Ex Vivo Activation of Red Blood Cell Senescence by Plasma from Sickle-Cell Disease Patients: Correlation between Markers and Adhesion Consequences during Acute Disease Events.

BACKGROUND: Blood transfusion remains a key treatment for managing occlusive episodes and painful crises in sickle-cell disease (SCD). In that clinical context, red blood cells (RBCs) from donors and transfused to patients, may be affected by plasma components in the recipients' blood. Senescence lesion markers appear on the red cells after transfusion, shortening the RBC lifespan in circulation. In the specific context of SCD, senescence signals can also trigger the occlusive painful events, typical of the disease. This work follows through our previous data that described a RBC senescence process, rapidly detected after challenge with SCD pathological plasmas. In this clinical context, we wanted here to further explore the characteristics and physiologic consequences of AA RBC lesions associated with senescence, as lesions caused by RBCs after transfusion may have adverse consequences for SCD patients. METHODS: Plasma samples from SCD patients, with acute symptoms (n = 20) or steady-state disease (n = 34) were co-incubated with donor AA RBCs from blood units for 24 to 48 h. Specific markers signing RBC senescence were quantified after the incubation with SCD plasma samples. The physiologic in-flow adhesion was investigated on senescent RBCs, an in vitro technic into biochips that mimic adherence of RBCs during the occlusive events of SCD. RESULTS: Senescence markers on AA RBCs, together with their in-flow adhesion to the plasma-bridging protein thrombospondin, were associated with the clinical status of the SCD patients from whom plasma was obtained. In these experiments, the highest values were obtained for SCD acute plasma samples. Adhesion of senescent RBCs into biochips, which is not reversed by a pre-treatment with recombinant Annexin V, can be reproduced with the use of chemical agents acting on RBC membrane channels that regulate either Ca2+ entry or modulating RBC hydration. CONCLUSION: We found that markers on red cells are correlated, and that the senescence induced by SCD plasma provokes the adhesion of RBCs to the vessel wall protein thrombospondin. In-flow adhesion of senescent red cells after plasma co-incubations can be reproduced with the use of modulators of RBC membrane channels; activating the Piezo1 Ca2+ mechanosensitive channel provokes RBC adhesion of normal (non-senescent) RBCs, while blocking the Ca2+-dependent K+ Gardos channel, can reverse it. Clinically modulating the RBC adhesion to vascular wall proteins might be a promising avenue for the treatment of painful occlusive events in SCD.

Cytokine changes in sickle-cell disease patients as markers predictive of the onset of delayed hemolytic transfusion reactions.

BACKGROUND: Changes in cytokine production are known to contribute to the pathogenesis of sickle-cell disease (SCD), particularly in painful acute complications (crises) and episodes of post-transfusion hemolysis. Little is known about cytokine profiles in patients with these complications. STUDY DESIGN AND METHODS: We investigated possible associations between cytokine profile and the onset of delayed hemolytic transfusion reactions (DHTRs), particularly during acute-phase episodes, to improve characterization of the biological parameters predictive of such events. We included SCD patients with severe acute symptoms (n = 36) or steady-state disease (n = 31), both possibly leading to a DHTR (n = 18) event. Luminex® technology was used to determine the plasma concentrations of 23 cytokines. RESULTS: Regardless of clinical context, the concentrations of interleukin (IL)-6, IL-10, inducible protein-10, and macrophage inflammatory protein-1ß were higher in plasma samples from SCD patients than in those from healthy controls. IL-6 and IL-10 concentrations were even higher in acute-phase plasma samples from SCD patients. In addition, IL-27 and TNFα levels were higher, and IL-6 and RANTES levels were lower in acute-phase SCD patients just before the onset of DHTR than in patients experiencing painful occlusive episodes. CONCLUSION: In addition to reporting the plasma cytokine profiles of SCD patients in various clinical phases of the disease, we provide the first evidence of a significant association between low plasma TNFα concentration, high plasma IP-10 concentration and the onset of DHTR in SCD patients.

Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease.

Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD. For HbS polymerization inhibition, the hypothesis of an "HbF per red blood cell (HbF/RBC) threshold" requires accurate measurement of HbF in individual RBC. To date, HbF detection methods are limited to a qualitative measurement of RBC populations containing HbF - the F cells, which are variable. We developed an accurate method for HbF quantification in individual RBC. A linear association between mean HbF content and mean RBC fluorescence by flow cytometry, using an anti-Human-HbF antibody, was obtained from non-SCD subjects presenting homogeneous HbF distribution. This correlation was then used to measure HbF/RBC. Hydroxyurea (HU) improves SCD clinical manifestations, mainly through its ability to induce HbF synthesis. The HbF distribution was analyzed in 14 SCD patients before and during HU treatment. A significant decrease in RBC population containing less than 2 pg of HbF/RBC was observed. Therefore, we tested associations for %RBC above different HbF/RBC thresholds and showed a decrease in the pathognomonic vaso-occlusive crisis incidence from the threshold of 4 pg. This quantity was also correlated with the level of sickle RBC after in vitro deoxygenation. This new method allows the comparison of HbF/RBC distributions and could be a useful tool to characterize baseline patients HbF distribution and therapeutic response to HbF inducers.

Complement activation in sickle cell disease: Dependence on cell density, hemolysis and modulation by hydroxyurea therapy.

The complement system is an innate immune defense cascade that can cause tissue damage when inappropriately activated. Evidence for complement over activation has been reported in small cohorts of patients with sickle cell disease (SCD). However, the mechanism governing complement activation in SCD has not been elucidated. Here, we observe that the plasma concentration of sC5b-9, a reliable marker for terminal complement activation, is increased at steady state in 61% of untreated SCD patients. We show that greater complement activation in vitro is promoted by SCD erythrocytes compared to normal ones, although no significant differences were observed in the regulatory proteins CD35, CD55, and CD59 in whole blood. Complement activation is positively correlated with the percentage of dense sickle cells (DRBCs). The expression levels of CD35, CD55, and CD59 are reduced in DRBCs, suggesting inefficient regulation when cell density increases. Moreover, the surface expression of the complement regulator CD46 on granulocytes was inversely correlated with the plasma sC5b-9. We also show increased complement deposition in cultured human endothelial cells incubated with SCD serum, which is diminished by the addition of the heme scavenger hemopexin. Treatment of SCD patients with hydroxyurea produces substantial reductions in complement activation, measured by sC5b-9 concentration and upregulation of CD46, as well as decreased complement activation on RBCs in vitro. In conclusion, complement over activation is a common pathogenic event in SCD that is associated with formation of DRBCs and hemolysis. And, it affects red cells, leukocytes and endothelial cells. This complement over activation is partly alleviated by hydroxyurea therapy.

Alloimmunization risk associated with amino acid 223 substitution in the RhD protein: analysis in the light of molecular modeling.

BACKGROUND: Partial D status is a major concern for transfusion and pregnancy, due to the possibility of carriers becoming immunized. When known carriers of a D variant have never been exposed to complete D, they are assumed to have D partial status based on the position of the amino acid substituted. New approaches for predicting immunization risk are required. We built a three-dimensional (3D) structural model to investigate the consequences of substitutions of Amino Acid 223 involved in a large number of D variants. STUDY DESIGN AND METHODS: Homology modeling was performed with multiple templates. The model was evaluated by comparing the interactions of the known p.Phe223Val variant (RHD*08.01) and a new p.Phe223Ser variant (RHD*52) to RhD reference allele (p.Phe223). The consequences predicted by modeling the variants were compared with serologic data. RESULTS: The 3D structural model was generated from two related protein structures and assessed with state-of-the-art approaches. An analysis of the interactions of the variant Residue 223 in the proposed 3D model highlighted the importance of this position. Modeling predictions were consistent with the serologic and clinical data obtained for the D antigen with a substitution of Amino Acid 223. CONCLUSION: We used a 3D structural model to evaluate the effect of the p.Phe223 substitution on the conformation of the RhD protein. This model shed light on the influence of substitutions on the structure of the RhD protein and the associated alloimmunization risk. These initial findings indicate that the p.Phe223Ser variant can be considered partial.

Red blood cells for transfusion in patients with sepsis: respective roles of unit age and exposure to recipient plasma.

BACKGROUND: Red blood cell (RBC) storage in blood banks is not exempt from cellular injury. Alterations not observed on RBCs freshly isolated from units can rapidly appear in circulation. The transfusion of old blood units, even if this is a controversial issue, could therefore have adverse effects on the recipient. We wanted to determine the respective effects of storage duration and recipient plasma on RBCs for transfusion into patients with severe sepsis. STUDY DESIGN AND METHODS: Eleven stored RBC units were sampled at various time points, approximately Days 3 to 8 (referred to as fresh RBCs) and Days 38 to 42 (old RBCs) and tested in coincubation experiments with plasma obtained from 13 patients with severe sepsis and 17 healthy donors as controls. RBCs were tested after 24 or 48 hours at 37°C for the detection of senescence markers (phosphatidylserine exposure, calcium influx, and reactive oxygen species detection and decrease in size) with or without exposure to plasma. RESULTS: We confirmed that a 42-day refrigerated storage of RBCs alone (without any incubation in plasma) had no significant effect on RBCs and no senescence marker detected. By contrast, ex vivo exposure to plasma samples altered both fresh and old RBCs, with a much larger effect for old RBCs, regardless of the plasma used (sepsis vs. control). CONCLUSION: We show that the main factor affecting the senescence of RBCs for transfusion into patients with severe sepsis is the age of the stored units rather than the clinical status of the recipient.

Severe hemolysis after plasma transfusion in a neonate with necrotizing enterocolitis, Clostridium perfringens infection, and red blood cell T-polyagglutination.

BACKGROUND: Red blood cell (RBC) Thomsen-Friedenreich antigen exposure (T activation) in infants with necrotizing enterocolitis (NEC) has occasionally been associated with posttransfusional intravascular hemolysis thought to be due to anti-T antibodies in the donor plasma. STUDY DESIGN AND METHODS: We describe an infant with NEC and Clostridium perfringens infection complicated by severe hemolysis after plasma transfusion. After this case, infants with confirmed NEC were prospectively evaluated for T activation. We checked for hemolysis in patients with T activation receiving plasma-containing blood products. RESULTS: The infant had received 80 mL of fresh-frozen plasma (FFP). His RBCs displayed strong T activation, and agglutination was observed with four of six ABO-compatible FFP units. A direct antiglobulin test was negative. IgM-class anti-T antibodies were present in small amounts (titer of 8) in the transfused FFP. Anti-T antibodies from the blood donor were not hemolytic in vitro. In the prospective study, T activation was observed in three of 28 infants with NEC (11%). One infant presented moderate T activation and two infants presented very strong T activation but only moderate decreases in sialic acid expression on the RBC membrane. These three infants presented no signs of hemolysis after transfusion with unwashed blood products or FFP. CONCLUSION: Anti-T antibodies are unlikely to be the etiologic factor for the hemolytic reactions observed in infants with NEC and T activation. Massive RBC desialylation and the direct action of bacterial toxins are more probable causes. Strict avoidance of plasma-containing blood products does not seem justified in these infants.

Neuraminidase enhances in vitro expansion of human erythroid progenitors.

In spite of recent key improvements, in vitro mass production of erythrocytes from human stem cells is still limited by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, such progenitors are as scarce in the bone marrow as in peripheral blood. We used a two-step culture model of human cord blood-derived erythroid progenitors in the presence or absence of high-purity neuraminidase, in a serum-free, defined culture medium. Granulocytic and megakaryocytic progenitor cell expansions were also studied. We show that significant enhancement of erythroid cell generation is obtained when CD34(+) human hematopoietic progenitors are cultured in the presence of neuraminidase. Interestingly, in so doing, expanded red cell progenitors remained erythropoietin-dependent for further expansion and survival, and cells thus generated displayed a normal phenotype. Moreover, the activity of neuraminidase on these cells can be reversed by simple cell washing. Finally, growth of cells of the other myeloid lineages (granulocytes and megakaryocytes) is either decreased or unchanged in the presence of neuraminidase. This specific feature of neuraminidase, that of stimulation of human red cell progenitor proliferation, provides a safe technique for producing greater numbers of in vitro-generated red blood cells for both basic research and transfusion use.

Evidence of benefits from using fresh and cryopreserved blood to transfuse patients with acute sickle cell disease.

BACKGROUND: The transfusion of red blood cell (RBC) concentrates is the main treatment for acute vaso-occlusive symptoms in sickle cell disease (SCD). Units of packed RBCs (pRBCs) must retain optimal characteristics for transfusion throughout the storage period. Transfused RBCs interact with the plasma and the endothelium that lines blood vessels and may be the target of immune-hematologic conflict if the patient produces antibodies against RBCs. Questions remain concerning the benefit-risk balance of RBC transfusions, in particular about the shelf-life of the units. STUDY DESIGN AND METHODS: Plasma samples from 33 hemoglobin SS patients with SCD who had severe acute-phase symptoms or were in steady-state were put in contact with 10 fresh-stored and older stored samples from the same 10 RBC units. The factors affecting RBC survival (phosphatidylserine exposure, cytosolic calcium influx, cell size reduction) were analyzed. RESULTS: We show that the effects of plasma samples from patients with SCD on pRBCs depend on the clinical condition of the patients and the duration of red cell storage. Signs of RBC senescence were correlated with the clinical status of the patient from whom the plasma sample was obtained. A decrease in RBC size and an increase in phosphatidylserine exposure were correlated with the duration of RBC storage. The behavior of cryopreserved pRBCs was similar to that of fresh refrigerated RBCs when challenged with patient plasma samples. CONCLUSION: The key points of this study are that the clinical condition of patients with SCD can negatively affect the integrity of pRBCs for transfusion, and those effects increase with longer storage. Also, cryopreserved pRBCs behave similarly to fresh RBCs when challenged with plasma samples from patients with SCD in acute phase. Our data provide the first evidence that fresh RBCs stored for short periods may be of greater benefit to patients with SCD than RBCs that have been refrigerated for longer periods, particularly for those who have acute symptoms of SCD.

Red blood cell Thomsen-Friedenreich antigen expression and galectin-3 plasma concentrations in Streptococcus pneumoniae-associated hemolytic uremic syndrome and hemolytic anemia.

BACKGROUND: Pneumococcal hemolytic uremic syndrome (P-HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking. STUDY DESIGN AND METHODS: A prospective study was conducted on 10 children with culture-confirmed IPD. Five presented with full-blown P-HUS, three had an incomplete form with hemolytic anemia and mild or no uremia (P-HA), and two had neither HUS nor HA. Thomsen-Friedenreich (T), Th, and Tk cryptantigens and sialic acid expression were determined on red blood cells (RBCs) with peanut (PNA), Glycine soja (SBA), Bandeiraea simplicifolia II, and Maackia amurensis lectins. Plasma concentrations of the major endogenous T-antigen-binding protein, galectin-3 (Gal-3), were analyzed. RESULTS: We found that RBCs strongly reacted with PNA and SBA lectins in all P-HUS and P-HA patients. Three P-HUS and three P-HA patients showed also concomitant Tk activation. Direct antiglobulin test (DAT) was positive in three P-HUS (one with anti-C3d and two with anti-IgG) and two P-HA patients (one with anti-C3d and one with anti-IgG). RBCs derived from the two uncomplicated IPD patients reacted with PNA but not with SBA lectin. Gal-3 plasma concentrations were increased in all P-HUS patients. CONCLUSIONS: The results indicate high levels of neuraminidase activity and desialylation in both P-HUS and P-HA patients. T-antigen activation is more sensitive than DAT for P-HUS diagnosis. Combining PNA and SBA lectins is needed to improve the specificity of T-antigen activation. High concentrations of Gal-3 in P-HUS patients suggest that Gal-3 may contribute to the pathogenesis of P-HUS.

In vitro characterization of patches of human mesenchymal stromal cells.

Stem cells may represent an excellent strategy to improve the healing of skin ulcers. Today the administration mode of stem cells to skin defects remains unsatisfactory. Delivering stem cells with topical treatments represents a new strategy and answering the patients' need. Mesenchymal stromal cells (MSC) have been shown to improve wound healing of cutaneous lesions and amniotic membrane (AM) is known to represent a natural scaffold for cells. The aim of this study is to develop a tissue-engineered product combining MSC and AM for clinical use. In this work we investigated whether the stromal matrix of intact human AM could constitute a scaffold for human MSC derived from either bone marrow (BM) or adipose tissue (AT). For this purpose, clinical-grade AM, MSC, and culture medium were used. We performed experiments of short-term adherence and proliferation for 15 days after the seeding of the cells. Morphological aspects and secretion profiles of MSC onto AM were studied, respectively, by scanning electron microscopy and Luminex analysis. Results demonstrated that the stromal matrix allow the adherence in much greater amount of MSC from BM or AT compared to 2D material. Experiments of proliferation showed that both kinds of MSC could proliferate on the stromal matrix and remain viable 15 days after the seeding of the cells. The 3D analysis of MSC culture demonstrated that both types of MSC invaded the stromal matrix and grew in multiple layers while retaining their fibroblastic morphology. By studying the secretion profile of MSC onto the stromal matrix, we found that both kinds of MSC secrete important cytokines and growth factors for wound healing of cutaneous lesions, such as vascular endothelial growth factor, hepatocyte growth factor, and basic fibroblast growth factor. In conclusion, these results suggest that the stromal matrix of AM seeded with MSC represents a bioactive scaffold that should be evaluated in patients with a nonhealing cutaneous wound.

Platelet lysate coating on scaffolds directly and indirectly enhances cell migration, improving bone and blood vessel formation.

Suitable colonization and vascularization of tissue-engineered constructs after transplantation represent critical steps for the success of bone repair. Human platelet lysate (hPL) is composed of numerous growth factors known for their proliferative, differentiative and chemo-attractant effects on various cells involved in wound healing and bone growth. The aim of this study was to determine whether the delivery of human mesenchymal stromal cells (hMSC) seeded on hPL-coated hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) scaffolds could enhance vascularization and bone formation, as well as to investigate the mechanisms by which hMSC participate in tissue regeneration. Our study demonstrates that hPL can be coated on HA/ß-TCP scaffolds, which play direct and indirect effects on implanted and/or resident stem cells. Effectively, we show that hPL coating directly increases chemo-attraction to and adhesion of hMSC and endothelial cells on the scaffold. Moreover, we show that hPL coating induces hMSC to produce and secrete pro-angiogenic proteins (placental growth factor and vascular endothelial growth factor) which allow the proliferation and specific chemo-attraction of endothelial cells in vitro, thus improving in vivo neovascularization and new bone formation. This study highlights the potential of functionalizing biomaterials with hPL and shows that this growth factor combination can have synergistic effects leading to enhanced bone and blood vessel formation.

IgA-mediated human autoimmune hemolytic anemia as a result of hemagglutination in the spleen, but independent of complement activation and FcαRI.

Autoimmune hemolytic anemia (AIHA) due to warm-acting IgA autoantibodies is rare. We explored the pathogenic mechanisms underlying destruction of red blood cells (RBCs) in a patient with severe AIHA mediated exclusively by polymeric immunoglobulin A (pIgA) anti-Band 3 autoantibodies. The follow-up period was 17 months. RBCs were not destroyed by complement activation as no deposition of complement was observed on the patient's RBCs. pIgA eluted from the patient's RBCs did not induce RBC destruction through phagocytosis by monocytes or antibody-dependent cell-mediated cytotoxicity by natural killer cells. Induction of eryptosis (ie, RBC apoptosis) due to direct alteration of the RBC membrane by pIgA autoantibodies was also excluded. By contrast, upon incubation with pIgA-opsonized RBCs, substantial RBC membrane transfers (ie, trogocytosis) to monocytes were observed that might contribute to RBC immune destruction. This effect was poorly inhibited by blockers of Fc receptors, excluding a major contribution of FcαRI to this process. Histologic analysis revealed a massive accumulation of agglutinated RBCs with little sign of erythrophagocytosis in the spleen. These results, together with the efficacy of splenectomy 17 months after AIHA onset, suggest that the trapping and subsequent sequestration of agglutinated RBCs in the spleen are the principal pathogenic mechanisms of pIgA-mediated AIHA.

Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate.

Culture of expanded mesenchymal stem cells (MSCs) seeded on biomaterials may represent a clinical alternative to autologous bone graft in bone regeneration. Foetal bovine serum (FBS) is currently used for MSC expansion, despite risks of infectious disease transmission and immunological reaction due to its xenogenic origin. This study aimed to compare the osteogenic capacities of clinical-grade human MSCs cultured with FBS or allogenic human platelet lysate (PL). In vitro, MSCs cultured in PL both accelerate the expansion rate over serial passages and spontaneously induce osteoblastic gene expression such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (Op) and bone morphogenetic protein-2 (BMP-2). In vivo, ectopic bone formation is only observed on ceramics seeded with MSCs grown in PL medium implanted under the skin of immunodeficient mice for 7 weeks. In conclusion, allogenic human PL accelerates MSC proliferation and enhances MSC osteogenic differentiation.