Robert S. Franco

Red cell life span heterogeneity in hematologically normal people is sufficient to alter HbA1c

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.

Evidence for Interindividual Heterogeneity in the Glucose Gradient Across the Human Red Blood Cell Membrane and Its Relationship to Hemoglobin Glycation

OBJECTIVE—To determine whether interindividual heterogeneity in the erythrocyte (red blood cell [RBC]) transmembrane glucose gradient might explain discordances between A1C and glycemic control based on measured fructosamine. RESEARCH DESIGN AND METHODS—We modeled the relationship between plasma glucose and RBC glucose as the concentration distribution (Ci-to-Co ratio) of a nonmetabolizable glucose analog 14C-3-O-methyl glucose (14C-3OMG) inside (Ci) and outside (Co) RBCs in vitro. We examined the relationship between that distribution and the degree of glycation of hemoglobin in comparison with glycation of serum proteins (fructosamine), the glycation gap. A1C, fructosamine, and in vitro determination of the 14C-3OMG distribution in glucose-depleted RBCs were measured in 26 fasted subjects. RESULTS—The Ci-to-Co ratio 0.89 ± 0.07 for 3-O-methyl-d-glucopyranose (3OMG) ranged widely (0.72–1.04, n = 26). In contrast, urea Ci-to-Co (1.015 ± 0.022 [range 0.98–1.07], P < 0.0001) did not. Concerning mechanism, in a representative subset of subjects, the Ci-to-Co ratio was retained in RBC ghosts, was not dependent on ATP or external cations, and was reestablished after reversal of the glucose gradient. The 3OMG Ci-to-Co ratio was not correlated with serum fructosamine, suggesting that it was independent of mean plasma glucose. However, Ci-to-Co did correlate with A1C (R2 = 0.19) and with the glycation gap (R2 = 0.20), consistent with a model in which differences in internal glucose concentration at a given mean plasma glucose contribute to differences in A1C for given level of glycemic control. CONCLUSIONS—The data demonstrate interindividual heterogeneity in glucose gradients across RBC membranes that may affect hemoglobin glycation and have implications for diabetes complications risk and risk assessment.

The measurement and importance of red cell survival

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. Am. J. Hematol., 2009.

Time-dependent changes in the density and hemoglobin F content of biotin-labeled sickle cells.

Sickle red blood cells (RBC) are subject to a number of important cellular changes and selection pressures. In this study, we validated a biotin RBC label by comparison to the standard 51Cr label, and used it to study changes that occur in sickle cells as they age. Sickle RBC had a much shorter lifespan than normal RBC, but the two labels gave equivalent results for each cell type. A variable number of sickle, but not normal, RBC disappeared from the circulation during the first few hours after reinfusion. The number of biotinylated sickle reticulocytes was decreased by 50% after 24 h and 75% after 48 h, with a gradual decrease in the amount of reticulum per cell. The labeled sickle cells exhibited major density increases during the first 4-6 d after reinfusion, with smaller changes thereafter. A small population of very light, labeled sickle RBC was essentially constant in number after the first few days. Fetal hemoglobin (HbF) content was determined in isolated biotinylated sickle RBC after reinfusion, allowing an estimate of lifespan for RBC containing HbF (F cells) and non-F cells. The lifespan of sickle biotinylated RBC lacking HbF was estimated to be approximately 2 wk, whereas F cells survived 6-8 wk.

International seminar on the red blood cells as vehicles for drugs.

The first human transfusion was performed by the pioneer Dr Jean-Baptiste Denis in France in 1667 and now, three centuries later, around 50 millions blood units are transfused every year, saving millions of lives. Today, there is a new application for red blood cells (RBCs) in cellular therapy: the effective use of erythrocytes as vehicles for chemical or biological drugs. Using this approach, the therapeutic index of RBC-entrapped molecules can be significantly improved with increased efficacy and reduced side effects. This cell-based medicinal product can be manufactured at an industrial scale and is now used in the clinic for different therapeutic applications. A seminar dedicated to this field of research, debating on this inventive formulation for drugs, was held in Lyon (France) on 28 January 2011. Drs KC Gunter and Y Godfrin co-chaired the meeting and international experts working on the encapsulation of drugs within erythrocytes met to exchange knowledge on the topic ‘The Red Blood Cells as Vehicles for Drugs’. The meeting was composed of oral presentations providing the latest knowledge and experience on the preclinical and clinical applications of this technology. This Meeting Highlights article presents the most relevant messages given by the speakers and is a joint effort by international experts who share an interest in studying erythrocyte as a drug delivery vehicle. The aim is to provide an overview of the applications, particularly for clinical use, of this innovative formulation. Indeed, due to the intrinsic properties of erythrocytes, their use as a drug carrier is one of the most promising drug delivery systems investigated in recent decades. Of the different methods developed to encapsulate therapeutic agents into RBCs [1,2,] the most widely used method is the lysis of the RBCs under tightly controlled hypotonic conditions in the presence of the drug to be encapsulated, followed by resealing and annealing under normotonic conditions (Figure 1). This results in uniform encapsulation of the material into the cells and a final product with good stability, reproducibility and viability. This process, which has now been developed to an industrial scale, is the technique chosen by the majority of the experts presenting their work in this seminar (by R Franco). Figure 1 The process of reversible hypotonic lysis of RBCs to entrap molecules Keywords: carriers, drug delivery, erythrocytes, red blood cells, targeting 1. Therapeutic enzyme-loaded RBCs Therapy using RBC encapsulated enzymes has the advantage of prolonging the half-life of the enzyme and maintaining therapeutic blood levels, reducing the dosage and frequency of therapeutic interventions, and preventing the need for expensive chemical modification [3]. The therapeutic index can be strongly improved, especially by reducing immunogenic reactions, which are often observed in enzyme replacement (Figure 2). Figure 2 The red blood cell as a bioreactor: the substrate (yellow) contained in the plasma permeates the erythrocyte membrane, with the entrapped enzyme (green) catalyzing the metabolism of the substrate to its normal product inside the red cell 1.1 L-asparaginase-loaded RBCs for ASNS-deficient tumor (by E Dufour) L-asparaginase has been used in the treatment of acute lymphoblastic leukemia for > 40 years. This enzyme converts plasmatic L-asparagine (L-Asn) into L-aspartate plus ammonia. Its use is motivated by the fact that malignant cells (especially leukemic) are deficient in asparagine synthetase (ASNS). Because these cells are unable to synthesize L-Asn to meet metabolic demands, L-Asn deprivation, due to L-asparaginase activity, kills the cancerous cells. However, L-asparaginase can also be responsible for adverse events such as hypersensitivity reactions or blood coagulations disorders, in addition to L-Asn depletion. An approach to decreasing side effects of free L-asparaginase in vivo is to entrap the enzyme in RBCs. Reversible hypotonic dialysis remains the most controlled and reproducible method. Indeed, with this process, human RBCs can be loaded with 116 ± 15 IU of L-asparaginase per milliliter of red cells. The resulting product acts as a bioreactor allowing transport of L-Asn through the RBC membrane where L-asparaginase hydrolyzes it. Due to the RBC membrane, the enzyme is protected from rapid catabolism as well as from potential neutralizing antibodies, resulting in an increased half-life and a reduction in hypersensitivity reactions. A Phase I–II trial testing GRASPA® (ERYTECH Pharma, France) on 24 patients in relapsed acute lymphoblastic leukemia showed a strong reduction in hypersensitive reactions, coagulation disorders and hepatic dysfunctions [4]. The L-asparaginase half-life is enhanced (40 days vs 1 day with the free form) and the mean duration of L-Asn depletion is 18.57 days at a dose of 150 IU/kg in a single injection that corresponds to eight injections of Escherichia coli native L-asparaginase. This improvement in tolerance allows the introduction of L-asparaginase treatment to other hematological malignancies, such as acute myeloid leukemia, and also in solid tumors. Indeed, the level of expression of L-ASNS, the enzyme responsible for the synthesis of L-Asn in mammalian cells, provides a rationale for testing L-asparaginase in several cancers. For example, about 30 and 40% of pancreatic ductal adenocarcinoma patients (85 – 90% of all pancreatic cancer subjects) have no or low level of expression of ASNS, respectively. A Phase I clinical study is ongoing with pancreatic adenocarcinoma patients.

Changes in the properties of normal human red blood cells during in vivo aging

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. Am. J. Hematol. 2013.

Effects of a vasopressin antagonist with combined antipressor and antiantidiuretic activities in rats with left ventricular dysfunction.

These experiments assessed the hemodynamic and aquaretic effects of an arginine vasopressin (AVP) antagonist with dual V1V2-receptor inhibiting properties in rats with congestive heart failure resulting from ischemic cardiomyopathy. The compound d(CH2)5-D-Tyr(Et)VAVP was used in these studies. Rats with limited or extensive myocardial infarcts (i.e., with less than 50% or greater than 66% necrosis of the left ventricular wall, respectively, induced by left coronary ligation) and sham-operated controls received the AVP antagonist (100 micrograms/kg i.v.) 4 weeks later. This agent produced an 18% increase in cardiac output (p less than 0.05) and 13% decrease in systemic vascular resistance in the severely damaged rats, both changes being significantly different from those seen in the normal controls or the rats with limited infarcts. All animals exhibited increases in urinary output of 4-10-fold over baseline. We conclude that the hemodynamic and renal effects of this agent are beneficial in animals with left ventricular dysfunction.

Splice variants of Tissue Factor promote monocyte-endothelial interactions by triggering the expression of cell adhesion molecules via integrin-mediated signaling

Summary.  Background: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. Objective: To examine the role of integrin signaling in TF‐dependent recruitment of monocytes by endothelial cells. Methods: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ‐TF) and recombinant asTF were assessed. Results: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid‐rich plaques. Tumor lesions, as well as stromal CD68+ monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ‐TF, and this was completely blocked by anti‐β1 integrin antibody. asTF‐ and LZ‐TF‐treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF‐elicited adhesion was more pronounced than that elicited by LZ‐TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E‐selectin, ICAM‐1 and VCAM‐1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ∼3‐fold under MCP‐1 gradient. Conclusions: TF splice variants ligate β1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non‐proteolytic, integrin‐mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+ exchange

KCl cotransport activated by swelling of sickle red blood cells (SS RBC)is inhibited by deoxygenation. Yet recent studies found a Cl--dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl--dependent component of the deoxygenation-induced net K+ efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl--dependent K+ efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+ at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+ or Rb+ minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+ exchange activity, known to be modulated by cytosolic Ca2+ elevation and cell shrinkage, remained silent under deoxygenation conditions.

KCl cotransport activity in light versus dense transferrin receptor-positive sickle reticulocytes.

A subset of sickle cells becomes K(+)-depleted and dehydrated before or soon after leaving the bone marrow. These young cells may be identified in blood as transferrin receptor-positive (TfR+) dense reticulocytes. KCl cotransport, which is normally active in young erythroid cells with a maximum at pH 6.8, is a candidate pathway for K+ depletion of sickle reticulocytes. In this investigation, KCl cotransport activity was evaluated in young, TfR+ cells which had become dense in vivo and in age-matched cells which had retained normal hydration. Sickle erythrocytes were first separated into three primary density fractions, with care taken to preserve the in vivo hydration state. After normalization of intracellular hemoglobin concentration with nystatin, the cells were incubated at 37 degrees C for 20 min at pH 6.8 and 7.4. Before and after incubation, each primary fraction was separated into four secondary density fractions. The percentage of TfR+ cells in each secondary fraction was measured and a density distribution for TfR+ cells was determined for each primary fraction before and after incubation. The density shift during incubation was a measure of KCl cotransport. TfR+ cells from the denser primary fractions II and III had significantly more density shift than TfR+ cells from the light fraction I. Although the shifts were larger at low pH, differences between primary fractions were also observed at pH 7.4. These data indicate that the cells which become dense quickly in vivo have more KCl cotransport activity than those which remain light in vivo, and support this pathway as a primary mechanism for dehydration of young sickle cells.