Purpose: We assess the diagnostic performance of the Molecular Microscope Diagnostic System (MMDx) and the AlloSure donor-derived cellfree DNA Fraction (dd-cfDNA%) in diagnosing heart transplant (HT) rejection compared to the gold standard of histopathology (HP).Methods: We reviewed biopsies from HT patients at Houston Methodist Hospital who had MMDx, HP and peripheral blood sample of donorderived cell-free DNA (dd-cfDNA) drawn at the same time point as the endomyocardial biopsy (EMBx).We evaluated the additive performance of MMDx combined with elevated dd-cfDNA% in diagnosing rejection in HT patients with elevated dd-cfDNA% when compared to EMBx HP.Results: We included 113 biopsies from 54 HT patients.The median time of biopsy post-transplantation was 108.0 (IQR 29.0, 307.0) days.Median age was 57.0 (IQR 48.0, 63.0) years and 38/54 (70.4%) were males.Rejection was classified on EMBx in 16 specimens (n=13 ACR ≥ 2R, n=3 AMR ≥ 50% C4d) and on MMDx in 18 specimens (n=9 ACR, n=8 AMR, n=1 ACR+AMR).The AlloSure dd-cfDNA was ≥0.12% in n=48 (42.5%) biopsies and out of these 11 (9 ACR, 2 AMR) had HP rejection on EMBx and 13/48 had rejection on MMDx (7 AMR, 5ACR and 1 mixed rejection) The diagnostic performance of MMDx to predict rejection showed high specificity and negative predictive value (NPV) compared to sensitivity and positive predictive value (PPV).When testing the additive effect of Allo-Sure to MMDx in diagnostic performance to predict rejection compared with HP, MMDx alone showed higher specificity compared to AlloSure ≥0.12% alone.Although the addition of AlloSure to MMDx does not increases the sensitivity, it appears to increase the specificity (Table 1).Conclusion: MMDx and blood-based dd-cfDNA add diagnostic value to the histopathological diagnosis of rejection in HT recipients.

An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling.

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

Phospholipids (PLs) in erythrocyte membranes are asymmetrically distributed in the bilayer; amino PLs such as PS and PE reside exclusively in the inner leaflet and the outer leaflet is thus enriched with neutral PLs such as PC and SM. The localization of PS and PE is principally maintained by an activity of ATP-dependent Flippase. However, the asymmetric distribution is abolished by PL scrambling at critical cellular events like cell senescence. A detailed mechanism of PL scrambling is yet to be understood. Phospholipid scramblase 1 (PLSCR1), a 37 kDa transmembrane protein, has been reported to be responsible for Ca2+-dependent, PL nonselective bidirectional movement between the outer and inner leaflets. Based on the fact that this protein is present in cholesterol-rich raft I examined the effect of cholesterol on scrambling activity. In the present symposium using human erythrocyte membranes and liposomes reconstituted with purified PLSCR1 or its transmembrane peptides I could show that (1) cholesterol removal from erythrocyte membranes resulted in PS exposure to the outer leaflet in Ca2+-independent manner and (2) presence of cholesterol in the reconstituted liposomes suppressed scrambling activity. These results strongly suggest that cholesterol inhibits scrambling activity of PLSCR1 in human erythrocytes.

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.

The erythrocyte membrane, the entry site of the malaria parasite, has lipid microdomains referred to as the lipidrafts, which are enriched in sphingomyelin and cholesterol. Evidences have demonstrated the importance of choles-terol in structure and functions of the lipid rafts and malaria parasite invasion into erythrocytes, while that of sphin-gomyelin is poorly understood. In this study, we examined the influence of sphingomyelinase treatment on erythro-cyte lipid rafts and malaria parasite invasion into erythrocytes. Sphingomyelin was decreased by about 65% aftersphingomyelinase treatment, and sphingomyelin, cholesterol and flotillin–1, a lipid raft marker protein, were absentfrom lipid raft fractions. The treatment also significantly reduced the association of Gsαin lipid raft fractions, sug-gesting that Gsα–mediated signal transduction was impaired. More importantly, malaria parasite invasion was pre-vented by this treatment. Our study illustrates that sphingomyelin, like cholesterol, is essential for the structure andfunctions of the lipid rafts in the erythrocyte membrane and malaria parasite invasion.

Membrane microdomains enriched in cholesterol and sphingolipids and containing specific membrane proteins are designated as lipid rafts. Lipid rafts have been implicated in cell signaling pathways in various cell types. Heterotrimeric guanine nucleotide‐binding protein (Gsα) has been shown to be a raft component of erythrocytes and has been implicated in cell signaling. Rafts are isolated as detergent‐resistant microdomains (DRMs) for biochemical analysis. Cholesterol depletion is widely used to disrupt raft structures to study their function in biological membranes. In the present study, we developed an alternate strategy for disrupting raft structures without altering membrane cholesterol content. Lidocaine hydrochloride, an amphipathic local anesthetic, is shown to reversibly disrupt rafts in erythrocyte membranes and alter the Gsα dependent signal transduction pathway. These findings provide evidence for the presence of rafts while maintaining normal cholesterol content in erythrocyte membranes and confirm a role for raft‐associated Gsα in signal transduction in erythrocytes. Am. J. Hematol., 2008. © 2008 Wiley‐Liss, Inc.

Normal erythrocytes have biconcave discoid shape that presents large surface area with higher cell surface to volume ratio than that of spherical shape. This appears to allow membrane internalization required for Plasmodium falciparum (Pf) invasion into erythrocytes. Indeed abnormal erythrocyte shape with decreased surface area to volume ratio such as hereditary spherocytosis limits invasion of the parasite. In the present study, using several agents to induce erythrocyte shape changes, we examined whether echinocytic shape change with membrane projections in opposite direction to membrane internalizations and/or stomatocytic shape change with decreased surface area to volume ratio that would be required for internalization, prevent Pf invasion. Having microscopically confirmed echinocytic and/or stomatocytic shape changes and also measured extensibility using an ektacytometer of the treated cells, subsequent Pf invasion assay was performed and parasitaemia determined. Both sodium flouride (NaF) and phospholipase A2 (PLA2) induced echinocytic change whereas phospholipase D (PLD), sphingomyelinase (SMase) and chlorpromazine (CPZ) caused stomatocytic change with decreased extensibility of erythrocytes. In both situations, Pf invasion was prevented, indicating that biconcave discoid shape of normal erythrocytes with high surface to volume ratio is required for membrane internalization when Pf invades into erythrocytes.

Proteins are synthesized by translation of mRNA. Their functions are regulated by posttranslational modifications such as phosphorylation due to change in their structure as well as the physical and chemical properties. In this review article, we introduced in our experiences that phosphorylation and de-phosphorylation of erythrocyte membrane skeletal proteins such as spectrin and protein 4.1 (4.1R) change their interactions with binding partners with modification of membrane mechanical functions.

BACKGROUND:Adaptation to problem-based learning (PBL) is a difficult process for high school graduates who are not used to self-directed learning, especially in the freshmen year of medical school. The difficulty includes finding problems from a given case.PURPOSE:Evaluate the effect of an intervention to facilitate case-based problem finding among medical school freshmen undergoing a PBL tutorial.METHODS:Medical school freshmen in 2000 (nonintervened group) and 2001 (intervened group) participated in the study. The intervened group received the modified problem-based program by (a) having briefings on the importance of problem finding, (b) encouragement by the tutors in problem finding, and (c) reinforcement using a self-assessment sheet. At the end of the year, the ability of students to extract problems from a short case was evaluated and compared with the nonintervened students.RESULTS:The intervened group extracted a significantly greater number of problems than the nonintervened group. When extracted problems were categorized, the intervened group was able to generate more questions in a greater number of specified categories.CONCLUSIONS:Interventions to foster problem finding significantly facilitated acquisition of problem extraction skills among young medical students.

Lipid rafts have been implicated in the signaling processes. Anesthesia has been postulated to represent a transient, drug-induced interruption of signal transduction. In examining the effects of lidocaine on lipid rafts, we found that the agent abolished raft formation in a reversible manner in an erythrocyte membrane model. Our results suggested that raft-related signal transduction is involved in the anesthetic mechanism.

Red blood cell (RBC) membranes are stable and deformable due to its mechanical properties maintained by the spectrin-based submembranous skeletal network . Therefore, RBC appears not to accept Plasmodium falciparum (Pf) invasion easily. However, Pf does invade RBC probably due to change in membrane mechanical properties at the area of Pf contact. In the present study we examined whether increase in membrane rigidity may prevent Pf invasion into RBC. Wheat germ agglutinin (WGA) was utilized to increase membrane rigidity and double fluorescence assay with a flow cytometer was established; the biotin-WGA-treated RBC (B-WGA-treated RBC) was mixed with Pf-infected RBC followed by staining with streptavidin-FITC and propidium iodide (PI) to detect BWGA-treated RBC and Pf-infected RBC, respectively. Having confirmed that membrane rigidity of B-WGA-treated RBC increased significantly using an ektacytometer, we could show that these cells were not infected by Pf using double fluorescence assay. The target molecule of B-WGA was also identified to be band 3 by pull-down assay . These results enabled us to suggest that B-WGA binding to band 3 molecule may induce conformational change in band 3-ankyrin-spectrin , sequentially, resulting in increase in membrane rigidity and thus prevention of Pf invasion into RBC .

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of alpha- and beta-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, beta-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties.

This study evaluates a novel application of a method for measuring serum antioxidant activity, based on the detection of erythrocyte membrane lipid peroxidation in cases of uremia. A human erythrocyte ghost membrane in Tris-HCl was mixed with adenosine 5'-diphosphate and iron chloride (FeCl3; ADP/Fe3+) solution (at a molar ratio of 17:1), and the mixture was incubated under aerobic conditions at 37 degrees C for 2 hours. The concentration of erythrocyte membrane thiobarbituric acid-reactive substances increased proportionally with respect to ADP/Fe3+ concentration, and this increase was inhibited by serum albumin in a dose-dependent manner. In patients undergoing chronic hemodialysis therapy, predialytic sera contained in this reaction mixture were weaker than postdialytic sera in terms of inhibitory effect against erythrocyte membrane lipid peroxidation, whereas serum albumin contents remained at levels equivalent to those of the normal control. A gradual increase in human mercaptalbumin nonmercaptalbumin ratio during hemodialysis treatment might be one of the major factors that leads to the recovery of decreased serum antioxidant activity. We clearly showed that the serum scavenging activity against erythrocyte membrane lipid peroxidation in hemodialysis patients decreases markedly, and this pathological condition is improved by hemodialysis.

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin–actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.

A boy had infantile-onset systemic inflammation, growth failure, hepatosplenomegaly, anemia, leukocytopenia, progressive muscular dystrophy, and hypercalprotectinemia, resulting in marked hyperzincemia. His mother had a history of chronic arthritis since childhood and also showed hypercalprotectinemia/hyperzincemia. We postulate an inherent defect in calprotectin metabolism.

Erythrocyte protein 4.1R is a multifunctional protein that binds to various membrane proteins and to phosphatidylserine. In the present study, we report two important observations concerning 4.1R-phosphatidylserine interaction. Biochemically, a major finding of the present study is that 4.1R binding to phosphatidylserine appears to be a two-step process in which 4.1R first interacts with serine head group of phosphatidylserine through the positively charged amino acids YKRS and subsequently forms a tight hydrophobic interaction with fatty acid moieties. 4.1R failed to dissociate from phosphatidylserine liposomes under high ionic strength but could be released specifically by phospholipase A(2) but not by phospholipase C or D. Biochemical analyses showed that acyl chains were associated with 4.1R released by phospholipase A(2). Importantly, the association of acyl chains with 4.1R impaired its ability to interact with calmodulin, band 3, and glycophorin C. Removal of acyl chains restored 4.1R binding. These data indicate that acyl chains of phosphatidylserine play an important role in its interaction with 4.1R and on 4.1R function. In terms of biological significance, we have obtained evidence that 4.1R-phosphatidylserine interaction may play an important role in cellular sorting of 4.1R.

Erythrocyte protein 4.1R is a multifunctional protein that binds to various membrane proteins and to phosphatidylserine. In the present study, we report two important observations concerning 4.1R-phosphatidylserine interaction. Biochemically, a major finding of the present study is that 4.1R binding to phosphatidylserine appears to be a two-step process in which 4.1R first interacts with serine head group of phosphatidylserine through the positively charged amino acids YKRS and subsequently forms a tight hydrophobic interaction with fatty acid moieties. 4.1R failed to dissociate from phosphatidylserine liposomes under high ionic strength but could be released specifically by phospholipase A2 but not by phospholipase C or D. Biochemical analyses showed that acyl chains were associated with 4.1R released by phospholipase A2. Importantly, the association of acyl chains with 4.1R impaired its ability to interact with calmodulin, band 3, and glycophorin C. Removal of acyl chains restored 4.1R binding. These data indicate that acyl chains of phosphatidylserine play an important role in its interaction with 4.1R and on 4.1R function. In terms of biological significance, we have obtained evidence that 4.1R-phosphatidylserine interaction may play an important role in cellular sorting of 4.1R.