Reactive Lysis Research Articles

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.

Effectiveness of Phytoactive Molecules on Transcriptional Expression, Biofilm Matrix, and Cell Wall Components of Candida glabrata and Its Clinical Isolates.

Toxicity challenges by antifungal arsenals and emergence of multidrug resistance scenario has posed a serious threat to global community. To cope up with this alarming situation, phytoactive molecules are richest, safest, and most effective source of broad spectrum antimicrobial compounds. In the present investigation, six phytoactive molecules [cinnamaldehyde (CIN), epigallocatechin, vanillin, eugenol (EUG), furanone, and epigallocatechin gallate] were studied against Candida glabrata and its clinical isolates. Among these, CIN and EUG which are active components of cinnamon and clove essential oils, respectively, exhibited maximum inhibition against planktonic growth of C. glabrata at a concentration of 64 and 128 μg mL–1, respectively. These two molecules effectively inhibited and eradicated approximately 80% biofilm of C. glabrata and its clinical isolates from biomaterials. CIN and EUG increased reactive oxygen species generation, cell lysis, and ergosterol content in plasma membrane and reduced virulence attributes (phospholipase and proteinase) as well as catalase activity of C. glabrata cells. Reduction of mitochondrial membrane potential with increased release of cytochrome c from mitochondria to cytosol indicated initiation of early apoptosis in CIN- and EUG-treated C. glabrata cells. Transcriptional analysis showed that multidrug transporter (CDR1) and ergosterol biosynthesis genes were downregulated in the presence of CIN, while getting upregulated in EUG-treated cells. Interestingly, genes such as 1,3-β-glucan synthase (FKS1), GPI-anchored protein (KRE1), and sterol importer (AUS1) were downregulated upon treatment of CIN/EUG. These results provided molecular-level insights about the antifungal mechanism of CIN and EUG against C. glabrata including its resistant clinical isolate. The current data established that CIN and EUG can be potentially formulated in new antifungal strategies.

RO7112689, a pH-switch anti-C5 mAb, for efficient cross-species C5 purification and inhibition of classical and reactive lysis

In view of realizing the economic viability, we fabricate a solar cell device containing low band gap and easily processable polymer 5-yl-8-(thiophene-2,5-diyl)-2,3-bis(3-(octyloxy)phenyl) quinoxaline (TQ1) and CdSe nanoparticles (NPs) and investigate its charge transport properties. When the TQ1 is combined with the CdSe NPs a strong photoluminescence quenching and shortening of photoluminescence lifetime of the TQ1 is observed indicating exciton transfer from TQ1 to the CdSe NPs. The time-resolved photoluminescence further reveals that the exciton transfer from the polymer to CdSe NPs is very efficient (68%) and it occurs in <1 ns. The exciton transfer from TQ1 to the NPs and electron-hole pair separation followed by hole transfer from the NPs to the TQ1 at the interface indeed increases the lifetime of the charge carriers. This in turn increases the efficiency of the solar cell as compared to polymer only device. These observations suggest the importance of other II-VI semiconductor NPs to achieve higher efficiency for photovoltaic devices containing TQ1 polymer.

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.

Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis

SummaryC5 plays a major role in complement activation; C5 convertase cleaves C5 into the pro‐inflammatory C5a, and C5b, the nidus for the formation of the lytic membrane attack complex. C5 is a major target for anti‐complement drugs, necessitating better methods for the study of C5 function. Purification of C5 is complicated; classical methods involve precipitation or pH shifts that result in functional loss and low yield. We here present a method for C5 purification using a novel anti‐C5 monoclonal antibody (mAb); RO7112689 (C5i mAb, SKY59), pH‐switch engineered to induce antibody–antigen dissociation in the acidic endosome (~ pH 5·5). RO7112689 was bound on an affinity column; applied serum was completely depleted of C5. Elution at pH 5 produced fully active C5 at 98% yield. The mAb also bound C5b in the C5b6 complex, preventing C5b6 binding to target membranes and enabling purification of C5b6 from activated serum. RO7112689 inhibited C5 in mouse serum and efficiently purified mouse C5. Used as capture, RO7112689 produced sensitive and specific assays for human and mouse C5. This novel antibody enables efficient production of intact, fully active, pure human and mouse C5, and quantification of C5 in these species. The demonstration that RO7112689 binds C5b6 adds an additional mechanism of membrane attack complex inhibition by this mAb.

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.

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-(2)H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the (2)H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Stable-isotope probing and metagenomics reveal predation by protozoa drives E. coli removal in slow sand filters.

Stable-isotope probing and metagenomics were applied to study samples taken from laboratory-scale slow sand filters 0.5, 1, 2, 3 and 4 h after challenging with (13)C-labelled Escherichia coli to determine the mechanisms and organisms responsible for coliform removal. Before spiking, the filters had been continuously operated for 7 weeks using water from the River Kelvin, Glasgow as their influent source. Direct counts and quantitative PCR assays revealed a clear predator-prey response between protozoa and E. coli. The importance of top-down trophic-interactions was confirmed by metagenomic analysis, identifying several protozoan and viral species connected to E. coli attrition, with protozoan grazing responsible for the majority of the removal. In addition to top-down mechanisms, indirect mechanisms, such as algal reactive oxygen species-induced lysis, and mutualistic interactions between algae and fungi, were also associated with coliform removal. The findings significantly further our understanding of the processes and trophic interactions underpinning E. coli removal. This study provides an example for similar studies, and the opportunity to better understand, manage and enhance E. coli removal by allowing the creation of more complex trophic interaction models.

A universal method for measuring functional activity of complement in humans, laboratory, domestic, and agricultural animals, amphibians, and birds.

A new universal method for measuring activity of the serum complement system in humans, laboratory, domestic, agricultural animals, birds and amphibians is based on automated evaluation of the mortality of ciliate Tetrahymena pyriformis under the effect of the complement system. In contrast to the hemolytic method, measured activity of the complement shows no erroneously high results caused by reactive lysis in febrile patients. The method can be used for studies of the complement system in humans and animals without species-specific adaptation.

Hyperhaemolysis syndrome responsive to splenectomy in a patient with δβ-thalassaemia: a discussion on underlying mechanisms.

Hyperhaemolysis syndrome responsive to splenectomy in a patient with δβ-thalassaemia: a discussion on underlying mechanisms.

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

A multicenter study of cellulite treatment with a variable emission radio frequency system

Radio frequency (RF) systems have been reported as producing electrothermally mediated and subcutaneous effects. The present study evaluates a new approach to treat cellulite with a bipolar RF device. The buttocks of 50 patients were treated, 10 from each of five multinational centers with a novel bipolar RF technology set at 6 J/cm(3), which changes its frequency between 0.6 and 2.4 MHz according to impedance of tissue. Twelve weekly sessions were given for 12 minutes on each buttock, with a treatment end point of 42 degrees C external skin temperature. Cellulite changes and tissue condition were assessed before and immediately after the first session, before the final 12th session, and 2 months thereafter. The patient Satisfaction Index was recorded. Objective evaluation involved clinical photography, three-dimensional optical skin surface measurement, and histological findings. Almost all patients noted improvement of cellulite and body silhouette at the final session, which slightly decreased at the 2-month assessment. Improved skin appearance was objectively detected. Histological findings following the first session showed reactive edema and lysis of adipocyte membranes, possibly implicated in the final effects achieved. The RF technology used in the present multicenter study improved the general aspect of skin and cellulite, with high patient Satisfaction Index. Maintenance sessions might lead to even better and longer-lasting results.

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.