, and the technologies used to obtain them. The described technologies can produce highly sensitive measurements of the ability of current treatments to improve blood rheology of SCD patients. The goal of curative therapies should be to achieve blood rheology biomarkers measurements in the range of sickle cell trait individuals (HbAS). The use of the panel of rheological biomarkers proposed in this review could significantly accelerate the development, optimization, and clinical translation of novel therapies for SCD.
In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading ...to vaso‐occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index ‐ EI) as a function of pO2. Upon a standardized, 22 minute, automated cycle of deoxygenation (pO2 median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3‐diphosphoglycerate showed a significant correlation (respectively R = ‑0.993 and R = 0.980) with Point of Sickling (PoS5%), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left‐shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state‐of‐the‐art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.
Summary
The hexokinase (HK) enzyme plays a key role in red blood cell energy production. Hereditary non‐spherocytic haemolytic anaemia (HNSHA) caused by HK deficiency is a rare disorder with only 12 ...different disease‐associated variants identified. Here, we describe the clinical features and genotypes of four previously unreported patients with hexokinase 1 (HK1)‐related HNSHA, yielding two novel truncating HK1 variants. The patients' phenotypes varied from mild chronic haemolytic anaemia to severe infantile‐onset transfusion‐dependent anaemia. Three of the patients had mild haemolytic disease caused by the common HK1 promoter c.‐193A>G variant combined with an intragenic HK1 variant, emphasizing the importance of including this promoter variant in the haemolytic disease gene panels. HK activity was normal in a severely affected patient with a homozygous HK1 c.2599C>T, p.(His867Tyr) variant, but the affinity for ATP was reduced, hampering the HK function. In cases of HNSHA, kinetic studies should be considered in the functional studies of HK. We reviewed the literature of previously published patients to provide better insight into this rare disease and add to the understanding of genotype–phenotype correlation.
The hexokinase 1 gene (HK1) encodes the hexokinase 1 enzyme, which plays a key role in red blood cell energy production. Biallelic pathogenic variants in HK1 cause hexokinase 1 deficiency and hereditary non‐spherocytic haemolytic anaemia (HNSHA). The clinical phenotype of hexokinase 1 (HK1)‐related HNSHA varies from mild chronic haemolytic anaemia to severe infantile‐onset disease. HK1 promoter variant c.‐193A>G combined with intragenic loss‐of‐function variants, such as c.372+1G>A, c.2361_2362del or c.2599C>T, is associated with predominantly mild HNSHA. Homozygous HK1 c.2599C>T p.(His867Tyr) missense variant results in severely hampered HK1 enzyme function and infantile‐onset HNSHA requiring regular red blood cell transfusions.
Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport ...phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient‐derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS‐stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol‐enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C‐deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.
ATP11C deficiency is associated with reduced red blood cell flippase activity and increased phosphatidylserine surface exposure
Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red blood cell (RBC) glycolysis, causing changes in metabolism including a deficiency in adenosine triphosphate (ATP). This ...affects red cell homeostasis, promoting premature removal of RBC from the circulation. In this study, we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBC and erythroid precursors from PK-deficient patients. In 15 patients,
ex vivo
treatment with AG-348 resulted in increased enzymatic activity in all patients' cells after 24 hours (h) (mean increase: 1.8-fold; range: 1.2-3.4). ATP levels increased (mean increase: 1.5-fold; range: 1.0-2.2) similar to control cells (mean increase: 1.6-fold; range: 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBC.
Ex vivo
treatment with AG-348 increased residual activity from 1.4- to >10-fold more than residual activity of vehicle-treated samples. Protein analyses suggest that a sufficient level of PK protein is required for cells to respond to AG- 348 treatment
ex vivo
, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients,
ex vivo
treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG- 348 effectively up-regulates PK enzymatic activity and increases stability in PK-deficient RBC over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. Registered at
clinicaltrials.gov identifiers: NCT02476916, NCT03853798, NCT03548220, NCT03559699
).
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