Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis

BackgroundDrug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin’s lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression.MethodsImmunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH.ResultsWe demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues.ConclusionsOur findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.

Altered Expression Levels of CD59, but Not CD55, on Red Blood Cells in Stored Blood

Objective: Red blood cells (RBCs) in storage undergo structural and biochemical changes that may cause functional effects. Studies exploring structural changes affecting the expression levels of CD55 and CD59 on RBCs are limited. The aim of this study was to investigate the pattern of CD55 and CD59 expression on RBCs in stored blood from Arab donors. Materials and Methods: Flow-cytometric analysis was performed on RBCs from 92 packed RBC (PRBC) units, stored for varying times, and from 56 nonstored RBC from healthy controls using the commercial REDQUANT kit. Results: The proportions of CD55- and CD59-deficient RBCs from stored PRBC units did not significantly differ when compared with those from healthy controls; however, the mean fluorescent intensity (MFI) of CD59 expression, but not MFI of CD55 expression, on RBCs from stored PRBC units was significantly reduced when compared to the expression of RBCs from healthy controls (p = 0.02). MFI of CD55 expression on RBCs from PRBC units did not significantly differ among the 3 groups of stored RBC; however, there was a statistically significant time-dependent preferential decline in MFI of CD59 expression on RBCs from stored PRBC units (p < 0.01). Conclusion: There is a preferential time-dependent decline in the expression of CD59, but not of CD55, on stored RBCs, the in vivo significance of which in relation to the response to PRBC transfusion needs further investigation.

Expression Pattern of CD55 and CD59 on Red Blood Cells in Sickle Cell Disease

Background: Altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, might result in increased susceptibility of red blood cells (RBCs) to complement-mediated lysis. Limited information is available on the pattern of CD55 and CD59 expression on RBCs of sickle cell disease (SCD) patients. Methods: Flow cytometric analysis was performed on RBCs from 71 adult SCD patients and 53 healthy controls, using the commercial REDQUANT kit. Results: CD59 deficiency was significantly higher among SCD patients than among healthy controls. The proportions of CD55-deficient and CD59-deficient RBCs from SCD patients were significantly higher when compared with those from healthy controls (0.17 vs. 0.09 and 2.1 vs. 1.2, respectively). The MFI of CD55 and CD59 expression on RBCs in SCD was significantly reduced when compared to the expression healthy controls (5.2 vs. 6.4 and 19.4 vs 20.3, respectively). The pattern of CD55 and CD59 expression was not correlated with anemia, biomarkers of hemolysis, erythropoietin level or other pro-inflammatory markers. Conclusions: There is an altered pattern of CD55 and CD59 expression on RBCs of SCD Patients; however, it does not seem to play a causal role in the pathophysiology of anemia, and is unlikely to be influenced by the level of erythropoietin or other inflammatory mediators. Disclosures No relevant conflicts of interest to declare.

Analysis of Platelets By Flow Cytometry in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by a clonal population of hematopoietic stem cells with an acquired somatic mutation in the PIG-A gene, giving rise to populations of circulating mature cells that are unable to synthesize glycosylphosphatidylinositol (GPI). The disease is most readily diagnosed by flow cytometry analysis of red blood cells, using antibodies specific for the GPI-linked protein CD59, or analysis of granulocytes, using antibodies specific for the GPI-linked protein CD24, along with the FLAER reagent, a fluorescent protein that binds to the GPI structure and which is detected only on the surface of GPI (+) cells. However, other mature blood lineages can be derived from the PNH clone. Notably, thrombosis is a major life threatening complication of PNH and may be triggered by complement activation on platelets that belong to the GPI-negative stem cell clone. The PNH clone size generally predicts thrombosis, but sometimes the proportion of PNH red cells and granulocytes are highly discordant, in which case there might be a role for the determination of the proportion of PNH platelets. Historically, flow cytometry analysis of platelets in patients with PNH has been technically difficult. Here is described a method to do this that avoids technical challenges by using aspirin and sepharose gel filtration of platelets to prevent their activation as well as simultaneous determination of CD59 expression and uptake of the FLAER reagent. Red cells were analyzed based on CD59 expression and granulocytes based on CD24 and FLAER. We analyzed blood samples from 48 patients with PNH and or AA/PNH who provided informed consent, 16 of whom had a prior history of thrombosis. To separate platelet rich plasma (PRP), whole blood collected in EDTA tubes was centrifuged at 200g for 7 minutes at room temperature with the brake turned off. After this step, there was no further centrifugation or vortexing of the platelets. A solution of aspirin was made up immediately prior to use and was added to the PRP at a final concentration of 0.5mMolar. Aspirinated PRP was then loaded on top of a sepharose-2B column using Tyrode's buffer. The platelet-rich turbid drops were collected, to isolate platelets from red cells and coagulation proteins. 50 ul of platelet rich buffer was then incubated with FLAER-Alexa-488 (Pinewood, 1:20 dilution) and CD59-PE (Serotec, 1:10 dilution) in the dark for 30' at room temperature. To prevent doublet events from confounding the analysis, the platelet suspension was diluted 1:200 in Hanks with 0.1% BSA. The sample was passed through a 35 uM Falcon cell strainer, and platelets were identified by forward/side scatter acquired on a log-log scale on a BD Facscan. The median proportion of PNH red cells, granulocytes and platelets was 24%, 86%, 76% respectively in the group without a history of thrombosis and 23% ,82%, and 65% in the group with a history of thrombosis. The proportion of PNH platelets was highly correlated with the proportion of PNH granulocytes (r=0.84). In two patients with almost undetectable PNH red cells and over 90% PNH granulocytes, the proportion of PNH platelets was over 90%; both were on prophylaxis and neither had thrombosis. It is predicted that this technique may be useful for determining thrombosis risk, particularly when the results from the analysis of rbc's and granulocytes are discordant. Disclosures No relevant conflicts of interest to declare.

Presence Of CD55- and/Or CD59-Deficient Erythrocytes In Patients With Rheumatic Diseases: An Immune-Mediated Phenomenon?

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by the somatic mutation of X-linked gene PIG-A, required for glycosylphosphatidylinositol (GPI)-anchor biosynthesis. This results in absent or decreased expression of all membrane proteins normally anchored by GPI - including CD55 and CD59 - in all circulating cells, leading to an unusual sensitivity of red blood cells (RBCs) to complement lysis and subsequently intravascular hemolysis and hemoglobinuria. According to the “dual pathogenesis” model, there is an immunoregulatory selection in favor of PNH clones to proliferate preferentially over normal hemopoiesis on a microenvironment of bone marrow failure. The incidence of “PNH-like” defect has been also demonstrated in many hematological diseases and on peripheral blood cells (PBC) of normal individuals. Complement system is recognized as having the potential to provoke severe impairment to host tissues. This is extensively demonstrated in autoimmune disease setting. Multiple regulatory and inhibitory enzymes, such as CD55 and CD59, known as complement regulatory proteins, adjust the progression of complement cascade at all levels, protecting the autologous cells. Complement activation and cytopenias have been associated with diminished CD55 and/or CD59 expression on PBC membranes. The aim of this study was to evaluate the presence of “PNH-like” red-cell populations in patients with rheumatic diseases and investigate possible correlations with clinical or laboratory parameters. CD55 and CD59 expression was evaluated in erythrocytes of 113 patients (94 females, 19 males, median age: 64 years) with rheumatic diseases: 38 with rheumatoid arthritis, 25 with systemic lupus erythematosus, 17 with Sjögren’s syndrome, 7 with systemic sclerosis, 12 with vasculitis, 2 with dermatomyositis, 1 with ankylosing spondylitis and 11 with mixed connective tissue diseas, using the sephacryl-gel microtyping system, a semi-quantitative, inexpensive and simple method useful in screening “PNH-like” red-cell defect, with sensitivity comparable with that of flow cytometry. One hundred and twenty-one (121) healthy blood donors of similar age and gender and 10 patients with PNH were also studied, as control groups. In all samples with CD55- and/or CD59- negative RBCs, Ham and sucrose tests were also performed. Interestingly, the majority of patients (104/113, 92%) demonstrated “PNH-like” erythrocytic populations: 47 (41.6%) with concomitant deficiency of CD55 and CD59, 50 (44.2%) with isolated deficiency of CD55 and 6 (6.2%) with isolated deficiency of CD59. In healthy donors, only 2 (1%) had red cells with concomitant CD55/CD59 negativity and 3 (2%) with isolated CD55 or CD59 deficiency. “PNH-like” erythrocytic clones never surpassed 25% of the total red-cell population, while the most common proportion of deficiency for both antigens was 10%. All PNH patients exhibited simultaneous CD55/CD59 deficiency. Moreover, it should be high-lightened that we found an unprecedented relation between patients' hemoglobin (Hb) and CD55 expression on RBCs (rs= -0.205, p=0.029), while there was a significant difference (δ) when the mean concentration of Hb was compared between patients with normal expression of CD55 and those with deficiency of this protein (δ=-1.4534 g/dl, p=0.0151). There was no clinical or laboratory evidence of hemolysis in our patients. There was no association between the presence of “PNH-like” red-cell populations and cytopenias or specific treatment for the autoimmune disorder. Positive Ham and sucrose tests were found only in PNH patients. In conclusion, this study provides evidence supporting the presence of erythrocytes with CD55- and/or CD59- deficiency in patients with rheumatic diseases. The pre-existence of small PNH clones in the bone marrow of these patients, that acquire a survival advantage to proliferate against normal hemopoietic tissue and become detectable with our methodology, may be the underlying cause for this phenomenon. Moreover, it was demonstrated that CD55- deficiency on RBCs influences the levels of Hb, in these patients. Further studies, using molecular techniques, will be required, to clarify the exact pathophysiologic mechanisms for this deficiency. Disclosures: No relevant conflicts of interest to declare.