[Evaluation of Low Concentration Dithiothreitol for Negating the Monoclonal Anti-CD38 Interference with Transfusion Compatibility Testing].

OBJECTIVE: To investigate the effectiveness and safety of low concentration dithiothreitol (DTT) in removing the interference of monoclonal anti-CD38 on transfusion compatibility testing, and develop a reasonable clinical transfusion strategy. METHODS: The blood type, direct antiglobulin testing (DAT) and antibody screening were tested according to standard methods. Antibody screening cells and donor's red blood cells were treated by DTT 0.2, 0.1, 0.05, 0.02, 0.01 and 0.005 mol/L, and antibody screening and cross-matching of serums after monoclonal anti-CD38 treatment were performed by anti-human globulin card. RESULTS: The 0.01 mol/L DTT at 37℃ for 30 minutes could remove the effect of monoclonal anti-CD38 on antibody screening and cross-matching, meanwhile retain their effectiveness in detecting anti-K, anti-LW, anti-JMH, anti-Lub, anti-e, anti-Dia and anti-Jka alloantibodies. All the 10 patients had no acute or delayed haemolytic transfusion reactions and their routine blood tests showed that the red blood cells transfusion was effective. CONCLUSION: The 0.01 mol/L DTT is a safe and effective method for removing the interference of monoclonal anti-CD38 with transfusion compatibility testing, while retaining the ability to detect most alloantibodies.

A single-center investigational study of CD36 antigen deficiency and platelet alloantibody distribution in different populations in Northern China as well as platelet alloantibodies effect on pregnancy.

BACKGROUND: Platelet antibodies can lead to clinical diseases such as platelet transfusion refractoriness (PTR), fetal/neonatal alloimmune thrombocytopenia (FNAIT), etc. This study is aimed at understanding CD36 expression, platelet alloantibody distribution in different populations in Northern China, and effects of platelet alloantibodies on pregnancy. STUDY DESIGN AND METHODS: Whole blood samples of 612 subjects including hematological patients, pregnant women, and blood donors were collected at a single center, then CD36 expressions were determined, followed by platelet antibody screening and characterization of platelet antibody specificity. A retrospective analysis was performed in 1552 pregnant women admitted to Department of Obstetrics, in order to investigate FNAIT occurrence. RESULTS: Rate of CD36 deficiency expression was 2.12% (13/612), all cases exhibited type II deficiency without type I deficiency being detected, and such rate is lower than that in Southern China (3.43%), Japanese (4.87%) and in the black people (4.18%), and higher than that in the White people (0.09%). Positive rates of platelet antibody screening in hematological patient group (6.86%, 14/204) and in pregnant women group (6.31%, 13/206) are higher than that in blood donor group (0.49%, 1/202), P < .01. Out of 1552 pregnant women, there were not children with FNAIT. CONCLUSION: The frequency of CD36 deficiency in northern China was low, all of them were type II deficiency, and no CD36 antibody was detected. It is speculated that the risk of immune-related thrombocytopenia caused by CD36 deficiency in this population is very low. Platelet antibodies should be monitored early in patients with hematological and multiple miscarriages pregnant.

Anti-HLA antibodies with complementary and synergistic interaction geometries promote classical complement activation on platelets.

High titers of HLA antibodies are associated with platelet refractoriness, causing poor platelet increments after transfusions in a subset of patients with HLA antibodies. Currently, we do not know the biological mechanisms that explain the variability in clinical responses in HLA alloimmunized patients receiving platelet transfusions. Previously we showed that a subset of anti-HLA IgG-antibodies induces FcγRIIa-dependent platelet activation and enhanced phagocytosis. Here, we investigated whether anti-HLA IgG can induce complement activation on platelets. We found that a subset of anti-HLA IgG induced complement activation via the classical pathway, causing C4b and C3b deposition and formation of the membrane-attack complex. This resulted in permeabilization of platelet membranes and increased calcium influx. Complement activation also caused enhanced α-granule release, as measured by CD62P surface exposure. Blocking studies revealed that platelet activation was caused by FcγRIIa-dependent signaling as well as HLA antibody induced complement activation. Synergistic complement activation employing combinations of monoclonal IgGs suggested that assembly of oligomeric IgG complexes strongly promoted complement activation through binding of IgGs to different antigenic determinants on HLA. In agreement with this, we observed that preventing anti-HLA-IgG hexamer formation using an IgG-Fc:Fc blocking peptide, completely inhibited C3b and C4b deposition. Our results show that HLA antibodies can induce complement activation on platelets including membrane attack complex formation, pore formation and calcium influx. We propose that these events can contribute to fast platelet clearance in vivo in patients refractory to platelet transfusions with HLA alloantibodies, who may benefit from functional-platelet matching and treatment with complement inhibitors.

Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis.

BACKGROUND/AIMS: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism. METHODS: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry. RESULTS: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription. CONCLUSION: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN.

Glutathione inhibits antibody and complement-mediated immunologic cell injury via multiple mechanisms.

Antioxidant glutathione (GSH) plays an important role in the regulation of immunity. However, little is known about its effects on humoral immunity, especially its action on effector molecules like antibody and complement. Given that these molecules contain abundant disulfide bonds, we speculated that GSH might influence the action of these proteins via its thiol function. Using a model of a glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we addressed this hypothesis. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused a complement-dependent cell lysis, which was completely blocked by GSH. Moreover, GSH potently prevented the antibody-mediated agglutination of red blood cells and aggregation of antibody-sensitized microspheres. Further analysis revealed that GSH inhibited antibody binding to antigens and promoted the conversion of the antibodies to its reduced forms. GSH also potently inhibited the formation and deposition of C5b-9 in MCs and suppressed both the classic and alternative complement activation pathway. Lastly, GSH attenuated P38 activation, an oxidative sensitive kinase that partially mediated the antibody- and complement-dependent MC lysis. Depletion of GSH via inhibiting gamma-glutamylcysteine synthetase or xCT transporter augmented P38 activation and sensitized MCs to the cell lysis. Collectively, our results indicate that GSH protects cells from immunological cell damage via mechanisms involving inhibition of antibody binding to the antigens, suppression of complement activation and augmentation of cellular defense mechanism. Our study provides novel mechanistic insights into the actions of GSH in the regulation of immune responses and suggests that GSH might be used to treat certain immune disorders.

Epithelial IL-23R Signaling Licenses Protective IL-22 Responses in Intestinal Inflammation.

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.

Anti-HI can cause a severe delayed hemolytic transfusion reaction with hyperhemolysis in sickle cell disease patients.

BACKGROUND: Delayed hemolytic transfusion reaction (DHTR) is a life-threatening condition in sickle cell disease (SCD) patients that is frequently complicated by hyperhemolysis. Antibodies resulting from antigen disparity between donors of European ancestry and patients of African ancestry are common, but situations involving antibodies not classically of clinical significance are also encountered. Anti-HI is generally considered to be an innocuous naturally occurring antibody. STUDY DESIGN AND METHODS: We describe two cases of hyperhemolysis with anti-HI and provide details of the reported cases. RESULTS: Both SCD patients were polyimmunized and belonged to blood group B. They developed anti-HI that was reactive at 37°C, after the transfusion of group O red blood cell units matched for all known and produced antibodies classically considered to be clinically significant. Both patients developed DHTR with hyperhemolysis. In the first case, a pregnant woman, a second transfusion was unavoidable and the patient died from cardiac arrest. The state of the second patient improved without the need for further transfusion. CONCLUSION: Three other cases of DHTR with anti-HI have been described in the literature in SCD patients. The two additional cases reported here definitively demonstrate that anti-HI is dangerous in SCD patients. As a result, ABO-identical matching (including A1 status) must be considered in SCD patients with anti-HI.

Suramin inhibits antibody binding to cell surface antigens and disrupts complement-mediated mesangial cell lysis.

Suramin inhibits immune responses and protects cells against inflammatory cell injury. However, little is known about its mechanisms. Using an in vitro model of glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we investigated the potential protective effects and mechanisms of suramin on immunologic cell injury. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused complement-dependent cell lysis, which was blocked by suramin and its structural analogue NF023 and NF049, but not by PPADS, an antagonist of purinergic receptors. Addition of exogenous ATP also failed to affect MC lysis. Further analysis revealed that suramin interfered with antibody binding to cell membrane antigens and suppressed antibody-induced phosphorylation of several proteins, including p38. Inhibition of p38 with chemical inhibitor significantly attenuated cell injury. Collectively, our results indicate that suramin protects cells against antibody-initiated and complement-dependent cell injury through inhibition of antibody binding to cell surface antigens and suppression of p38 activation. Our study thus provides novel mechanistic insights into the actions of suramin and suggests that suramin might be used to treat certain immune diseases.

Epidermal growth factor receptor inhibition with erlotinib ameliorates anti-Thy 1.1-induced experimental glomerulonephritis.

BACKGROUND: Mesangial proliferative glomerulonephritis is a common glomerular disorder that may lead to end-stage renal disease. Epidermal growth factor (EGF) plays an important role in the regulation of cell growth, proliferation, and differentiation and in the pathology of various renal diseases. Erlotinib is a novel, oral, highly selective tyrosine kinase inhibitor of the EGF receptor. It is clinically used to treat non-small cell lung and pancreatic cancers. Here, we investigated the effect of erlotinib on the progression of mesangioproliferative glomerulonephritis in an experimental model. METHODS: Mesangial glomerulonephritis was induced with anti-rat Thy-1.1 antibody in male Wistar rats weighing 150-160 g. Rats were treated with erlotinib (10 mg/kg/day p.o.) or vehicle only (polyethylene glycol). Native Wistar rat kidneys were used as histological controls. Serum creatinine levels were measured at day 7. Kidneys were harvested 7 days after antibody administration for histology. RESULTS: Native controls showed no histological signs of glomerular pathology. In the vehicle group, intense glomerular inflammation developed after 7 days and prominent mesangial cell proliferation and glomerular matrix accumulation was seen. Erlotinib was well tolerated and there were no adverse effects during the follow-up period. Erlotinib significantly prevented progression of the glomerular inflammatory response and glomerular mesangial cell proliferation as well as matrix accumulation when compared with the vehicle group. Erlotinib also preserved renal function. CONCLUSION: These results indicate that erlotinib prevents the early events of experimental mesangial proliferative glomerulonephritis. Therefore, inhibition of the EGF receptor with erlotinib could prevent the progression of glomerulonephritis also in clinical nephrology.

A monoclonal antibody with anti-D-like activity in murine immune thrombocytopenia requires Fc domain function for immune thrombocytopenia ameliorative effects.

BACKGROUND: The mechanism of action of anti-D in ameliorating immune thrombocytopenia (ITP) remains unclear. The monoclonal antibody (MoAb) Ter119, which targets murine red blood cells (RBCs), has been shown to mimic the effect of anti-D in improving antibody-mediated murine ITP. The mechanism of Ter119-mediated ITP amelioration, especially the role of the antigen-binding and Fc domains, remains untested. A functional Fc domain is crucial for many therapeutic MoAb activity; therefore, the requirement of Ter119 Fc domain in ITP amelioration is investigated using outbred CD-1 mice. STUDY DESIGN AND METHODS: Ter119 variants, including Ter119 F(ab')2 fragments, deglycosylated Ter119, and afucosylated Ter119, were generated to test their effect in ameliorating antibody-induced murine ITP. In vivo inhibition of FcγRIII and FcγRIIB was achieved using the Fab fragment of the FcγRIII/FcγRIIB-specific MoAb 2.4G2. RESULTS: Ter119 F(ab')2 fragments and deglycosylated Ter119 were unable to ameliorate murine ITP or mediate phagocytosis of RBCs by RAW264.7 macrophages in vitro. Inhibition of FcγRIII and FcγRIIB, as well as Ter119 defucosylation, do not affect Ter119-mediated ITP amelioration. CONCLUSION: The Fc domain of Ter119, as well as its Fc glycosylation, is required for Ter119-mediated ITP amelioration. Moreover, both Fc and Fc glycosylation are required for Ter119-mediated phagocytosis in vitro. These findings demonstrate the importance of the Fc domain in a therapeutic MoAb with anti-D-like activity.