Membrane Inhibitor Of Reactive Lysis Research Articles

Hypoxic Stress Induces Complement-Mediated Lysis of Mesenchymal Stem Cells by Downregulating Factor H and CD59

Background:Factor H and membrane inhibitor of reactive lysis (CD59) are key regulators of complement activation. Mesenchymal stem cells (MSCs) secrete Factor H and express CD59 to protect themselves from complement-mediated damage. Severe hypoxia found to decrease the survival chances of MSCs after transplantation; however, little is known about the impact of severe hypoxia on modulating the complement system activity and its effect on MSCs survival. Our study seeks to explore the effect of severe hypoxia on modulating the complement cascade in MSCs.Methods:Human adipose tissue-derived MSCs (hAD-MSCs) were cultured under severe hypoxia using 400 μM Cobalt Chloride (CoCl2) for 48 h. The protein expressions of survival marker; Phosphoinositide 3-kinases (PI3K), and pro-apoptotic marker; Caspase-3 were assessed using western blotting. The level of complement system related factors; Factor H, CD59, C3b, iC3b, C5b, C9, and the complement membrane attack complex (MAC) were analyzed using Elisa assays, western blotting, and immunocytochemistry.Results:Our results showed for the first time that severe hypoxia can significantly impair Factor H secretion and CD59 expression in MSCs. This has been associated with upregulation of MAC complex and increased level of cell lysis and apoptosis marked by downregulation of PI3K and upregulation of Annexin v and Caspase-3.Conclusion:The loss of Factor H and CD59 in hypoxic MSCs can initiate their lysis and apoptosis mediated by activating MAC complex. Preserving the level of Factor H and CD59 in MSCs has significant clinical implication to increase their retention rate in hypoxic conditions and prolong their survival.

Thrombotic risk in paroxysmal nocturnal hemoglobinuria-like (PNH-like) phenotype.

The complement system is an essential component of the innate immune defence that, if overly activated, may damage organs and tissues. For this reason, there is a fine complement regulatory system. The complement modulation system includes two proteins with important regulatory activity, CD55 or decay accelerating factor (DAF) and CD59 or membrane inhibitor of reactive lysis (MIRL).The paroxysmal nocturnal hemoglobinuria (PNH) is a clonal and non-neoplastic disease characterized by intravascular haemolysis, occurrence of thrombosis and bone marrow failure.In clinical practice, in opposition to PNH, a variety of pathological conditions have been observed with an acquired and non-genetic deficiency of the regulatory proteins CD55 and CD59. This abnormal, non-clonal, reduced expression of complement regulatory proteins configures what we may define as PNH-like phenotype.Similarly to PNH, even in the PNH-like phenotype diseases there has been a greater exposure to the mediated complement cellular lysis and, a likely increased risk of thromboembolic events.Therefore, the knowledge of the potential roles of the complement system becomes necessary for a deeper understanding of several pathological conditions and for an improved clinical management of the patients.

Evaluation of Patients with PNH Treated By Eculizumab: Real World Data from Turkey

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease which is characterized with complement mediated intravascular hemolysis, bone marrow failure and thrombosis. The prevelance of PNH is estimated 1 to 16 cases per million in USA. X-linked somatic mutation of phosphatidylinositol glycan‐A (PIGA) gene in hematopoetic stem cells causes an impairment of glycosylphosphatidylinositol (GPI) anchored proteins. The absence of GPI‐dependent molecules that are CD55 (decay accelerating factor; DAF) and CD59 (membrane inhibitor of reactive lysis; MIRL), normally protect the cell from complement‐mediated hemolysis by preventing the formation of the membrane attack complex. The diagnosis is based on flow cytometry which can detect the deficiency of these two complement regulary proteins. We report a retrospective analysis of demographic and clinical characteristics of PNH patients from different centers. Material and methods: We conducted a retrospective analysis of the patients' recorded data. Patients' demographics, medical and treatment history, comorbid conditions, PNH clone size, disease characteristics and outcomes, symptoms, PNH-specific treatments, PNH-related events, morbidity (including myeloproliferative disease, other malignancies, and infections), mortality. Clinical data captured include lactate dehydrogenase (LDH) levels, PNH clone size, hemoglobin levels, thrombotic events, renal functional tests at the time of diagnosis, and other laboratory data. Specific information collected for eculizumab-treated patients includes dosage and dose adjustments and blood cell counts, reticulocyte count and LDH level after eculizumab treatment. Results: 138 patients were included from 28 different centers. All patients were diagnosed by flow cytometry for GPI-linked antigens on red cells and neutrophils. The number of male (69/138) and female (69/138) patient was equal and the median age was 41 years. Median hemoglobin (hb) level was 8.75±2.13 gr/dL; Platelet (plt) level was 131× 109/L at the time of diagnosis. Overall, 49(35,5%) of the patients had been diagnosed with bone marrow failure, including aplastic anemia or hypoplastic anemia (n=31; 22,5%), myelodysplastic syndromes (n=18; 13%). A history of any prior thrombotic event was reported in 45 patients (32,6%). At the time of analysis, 12 (8,7%) patients had pulmonary hypertension. The median granulocyte and monocyte clone size was 63,6% (±32.26) and 66.76 ±28.75 respectively. Fatigue (58%) is the most commonly reported symptom and abdominal pain was seen in 8% of patients. After the eculizumab therapy, the median time for normalization of Hb and LDH level were 7 and 14,6 months, respectively. There was no correlation between thrombosis and clone size, hb, plt, LDH level at the time of diagnosis. LDH level was higher in fatigue patients compared with the patients who were not fatigue (p=0.021). Discussion: PNH is a clonal but non malignant disease that is very rare and knowledge on large case series is really limited. The clinical findings and symptoms could be variable and unfortunately it takes very long time to diagnose because of unawareness of physicians. Eculizumab is a good option in treatment of PNH for improving symptoms of intravascular hemolysis but we still need better understanding of thrombosis mechanism of PNH to better management. In the future, novel inhibitors of the alternative pathway of complement will be used to improve survival and quality of life for PNH patients. Disclosures No relevant conflicts of interest to declare.

The effect of splenectomy on complement regulatory proteins in erythrocytes in β-thalassemia major.

IntroductionHemolysis due to ineffective erythropoiesis is a serious problem β-thalassemia major (β-TM) patients. The role of complement system in the etiopathogenesis of hemolysis observed in β-TM were released. Hemolysis induced by activation of complement system is prevented by complement regulatory proteins. Decay accelerating factor (CD55), membrane inhibitor of reactive lysis (CD59), and complement reception 1 (CR1, CD35) are among these proteins. The absence of these proteins thus accounts for the increased susceptibility of erythrocytes to complement lysis. Splenomegaly and hypersplenism are common complications among thalassemia major patients necessitating splenectomy.Material and methodsIn this study we investigated how splenectomy effects complement regulatory system in erythrocytes. We analysed CD35, CD55, and CD59 levels on erythrocytes in β-TM by flow cytometry.ResultsThe overall mean percentage of CD55 and CD35 positive RBCs of group 1 (22 β-TM with splenectomy) was significantly lower than group 2 (23 β-TM without splenectomy) and group 3 (healthy controls) (p < 0.05). The overall mean percentage CD59 positive RBCs of patients was no significantly different in all groups. The levels of CD35 and CD55 expression on the erythrocytes of splenectomized patients was significantly lower than non-splenectomized patients (p < 0.05).ConclusionsIncreased erythrocyte destruction and iron deposition in organs due to deficiency of these regulatory proteins may be the underlying mechanism of organ damage developing in β-TM patients.

Structural Analysis of CD59 of Chinese Tree Shrew: A New Reference Molecule for Human Immune System Specific CD59 Drug Discovery.

Chinese tree shrews (Tupaia belangeri chinensis) bear several characteristics that are considered to be very crucial for utilizing in animal experimental models in biomedical research. Subsequent to the identification of key aspects and signaling pathways in nervous and immune systems, it is revealed that tree shrews acquire common as well as unique characteristics, and hence offer a genetic basis for employing them as a prospective model for biomedical research. CD59 glycoprotein, commonly referred to as MAC-inhibitory protein (MAC-IP), membrane inhibitor of reactive lysis (MIRL), or protectin, is encoded by the CD59 gene in human beings. It is the member of the LY6/uPAR/alpha-neurotoxin protein family. With this initial point, the objective of this study was to determine a comparative composite based structure of CD59 of Chinese tree shrew. The additional objective of this study was to examine the distribution of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, hydrophobicity molecular surface analysis and electrostatic potential analysis with the assistance of several bioinformatical analytical tools. CD59 Amino acid sequence of Chinese tree shrew was collected from the online database system of National Centre for Biotechnology Information. SignalP 4.0 online server was employed for detection of signal peptide instance within the protein sequence of CD59. Molecular model structure of CD59 protein was generated by the Iterative Threading ASSEmbly Refinement (I-TASSER) suite. The confirmation for three-dimensional structural model was evaluated by structure validation tools. Location of negatively and positively charged amino acid over molecular modeled structure, distribution of secondary structural elements, and hydrophobicity molecular surface analysis was performed with the help of Chimera tool. Electrostatic potential analysis was carried out with the adaptive Poisson-Boltzmann solver package. Subsequently validated model was used for the functionally critical amino acids and active site prediction. The functionally critical amino acids and ligand- binding site (LBS) of the proteins (modeled) were determined using the COACH program. Analysis of Ramachandran plot for Chinese tree shrew depicted that overall, 100% of the residues in homology model were observed in allowed and favored regions, sequentially leading to the validation of the standard of generated protein structural model. In case of CD59 of Chinese tree shrew, the total score of G-factor was found to be -0.66 that was generally larger than the acceptable value. This approach suggests the significance and acceptability of the modeled structure of CD59 of Chinese tree shrew. The molecular model data in cooperation to other relevant post model analysis data put forward molecular insight into protecting activity of CD59 protein molecule of Chinese tree shrew. In the present study, we have proposed the first molecular model structure of uncharted CD59 of Chinese tree shrew by significantly utilizing the comparative composite modeling approach. Therefore, the development of a structural model of the CD59 protein was carried out and analyzed further for deducing molecular enrichment technique. The collaborative effort of molecular model and other relevant data of post model analysis carry forward molecular understanding to protecting activity of CD59 functions towards better insight of features of this natural lead compound.

Potential impact of complement regulator deficiencies on hemolytic reactions due to minor ABO-mismatched transfusions

AbstractMinor ABO-mismatched transfusions are a common occurrence, although infrequent transfusion reactions occur. We sought to investigate the regulation of complement C3 activation induced by anti-A. In vitro complement C3 activation was observed with 10 of 30 group O samples and correlated with immunoglobulin M (IgM) anti-A titers. We developed an in vitro paroxysmal nocturnal hemoglobinuria (PNH) model of hemolysis in which group A1 red blood cells (RBCs) were chemically treated with 2-aminoethylisothiouronium (AET) to alter regulators of complement C3 activation. Intravascular hemolysis was simulated by incubating an IgG nonhemolytic group O plasma (titer = 32) with A1 RBCs. IgG was detected on the RBCs, but hemolysis was observed with AET-treated RBCs only. When treated and untreated RBCs were tested together (1:4), we determined that the failure to observe C3b/d deposition on RBCs was due to the complete hemolysis of the AET-treated minor RBC population. A group O patient with a 9% CD59-deficient PNH clone was sensitized with an IgM anti-I. Hemolysis, with a weak positive direct antiglobulin test (DAT) resulting from C3b/d, was observed after incubation with fresh AB serum. Flow cytometry showed an 86% reduction of the PNH clone. Our work indicates that the transfusion of minor ABO-mismatched plasma could cause hemolysis with a negative DAT C3b/d. We propose that the presence of a PNH clone is 1 possible cause of unexplained anemia for recipients of ABO-mismatched product. This work suggests that other acquired or inherited defects of decay-accelerating factor and membrane inhibitor of reactive lysis could be responsible for infrequent but clinically important hemolysis after ABO-mismatched transfusions.

Renal Manifestations in Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired chronic disorder characterized by a triad of clinical features - hemolytic anemia, pancytopenia, and thrombosis. Not many reports of renal involvement in PNH are available in literature. We present a case series of PNH with renal involvement. We present the data of PNH patients who attended to Departments of General Medicine and Nephrology at a government-run tertiary care institute in South India. The diagnosis of PNH in these patients during initial phase, between 1998 and 2004 was based on sucrose lysis and Ham's test. After 2004, the diagnosis was based on flow cytometry to detect CD59 (membrane inhibitor of reactive lysis), a glycoprotein, and CD55 (decay accelerating factor) in regulation of complement action. The patient data were collected from 1998 to 2014. There were 14 patients of PNH in this period. The mean age was 37 years and the range was 16–68 years. There were eight females. Acute kidney injury (AKI) was noted in six patients. Dialysis was performed in four of them. The mean serum creatinine and urea at the initiation of dialysis were 5.4 ± 0.6 and 64.1 ± 6.1 mg/dl, respectively. The median number of hemodialysis sessions done was four. Renal biopsy was done in four patients. In three patients, the urinalysis and serum chemistry were suggestive of Fanconi syndrome. In our patients, three renal manifestations of PNH were identified. They were AKI, renal vessel thrombosis, and Fanconi syndrome. Chronic renal failure was not identified.

Recommendations for the diagnosis of paroxysmal nocturnal hemoglobinuria: a German-Austrian Consensus1)

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening hematological disease. An acquired mutation in the PIG-A gene in hematopoietic precursor cells leads to a deficiency of glycosylphosphatidylinositol (GPI), a common membrane anchor for certain cell surface proteins. The consequence is a deficiency of the decay-accelerating factor (recognized by antibodies of the cluster CD55) and of the membrane inhibitor of reactive lysis (MIRL, recognized by CD59 antibodies) on the surface of blood cells leading to dysregulation of the complement cascade resulting in characteristic intravascular hemolysis and thromboembolic complications. The analysis of GPI-anchored proteins on blood cells is the recognized diagnostic gold standard. Some aspects of the recently published international guidelines are still a matter of discussion. On the basis of these guidelines, we discussed practical recommendations, which led to the German-Austrian consensus paper presented here. It contains specific proposals for indication of the test, preanalytical aspects, how to perform immunophenotyping, and for result interpretation. We propose a two-step diagnostic approach. Step 1 is a screening and step 2 is a confirmation of the diagnosis. The latter should be restricted to expert laboratories’ experience with PNH. According to this algorithm for a stepwise diagnostic approach, we recommend which antibody panel should be used in which step. The most frequent sources of error and how to avoid them are also summarized. This consensus should support medical laboratories to make an early and correct diagnosis of what is important to therapy and prognosis of the patient. Aspects of economic efficiency and feasibility were also included in our considerations.

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases

Exosomes are 40–100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.

Frequency of paroxysmal nocturnal hemoglobinuria in patients attended in Belém, Pará, Brazil

Background Paroxysmal nocturnal hemoglobinuria is a hematological disease with complex physiopathology. It is genetically characterized by a somatic mutation in the PIG-A gene (phosphatidylinositol glycan anchor biosynthesis, class A), in which the best known antigens are DAF (decay accelerating factor or CD55) and MIRL (membrane inhibitor of reactive lysis or CD59). Objective To determine the frequency of paroxysmal nocturnal hemoglobinuria in patients attended at the HEMOPA foundation from November 2008 to July 2009. Method Thirty patients, with ages ranging from two to 79 years old and suspected of having paroxysmal nocturnal hemoglobinuria were examined. All patients were immunophenotyped by flow cytometry for the CD5, CD59, CD16 and CD45 antigens. Results Paroxysmal nocturnal hemoglobinuria was identified in nine of the thirty patients investigated. Another 3 cases had inconclusive results with CD59-negative labeling only for neutrophils. The highest frequency of paroxysmal nocturnal hemoglobinuria patients (7/9) and inconclusive cases (2/3) were between 19 years old and 48 years old, with a median of 28 years. Conclusion These results show the importance of flow cytometry to identify cases in which patients are deficient in only one antigen (CD59).

A Novel Approach to Treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH): Both Hemolysis and C3 Deposition Are Blocked by a Monoclonal Antibody (mAb) Specific for the Alternative Pathway of Complement (APC) C3/C5 Convertase.

Abstract 157In PNH, red blood cells (RBCs) lack key complement control proteins, CD55 and CD59 and are therefore sensitive to complement activation and intravascular hemolysis. These regulatory proteins function at two different steps in the complement cascade; CD55 (decay accelerating factor, DAF) controls the formation and stability of the APC C3 and C5 convertases, while CD59 (membrane inhibitor of reactive lysis, MIRL) blocks formation of the cytolytic membrane attack complex (MAC). The intravascular hemolysis of PNH can be inhibited in vivo by eculizumab, a humanized mAb that binds complement C5, thereby preventing formation of the MAC. However, PNH patients treated with eculizumab continue to manifest evidence of ongoing hemolysis as they remain anemic with an elevated reticulocyte count and low serum haptoglobin concentration, and approximately 50% of eculizumab-treated patients require transfusion. This observation is consistent with the hypothesis that, in patients treated with eculizumab, PNH RBCs undergo extravascular hemolysis as a consequence of C3 opsonization because eculizumab does not compensate for deficiency of DAF. Recent studies (Risitano et al., Blood, 2009) support this hypothesis as patients undergoing treatment with eculizumab were found to have a positive Coombs test for C3 but not IgG, and flow cytometry demonstrated C3 activation and degradation products bound to the PNH RBCs. This process appeared clinically relevant as transfusion requirement correlated with the percentage of C3 opsonized PNH RBCs. These observations suggest that blocking the APC C3/C5 convertase would be a better way to treat the hemolysis of PNH because this approach has the advantage of blocking both extravascular hemolysis by inhibiting C3 opsonization and preventing intravascular hemolysis by inhibiting MAC generation. We have developed a mAb 3E7 and its deimmunized chimeric humanized derivative H17 that specifically block the APC C3/C5 convertase by binding to a neoepitope expressed when complement C3 is activated. In vitro, 3E7/H17 prevents APC-mediated lysis of rabbit RBCs in human serum and blocks deposition of human C3 activation fragments on APC activator substrates such as zymosan (Mol Immunol, 2006; J Immunol, 2007). We now report that mAb H17/3E7 blocks lysis in acidified normal human serum (aNHS) (a process mediated by the APC) of RBCs from patients with PNH (n=5). Representative results for patients 1 and 2 are as follows: 60% and 40% of RBCs were lysed after a one hour incubation at 37°C; lysis was reduced to 10% and 6%, respectively, at 80 ug/ml of mAb H17, and to 1% lysis (both patients) at 170 ug/ml of mAb H17. We also showed that mAb H17/3E7 blocks deposition of C3 activation fragments on PNH RBCs. After lysis in aNHS, blood samples from PNH patients were probed with Al488 mAb 1H8, specific for C3b/iC3b/C3dg. Flow cytometry experiments revealed C3 fragment deposition on lysed cells corresponding to 30,500 molecules of equivalent soluble fluorochrome (MESF) compared to a background signal of 225 MESF on unlysed RBCs in the same sample. Addition of mAb H17 blocked C3 fragment deposition not only on the unlysed cells but also on the small number of recovered ghosts . Importantly, mAb H17/3E7 inhibits the APC specifically. PNH RBCs, opsonized with IgM in serum from a patient with chronic cold agglutinin disease, were lysed in NHS by the classical complement pathway, and this lysis was not inhibited by mAbH17/3E7. Together, these experiments demonstrate that both hemolysis and C3 opsonization of PNH RBCs can be inhibited by a novel mAb that specifically blocks the APC C3/C5 convertase while leaving intact the classical pathway of complement. These findings suggest an approach to therapy of PNH in which both intravascular and extravascular hemolysis can be inhibited while preserving the important immune functions of the classical pathway of complement. Disclosures:No relevant conflicts of interest to declare.

Expression of membrane inhibitor of reactive lysis in non-small cell lung carcinoma and its clinical significance

Objective To survey membrane inhibitor of reactive lysis(MIRL) expression in non-small cell lung careinoma(NSCLC) and to analyze the relationship between MIRL expression and clinical staging, adjuvant chemotherapy and disease-free survial. Methods The expression of MIRL in 8 adjacent tissues and 36 NSCLC sam-pies were determined by immunohistochemistry. Furthermore, the relationship between MIRL expression and clinical stage ,adjuvant chemotherapy and disease-free survival was assayed by follow-up. Results Among 36 samples of non-small-cell lung cancer,there were 10(27.8%) samples expressing MIRE. Out of 18 samples of squamous carcinoma, 4(22.2%) expressed MIRL,while 6(37.5%) expressed it in 16 samples of adenocarcinoma,there was no statistical significance between them(P>0.05). There were no expression in 2 samples of large cell carcinoma. There was no correlation between MIRL expression and disease-free survival(P>0.05). MIRL positive expression rate in patients with preoperational adjuvant chemotherapy was significantly lower than that of those without preoperational adjuvant chemotherapy(P<0.05). Conclusions There is great percentage of MIRE expression in NSCLC. Our present study suggests that the immunological inhibition of MIRL should be blocked when monoclonal antibody is used in the treat-merit of NSCLC. Key words: Antigens,CD59; Cytotoxicity tests,immunologic; Careinoma,non-small cell lung

40 years of heart transplantation and the DFG‐Transregio Research Group Xenotransplantation

40 years of heart transplantation and the DFG‐Transregio Research Group Xenotransplantation

Expression of CD47, CD35, CD55 and CD59 on Red Cells from Patients with Warm Autoimmune Hemolytic Anemia.

Autoimmune hemolytic anemia (AIHA) is defined as an increased destruction of red cells (RBC) in the presence of anti-RBC autoantibodies. CD47 is an integrin-associated protein expressed on all cells including RBCs. Animal models show that CD47 deficiency contributes to accelerated development of AIHA, while CD35 (CR1- complement receptor 1), CD55 (decay accelerating factor), and CD59 (membrane inhibitor of reactive lysis) are complement inhibitory proteins. Using flow cytometry analysis, in this study we evaluated the expression of CD47, CD35, CD55, and CD59 on RBCs of patients with warm AIHA before any treatment had been initiated. The study population consisted of 12 patients with active AIHA [M:F = 6:6, median age: 32 yrs (3 – 73)], and 20 healthy controls [M:F = 9:11, median age: 36 yrs (25 – 71)]. Ten patients presented idiopathic AIHA while 2 subjects had secondary AIHA (systemic lupus erythematosus and non-Hodgkin's lymphoma). At presentation the median Hb level was 6.6 mg/dL (range: 2.9 to 10 mg/dL), and the median absolute reticulocyte count was 324 × 109/L (range: 215 to 756 × 109/L). At the time of the analyses, all 12 patients had a positive direct antiglobulin test (DAT), 12 (100%) had IgG on their RBCs, 5 (41.7%) had IgG plus C3, and none had C3 alone. The strength of agglutination of all positive DATs showed a strong reaction. The RBC eluates prepared by a dichloromethane technique from the cell samples were positive in all 12 patients, but the retrieved autoantibodies were pan-reactive showing no specific reactivity. The mean fluorescence intensity (MFI) of the expression of CD47, CD35 and CD55 on RBCs of AIHA patients and healthy individuals were not statistically different (CD47 = 464.4 and 464.4; CD35 = 186.8 and 194.3; CD55 = 396.9 and 381.1, respectively). Four patients with life-threatening AIHA were treated with high dose of steroids and RBC transfusions, but 3 patients evolved to death. Two patients who died presented low CD55 expression on their RBCs at diagnosis. AIHA patients showed significant lower CD59 expression on RBCs than healthy controls (MFI = 512.3 ± 28.0 and 553.7 ± 36.6, P = .03). Although CD59 expression in patients that evolved to remission was not significantly different from healthy controls (MFI = 538.5 ± 14.4 and 553.7 ± 36.6), the expression of CD59 on RBCs of 3 AHAI patients who died were significantly lower than that seen on RBCs of healthy controls (MFI = 433.6 ± 69.6 and 553.7 ± 36.6, P = .0001). Although experimental studies have suggested that CD47 has a profound influence on the severity of AIHA in mice, our preliminary data on 12 patients with AIHA did not demonstrate difference on the expression of CD47 on RBCs of patients with warm AIHA or healthy indibiduals. On the other hand, complement regulatory proteins (CD35, CD55, and CD59) may play an important role in protecting RBC destruction through the activation of complement. Our results suggest that patients with life-threatening warm AIHA may present significant CD59 deficiency on their RBCs that may increase the susceptibility of cells to complement-mediated lysis resulting in severe clinical hemolysis.

In vitro RBC exposure to Plasmodium falciparum has no effect on RBC antigen expression.

Severe malarial anaemia is a leading cause of death in African children younger than 3 years of age who are infected with Plasmodium falciparum. The pathogenesis of this anaemia is not understood. The purpose of this study was to determine if P. falciparum induces changes in RBC membranes that contribute to the immune destruction of RBCs. RBCs were collected from healthy subjects and tested using standard haemagglutination assays for 45 antigens representing 21 blood group systems/collections before and after exposure to P. falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite, and blinded serologic studies were completed. CD35 (complement receptor 1), CD55 (decay-accelerating factor), CD59 (membrane inhibitor of reactive lysis) and CD47 (integrin-associated protein) flow cytometric assays were compared for infected and uninfected RBCs. The percentage of parasitaemia was determined using Giemsa-stained thin blood films. Two (Ch, Lub) of the 45 antigens had differing strengths of agglutination between infected and uninfected RBCs, but these differences were resolved with a second source of antisera. Forty-three antigens showed no significant differences in the strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no significant differences. P. falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. The pathogenesis of the haemolytic episode that occurs in these children remains unclear.

Flowcytometric detection of PNH defect and response to therapy in aplastic anemia patients.

Clonal hemopoietic stem cell disorders, characterized by features of bone marrow failure, include aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) [1]. These diseases differ in the etiopathogenesis and clinical presentations, although treatment options for both still remain a clinician’s nightmare. Damashek, way back in 1960s, first appreciated the common link between AA and PNH i.e., one disorder can evolve into the other. The ‘PNH defect’, whether de novo or secondary (arising in the background of other hematological disorders like AA) is characterized by deficiency or complete lack of more than 20 different membrane proteins, which are covalently attached to the cell membrane via a glycosyl phosphatidyl inositol (GPI) anchor [2]. The absence of GPI anchored complement regulating proteins such as membrane inhibitor of reactive lysis (MIRL or CD 59) and decay accelerating factor (DAF or CD55) is responsible for the phenotype of the disease and same feature is used for diagnostic purposes as well [2]. There is scarcity of data in the literature regarding the frequency and prognostic implications of PNH defect in AA patients [2–4]. Here we elaborate upon our experience on this subject, published earlier as a preliminary communication [4].

A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria

Abstract Paroxysmal nocturnal hemoglobinuria (PNH), an acquired stem cell defect, is underdiagnosed because of its atypical symptoms in some patients and because available methods, which are time consuming and complicated, are not widely used. The hemolysis of PNH red blood cells (RBCs) is attributed to their enhanced susceptibility to complement lysis caused by a deficiency in glycosylsphos-phatidylinositol (GPI)-anchored complement regulatory membrane proteins, especially membrane inhibitor of reactive lysis (MIRL [CD59]). We evaluated the diagnostic value of a simple hemagglutination test using the gel microtyping system by comparing it with lytic tests (the Ham test and the sucrose lysis test) and with flow cytometry (FC) assessment of expression of GPI-anchored proteins (CD59 and CD55). Examining 51 blood samples from 48 patients, we found that the gel test is useful as a screening test for PNH diagnosis and can replace the Ham test and the sucrose lysis test. The threshold of the gel test is about 10 percent of defective RBCs detected by FC. It should, however, be supplemented with FC so as to analyze precisely the defective RBCs and granulocytes in patients with positive gel test results, and, in case of negative results, to detect a small clone of defective cells in atypical cases. Due to the simplicity of the gel test, its wide use can facilitate the diagnosis of PNH. Immunohematology 2002;18:9–12.

Complement-regulatory protein expression and activation of complement cascade on erythrocytes from patients with rheumatoid arthritis (RA).

It has been previously reported that the expression of the complement receptor, CR1, on erythrocytes is reduced in patients with RA and that the reduced expression of CR1 is related to disease activity. In this study we investigate the role of other regulatory proteins, i.e. decay-accelerating factor (DAF) and CD59 (membrane inhibitor of reactive lysis), in the pathogenesis of RA by checking the expression of DAF and CD59 on erythrocytes of RA patients to establish whether reduced expression of DAF and CD59 on erythrocytes could be related to increased ability of erythrocytes to activate complement in RA. Flow cytometry was used to measure the expression of DAF and CD59 on erythrocytes from RA patients as well as the deposition of C3 fragments occurring in vivo or after in vitro complement activation. Significantly reduced expression of DAF and CD59 was observed on erythrocytes of RA patients. A significant inverse relationship was observed between DAF expression and in vitro complement activation, whereas no significant relationship between CD59 and complement activation was observed. Finally, we demonstrated an inverse relationship between CH50 activity and DAF expression. Thus, determination of DAF on erythrocytes can emerge as an additional tool in the assessment of extent of complement activation in RA.

Tears contain the complement regulator CD59 as well as decay-accelerating factor (DAF).

Previous studies have shown that DAF (or CD55), a cell surface inhibitor of autologous C3 activation, is present in tears and that > 90% of the C3 convertase regulatory activity in tear fluid resides in this protein (Lass JH et al., Invest Ophth Vis Sci 1990; 31:1136-48). This study investigated whether (i) the membrane cofactor protein (MCP or CD46), an additional factor that regulates C3 activation, and (ii) the membrane inhibitor of reactive lysis (MIRL or CD59), a cell surface regulator that acts to prevent formation of the membrane attack complex, are also present in tears, and if so, are functional. Two-site immunoradiometric assays showed that MCP is present in tears at low levels (42 + 8 ng/ml, n = 8) while CD59 is present at levels (222 + 78 ng/ml, n = 14) comparable to those of DAF (325 + 289 ng/ml, n = 12). The concentrations of CD59 (i) were increased two-fold or more in closed eye tears, and (ii) were decreased in reflex tears. Western blotting showed that CD59 protein in tears migrates with an apparent mol. wt similar to membrane CD59 protein. Phenyl-Sepharose adsorption and Triton X-114 partitioning of tear CD59 as well as of tear DAF however, showed that both proteins are devoid of GPI anchors. Assays using cobra venom factor-activated human serum and guinea pig erythrocytes showed that CD59 is functionally active in inhibiting autologous C5b-9-mediated lysis and, under constitutive conditions, accounts for > 85% of the C9 inhibitory activity in tear fluid.

Expression of complement regulatory proteins on erythrocytes from patients with idiopathic focal segmental glomerulosclerosis

SUMMARY:Focal segmental glomerulosclerosis (FSGS) is a heterogeneous group of disorders with respect to aetiology, morphology, clinical course and response to treatment. The present study assessed the expression of complement receptor 1 (CR1), decay accelerating factor (DAF) and membrane inhibitor of reactive lysis (CD59) on the erythrocytes of FSGS patients using flow cytometry compared with their expression on the erythrocytes of minimal change nephrotic syndrome (MCNS) patients. Significantly reduced expression of CR1, DAF and CD59 was observed on the erythrocytes of FSGS patients compared with the reduced expression of CR1 and enhanced expression of DAF on the erythrocytes of MCNS patients. A significant inverse relationship was demonstrated between CR1 expression and proteinuria levels in FSGS patients (r = 0.55, P &lt; 0.01). A follow‐up study of 12 patients with FSGS after immunosuppressive therapy showed that the levels of complement regulatory proteins are significantly increased when the disease goes into remission. However, in patients not responding to immunosuppressive therapy, levels of these proteins remained low. MCNS patients showed significant increases in CR1 and decreases in DAF expression during remission of the disease.